Using Novel Spectroscopy Tool to Study Organized Self

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8-2019

Using Novel Spectroscopy Tool to Study Organized Self-Using Novel Spectroscopy Tool to Study Organized Self-

Assemblies Assemblies

Mohammad Rafe M Bin Hatshan Western Michigan University, [email protected]

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USING NOVEL SPECTROSCOPY TOOL TO STUDY ORGANIZED

SELF-ASSEMBLIES

by

Mohammad Rafe M Bin Hatshan

A dissertation submitted to the Graduate College

in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Chemistry

Western Michigan University

August 2019

Doctoral Committee:

Ramakrishna Guda, Ph.D., Chair

Ekkehard Sinn, Ph.D.

Sherine Obare, Ph.D.

Pamela Hoppe, Ph.D.

Copyright by

Mohammad Rafe M Bin Hatshan

2019

ii

DEDICATION

This dissertation is lovingly dedicated to my mother and my father, for their support,

encouragement, prayer and constant love that has sustained me throughout my life. This

dedication is less than my parent right on me. Dear parent, you are the reasons behind my

success.

To my family - my wife and my kids - for their support, love, and for providing a good

environment in which to work. I know you were the most affected by my PhD journey, my

success is because your sacrifices and support. The dream of my life is to please you and make

you proud of me.

iii

ACKNOWLEDGEMENTS

I indeed could not have written this dissertation without the help of Allah “God

Almighty”, who is the best of helpers. I am indebted to my committee members for their

guidance, the prayers of my parents, and support from my family and friends. Without any of

these, it was beyond my capacity to finish this journey.

Foremost I would like to express my sincere and deepest gratitude and appreciation to my

advisor, Dr. Ramakrishna Guda for his excellent guidance, continuous unlimited support,

motivation, enthusiasm, patience and immense knowledge which enabled me to accomplish this

work. It has been an honor for me that I worked under a distinguished advisor who believes in

his students and encourages them to be a scientist. Dear Dr Guda, I have learned from you many

things that hard to list here, you were a great mentor and friend, I hope to be like you one day,

especially in your passion for teaching and your constant concern and care for those around you.

I would like to acknowledge with much appreciation my committee members Dr.

Ekkehard Sinn, Dr. Sherine Obare, Dr. Pamela Hoppe for their kind encouragement, great

thoughts and insightful comments that made this work the best it could be. Thanks for your

presence and cooperation despite your busy work.

My heartfelt appreciation goes out to Allah and then to my family members: my parent

my wife my kids, and my brothers and sisters for their prayers, endless love and extremely

supportive all over PhD journey.

iv

Acknowledgements—Continued

My appreciation goes to King Saud University for giving me the opportunity to pursue

this degree and to all my work colleagues who made it possible. I am grateful to the faculty and

staff of the Chemistry Department at Western Michigan University at Kalamazoo for being

supportive during my time here. Special thanks are due to Pamela A McCartney, Robin K

Lenkart, Michelle McBarnes and Lisah N Crall for their support regarding the administration

stuff.

In my daily work I have been blessed with a friendly and cheerful group of fellow

students. I am grateful for the past and present members of both Dr. Guda’s research group who

have been invaluable for practical help, support and suggestion, include: Dr. Ibtesam Alja’afreh

Dr. Jameel Hasan, Dr. Edwin Mghanga, Dr. Saad Alotaibi, Dr. Viraj Thanthirige, Dr. Abu Bakar

Abu Hagar. In addition, I have had a pleasure of working with undergraduate students (Amanda

Osborne, Jasmine Sarmiento, Arju Patel, Jason Hugan) and high school students (Natasha

Goenawan, Ziyan Mo, Nathaniel Goenawan, Liya Jin, Karthik Subramanian).

Finally, for anyone wished me success, thank you.

Mohammad Rafe Bin Hatashan

USING NOVEL SPECTROSCOPY TOOL TO STUDY ORGANIZED

SELF-ASSEMBLIES

Mohammad Rafe M Bin Hatshan, Ph.D.

Western Michigan University, 2019

Organized self-assemblies are the cornerstones for countless biological processes and are

integral parts of lipids, proteins, carbohydrates, nucleic acids, and cell membranes. Several man-

made organized assemblies also play a vital role in interdisciplinary sciences that include

micelles, reverse micelles, polymers, polyelectrolytes etc. Weak chemical interactions such as

hydrogen bonding, p-p stacking, hydrophilic-hydrophobic and electrostatics often result in

interesting organized self-assemblies. Understanding organized self-assemblies provides a huge

opportunity to mimic naturally occurring biological macromolecules, design materials and

develop strategies for specific applications. Several techniques are routinely used to understand

the organized self-assemblies, and optical techniques play an important role in most of them.

However, the search for novel optical techniques to monitor organized self-assemblies is on-

going, which is the main motivation behind the investigations carried out in this study. The

research approach is to use the power of two-photon absorption (2PA) spectroscopy to monitor

the organized assemblies.

The hypothesis is that specific non-canonical forms of organized self-assembly provide

unique local electric fields and the 2PA cross-sections of chromophores solubilized in this

environment can be altered by these local electric fields providing an efficient probe to study

them. To test the hypothesis, investigations were carried out using known DNA binders with two

non-canonical structures of DNA that were implicated for cancer, G-Quadruplex and G-Triplex

DNA, in an attempt to use the 2PA cross-sections of the binders to monitor the melting and

stability of these organized self-assemblies. The results have conclusively shown that 2PA cross-

sections of the chromophores were sensitive to monitor the melting transitions in G-Quadruplex

and G-Triplex with different enhancements that can be assigned to electrostatic and aromatic

interactions of the drug binders with DNA. These results have enabled us to use the power of

2PA spectroscopy to monitor protein folding and aggregation of a model protein, bovine-serum

albumin (BSA), and a protein implicated in Amyotrophic Lateral Sclerosis (ALS) disease

superoxide dismutase (SOD1). The results have shown that the 2PA cross-sections of

fluorescamine bound to these proteins is a valuable probe to monitor the folding and unfolding as

well as follow the early onset of aggregation in these proteins. To build on understanding the

organized self-assemblies using two-photon fluorescence spectroscopy, a novel one- and two-

photon fluorescence-based biosensor with a specific oligonucleotide that forms a G-Quadruplex

was developed to selectively and with sensitivity detect toxic metal ions such as Pb2+ and Hg2+.

Guanine-rich nucleotide T30695 was designed to bind to Pb2+ and Hg2+ selectively and used the

one- and two-photon fluorescence of cyanine chromophore to detect different levels of these

toxic metal ions, and a detection limit of 4.5 ppb for Pb2+ and 5.0 ppb for Hg2+ was realized from

the studies. We have also used the power of 2PA spectroscopy to monitor the local environment

in synthetic organized self-assemblies like polyelectrolytes. The electrostatic interactions

between the chromophore and polyelectrolytes were able to enhance the 2PA cross-sections of

chromophores and have shown that this technique is quite universal and can be used to monitor

several organized self-assemblies.

v

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................................... iii

LIST OF TABLES ....................................................................................................................... xvi

LIST OF FIGURES .................................................................................................................... xvii

1. INTRODUCTION .......................................................................................................................1

1.1 Organized Self-Assemblies ...............................................................................................1

1.2 Chemical Interactions in Organized Self-Assemblies ......................................................3

1.2.1 Hydrogen Bonding ................................................................................................4

1.2.2 Ionic Bonds ...........................................................................................................5

1.2.3 π-π Stacking ..........................................................................................................5

1.2.4 Van der Walls Interaction .....................................................................................6

1.2.5 Hydrophilic/Hydrophobic Interactions .................................................................7

1.2.6 Electrostatic Interactions .......................................................................................8

1.3 Organized Self-Assemblies—Examples ...........................................................................9

1.3.1 Micelles .................................................................................................................9

1.3.2 Reverse Micelles .................................................................................................10

1.3.3 Lipid Bilayers......................................................................................................11

1.3.4 DNA ....................................................................................................................12

1.3.5 Proteins ...............................................................................................................13

1.3.6 Polymers and Polyelectrolytes ............................................................................15

1.4 Why Are Organized Self-Assemblies Important? ..........................................................15

vi

Table of Contents—Continued

CHAPTER

1.5 Existing Techniques to Monitor Organized Self-Assemblies .........................................16

1.5.1 Affinity Capillary Electrophoresis ......................................................................16

1.5.2 Enzyme-Linked Immunosorbent Assay ..............................................................17

1.5.3 Fluorescence Resonance Energy Transfer ..........................................................17

1.5.4 Isothermal Calorimetry .......................................................................................18

1.5.5 Ultraviolet Visible Spectroscopy ........................................................................19

1.5.6 Nuclear Magnetic Resonance .............................................................................19

1.5.7 Surface Plasmon Resonance ...............................................................................20

1.6 Research Motivation .......................................................................................................20

1.7 Sensing Organized Self-Assemblies with Dye Molecules and 2PA Spectroscopy ........21

1.8 Research Approach Using 2PA Cross-Sections .............................................................22

1.8.1 Principle ..............................................................................................................22

1.8.2 Applications of 2PA ............................................................................................23

1.9 Aims and Objectives of the Dissertation ........................................................................25

1.10 Outline of the Dissertation ............................................................................................26

1.11 Chapter 1 Summary ......................................................................................................28

1.12 References .....................................................................................................................30

2. EXPERIMENTAL TECHNIQUES ...........................................................................................44

2.1 Lasers ..............................................................................................................................44

2.1.1 Basic Components of Laser ................................................................................44

vii


CHAPTER

2.1.1.1 Optical Cavity .........................................................................................45

2.1.1.2 Gain Medium ..........................................................................................46

2.1.1.3 Pumping Mechanism ..............................................................................46

2.1.2 Properties of Laser Light ....................................................................................47

2.1.2.1 Monochromaticity ...................................................................................47

2.1.2.2 Coherence ...............................................................................................48

2.1.2.3 Directionality ..........................................................................................49

2.1.2.4 Ultrashort Pulse Duration .......................................................................50

2.2 Steady-State Methods .....................................................................................................53

2.2.1 Optical Absorption ..............................................................................................53

2.2.2 Steady-State and Time-Resolved Fluorescence Measurements .........................54

2.3 Two-Photon Absorption..................................................................................................58

2.3.1 Measuring 2PA Cross-Sections ..........................................................................59

2.4 Circular Dichroism..........................................................................................................61

2.5 Dynamic Light Scattering ...............................................................................................64

2.5.1 How DLS Works.................................................................................................66

2.6 Chapter 2 Summary ........................................................................................................68

2.7 References .......................................................................................................................70

3. ANALYZING THE STABILITY AND INTERACTIONS OF G-QUADRUPLEX

AND G-TRIPLEX DNA IN DRUG SYSTEMS BY USING NOVEL OPTICAL

SPECTROSCOPY TECHNIQUES ...............................................................................................79

3.1 Introduction .....................................................................................................................79

viii


CHAPTER

3.2 Materials and Methods ....................................................................................................86

3.2.1 Materials .............................................................................................................86

3.2.2 Optical Methods ..................................................................................................86

3.3 Results and Discussion ...................................................................................................87

3.3.1 Naked G-Quadruplex ..........................................................................................87

3.3.1.1 Optical Absorption Measurements .........................................................87

3.3.1.2 CD Measurements ...................................................................................87

3.3.2 Naked G-Triplex ................................................................................................89

3.3.2.1 Optical Absorption Measurements .........................................................89

3.3.2.2 CD Measurements ...................................................................................89

3.3.3 Interaction with ThT ..........................................................................................90

3.3.3.1 G-Quadruplex/ThT .................................................................................90

3.3.3.1.1 Optical Absorption Measurements .............................................90

3.3.3.1.2 Steady-State Fluorescence Measurements ..................................91

3.3.3.1.3 Two-Photon Fluorescence Measurements ..................................92

3.3.3.1.4 Relative 2PA Enhancements .......................................................93

3.3.3.2 G-Triplex/ThT.........................................................................................94




ix


CHAPTER

3.3.3.2.4 Relative 2PA Measurements .......................................................97

3.3.4 Interaction with ThO ...........................................................................................98

3.3.4.1 G-Quadruplex/ThO .................................................................................98




3.3.4.1.4 Relative 2PA Cross-Sections ....................................................100

3.3.4.2 G-Triplex/ThO ......................................................................................101

3.3.4.2.1 Optical Absorption Measurements ...........................................101

3.3.4.2.2 One-Photon Fluorescence Measurements .................................102

3.3.4.2.3 Two-Photon Fluorescence Measurements ................................102


3.3.5 Interaction with DAPI .......................................................................................105

3.3.5.1 G-Quadruplex/DAPI .............................................................................105


3.3.5.1.2 Steady-State Fluorescence Measurements ................................106



3.3.5.2 DAPI/Triplex ........................................................................................108


x


CHAPTER




3.3.6 Interaction with EtBr.........................................................................................111

3.3.6.1 G-Triplex/EtBr ......................................................................................111





3.3.7 Interaction with TMPyP4 ..................................................................................115

3.3.7.1 G-Quadruplex/TMPyP4 ........................................................................115




3.3.7.1.4 Relative 2PA Measurements .....................................................118

3.3.7.2 G-Triplex/TMPyP4 ...............................................................................118





xi


CHAPTER

3.3.8 Interaction with Doxorubicin ............................................................................121

3.3.8.1 G-Triplex/Doxorubicin .........................................................................121





3.3.9 Mechanism Why Are 2PA Cross-Sections Showing Melting Transition? .......124

3.4 Conclusion ....................................................................................................................129

3.5 Chapter Summary .........................................................................................................130

3.6 References .....................................................................................................................132

4. MONITORING PROTEIN AGGREGATION VIA TWO-PHOTON SPECTROSCOPY:

EARLY DETECTION OF AGGREGATES ...............................................................................139

4.1 Introduction ...................................................................................................................139

4.2 Materials and Methods ..................................................................................................144

4.2.1 Materials ...........................................................................................................144

4.2.2 Optical Methods ................................................................................................144

4.3 Results and Discussion .................................................................................................145

4.3.1 Fluram Binding with BSA and SOD1 ..............................................................145

4.3.2 Protein Folding/Unfolding ................................................................................145

4.3.2.1 BSA-Fluram ..........................................................................................146

4.3.2.2 SOD1-Fluram ........................................................................................147

xii


CHAPTER

4.3.3 Protein Aggregation ..........................................................................................149

4.3.3.1 BSA-Fluram with GuHCl and Temperature .........................................150

4.3.3.2 SOD1-Fluram with GuHCl ...................................................................151

4.3.4 Dynamic Light Scattering .................................................................................152

4.3.5 Time-Resolved Fluorescence Measurements ...................................................153

4.4 Conclusions ...................................................................................................................154

4.5 Chapter Summary .........................................................................................................155

4.6 References .....................................................................................................................157

5. SINGLE AND TWO-PHOTON DNA-BASED FLUORESCENCE SENSORS FOR

Pb2+ AND Hg2+ ............................................................................................................................163

5.1 Introduction ...................................................................................................................163

5.2 Materials and Methods ..................................................................................................168

5.2.1 Materials ...........................................................................................................168

5.2.2 Spectroscopic Methods .....................................................................................169


5.3.1 Interaction of G-Quadruplex DNA with Pb2+ ...................................................170

5.3.1.1 Optical Absorption Measurements .......................................................170

5.3.1.2 One-Photon Fluorescence Measurements .............................................171

5.3.1.3 Two-Photon Fluorescence Measurements ............................................172

5.3.2 Interaction of G-Quadruplex DNA with Hg2+ ..................................................173

5.3.2.1 Optical Absorption Measurements .......................................................173

xiii


CHAPTER

5.3.2.2 One-Photon Fluorescence Measurements .............................................174

5.3.2.3 Two-Photon Fluorescence Measurements ............................................175

5.3.3 Relative 2PA Enhancements .............................................................................176

5.3.4 Selectivity .........................................................................................................178

5.3.5 Forming G-Quadruplex Structure .....................................................................180

5.4 Conclusions ...................................................................................................................181

5.5 Chapter Summary .........................................................................................................182

5.6 References .....................................................................................................................184

6. TWO-PHOTON ABSORPTION PROPERTIES OF CHROMOPHORES IN

POLYELECTROLYTES .............................................................................................................191

6.1 Introduction ...................................................................................................................191

6.2 Experimental .................................................................................................................195

6.2.1 Materials ...........................................................................................................195

6.2.2 Methods.............................................................................................................195


6.3.1 Interactions with AcrO ......................................................................................196

6.3.1.1 Interactions of MPS-PPV with AcrO ....................................................196


6.3.1.1.2 One- and Two-Photon Fluorescence Measurements ................197

6.3.1.1.3 Relative 2PA Cross-Sections Measurements ............................198

6.3.1.2 Interactions of PSS with AcrO ..............................................................198

xiv


CHAPTER




6.3.2 Interactions with Hoe ........................................................................................201

6.3.2.1 Interactions of MPS-PPV with Hoe ......................................................201




6.3.2.2 Interactions of PSS with Hoe ................................................................203




6.3.3 Interactions of Polyelectrolytes with ThT.........................................................206

6.3.3.1 Interactions of MPS-PPV with ThT ......................................................206




6.3.3.2 Interaction of PSS with ThT .................................................................208



xv


CHAPTER


6.3.4 Interactions with C-485.....................................................................................211

6.3.4.1 Interactions of MPS-PPV with C-485...................................................211




6.3.4.2 Interactions of PSS with C-485.............................................................213




6.3.5 Comparison of 2PA Cross-Sections..................................................................216

6.4 Conclusion ....................................................................................................................218

6.5 Chapter Summary .........................................................................................................220

6.6 References .....................................................................................................................221

7. OVERALL SUMMARY AND FUTURE OUTLOOK ...........................................................228

7.1 Overall Summary ..........................................................................................................228

7.2 Future Outlook ..............................................................................................................233

xvi

LIST OF TABLES

5.1 Possible health effects corresponding to lead and mercury metals ........................................165

xvii

LIST OF FIGURES

1.1 Schematic illustrations how large number of bonds can stabilize the organized self-

assemblies ........................................................................................................................................3

1.2 Hydrogen bonds between in the A·T nucleic acid- Watson-Crick DNA bases .........................4

1.3 Ionic bond is one of the important non-covalent bonds that stabilizes organized self-

assemblies ........................................................................................................................................5

1.4 Schematic illustration of possible orientations for π–π stacking interactions............................6

1.5 Schematic of van der Walls weak interaction ............................................................................7

1.6 The hydrophilic/hydrophobic interactions are highly important factor governed

organized self-assemblies ................................................................................................................8

1.7 Schematic illustrating the electrostatic interactions between Lysine and Glutamic acids.........9

1.8 Schematic representation the micellar structure in aqueous solution ......................................10

1.9 Reverse micelle structure in aqueous solution .........................................................................11

1.10 Schematic structure for lipid bilayers ....................................................................................12

1.11 The structure of DNA which consisted of nucleotide bases A, T, C and G, that are

linked to deoxyribose sugar units and phosphates group...............................................................13

1.12 Schematic for the native folded protein, unfolded protein, and aggregated protein ..............14

1.13 Schematic of the three classes of polyelectrolytes .................................................................15

1.14 Comparison between energy diagrams of two-photon absorption (2PA) and one-photon

absorption (1PA) ............................................................................................................................22

1.15 Illustration of the applications of 2PA ...................................................................................24

2.1 Schematic depicting the basic components of a laser ..............................................................45

xviii

List of Figures—Continued

2.2 This figure illustrates the advantage of laser light monochromatic properties over

ordinary light ..................................................................................................................................48

2.3 This figure shows how coherence is one of the unique properties of laser light

comparing to ordinary light source ................................................................................................49

2.4 Directionality of the beam displaying minor divergence of compact light waves...................50

2.5 Schematic diagram of depicting fluorescence. ........................................................................55

2.6 Schematic diagram of TCSPC setup ........................................................................................57

2.7 Schematic of the setup used to determine the 2PA cross-sections ..........................................61

2.8 Schematic representation of speckle pattern of the setup used to determine the DLS ............66

3.1 Biological roles of the telomeric DNA ....................................................................................81

3.2 Polymorphic G-Quadruplexes .................................................................................................81

3.3 Schematic representation of G-tetrad, G-Quadruplex, G-Triad, and G-Triplex ................82

3.4 G–Quadruplex and G–Triplex sequences, and it illustrates how the DNA folds due to

Guanin and forms these structures ..............................................................................................84

3.5 Molecular structures of the investigated drug molecules ........................................................85

3.6 Optical absorption spectrum of naked Quad DNA as a function of temperature ....................88

3.7 (A) Molar ellipsity of naked Quadruplex DNA as a function of temperature;

(B) Molar Ellipsity at 246 nm, 267 nm and 292 nm ..................................................................88

3.8 Absorption spectrum of Triplex as a function of temperature ............................................89

3.9 (A) Molar ellipsity of naked Triplex DNA as a function of temperature; (B) Molar

ellipsity change of Triplex DNA at 255 and 290 nm .................................................................90

3.10 (A) Absorption spectrum of ThT with and without Quadruplex, (B) Absorption

spectrum of ThT/Quadruplex as a function of temperature ...........................................................91

xix


3.11 One-photon fluorescence spectrum of ThT/Quadruplex as a function of temperature .........92

3.12 Two-photon fluorescence spectrum of ThT/Quad as a function of temperature .............93

3.13 Relative 2PA cross-sections enhancement of ThT/Quadruplex as a function of

temperature ...................................................................................................................................94

3.14 (A) Absorption spectrum of ThT with and without Triplex, (B) Absorption

spectrum of ThT/Triplex as a function of temperature ..............................................................95

3.15 One-photon fluorescence spectrum of ThT/Triplex at different temperatures ......................96

3.16 Two-photon fluorescence spectrum of ThT/Triplex as a function of temperature ..........96

3.17 Relative 2PA cross-sections enhancement for ThT/Triplex as a function of

temperature ...................................................................................................................................97

3.18 (A) Absorption spectrum of ThO with and without Quadruplex DNA,

(B) Absorption spectrum of ThO/Quadruplex as a function of temperature ...........................98

3.19 Steady-state fluorescence spectrum of ThO/Quadruplex as a function of

temperature ...................................................................................................................................99

3.20 Two-photon spectrum of ThO/Quadruplex as a function of temperature ......................100

3.21 Relative 2PA cross-sections of ThO/Quadruplex as a function of temperature ............101

3.22 (A) Absorption spectrum of ThO with and without Triplex, (B) Absorption

spectrum of ThO/Triplex with temperature dependence .........................................................102

3.23 One-photon fluorescence spectrum of ThO/Triplex as a function of temperature ........103

3.24 Two-photon fluorescence spectrum of ThO/Triplex as a function of temperature ...........103

3.25 Relative 2PA cross-sections for ThO/Triplex as a function of temperature ..................104

3.26 (A) Optical absorption spectrum of DAPI with and without Quad DNA,

(B) Absorption spectrum of DAPI/Quad as a function of temperature ..................................105

3.27 One-photon fluorescence spectrum of DAPI/Quadruplex as a function of

temperature .................................................................................................................................106

xx


3.28 Two-photon fluorescence spectrum of DAPI/Quadruplex as a function of

temperature .................................................................................................................................107

3.29 Relative 2PA cross-sections for DAPI/Quadruplex as a function of temperature ........108

3.30 (A) Absorption spectrum of DAPI with and without Triplex, (B) Absorbance

spectrum of DAPI/Triplex at different temperatures ...............................................................109

3.31 One-photon fluorescence spectrum of DAPI/Triplex ......................................................110

3.32 Two-photon fluorescence spectrum of DAPI/Triplex as a function of temperature .....110

3.33 Relative 2PA cross-sections for DAPI/Triplex as a function of temperature ................111

3.34 (A) Optical absorption spectrum of EtBr with and without Triplex, (B) Absorption

spectrum of EtBr/Triplex as a function of temperature ...........................................................112

3.35 One-photon fluorescence spectrum of EtBr/Triplex as a function of temperature ........113

3.36 Two-photon fluorescence spectrum of EtBr/Triplex as a function of temperature .......114

3.37 Relative 2PA cross-sections for EtBr/Triplex as a function of temperature ..................115

3.38 (A) Absorption spectrum for TMPyP4 with and without Quadruplex, (B) Optical

absorption spectrum of TMPyP4/Quadruplex as a function of temperature .........................116

3.39 One-photon fluorescence spectrum of TMPyP4/Quadruplex as a function of

temperature .................................................................................................................................117

3.40 Two-photon fluorescence spectrum of TMPyP4/Quadruplex as a function of

temperature .................................................................................................................................117

3.41 Relative 2PA cross-sections of TMPyP4/Quad ...............................................................118

3.42 (A) Absorption spectrum of TMPyP4 with and without Triplex, (B) Optical

absorption spectrum of TMPyP4/Triplex as a function of temperature .................................119

3.43 One-photon fluorescence spectrum of TMPyP4/Triplex as a function of

temperature .................................................................................................................................120

xxi


3.44 Two-photon fluorescence spectrum of TMPyP4/Triplex as a function of

temperature .................................................................................................................................120

3.45 Relative 2PA cross-sections of TMPyP4/Triplex as a function of temperature ............121

3.46 (A) Absorption spectrum of Doxorubicin with and without Triplex, (B) Optical

absorption spectrum of Doxo/Triplex as a function of temperature .......................................122

3.47 Fluorescence spectrum of Doxo/Triplex as a function of temperature ..........................123

3.48 Two-photon fluorescence spectrum of Doxo/Triplex as a function of temperature ......124

3.49 Relative 2PA cross-sections for Doxo/Triplex as a function of temperature ................124

3.50 Molecules interacting with ssDNA, dupDNA, quadDNA, tripDNA and also interacting

with each of the electric fields produced by the backbone of the DNA ......................................126

3.51 Relative 2PA cross-sections of molecules/drugs interacting with Quadruplex

and Triplex ...................................................................................................................................127

4.1 Schematic showing the binding of Fluram with primary amines leading to a fluorescent

product .........................................................................................................................................142

4.2 Schematic representation of the protein systems studied in the investigation .......................143

4.3 (A) Optical absorption and fluorescence spectrum of Fluram binding to BSA .....................146

4.4 Schematic illustration of protein unfolding mechanism ........................................................146

4.5 (A) One photon fluorescence for BSA-fluram after excitation at 370 nm, inset shows

changes in fluorescence intensity at 479 nm for BSA-fluram as a function of temperature

from 25 to 75 oC ...........................................................................................................................148

4.6 (A) One photon fluorescence spectrum of SOD1-fluram after excitation at 370 nm,

inset shows changes in fluorescence intensity at 482 nm for SOD1-fluram as a function of

temperature from 25 to 95 oC.......................................................................................................149

4.7 Schematic showing the aggregation mechanism ...................................................................150

xxii


4.8 (A) Normalized fluorescence with one- and two-photon excitation to monitor

aggregation of BSA; (B) Relative 2PA cross-sections for BSA-Fluram monitoring

aggregation of BSA......................................................................................................................151

4.9 (A) Normalized fluorescence intensities as a function of temperature with one- and two-

photon excitation; (B) Relative 2PA cross-sections for SOD1-Fluram monitoring

aggregation of SOD1 ...................................................................................................................152

4.10 DLS size distribution at different temperatures for BSA and SOD1 ...................................153

4.11 (A) Time-resolved fluorescence of SOD1 and BSA; (B) Average lifetimes for

BSA-fluram and SOD1-fluram as function of temperature .........................................................154

5.1 Various sources of lead and mercury pollutions in the environment .....................................165

5.2 Schematic representation of the use of G-Quadruplex based DNA sensors to detect

presence of Pb2+ and Hg2+ ions through quenching the fluorescence of the fluorophore ............167

5.3 Molecular structure of the fluorophore, diethylthiacyanine iodide .......................................170

5.4 Optical absorbance spectrum of T-30695/DTI at varying Pb2+ concentrations .....................171

5.5 Steady fluorescence spectrum of T30695/DTI upon exposure to different Pb2+

concentrations ..............................................................................................................................172

5.6 Two-photon spectrum of T30695/DTI upon exposure to different Pb2+ concentrations .......173

5.7 Optical absorbance spectrum of T-30695/DTI at varying Hg2+ concentrations ....................174

5.8 Steady fluorescence spectrum of T30695/DTI upon exposure to different Hg2+

concentrations ..............................................................................................................................175

5.9 Two-photon spectrum of T30695/DTI after exposure to different Hg2+ concentrations .......176

5.10 (A) A comparison of the change in the single and two-photon fluorescence sensing

of Pb2+ at different concentrations................................................................................................178

5.11 A demonstration of T30695’s selectivity of Pb2+ and Hg2+ over other common metals

for both one-photon fluorescence and two-photon fluorescence excitations ...............................179

xxiii


5.12 The circular dichroism measurements of the T30695 oligonucleotide with K+, Hg2+,

and Pb2+ shows the formation of a G-Quadruplex structure ........................................................180

6.1 Schematic illustration of the polyelectrolytes changing folding/unfolding conditions .........192

6.2 The chemical structure for the investigated anionic polymer in this chapter MPS-PPV

and PSS ........................................................................................................................................194

6.3 Molecular structures of the investigated chromophores ........................................................194

6.4 Optical absorption spectrum of AcrO with increasing MPS-PPV concentrations ................196

6.5 (A) One photon fluorescence spectrum of AcrO with increasing MPS-PPV

concentrations ..............................................................................................................................197

6.6 Relative 2PA cross-sections of AcrO at different MPS-PPV concentrations ........................198

6.7 Optical absorption spectrum of AcrO with increasing PSS concentrations ..........................199

6.8 (A) One photon fluorescence spectrum of AcrO with increasing PSS concentrations..........200

6.9 Relative 2PA cross-sections of AcrO at different PSS concentrations ..................................201

6.10 Optical absorption spectrum of Hoe with increasing MPS-PPV concentrations .................202

6.11 (A) One photon fluorescence spectrum of Hoe with increasing MPS-PPV

concentrations ..............................................................................................................................203

6.12 Relative 2PA cross-sections of Hoe at different MPS-PPV concentrations ........................204

6.13 Optical absorption spectrum of Hoe with increasing PSS concentrations ...........................204

6.14 (A) One photon fluorescence spectrum of Hoe with increasing PSS concentrations ..........205

6.15 Relative 2PA cross-sections of Hoe at different PSS concentrations ..................................206

6.16 Optical absorption spectrum of ThT with increasing MPS-PPV concentrations ................207

6.17 (A) One photon fluorescence spectrum of ThT with increasing MPS-PPV

concentrations ..............................................................................................................................208

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6.18 Relative 2PA cross-sections of ThT at different MPS-PPV concentrations ........................209

6.19 Optical absorption spectrum of ThT with increasing PSS concentrations ..........................209

6.20 (A) One photon fluorescence spectrum of ThT with increasing PSS concentrations..........210

6.21 Relative 2PA cross-sections of ThT at different PSS concentrations ..................................211

6.22 Optical absorption spectrum of C-485 with increasing MPS-PPV concentration ...............212

6.23 (A) One photon fluorescence spectrum of C-485 with increasing MPS-PPV

concentrations ..............................................................................................................................213

6.24 Relative 2PA cross-sections of C-485 at different MPS-PPV concentrations .....................214

6.25 Optical absorption spectrum of C-485 with increasing PSS concentration .........................214

6.26 (A) One photon fluorescence spectrum of C-485 with increasing PSS concentrations ......215

6.27 Relative 2PA cross-sections of C-485 at different MPS-PPV concentrations .....................216

1

CHAPTER 1

INTRODUCTION

Nature uses organized self-assemblies for most processes. Proteins, enzymes, membranes

all rely heavily on the weak chemical interactions that constitute the organized self-assemblies.

Understanding organized self-assemblies has been an integral part of research interest for

interdisciplinary areas of sciences that include chemistry and biology. Among several tools to

monitor organized self-assemblies, spectroscopy plays a huge role and especially optical

spectroscopic techniques are vital to probe them. Organized self-assemblies are interdisciplinary

between biology, chemistry, material science. Understanding how organized self-assemblies

interact with one another will allow us to understand several biologically important phenomena.

The main goal of this dissertation is to develop and establish novel optical spectroscopic tools to

probe organized self-assemblies, their interactions and aggregation via applying the power of two-

photon absorption (2PA) spectroscopy. Our hypothesis is that the 2PA cross-sections of

chromophores in organized self-assemblies are sensitive to the local electric fields and their

orientation, that can be used to monitor the formation and aggregation of organized self-

assemblies. In this Chapter, a brief introduction about organized self-assemblies and how they are

important is provided. It is followed by a description of the techniques that are used to study the

organized self-assemblies and need for newer techniques to study them. Finally, the research

motivation and the work carried out for the dissertation is outlined.

1.1 Organized Self-Assemblies

Organized self-assembly refers to a spontaneous process where components either form

or aggregate without any external interference1,2. Although, this spontaneous process occurs

2

by weak non-covalent interactions, the final structure is known to be stable. These chemical

bonds include van der Walls interactions, electrostatic, π-π stacking interactions, and hydrogen

bonding1. Organized self-assembly is diffuse everywhere with varying sizes between molecular,

mesoscopic, and macroscopic scales, whose components span from atomic, ionic, and molecular

crystals to solar system and galaxies3–5. Molecular and mesoscopic organized self-assemblies

have attracted scientists and received enormous researcher attention because of their practical

application and potential to be compatible with the needs of humans. Organized self-assemblies

are normally affected by surrounding microenvironment such as solvents, pH, co-assembled

molecules, salinity and temperature6,7.

Organized self-assemblies can be classified into two main groups: static and dynamic

based on thermodynamic equilibrium conditions. Static organized self-assembly is a system that

do not dissipate energy and attains local or global equilibrium. In contrast, dynamic organized

self-assembly is a system that is required to dissipate energy to form them8,9. Most researchers

have focused on static organized self-assemblies rather than the dynamic because it includes the

biologically important processes and components and can be used for therapeutics and

diagnostics10,11. Example of static organized self-assembly include: atomic, ionic, molecular

crystals, organized self-assembled monolayers, lipid bilayers, liquid crystals and colloidal

crystals. On the other hands dynamic organized self-assembly examples include light-matter

interactions, oscillating reactions, diffusion reactions, bacterial colonies, swarms and spools,

weather patterns, solar systems, galaxies among many others4,5,12,13.

For the research work carried out in this dissertation, we have focused on the interactions

in static organized self-assemblies. The greatest merit of the static organized self-assemblies is

that the structural features of the final assemblies can be readily and finely tuned by the

3

molecular chemistry, assembling environment (solvents, pH, co-assembling molecules, salinity

and temperature), and assembly kinetics.

1.2 Chemical Interactions in Organized Self-Assemblies

Different chemical interactions make organized self-assemblies that include hydrogen

bonding, ionic bonding, π-π stacking, van der Walls interaction, hydrophilic/hydrophobic

interactions and electrostatic interactions. These strong and weak interactions help create a

unique property that creates self-assembly. All these are associated with specific forces that

adhere to their specific functions. Some weaker systems such as van der Walls and electrostatic

interactions are associated with other bonding functions14,15. Although non-covalent bonds are

weak in general, each organized self-assembly contains multiple non-covalent bonds that work

together to stabilize them as shown in Figure 1.1. A brief introduction about each one of the non-

covalent bonds is provided here.

Figure 1.1: Schematic illustrations how large number of bonds can stabilize the organized self-

assemblies.

4

1.2.1 Hydrogen Bonding

All life forms require hydrogen bonding to exist as water is a main component for life.

The hydrogen atom can bond covalently to a donor atom and also to an acceptor atom and is

considered a weaker bond with less energy. However, large number of these weak hydrogen

bonds make them important for many biological processed. The donor atoms are electronegative

producing a donor-to-hydrogen polar covalent bond. As for the acceptor atom, it too is

electronegative, has at least 1 bonding pair of electrons, and pulls a positive polar charge of a

hydrogen atom as describe by:14,15.

𝐷𝛿− − 𝐻𝛿+ +:𝐴𝛿− ⇄ 𝐷𝛿− − 𝐻𝛿+ ⋯𝐴𝛿− 1.1

Nitrogen and oxygen atoms of amino and hydroxyl groups in biochemical systems are

known to form strong hydrogen bonds. These nitrogen-to-hydrogen and oxygen-to-hydrogen

bonds perform as donor and acceptor molecules and are also polar in nature as shown in Figure

1.2. With these properties they are able to interact with hydrogen of water molecules. Moreover,

hydrogen bond produce stability among organized self-assemblies. For example, they are

responsible for stabilizing protein structures and nucleic acids in Deoxy ribonucelic acid

(DNA)14,15.

Figure 1.2: Hydrogen bonds between in the A·T nucleic acid- Watson-Crick DNA bases.

5

1.2.2 Ionic Bonds

Ionic bonds form due to the electronegativity difference between atoms (example of ionic

interaction is shown in Figure 1.3). They are essentially the resultant of cation and anion

coulombic interactions. The lattice energy is the force responsible for the stabilization of the

crystal structure produced by ionically bonded atoms e.g. sodium chloride (NaCl). In biological

systems, cations and anions are stabilized by the surrounding water molecule via energy of

hydration which has a higher energy than most lattice energy forces formed by crystals15.

Figure 1.3: Ionic bond is one of the important non-covalent bonds that stabilizes organized self-

assemblies.

1.2.3 π-π Stacking

π-π stacking is a non-covalent interaction between aromatic groups containing π bonds.

π-π stacking can be classified into three main classes based on the geometry of aromatic

interactions: (1) edge-to-face stacked (T-shaped), (2) offset stacked (3) face-to-surface stacked as

shown in Figure 1.4. For understanding the fundamental structure properties of organized self-

6

assemblies, there are many molecules that can generate π-π interactions and interact with

aromatic groups via π bonds16–18.

Figure 1.4: Schematic illustration of possible orientations for π–π stacking interactions.

1.2.4 Van der Walls Interaction

Described by physicist Johannes Diderk van der Walls, this weak force is created by two

atoms that nearly approach one another and is called van der Walls bond15. van der Walls forces

can be apparent between all kinds of atoms due to odd distribution of electrons via transient

electric dipoles19. These interactions can happen within polar and non-polar systems. For

example, in the case of the nonpolar liquid heptane there are weak van der Walls interactions.

Moreover, van der Walls interaction strength decreases as the distance between the two atoms

increases on the contrary if the atoms are too close, they will repel from the force of like negative

charges via outer electron shell. Furthermore, van der Walls interactions can assist in the binding

process of enzymatic systems to interact with substrates as well as antibody and antigen

interactions as shown in Figure 1.514,15,19.

7

Figure 1.5: Schematic of van der Walls weak interaction.

1.2.5 Hydrophilic/Hydrophobic Interactions

Hydrophobic bonds (Figure 1.6) are usually correlated with carbon-to-hydrogen bonding

as in the case of non-polar systems. These nonpolar bonds by hydrocarbons are insoluble in

water. However, in some case due to oxygen atoms some can be partially soluble in water. The

hydrophobic bond is the force responsible for the lack of hydrogen bonding with water and

prevalent in many biological processes. Phospholipids such as micelles, bilayer sheets, and

liposomes form to eliminate the presence of the surrounding water and is mainly due to the

hydrophobic interactions. Also, van der Waals forces are responsible for further stabilization of

the hydrocarbon systems15,20–22.

8

Figure 1.6: The hydrophilic/hydrophobic interactions are highly important factor governed

organized self-assemblies.

1.2.6 Electrostatic Interactions

The electrostatic interaction is non-covalent type of bond that is based on the electric

charges of an atom or bio-molecular entity (example of electrostatic interaction is provided in

Figure 1.7). It can be explained and understood by physics concepts. The Coulomb’s law can

define the electrostatic interaction with the formula:

𝐸 = 𝑘𝑞1𝑞2/𝐷𝑟 1.2

where the E is the energy; q1 and q2 are corresponding charges in the system or on the atoms via

electronic charge units. r is the distance between the two atoms in angstroms; D is the dielectric

constant (the effect of the intervening medium); k is the proportionality constant. Also, the

electrostatic interactions can happen between the two entities or atoms that have single opposing

charges and has a distance less than 3 angstroms between them 14,23,24.

9

Figure 1.7: Schematic illustrating the electrostatic interactions between Lysine and Glutamic

acids.

1.3 Organized Self-Assemblies—Examples

There are several kinds of organized self-assemblies and some of them are discussed

here.

1.3.1 Micelles

Micelles (shown in Figure 1.8) are the structures formed spontaneously under certain

conditions of temperature and concentration of amphiphilic molecules in aqueous solutions,

which drive them into a self-assembled structure composed of both hydrophobic and hydrophilic

parts. One simplest example that we see every day is the soap. Micelles normally have particle

size from ranging 5 to 100 nm, depending on the nature of head groups type and length of the

alkyl chains25. Micelles are known to be sensitive to monomeric amphiphilic concentration

where higher concentration will increase aggregation and lower concentration will cause

amphiphilic molecules to exist separately. Therefore, a critical micelle concentration (CMC) is

10

needed to form micelles25–28. Micelles can be shaped in different morphologies such as spheres,

lamellae, tubules, vesicles and rods based on environment at conditions such as: solvent, length

and nature of the blocker chain and temperature26,29. The micelles foremost characteristic is the

core-shell structure, where core is formed by van der Walls bonds while the shell is formed by

hydrophobic and hydrophilic interaction with surrounding environment26. The physical chemical

properties of micelles make it suitable for several biological applications. Recently, micelles

have shown a promising opportunity develop drug delivery systems due to several advantages

such as; reduced toxicity, improved permeability, improved penetration with small micelles and

improved targeting selectivity30. A recent study by our group has shown that the chromophores

localized in micelles can have greater 2PA cross-sections based on the electric field environment

provided by the micelles.

Figure 1.8: Schematic representation the micellar structure in aqueous solution.

1.3.2 Reverse Micelles

Reverse Micelles is another organized self-assembly and referred to molecular aggregates

of surfactants in a polar media. Reverse micelles are similar to micelles except that the surfactant

has polar head groups are heading to the inner core and form a polar core while the lipophilic

parts are heading outer toward surrounding non-polar solvent, which will create a shield to

protect the core part as shown in Figure 1.931,32. There are several applications for reverse

micelles. Reverse micelles are used as reaction medium for nanoparticles synthesis. Also, reverse

https://www.sciencedirect.com/topics/materials-science/toxicity

https://www.sciencedirect.com/topics/materials-science/permeability

11

micelles are used for protein extraction, where the protein molecule is immersed in the water

pool surrounded by a water-shell. In addition, reverse micelles used to transport and stabilize the

charges in nonpolar media33. Also, reverse micelles used to disperse aggregated colloids in

nonpolar suspensions. Moreover, formation of reverse micelles may affect solvent physical

properties, such as increased solvent viscosity34.

Figure 1.9: Reverse micelle structure in aqueous solution.

1.3.3 Lipid Bilayers

Lipid bilayers are thin polar molecules that form the core structure of cell membranes. It

is formed from two layers of amphipathic lipid molecules where the hydrophilic heads point

outward and hydrophobic tails toward the inner core as shown in Figure 1.10. Lipid

bilayers are highly important for living organisms to separate aqueous compartments and

selectively transfer molecules and information from cell to cell. Therefore, lipid bilayers are

essential for membrane functions35. Organized self-assembly interactions play a vital role in the

formation of lipid bilayer structures.

12

Figure 1.10: Schematic structure for lipid bilayers.

1.3.4 DNA

Deoxyribonucleic acid, commonly known as DNA consists of three different chemical

units which are (1) four different chemical types of nitrogen nucleotide bases which are (Adenine

(A), Thymine (T), Guanine (G), Cytosine (C) ), (2) deoxyribose sugar unit and (3) a phosphate

group36. The DNA chemical bases pair up in (C with G and A with T), which form what known

as base pairs. The DNA stored the cell information in code form in the four chemical nucleotide

bases (A,T,G and C). The sequences of chemical nucleotide bases are fundamental to build and

maintain the organism. Moreover, each base is attached to deoxyribose sugar molecule, and each

sugar unit is covalently attached via phosphodiester linkages to phosphate ion groups as shown

in Figure 1.11. All the base pairs, deoxyribose sugar and phosphate are referred as nucleotide

(single-stranded DNA molecule). Two nucleotides are organized in two long single strands thus

forming what is known as a double helix (double-stranded DNA molecule). Therefore, DNA

exists as a biopolymer where the deoxyribose sugar molecule and phosphate residues linked

together to form the DNA backbone. A distinctive DNA property is that it contains the needed

instructions to replicate and reproduce needed protein for an organism36,37. The first accurate

three-dimensional structure of DNA was determined by Francis Crick and James Watson in

13

1953. Since then an enormous progress was made in studying DNA sequencing, manipulation,

and synthesis36.

Figure 1.11: The structure of DNA which consisted of nucleotide bases A, T, C and G, that are

linked to deoxyribose sugar units and phosphates group. Each of the base pairs are boned to

each other by weak hydrogen bonding interactions.

1.3.5 Proteins

Protein can be considered a polymer consisting of amino acids. There are 20 different

types of amino acids, which bind together through covalent peptide bonds in a certain sequence.

Peptide bonds are a chemical bond between amino acids where one amino acid with carbonyl

group in bonded to another amino acid’s amine functional group. Due to numbers of non-

covalent bonds such as hydrogen bonds, ionic bonds, and van der Walls attractions, and different

polarity of amino acids, proteins can fold into a compact three-dimensional conformation, which

is determined by the amino acids order in its chain. The importance of protein folding

14

governs the biological function for cells such as maintenance, defense, replication, and

reproduction36,37.

Proteins exist in folded native state conformation that meet the lowest configurational

entropy energy state. The process of folding has been investigated actively to study how the

polypeptide chains find them into the lowest conformation energy state. Although thousands of

bonds form the folded protein conformation, most of them are non-covalent (weak) bonds. The

folded structure could be unfolded or aggregate under specific condition that include : temperate,

chemical denaturants, pressure and force as shown in Figure 1.1238–41.

Figure 1.12: Schematic for the native folded protein, unfolded protein, and aggregated protein.

15

1.3.6 Polymers and Polyelectrolytes

Polyelectrolytes are types of polymers with repeating groups that are linked covalently

when they are dissolved in polar solvent. Polyelectrolyte classified to polycations, polyanions

and polyampholyte depending on the carrier positive charges, negative charges or both charges

as shown in Figure 1.1342,43. Polyelectrolytes can be naturally occurring or synthetic. Famous

examples for naturally occurring polyelectrolytes are nucleic acids, DNA, RNA, and

polysaccharides. The ionic group and its counter ion in the polymer chain determine the

polyelectrolyte chemical and physical properties depending on how strong electrostatic

interaction between the charged dissociating groups are. There are several applications for

polyelectrolytes that include improved aqueous colloidal stability, flocculation and flow

modification, superabsorbent gels, implant coatings and drug delivery42–46.

Figure 1.13: Schematic of the three classes of polyelectrolytes.

1.4 Why Are Organized Self-Assemblies Important?

Organized self-assemblies have received increasing research attention over the past

decades for several reasons. First, organized self-assemblies are significantly important in

several biological processes as the organization of the cell (fundamental unit of life)

16

encompasses countless examples of self-assembled macromolecules that make it work. One such

example is the cell membrane which is formed from the organization of lipid bilayers. Secondly,

knowing how organized self-assemblies are formed will help for one to mimic the nature’s

biological processes to fabricate new materials for applications that meet specific requirements.

Thirdly, understanding organized self-assemblies can provide opportunities to make novel

sensing and diagnostic tools out of them. Finally, organized self-assemblies provide the best

practical strategies for generating nanostructures for applications such as light harvesting,

catalysis and imaging3–5,12,47–50. For all these reasons, organized self-assemblies play an

important role in interdisciplinary areas of sciences that include biology, physics, chemistry,

nanoscience, materials science, and manufacturing. Also, mimicking nature’s organized self-

assemblies is a promising strategy to solve important problems facing mankind such as

alternative energy, sustainability and environment. Thus, it is important to understand how the

organized self-assemblies operate.

1.5 Existing Techniques to Monitor Organized Self-Assemblies

As discussed in the earlier sections , it is important to understand how organized self-

assemblies operate and interact. Researchers have routinely used several analytical techniques to

monitor the organized self-assemblies. Although, these methods help with understanding self-

assembly interactions, they still have their own limitations. Some of the current analytical

techniques are discussed here.

1.5.1 Affinity Capillary Electrophoresis (ACE)

ACE work through separation by capillary electrophoresis which depend on molecule

migration or mobility characteristics. The self-assembly interactions relative to the size, shape,

and charge will influence the migration patterns via electrophoretic matrix. These non-covalent

17

interactions can be studied by mapping the electrophoretic behavior of a molecular system

fashioned with a marked biomolecule along with a labeled affinity probe. This system typically

contains the electrophoresis buffer with a receptor. The receptor will interact among the marked

self-assembled analytes. Advantage of ACE include: automation, separation efficiency, ability to

use less reagent, studying system at varied conditions such pH levels and temperatures, and short

analysis time. 27,28,33,34 A disadvantage of ACE is that it cannot handle specification for

adsorption of proteins on capillaries, and also it requires a high purity samples51–54. This

technique cannot understand the micro-environments in organized self-assemblies.

1.5.2 Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is generally used to recognize how much antigen is within a sample and often can

probe biologically important self-assembled macromolecules. To do this, a sample is placed on a

solid motionless plate and incubated along with an antibody. This antibody will bind to an

antigen that is covalently conjugated to an enzyme giving an enhanced signal. The bound

enzyme gives a fluorogenic reaction with the substrates yield a signal indicating the

concentration of antigen in the sample. The advantage of ELISA includes easy to setup, rapid

detection and economical cost. However, ELISA cannot detect specific antigen immobilization

and so fails to detect low affinity antibodies 55–58. Also, ELISA is specific to certain kinds of

organized self-assemblies and is not quite general.

1.5.3 Fluorescence Resonance Energy Transfer (FRET)

(FRET) is a non-radiative process that can be used as a marker to study the distances,

protein folding and aggregation in biologically important systems. FRET requires the labelling of

the organized self-assemblies with different fluorophores marking one as donor and another as

acceptor. Emission wavelengths for the two labeling systems requires the excitation and

https://pubs.rsc.org/en/Content/ArticleLanding/2012/CS/C1CS15168A#cit27

18

emission spectruml regions to overlap. Where the donating systems give fluorescence that can be

absorbed by acceptor and gives rise to emission from the acceptor. The ratio of donor

fluorescence to acceptor fluorescence can be used as a ruler to study several biological important

phenomena. This is mainly used for studying the distance between two biological entities. FRET

has its own advantages and disadvantages. FRET is powerful for imaging and following the

distances. However, it is limited to monitoring self-assembly interactions that are within 10 nm

of each other, and requires labeling of biological molecules which can be tedious. The major

limitation is it is insensitive to microenvironments such as changes in pH, temperature, oxidation

and ionic concentrations. In addition, FRET suffers from the low signal-to-noise ratio37,59–61.

1.5.4 Isothermal Calorimetry (ITC)

Theoretically, any reaction involves either generating or absorbing energy. ITC is a

technique that measures the thermodynamic change during a chemical reaction. The ITC

instrument contains two compartments. The two compartments must maintain the thermal

equilibrium in the solution. Fundamentally, one compartment is referred as the reference cell and

is maintained with water or buffer solution, while the other compartment contains a component

of the system that is being observed. The change in enthalpy occurs as reactions components are

placed in the sample cell. The change in temperature is noted via thermo-circuits in the system

and detect changes in cells37,62–65. ITC is used extensively because it easy to use with good

sensitivity. Although ITC is used to gather information from many systems such as ligand

binding, protein-protein, enzymatic activity, lipid-lipid, drug development and protein-receptor,

it has several limitations. The limitations include issues with precision in the temperature

changes as well as whether the interactions can be measured successfully via enthalpic effects,

19

concentration influence in data inaccuracy63,65,66. Also, ITC does not give specific information

about the microenvironments or the orientations.

1.5.5 Ultraviolet Visible Spectroscopy (UV-vis)

Another method that used to routinely used to study organized self-assembly is UV-vis

spectroscopy. UV-vis analyzes the absorption of a molecule or material. UV-vis works by using

the electron magnetic radiation from the visible to ultraviolet range to energize a sample and then

scan how much light is absorbed. The concentration of the absorbing species can be calculated

from Beer-Lamber Law. The changes in absorption are often associated with changes in

confirmations of organized self-assemblies. Also, information how a chemical molecule is

bonded to another molecule or self-assembled material can be studies. UV-vis spectroscopy is

routinely used for many applications as it is easy to use and affordable. However, it has low

resolution, the changes in spectruml shifts of the wavelength maximum can give low

specificity67,68.

1.5.6 Nuclear Magnetic Resonance (NMR)

NMR uses the chemical species information from the atomic nuclei to probe the structure

of organized self-assemblies. NMR spectroscopy is used to determine structures of proteins or

drug-protein or drug-DNA interactions. The frequency from the resonating atoms help produce

a code to compute the molecular formulations and structure, as well as the conformational and

intermolecular responses68–71. NMR is a powerful analytical method due to high resolution, deep

information on self-assemblies conformational changes and information on structure67. However,

for NMR to be used, the molecular weight for sample solutions should be less than 40 kDa. It

may requires 15N and 13C which is expensive and large amount of sample is required for the

process66,69,72. Also, the technique is expensive as well as difficult to use and analyze the data.

20

1.5.7 Surface Plasmon Resonance (SPR)

SPR is a powerful technique which is based on surface plasmon resonances of metal

nanomaterials. It is an optical-sensing method to detect the changes in refractive index of a thin-

metal-film surface component with the onset of binding of a biochemical species. In terms of the

refractive index, a change occurs of the resonance angle of light as cells surface change.

Understanding this allows SPR to monitor of organized self-assemblies that can be monitored by

analyzing the shift in resonance of the SPR absorption and which can be used to monitor the

concentration of biological samples. SPR has the ability to allow for label-free and sub-

nanogram detections. The functionalization of metal surface is desired for a specific biochemical

interaction and has real-time measurement capability to monitor steady-state and dynamic

processes73–77. SPR is a high sensitivity technique and can work with small amount of sample.

Although SPR has some great qualities, it is limited in its use except for studying antigen-

antibody interactions, concentrations of cells and so on. It cannot be used to study the

microenvironments in self-assembled systems66,67.

1.6 Research Motivation

As discussed above, there are several techniques that are currently in use to monitor the

organized self-assemblies. All of them have their merits but most suffer from inherent

disadvantages such as inability to probe micro-environments, lower sensitivity and ease of use.

We can point out following important characteristics that are needed for a spectroscopic tool to

be routinely used for probing organized self-assemblies. Firstly, they should be sensitive to

micro-environment changes such as pH, drug binding, structural change etc. Secondly, they

should be very easy to use and analyze. Thirdly, the technique should be general in the sense that

they can be used for variety of organized self-assemblies. Developing such an analytical tool and

21

using it to study organized self-assembled materials form the main motivation behind the work

carried out in this dissertation. In recent years, it was shown that two-photon absorption cross-

sections of chromophores can act as markers for local electric fields in proteins or micelles etc.

We propose to use this property of 2PA cross-sections of chromophore as a tool to study the

organized self-assemblies and their interactions. The main dissertation objective is to use two-

photon fluorescence and corresponding 2PA cross-sections to study organized self-assembly

interactions and probe micro-environmental changes in them, and use this tools to develop novel

sensing and diagnostic tools.

1.7 Sensing Organized Self-Assemblies with Dye Molecules and 2PA Spectroscopy

The development of novel spectroscopic tools is necessary for monitoring organized self-

assemblies. There two main analytical fundamental optical techniques that are used to sense

absorption and fluorescence techniques using dye molecules as probes. It is important to know

and study how self-organized assembly bind with the dyes and how such signals can be used to

monitor organized self-assemblies. Electrostatic interaction with dye molecules gives rise to

significant changes in fluorescence. For example, fluorescence enhancement has been used as a

tool to study the binding of molecule with DNA. The dyes can intercalate with the DNA and

rigidify it to give rise to enhanced signal. Intercalation is possible due a two the hydrophobic

energy as well as van der Waal forces producing noncovalent bondages where the dye molecule

is bound to self-assembled. Investigations of self-organized assembly sensing have limitations in

regard to scattering in complex sample mediums. However these boundaries can be breeched by

new analytical techniques such as two-photon fluorescence and 2PA cross-sections which is the

focus of the work carried out in this dissertation1,3,8,11,73,78–84.

22

1.8 Research Approach Using 2PA Cross-Sections

2PA fluorescence spectroscopy has attracted more attention in recent years as inherent

characteristics of 2PA that include greater penetration depth, ability to monitor complex

environments and minimum photodamage for biological molecules. A brief discussion about the

principle of 2PA, properties and how it can monitor the local electric fields and thereby

organized self-assemblies is provided below.

1.8.1 Principle

2PA requires simultaneous absorption of two-photons to progress to an excite state84–86.

The summation of these two photon energies is equivalent to the energy difference between the

upper and lower energy states (Figure 1.14). This is a third-order nonlinear process and its signal

is proportional to the square of incident light intensity. Furthermore, it requires no intermediate

transition state during the excitation processes. The efficiency of two-photon absorption is

characterized by 2PA cross sections and has the units of Goppert Mayer87–90.

Figure 1.14 Comparison between energy diagrams of two-photon absorption (2PA) and one-

photon absorption (1PA).

23

1.8.2 Applications of 2PA

2PA uses intense near infrared radiation and because of that it has some special

characteristics that include greater penetration depth, less scattering, less photo damage. These

unique characteristics of 2PA made them ideal for several applications. Some are optical-power

limiting, laser upconversion, microfabrication, microscopy, three-dimensional data storage; and

photodynamic therapy for biological materials.91

Optical limiting works as goggles for optical sensor materials where in the output energy

will always be smaller despite greater input energy. This property makes it valuable for optical

sensing and electromagnetic radiation lenses92. Also, 2PA is useful for the microscopic imaging

of biologically tissues as well as cellular moieties without damage for a protracted time span and

high penetration of energy93. Reduction of storage space is a very important property 2PA has

the ability to assist with optical data storage giving decreased focus-volumes, increase

penetration depth and reduction of scattering88,92. Laser excitation provides focal properties

involved with 2PA allowing for chemical polymerization of three-dimensional object in minute

spaces.

Exceptional 2PA cross-sections molecules can be used for photodynamic cancer therapy.

The photosensitizers are linked chromophores that are used for the 2PA as they produce a singlet

state oxygen molecule that can kill tumors94,95. Newer applications of 2PA are arising, such as

the use of 2PA spectroscopy to monitor local electric fields in biological systems. 2PA

spectroscopy has a special sensitivity to the local electric field of fluorescent proteins as

explained in earlier studies composed by Rebane and co-workers96. Moreover, Guda and co-

workers have demonstrated that micelles local electric fields can be monitored with 2PA

spectroscopy 97–99.

24

Figure 1.15 Illustration of the applications of 2PA.

Why 2PA spectroscopy can be sensitive to local electric fields can be explained as

follows. The 2PA cross-sections can be given by the following expression:100,

𝛿 =2(2𝜋)4𝑓𝐿

4

15(𝑛ℎ𝑐)2 (1 + 2𝑐𝑜𝑠2𝜃)|𝑀𝑔𝑒|

2|∆𝜇𝑔𝑒|

2𝑔(2𝜈) 1.3

where δ is the two-photon absorption cross-sections; Mge is the transition dipole moment vector

that exist between the ground state (g) and the excited ( e) states; θ is the angle between the

vector Mge and Δμge; Δμge is the difference between the permanent dipole moments that exist in

the ground and excites states; g(2ν) is the normalized line shape function; c is the speed of light;

n is the refractive index; h is the Plank’s constant; and fL is the local field factor which is shown

in the following equation97,100,

𝑓𝐿 =(𝑛2+2)

3 1.4

25

Furthermore, the resulting equation below represents the resulting change in the local

electric field in their respective dipole moments from the increase of the induced dipole moment

∆𝜇𝑔𝑒 = ∆𝜇𝑔𝑒° + 0.5∆𝜇𝑔𝑒

𝑖𝑛𝑑 = ∆𝜇𝑔𝑒° ± 0.5(∆𝛼 ). �� = ∆𝜇𝑔𝑒

° ± 0.5(∆𝛼)𝐸 cos 𝜃 1.5

for these functions, Δα = αe – αg is the change in the polarizability between the excited state and

the ground state; and θ represent the angle between the transition dipole and moment of the

molecule and the electric field vector. As Δμ°ge represents the change in the permanent dipole

moment in a zero-field outcome and E is presentative of the local electric filed vector than the

2PA cross-sections will be directly relational to the square of the change in the permanent dipole

moment along with the change in the dipole-moment.

The δ has a sensitivity to the electric field if the chromophore is localized in it.

Moreover, the orientation of the chromophore with respect to the electric field orientation can

alter the 2PA cross-sections96,99–101. For all these reasons, we feel that 2PA spectroscopy can be a

valuable tool to monitor the organized self-assemblies.

1.9 Aims and Objectives of the Dissertation

1. Development of 2PA spectroscopy as a tool to probe change in electrical fields in

organized self-assemblies.

2. Using 2PA spectroscopy as a tool to monitor melting and stabilization of non-canonical

structures of DNA.

3. Use of 2PA spectroscopy as to monitor folding/unfolding and aggregation of proteins.

4. Design and development of novel sensing platforms based on 2PA spectroscopy.

5. Using 2PA spectroscopy to monitor the micro-environment in synthetic organized

assemblies and obtain materials with enhanced 2PA cross-sections.

26

1.10 Outline of the Dissertation

This dissertation is focused on the study of organized self-assemblies, their formation,

stability, folding/unfolding, aggregation and interactions. The thesis is comprised of seven

chapters. The first chapter is a brief introduction to organized self-assemblies, examples,

importance and the gaps in the field. The different chemicals interactions that rule organized

self-assemblies, chemical and physical properties are discussed. Current methods that are used

for monitoring various organized self-assemblies interactions like ACS, ELISA, FRET, ITC,

UV-vis, NMR and SPR is provided. A brief introduction about the motivation behind developing

of novel techniques used in the dissertation is presented.

The second Chapter discusses the experimental techniques that are used to carry out the

investigations. The basic laser components and the laser light radiation significant characteristics

is provided. Other experimental techniques that are discussed include UV-vis, one-photon

fluorescence, 2PA cross-sections, time-resolved spectroscopic techniques, circular dichroism,

dynamic light scattering. A brief description of each experimental technique is provided.

The third Chapter of the dissertation is focused on studying the melting and stability of

the G-Quadruplex and G-Triplex systems with different chromophores. For this investigation,

ThT, ThO, DAPI, TMPyP4, EtBr and DoXo chromophores are chosen, as they all well-known to

bind to investigated G-Quadruplex and G-Triplex systems. The interaction of these dyes was

studied with temperature-dependent UV-Vis, temperature-dependent one photon fluorescence

and temperature-dependent 2-photon fluorescence, Circular Dichroism. From this study, we were

able to show that relative 2PA cross-sections is a powerful tool to monitor organized self-

assemblies.

27

Chapter four of the thesis further establishes the technique of 2PA cross-sections as

powerful tool to monitor proteins folding/unfolding and aggregation. To establish this technique,

measurements were carried out with well-investigated proteins which are bovine serum albumin

(BSA) and superoxide dismutase 1 (SOD1). In addition, fluorescamine dye is chosen due to its

ability to selectively bind to the amine group of lysine in proteins to give rise of fluorescence in

the visible region. The investigation were carried out using a range of techniques including

temperature dependent UV-Vis absorption, temperature dependent one and two-photon excited

fluorescence, temperature dependent time-resolved fluorescence, and temperature dependent

dynamic light scattering. The studies have shown that 2PA spectroscopy can be sensitively used

to study protein folding and aggregation.

In the fifth Chapter we developed a single and two-photon fluorescence-based biosensors

that rely upon specific oligonucleotides T30695 that known to show sensitivity and selectivity

toward specific toxic heavy metal ions (Pb2+ and Hg2+). T30695 known to form stable G-

Quadruplex when it mixed with Pb2+ and Hg2+. The investigation were carried through UV-Vis

absorption, one photon and of two-photon fluorescence and circular dichroism. The study was

able to provide a novel one and two-photon based biosensor for sensitive and selective detection

of toxic metal ions that can also be used for on-site detection.

In Chapter six, we study the 2PA cross-sections of chromophores in polyelectrolytes to

probe how anionic polyelectrolytes will interact with different chromophores and influence their

2PA cross-sections. For this investigation, two well-known anionic polyelectrolytes, MPS-PPV

and PSS where chosen. The investigation was carried out with cationic dyes AcrO, Hoe, ThT and

neutral dye C-485. UV-Vis absorption, one photon and of two-photon fluorescence and real 2PA

cross-sections used all to study the charge effect on local electrostatic environment of Organized

28

self-assemblies. 2PA enhancement is observed for chromophores when they are soluble in

electrostatic environments. But the enhancement varied with the type of chromophore.

Chapter 7 presents the overall summary of the research results, along with an outlook of

the future work.

1.11 Chapter 1 Summary

• Organized self-assembly forms in a spontaneous manner where components either form

or aggregate without any external interference.

• Organized self-assemblies are sensitive to change in their microenvironment such as

polarity, pH, co-assembled molecules, salinity and temperature.

• Self-assemblies are formed mainly by weak noncovalent bonds; however, they are

considered stable due to numerous of noncovalent bonds that work together to increase

stability.

• Chemical interactions for organized self-assemblies include hydrogen bonding, ionic

bonding, π-π stacking, van der Walls interaction, hydrophilic/hydrophobic interactions

and electrostatic interactions.

• Examples of self-assemblies include micelles, reverse micelles, lipid bilayers, DNA,

RNA, protein, polymer and polyelectrolyte.

• Organized self-assemblies are studied and monitored through several techniques include

affinity capillary electrophoresis, enzyme-linked immunosorbent assay, fluorescence

resonance energy transfer, isothermal calorimetry, ultraviolet visible spectroscopy,

nuclear magnetic resonance and surface plasmon resonance.

• In search of novel spectroscopic techniques to monitor self-organized assembly

interactions and sensing, 2PA spectroscopy was introduced.

29

• The research approach is that self-assemblies possess aligned electric fields and the 2PA

cross-sections of chromophores whose dipolar orientation is parallel or perpendicular

with this electric field can be used to measure the change in the electrical fields and

monitor them.

30

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CHAPTER 2

EXPERIMENTAL TECHNIQUES

Chapter 2 covers different experimental techniques that were used to carry out the

investigations detailed in this dissertation. The main experimental techniques used are steady-

state, time-resolved and two-photon fluorescence measurements. Circular dichroism

measurements and dynamic light scattering measurements are used for additional

characterization. The majority of the analytical techniques are related to the emission of radiation

that use high-intensity laser pulses to perform the measurements. The overall objective of this

dissertation is to use all these novel spectroscopic techniques to study and understand micro-

environment in self-assembled systems. A brief description of the experimental techniques is

presented in this chapter.

2.1 Lasers

“Light Amplification by Stimulated Emission of Radiation” is the acronym for what is

now referred to as a laser. Lasers are defined as sources of radiation that result in a highly intense

light source1,2. To further describe them, they are constructed in such a way that they can result

in a highly intense light source with monochromaticity, directionality and ultrashort pulse

duration. The unique properties of this laser radiation that make them useful for studying

different optical phenomena is presented here1.

2.1.1 Basic Components of Laser

The intense light created by lasers is useful for studying the 2PA properties of

molecules3. Moreover, laser is a very powerful tool for probing both linear and nonlinear optical

properties of molecules and materials4,5. The basic makeup of a laser has three major

45

components. These components are:  an optical cavity (optical resonator); a pump source which

is used for particles excitation which are located in the gain medium; and most important is the

gain medium6–9. The basic components of a laser are shown in figure 2.1.

Figure 2.1: Schematic depicting the basic components of a laser.

2.1.1.1 Optical Cavity

Optical cavities are composed of two highly reflective mirrors which make the light

bounce and forth between them amplifying the stimulated emission of the radiation that was

generated in the gain medium. One cavity mirror contains a highly reflective mirror (100%)

which would deflect all stimulated emission back to the gain medium. The second mirror has

a partially reflective property which referred to as an output-coupler (95%). The main function of

this output-coupler is to reflect back most but not all of the stimulated emission which causes

transmission losses to which will generate the laser beam out of the cavity7–11. As the gain

medium generates energy a laser output can be sustained out of the cavity.

46

2.1.1.2 Gain Medium

The stimulated emission is generated in the gain medium. The population inversion is

created in the gain medium using pumping mechanism. The generated stimulated emission is

amplified in the optical cavity when the light bounces back and forth inside it, and undergoes

amplification of number of times inside the gain medium so as to give rise to an intense laser

output9,12. The amplified light maintains the same direction and phase giving directionality and

coherence to the laser light output. This is due to the stimulated emission of the radiation which

was generated in the gain medium9,13. The particles located in the gain medium should be in a

state of population inversion as to attain an efficient laser output. For the population inversion to

take place a pumping mechanism in needed. An example of a gain medium can be Ti:Sapphire

or Nd:Yttrium Aluminum Garnet etc. In this dissertation Ti:Sapphire (Ti3+:Al2O3) was the gain

medium which is a transition-metal-doped widely used as gain medium for femtosecond solid-

state and tunable lasers.

Ti: Sapphire was first introduce in 198613. Sapphire has a great thermal

conductivity which improving the thermal effects for high laser intensities and powers. While the

titanium ion used due the large bandwidth which appropriate to generate a very short

pulses and wide wavelength tunability.

2.1.1.3 Pumping Mechanism

Pump Source is considering the energy source for the laser to populate a high energy

level of the laser. Therefore, pumping mechanism is essential to achieve appropriate and

adequate population inversion, which is a state where the population in the higher energy state

is higher than the lower energy state. The pump Source could be from optical, electrical or

chemical source. The laser that been used in this dissertation is based in optical pumping

47

mechanisms9,14. For the Ti:Sapphire laser, the pumping mechanism used was an optical pumping

by Nd:YVO4 laser. The Nd:YVO4 has a pumping source of a diode laser. The diode laser is

pumped by electrical input.

2.1.2. Properties of Laser Light

The laser radiation has several unique properties, which makes it a powerful

tool for probing linear and nonlinear optical phenomena. Some of the characteristics are

discussed here.

2.1.2.1 Monochromaticity

Monochromaticity is a perfectly functioning single wavelength of laser radiation. This

monochromatic radiation also possesses an extremely narrow bandwidth comparable to

conventional sources of radiation that can comprise of different variations of wavelengths of

radiation as shown in figure 2.2. Laser radiations not perfectly monochromatic; however, it has

capabilities of emitting a narrow bandwidth around a single wavelength9. The monochromaticity

characteristic is based on the gain mechanism that depends on stimulated emission of radiation.

There are several different laser sources that can produce an array of wavelengths and frequency

bandwidths. Because of the stimulated emission of radiation, one wavelength of radiation gets

amplified in the resonant cavity resulting in very narrow line bandwidth having a specific

wavelength or frequency 9,12.

48

Figure 2.2: This figure illustrates the advantage of laser light monochromatic properties over

ordinary light.

2.1.2.2 Coherence

One of the fundamental property of laser radiation is its coherence

Electromagnetic radiation is said to be coherent if it has the same phase throughout the beam of

radiation9. Radiation is termed as a coherent at that particular moment when the trough and crest

of emitted photons are linked with one another (Figure 2.3).

There are two types of coherence. These systems are referred to as temporal and spatial

coherence9,15,16. The temporal coherence possesses a frequency of a wave at difference time

period which will remain constant while holding monochromaticity. The spatial coherence has

a constant phase relationship between two waves in a wave-front at different places in space.

One consequence of spatial coherence is that it possessed minimum divergence9,15,16.

49

Figure 2.3: This figure shows how coherence is one of the unique properties of laser light

comparing to ordinary light source.

2.1.2.3 Directionality

Directionality is one of the important properties of laser radiation which causes its

propagation in a single direction with suitable orientation12. This property enforces outcome laser

radiation to be in single direction because the amplification and sustaining of waves in the gain

medium can only happen to the waves that propagate in a direction parallel to the gain medium

itself. It is the property of the laser radiation that makes it propagate in single

direction possessing pure collimation. Moreover, it is the degree to which the rays remain

parallel as they propagate as shown in figure 2.4. Most of the conventional radiation sources,

light is emitted in all directions as the process is spontaneous. On the other hand, the laser

radiation will produce a radiation that displays minimum divergence. This can be ascribed to the

stimulated emission of radiation and a divergence of milli-radian which can be obtained from

50

these systems. A collimated laser beam fundamentally emits efficient power density. This

produces a tiny spot size via diffraction limit as compared to a conventional light source and

makes it important for nonlinear optical applications9,13.

Figure 2.4: Directionality of the beam displaying minor divergence of compact light waves.

2.1.2.4 Ultrashort Pulse Duration

The majority of the laser sources are of a continuous wave type. However, a short pulse

system is needed to study the nonlinear optical phenomenon. The laser sources will be able

to generate short pulses if they are appropriately designed and tuned in. The short time durations

are moments of time that exist between two consecutive pulses that are separated with

duration on the time frame of nanoseconds to femtoseconds9,17,18. The ultrafast pulse durations of

the lasers can provide the information needed to study the nonlinear optical phenomenon such as

two-photon absorption (2PA). The ultrafast laser pulses can be attained n different manner one of

51

which is cavity dumping. Other forms include, Q-switching, mode locking, and

several others17,19. In this dissertation we used of mode-locking method which is employed for

the generation of ultrashort pulses.

The mode-locking technique is one of the routinely used methods for the production of

ultrashort laser radiation pulses. Overall, this technique is needed to provide a constant phase

correlation among various modes that are produced in the resonant cavity20. This system is

similar to phase-locking technique. As there is a generation of various frequencies due to the

emission coming from the active medium. The overall rate of locking of a certain mode has some

effects on the lasers’ pulse duration. The materials which need high intensity laser sources need

ultrashort laser pulses17,19–21.

If the longitudinal modes of a laser have a fixed and properly defined phase relationship,

then the laser is understood to be in a state of mode-locked21. The derivation of the mode-

locking procedure can be useful in comprehending how the technique works. Considering the

mode-locking where the oscillating modes all resonate at relative amplitudes in a resonation

cavity, where E0 refers to equal amplitude, Δω delta omega represents the change in

frequency, ωo represents the central frequency and N is the total number of modes which are

involved. The electromagnetic field which happens due to the 2n+1 mode which are equally

spaced and have a similar amplitude is obtained by :

E(t)=∑ = − nq n Eqei[(ω

o + q Δω) t + φ

q] 2.1

Equation 2.1 is a representation of the total summation of all possible nodes. In instances

where there locked phases exist and similar amplitudes equation 2.1 can be reduced to

E(t)=Eoeiωot ∑ =nq

− neiqΔωt 2.2

52

Equation 2.2 can be equated to

E(t)=A(t)eiωot 2.3

Where A(t) is calculated by

A(t) = Eo ∑ =n

q − neiqΔωt 2.4

Furthermore equation 2.4 can be expressed in terms of viewing the intensity by a function

of time, which gives:

I(t) = α [A(t)]2 = ( sin2 [(2n+1) Δωt/2]) / sin2 (Δωt/2) 2.5

As can be seen, an increase in the number of modes will cause a reduction in the pulse

duration and a rise in the amplitude. The different types of laser sources can be used depending

on what amplitude is needed as well as the pulse duration needed. Wherever there is a presence

of continuum vibrating mode this means that there are oscillating instances. The distribution of

their amplitude usually exhibits a Gaussian-like curve. For this situation A(t) can be expressed as

follows by equation 2.6

A(t) = ∑ nq = − n Eqe iqΔωt 2.6

The amplitude is not a constant; however, the above equation is integrated, and the limits

are considered to be infinite as the amplitude En is zero. This leads to the Fourier transformation

function as:

A(t) = ∫ − Eqe iqΔωt dq 2.7

53

The multi-mode output of the amplitude is obtained through the transformation of the

mode distribution in the frequency domain. Special characteristics of the laser sources will make

them ideal for monitoring the time-resolved and two-photon fluorescence measurements. The

measurements, time-resolved and two-photon fluorescence, use broadband 100 fs pulse width

Ti:Sapphire laser radiation that can be tunable between 710 to 900 nm with a fundamental

wavelength of 800 nm. Additionally the steady-state optical measurements used will be

discussed here.

2.2 Steady-State Methods

Optical absorption and steady-state fluorescence techniques are mostly used for the

analysis of molecules and the environments in organized self-assemblies. Steady-state

measurements work by exciting the molecules via the absorption of radiation to reach the higher

excited state then record the emitted radiation for fluorescence measurements.

2.2.1 Optical Absorption

The optical absorption spectroscopy (known also as s ultraviolet-visible spectroscopy) is

valuable technique to study the ground state (low energy state) absorption of the molecule or

material22,23. The absorption of radiation is often used to understand the transitions involved in

the molecule and the influence of environment on it. From Lambert-Beer's law (equation 2.8),

the absorbance can be used to calculate the concentration if the molar extinction coefficient is

known. Often absorption spectrum of a molecule is affect by solvent polarity, molecular

concentration, unclear or colloidal samples, compound functional group and electronic

arrangement of molecules22–25.

54

The absorbance of a molecule can be expressed by Lambert-Beer's law:

A = log(I0/I) = ελCl 2.8

where, I0 represent the intensity of the incident radiation while I represent the transmitted

radiation, the path length of the cuvette is l. In this dissertation, all optical absorption

measurement was carried out using Shimadzu UV-160 absorption spectrophotometer.

2.2.2 Steady-State and Time-Resolved Fluorescence Measurements

Fluorescence is a radiative decay from the excited state of a molecule to a lower energy

state of same spin state. Fluorescence process relies on three fundamental steps: excitation,

excited-state relaxation, and radiative decay (Figure 2.5). Upon excitation, the molecule absorbs

appropriate photon energy and will excite molecule’s valence electron to temporarily leave to a

higher electronic excited state. The electron stays in the excited state which usually lasts for few

nanoseconds, then the excited electron will lose acquired energy through radiative decay to relax

back to a lower energy state24,26–28 giving rise to fluorescence 28. The obtained fluorescence

emission spectrum has distinguishing characteristics for the molecule and its intensity will

change because fluorescence emission is proportional to concentration. Shifts in fluorescence are

often observed which is known as Stoke’s shift. The Stoke’s shift is referred to energy difference

between absorption spectrum and emission spectrum of radiation that is usually affected by self-

assembly interactions24.

55

Figure 2.5: Schematic diagram of depicting fluorescence.

Fluorescence measurements can be classified into two main groups. These two groups are

steady state and time-resolved measurements. Between these two the most common one is

steady-state measurements in which there is a continuous beam of radiation that is used to

illuminate the sample and the intensity of the emission will be monitored. For the measurements

carried out in the dissertation, a Edinburgh F900 spectrolfuorimeter was used. A Xe450W lamp

was used as the excitation source and a R2618P PMT was used as the detector.

For time-resolved fluorescence measurements, the fluorescence intensity decay as a

function of time is monitored. Furthermore, for time-resolved measurements, the samples are

exposed to a pulse of radiation in which the pulse-width is typically shorter than the decay time

of the fluorescence. In this case, a high-speed detection system to measure the intensity in the

nanosecond timescale or faster is required. Both time-resolved and steady-state measurements

56

are relative because the steady-state observation is the average of the time-resolved phenomena

over the complete intensity decay of the sample24,29,30.

The excited state lifetimes of the organized assemblies were measured with time-resolved

techniques. Also, time-resolved anisotropy techniques were done to provide the information

about binding detail in the organized assemblies31. Lifetimes of the different systems required

both time-resolved fluorescence measurements. These include the time-correlated single photon

counting (TCSPC), which provide different time resolutions as the TCSPC has a resolution of

250 ps.31,32

Time -correlated-single photon counting (TCSPC) is used in this dissertation to monitor

the fluorescence lifetimes with diode laser excitation. A schematic of TCSPC setup is provided

in Figure 2.6. For TCSPC, sample is excited with diode laser pulses, then an appropriate detector

system will record the time difference between the first fluorescence photon of the

sample and the excitation pulse. In this technique, only one photon is required to observe a large

number of excitation pulses. It is imperative that a very low count rate is set to operate the

system in a single photon counting mode. In this condition, statistics of poisson distribution and

were followed as fluorescence is spontaneous30,31,33. Time-resolved fluorescence measurements

were carried out using Edinburgh F900 spectrofluorimeter with PicoQuant diode lasers as

excitation sources.

57

Figure 2.6: Schematic diagram of TCSPC setup.

Following the schematic, an excitation pulse is split into two fractions, one part is used to

excite the sample and the other is used to generate a start pulse in the start PMT or

photodiode. This optical signal at the PMT will initiate an electrical start pulse, that runs through

a constant fraction discriminator (CFD) to start the input of time to amplitude converter(TAC) to

initialize the charging operation. A second part of the optical pulse will excite the sample and

giving rise to a n emission of photons. The emitted photons are detected by stop-PMT which will

generate an electrical stop pulse. This stop pulse will pass through another CFD along with a

variable delay line before it is routed to the TAC. the TAC stops it charging operation after

receiving the stop signal. It then generates an electrical output, having an amplitude proportional

58

to the time difference (Δt) between the start and stop pulses extending to the TAC. this TAC

output pulse is then directed to the input of a multi-channel analyzer (MCA) through an analogue

into a digital converter (ADC).

This ADC will generate a numerical value that corresponds to the TAC output pulse

which is selected to an appropriate channel of the MCA and the count is added to the channel.

The circulation of the excitation-to-data-storage is repeated a large number of times. As

a result, a histogram of the counts-versus-the-channel-number of the MCA is produced. This

represents the true emission decay as the collection rate of emission of photons by the stop pulse

is very low this visible emission decay will then be deconvoluted with the instrument response to

get the corresponding lifetime. The instrument response function (IRF) is measured using a

scattered put into the place of the sample. Furthermore, the width of the IRF function is

dependent on excitation source and detection systems used14,34–39.

2.3 Two-Photon Absorption

The two-photon absorption process is a non-linear process where two photons

are absorbed simultaneously to excite the molecule from its ground state into a higher state, with

total energy is equal to the energy difference between the ground and excited state. This process

takes place at higher optical intensities and a femtosecond laser excitation is needed to observe

two-photon absorption and two-photon excited fluorescence. This phenomenon was first

introduced theoretically by Maria GöppertMayer in 1931 in her doctoral dissertation3,40. Soon

after laser innovation in 1961, two-photon absorption was first demonstrated experimentally by

Franken and others who experimentally demonstrated the process in a Europium-doped crystal

41,42. In honor of Göppert-Mayer, the unit for 2PA cross-sections is named GM after her, also she

was awarded Physics Nobel Prize in 196343–45.

59

The main advantage of two-photon absorption over conventional one-photon absorption

is it involves two photons absorbed simultaneously and uses much smaller energy photon. To

completely understand, two-photon absorption is a rare phenomenon that requires an intense

laser source to observe its effect. The rate equation for the 2PA is a second-order in the photon

flux and a first-order in the concentration of molecules, equation (2.9)

(dNTP /dt) = ½ δ Ngs F2 2.9

where, NTP is the number of molecules per unit volume in the excited state, t is the time.

δ represent the two-photon absorption cross sections, while Ngs is the number of molecules in the

ground state and finally F2 is the photon flux( the number of photons per unit are and time, light

intensity).

As discussed above, the 2PA has the advantage as it uses near infrared wavelength which

will lower the scattering effect when compared to 1PA. In Rayleigh’s law of scattering via

equation 2.10, the excitation wavelength (λ) can reduce the scattering effect (σs) by up to 16

times for two-photon excitation46–49.

σs α 1/λ4 2.10

Another advantage of 2PA over 1PA is the ability to penetrate deeper, so that it can be

used for 3D microfabrication and multi-photon imaging48,49.

2.3.1 Measuring 2PA Cross-Sections

There are several techniques that can be used to measure the 2PA cross sections s. The

most common way is to use nonlinear transmission techniques. For example, open-aperture-z-

scan.  As researchers used this technique, they found that it can be erroneous because of overlap

60

from the excited state absorption. To overcome this issue researchers have used fluorescence-

based techniques. The absolute 2PA-cross-sections can be measured with knowing the incident

radiation power and the emitted radiation power. Moreover, measuring the 2PA-cross sections s

are difficult and complicated, therefore; measurements using a relative two-photon excited

fluorescence (TPEF) technique is used to determine the 2PA cross-sections, which is used

during this dissertation 50,51.

TPEF (Figure 2.7 gives a schematic of a TPEF setup) is one of the simplest methods

that can be employed to determine the 2PA cross-sections of molecules.  It was originally used

for the determination of the 2PA properties of CaF2:Eu2+ crystal43. Directly measuring the 2PA

cross-sections is a difficult task due to the small portion of photons that undergo absorption in

the excitation process by two photons. To overcome this issue and facilitate measuring 2PA, the

measurement should be concluded through a particular chromophore that is quite fluorescent,

and must possess higher fluorescence quantum efficiency and known 2PA cross-sections . This

technique is definitely limited to the study of fluorescent molecules because fluorescence

quantum yield is a material intrinsic property31.

Rebane and associates, have carried out measurements of 2PA cross-sections for several

dyes in a broad wavelength region of 650 nm and 1500 nm which used as a reference for this the

measurements carried out in this dissertation51,52. For the two-photon fluorescence experiments

in this dissertation, Ti:Sapphire laser was used (Tsunami, 100 fs, 700 to 900 nm) as the

excitation source53.  In addition, a rotating neutral density filter was used to change the excitation

power. The laser light was focused by a lens onto the sample cuvette placed in the Edinburgh

fluorimeter, then fluorescence was collected perpendicularly to the excitation direction using a

photomultiplier tube. The fluorescence produced after two-photon excitation was focused on the

61

slit of the monochromator, then it was detected with a cooled Hamamatsu R2618P PMT detector.

 To reduce the fluorescence self-absorption, beam is moved close to the sample cells edge beside

the the direction of measurement collection. This also reduces the fluorescence path length which

help to reduce possible noise from the incident beam.

For measurements used in this dissertation, an Edinburgh F900 spectrofluorimeter was

used for two-photon fluorescence measurement with Ti:Saphhore as the excitation laser source.

2PA cross-sections measurements used Coumarin 485 in methanol as a standard based

on Rebane and co-workers51,52. Moreover, the TPEF has its own advantages when compared to

the nonlinear transmission methods such as: easy to use and more efficient without

interferences directed from both excite absorption and thermal lensing51,52.

Figure 2.7: Schematic of the setup used to determine the 2PA cross-sections.

ND is neutral density filter. LI and L2 are lenses.

2.4 Circular Dichroism

Another absorption spectroscopy technique that used in this dissertation is circular

dichroism (CD). CD measurements are used to study the conformations of organized self-

assemblies, especially chiral molecules of all types and sizes in solution such as proteins, DNA

62

etc. CD primarily is used because it is sensitive to change in confirmation around organized

assemblies, pH and temperature. Also, CD is used to monitor the interactions of organized

assemblies with molecules. Moreover, CD can be used to study kinetic and thermodynamic

information. CD spectroscopy has advantages that include: ease of use, sensitivity,

nondestructive method for the sample, not consuming large amount of sample and ability to run

measurements in solution54–56. CD uses circularly polarized light and is the difference in the

absorption between the left-side and right-side of circularly polarized components. This is

represented in equation 2.11

ΔA(λ) = ALCPL – ARCPL 2.11

where A is the absorbance and λ is the wavelength. The CD technique is usually used in a range

of wavelengths to analyze the data. The CD signal can be positive or negative, depending on the

selective absorption of the circular polarized lighting source whether light absorbed is greater

extent to left circular polarized light (LCPL), where CD signal will be negative, or the right

circular polarized light (RCPL), where CD signal will be positive57–59.

This radiation source for CD is produced when two linear polarized radiations are mixed

with respect to each other phase by a quarter wave plate. By the use of a quarter wave plate, this

will slow one of the linear components with respect to the other by a quarter wave and change

their refractive indices. The will result in the left circular polarized light (LCPL) or the right

circular polarized light57.

Chiral molecules are known to be existed as pairs or mirror image isomers,

“enantiomers”, and are not superimposable mirror images. Enantiomers are identical in their

chemical physical properties; however, not for their interactions with polarized light and other

chiral molecules. The optical rotation is measured for chiral molecules as a function of

63

wavelength giving a result referred to as optical rotatory dispersion spectroscopy (ORD). ORD is

used to determine chiral molecules concentration and purity. However, CD has a better

spectruml resolution when compared to ORD, therefore, CD is used for understanding the

organized self assemblies24,60.

As the linear polarized light passes thought the optically active sample, amplitude of the

less absorbed component will be larger than the more strongly absorbed component. As the

amplitudes are combined an ellipse will form. The degree of ellipticity ө is than defined as the

tangent ratio between minor and major axes elliptic. The linearly polarized radiation possesses

zero degree of ellipticity while LCPL degrees will be + 45 and - 45 degrees for RCPL 30,61–65.

CD is relative to the change in the absorbances of LCPL and RCPL as is only observed at

the wavelengths were the chiral molecule absorbs light. The CD and ORD can be interconverted

by the Kramer-Kronig transformation relation equation.

ΔA = ө / 32.982 2.12

The molar ellipticity is valued in deg.cm2 by dmol-1 (deg.M-1 by m-1) and calculated as in

equation (2.13)

[ө]M = 10 (ө / C * l ) 2.13

where ө is in mdeg, C is the concentration is molar, and l is the path length in cm. Molar

values can be converted to a molar extinction coefficient from the equation:

Δε = [ө]M / 3298.2 2.14

Most biological self-assemblies consist of chiral structure and CD is often used to study

them. The CD spectrum can be influenced by the three dimensional structure of the protein or the

64

DNA, so the spectrum is not a simple reflection of the individual residues or bases23,61–63,66. The

CD signal can be used to identify the structure depending on the average of the molecule. In

addition, CD can be used as well to as well to monitor changes in self-assembly structure.

However, it is difficult to identify which specific residues are responsible in each structure60,67.

The CD signal for protein can be calculated through the mean residue ellipticity [ө]MR

from equation 2.15. [ө]MR is responsible for individual protein residues which could be used to

compare different proteins with different molecular weight.

[ө]MR = 100 ( ө / CMR * l ) 2.15

where CMR is the mean residue concentration which can be calculated in two different ways. It

is a product of the molar concentration of the protein and the number of residues (N). The other

calculation can be done is by dividing the concentration of protein in unit of gram per liter over

the average amino acid residue weight via 113 Daltons 60.

CMR = C (molar) / N or CMR = C ( g/l ) / 113 2.16

For the measurements carried out in the dissertation Jasco J-815 Circular Dichroism (CD)

Spectropolarimeter was used.

2.5 Dynamic Light Scattering

Dynamic light scattering (DLS), also known as photon correlation spectroscopy, is a

powerful technique for measuring particle sizes through monitor solution dynamics. Self-

assembly size information can be obtained within few minutes with a few nanometer sensitivity.

As the light interaction with matter, the affected electric fields of the energy source will induce

an oscillating polarization on the matter. These molecules would release secondary source of

65

light energy and scatter the radiation. The type of light source can definitely have an effect on

the molecule. For instance, if there is a monochromatic and coherent light source such as laser

source, the time dependent fluctuation will be observed in the scattered intensity.

The observed fluctuation is due an interaction called Brownian motion. The Brownian

motion creates the distance between the scattered molecules within a solution as they are

changing continuously with time. Brownian motion is understood to be the random

phenomenon of particles in a solution from collisions initiated by the bombardment of a solvent

molecules that surrounds the particles. This can result in a Doppler shift between the frequency

of incident light as well as the frequency of the scattered light which are all due to the particles

size68,69. The obtained data could be studied through electrodynamics and time

dependent statistical mechanics theories.

To measure the size of the particles much smaller than the wavelength of the light, a non-

invasive technique via DLS can be used. The Brownian motion of samples can be caused by the

size of the particle, sample viscosity, as well as the temperature68. In the Brownian motion, the

velocity can be described by the translational diffusion coefficient (D). This can then be

converted to particle size via Stokes-Einstein equation (Equation 2.17)

dh = KBT / 6 π η D 2.17

where dh is the hydrodynamic radius, kB is the Boltzmann’s constant, T is the temperature in the

Kelvin (K) and the viscosity is η 68. The translational diffusion coefficient (D) is essentially

determined by particle size, surface structure, with ions in the medium and their concentration.

Understanding further, ions in addition to their concentrations affect the thickness of the electric

double layers (Debye length, K-1), which reflect on particle diffusion speed and size.

Furthermore, the particle sizes defined from the amount of scattered light, while the particle

66

diameter defined from scattered light intensity68,69. The static scattering and dynamic light

scattering are both used to study the scattering of light. As in static light scattering, the time-

average intensity of the scattering light is monitored while fluctuations of the light intensity are

monitored70.

2.5.1 How DLS Works

As the sample is illuminated by a laser in the quartz cuvette, a classical speckle pattern

can be observed. The interference of speckle presents a dark and bright bubble shape cause by

the destructive and constructive phases from the scattered light (Figure 2.8). The fluctuations in

the samples exhibit Brownian motion giving observation to the spots on the speckles can be seen

are in continuous motion. Furthermore, this is due to the phase addition from the moving

particles which is constantly forming into differentiating patterns where the rates of change are

faster in smaller particles and vice versa for larger ones. In the DLS system, there is a device

called a digital auto correlator which measures similarity degree of two or more signals with

different time intervals68,71.

Figure 2.8: Schematic representation of speckle pattern of the setup used to determine the DLS.

67

The scattered intensity is proportional to the sixth power of the molecular radius making

DLS a useful sensitive technique to trace the sizes self-assembly aggregates. With the presence

of a small amount of aggregate, significant changes will occur in the mean hydrodynamic radius.

Also, DLS seems useful in thermal stability studies by the measuring the change in size and

scattering intensity as a temperature function. Furthermore, the heat influences the denaturation

of self-assembly which leads to larger aggregates which will leads to increase scattering intensity

and the size of the particle.

It’s crucial for DLS measurement to dispersed sample well in the suspending medium,

therefore; filtration the sample is required to eliminate large particles or contaminants that could

cause not representative scattering. In addition, the refractive-index has to be different between

the dispersed phase and suspending medium. Moreover, the solvent refractive index and

viscosity must also be known to apply the Stokes-Einstein relationship (2-17) to calculate the

hydrodynamic particle diameter24.

Another important condition to run DLS is the sample concentration. Chosen sample

concentration depends on other factors: laser power, particle size and refractive index. If the

sample exceed the concentration limit, that will cause multiple scattering phenomena where

scattered light from one particle is re-scattered by another particle. The concentrated

charged self-assembly may increase the repulsion forces among molecule due to the reduction

in the intermolecular distance. This enables to an increase in the diffusion speed decreasing the

apparent size. On the other hand, if the sample was below concentration limit, that will lead to

reduced number of particles in the scattering volume which could be insufficient to be

detected68,69. The scattered light is measured at a 90° angle by photomultiplier tube detector. The

68

particle sized carried-out through correlation analysis of the processed signal as previously

discussed. Furthermore, to avoid possible multiple scattering phenomena, it is necessary to use

dual photodetectors. The dynamic light scattering performed using a DynaPro Titan Batch

instrument from Wyatt Technology Corporation. It is operates using a micro cuvette.

2.6 Chapter 2 Summary

• In this dissertation, ultrafast laser spectroscopy used extensively to monitoring changes in

local electric field of self-organized assembly system. Therefore, the laser basic

components and properties was introduced.

• Laser main components are optical cavity, pump source and gain medium.

• Optical absorption and steady-state fluorescence techniques are generally used to study

self-organized assembly either by study the absorbance radiation for UV-vis or study the

emission radiation for fluorescence which will used to determining the energy of a

molecule's excited state and studying the interaction between self-organized assembly

and dye molecules

• Appling two photon absorption help to excite a biological marker (dye molecule). There

are several advantages for two photon absorption over single-photon absorption as a

means of exciting a molecule such allowed Some forbidden transitions, better spatial

resolution, less photodegradation, less scattering, 3D imaging, higher excitation energies

deep penetrate and work in complex environment.

• Circular dichroism is a tool that explores the interactions of chiral molecules with

circularly polarized light. CD used to study the conformations structure of organized self-

assemblies and monitor the interactions with dye molecules.

69

• Dynamic light scattering used to measure organized self-assemblies sizes through

monitor solution dynamics. It used in this dissertation to measure protein aggregation.

70

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79

CHAPTER 3

ANALYZING THE STABILITY AND INTERACTIONS OF G-QUADRUPLEX AND

G-TRIPLEX DNA IN DRUG SYSTEMS BY USING NOVEL OPTICAL

SPECTROSCOPY TECHNIQUES

3.1 Introduction

Among different organized self-assemblies, deoxy ribonucleic acid (DNA) is one of the

interesting self-assemblies that can organize into different canonical forms, most famous being

the double helical shape visualized by Watson and Crick. In this dissertation, we have studied

different canonical forms of DNA using optical spectroscopic techniques.

It is well known that DNA contains the genetic information needed for all known

organisms to function. These genetic codes consist of instruction that are needed by cells to

replicate, transcribe, and translate that then helps organisms regulate their bodily functions. But

when the DNA is altered through mutagenic factors, it may lead to the dysfunction of the cell.

This dysfunction of the cell can lead to diseases such as cancer1,2. With the development of drugs

and technology, treatments have evolved to better treat these conditions, targeting DNA and

lysing cells that have become cancerous. Though these anticancer drugs are effective in their

treatment, causing the tumor cells to die, they have adverse effects that increase the chance of

another tumor to grow due to these drugs’ toxicities3,4. Current chemotherapeutic drugs bind to

highly active duplex DNA in cancer cells which is non-specific binding, but these drugs can bind

to negatively charged backbone of DNA as well. This led to the development of targeting non-

canonical structures using properties of the DNA backbone. 5–7.

One recent strategy of cancer treatment relies on targeting telomeres. Telomeres are at the

ends of chromosomes, playing the role of protecting the genome from degradation8. After each

replication cycle, the telomere shortens and with this continued shortening, DN start to become

80

damaged, eventually leading to cell death. But in the case of cancer cells, an enzyme known as

telomerase becomes highly active, adding nucleic acids to the ends of the chromosomes and

prevent cell death9–11. These enzymes are usually active in germline and hemapoetic cells, but

not in somatic cells12. An ideal way to target most cancers is to stop the telomerase activity by

targeting it in the DNA within the cancerous cell. About 90% of cancers have elevated

telomerase activities13; therefore, by coming up with a therapeutic way to target the cells with

elevated telomerase activity, one can effectively destroy tumors through minimal adverse

effects11. The key to this is that the telomeres have a stretch of single stranded DNA (ssDNA)

that has been elongated by telomerase14. It is known that these ssDNA can develop into non-

canonical structures, thus increasing the capability of targeting these cancer cells specifically10.

A well-known non- canonical structure is the G-Quadruplex along with an intermediate folding

structure called a G-Triplex15,16. These structures are possible due to the high number of

guanines that are added in by the telomerase and can prevent telomerase from elongating the

DNA strand (Figure 3.1)17.

The structure of the DNA plays a key role on how these drugs bind to the DNA, and

furthermore how the stability of the structure is affected after such binding. In the case of G-

Quadruplex structures, they contain purine-rich strands, which are mostly guanine, allowing such

non-canonical structures to form18. Quadruplex DNA contains stacked tetrads that help with the

stabilization of the structure, which is done through cyclic hoogsteen binding arrangements that

aids with the current hydrogen bonds18. Adding monovalent cations such as sodium or potassium

has been shown to stabilize the structure further by “coordinating with eight carbonyl oxygen

atoms present between stacked tetrads.”19 It has also been shown through multiple studies that G-

Quadruplexes have multiple structures that they can take up and still be stable (Figure 3.2)17,20.

81

Figure 3.1: Biological roles of the telomeric DNA. G-Quadruplexes can prevent the elongation

reaction in tumor cells17. Telomeric DNA shortens after each cycle of division, but in a tumor

cell, an enzyme called Telomerase elongates the telomere. With G-Quadruplex formation, the

telomerase activity is inhibited.

Figure 3.2: Polymorphic G-Quadruplexes17. Different types of G-Quadruplexes with the need of

certain ions in order to be stable along with their DNA sequence.

82

Similar to the G-Quadruplex, there exists a G-Triplex that displays G-rich triad planes

which are also stabilized with Hoogsteen binding and also have alternative many structures21. In

a previous study, a truncated version of the DNA that formed the G-Quadruplex was used to

form a stable intermediate known as the G-Triplex (Figure 3.3)21,22. It has been hypothesized by

scientists that these G-Triplex exists in various parts of our genome, expressing functions that are

yet to be fully discovered 23,24.

Figure 3.3: Schematic representation of G-tetrad, G-Quadruplex, G-Triad, and G-Triplex22.

It displays the guanines functional groups involved in creating the structure.

The way to stabilize these non-canonical structures is by binding small molecules such as

drugs to stabilize them. Several techniques are normally used to monitor the stability of the non-

canonical structures that include circular dichroism, fluorescence etc. However, they are not as

effective and selective. There is a need to develop novel techniques to monitor the stability of the

non-canonical DNA structures. This forms the main motivation behind this study. In this

83

investigation, Thrombin Binding Aptamer (TBA) consisting of 15-mer oligonucleotide (5′-

GGTTGGTGTGGTTGG-3′) was used because it forms a G-Quadruplex22,25,26. The shortened

form of TBA, (TBA11) consist from 11-mer oligonucleotide (5′-GGTTGGTGTGG-3′) has been

shown to form a relatively stable G-Triplex structure (Figure 3.4) 21,22,27. Both G-Quadruplex

and G-Triplex structure formed through the hydrogen bonding interactions in the presence of

potassium28,29.The stability of G-Quadruplex and G-Triplex structures can be estimated by

monitoring the DNA melting transitions (the temperature at which 50% DNA is melted)30.

Melting or denaturation of DNA can be induced by many factors such as temperature, pH and

organic molecules such as urea. Most investigations have used temperature to induce the melting

of the DNA as hydrogen bonds tend to get broken with an increase in temperature. According to

Jiang et all, TBA G-Quadruplex is relatively stable in K+ buffer, where the melting temperature

(Tm) is at 48.9 °C under the molecular dilution condition. While for G-Triplex (TBA11), Tm is

determined to be 26.4 °C 21. This low Tm in G-Triplex could indicate that G-Triplexes may not

be stable at physiological temperatures15. If drugs can increase the Tm, it would be ideal.

There are several ways to measure DNA binding and stability such as CD and optical

absorption. These techniques shown promise for analyzing and monitoring the binding of

different drug molecules to DNA structures, including the present non-canonical structures33,34.

In this study we used the power of two-photon absorption (2PA) spectroscopy to monitor

molecule-DNA interactions. Our hypothesis is that the 2PA cross-sections of molecules are

sensitive to local electric fields and their orientation, which will be used to monitor the stability

of these DNA structures.

84

Figure 3.4: G–Quadruplex and G–Triplex sequences, and it illustrates how the DNA folds due

to Guanin and forms these structures.

The drugs used in these systems are known to bind with DNA using hydrogen bonding,

pi-pi stacking interactions, and/or the electrostatic backbone of DNA. Thioflavin T (ThT) has

been shown to bind preferentially to non-canonical structures through intercalation and external

binding35. 4',6-Diamidino-2-phenylindole (DAPI) preferentially binds to dsDNA rather than non-

canonical structures through minor groove binding and thus it would be interesting to see how it

binds to non-canonical structures36. Thiazole Orange (ThO) has been shown to bind

preferentially to non-canonical structure as supposed to dsDNA, but has been difficult to show

the binding orientation of the molecule with non-canonical structure37. Doxorubicin has been

shown in previous literature to be able to bind to G – Quadruplex structures through pi-pi

stacking interactions along with hydrogen bond interactions 38,39. 5,10,15,20-tetra-(N-methyl-4-

pyridyl)-porphine (TMPyP4) has been shown to contain the proper attributes in physical

85

properties to bind with G- Quadruplex structures, either intercalating or interacting with the

stacking interactions17. TMPyP4 is the most extensively dye used to study G-Quadruplex. The

structural features of TMPyP4 that consist of four modified pyrrole components and four

cationic functional groups give TMPyP4 ability to bind to a human telomeric G-Quadruplex with

high affinity through either p–p stacking or electrostatic interactions40,41. Ethidium derivatives

such as ethidium bromide (EtBr) has shown to bind with non-canonical structures. EtBr as well

binds with G- Quadruplexes, in a similar way to as the previous drugs, by interacting with the

stacking interactions but has been shown to bind better to G-Triplex DNA35. The stability of G-

Quadruplex and Triplex with these drug molecules was studied in the investigation. The idea is

to find which drug can better stabilize these non-canonical structures.

Figure 3.5: Molecular structures of the investigated drug molecules.

86

3.2 Materials and Methods

3.2.1 Materials

G–Quadruplex and G–Triplex systems were obtained from Integrated DNA technology,

San Diego, CA, and buffer was combined to make a DNA stock solution. Tris (hydroxymethyl)

aminomethane hydrochloride, KCl was obtained from Sigma-Aldrich and used as received.

Nanopure water from Millipore was used. A buffer solution was made with Tris-HCl (10 mM),

Na2EDTA (0.5 mM), KCl (100 mM), and MiliQ water (200 mL) at a pH of 7.4 was used for both

G – Quadruplex and G – Triplex. Thermal annealing technique was used on the DNA/buffer

stock solution to allow the intramolecular bonds to form.

5,10,15,20-tetra(N-methyl-4-pyridyl)-porphine (TMPyP-4), Thiazole Orange (ThO) 4',6-

Diamidino-2-Phenylindole (DAPI), Ethidium Bromide (EtBr), Thioflavin T (ThT) and

Doxorubicin (DoXo) drugs were obtained from Sigma-Aldrich and used as received. Dyes were

dissolved in Dimethyl Sulfoxide (DMSO) and diluted with MilliQ water to make 10 mL of 1

mM of stock solution. Each sample (1 mL) used for measurements contained both the drug (20

μM) and the DNA buffer solution (980 µL). This process was repeated for all the investigated

drug molecules.

3.2.2 Optical Methods

Optical absorption measurements were carried out using a Shimadzu UV2101 PC

spectrophotometer. An Edinburgh F900 spectrofluorimeter was used for one-photon fluorescence

spectroscopy. The excitation source consisted of a 450 W Xe lamp and Hamamatsu R2518P

detector. Two-photon excited fluorescence technique was used for measuring 2PA cross-

sections. The output from Tsunami laser at 800 nm, 100 fs was used as excitation and the power

was varied using a neutral density filter. The fluorescence obtained was collected in an

87

Edinburgh F900 spectroflourimeter. Circular Dichroism (J1100 CD) was measured under N2 and

analyzed to find the molar ellipsity. All spectroscopy measurements were carried out as a

function of temperature.

3.3 Results and Discussion

3.3.1 Naked G-Quadruplex

3.3.1.1 Optical Absorption Measurements

The absorption spectrum of G-Quadruplex was measured between 220 nm and 350 nm at

different temperatures and shown in Figure 3.6. The spectrum has shown maximum at 257 nm

and some changes were observed when the temperature changed from 20 °C to 90 °C. At around

40 °C, the absorbance has increased which is then stabilized at 70 °C. After 70 °C it began to

further unfold, eventually leading to a single stranded DNA. These shifts are well represented as

the change in absorbance at 257 nm are shown as the inset in Figure 3.6. The changes on

absorbance at 257 nm are often used as the indicators for unfolding of DNA structures. Even, the

absorbance at 292 nm is also used for monitoring the DNA melting transition and is shown in the

inset of Figure 3.6.

3.3.1.2 CD Measurements

CD measurements were carried out to monitor the melting of G-Quadruplex. The molar

ellipticity shown in Figure 3.7 (A), shows us where the G-Quadruplex starts to melt. Molar

Ellipsity of Quadruplex DNA at 246 nm, 267 nm and 290 nm as a function of temperature are

shown in Figure 3.7(B). The CD spectrum of TBA in a buffer contained K+ show a positive peak

88

around 291 nm and a negative peak around 268 nm, this result indicates that the TBA is forming

antiparallel G-Quadruplexes.22,27,43

250 300 3500.0

0.5

1.0

1.5

Ab

so

rba

nc

e

Wavelength nm

Quad

20 30 40 50 60 70 80 901.16

1.18

1.20

1.22

Ab

s. @

257

Temperature oC

0.15

0.18

0.21

0.24

Ab

s. @

29

2

Figure 3.6: Optical absorption spectrum of naked Quad DNA as a function of

temperature. Inset shows the variation in absorbance at 257 nm and at 292 nm.

200 220 240 260 280 300 320 340-10

0

10

20

30

Mo

lar

Ell

ips

ity

wavelength nm

Quad DNAA

10 20 30 40 50 60 70 80 90 100-10

-5

0

5

10

15

CD@246 nm

CD@267 nm

CD@292 nm

Tempreature oC

Mo

lar

Ellip

sit

y

QuadB

0

5

10

15

20

25

30

Figure 3.7: (A) Molar ellipsity of naked Quadruplex DNA as a function of temperature; (B)

Molar Ellipsity at 246 nm, 267 nm and 292 nm.

89

The optical absorption and CD measurements show melting of G-Quadruplex at around

45 °C matched well with literature reports.

3.3.2 Naked G-Triplex


The optical absorption spectrum of G-Triplex as a function of temperature was measured

between 230 nm and 330 nm and shown in Figure 3.8. The spectrum has a maximum at 255 nm

and changes were observed as a function of temperature. Inset of Figure 3.8 shown changes at

255 nm and at 290 nm. From the data, melting transition of 26 °C was determined.

250 300 3500.0

0.3

0.6

0.9

1.2

Ab

so

rban

ce

Wavelength nm

Triplex DNA

0 20 40 60 80

1.10

1.15

1.20

Ab

s.

@29

0 n

m

Ab

s.

@25

5 n

m

Temperature o

C

0.08

0.10

0.12

0.14

Figure 3.8: Absorption spectrum of Triplex as a function of temperature. Inset shows the

variation in absorbance at 255 nm and at 290 nm.

3.3.2.2 CD Measurements

CD spectrum of naked G-Triplex DNA as a function of temperature are shown in Figure

3.9A. Figure 3.9B shows the decrease in molar ellipsities at 255 nm and 290 nm and both show

melting transition around 25 °C and matched well with literature reports of melting transition of

90

TBA1143,22,27.. In addition, it can be noticed that the melting of G-Triplex is lower than G-

Quadruplex signifying that the possibility of forming G-Triplex is less than a G-Quadruplex.

240 260 280 300 320 340

0

10

20

Mo

lar

Ell

ipsit

y

Wavelength nm

Triplex DNAA

10 20 30 40 50 60 70 80 90 100-2

0

2

4

6

CD@255 nm

CD@290 nm

Temperature oCM

ola

r E

llip

sit

y

TriplexB

4

8

12

16

Figure 3.9: (A) Molar ellipsity of naked Triplex DNA as a function of temperature; (B) Molar

ellipsity change of Triplex DNA at 255 and 290 nm.

The optical absorption measurement and CD measurement for Triplex DNA show that

similar melting transition around 25 °C. It will be interesting to see if the drugs can increase the

melting transition so that it is stable in physiological temperatures.

3.3.3 Interaction with ThT

3.3.3.1 G-Quadruplex/ThT

3.3.3.1.1 Optical Absorption Measurements

Optical absorption was carried out for Quadruplex DNA with and without ThT at room

temperature as shown in Figure 3.10 A. Absorption maximum has shifted from 412 nm to 417

nm when ThT is bound to Quadruplex. Figure 3.10B shows the absorption spectrum of

ThT/Quadruplex as a function of temperature. The inset of Figure 3.10B shows the change in

91

absorbance at 257 nm and 415 nm and both of them match well with each other. The melting

transition seems to become higher when ThT is bound to G-Quadruplex. Further fluorescence

measurements were carried out to study the stability of G-Quadruplex upon binding to ThT.

200 300 400 500

0.0

0.5

1.0

1.5

Ab

so

rba

nc

e

Wavelength nm

ThT

ThT-Quad

A

300 400 500 6000.0

0.2

0.4

0.6

0.8

Ab

so

rban

ce

Wavelength nm

ThT/QuadB

20 30 40 50 60 70 80 900.30

0.45

0.60

0.75

Ab

s @

415 n

m

Ab

s @

257 n

m

Temperature oC

0.12

0.16

0.20

Figure 3.10: (A) Absorption spectrum of ThT with and without Quadruplex, (B) Absorption

spectrum of ThT/Quadruplex as a function of temperature. Inset shows the change in

absorbance at 257 nm and 415 nm.

3.3.3.1.2 Steady-State Fluorescence Measurements

ThT is known to bind to G-Quadruplex DNA with an increase in fluorescence counts

upon binding44,45,46. Steady-state fluorescence measurements for ThT/Quadruplex as a function

of temperature are shown in Figure 3.11. The maximum of fluorescence was observed at 483 nm

at 20 °C and then counts decreased with increase in temperature. The change in fluorescence

counts at 483 nm is shown in the inset of Figure 3.11. The decrease in fluorescence counts

suggest that ThT is coming out of the G-Quadruplex DNA, but it could also be a combination

effect from increased non-radiative deactivation with increase in temperature. The decrease was

unable to clearly predict the melting transition.

92

450 500 550 600 6500

100

200

300

400

Fl C

ou

nts

Wavelength nm

ThT/Quad

20 40 60 80

0

150

300

450

Fl @

48

3 n

m

Temperature oC

Figure 3.11: One-photon fluorescence spectrum of ThT/Quadruplex as a function of

temperature. The inset shows the variation in the fluorescence intensity at 483 nm.

3.3.3.1.3 Two-Photon Fluorescence Measurements

As the main objective of the study is to accurately determine the melting transition so that

we can assess the stability of G-qaudruplex binding with ThT. Two-photon fluorescence

measurements were measured as a function of temperature from 20 °C to 90 °C after 800 nm

excitation and shown in Figure 3.12. The inset shows the decrease in 2P fluorescence intensity at

483 nm and as in the case of one-photon fluorescence, 2P fluorescence intensity also decreased

dramatically with increase in temperature. Even 2P fluorescence was unable to accurately show

the melting transition of G-Quadruplex.

93

450 500 550 600 6500

1000

2000

3000

4000

5000

6000

Fl C

ou

nts

Wavelength nm

ThT/Quad

20 40 60 80

0

1500

3000

4500

6000

2P

@4

83

nm

Temperature oC

Figure 3.12: Two-photon fluorescence spectrum of ThT/Quad as a function of temperature.

The inset shows the change in fluorescence at 483 nm.

3.3.3.1.4 Relative 2PA Enhancements

The relative 2PA cross-sections were determined from the ratio of 2PA fluorescence to 1PA

fluorescence with the normalization at the lowest temperature. As the one-photon and two-photon

fluorescence did not provide any information about the melting of the Quadruplex, we resorted to

the use of relative 2PA enhancement that is affected by the local electric fields offered by the

DNA. Shown in Figure 3.13 are the relative 2PA cross-sections of ThT/Quad as a function of

temperature. Note from Figure that there is a transition around 35 °C and a transition at much later

temperature for ThT/Quad. It is expected that 2PA cross-sections of ThT decreases when Quad

melts as ThT orientation will be different when the Quad is melting. The data suggest an interesting

intermediate during the melting of the Quad in the presence of ThT. The presence of the

intermediate suggests that ThT is probably a best drug to stabilize the Quadruplex DNA and this

information would not have been obtained if we had not used the 2PA cross-sections.

94

20 30 40 50 60 70 80 90

0.2

0.4

0.6

0.8

1.0

Rel 2P

A C

ross-s

ecti

on

Temperature oC

ThT/Quad

Figure 3.13: Relative 2PA cross-sections enhancement of ThT/Quadruplex as a function of

temperature.

3.3.3.2 G-Triplex/ThT


The absorption spectrum of ThT with and without Triplex is shown in Figure 3.14A.

There is a shift from 412 nm to 415 nm suggesting that ThT is bound to Triplex. Figure 3.14B

shows the absorption spectrum of ThT/Quad as a function of temperature. The inset of Figure

3.14B shows the variation of ThT absorption at 413 nm, as well as absorbance at 254 nm of

Triplex as a function of temperature. Both the data shows a first transition at low temperature

followed by further decrease at high temperature. Here again, the absorption spectrum is unable

to predict the melting transition of the Triplex clearly. Further one- and two- photon fluorescence

measurements were carried out to monitor the melting and stability of the Triplex.

95

300 400 5000.0

0.5

1.0A

bs

orb

an

ce

Wavelength nm

ThT

ThT-Triplex

A

300 400 500 6000.0

0.2

0.4

0.6

0.8

Ab

so

rban

ce

Wavelength nm

ThT/Triplex

B

20 40 60 80

0.6

0.7

0.8

0.9

Ab

s @

41

3 n

m

Ab

s @

25

4 n

m

Temperature o

C

0.19

0.20

0.21

0.22

0.23

Figure 3.14: (A) Absorption spectrum of ThT with and without Triplex, (B) Absorption

spectrum of ThT/Triplex as a function of temperature. The inset shows the variation

in absorbance at 254 nm and 413 nm.


Steady-state fluorescence measurements for ThT/Triplex were carried out as a function of

temperature and shown in figure 3.15. The maximum fluorescence was observed at 483 nm at 20

°C, then counts started to decrease. The inset shows the change in fluorescence counts at 483 nm

and it can be observed that there is a dramatic decrease with increase in temperature for ThT that

can arise from ThT coming out of the Triplex because of melting or also increased non-radiative

channels for ThT. The data seems to show that Triplex is melting quicker.


Two-photon fluorescence spectrum of ThT/Triplex as a function of temperature are

shown in Figure 3.16. Similar to what we saw in the one-photon fluorescence, the 2P

fluorescence counts also decreased rather dramatically with increase in temperature. The inset of

Figure 3.16 shows the variation of ThT fluorescence at 483 nm. It is interesting to see rather

faster decrease in 2P fluorescence when compared to 1P fluorescence.

96

450 500 550 600 6500

100

200

300

400

Fl C

ou

nts

Wavelength nm

ThT/Triplex

20 40 60 800

100

200

300

400

Fl @

483

nm

Temperature oC

Figure 3.15: One-photon fluorescence spectrum of ThT/Triplex at different temperatures. The

inset shows the variation the fluorescence intensity at 483 nm.

450 500 550 600 6500

300

600

900

1200

Fl C

ou

nts

Wavelength nm

ThT/Triplex

20 40 60 800

400

800

1200

Fl @

483 n

m

Temperature oC

Figure 3.16: Two-photon fluorescence spectrum of ThT/Triplex as a function of

temperature. The inset shows variation of fluorescence intensity at 483 nm.

97

3.3.3.2.4 Relative 2PA Measurements

The relative 2PA cross-sections were obtained from the ratio of two-photon fluorescence

to one-photon fluorescence. Figure 3.17 shows the relative 2PA cross-sections for ThT/triples as

a function of temperature. The 2PA cross-sections for this interaction displays a slight decrease

in beginning from 10 ˚C till 30 ˚C, then an increase in 2PA cross-sections . First decrease

suggests faster melting to give rise to an intermediate state and total melting happens around 45

°C. Again, ThT does not seem to increase the stability although it binds to the Triplex. Not all

drugs that bind can add to the stability of the non-canonical structures.

20 40 60 80

0.5

1.0

1.5

2.0

2.5

Rel 2P

A c

ross-s

ecti

on

Temperature oC

ThT/Triplex

Figure 3.17: Relative 2PA cross-sections enhancement for ThT/Triplex as a function of

temperature.

98

3.3.4 Interaction with ThO

3.3.4.1 G-Quadruplex/ThO


Optical absorption spectrum of ThO with and without Quad DNA at room temperature is

shown in Figure 3.18A. Absorption maximum has shifted from 480 nm to 484 nm when it was

bound to Quadruplex. Figure 3.18B shows the absorption spectrum of ThO/Quad as a function of

temperature. The spectrum consisted of maximum at 257 nm for the DNA and 480 nm for ThO.

The inset of Figure 3.18B shows the changes at around 65 ˚C, the Quadruplex begins to shift to

an intermediate as absorbance decreases all the way till 90 ˚C. There is a small shift of the

maximum around 484 nm to 503 nm suggesting that there is an interaction between the drug and

the DNA due to the alternation of the environment as they bind.

300 400 500 600

0.0

0.5

1.0

Ab

so

rba

nc

e

Wavelength nm

ThO

ThO-Quad

A

300 400 500 6000.0

0.2

0.4

0.6

0.8

1.0

Ab

so

rban

ce

Wavelength nm

ThO / Quad

20 40 60 80 1000.45

0.60

0.75

0.90

Ab

s @

48

0 n

m

Ab

s @

25

7 n

m

Temperature oC

B

0.24

0.28

0.32

Figure 3.18: (A) Absorption spectrum of ThO with and without Quadruplex DNA, (B)

Absorption spectrum of ThO/Quadruplex as a function of temperature. The inset

shows variation in absorbance at 257 nm and 480 nm.

99


Several studies have shown that ThO binds effectively with G-Quadruplex and G-

Triplex structures than double stranded DNA which enables them good drugs for stabilizing the

non-canonical structures37. Steady-state fluorescence measurements for ThO/Quadruplex were

carried out as a function of temperature from 20 °C to 90 °C after 470 nm excitation and shown

in Figure 3.19. The maximum fluorescence was observed at 541 nm and a decrease in

fluorescence was observed with increase in temperature. The inset of Figure 3.19 shows the

decrease in fluorescence at 541 nm. The constant decrease with increase in temperature was

unable to pinpoint the melting transition of the Quad DNA.

500 550 600 650 7000

100

200

300

400

500

Fl C

ou

nts

Wavelength nm

ThO/Quad

20 40 60 80

0

150

300

450

Fl @

54

1 n

m

Temperature oC

Figure 3.19: Steady-state fluorescence spectrum of ThO/Quadruplex as a function of

temperature. The inset shows the in fluorescence at 541 nm.


The two-photon fluorescence spectrum of ThO/Quad as a function of temperature are

shown in Figure 3.20. Like one-photon fluorescence, 2P fluorescence also quenched quite

100

dramatically with increase in temperature. The inset of Figure 3.20 shows the decrease in 2P

fluorescence at 540 nm, and it decreased much faster than what was observed with 1P

fluorescence. Even the 2P fluorescence spectrum was unable to predict the melting transition in

Quad DNA.

520 560 600 6400

2500

5000

20 40 60 80

0

1000

2000

3000

4000

2P

A C

ou

nts

Wavelength (nm)

ThO/Quad

2P

A@

54

1 n

m

Temperature oC

Figure 3.20: Two-photon spectrum of ThO/Quadruplex as a function of temperature. The

inset shows change in fluorescence at 540 nm.

3.3.4.1.4 Relative 2PA Cross-Sections

Relative 2PA cross-sections were determined from the ratio of 2P fluorescence to 1P

fluorescence after normalizing the intensities at the lowest temperature and shown in Figure

3.21. Although the red fitted curve shows a transition greater than 60 °C. The melting of Quad

DNA follows through an intermediate where the first transition occurs at around 50 °C

suggesting that ThO was able to stabilize the Quad DNA. It is again interesting to see that even

though both 1P and 2P fluorescence were unable to predict the melting transition, the relative

101

2PA enhancement was able to accurately show the melting of Quad DNA with ThO and it was

also shown that ThO is better drug to stabilize the Quad DNA.

20 30 40 50 60 70 80

0.0

0.4

0.8

1.2

ThO / Quad

Rel 2P

A c

ross-s

ecti

on

Temperature oC

Figure 3.21: Relative 2PA cross-sections of ThO/Quadruplex as a function of temperature.

3.3.4.2 G-Triplex/ThO


Figure 3.22A shows the optical absorption spectrum of ThO with and without Triplex

DNA. The shift in absorption maximum indicates the binding of ThO with tripex DNA. The

melting transition of ThO/Triplex is monitored as a function of temperature using UV-Vis

spectroscopy and shown in Figure 3.22B. The inset of Figure 3.22 shows the change in

absorbance at 257 nm for the Triplex DNA and 480 nm for ThO. Interesting tends are observed

where two transitions were observed, one around 35 ˚C and another greater than 55 ˚C. The

absorption spectrum suggest that ThO binds with Triplex DNA but also stabilizes it with

observed changes in absorbance.

102

300 400 500 600

0.0

0.5

1.0A

bso

rban

ce

Wavelength nm

ThO

ThO-Triplex

A

300 400 500 600 7000.0

0.2

0.4

0.6

0.8

Ab

so

rba

nc

e

Wavelength nm

ThO/Triplex

B

20 40 60 80

0.70

0.72

0.74

0.76

0.78

Ab

s @

480 n

m

Ab

s @

25

7 n

m

Temperature oC

0.28

0.30

0.32

0.34

Figure 3.22: (A) Absorption spectrum of ThO with and without Triplex, (B) Absorption

spectrum of ThO/Triplex with temperature dependence. The insets show variation

in absorbance at 257 nm and 480 nm as a function of temperature.

3.3.4.2.2 One-Photon Fluorescence Measurements

Steady-state fluorescence spectrum of ThO/Triplex as a function of temperature is shown

in Figure 3.23. The maximum of fluorescence was observed at 541 nm at and it decreased with

increase in temperature. The inset of Figure 3.23 shows the change in fluorescence intensity as a

function of temperature and the data shows a transition around 30 °C for ThO/Triplex. This can

be attributed to the melting transition of Triplex in the presence of ThO.


Figure 3.24 shows the 2P fluorescence spectrum of ThO/Triplex as a function of

temperature. Similar to steady-state fluorescence, 2P fluorescence counts also decrease with

increase in temperature and the inset of Figure 3.24 shows the change in 2P counts as a function

of temperature. The decrease in 2P fluorescence counts is more rapid when compared to 1P

fluorescence counts. The trend of Triplex melting is not clear in the case of 2P fluorescence

measurements.

103

500 550 600 650 7000

50

100

Fl C

ou

nts

Wavelength nm

ThO/triplex

0 20 40 60 80

0

50

100

Fl@

541 n

m

Temperature oC

Figure 3.23: One-photon fluorescence spectrum of ThO/Triplex as a function of

temperature. Inset shows change in fluorescence at 541 nm.

500 520 540 560 580 600 620 640 660 6800

2000

4000

Fl C

ou

nts

Wavelength nm

ThO/Triplex

0 20 40 60 80

0

1000

2000

3000

Fl.@

54

1 n

m

Temperature oC

Figure 3.24: Two-photon fluorescence spectrum of ThO/Triplex as a function of temperature.

The inset shows variation in fluorescence at 541 nm.

104


Relative 2PA cross-sections were obtained from the ratio of two-photon fluorescence to

one-photon fluorescence and corresponding data for ThO/Triplex as a function of temperature is

provided in Figure 3.25. The 2PA cross-sections show an interesting trend where small decrease

was observed at initial stages, the data clearly shows a melting transition at around 40 °C . This

interesting trend shows that ThO stabilizes Triplex well and as good as it stabilizes the

Quadruplex DNA. This can be ascribed to the molecular structure and how it better -stacks with

DNA base pairs.

0 10 20 30 40 50 60 70 80 900.0

0.2

0.4

0.6

0.8

1.0

Re

l. 2

PA

Cro

ss

-se

cti

on

Temperature oC

ThO/Triplex

Figure 3.25: Relative 2PA cross-sections for ThO/Triplex as a function of temperature.

105

3.3.5 Interaction with DAPI

3.3.5.1 G-Quadruplex/DAPI


Figure 3.26A shows the optical absorption spectrum of DAPI with without Quad DNA.

The absorption maximum has shifted from 343 nm to 350 nm when it is bind to Quadruplex. A

shift in absorption was observed for DAPI when mixed with Quad suggesting that DAPI binds

with Quad DNA. Figure 3.26B shows the change in absorption spectrum of Quad/DNA as a

function of temperature. A small increase in absorbance is observed at 257 nm as well as at 343

nm that corresponds to the absorption of DAPI. The inset of Figure 3.26B shows the change in

absorbance at 257 nm and 343 nm, showing an increase. More over the absorption spectruml

shape of DAPI is changed from low temperature to high temperature. But, no clear cut melting

transition is observed from optical absorption measurements.

300 4000.0

0.5

1.0

Ab

so

rba

nc

e

Wavelength nm

DAPI

DAPI-Quad

A

250 300 350 400 450 5000.0

0.2

0.4

0.6

0.8

1.0

Ab

so

rban

ce

Wavelength nm

DAPI/QuadB

20 40 60 80

0.88

0.90

Ab

s @

34

3 n

m

Ab

s.

@2

57

nm

Temperature OC

0.22

0.24

0.26

Figure 3.26: (A) Optical absorption spectrum of DAPI with and without Quad DNA, (B)

Absorption spectrum of DAPI/Quad as a function of temperature. The inset shows

variation in absorbance at 257 nm and 343 nm as a function of temperature.

106


Several studies have shown that DAPI preferentially binds with dsDNA rather than

Quadruplex DNA36,37. Steady-state fluorescence spectrum of DAPI/Quadruplex as a function of

temperature is shown in Figure 3.27. A decrease in fluorescence is observed with increase in

temperature. But, the decrease is only marginal when compared to the fluorescence decrease

observed for other dyes with DNA. The inset of Figure 3.27 shows the variation of fluorescence

intensity at 475 n which shows a decrease of around 40% and decrease happening around 50 °C .

400 500 600 7000

100

200

300 DAPI/Quadruplex

Fl C

ou

nts

Wavelength nm

20 40 60 80

200

240

280

Fl @

475 n

m

Temperature oC

Figure 3.27: One-photon fluorescence spectrum of DAPI/Quadruplex as a function of

temperature. Inset shows the variation in fluorescence at 475 nm.


Intrinsically, DAPI has low fluorescence quantum yield and the 2PA cross-sections is

also low. That is the reason, its two-photon fluorescence counts are low. Shown in Figure

3.28 are the 2P fluorescence spectrum of DAPI/Quad as a function of temperature. Here

again, a decrease in two-photon counts is observed. The decrease in 2P counts at 475 nm as

107

a function of temperature is shown in the inset of Figure 3.28. There is a clear melting

transition at around 50 °C observed from 2P fluorescence data, consistent with what was

observed using one-photon fluorescence.

400 500 600

200

400

600

800

Fl C

ou

nts

Wavelength nm

DAPI/Quadruplex

20 40 60 80

150

300

450

2P

A @

475 n

m

Temperature oC

Figure 3.28: Two-photon fluorescence spectrum of DAPI/Quadruplex as a function of

temperature. The inset shows variation in fluorescence at 475 nm.


Relative 2PA cross-sections were determined from the ratio of of 2P fluorescence to 1P

fluorescence and shown in Figure 3.29. The data clearly shows a melting transition at around 50

°C suggesting that DAPI was able to stabilize the Quad DNA.

108

20 30 40 50 60 70 80 90

0.4

0.8

1.2

DAPI/Quadruplex

Real 2P

A C

ross-s

ecti

on

Temperature oC

Figure 3.29: Relative 2PA cross-sections for DAPI/Quadruplex as a function of temperature.

3.3.5.2 DAPI/Triplex


Optical absorption spectrum of DAPI with and without G-Triplex is shown in Figure

3.30 A, where absorption maximum of DAPI has shifted from 342 nm to 355 nm when it is with

Triplex suggesting that DAPI binds with Triplex DNA. Temperature-dependent absorption

measurements were carried out to study the melting of Triplex with DAPI and shown in Figure

3.30B. An increase in absorbance is observed at 257 nm and 350 nm and the inset of Figure

3.30B. Melting transition around 40 ˚C is determined for the Triplex melting with DAPI.

109

300 4000.0

0.5

1.0A

bs

orb

an

ce

Wavelength nm

DAPI

DAPI-Triplex

A

300 400 500 6000.0

0.2

0.4

0.6

0.8

1.0

Ab

so

rban

ce

Wavelength nm

DAPI/TriplexB

10 20 30 40 50 60 70 80

0.20

0.24

0.28

Ab

s @

35

0

nm

Ab

s @

25

7 n

m

Temperature oC

0.80

0.84

0.88

0.92

Figure 3.30: (A) Absorption spectrum of DAPI with and without Triplex, (B) Absorbance

spectrum of DAPI/Triplex at different temperatures. The inset shows variation in

optical density at 257 nm and 350 nm.


Steady-state fluorescence spectrum of DAPI/Triplex as a function of temperature are

shown in Figure 3.31. The maximum fluorescence was observed at 475 nm and a slight increase

and decrease was observed. Inset of Figure 3.31 shows the change in fluorescence intensity at

475 nm. No clear melting transition is observed.


Figure 3.32 shows the 2P fluorescence spectrum of DAPI/Triplex as a function of

temperature after excitation at 800 nm. Here again, DAPI shows low counts but a decrease

in 2P fluorescence is observed and is plotted as a function of temperature in the inset of

Figure 3.30. The decrease in 2P fluorescence is significantly greater than what was observed

with 1P fluorescence. It is interesting to note that both one and two-photon fluorescence

were unable to judge the melting transition in Triplex.

110

400 500 600 7000

50

100

150

200

250

Fl C

ou

nts

Wavelength nm

DAPI/Triplex

0 20 40 60 80

200

220

240

Fl @

475 n

m

Temperature o

C

Figure 3.31: One-photon fluorescence spectrum of DAPI/Triplex. The insets show variation

in fluorescence at 475 nm as a function of temperature.

350 400 450 5000

400

800

1200

DAPI/Triplex

Fl C

ou

nts

Wavelength nm

0 20 40 60 80

150

300

450

600

2P

A @

47

5 n

m

Temperature o

C

Figure 3.32: Two-photon fluorescence spectrum of DAPI/Triplex as a function of


111


Relative 2PA cross-sections were determined from the ratio of 2P fluorescence and 1P

fluorescence for DAPI/Triplex and plotted as a function of temperature in an effort to better

judge the melting transition (Figure 3.33). A melting transition around 30 °C which is a marginal

stabilization for DAPI when it binds to Triplex. Here again, relative 2PA measurements were

able to better probe the melting transitions.

10 20 30 40 50 60 70 80

0.4

0.8

1.2

Real 2P

A C

ross-s

ecti

on

Temperature oC

Figure 3.33: Relative 2PA cross-sections for DAPI/Triplex as a function of temperature.

3.3.6 Interaction with EtBr

3.3.6.1 G-Triplex/EtBr


EtBr did not interact with Quad DNA, that is the reason that data is not discussed in

the dissertation. Part A of Figure 3.34 shows the absorption spectrum of EtBr with and

without Triplex DNA. Absorption maximum of EtBr has shifted from 479 nm to 485 nm

112

upon interaction with Triplex suggesting that EtBr binds with Triplex. Figure 3.34B shows

the temperature-dependent absorption spectrum of EtBr/Triplex as a function of

temperature. The inset of Figure 3.34B shows the change in absorption at 257 nm of Triplex

and absorption at 485 nm of EtBr. EtBr absorption shows a lower melting transition, while

the absorbance of Triplex has shown higher melting transition. It will be interesting to see

which one the actual melting transition is.

300 400 500 6000.0

0.5

1.0

1.5

Ab

so

rban

ce

Wavelength nm.

EtBr

EtBr-Triplex

A

250 300 350 400 450 500 550 6000.0

0.5

1.0

1.5

Ab

so

rba

nc

e

Wavelength nm

EtBr/TriplexB

0 20 40 60 80

1.350

1.375

1.400

1.425

Ab

s.

@257

nm

Temperature oC

0.04

0.06

0.08

Ab

s. @

485 n

m

Figure 3.34: (A) Optical absorption spectrum of EtBr with and without Triplex, (B)

Absorption spectrum of EtBr/Triplex as a function of temperature. The inset shows

variation in absorbance at 257 nm and 485 nm.


EtBr is a known intercalator and binds through terminal end stacking42. Steady-state

fluorescence measurements were carried out as a function of temperature to determine the

melting transition in EtBr/Triplex. One-photon fluorescence of EtBr/Triplex as a function of

temperature is shown in Figure 3.35. The maximum fluorescence was observed at 611 nm at 10

°C. After 20 ˚C, the fluorescence began to decrease until stabilizing at 60 ˚C and corresponding

change in fluorescence at 611 nm is shown in the inset of Figure 3.35. As the structure of the

Triplex changes with increasing temperature, changing into different intermediates, EtBr

113

becomes less bound, thus emitting less fluorescence. One-photon fluorescence has shown a

melting transition around 40 °C for EtBr/Triplex.

550 600 650 700 750 8000

1x105

2x105

3x105

4x105

EtBr/Triplex

Fl C

ou

nts

Wavelength nm

0 20 40 60 80

2.0x105

2.5x105

3.0x105

3.5x105

Fl @

61

1 n

m

Temperature o

C

Figure 3.35: One-photon fluorescence spectrum of EtBr/Triplex as a function of

temperature. The insets show variation in fluorescence at 611 nm.


Two-photon fluorescence measurements were carried out as a function of temperature for

EtBr/Triplex and corresponding data is shown in Figure 3.36. As the inherent 2P cross-

sections of EtBr is low, and its 2P counts are also rather low. However, the fluorescence

counts decreased with increase in temperature. Inset of Figure 3.36 shows the variation in

2P fluorescence as a function of temperature. Here again, the data seems to indicate that the

melting transition temperature of Triplex is increased when it is bound to EtBr.

114

550 600 650 7000

200

400

600

2P

A C

ou

nts

Wavelength nm

EtBr/Triplex

0 20 40 60 80

100

200

300

400

2P

A @

611 n

m

Temperature o

C

Figure 3.36: Two-photon fluorescence spectrum of EtBr/Triplex as a function of



Both 1P and 2P fluorescence data have shown increased stability for Triplex when it is

bound to EtBr. Further relative 2PA cross-sections were determined from the ratio of 2P to 1P

fluorescence to check if it can show the transition. Corresponding relative 2PA cross-sections of

EtBr/Triplex is shown in Figure 3.37. It is clear from the figure that relative 2PA cross-sections

were also able to show that the melting transition of Triplex is around 40 °C when it is bound to

EtBr suggesting that it is a good drug to stabilize Triplex.

115

10 20 30 40 50 60 70 80

0.4

0.8

1.2

EtBr/Triplex

Real 2P

A C

ross-s

ecti

on

Tempreature oC

Figure 3.37: Relative 2PA cross-sections for EtBr/Triplex as a function of temperature.

3.3.7 Interaction with TMPyP4

3.3.7.1 G-Quadruplex/TMPyP4


TMPyP4 is a flat molecule well known to bind Quadruplex DNA. To understand how it

interacts with tis Quadruplex DNA, optical measurements were carried out. Optical absorption

spectrum of TMPyP4 with and without Quad are shown in Figure 3.38A. Absorption maximum

of TMPyP4 has shifted from 422 nm to 428 nm when it is bound to Quadruplex. Figure 3.38B

shows the absorption spectrum of TMPyP4/Quad as a function of temperature. With increasing

temperature, the absorbance of Quadruplex at 257 nm changed and is shown in the inset.

Similarly, the absorbance of TMPyP4 at 428 nm changed suggesting a melting temperature of

around 46 °C .

116

300 400 500 6000

1

2

3

Ab

so

rba

nc

e

Wavelength nm

TMPyP4

TMPyP4-Quad

A

300 400 500 600 7000.0

0.5

1.0

1.5

2.0

Ab

so

rban

ce

Wavelength nm

TMPyP4/QuadB

20 40 60 80

1.26

1.28

1.30

1.32

1.34

1.36

Ab

s. @

257 n

m

Temperature oC

1.40

1.45

1.50

1.55

1.60

Ab

s.

@4

28

nm

Figure 3.38: (A) Absorption spectrum for TMPyP4 with and without Quadruplex, (B)

Optical absorption spectrum of TMPyP4/Quadruplex as a function of temperature.

The inset shows variation in the optical density at different wavelengths.


TMPyP4 is known to bind well with Quadruplex DNA and has also been shown to act as

both an intercalator and an external binder40. Steady-state fluorescence measurements for

TMPyP4 /Quadruplex were carried out as a function of temperature and shown in figure 3.39. A

maximum at 650 nm is observed that decreased with increase in temperature and shown in the

inset. There is a clear melting transition from 1P fluorescent at a temperature of around 48 °C,

suggesting that TMPyP4 stabilizes the Quadruplex.


Two-photon fluorescence spectrum of TMPyP4/Quadruplex as a function of

temperature are shown in figure 3.40. Here again, there is a decrease in 2P fluorescence with

increase in temperature at around 650 nm and shown in the inset. A melting transition of

around 45 °C is observed from the data.

117

600 650 700 7500.0

2.0x105

4.0x105

6.0x105

Fl C

ou

nts

Wavelength nm

TMPyP4/Quadruplex

20 40 60 80

1.50x105

3.00x105

4.50x105

6.00x105

Fl

@ 6

50

nm

Tempreature o

C

Figure 3.39: One-photon fluorescence spectrum of TMPyP4/Quadruplex as a function of


600 650 700 7500

2000

4000

6000

TMPyP4/Quadruplex

Fl C

ou

nts

Wavelength nm

20 40 60 800

2000

4000

6000

2P

A @

650 n

m

Temperature o

C

Figure 3.40: Two-photon fluorescence spectrum of TMPyP4/Quadruplex as a function of


118

3.3.7.1.4 Relative 2PA Measurements

Relative 2PA cross-sections were obtained from the ratio of 2P to 1P fluorescence for

TMPyP4/Quad and shown in Figure 3.41. No clear melting transition is observed from the data.

This is the first observation where relative 2PA cross-sections were unable to show clear melting

transition and is ascribed to the orientation of TMPyP4 without clear dipole in it. Interestingly,

1P and 2P fluorescence were able to show clear melting transitions which suggested that

TMPyP4 stabilizes Quad DNA.

20 30 40 50 60 70 80 900.2

0.4

0.6

0.8

1.0

1.2

TMPyP4/Quadruplex

Real 2P

A C

ross-S

ecti

on

Temperature oC

Figure 3.41: Relative 2PA cross-sections of TMPyP4/Quad.

3.3.7.2 G-Triplex/TMPyP4


The absorption spectrum of TMPyP4 with and without the G-Triplex is shown in figure

3.42A where absorption maximum shifts from 422 nm to 432 nm when it is bound to Triplex.

Figure 3.42B shows the change in absorbance of TMPyP4/Triplex as a function of temperature.

119

Both the absorbance of Triplex DNA at 257 nm and the absorbance of TMPyP4 at 432 nm

changed with temperature and the variation is shown in the inset. An increase in melting

transition is observed suggesting that TMPyP4 can stabilize the Triplex DNA.

200 300 400 500 600 700 800

0

1

2

3

Ab

so

rban

ce

Wavelength nm

TMPyP4

TMPyP4-Triplex

A

300 400 500 600 7000

1

2

Ab

so

rban

ce

Wavelength nm

TMPyP4/Triplex

B

0 20 40 60 80

1.330

1.368

1.406

1.444

Ab

s. @

257 n

m

Temperature oC

1.4

1.5

Ab

s. @

432 n

m

Figure 3.42: (A) Absorption spectrum of TMPyP4 with and without Triplex, (B) Optical

absorption spectrum of TMPyP4/Triplex as a function of temperature. The inset

shows variation in the optical density at representative wavelengths.


1P fluorescence spectrum of TMPyP4/Triplex as a function of temperature is shown in

figure 3.43. Fluorescence maximum was observed at 650 nm and it decreased with increase in

temperature. The inset of Figure 3.43 shows variation of fluorescence intensity at 650 nm. A

clear melting transition of 35 °C, suggesting that TMPyP4 stabilizes the Triplex DNA.


2P fluorescence measurements were carried out for TMPyP4/Triplex after excitation

at 800 nm and corresponding spectrum are shown in Figure 3.44. As observed in the case of

1P fluorescence, a maximum was observed at 650 nm that changed with increase in

120

temperature as shown in the inset. A decrease in fluorescence intensity was observed but no

clear transition is observed from the data.

600 650 700 7500

1x105

2x105

3x105

4x105 TMPyP4/Triplex

Fl C

ou

nts

Wavelength nm

0 20 40 60 80

1x105

2x105

3x105

4x105

Fl @

650 n

m

Temperature o

C

Figure 3.43: One-photon fluorescence spectrum of TMPyP4/Triplex as a function of

temperature. The inset shows variation in the fluorescence at 650 nm.

600 650 7000

3000

6000

9000TMPyP4/Triplex

Fl C

ou

nts

Wavelength nm

0 20 40 60 80

3000

4000

5000

2P

A @

65

0 n

m

Temperature oC

Figure 3.44: Two-photon fluorescence spectrum of TMPyP4/Triplex as a function of


121


Relative 2PA cross-sections were determined from the ratio of 2P fluorescence to 1P

fluorescence and shown in Figure 3.45. The data has shown interesting trend where the 2PA

cross-sections with Triplex melting suggesting that TMPyP4 binds to Triplex in a manner such

that 2P cross-sections are decreased. With increase in temperature, Triplex melts and the cross-

cross-sections increases. From the data, a clear melting transition is observed with TMPyP4,

stabilizing the Triplex better than Quadruplex.

10 20 30 40 50 60 70 80 900.5

1.0

1.5

2.0

2.5

TMPyP4/Triplex

Real 2P

A C

ross-S

ec

tio

n

Temperature oC

Figure 3.45: Relative 2PA cross-sections of TMPyP4/Triplex as a function of temperature.

3.3.8 Interaction with Doxorubicin

3.3.8.1 G-Triplex/Doxorubicin


Doxorubicin is known to preferably bind to dsDNA than other non-canonical DNA

structures. But a recent study has shown that Doxo can bind to certain non-canonical DNA such

as a G-Quadruplex with some stability39. In this investigation, we have attempted to see if Doxo

122

can bind and stabilize Triplex DNA. Shown in Figure 3.46A are the absorption spectrum of

Doxo with and without Triplex. An absorption maximum was observed at 478 nm that shifted to

489 nm when it is bound to Triplex. The absorption spectrum of Doxo/Triplex as a function of

temperature were measured to monitor the melting transition in Triplex and the corresponding

spectrum are shown in Figure 3.46B. The inset of Figure 3.46B plotted the change in the

absorbance at 257 nm for Triplex and 480 nm for Doxorubicin as a function of temperature.

Both of them followed a similar trend with a transition temperature around 40 °C suggesting

Doxo can stabilize Triplex DNA.

300 400 5000.0

0.5

1.0

1.5

Ab

so

rba

nc

e

Wavelength nm

DoXo

DoXo-Triplex

A

250 300 350 400 450 500 550 6000.0

0.5

1.0

1.5

Ab

so

rba

nc

e

Wavelength nm

Doxo/TriplexB

0 20 40 60 80

1.32

1.43

1.54

Ab

s. @

257

nm

Temperature oC

0.08

0.10

0.12

Ab

s. @

480

nm

Figure 3.46: (A) Absorption spectrum of Doxorubicin with and without Triplex, (B) Optical

absorption spectrum of Doxo/Triplex as a function of temperature. The inset shows

variation in the absorption at representative wavelengths 257 nm and 480 nm.


Steady-state fluorescence spectrum of Doxorubicin /Triplex as a function of temperature

are shown in figure 3.47. The fluorescence began to increase with increase in temperature (inset

of Figure 3.47), which is one of the first observation made in the molecules that were studied in

this investigation. This is unique in the sense that Doxo fluorescence is quenched with it is bound

123

to but increases when it comes out of the Triplex. The data suggests that Doxo binds with

Triplex.

500 550 600 650 700 750 8000.0

3.0x105

6.0x105

9.0x105

DoXo/Triplex

Fl C

ou

nts

Wavelength nm

0 20 40 60 802.0x105

4.0x105

6.0x105

8.0x105

Fl @

589

nm

TempreatureoC

Figure 3.47: Fluorescence spectrum of Doxo/Triplex as a function of temperature. The inset

shows variation in the fluorescence at 580 nm.


Similar to 1P fluorescence, 2P fluorescence also increased with increase in

temperature (Figure 3.48). Inset of Figure 3.48 provides the variation in 2P fluorescence at

580 nm as a function of temperature which clearly shows an increase with a melting

transition around 30 °C .


Relative 2PA cross-sections were determined for Doxorubicin /Triplex from the ratio of

2P fluorescence to 1P fluorescence and shown in Figure 3.49. Although both 2P and 1P

fluorescence increase with increase in temperature, relative 2PA cross-sections have shown a

clear transition suggesting that Doxo not only binds to Triplex but also stabilizes it.

124

500 550 600 650 7000

1500

3000

4500

2P

A C

ou

nts

Wavelength nm

DoXo/Triplex

0 20 40 60 801500

3000

4500

2P

A @

58

9 n

m

TempreatureoC

Figure 3.48: Two-photon fluorescence spectrum of Doxo/Triplex as a function of


0 10 20 30 40 50 60 70 80 90

0.8

1.0

1.2

Real 2P

A C

ross-s

ecti

on

Temperature oC

DoXo/Triplex

Figure 3.49: Relative 2PA cross-sections for Doxo/Triplex as a function of temperature.

3.3.9 Mechanism Why Are 2PA Cross-Sections Showing Melting Transition?

Overall, different optical spectroscopic techniques were used to follow the melting and

stabilization of Quadruplex and Triplex DNA with small molecule drugs. G-Quadruplex and G-

125

Triplex melting studied by UV-Vis Absorbance, 1P and 2P fluorescence and relative 2PA cross-

sections with different dye molecules. The result was different depending on the dye molecules

used. Moreover, the obtained data showed that new technique of relative 2PA cross-sections can

monitor the melting transition better than conventional optical spectroscopic techniques.

techniques can monitor the melting sensitively. Among all the techniques, the ones that can

provide the relationship between how the drug binds to phosphate backbone and its unfolding

transition is the relative 2PA cross-sections of the drugs. How this technique may be able to

monitor the same can be explained if we follow the relation between the 2PA cross-sections and

the local electric fields present in the phosphate backbone of the DNA. The 2PA cross-sections

is proportional to the square of change in dipole moment and transition dipole moment in a

simple two-state formalism given by:

𝛿2−𝑠𝑡𝑎𝑡𝑒 =2(2𝜋)4𝑓𝐿

4

15(𝑛ℎ𝑐)2(1 + 2𝑐𝑜𝑠2𝜃)|𝑀𝑔𝑒

|2|∆𝜇𝑔𝑒 |

2𝑔(2𝜈) 3.1

Mge is shown to be the dipole moment at the transition between the ground state and the excited

state. (2𝜈) is where the normalized line function is placed into, and ∆𝜇ge represents the difference

between excited state and ground state, in which the permanent dipole encompasses each. The

angle is between those two permanent dipole vectors, n is a refractive index17. From the

equation, it can be seen that the 2PA cross-sections of the chromophores used in the experiment

is proportional to the square of ∆𝜇ge. However, ∆𝜇ge is related to the electric field and can be best

described by:

∆𝜇𝑔𝑒 = ∆𝜇𝑔𝑒0 + 0.5∆𝜇𝑔𝑒

𝑖𝑛𝑑 = ∆𝜇𝑔𝑒0 + 0.5(∆𝛼)𝐸 3.2

Where ∆𝛼 is the change in polarizability between the ground and excited state, E is the

local electric field, and θ is the angle between the transition dipole vector and the electric field

126

vector. So, the alignment of the molecular or drug dipole is very important. If this angle is zero,

we interpret it as the drug being parallel to the local electric field of the DNA. This parallel

orientation causes an increase in the 2PA cross-sections , but if the angle is 90 degrees, then

there is no change in the 2PA cross-sections and the drug is bound perpendicularly to the DNA

electric field. Lastly if the angle is anywhere between 90 degrees and 270 degrees then this will

cause a decrease in the 2PA cross-sections , which means that the drug is bound anti-parallel to

the DNA electric field. So, the relative 2PA cross-sections can give information about the

orientation of the local electric field and the drug’s transition dipole.

The electric field in DNA arises from the phosphate backbone as it possesses mini

dipoles coming out of the phosphate anions and the positive metal ion from the salt. The electric

field of the DNA changes direction as the conformation of the DNA changes, since the electric

field is produced from the phosphate sugar backbone. The binding of the drug changes that, and

by looking at this schematic below (Figure 3.50), it provides a representation of the possible

orientations that we can analyze of the drugs bound to Quadruplex and Triplex structures.

Figure 3.50: Molecules interacting with ssDNA, dupDNA, quadDNA, tripDNA and also

interacting with each of the electric fields produced by the backbone of the DNA.17

127

10 20 30 40 50 60 70 80 900.0

0.3

0.6

0.9

Re

l. 2

PA

cro

ss

-se

cti

on

Temperature oC

0

1

2

3 Quad

Triplex

ThT

10 20 30 40 50 60 70 80 90

0.0

0.4

0.8

1.2

Triplex

QuadRe

l. 2

PA

En

ha

nc

em

en

t

Temperature o

C

ThO

0.0

0.3

0.6

0.9

1.2

10 20 30 40 50 60 70 80 90

0.4

0.8

1.2

Quad

Triplex

Temperature oC

Rea

l 2P

A C

ross-s

ecti

on

DAPI

0.4

0.8

1.2

10 20 30 40 50 60 70 80 90 1000.2

0.4

0.6

0.8

1.0

1.2

Quad

Triplex

Temperature 0C

Re

al 2

PA

Cro

ss

-Se

cti

on

1.0

1.5

2.0

2.5TMPyP4

10 20 30 40 50 60 70 80

0.4

0.8

1.2

EtBr/Triplex

Rea

l 2P

A C

ross-s

ecti

on

Tempreature 0C

EtBr

0 10 20 30 40 50 60 70 80 90

0.8

1.0

1.2

Rea

l 2P

A C

ross-s

ecti

on

Temperature oC

DoXo/Triplex

DoXo

Figure 3.51: Relative 2PA cross-sections of molecules/drugs interacting with

Quadruplex and Triplex.

Although relative 2PA cross-sections are able to monitor the melting and stabilization of

Quadruplex and Triplex, each drug or molecule stabilization interaction is different. Relative

2PA cross-sections of each molecule interacting with G-Quadruplex and G-Triplex are shown in

Figure 3.51. Interesting trends can be observed here. ThT seems not to stabilize Quadruplex but

128

was able to stabilize Triplex. The melting temperature was found to 40 °C for ThT/Quadruplex

and 45 °C for ThT/Triplex. Interestingly, the interaction of ThT with Quadruplex and Triplex

seems to be different as there is a 2PA cross-sections decrease for ThT/Quadruplex while it

increased for ThT/Triplex when the DNA melted. This result suggests that ThT binds parallel

with phosphate backbone in the case of Quadruplex but anti-parallel in the case of Triplex.

In contrast, ThO seems to stabilize both Quadruplex and Triplex with melting

temperatures of around 40 oC and 52 oC for Triplex and Quadruplex, respectively. Both of them

are significantly better than individual melting temperatures of Quadruplex and Triplex. Also,

ThO alignment with phosphate backbone is parallel for both Quadruplex and Triplex as the

cross-sections decreased with melting of the non-canonical structures. DAPI, a drug molecule

that is known to bind to DNA seems to not to increase the stability of either the Triplex or

Quadruplex. The melting temperatures for DAPI/quad and DAPI/Triplex seems to be around 50

oC and 28 oC, marginally better than individual melting of Quadruplex and Triplex. However, the

orientation of DAPI binding to Quad and Triplex seems to be parallel to the backbone of the

DNA.

Among all molecules, TMPyP4 is an anomaly where the relative 2PA cross-sections of

TMPyP4 binding to Quadruplex did not produce a good melting temperature curve while

individual 1P and 2P fluorescence have shown a decrease in fluorescence consistent with melting

temperature of around 45 oC, which is not exceptional. On the other hand, TMPyP4/Triplex has

increased the melting temperature and stabilized the Triplex. Also, TMPyP4 orientation with

DNA backbone is similar to that of ThT where TMPyP4 binds anti-parallel with DNA backbone

in the case of Triplex but parallel in the case of Quadruplex. Increased stabilities were observed

for EtBr with Triplex (45 oC) as well as Doxo with Triplex (50 oC). Both EtBr and Doxo

129

stabilized the Triplex and the melting temperature is greater than the physiological temperature

suggesting, that these drugs can be used to stabilize the Triplex in-vivo. Overall, from the

investigations carried out in the study, we were able to show a new technique based on the

relative 2PA cross-sections to monitor melting and stabilization of DNA. Also, from the study

we can predict the orientation of molecule or drug with these non-canonical forms of DNA.17,39

3.4 Conclusions

A novel spectroscopic tool based on relative 2PA cross-sections is employed to study the

melting and stabilization of an organized self-assembly of non-canonical structures of DNA.

Two DNA oligonucleotide were selected for their ability to form a stable Quadruplex and

Triplex DNA canonical structures. Six drugs were studied that can bind to DNA at varying

interaction strengths that included ThT, ThO, TMPyP4, DAPI, EtBr and Doxo. One common

thing in all these dyes that they are cationic and can bind to the phosphate backbone of the DNA.

However, they vary by the structure of the drug, hydrogen bonding interactions, aromatic and

electrostatic interaction strengths. Due to DNA backbone’s local electrostatic environment after

interacted with dyes, it can be altered, and this alteration can be measured through several

spectroscopy techniques in order to understand how they bind, and also see the effect of each

drug on G-Quadruplex and G-Triplex DNA stability.

From all the studies, we have shown that relative 2PA cross-sections of molecules is a

better tool to monitor the melting and stability of Quadruplex and Triplex. This is because 2PA

cross-sections of molecules are sensitive to local electric fields and the phosphate backbone in

the DNA offers a local electrostatic environment and the orientation of the drug with the

backbone will be different in the organized self-assembly form when compared to un-folded

form. However different molecules stabilized the non-canonical structures at varying abilities.

130

ThT seems to stabilize Triplex while ThO stabilized both Quadruplex and Triplex significantly.

DAPI’s effect on the stabilizing Quadruplex and Triplex is marginal at best. In contrast, TMPyP4

did not stabilize Quadruplex better but was able to stabilize Triplex better. Also, the drugs EtBr

and Doxo were able to stabilize the Triplex much beyond the physiological temperature. In

addition, the new technique can also provide the orientation of the drug with the DNA backbone.

However, more investigations and better characterization of the intermediates are needed to

complete the understanding of the systems. This study was able to make inroads in the

development of novel 2PA cross-sections technique to monitor drug-DNA interactions.

3.5 Chapter Summary

• For this investigation, two DNA sequences used which are Thrombin Binding Aptamer

(TBA) and shortened form of TBA, (TBA11) to form two non-canonical structure G-

Quadruplex and G-Triplex structure.

• Six drugs were chosen to carry out the investigation which are ThT, ThO, TMPyP4,

DAPI, EtBr and Doxo. All chosen dyes are cationic and well known to bind to DNA at

varying interaction strengths.

• The interaction of these dyes was studied with temperature-dependent UV-Vis,

temperature-dependent one photon fluorescence and temperature-dependent 2-photon

fluorescence, Circular Dichroism.

• The stability trends were obtained for all drugs which correlated with drug structure and

DNA binding modes.

• The results show that relative 2PA cross-sections of all chosen chromophores is a

sensitive technique to monitor the melting transitions in G-Quadruplex and G-Triplex.

131

Also, this technique was able to provide the knowledge of orientation of the drugs or

molecules with respect to phosphate backbone of the DNA.

• ThO seems to stabilize both Quadruplex and Triplex better. But the stabilization offered

by DAPI is rather marginal. EtBr and Doxo drugs were able to stabilize Triplex beyond

physiological temperature.

132

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CHAPTER 4

MONITORING PROTEIN AGGREGATION VIA TWO-PHOTON SPECTROSCOPY:

EARLY DETECTION OF AGGREGATES

4.1 Introduction

In the previous Chapter, investigations have focused on an organized self-assembly of

non-canonical structures, G-Quadruplex and G-Triplex and their structure stabilization using

molecules and drugs monitored via relative two-photon absorption (2PA) cross-sections. In this

Chapter, the studies are focused on another interesting organized self-assembly- protein and in

particular, aspects of protein folding and unfolding, protein aggregation via the use of 2PA

spectroscopy.

Proteins are formed from several amino acids that linked to each other through peptide

bonds (also known as amide bonds) which form when two amino acids are attached by losing a

water molecule1,2. Proteins are classified as organized self-assemblies due to their spontaneous

well-ordered oligomers through multiple noncovalent bonds. Furthermore, the numbers and

sequences of amino acids play significant role in folding of polypeptide chains (protein)3.

Essentially, polypeptide the bond is planar. However, protein will fold into well-defined

conformation structures that has the lowest energy due to numerous hydrophobic and hydrophilic

interactions2. The remarkable mechanism of folding and unfolding protein is still under

investigation and not completely understood as different proteins behave differently4. There were

many different techniques used to monitor change in protein folding such as UV-vis, one photon

fluorescence, circular dichroism and NMR. In this project, we aim to apply the power of 2PA

cross-sections to monitor the protein folding as 2PA spectroscopy was proved to be more

140

sensitive than other optical techniques because it can sense minute changes in electric fields

around the fluorophore during unfolding5,6.

Protein folding/unfolding is a biophysical process that makes the proteins to fold in a

particular way that gives rise to their function. Misfolded proteins are often causing of diseases

and can cause aggregation of proteins. Protein aggregation is often implicated in several

neurodegenerative diseases. One among them is Amyotrophic lateral sclerosis, commonly known

as ALS, is a neurodegenerative disease that targets motor neurons7. As the motor neurons

degenerate, they can no longer relay impulses from the spinal cord and brain to the muscles,

eventually resulting in paralysis7,8. The most common form of ALS is sporadic which affects 90-

95% of all cases, the remaining 5-10% are familial ALS (FALS) which means that the disease is

inherited9–13. There is no cure for ALS; treatment focuses primarily on treating the symptoms

rather than the disease2. Furthermore, ALS is difficult to diagnose and it can take up to a year to

be certain of an ALS diagnosis13,14.

There are many genes that have to been found to be linked to ALS as well as aggregates

of proteins, starting with Superoxide dismutase 1 (SOD1) that was discovered in 19938,15,16.

SOD1 is a protein that destroys free radicals by converting toxic superoxide anions to hydrogen

peroxide17,18. Mutations in the SOD1 gene was implicated in 20-25% of all ALS cases11. These

mutations will alter the amino acid sequence of the SOD1 protein, causing the protein to

improperly fold and aggregate18. Once secreted, the SOD1 aggregates will provoke a neuro-

inflammatory response around the motor neurons19. Recognizing and detecting SOD1

aggregation can lead to an early diagnosis of ALS, which can in turn lead to a more efficient

treatment and result in a more positive prognosis18–22.

141

The harmful effects of SOD1 mutations in familial ALS does not result from a loss of

dismutase function of the enzyme, but rather from the gain of an adverse property of the mutant

protein12,23,24. The presence of SOD1 aggregates has been linked with ALS. The presence of

inclusion bodies, which are abnormal structures in the nucleus or cytoplasm of a cell, in

degenerating neurons and surrounding astrocytes is a pathological hallmark of ALS.

Furthermore, the results of a study involving transgenic mice expressing human mutant SOD1

revealed that SOD1 insoluble protein complexes (IPCs) are detectable in spinal cord extracts

from the mice several months before inclusion body formation and motor neuron degeneration

was apparent 25–27. If one can be able to identify the misfolded forms of SOD1 or the early

soluble aggregates form of SOD1, it is possible to detect ALS early and provide intervention at

this stage to cure the disease. Once the insoluble aggregates are formed, they tend to destroy the

neurons. This forms the main motivation behind the work carried out in this study.

The folding/unfolding and aggregation of SOD1 was investigated using fluorescence

technique 13,28–33. A myriad of techniques were used for monitoring folding/unfolding and

aggregation of proteins in literature34. Among them, fluorescence-based techniques are found to

be better and easier. Proteins are often mixed with chromophores to give rise to fluorescence

signals. There are two ways where chromophores are normally bound to proteins, covalent and

non-covalent binding. Non-covalent binding of chromophores cannot specifically target the

location of the chromophore. On the other hand, covalent binding allows one to know the

location of the chromophore that allows one to make better structure-function relationships. One

way of covalent binding is binding a dye (Fluram) to the protein and follows its fluorescence as a

function of protein aggregation and folding/unfolding35. Fluram is a non-fluorescent compound;

however, it will react with primary amines to form a highly stable fluorescent compound (Figure

142

4.1) to with low background due to hydrolysis36,37. In this study, we have used Fluram bound to

SOD1 as well as BSA to establish two-photon fluorescence technique as a way to follow the

misfolding and aggregation of proteins.

Figure 4.1: Schematic showing the binding of Fluram with primary amines leading to a

fluorescent product.

BSA is used as standard because its stability and lack of interference with other

biological reactions. The hypothesis behind the study is that the 2PA cross-sections of the

chromophore can probe the local electric fields in proteins which can change during protein

folding/unfolding and aggregation. The structures of proteins are provided in Figure 4.2.

143

Figure 4.2: Schematic representation of the protein systems studied in the investigation.30,38

The unfolding of BSA and SOD1 was carried out as a function of temperature (from 25

to 90 oC) and was monitored by one and two-photon excited fluorescence of Fluram bound to the

proteins. Fluram, a primary amine binding dye readily binds to lysine of proteins to give rise to

fluorescence in the visible region. Interestingly, 2PA cross-sections (ratio of two-photon to one-

photon fluorescence) were able to monitor unfolding sensitively over other optical techniques.

The mechanism was attributed to the decrease in electric fields around the fluorophore during

unfolding. The aggregation of SOD1 and BSA was studied by adding 1 M guanidine

hydrochloride GuHCl to soften the protein and temperature variation to monitor it39. UV-vis

absorption, one and two-photon excited fluorescence, time-resolved fluorescence and dynamic

light scattering techniques were used to investigate proteins aggregation as a function of

temperature.

144


4.2.1 Materials

Superoxide Dismutase from bovine erythrocytes (SOD1) and Bovine serum albumin

(BSA) were obtained from Sigma Aldrich and used as received. A stock solution was prepared

using 50 mM sodium phosphate buffer (NaP buffer) at pH 8 with. A stock solution of the dye (1

mM), Fluorescamine (Fluram) was prepared in 1 mL of acetonitrile and diluted with Milli-Q

water to make 10 mL of 1 mM stock solution. For the unfolding of BSA and SOD1, several

sample solutions were made by adding 100 μL of the protein stock solution to10 μL of the

Fluram dye (10 μM) and 890 μL of the NaP buffer. GuHCl was obtained from Sigma Aldrich

and a 10 M stock solution was prepared.

4.2.2 Optical Methods

UV-Vis absorption spectroscopy was used to perform measurements through the use of

Shimadzu UV2101 PC spectrophotometer. Edinburgh F900 spectrofluorometer was used for

one-photon fluorescence measurements. The excitation source consisted of the 450 W Xe lamp

and Hamamatsu R2518P detector. The sample was excited at 370 nm and a fluorescence was

monitored from 380 nm to 600 nm. For two-photon fluorescence measurements, a Ti: Sapphire

laser at 800 nm was focused onto the sample and the resulting fluorescence was collected in an

Edinburgh F900 spectrofluorimeter. The relative 2PA cross-sections were obtained from the ratio

of two-photon to one-photon fluorescence of the sample. Time-resolved fluorescence

measurements were carried out in an Edinburgh FLS900 spectrofluorometer after excitation at

370 nm with a diode laser. Lastly, the Dynamic Light Scattering technique (DynaPro

Temperature-Controlled Microsampler) was used to measure the particle size of aggregates. The

sample solutions were initially filtered to remove any extraneous particles before DLS

145

measurements. All spectroscopy measurements were carried out under temperature dependence

to monitor BSA and SOD1 folding, unfolding and aggregation.


4.3.1 Fluram Binding with BSA and SOD1

The first step was to prove the Fluram is binding to investigated BSA and SOD1via

optical absorption and steady fluorescence measurements. Fluram is known to bind to the

Lysines in the protein and forms a covalent bond with the protein40. Therefore, the expected data

should show an increase in the absorbance and fluorescence when the dye is bonded to the

Lysine in the respective proteins. Parts A and B of Figure 4.3 shows the absorbance and

fluorescence data of Fluram binding to BSA and SOD1 respectively. Optical absorption

measurements were carried out for Fluram alone and with BSA and SOD1 at room temperature.

Fluram does not have any absorbance at 370 nm by itself but after binding to, a strong

absorbance with a maximum at 370 nm was seen for both BSA-fluram and SOD1-fluram (Figure

4.3A and B). In addition, the steady fluorescence data illustrate that the Fluram (which is non-

fluorescent reagent) has a strong luminescence after binding to BSA and SOD1 with maximum

at 470 nm and 478 nm, respectively. The result shows that the Fluram is covalently bound to

both BSA and SOD1.

4.3.2 Protein Folding/Unfolding

The folding and unfolding of BSA and SOD1 was studied in order to determine whether

2PA spectroscopy is an effective tool for monitoring the folding and unfolding of proteins. The

hypothesis is that the local electric fields are higher in the folded form and lower in the unfolded

146

form. When a protein unfolds, it stretches out and loses its tertiary structure; there is more free

space around the protein, causing the electric field around the dye to decrease.

300 400 500 6000.0

0.2

Fl

Co

un

ts

Fluram

BSA

BSA-Fluram

Ab

so

rba

nc

e

Wavelength (nm)

A

0

100

200

300

400

300 400 500 6000.00

0.03

0.06

0.09

0.12

0.15B

Fl

Co

un

ts

Fluram

SOD1-Fluram

Ab

so

rba

nc

e

Wavelength (nm)

0

50

100

150

200

Figure 4.3: (A) Optical absorption and fluorescence spectrum of Fluram binding to BSA. Note

new absorption feature after Fluram bound to BSA. (B) Optical absorption and fluorescence

spectrum of SOD1 binding to fluram. New absorption and fluorescence features

confirm binding of Fluram to SOD1.

Figure 4.4: Schematic illustration of protein unfolding mechanism.

4.3.2.1 BSA-Fluram

Folding and unfolding BSA were studied as a function of temperature. Both one-photon

fluorescence and two-photon fluorescence measurements were carried out as a function of

temperature. Figure 4.5A shows the change in one-photon fluorescence intensity decrease at 479

147

nm for BSA-fluram as a function of temperature from 25 to75 oC. A similar trend was observed

for two-photon fluorescence as shown in Figure 4.5B.

The obtained data showed that fluorescence intensity decreased for both one and two-

photon excitation and did not show any special transition about the folding and unfolding of the

protein. On the other hand, the relative 2PA cross-sections that was determined from the ratio of

two-photon to one-photon fluorescence showed a clear indication of when folding and unfolding

occurred. The 2PA cross-sections were able to show the melting transition for BSA. From Figure

4.5C it is obvious that the BSA starts to unfold around 50-60 oC. This data matched well with the

unfolding transition monitored using circular dichroism measurements.

4.3.2.2 SOD1-Fluram

The folding and unfolding of SOD1-fluram was also monitored as a function of

temperature. The SOD1 result was shows similar outcomes to BSA, where both one and two-

photon excitation result didn’t show any interesting transition to monitor the unfolding state in

SODI. Both one and two-photon excitation show decrease in fluorescence as temperature

function Figure 4.6A and 4.6B. While the 2PA cross sections s showed a capability to monitor

SOD1 unfolding transition Figure 4.6C, which demonstrate the importance of using 2PA cross

sections s tool to study protein folding/unfolding states.

148

400 450 500 550 600 650

0

100

200

300

400BSA-Fluram

Fl

Co

un

ts

Wavelengths nm

A

20 40 60

200

250

300

350

400

FL

@ 4

79

nm

Temperature oC

450 500 550 6000

5000

10000

15000

BSA-Fluram

2P

A C

ou

nts

Wavelengths nm

B

20 40 60 805000

10000

15000

2P

A@

47

9 n

m

Temperature oC

20 30 40 50 60 70 80

0.85

0.90

0.95

1.00

1.05C

Rel

2P

A c

ross-s

ecti

on

Temperature oC

Temperature-dependent

unfolding

BSA-Fluram

Figure 4.5: (A) One photon fluorescence for BSA-fluram after excitation at 370 nm, inset shows

changes in fluorescence intensity at 479 nm for BSA-fluram as a function of temperature from

25 to 75 oC. (B) Two photon fluorescence for BSA-fluram after excitation at 800 nm,

inset show changes in fluorescence intensity at 479 nm for BSA-fluram as a

function of temperature from 25 to75 oC. (C) Relative 2PA cross-sections

showing the trend of unfolding transition for BSA as a function of

temperature from 25 to75 oC.

149

450 500 550 6000

250

500

750

1000SOD1-Fluram

FL

Co

un

ts

Wavelengths nm

A

20 40 60 80

300

600

900

FL

@ 4

82

nm

Temperature oC

450 500 550

1000

2000

3000

4000

BSOD1-Fluram

2P

A C

ou

nts

Wavelength nm

20 40 60 800

1000

2000

3000

4000

2P

A@

48

2 n

m

Temperature oC

30 40 50 60 70 80 90

0.8

0.9

1.0

1.1C

Rel

2P

A C

ross

-secti

on

Temperature (0C)

SOD1-Fluram

Temperature-dependent

unfolding

Figure 4.6: (A) One photon fluorescence spectrum of SOD1-fluram after excitation at 370 nm,

inset shows changes in fluorescence intensity at 482 nm for SOD1-fluram as a function of

temperature from 25 to 95 oC. (B) Two photon fluorescence spectrum of SOD1-

fluram after excitation at 800 nm, inset show changes in fluorescence intensity

at 482 nm for SOD1-fluram as a function of temperature from 25 to 95 oC.

(C) (B) Relative 2PA cross-sections for SOD1-fluram again following the

unfolding transition as a function of temperature from 25 to 95 oC.

4.3.3 Protein Aggregation

After we studied the unfolding pathway for both systems (BSA and SOD1) as a function

of temperature. We have monitored the aggregation of proteins by first softening the proteins

with Gu-HCl and studying the aggregation as a function of temperature (Figure 4.7).

150

Figure 4.7: Schematic showing the aggregation mechanism.

4.3.3.1 BSA-Fluram with GuHCl and Temperature

To achieve the objective of monitoring aggregation of BSA, BSA was softened with 1 M

GuHCl, and temperature was used change to achieve the aggregation of the proteins. BSA

aggregation was tracked with one photon excited fluorescence, two-photon excited fluorescence

and relative 2PA cross-sections. It can be noticed from Figure 4.8A, that one photon excitation

was not sensitive tool to study BSA aggregation, while, two-photon excitation did sense the

aggregation with increasing the temperature. However, the results prove that the local

environment, via the local electric fields in proteins, is enhancing the 2PA cross-sections of the

chromophores, as shown in Figure 4.8B, the electric field increased as the proteins started to

aggregate because there were more dye molecules available.

151

30 40 50 60 70 80

0.5

1.0

1.5

2.0

2.5

3.0BSA-Fluram/1M GuCl Aggregation

One-photon excitation

Two-photon excitation

I/I 0

Temperature (0C)

A

30 40 50 60 70 80

1

2

3

4

B

Enhancement for

larger aggregates

Re

l 2

PA

cro

ss

-se

cti

on

Temperature (0C)

BSA-Fluram/1M GuCl Aggregation

Decrease

in the beginning

Figure 4.8: (A) Normalized fluorescence with one and two-photon excitation to monitor

aggregation of BSA; (B) Relative 2PA cross-sections for BSA-Fluram monitoring

aggregation of BSA. The results show that relative 2PA cross-sections

is a sensitive technique.

4.3.3.2 SOD1-Fluram with GuHCl

SOD1 aggregation was similarly monitored via one photon excitation, two-photon

excitation and 2PA cross-sections as is shown in Figure 4.9A. the carried-out data for one and

two-photon excitation illustrate that both are unable to track aggregation in SOD1, where both

one and two-photon excitation decreased with increasing temperature without any interesting

transition. On the other hand, the relative 2PA cross-sections as a function of temperature (Figure

4.9B) shows that there is more than one stage in the aggregation of SOD1 and it is possible to

use the 2PA cross-sections as tools to monitor such aggregation process.

152

30 40 50 60 70 80 90

0.2

0.4

0.6

0.8

1.0

SOD1-Fluram/1 M GuCl

Aggregation

One-photon excitation

Two-photon excitation

I/I 0

Temperature (0C)

A

30 40 50 60 70 80 90

0.9

1.2

1.5

1.8

2.1

2.4

B

Rel.

2P

A C

ross

-secti

on

Temperature (0C)

SOD1-Fluram/1 M GuCl

Aggregation

Figure 4.9: (A) Normalized fluorescence intensities as a function of temperature with one and

two-photon excitation; (B) Relative 2PA cross-sections for SOD1-Fluram monitoring

aggregation of SOD1. The results show that relative 2PA cross-sections is a

sensitive technique.

4.3.4 Dynamic Light Scattering

Further aggregation studies were carried out with dynamic light scattering. The DLS

results agreed with the 2PA data; DLS showed that the size increased as a function of

temperature (Figure 4.10A) The sample solutions were initially filtered to remove any extraneous

particles through 0.22 μm filter. DLS measurements demonstrate an increase in size from 5 to 20

nm was observed for BSA, while size changed from 3.8 nm to 7.9 nm for SOD1 (Figure 4.10B).

The trends observed matched well with relative 2PA cross-sections data. The data clearly

showed that present relative 2PA cross-sections measurements can used successfully to monitor

the early aggregates of SOD1 protein. The mechanism can be ascribed to the fact that the local

electric fields changes, when the proteins aggregate and the relative 2PA cross-sections are

sensitive to changes in the electric fields and thereby able to monitor the aggregation of proteins.

153

0 20 40 60 80 1000

5

10

15

20

25

25 0C

65 0C

90 0C

Am

pli

tud

e

Size (nm)

BSA/1 M GuHCl Aggregation

20 40 60 800

5

10

15

20

25

Av

g S

ize

(n

m)

Temperature (Cels)

A

0 8 16 24 32 400

2

4

6

8

10B

30 0C

50 0C

90 0C

Am

pli

tud

e

Size (nm)

SOD1/1 M GuHCl - Aggregation

20 40 60 800

2

4

6

8

10

12

Av

g S

ize

(n

m)

Temperature (0C)

Figure 4.10: DLS size distribution at different temperatures for BSA and SOD1. Inset shows

average size as a function of temperature.

4.3.5 Time-Resolved Fluorescence Measurements

Furthermore, protein aggregation studies were carried out with time-resolved

fluorescence measurements. The time-resolved fluorescence data showed that the structure of the

Fluram bound to SOD1 is different from the structure of the Fluram bound to BSA, it is

interesting to note the lower lifetime for SOD1 when compared to BSA. Because the

fluorescence lifetimes were quite different in both systems, indicating environmental effects on

their lifetimes.

Based on the above measurements, we can conclude the following. Fluram was

successfully bound to lysines in both BSA and SOD1 as observed from new peak at 380 nm in

absorption and fluorescence at 480 nm. Thermal unfolding of BSA-Fluram and SOD1-Fluram

was monitored with one-and two-photon excited fluorescence techniques and the results show

that relative 2PA cross-sections is a sensitive technique to follow the unfolding. The mechanism

is attributed to changes in local electric fields around the chromophore. Aggregation of SOD1

and BSA was induced by softening the proteins with 1M GuHCl and varying temperature and

was monitored with one and two-photon excited fluorescence techniques. The results have

154

shown that relative 2PA cross-sections again are quite sensitive to aggregation as opposed to

individual fluorescence intensities. Aggregation was also followed by DLS measurements and an

increase in size from 5 to 20 nm was observed for BSA while size changed from 3.8 nm to 7.9

nm for SOD1. The trends observed matched well with relative 2PA cross-sections

measurements.

10 20 30 40 50

10

100

1000

A SOD1-Fluram

BSA-Fluram

Fl C

ou

nts

Time (ns)

20 40 60 80 100

2

3

4

5

6

7

8

9B

BSA-Fluram

SOD1-Fluram

Avg

Lif

eti

me (

ns)

Temperature (0C)

Figure 4.11: (A) Time-resolved fluorescence of SOD1 and BSA; (B) Average lifetimes for

BSA-fluram and SOD1-fluram as function of temperature.

Our results prove the principle that 2PA cross-sections imaging (ratio of two-photon to

one-photon imaging) can be a valuable analytical tool to monitor folding and aggregation and

can be used to follow the early aggregation in proteins (such as that in ALS). This approach

works because two-photon absorption (2PA) cross-sections probe the local electric fields in

proteins which follow patterns in the folded and unfolded states of proteins.

4.4 Conclusions

Two-photon absorption (2PA) cross-sections of dye chemically bound to protein were

used as markers to monitor unfolding and aggregation transitions in bovine serum albumin

(BSA) and superoxide dismutase 1 (SOD1). SOD1 is a protein that was implicated for familial

155

form of Amyotropic Lateral Scelrosis (ALS). Aggregation of SOD1 considered to be a major

reason for the death of neurons responsible for muscle function. In search of developing such

analytical tools, the feasibility of 2PA cross-sections of dyes bound to proteins was investigated

for probing the folding and aggregation of SOD1. The hypothesis behind the project is that two-

photon absorption (2PA) cross-sections probe the local electric fields in proteins which follow

patterns in the folded and unfolded states of proteins. Therefore, it can be used to probe different

conformations of proteins and can be used to image early aggregation in SOD1. The unfolding of

BSA and SOD1 was carried out by varying the temperature (from 25 to 90 0C) and was

monitored by one and two-photon excited fluorescence of Fluram bound to the proteins. Fluram,

a primary amine binding dye readily binds to lysine of proteins to give rise fluorescence in the

visible region. Interestingly, 2PA cross-sections (ratio of two-photon to one photon fluorescence)

were able to monitor unfolding sensitively over other optical techniques. The mechanism was

attributed to the decrease in electric fields around the fluorophore during unfolding. The

aggregations of SOD1 and BSA were studied by adding 1 M GuHCl to soften the protein and

temperature variation to monitor it. We used UV-vis absorption, one and two-photon excited

fluorescence, time-resolved fluorescence and dynamic light scattering techniques. The results

have shown that 2PA cross-sections of Fluram bound to proteins can selectively monitor the

aggregation of proteins and the mechanism is again ascribed to the changes in local electric

fields around the probe during aggregation.

4.5 Chapter Summary

• Two known protein were chosen for this investigation which are BSA and SOD1.

• Fluram is non-fluorescent dye but it will it will fluorescent when it binds with primary

amines.

156

• Fluram was successfully bound to Lysines in both BSA and SOD1 as observed from a

new peak in absorption at 380 nm and fluorescence at 480 nm.

• The Fluram bound to SOD1 is different from that of Fluram bound to BSA as the

fluorescence lifetimes are quite different in both systems, indicating that the environment

influences their lifetimes.

• Thermal unfolding of BSA-Fluram and SOD1-Fluram was monitored with one-and two-

photon excited fluorescence techniques and the results show that relative 2PA cross-

sections is a sensitive technique to follow the unfolding. The mechanism is attributed to

changes in local electric fields around the chromophore.

• Aggregation of SOD1 and BSA was induced by softening the proteins with

1M GuHCl and varying temperature and was monitored with one and two-photon excited

fluorescence techniques. The results have shown that relative 2PA cross-sections again

are quite sensitive to aggregation as opposed to individual fluorescence intensities.

• Aggregation was also followed by DLS measurements and an increase in size from 5 to

20 nm was observed for BSA while size changed from 3.8 nm to 7.9 nm for SOD1. The

trends observed matched well with relative 2PA cross-sections measurements

• Our results prove the principle that 2PA cross-sections imaging (ratio of two-photon to

one-photon imaging) can be a valuable analytical tool to monitor folding and

aggregation.

157

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163

CHAPTER 5

SINGLE AND TWO-PHOTON DNA-BASED FLUORESCENCE SENSORS FOR Pb2+

AND Hg2+

5.1 Introduction

In Chapters 3 and 4, we have focused on the use of relative 2PA cross-sections of

chromophores solubilized in organized self-assemblies as a tool to monitor the stabilization of

non-canonical structures of DNA as well as following the protein folding and aggregation. In this

Chapter, we have focused on using this knowledge to develop a one- and two-photon

fluorescence-based biosensor for the detection of toxic heavy metal ions.

Heavy metals are abundant, naturally occurring elements with densities at least five times

greater than water1. Their many applications in various fields of modern science have led to their

abundant distribution, as well as for their widespread environmental contamination. Natural

events, such as volcanic activity, weathering, atmospheric deposition, and soil erosion of metal

ions all can contribute to heavy metal pollution and do so in significant respects2. But while often

heavy metals receive a negative reputation for their toxic nature, their contribution towards the

poisoning of various environments has been largely catalyzed by anthropogenic activities such as

mining, industrial production, industrial emissions from power plants and incinerators, fertilizer,

forest fires, and even the modern lifestyle consumables1,3,4. Whatever were the heavy metals

contamination reasons and sources, it is becoming an increasingly prominent ecological and

global health concern. The severity with which heavy metal ions pollute even in relatively small

concentrations, their impact is much more severe for human health. Some of these heavy metals

play vital biological functions in living organisms mechanisms such as iron, copper, manganese,

cobalt, zinc and molybdenum. However, all heavy metals are considered toxic at higher

164

concentrations1,5,6. Heavy metals impact living organisms in two ways: through their

accumulation in specific organ or binding to a protein and replacing the metal in their original

place6,7.

Some heavy metals, especially those such as lead (Pb2+) and mercury (Hg2+), have no

known beneficial effects on organic life, and can cause serious, even fatal illness over time as

shown in table 5.16,8–10. The degree of toxicity of lead and mercury result from their ability to

bind with cell components such as DNA and protein sites, by displacing other vital minerals

from their natural binding locations, and confusing the coherence of the system; such inorganic

heavy metals can readily cause cell malfunction, DNA damage, and very grave health

concerns2,6,8. With regards to lead, chronic exposure to and subsequent bioaccumulation been

proven to impact the body’s ability to function efficiently, effectively, and healthily, impacting

the mechanisms of the kidneys and liver as well as the central nervous, hematopoietic, endocrine,

and reproductive systems11–13. For an example, lead absorbed by a pregnant mother is readily

transferred to the developing fetus, and prenatal exposure to lead has been linked with lower

birth weights and early, preterm delivery alongside neuro-developmental abnormalities14. The

greatest danger; however, awaits children: due to their smaller blood capacity and biomass14.

Pb+2 has attracted more attention due to its variety of resources and consumption.

However, Hg+2 is very important because, despite its limited contaminated resources compared

to Pb+2, it remains highly hazardous because of its severity effect even at low concentrations.

Studies show that enzymatic activity within those exposed to mercury produce genotoxic

alterations and suggest that even relatively low levels of exposures to mercury inhibits enzyme

activity and induces oxidative stress in the cells. Many contests that mercurial exposure has a

165

direct relationship with carcinogenesis, and studies suggest that the DNA’s susceptibility to

damage exists as a consequence of cellular exposure to mercury6,15.

Figure 5.1: Various sources of lead and mercury pollutions in the environment.

Table 5.1: Possible health effects corresponding to lead and mercury metals6.

Metal Forms Sources Route of Entry Health Effects

Mercury Hg, Hg2+,

Hg+, Hg-

organic

Oxidation

state:

+1, +2

Fossil fuel combustion,

mining, smelting, solid waste

combustion, fertilizers

industrial wastewater, use in

electrical switches,

fluorescent bulbs, Mercury

arc lamps, incineration of

municipal wastes, emissions

from mercury products:

batteries, thermometers,

Mercury amalgams

Inhalation,

ingestion and

absorption

through skin

Disruption of the

nervous system,

damage to brain

functions, DNA

damage and

chromosomal

damage, allergic

reactions, tiredness

and headaches,

negative reproductive

effects, such as sperm

damage, birth defects

and miscarriages

Lead Pb2+,

Oxidation

state:

+2, +4

Application of lead in

gasoline, fuel combustion,

industrial processes, solid

waste combustion, used in

paints, used in ceramics and

dishware, Lead is used in

some types of PVC mini-

blinds

Inhalation and

ingestion

Anemia (less Hb),

hypertension, kidney

damage, miscarriages,

disruption of nervous

systems, brain

damage, infertility,

intellectual disorders

166

Consequently, the need is there for developing sensitive, accurate, and selective method

of detecting the presence of heavy metal ions especially Pb2+ and Hg2+. While conventional

methods used for Pb2+ and Hg2+ detections such as inductively coupled plasma-mass

spectroscopy (ICP-MS), anodic stripping voltammetry (ASV) inductive coupled plasma atomic

emission spectrometry (ICP-AES) and atomic absorption spectroscopy have been proven to

demonstrate accuracy and sensitivity, these methods often require complicated multistage sample

preparation and sophisticated, expensive equipment, failing to account for the more realistic

aspects of on-site detection16–20. Therefore, there is a need to develop novel approaches for

sensitive, selective methods for the detection of Pb2+ and Hg2+ that can be used for on-site

detection.

In recent years, the use of functional nucleic acid-based sensors has shown increasing

potential in the detection of heavy metal ions, where specific oligonucleotide ligand is designed

for particular heavy metal ion selectively. This method of sensing is based primarily upon the

observation of shifts in the fluorescence spectrum that take place within the system of a G-

Quadruplex that forms at room temperature upon encounter with the particular metal ion20–24.

One of the most promising of non-canonical structure that is used in biosensor is G-Quadruplex,

as it is one of the rigid structures and needs a metal ion to form it. The binding of particular

heavy metal ions within the G-Quadruplex structure would thus provide an effective mode of

detection10,25,26. In G-Quadruplex, four guanine bases will form a guanine tetrad through

Hoogsteen hydrogen-bonding, then another guanine tetrads (based on the number and location of

guanine bases) will be form in the top of each other and form G-Quadruplex27,28. Due to the

unique structure of G-Quadruplex, it is extensively used as signal amplifier in numerous

biosensors28–31.

167

For the detection of Pb2+ and Hg2+ in this study, we employed a specific oligonucleotide

that displays sensitivity and selectivity for specific heavy metal ions, which is T30695

oligonucleotide for the case of mercury and lead22,32–36. Due to guanine (G)-rich base pair in

T30695 oligonucleotide, it will lead to formation of a G-Quadruplex structure, resulting in

significant, measurable change in fluorescence of attached chromophores (Figure 5.2) 37–39. The

research approach used in the present study is to develop a technique for on-site detection of Pb2+

and Hg2+ using single and/or two-photon fluorescence-based DNA sensors with non-covalently

bound chromophores36. A wide variety of fluorophores, including Diethylthiacyanine Iodide

(DTI), Thiazole Orange, Methyl Orange, Thioflavin-T, Acridine Orange, 5,10,15,20-Tetra(N-

Methyl-4-Pyridyl)Porphine (TMPyP4), were studied to determine which would demonstrate the

best sensitivity and selectivity for the detection of lead and mercury. From the studies, DTI was

found to be the optimal fluorophore for the sensitivity and selectivity as it is quite planar and

binds to DNA base pairs via - stacking.

Figure 5.2: Schematic representation of the use of G-Quadruplex based DNA sensors to detect

presence of Pb2+ and Hg2+ ions through quenching the fluorescence of the fluorophore.

168


5.2.1 Materials

For this investigation, T30695 primary oligonucleotides was selected due to its ability to

form a stable G-Quadruplex and bind with both Pb2+ and Hg2+. T30695 was synthesized by

Integrated DNA Technologies and the sequence is as follows:

T30695: 5'-GGGTGGGTGGGTGGGT-3'

In addition, a tris-acetate buffer (10 mM, pH 8.0) buffer was prepared from tris hydrochloride,

sodium acetate, and ultrapure water obtained from the Milli-Q system. The DNA stock solutions

were then prepared and annealed with 100 mM of KCl in tris buffer. Aqueous metal ion stock

solutions were also prepared in tris buffer for Pb2+, Hg2+, Zn2+, Cd2+, Na+, Fe2+, Cu2+, K+, Co2+,

Ni2+, and Mn2+.

Ultimately, diethylthiacyanine iodide (DTI, 1 mM) was determined to be the most

effective fluorophore and was consequently added to each of the examined samples, illuminating

the shifts or lack thereof in the fluorescence of the DNA sequences upon their exposure to both

Pb2+ and Hg2+ and the other prepared metals. For this investigation, three classes of samples were

prepared throughout the experiment: native T30695 DNA samples (those devoid of the heavy

metals) as a control, and Pb2+ and Hg2+ samples with T30695 used for the testing of the DNA

sensitivity, various metal ion samples used for selectivity tests. Each sample used for the

measurements was prepared to reach 1 mL, and contained both DTI (20 μM) and 100 μL of an

T30695 oligonucleotide solution and a certain volume of the prepared tris buffer, which was

introduced as a sample’s final component in order to bring the total volume of each to 1 mL. The

naked samples included no additional components besides the aforementioned three. The

standard metal ion samples; however, contained an additional 100 μL of a 1000 ppb metal ion

169

stock solution (a concentration of 100 ppb), changing the final volume of buffer added: these

solutions were formed with the cationic compounds Pb2+, Hg2+, Zn2+, Cd2+, Na+, Fe2+, Cu2+, K+,

Co2+, Ni2+, and Mn2+. Finally, the samples used for testing the system’s sensitivity incorporated

various concentrations of Pb2+ and Hg2+ (samples at 5, 10, 20, 30, 50, 80, 100, 200, 300 ppb),

which again changed the final volume of buffer added. This process was repeated for all the

investigated drugs molecules

5.2.2 Spectroscopic Methods

A shimadzu UV-2160 UV/Vis absorption spectrophotometer was used for the optical

absorption measurements. For one-photon fluorescence measurements, an Edinburgh F900

spectrofluorimeter was used with an Xe-450W lamp for excitation and R2158P as detector. For

two-photon fluorescence measurements, Ti:Sapphire laser centered on 800 nm was used as the

excitation source and the resulting fluorescence is collected on an Edinburgh F900

spectrofluorimeter. A Jasco J-815 Circular Dichroism (CD) Spectropolarimeter were used to

document the DNA’s structural change in the presence and absence of heavy metal ions,

especially in terms of G-Quadruplex formation. A wide variety of fluorophores, including DTI,

Thiazole Orange, Methyl Orange, Thioflavin-T, Acridine Orange, TMPyP4, were under

investigation to determine which would demonstrate the most evident alterations in magnitude of

fluorescence as the oligonucleotides bound with the toxic heavy metal ions. In determining

which dye was best suited for the system, all dyes were subjected to the UV/Vis absorbance

measurements as well as single and two-photon spectroscopy scans; these test samples contained

20 μM of the dye, 100 μL of an oligonucleotide (T30695), Pb2+ and Hg2+ concentrations of 100

ppb, and the appropriate volume of tris buffer to raise the sample’s volume to 1000 μL. DTI,

when coupled with the oligonucleotides, demonstrated highest change in fluorescence intensity

170

upon exposure to Pb2+ and Hg2+ ions, and thus was concluded to be the optimal fluorophore for

the sensitivity and selectivity for toxic metal ions.

Figure 5.3: Molecular structure of the fluorophore, diethylthiacyanine iodide (DTI dye).


5.3.1 Interaction of G-Quadruplex DNA (T30695/DTI) with Pb2+


T30695 is well-known to form G-Quadruplex structure when induced through

appropriate metal ion such as K+ that is added to the buffer during these experiments. In addition,

Zhan et al found that T30695 is selective to recognize Pb2+ , and moreover, Pb2+ can stabilize and

strengthen the G-Quadruplex when it replaces the K+ 11. Figure 5.4 shows the optical absorbance

spectrum of the T30695/DTI in 10 mM tris buffer (pH 8.0) at varying concentrations of Pb2+. It

can be noticed that there is almost no change in both DTI and T30695 DNA absorbances at 420

nm and 255 nm respectively, suggesting that there is no significant structural change in the G-

Quadruplex of T30695 as it interacts with Pb2+, which supports the proposal that T30695 G-

Quadruplex structure is maintained upon binding to Pb2+ (Figure 5.4).

171

250 300 350 400 450 500

0.00

0.05

0.10

0.15

0.20

Ab

so

rban

ce

Wavelength nm

T-30695/DTI (diff concentrations of Pb2+)

No change in

ground state absorption

Figure 5.4: Optical absorbance spectrum of T-30695/DTI at varying Pb2+ concentrations. Lack

of significant change in absorption of both the dye and DNA suggests no structural

changes to Quadruplex DNA.

5.3.1.2 One-Photon Fluorescence Measurements

The steady-state fluorescence measurements for T-30695/DTI were monitored with

increasing Pb2+ concentrations from 0 ppb till 300 ppb after 420 nm excitation, and

corresponding data is shown in Figure 5.5. The maximum fluorescence was found to be at 475

nm where fluorescence started to quench with increase in Pb2+ concentrations as shown in the

inset of Figure 5.5. Thus, steady-state fluorescence measurements results show fluorescence

intensity could be decreased due to T-30695 hairpin where deoxyguanosines become close to

DTI which induced the photo-electron transfer from DTI to G-quadruplet which will lead to

increased quenching efficiency. The fluorescence quenched by 80 % from the free T-30695 to

the 300 ppb Pb2 and has a limit of detection (LOD) of 12 ppb which was determined from the

change in fluorescence intensity changes by calculating standard deviation of three blank

measurements40.

172

450 500 550 600 650

0

1x105

2x105

3x105

4x105

T-30695/DTI (diff concentrations of Pb2+) - 420 nm ex.

Fl

Co

un

ts

Wavelength (nm)

0 50 100 150 200 250 3002.6x10

5

2.8x105

3.0x105

3.2x105

3.4x105

Detection limit = 12 ppb

FL

. In

ten

sit

y

[Pb2+] (ppb)

Figure 5.5: Steady fluorescence spectrum of T30695/DTI upon exposure to different Pb2+

concentrations. One-photon excitation begins at 420 nm, and sensitive change in fluorescence

takes place at 475 nm and is shown in the inset.

5.3.1.3 Two-Photon Fluorescence Measurements

Similar to one-photon fluorescence, two-photon fluorescence measurements were also

carried out for T30695/DTI as a function of Pb+2 concentration after excitation at 800 nm. Shown

in Figure 5.6 are the corresponding fluorescence spectrum as a function of increasing Pb+2

concentration. The decrease in two-photon fluorescence intensity is similar to that of one photon

fluorescence. The normalized fluorescence at 475 nm is shown in the inset of Figure 5.6 and a

quenching in two-photon fluorescence was also observed which was much greater than the

change in one-photon fluorescence quenching. Close to 250% quenching was observed with two-

photon excitation. The results do confirm that two-photon fluorescence measurements provide a

higher sensitive detection tool and a LOD of 4.5 ppb was determined from the data.

173

450 500 550 600 650

0

1000

2000

3000

4000

5000

T-30695/DTI (diff concentrations of Pb2+) - 800 nm ex.

Fl

Co

un

ts

Wavelength nm

0 100 200 300

2000

3000

4000

5000

2P

Fl

Int.

[Pb2+

] (ppb)

Detection limit = 4.5 ppb

Figure 5.6: Two-photon spectrum of T30695/DTI upon exposure to different Pb2+

concentrations. Two-photon excitation begins at 800 nm, and sensitive change in

fluorescence takes place at 475 nm as shown in the inset.

5.3.2 Interaction of G-Quadruplex DNA (T30695/DTI) with Hg2+


The optical absorbance measurement of the T30695/DTI system in 10 mM tris buffer (pH

8.0) at varying concentrations of Hg2+ is shown in Figure 5.7 Lack of significant changes in

absorption in DTI at 420 nm and T30695 DNA at 255 nm suggests no structural changes as it

interacts with increasing Hg2+ concentrations from 0 ppb to 300 ppb. Here again, T30695 G-

Quadruplex structure is maintained at higher Hg2+ concentrations.

174

300 400 500

0.00

0.05

0.10

0.15

0.20T-30695/DTI (diff concentrations of Hg2+)

Ab

so

rban

ce

Wavelength (nm)

Figure 5.7: Optical absorbance spectrum of T-30695/DTI at varying Hg2+ concentrations. Lack

of significant changes in absorption suggests no structural changes.

5.3.2.2 One-Photon Fluorescence Measurements

Similarly, to Pb2+, Hg2+ is known to form G-Quadruplex when T30695 interact with Hg2.

However, the fluorescence intensity of T30695/DTI did not show a significant change with the

increasing Hg2+ concentrations at 420 nm excitation, as shown in Figure 5.8. The data of one-

photon fluorescence change is quite not sensitive and did not show any trend for Hg2+. This

study shows that one-photon fluorescence change of T3095/DTI system is quite selective for

Pb2+ and not to that of Hg2+. This has something to do with how Hg2+ is interacting with the

chromophore. Pb2+ can induce change in fluorescence intensity via enhancing the intersystem

crossing while Hg2+ cannot do the same to that extent.

175

450 500 550 600 650

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

T-30695/DTI (diff concentrations of Hg2+) - 420 nm ex.

Fl

Co

un

ts

Wavelength (nm)

0 50 100 150 200 250 3002.0x105

2.5x105

3.0x105

3.5x105

4.0x105

Fl @

47

0 n

m

[Hg2+

] (ppb)

No sensitivity

Figure 5.8: Steady fluorescence spectrum of T30695/DTI upon exposure to different Hg2+

concentrations. One-photon excitation is at 420 nm, and non-sensitive change in


5.3.2.3 Two-Photon Fluorescence Measurements

In contrast to one-photon fluorescence, two-photon fluorescence measurements were

more sensitive to Hg2+ for the T30695/DTI system as shown in Figure 5.9. In the presence of

Hg2+, the binding of Hg2+ with T30695/DTI significantly quenches the fluorescence of DTI as

Hg2+ concentrations increased from 0 ppb to 300 ppb. A limit of detection of 5 ppb was

determined from the change in two-photon fluorescence intensity for T30695/DTI towards Hg2+

(Inset of Figure 5.9). It is interesting to see that T30695/DTI has shown better sensitivity with

two-photon excitation than one-photon excitation. This can be assigned to the fact that Hg2+ can

alter the local electric field environment the G-Quadruplex, which can be observed only with

two-photon excitation and not one-photon excitation.

176

450 500 550 600 650

0

300

600

900

1200

1500

T-30695/DTI (diff concentrations of Hg2+) - 800 nm ex.

Fl C

ou

nts

Wavelength nm

0 50 100 150 200 250 300300

600

900

1200

1500

Fl @

475

nm

[Hg2+

] (ppb)

Detection limit = 5 ppb

Figure 5.9: Two-photon spectrum of T30695/DTI after exposure to different Hg2+

concentrations. Two-photon excitation begins at 800 nm, and sensitive change in


5.3.3 Relative 2PA Enhancements

The T30695-DNA was prepared by adding 100 mM KCl, which highly important

because a cation such K+ will bind to the negative charge in phospho-diester linkages in T30695-

backbone, which will lead to the generation of a local electrostatic dipole in T30695 backbone.

In addition, K+ will induce the single strand T30695-DNA to form a G-Quadruplex41,42. It is

important to note that with increase the concentrations of Pb2+ and Hg2+ from 0 ppb to 300 ppb,

there was no change in the ground state absorbance observed, which indicate that the T30695 G-

Quadruplex structure is highly stable.

Parts A and B of Figure 5.10 shows a comparison between the effectiveness of one-

photon fluorescence and two-photon fluorescence by comparing their change in fluorescence

intensities (I0/I) for Pb2+ and Hg2+, respectively. As can be clearly observed from the Figures, the

two-photon excitation gave better LODs when compared to one-photon excitation, this can be

ascribed to the altered local electric field changes around the G-Quadruplex when heavy metal

177

ions like Hg2+ and Pb2+ have replaced the K+ ion. Relative 2PA cross-sections were monitored by

taking a ratio of two-photon intensity changes over one-photon fluorescence intensity changes

and plotted in Figure 5.10C. As the Figure depicts an extremely clear relationship between the

concentration of the heavy metal present and the relative two-photon fluorescence change in the

presence of both Pb2+ and Hg2+. Again, the relative 2P changes were shown to be better

indicators for the sensor response suggesting that the 2P spectroscopy can be used to monitor the

concentrations of toxic metal ions like Hg2+ and Pb2+. Moreover, with the inherent advantages of

two-photon excitation of greater penetration depth, low scattering and ability to work in cloudy

environments, this can be used for on-site detection.

In this chapter, we have developed a technique for continuous detection of Pb2+ and Hg2+

ions rely on single and two-photon fluorescence-based DNA sensors through significant changes

in fluorescence. The presence of Pb2+ and Hg2 ions is expected to open the hairpin T30695,

which will cause a change in fluorescence that can be measured. With increasing the

concentrations of Pb2+ and Hg2+ with T30695, one photon fluorescence and two-photon

fluorescence measurement and relative two-photon cross-sections carried out. The obtained data

demonstrate that two photon fluorescence is a superior technique in detection filed due to two

photon measurement ability to probe the small change in electrical field around DNA, which

make it a powerful tool that should be used in detection methods.

178

0 50 100 150 200 250 300

1.0

1.5

2.0

2.5

3.0T-30695/DTI

2P Fluorescence

(Detection limit = 4.5 ppb)

1P Fluorescence

(Detection limit = 12 ppb)

I 0/I

[Pb2+] (ppb)

A

0 50 100 150 200 250 300

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 T-30695/DTI

2P Fluorescence

(Detection limit = 5 ppb)

1P Fluorescence

(No sensitivity to Hg2+)

I 0/I

[Hg2+] (ppb)

B

0 50 100 150 200 250 300

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

T-30695/DTI (Pb2+)

T-30695/DTI (Hg2+)

Re

l. 2

P C

ha

ng

e

[Pb2+] [Hg2+](ppb)

C

Figure 5.10: (A) A comparison of the change in the single and two-photon fluorescence sensing

of Pb2+ at different concentrations. Better sensitivity was observed with two-photon excitation.

(B) A comparison of the change in the single and two-photon fluorescence sensing of Hg2+ at

different concentrations. Better sensitivity was observed with two-photon excitation. (C) A

comparison of 2P/1P cross-sections ratio in determining sensitivity of T30695

for Pb2+ and Hg2+ detection.

5.3.4 Selectivity

One of the most important factors must be examined is the selectivity of T30695/DTI

toward Pb2+ and Hg2+ and to ensure there is no interference with other possible contaminates.

Therefore, the specificity of T30695/DTI sensing system for Pb2+ and Hg2+ detection was

investigated with a variety of environmentally possible relevant metal ions, such as Cd2+, Co2+,

Cu2+, Fe2+, K+ , Mg2+, Mn2+, Na+ , Ni2+, Zn2+, and free T30695 (as control) to evaluate the

specificity of the system. All metals ion samples were prepared at 100 ppb under identical

179

conditions with using 10 mM tris (pH 8.0) buffer. All solutions were prepared with MQ water

under Nitrogen gas and run as soon it settled down to avoid possible oxidation. Moreover, the

examination was done through one-photon fluorescence and two-photon fluorescence

measurements. As shown in Figure 5.11, under one-photon spectroscopy, the T30695/DTI

system showed notable selectivity for Pb2+ only among other metals such as iron, copper, and

cadmium; however, no selectivity was shown for Hg2+. On the other hand, the system showed

extremely clear selectivity for Pb2+ as well as Hg2+ under two-photon fluorescence. On the other

hands, further investigations are needed at higher concentrations to ensure that there is no

interference of environmentally metal ions at high concentrations. The result suggests that the

T30695/DTI system for detection Pb2+ and Hg2+ have a high selectivity for identification Pb2+

and Hg2+, this may be due to hairpin structure of T30695 that play important role in having better

specificity.

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Zn2+

Pb2+

Hg2+

1-I

/I0

[M2+]

One-Photon fluorescence

Two-Photon fluorescence

Pb2+

Ni2+FreeNa

+Mn

2+Mg

2+K

+Hg

2+Fe

2+Cu

2+Co2+

Cd2+

T-30695/DTI - selectivity

Figure 5.11: A demonstration of T30695’s selectivity of Pb2+ and Hg2+ over other common

metals for both one-photon fluorescence and two-photon fluorescence excitations.

180

5.3.5 Forming G-Quadruplex Structure

The T30695/DTI system formed a G-Quadruplex structure upon binding with the K+ ion

in the salt used in the preparation of the DNA sample at room temperature, and thus remained in

its G-Quadruplex form as it encountered Pb2+ and Hg2+, which can be proven from circular

dichroism (CD) spectrometer Figure 5.12. Circular dichroism (CD) was used to identify the

structural changes of T30695/DTI system. The sample was run in 1 mL volume total under dry

purified nitrogen. In addition, the buffer solution background was subtracted from the circular

dichroism data for T30695/DTI-K+, T30695/DTI-Pb2+ and T30695/DTI-Hg2+. The obtained data

showed that Pb2+ and Hg2+ indeed form G-Quadruplex and the fluorescence changes were

attributed to Pb2+ and Hg2+ influence. Furthermore, parallel” G-Quadruplex structure was formed

in T30695/DTI-K+, T30695/DTI-Pb2+ and T30695/DTI-Hg2+, which can be conclude from the

positive peak around 263 nm and the negative peak in around 240 nm as what shown in Figure

5.12 11,23,43–46.

250 300 350 400 450 500

-2

0

2

4T30695/K+

T30695/Pb2+

T30695/Hg2+

Mo

lar

Ellip

sit

y

Wavelength (nm)

G-Quadruplex formation

Figure 5.12: The circular dichroism measurements of the T30695 oligonucleotide with K+, Hg2+,

and Pb2+ shows the formation of a G-Quadruplex structure.

181

From the studies carried out in this Chapter, we have designed and developed a two-

photon fluorescence-based biosensors that rely upon specific oligonucleotides, coupled with a

fluorophore, that form stable G-Quadruplex structures at room temperature. Upon exposure to

Pb2+ or Hg2+, the DNA’s existing metal ion is replaced by the heavy metal ion, quenching the

fluorescence of the dye; the degree of decrease in fluorescence intensity is the figure of merit for

assessing the sensitivity and the selectivity of the sensor. As such, the oligonucleotide T30695

demonstrated high selectivity for Pb2+ and Hg2+ ions with detection limits of 4.5 and 5 ppb

respectively. In addition, two-photon spectroscopy was shown to be better for sensitivity, and

due to its near-infrared excitation and low scattering, is still effective in muddy and opaque

waters. In summary, the carried-out result shows a significant promise for development of an

effective, single and two-photon, fluorescence-based DNA biosensor which will allow for

continuous monitoring of toxic heavy metal ions in water sources for the purpose of avoiding

potential disasters. Also, these results have proven again the powerful of two-photon,

fluorescence and relative 2PA cross sections as tool to monitor and measure the change in

electrical field around the self-assemblies in complex environments.

5.4 Conclusions

T30695 DNA oligonucleotide sequences was selected for it ability to form G-Quadruplex

structures in order to detect the toxic heavy metal ions Pb2+ and Hg2+ with greater sensitivity and

selectivity. DTI dye was found to be the most optimal fluorophore for this mode of detection,

with the ability to interact with the DNA via intercalation and to demonstrate notable

fluorescence changes upon binding to Pb2+ and Hg2+ especially with two-photon excitation. The

oligonucleotide structure demonstrated high sensitivity to Pb2+ through both one-photon (12 ppb)

and two-photon fluorescence (4.5 ppb); sensitivity under two-photon excitation was far better

182

than that under one-photon excitation, which may be ascribed to the enhancing of two- photon

cross-sections by aligning better with the DNA’s backbone electric field. The limit of detection

under two-photon spectroscopy, 4.5 ppb, falls below the World Health Organization’s minimum

for hazardous Pb2+ concentrations, which is 10 ppb47. T30695/DTI lacked sensitivity to Hg2+

under one-photon excitation; far better sensitivity was observed with two-photon excitation (5

ppb). This again can be ascribed to the orientation of the G-Quadruplex structures’ electric fields

along with the orientation of the fluorophore. The limit of detection under two-photon

spectroscopy, 5 ppb, also falls below the World Health Organization’s minimum for hazardous

Hg2+ concentrations, which is 6 ppb48. T30695/DTI was found to be highly selective for only the

heavy metal ions Pb2+ or Hg2+, and far more particularly through the two-photon method than the

one-photon method. Such selectivity may be attributed to the size of the mercury and lead metal

ions, which likely more effectively stabilized the G-Quadruplex structure. In conclusion,

T30695/DTI system show a great selectivity and specificity for Pb2+ or Hg2+ detection over other

ion metals. This approach shows significant promise for development of an effective, single and

two-photon, fluorescence-based DNA biosensor which will allow for on-site monitoring of toxic

heavy metal ions in water sources; however, further investigations are needed to study the

interferences with higher concentrations and under different pH levels.

5.5 Chapter Summary

• A novel DNA oligonucleotide sequence, T30695 was selected for its ability to form G-

Quadruplex structures in order detect toxic heavy metal ions, Pb2+ and Hg2+.

• DTI dye was found to be a good fluorophore for this mode of detection, with the ability

to both interact with the DNA via intercalation and to demonstrate notable fluorescence

changes upon binding to toxic heavy metal ions.

183

• T30695/DTI showed sensitivity to Pb2+ with both one-photon (12 ppb) and two-photon

excitation (4.5 ppb); sensitivity under two-photon excitation was far better than that

under one-photon excitation, which may be ascribed to changes in two-photon cross-

sections.

• T30695/DTI lacked sensitivity to Hg2+ under one-photon excitation; far better sensitivity

was observed with two-photon excitation (5 ppb). This—again—can be ascribed to the

orientation of the G-Quadruplex structures’ electric fields along with the orientation of

the fluorophore.

• T30695/DTI was found to be highly selective for only the heavy metal ions Pb2+ and

Hg2+. Such selectivity may be attributed to the size of the mercury and lead metal ions,

which more effectively stabilized the G-Quadruplex structure.

184

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CHAPTER 6

TWO-PHOTON ABSORPTION PROPERTIES OF CHROMOPHORES IN

POLYELECTROLYTES

6.1 Introduction

In previous Chapters, studies were carried out on organized self-assemblies that are natural.

Likewise there are self-assembled materials that are man-made. One among them are

polyelectrolytes. In this part of the dissertation, we have attempted to use the power of two-photon

absorption (2PA) to understand the interactions in polyelectrolytes.

Polyelectrolytes are polymers with multi electrolyte groups1. One of the important

properties for polyelectrolytes is their ability to dissociate their electrolyte groups in polar solution

which generates charges in the solution and opposite charge in the polymer chain1–4. As a result,

for dissociate electrolyte groups; changing in folding/unfolding polymer state will accrue. These

ions and counter ions created electrical field around the polymer. This electrical field is responsible

for optical properties for the polymers3,5–9. Important improvements in studying polyelectrolytes

interactions with diverse ions in solution had been studied for last two decades, because the

significance of polymer physics/biophysics properties10–14. One of the main reasons for studying

polyelectrolytes is that they resemble many biological molecules such as polypeptides, proteins

and DNA.

Polyelectrolytes are highly sensitive to any changes around their environment conditions;

therefore, polyelectrolytes give great promise for use as biosensors similar to DNA5,15–17.

However, Polyelectrolytes are difficult to monitor due to their high sensitivity for any change in

their environment that could cause a change in behaviors and characteristics of the polyelectrolytes

because of amphiphilic interactions between the polyelectrolyte and counterions15,18–21.

192

Polyelectrolytes offer one such possibility where one can alter the electric fields by changing

folding/unfolding conditions as shown in Figure 6.1.

Figure 6.1: Schematic illustration of the polyelectrolytes changing folding/unfolding conditions.

Polyelectrolytes, in general, exhibit many properties that benefit the industrial as well as

research fields due to their attributes as well as they are nontoxic. In addition, polyelectrolytes

are used in many industrial applications because of their water solubility and cost-

effectiveness22. Polyelectrolytes have been used for applications such as drug release, surface

coating, as well as for chemical and biological sensing10,24,27,28. Furthermore, polyelectrolytes

have been used for applications relative to electrochemical sensors, photovoltaic cells, smart

windows, and light-emitting diodes29–33.

Polyelectrolytes are promising for biological sensors due to their attributes in optical

properties. These properties can include their effectiveness in sensing as they are very sensitive

to minute changes in the conformational movements relative to intra and interchain energy

transfer2,5,10,24. This ability allows for the amplification of sensing. Some of these sensing

techniques include direct fluorescence superquenching-based systems for monitoring amino

193

acids and FRET-based biosensing24,34,35. Also, they have been combined with nanoparticle

constituents to amplify fluorescence visibility.

Polyelectrolytes are mostly made from conjugated polymers. Conjugated polymers are

polyunsaturated compounds. They contain an alternating single and double bond along a

polymer chain. The backbone of the polymer chain contains atoms of sp and sp2 hydrization

which creates properties similar to that of semiconductor36–38. Conjugated polymers have a wide

band-gap allowing for efficient UV-visible electromagnetic radiation absorption and emission on

the band-gap36. This system helps maintain promising optical and electronic attributes for

conjugated polymers. Some current applications for these systems can consist of transistors,

antistatic coatings and modified electrodes36,39,40. Also they are promising component that can be

used in fluorescence investigations24,34,41. For example, they have been used in applications for

quenching metal-ion sensor making them very useful for detecting some mildly toxic metals42.

As polyelectrolytes resemble DNA, the knowledge gained from the previous studies can

be used to understand the interactions of chromophores with polyelectrolytes. In this Chapter, we

aim to study the 2PA cross-sections of chromophores in polyelectrolytes in an effort to probe the

local electrostatic interactions in polyelectrolytes. The investigations are carried out with cationic

(Acridine Orange, Hoechst 33258, Thioflavin T) and neutral (Coumarin 485) dye molecules

interacting with two well-known anionic polyelectrolytes, Poly [5-methoxy-2-(3-sulfopropoxy)-

1,4-phenylenevinylene]) MPS-PPV and (Poly (sodium 4-styrenesulfonate) PSS. Figure 6.2

shows their chemical structures. Figure 6.3 shows the chemical structures of the dye molecules

under investigation. These systems are chosen specifically to understand the influence of

electrostatic interactions and in turn the electric fields on the 2PA cross-sections of

chromophores. Our hypothesis is that the local electrostatic environment in polyelectrolytes can

194

enhance the 2PA cross-sections of chromophores due to the electric field in polymer chain. Also,

the enhancement can be different for chromophores as the polarizability of chromophores can be

varying.

Figure 6.2: The chemical structure for the investigated anionic polymer in this chapter MPS-

PPV and PSS.

Figure 6.3: Molecular structures of the investigated chromophores.

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6.2 Experimental

6.2.1 Materials

Poly [5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene]) MPS-PPV and Poly

(sodium 4-styrenesulfonate) PSS were obtained from Sigma-Aldrich. Both MPS-PPV and PSS

dissolved in 1:1 DMSO/MilliQ water to prepare stock solutions. The investigated dyes: Acridine

Orange (AcrO), Hoechst 33258 (Hoe), Thioflavin T (ThT) and Coumarin 485 (C-485) dye

molecules were purchased from Sigma Aldrich. Each dye was dissolved in appropriate solvent and

then diluted to make 10 mL of 1 mM of stock solution. Fourteen samples were made from the

stock solutions with increasing concentrations of MPS-PPV and PSS placed in each( 0, 0.002,

0.01, 0.02, 0.03, 0.06, 0.07, 0.17, 0.26 mM for MPS-PPV) and (0, 0.001, 0.01, 0.02, 0.05, 0.17,

0.23, 0.3, 0.39 mM for PSS), while keeping dye concentration at 10 μM, then diluting the sample

volume to be 1 mL. This process was repeated for all the investigated dye molecules.

6.2.2 Methods

The UV/Vis absorption spectrometric measurements were performed using a Shimadzu

UV2101 PC spectrophotometer. Steady-state fluorescence measurements were performed on

Edinburgh spectofluorimeter. The 2PA cross-sections were obtained using the two-photon excited

fluorescence technique Tsunami (Spectrum-Physics), 720 nm to 900 nm, 100 fs, with C485 in

methanol as a standard. The relative 2PA cross-sections were obtained from the ratio of one and

two-photon fluorescence from the samples.

196


6.3.1 Interactions with AcrO

6.3.1.1 Interactions of MPS-PPV with AcrO


Optical absorption was carried out for MPS-PPV with AcrO at room temperature with

increasing concentrations of MPS-PPV as shown in Figure 6.4. It can be noticed that there is no

change in the ground state absorption at low MPS-PPV concentration. However, there is a small

shift from 499 nm to 494 nm with increasing MPS-PPV concentrations. In addition, with

increasing MPS-PPV concentration scattering is observed. So, increasing MPS-PPV

concentrations will lead to increase in the viscosity of the solution and scattered more light.

Furthermore, there is a rise of dye dimer formation at 470 nm and increase with increase MPS-

PPV concentration, which could indicate to aggregation of the system with increase in MPS-

PPV.

300 400 500 600

0.0

0.1

0.2

0.3

0.4

Ab

so

rba

nc

e

Wavelength (nm)

MPS-PPV /AcrO

Figure 6.4: Optical absorption spectrum of AcrO with increasing MPS-PPV concentrations.

197

6.3.1.1.2 One- and Two-Photon Fluorescence Measurements

Figure 6.5A shows the fluorescence spectrum of AcrO at different MPS-PPV

concentrations after excitation at 440 nm. AcrO fluorescence intensity decreased with MPS-PPV

addition, which indicate that AcrO is interacting with MPS-PPV. The fluorescence maximum of

AcrO was at 530 nm with a small shifting in fluorescence to be 537 nm at higher MPS-PPV

concentration. The decrease in fluorescence intensity at 530 nm is normalized and plotted as a

function of MPS-PPV concentration as what shown in Figure 6.5A. Similar to one-photon

fluorescence, two-photon fluorescence measurements were carried out for AcrO as a function of

MPS-PPV concentration after laser excitation at 800 nm as shown in Figure 6.5B. The

normalized fluorescence at 530 nm is plotted, an increase in the beginning then decrease all the

way in two-photon fluorescence was observed. Interestingly, the decrease might also suggest that

there was an aggregation in system with increase in MPS-PPV concentrations which would

affect the AcrO binding and stability of MPS-PPV. But this decrease can also suggest the

interaction of positive dye with anionic polyelectrolyte.

500 550 600 650 7000

300

600

900

1200

Fl C

ou

nts

Wavelength (nm)

MPS-PPV/AcrO

0.0 0.1 0.2

0.2

0.4

0.6

0.8

1.0

1.2

I 0/I

MPS-PPV (mM)

A

500 550 600 650 7000

10k

20k

30k

40k

50k

2P

A C

ou

nts

Wavelength (nm)

MPS-PPV/AcrO

2P exc.

B

0.0 0.1 0.20.3

0.6

0.9

1.2

I 0/I

MPS-PPV (mM)

Figure 6.5: (A) One photon fluorescence spectrum of AcrO with increasing MPS-PPV

concentrations. Inset showed the normalized fluorescence data at 530 nm. (B) Two-

photon fluorescence spectrum of AcrO with increasing MPS-PPV concentrations.

Inset showed the normalized fluorescence data at 530 nm.

198

6.3.1.1.3 Relative 2PA Cross-Sections Measurements

As the main aim of the dissertation is to understand the effect of electrostatic interactions

and local electric fields on the 2PA cross-sections of chromophores, relative 2PA cross-sections

of AcrO were determined from the ratio of two-photon to one-photon fluorescence with

increasing concentrations of MPS-PPV. The corresponding relative 2PA cross-sections of AcrO

as a function of MPS-PPV concentration are shown in Figure 6.6. The data shows an

enhancement by almost 100% in 2PA cross-sections at highest polyelectrolyte concentrations.

This result confirms that the dye is binding to the anionic pocket of the polyelectrolyte and its

2PA cross-sections has increased with the local electric field developed in its vicinity.

0.00 0.05 0.10 0.15 0.20 0.25

1.0

1.5

2.0

2.5

Re

l. 2

PA

cro

ss-s

ecti

on

MPS-PPV (mM)

MPS-PPV/AcrO

Figure 6.6: Relative 2PA cross-sections of AcrO at different MPS-PPV concentrations.

6.3.1.2 Interactions of PSS with AcrO


Interaction of AcrO with another polyelectrolyte PSS are presented here. The optical

absorption spectrum of AcrO at room temperature and as function of increasing PSS

199

concentrations is shown in Figure 6.7. The absorption maximum of PSS has shifted minimally

from 496 nm to 500 nm when AcrO was bound to PSS. The obtained data suggest that there is no

major change in PSS when it’s bound to AcrO. There is a rise of dye dimer formation at 470 nm

and increase in PSS concentration, which could indicate the aggregation of polyelectrolyte.

400 450 500 5500.0

0.1

0.2

0.3

0.4

PSS/AcrO

Ab

so

rban

ce

Wavelength (nm)

Figure 6.7: Optical absorption spectrum of AcrO with increasing PSS concentrations.


Steady-state fluorescence measurements were carried out for AcrO at different PSS

concentrations after excitation at 440 nm. The maximum fluorescence was observed at 530 nm,

and no shift was observed with increasing PSS concentrations. Normalized data at 530 nm shows

a sharp decrease after adding PSS, then increasing again at 0.2 mM then dropping back as shown

in Figure 6.8A. Two-photon fluorescence measurements were carried out for AcrO as a function

of PSS concentration after laser excitation at 800 nm. The maximum fluorescence intensity was

at 530 nm with no shift as observed in the case of one-photon fluorescence. The normalized

fluorescence intensity at 530 nm also showed a similar decrease to one photon fluorescence in

200

the beginning; however, interestingly its increase in two-photon fluorescence then stabilized

without change as shown in the Figure 6.8B. The decrease in both one- and two-photon

fluorescence could suggest that there was an interaction of the dye with polyelectrolyte as

observed previously with PSS.

500 550 600 650 7000.0

400.0

800.0

1.2kA

PSS/AcrO

Fl C

ou

nts

Wavelength (nm)

0.0 0.2 0.40.0

0.5

1.0

1.5

2.0

I 0/I

PSS (mM)

500 550 600 650 7000

10k

20k

30k

40k

50k

60k

PSS/AcrO

2P exc.

2P

A C

ou

nts

Wavelength (nm)

0.0 0.2 0.40.0

0.4

0.8

1.2

1.6

2.0B

I 0/I

PSS (mM)

Figure 6.8: (A) One photon fluorescence spectrum of AcrO with increasing PSS concentrations.

Inset showed the normalized fluorescence data at 530 nm. (B) Two-photon fluorescence

spectrum of AcrO with increasing PSS concentrations. Inset showed the normalized

fluorescence data at 530 nm.


As the main objective of the study is to use 2PA cross-sections of the dye as a way to

monitor the aggregation of the polyelectrolytes. Relative 2PA cross-sections were obtained for

AcrO as function of increased PSS concentration and corresponding data is provided in Figure

6.9 . There was a slight increase in 2PA cross-sections similar to the obtained data for MPS-PPV.

Here again, the increase in 2PA cross-sections for AcrO with different polyelectrolytes is found

to be marginal and this might be ascribed to the molecular parameters such as change in

polarizability, among others.

201

0.0 0.2 0.40.5

1.0

1.5

2.0

2.5PSS/AcrO

Re

l. 2

PA

cro

ss-s

ecti

on

PSS (mM)

Figure 6.9: Relative 2PA cross-sections of AcrO at different PSS concentrations.

6.3.2 Interactions with Hoe

6.3.2.1 Interactions of MPS-PPV with Hoe


Shown in Figure 6.10 is the optical absorption at different MPS-PPV concentrations for

Hoe, a cationic dye well known to bind to minor-groove of DNA. It is observed that the

absorption maximum of Hoe shifted from 344 nm to 347 nm with increasing MPS-PPV

concentration. Also, it is noticeable the growth of a new maximum peak at 447 nm and increase

with increase MPS-PPV concentrations which is the absorption arising from the polyelectrolyte

itself.

202

300 400 500 6000.0

0.1

0.2

0.3

0.4MPS-PPV/Hoe

Ab

so

rba

nc

e

Wavelength (nm)

Figure 6.10: Optical absorption spectrum of Hoe with increasing MPS-PPV concentrations.


Steady-state fluorescence measurements were carried out with increasing MPS-PPV

concentrations of Hoe after excitation at 330 nm. There was no shift in fluorescence with

increasing MPS-PPV concentrations. Normalized data at 544 nm shows a sharp decrease after

adding MPS-PPV till the fluorescence intensity start to be steady at higher concentration as show

in Figure 6.11A. Two photon fluorescence measurements were carried out after laser excitation

at 800 nm and show a quite opposite result (Figure 6.11B). The maximum fluorescence intensity

was at 544. The normalized fluorescence intensity at 544 nm showed a huge enhancement in two

photon counts by 15-fold. This study again shows that Hoe strongly interacts with MPS-PPV and

probably the 2PA cross-sections are influenced by the polyelectrolyte.

203

350 400 450 500 550 6000

1k

2k

3kMPS-PPV/Hoe

Fl C

ou

nts

Wavelength (nm)

A

0.0 0.1 0.2 0.30.2

0.4

0.6

0.8

1.0

I o/I

MPS-PPV (mM)

400 450 500 550 600 6500

1k

2k

B

MPS-PPV/Hoe

2P exc.

Fl C

ou

nts

Wavelength (nm)

0.0 0.1 0.2 0.3

0

4

8

12

I o/I

MPS-PPV (mM)

Figure 6.11: (A) One photon fluorescence spectrum of Hoe with increasing MPS-PPV


photon fluorescence spectrum of Hoe with increasing MPS-PPV concentrations.



The relative 2PA cross-sections were obtained for Hoe at different MPS-PPV

concentrations and shown in Figure 6.12. It is evident from the data that there is almost 50-fold

enhancement in 2PA cross-sections for Hoe with MPS-PPV, suggesting that Hoe is a very good

chromophore to monitor the local electric fields in MPS-PPV. This has something to do with

molecule’s polarizability.

6.3.2.2 Interactions of PSS with Hoe


Shown in Figure 6.13 are the optical absorption spectrum of Hoe as a function of PSS

concentration. Hoe blank sample has absorption maximum at 347 nm and shift to 364 nm when

it is bound to PSS. With an increase in PSS concentration, a gradual shift to 350 nm is observed,

indicating the interaction of Hoe with PSS even in its ground-state absorption spectrum. The

shift might have also arisen from overlapping absorption of the PSS at higher concentrations.

204

0.00 0.05 0.10 0.15 0.20 0.25 0.30

0

10

20

30

40

50

MPS-PPV/Heo

Re

l. 2

PA

cro

ss

-se

cti

on

MPS-PPV (mM)

Figure 6.12: Relative 2PA cross-sections of Hoe at different MPS-PPV concentrations.

350 400 4500.00

0.25

0.50

0.75

1.00

PSS/Hoe

Ab

so

rban

ce

Wavelength (nm)

Figure 6.13: Optical absorption spectrum of Hoe with increasing PSS concentrations.


Steady-state and two photon fluorescence measurements were carried out as a function of

increasing PSS concentrations for Hoe after excitation at 330 nm for one-photon and 800 nm for

two photon fluorescence. Corresponding fluorescence spectrum and normalized fluorescence at

205

482 nm is shown in Figure 6.14A. The normalized data at 482 nm for one-photon fluorescence

measurements with Hoe shows a fluctuating shift with increase MPS-PPV concentrations. At the

beginning Hoe blank sample maximum at 487 nm, then intensity decreased with shift to 530 nm.

Two-photon fluorescence measurements show interesting enhancement by 15 folds as shown in

Figure 6.14B.

350 400 450 500 550 6000

1k

2k

3k

4k

5k

6k

PSS/Hoe

Fl

Co

un

ts

Wavelength (nm)

0.0 0.2 0.40.0

0.5

1.0

1.5

2.0I o

/I

PSS (mM)

A

400 450 500 550 6000

1k

2k

PSS/Hoe2P exc.

2P

A C

ou

nts

Wavelength (nm)

B

0.0 0.2 0.4

0

5

10

15

20

I o/I

PSS (mM)

Figure 6.14: (A) One photon fluorescence spectrum of Hoe with increasing PSS concentrations.


spectrum of Hoe with increasing PSS concentrations. Inset showed the normalized



From the ratio of two-photon to one-photon fluorescence intensities, relative 2PA

cross-sections were carried-out for Hoe and plotted as a function of PSS concentration. The

normalized Relative 2PA cross-sections at 482 nm is shown in Figure 6.15. Relative 2PA cross-

sections showed 20-folds enhancement, which indicate the strength of interaction between the

Hoe and PSS. Here again, Hoe has shown greater 2PA cross-sections enhancement with

polyelectrolytes when compared to AcrO.

206

0.0 0.1 0.2 0.3 0.4

0

5

10

15

20

25

PSS/Hoe

Rel. 2

PA

cro

ss-s

ecti

on

PSS (mM)

Figure 6.15: Relative 2PA cross-sections of Hoe at different PSS concentrations.

6.3.3 Interactions of Polyelectrolytes with ThT

6.3.3.1 Interactions of MPS-PPV with ThT


Electronic absorption spectrum of ThT at different concentrations of MPS-PPV are

shown in Figure 6.16. The optical absorption spectrum show an enormous increase in the

absorbance with increase MPS-PPV concentrations; also there was a red shift in absorbance from

418 nm without MPS-PPV to 431 nm at higher MPS-PPV concentration. This change in

absorption can be correlated to the absorption of MPS-PPV at the same wavelength as that of

ThT.

207

300 400 500 600

0.0

0.1

0.2

0.3

0.4

0.5

MPS-PPV/ThT

Ab

so

rban

ce

Wavelength (nm)

Figure 6.16: Optical absorption spectrum of ThT with increasing MPS-PPV concentrations.


Fluorescence spectrum of ThT with increasing MPS-PPV concentrations were obtained

by exciting the samples at 400 nm and corresponding spectrum are shown in Figure 6.17A.

Normalized one-photon at 551 nm shows corresponding increasing in fluorescence intensity by

10 folds. In addition, there was a shift in fluorescence from 454 nm to 527 nm. Similar to one-

photon fluorescence, two-photon fluorescence measurements were carried out for ThT as a

function of MPS-PPV concentration after laser excitation at 800 nm as Figure 6.17B. At 551 nm.

Normalized two photon fluorescence shows a huge increase in fluorescence intensity by 100

folds with increasing MPS-PPV concentrations. Current results suggest that ThT binds strongly

to MPS-PPV.

208

450 500 550 600 650 700 750

20

40

60

80

MPS-PPV/ThT

Fl C

ou

nts

Wavelength (nm)

A

0.0 0.1 0.2 0.30

3

6

9

I 0/I

MPS-PPV (mM)

450 500 550 600 650 700 7500.0

2.0k

4.0k

6.0k

8.0k

MPS-PPV/ThT

2P exc.

2P

A C

ou

nts

Wavelength (nm)

0.0 0.1 0.2 0.30

30

60

90

I 0/I

MPS-PPV (mM)

B

Figure 6.17: (A) One photon fluorescence spectrum of ThT with increasing MPS-PPV

concentrations. Inset showed the normalized fluorescence data at 551 nm. (B) Two-photon

fluorescence spectrum of ThT with increasing MPS-PPV concentrations. Inset

showed the normalized fluorescence data at 551 nm.


Relative 2PA cross-sections were obtained from the ratio of two photon to one photon

fluorescence intensities; the corresponding cross-sections as a function of MPS-PPV

concentration is shown in Figure 6.18. The obtained result show 10-fold 2PA enhancements,

which confirm that ThT binds strongly with anionic MPS-PPV and its cross-sections in

influenced by the local electric field of the polyelectrolyte.

6.3.3.2 Interaction of PSS with ThT


Optical absorption spectrum of ThT with increasing PSS concentrations is shown in

Figure 6.19. With increase in PSS concentration, there was a red shift from 418 nm to 421 nm at

higher PSS concentration which is very marginal. The data suggest that there is no ground state

interactions of ThT with PSS.

209

0.0 0.1 0.2 0.30

4

8

12

MPS-PPV/ThT

Rel. 2

PA

cro

ss-s

ecti

on

MPS-PPV (mM)

Figure 6.18: Relative 2PA cross-sections of ThT at different MPS-PPV concentrations.

350 400 450 500 5500.0

0.1

0.2

0.3

PSS/ThT

Ab

so

rban

ce

Wavelength (nm)

Figure 6.19: Optical absorption spectrum of ThT with increasing PSS concentrations.


One-photon and two-photon fluorescence measurements were carried out as a function

of PSS concentrations with ThT after excitation at 400 nm for steady-state fluorescence and 800

210

nm excitation for two photon measurements as shown in Figure 6.20A and 6.20B respectively.

Steady-state fluorescence showed a blue shift from 495 nm to 479 nm with increase PSS

concentrations. Normalized one-photon and two-photon fluorescence spectrum of ThT was

plotted at 495 nm with increasing concentrations of PSS. Interestingly, both one-photon and two-

photon spectrum follow the same trend, where both start with a sharp increase at lower PSS

concentration then stay steady. In addition, both one-photon and two-photon intensity,

interestingly, have same enhancement by 7 folds.

450 500 550 600 6500

100

200A

PSS/ThT

Fl

Co

un

ts

Wavelength (nm)

0.0 0.2 0.4

0

5

10

I o/I

PSS (mM)

450 500 550 6000

500

1000

PSS/ThT

2P exc.

2P

A C

ou

nts

Wavelength (nm)

B

0.0 0.2 0.4

0

5

10

I o/I

PSS (mM)

Figure 6.20: (A) One photon fluorescence spectrum of ThT with increasing PSS concentrations.


spectrum of ThT with increasing PSS concentrations. Inset showed the normalized



Relative 2PA cross-sections enhancement was determined from the ratio of two-photon

to one-photon fluorescence and plotted as a function of PSS concentration, shown in Figure 6.21.

Interestingly there is no change in 2PA cross-sections for PSS with ThT indicating that the dye

does not bind to the electrostatic pocket.

211

0.0 0.1 0.2 0.3 0.4

1

2

3

PSS/ThT

Re

l. 2

PA

cro

ss

-se

cti

on

[PSS] (mM)

Figure 6.21: Relative 2PA cross-sections of ThT at different PSS concentrations.

6.3.4 Interactions with C-485

6.3.4.1 Interactions of MPS-PPV with C-485


As a control, we have studied the interaction of neutral dye C-485 binding to

polyelectrolytes via hydrophilic-hydrophobic interactions. Shown in Figure 6.22 are the

absorption spectrum of C-485 as a function of MPS-PPV concentration. C-485 has an absorption

maximum at 410 nm which shifted to 435 nm with an increase in MPS-PPV concentration and

this can be ascribed to overlapping absorption of MPS-PPV polyelectrolyte. The data shows no

ground state interaction between the dye and the polyelectrolyte.

212

300 400 500 600

0.0

0.1

0.2

0.3

0.4

MPS-PPV/C-485

Ab

so

rban

ce

Wavelength (nm)

Figure 6.22: Optical absorption spectrum of C-485 with increasing MPS-PPV concentration.


One- and two-photon fluorescence measurements were carried out as a function of

increasing MPS-PPV concentrations for C-485 after excitation at 400 nm for one-photon and 800

nm for two-photon as shown in Figure 6.23A and 6.23B respectively. A blue shift was observed

in one-photon fluorescence from 533 nm to 522 nm with increase MPS-PPV concentrations.

One- and two-photon fluorescence were normalized at 519 nm. Steady fluorescence

measurements with C-485 show increase in intensity in the beginning then show no sensitivity,

while two photon show a gradual increase.

213

450 500 550 600 6500

100

200

300

400MPS-PPV/C-485

Fl C

ou

nts

Wavelength (nm)

A

0.0 0.1 0.2 0.3

0.5

1.0

1.5

2.0

I o/I

MPS-PPV (mM)

450 500 550 600 6500

1k

2k

3k

4k MPS-PPV/C-485

2P exc.

2P

A C

ou

nts

Wavelength (nm)

B

0.0 0.1 0.2 0.3

1

2

3

4

I o/I

MPS-PPV (mM)

Figure 6.23: (A) One photon fluorescence spectrum of C-485 with increasing MPS-PPV

concentrations. Inset showed the normalized fluorescence data at 519 nm. (B) Two-photon

fluorescence spectrum of C-485 with increasing MPS-PPV concentrations. Inset

showed the normalized fluorescence data at 519 nm.


Relative 2PAcross-sections were determined for C-485 and plotted as a function of MPS-

PPV concentration. The normalized Relative 2PA cross-sections at 519 nm, as shown in Figure

6.24. Relative 2PA cross-sections show marginal increase pointing to the fact that the neutral dye

was unable to solubilize in the area where there are significant local electric fields.

6.3.4.2 Interactions of PSS with C-485


Optical absorption spectrum C-485 at room with increasing PSS concentration is shown

in Figure 6.25. The maximum absorbance peak recorded at 405 nm, and there was no shift in

absorbance spectrum with increasing PSS concentration. This result suggests no ground state

interaction between the dye and polyelectrolyte.

214

0.00 0.05 0.10 0.15 0.20 0.25 0.300.0

0.5

1.0

1.5

2.0

2.5

3.0

MPS-PPV/C-485

Re

l. 2

PA

cro

ss

-se

cti

on

[MPS-PPV ] (mM)

Figure 6.24: Relative 2PA cross-sections of C-485 at different MPS-PPV concentrations.

350 400 450 500 5500.0

0.1

0.2

PSS/C485

Ab

so

rban

ce

Wavelength (nm)

Figure 6.25: Optical absorption spectrum of C-485 with increasing PSS concentration.


One- and two-photon fluorescence measurements were carried were carried o for C-485

as a function of PSS concentration, after excitation at 400 nm for one-photon and 800 nm for

215

two-photon and normalized fluorescence intensity at 519 nm, as shown in Figure 6.26A and

6.26B respectively. In fluorescence steady state measurement, there was a blue shift from 520

nm to 513 nm. Both one- and two-photon fluorescence were normalized at 519 nm; interestingly,

the increase in fluorescence intensity was found to be minimal suggesting weaker interaction

between the dye polyelectrolyte.

450 500 550 600 6500

100

200

300

400

PSS/C-485

Fl

Co

un

ts

Wavelength (nm)

0.0 0.2 0.4

1.0

1.5

2.0

I o/I

PSS (mM)

A

450 500 550 600 6500

1000

2000

3000B

PSS/C-485

2P exc.

2P

A C

ou

nts

Wavelength (nm)

0.0 0.2 0.4

1.0

1.2

1.4

I o/I

PSS (mM)

Figure 6.26: (A) One photon fluorescence spectrum of C-485 with increasing PSS


photon fluorescence spectrum of C-485 with increasing PSS concentrations.



Relative 2PA cross-sections were obtained by taking the ratio of two-photon fluorescence

to one-photon fluorescence. 2PA cross-sections were plotted for C-485 as a function of PSS

concentration. The normalized relative 2PA cross-sections at 519 nm shown in Figure 6.27.

Similar to one and two photon fluorescence results, relative 2PA cross-sections show little to no

enhancement.

216

0.0 0.1 0.2 0.3 0.40

1

2

3

PSS/C485

Rel. 2

PA

cro

ss-s

ecti

on

[PSS]

Figure 6.27: Relative 2PA cross-sections of C-485 at different MPS-PPV concentrations.

6.3.5 Comparison of 2PA Cross-Sections

The Main objective of this chapter is to establish 2PA spectroscopy as a tool to study

self-assemblies system such as polyelectrolytes. To achieve this objective, relative 2PA cross-

sections were carried out for all investigated dyes (AcrO, Hoe, ThT and C-485). AcrO, Hoe, ThT

are cationic dyes while C-485 is a neutral dye. The comparative relative 2PA cross-sections

enhancement for all dyes as a function of MPS-PPV and PSS concentrations are present in

Figure 6.28A and 6.28B for MPS-PPV and PSS respectively. One- and two-photon

measurements showed that all dyes are bound strongly with MPS-PPV and PSS. It is observed

from the Figure that cationic dye molecules have increased in 2PA cross-sections significantly

with both MPS-PPV and PSS while no change on the 2PA cross-sections enhancement is

observed with neutral dye molecule, which goes back to no electrostatic environment.

The enhancement was different for each chromophore, which relates to chromophore

polarizability, which is attributed to charge transfer from chromophores to the MPS-PPV and

217

PSS systems. When the chromophore are affected by external electric field E, this will alert the

electron which generate a dipole moment. The induced dipole moment ind is related to the

polarizability of chromophore and the strength of the electric field E and given by the following

equation

𝜇𝑖𝑛𝑑 = 𝛼𝐸 6.1

where is is the polarizability coefficient constants. If there is a different between chromophore

polarizabilities ground g and excited e states, that will make the change in the induced dipole

moment μgeind more sensitive and given with the following equation

∆𝜇𝑔𝑒 = ∆𝜇𝑔𝑒0 + 0.5∆𝜇𝑔𝑒

𝑖𝑛𝑑 6.2

From the equation 6.1 and 6.2

∆𝜇𝑔𝑒 = ∆𝜇𝑔𝑒0 + 0.5 (Δ𝛼𝑒−𝑔 )𝐸 6.3

This equation explains how the total change of dipole moment ∆𝜇𝑔𝑒 is very sensitive to the local

surface electric field (E) around the chromophores and enhancing 2PA cross-sections of

chromophore based on the different polarizability for each chromophore.

Hoe achieves the highest enhancement in both MPS-PPV and PSS by 50- fold and 20-

fold respectively. ThT give a good enhancement as 10-fold for MPS-PPV in 2PA cross-sections;

however, interestingly its show no change with PSS due to the transition dipole vector of the

ThT orientation with the PSS backbone. The relative 2PA cross-sections for AcrO show a slight

enhancement of 2-fold for both MPS-PPV and PSS, which can be attributed to how AcrO are

bound to both systems which affects the dipole moment of the molecule. In contrast, there were

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no enhancement for 2PA cross-sections of neutral C-485 dye for MPS-PPV and PSS systems,

due to insufficient amphiphilic interactions between MPS-PPV and PSS and neutral C-485

chromophores. It is noted that there is an influence of drug charge type, which will lead to

change the way that MPS-PPV and PSS interact with the dye. As both MPS-PPV and PSS are

anionic polyelectrolyte, they interact significant with cationic drugs and enhance the relative

2PA cross-sections compared to no enhancement in neutral dye.

0.00 0.05 0.10 0.15 0.20 0.25 0.30

0

10

20

30

40

50A

Re

l. 2

PA

cro

ss

-se

cti

on

MPS-PPV (mM)

AcrO-MPS-PPV Hoe-MPS-PPV

ThT-MPS-PPV

C-485-MPS-PPV

0.0 0.1 0.2 0.3 0.4

0

5

10

15

20

25 AcrO-PSS

Hoe-PSS

ThT-PSS

C-485-PSS

Re

l. 2

PA

cro

ss

-se

cti

on

PSS (mM)

B

Figure 6.28: Comparison relative 2PA cross-sections enhancement for AcrO, Hoe, ThT and C-

485 drugs with (A) MPS-PPV (B) PSS.

6.4 Conclusion

Due to polyelectrolytes physics/biophysics properties, there is an increasing interest to

investigate their properties and interactions. One of the main characteristics of polyelectrolytes is

their extreme sensitivity to changes in their environment. Polyelectrolytes offer one such

possibility where changing folding/unfolding conditions can alter the electric fields. In this

chapter, we aim to study the 2PA cross-sections of chromophores in polyelectrolytes in an effort

to probe the folding and unfolding of polyelectrolytes. The investigations are carried out with

cationic (Acridine Orange, Hoechst 33258, Thioflavin T) and neutral (Coumarin 485) dye

molecules interact with two anionic polyelectrolytes (Poly (sodium 4-styrenesulfonate) PSS, and

219

Poly [5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene]) MPS-PPV. These systems are

chosen specifically to understand the influence of electrostatic interactions and in turn on the

electric fields on the 2PA cross-sections of chromophores. Our investigation focuses on the study

of MPS-PPV and PSS behavior with different dyes by monitoring the electric fields through

changing folding/unfolding conditions in polymer. Our approach is to use two-photon absorption

(2PA) cross-sections of chromophores as markers to study chromophore polyelectrolytes

behavior with different dyes, such ability to drug based on the orientation of the electric field

vector, if it is parallel to chromophore electrical field, there will be enhancement in 2PA cross-

sections. But no change on 2PA cross-sections will record if the transition dipole vector is

perpendicular to chromophore electrical field.

Investigated cationic dye molecules bound effectively with negatively charge

polyelectrolytes as evidenced from optical absorption and fluorescence measurements. 2PA

cross-sections of cationic dye molecules have increased significantly with both PSS and MPS-

PPV while no change in 2PA cross-sections was observed with neutral dye molecule. This is

assigned to the effect of electric field on the 2PA cross-sections. In the cationic dyes,

enhancement as high as 50-fold was observed with Hoe for MPS-PPV and 20-folds enhancement

for PSS . In ThT there was an enhancement by 10-fold for MPS-PPV while only 2-fold

enhancement was observed for AcrO in both MPS-PPV and PSS. This is assigned to the strength

of interaction between the dye molecules and polyelectrolytes. Whereas in neutral dye (C-485)

there was no enhancement in both MPS-PPV and PSS. The results shed lights on the role of the

electrostatic interactions in enhancing the nonlinear optical properties of chromophores.

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6.5 Chapter Summary

• For this investigation two anionic polyelectrolytes, Poly [5-methoxy-2-(3-

sulfopropoxy)-1,4-phenylenevinylene]) MPS-PPV and (Poly (sodium 4-

styrenesulfonate) PSS were chosen to study the influence of electrostatic interactions

in turn the electric fields on the 2PA cross-sections of chromophores.

• The investigations are carried out with cationic (Acridine Orange, Hoechst 33258,

Thioflavin T) and neutral (Coumarin 485).

• Investigated cationic dye molecules bound effectively with negatively charge

polyelectrolytes as evidenced from optical absorption and fluorescence

measurements.

• 2PA cross-sections of cationic dye molecules has increased significantly with both

PSS and MPS-PPV while little change in 2PA cross-sections was observed with

neutral dye molecule. This is assigned to the effect of electric field on the 2PA cross-

sections.

• Enhancement as high as 40-fold was observed with Hoe while only 2-fold

enhancement was observed for AcrO. This is assigned to the strength of interaction

between the dye molecules and polyelectrolytes.

• In case of neutral dye (C-485), no enhancement were observed in both MPS-PPV and

PSS.

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CHAPTER 7

OVERALL SUMMARY AND FUTURE OUTLOOK

7.1 Overall Summary

A detailed summary of each study followed by a future outlook is provided in this

Chapter. The main goal in this dissertation is to use the power of two-photon spectroscopy to

understand the organized self-assemblies that are integral for several biological processes and

applications in interdisciplinary areas of sciences. The hypothesis is that the local electric fields

in organized self-assemblies can change the two-photon absorption (2PA) cross-sections of

chromophores stabilized in those environments and thereby providing a window to glean the

micro-environments as well as follow the organized self-assemblies.

In the first Chapter, a detailed introduction to several naturally available organized self-

assemblies, as well as synthetic organized self-assemblies, is provided. The need to monitor the

local environments in the self-assemblies is described, followed by the techniques that are

currently in use to monitor these organized self-assemblies. This led to understanding of the gap

in the field and the necessity to develop novel spectroscopic techniques to monitor the organized

self-assemblies, which is the main motivation behind the work carried out in this dissertation.

Chapter 2 covers different experimental techniques that were used to complete

experimental measurements presented in this dissertation. The analytical experimental

techniques that were carried out for this dissertation were optical absorbance, steady-state

fluorescence, time-resolved fluorescence, two-photon excited fluorescence to measure 2PA

cross-sections, dynamic light scattering to measure practical size and circular dichroism to

identify the secondary structure. A brief description of each of the experimental techniques is

provided.

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In Chapter 3, the stability of the non-canonical structures of DNA, G-Quadruplex and G-

Triplex with different drugs was probed using the one- and two-photon fluorescence of the drugs

bound to these DNA structures. In the process of evaluating the stability of these DNA

structures, we established a new spectroscopic technique based on relative two-photon

absorption to monitor drug-DNA binding interactions as well as understanding the non-canonical

structures. For these investigations, two DNA oligonucleotides, were selected due to their ability

to form a stable Quadruplex and Triplex DNA, named TBA and TBA11, respectively. For these

studies, temperature-dependent UV-Vis, temperature-dependent one-photon fluorescence and

temperature-dependent 2-photon fluorescence, Circular Dichroism and relative 2-photon cross-

sections were carried out with different small molecules. ThT, ThO, DAPI, TMPyP4, EtBr and

DoXo are the chosen drugs to investigate the binding and stability for both DNA structures. The

drugs used in these systems have been known to bind with DNA using hydrogen bonding, pi-pi

stacking interactions, and/or the electrostatic interaction to backbone of DNA. Interesting

stability trends were obtained for the drugs which correlated with drug structure and DNA

binding modes. 2PA cross-sections changes were observed for ThT and ThO when the DNA

structures were melted using temperature and they seem to stabilize these non-canonical

structures that can be assigned to the molecular structure of the drugs. Although TMPyP4

stabilized both structures, the trend in 2PA cross-sections enhancement was an increase with G-

Triplex while decreased with G-Quadruplex, suggesting that the mode of binding of this drug

with DNA structures is different. DAPI was found to be the least efficient drug to stabilize G-

Quadruplex and G-Triplex structures. Both EtBr and DoXo stabilized only the G- Triplex

structure. From this study, we were able to show that the 2PA cross-section of the drug

molecules is a viable technique to monitor DNA melting transitions as well as probe the

230

stabilization of the non-canonical structures of the DNA with drug molecules. This can be

ascribed to specific electric field orientation changes when the DNA melts that can be monitored

with relative 2PA cross-sections of attached drug molecules. This study has also shown that this

technique can be further used to monitor other organized self-assemblies.

Having found a novel technique to study organized self-assemblies, we have focused on

applying this spectroscopic technique to study another organized assembly, which is proteins and

especially the investigations centered on protein folding/unfolding and aggregation. Protein

misfolding and formation of soluble aggregates were found to be the common pathways for

numerous neurodegenerative diseases such as Alzhimers, Parkinsons and ALS. In this study, we

have focused on a model protein (Bovine serum albumin, BSA) and a protein implicated in ALS,

superoxide dismutase (SOD1). For this study, we have covalently attached Fluram to both BSA

and SOD1 and monitored protein folding/unfolding as a function of temperature using relative

2PA technique. The results show that relative 2PA cross-sections of the attached dye is a good

way to probe the folding/unfolding of the proteins as it can probe the local electric fields in

organized assemblies that can be altered with unfolding. To study the aggregation of BSA and

SOD1, both proteins were softened by adding 1 M GuHCl and changed the temperature to cause

aggregation. The aggregation was followed with UV-Vis absorption, one-photon and two-photon

fluorescence measurements as well as dynamic light scattering. From the ratio of two-photon to

one-photon fluorescence, we have determined the relative 2PA cross-sections of Fluram and the

trend was able to show different aggregates forming at different temperatures, which was support

with DLS to determined different aggregates size. Our results proved that relative 2PA cross-

sections is a valuable analytical tool to monitor and probe folding and aggregation.

231

From the previous studies, we have shown that 2PA spectroscopy can be used to monitor

the local environment in organized self-assemblies. We applied this technique to develop a two-

photon based biosensor for the detection of toxic metal ions such as Hg2+ and Pb2+. The

biosensor is based on the G-Quadruplex DNA structures which are sensitive to Hg2+ and Pb2+

and we used the power of relative 2PA cross-sections to detect trace amounts of toxic metal ions

in water. Two-photon sensing has its unique advantages as it can be used in complex

environments and for on-site detection, since the wavelength of excitation is in the near-infrared

region with low scattering. Thus, we propose a one-photon and two-photon fluorescence-based

biosensors that rely upon specific oligonucleotide T30695, which is known to show selectivity

for mercury and lead. After coupling T30695 with DTI fluorophore, a stable G-Quadruplex at

room temperature was formed with K+. When Pb2+ and Hg2+ interact with T30695 G-Quadruplex

DNA structure, K+ ion is selectively replaced by Pb2+ and Hg2+ with quenching of the

fluorescence of DTI as the heavy metal ions promote efficient intersystem crossing and the

quenching can be correlated to Pb2+ and Hg2+ concentrations. Circular dichroism measurements

have shown that a stable G-Quadruplex is formed with the metal ions. The oligonucleotide

showed a good sensitivity to Pb2+ through both one-photon (12 ppb) and better sensitivity with

two-photon fluorescence (4.5 ppb). While, Hg2+ showed no sensitivity under one-photon

excitation, but two-photon have a good sensitivity (5 ppb). The relative 2PA cross-sections of T-

30695/DTI shows good sensitivity for mercury and lead. In addition, the selectivity was found to

be good and it can be ascribed to the selectivity of T-30695 towards binding to heavy metal ions.

In conclusion, these results shown the power of 2PA spectroscopy for metal ion sensing.

Having established 2PA spectroscopy to monitor organized self-assemblies, especially

those biological in nature, we have studied their use in understanding synthetic analogues like

232

polyelectrolytes. Previous study from our group has shown that the 2PA cross-sections of

chromophores were enhanced when they are solubilized in micelles because of electrostatic

interactions. In this study, we have studied the 2PA cross-sections of chromophores solubilized

in polyelectrolytes. The idea is that polyelectrolytes are similar to DNA backbone and can

provide local electric fields that can be used to enhance the 2PA cross-sections of chromophores

when appropriate dyes are solubilized in those environments. The investigations were carried out

on two anionic polyelectrolytes, MPS-PPV and PSS. Several cationic dyes were chosen to

interact with the polyelectrolytes: namely, Acridine orange, ThT, Hoechst 33258. As a control,

investigations were also carried out with a neutral dye, C485 interacting with anionic

polyelectrolytes. The local electrostatic environment in polyelectrolytes can enhance the 2PA

cross-sections of chromophores. UV/Vis absorption, steady-state fluorescence and two-photon

fluorescence measurements were carried out on all the dye molecules with increasing

concentrations of polyelectrolytes. In addition, relative 2PA cross-sections were determined from

the ratio of one and two-photon fluorescence for all dyes with increasing concentrations of

polyelectrolytes. The results have shown that the cationic dyes bind strongly with both MPS-

PPV and PSS with negligible ground state interactions. But fluorescence enhancement was

observed for ThT when it was bound to MPS-PPV and PSS. Relative 2PA cross-sections

measurements have shown interesting trends wherein close to 50-fold 2PA enhancement was

observed for Hoechst with MPS-PPV and 20-fold with PSS. ThT also has shown enhancement

reaching 10-fold with MPS-PPV while 2-fold enhancement was observed for AcrO with

polyelectrolytes. Very small to negligible 2PA enhancement was observed when C485 interacted

with polyelectrolytes suggesting weaker binding of the dye with polyelectrolytes. The results are

rationalized based on differences in the polarizabilities of chromophores, where the chromophore

233

with greater change in polarizability has shown maximum 2PA enhancement. This study has

provided a framework for the design and development of chromophores that can show maximum

2PA enhancement with local electric fields.

Overall in the work carried out for the dissertation, a novel spectroscopic tool based on

relative 2PA cross-sections was developed to probe the local electrostatic fields in organized self-

assemblies. The 2PA cross-sections have changed with changing in microenvironment around self-

assembled materials. As the 2PA cross sections is proportional to the square of change in

permanent dipole moments and the change in permanent dipole moment is proportional to local

electric fields, they were able to probe the local environments in organized self-assemblies.

7.2 Future Outlook

The work carried out in the dissertation has shown the potential of 2PA spectroscopy to

understand organized self-assemblies with some examples. However, several biological important

organized self-assemblies are present that include vesicles, cell membranes and lipid bilayers.

Future studies can shed light on the use of this technique to probe them, and the holy grail of this

project is to develop a novel 2PA based imaging technique that will be powerful to probe the local

electric fields in organized self-assemblies with improved selectivity and sensitivity. This research

can be further extended to develop novel diagnostic tools as well as better sensors that can be work

in complex environments.

Documents

Using Novel Spectroscopy Tool to Study Organized Self