Detection of Cystic Fibrosis Transmembrane Conductance ......Karan Malhotra Master of Science Department of Chemistry University of Toronto 2018 Abstract Diagnostic technology that

Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic

Acid Hybridization Assay and a Smartphone Camera

by

Karan Malhotra

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

copy Copyright by Karan Malhotra 2018

ii



Karan Malhotra

Master of Science

Department of Chemistry

University of Toronto

2018

Abstract

Diagnostic technology that utilizes paper substrates and device cameras offers opportunities for

development of cost effective point-of-care technologies The translation of assays operating in

aqueous solution require further development for implementation in paper substrates This report

presents and compares two methods for determination of oligonucleotides that serve as indicators

of Cystic Fibrosis differentiating wild type and mutant type sequences containing a 3-base

deletion The transduction strategy operates by selective hybridization of dye-labelled

oligonucleotides (target or reporters) to capture probes immobilized on quantum dots and

hybridization results in emission of dyes via resonance energy transfer Detection is based on

hybridization of fluorophore labelled target or hybridization of unlabelled target and labelled

reporter in a sandwich assay format Selectivity to determine mismatched sequences required

control of stringency conditions using formamide as a chaotrope It was determined that both

formats can distinguish between wild type and mutant type samples on paper substrates

iii

Acknowledgments

I would like to begin by expressing my gratitude to Professor Ulrich J Krull for his guidance and

mentorship throughout my graduate career I am privileged to have worked in his lab with other

motivated and driven graduate students I have learned a lot at my time in the Chemical Sensors

Group (CSG) and I will always cherish my experience and memories here I am also grateful to

Professor Aaron R Wheeler who has graciously agreed to be the second reader for this thesis I

would also like to acknowledge Professor Paul A E Piunno with whom I have had many

conversations about research graduate work and judo Special thanks are also extended to Dr M

Omair Noor for his mentorship I have learned a lot about research from him that I would have

never have learned otherwise

I am grateful to numerous members of the University of Toronto Community for their help

Members of the UTM stores Microelectronics Academic workshop are sincerely thanked for their

support I would also like to thank the administrative staff at the UTM campus for their support

during my graduate work including Carmen Bryson Jessica Bailey Michelle Bae Christina M

Fortes and Roxana Moreira-Diaz

Much of the work in this thesis would not have been possible without the support of members from

the Department of Chemical and Physical Science and CSG I am forever grateful to Dr Abootaleb

Sedighi Dr Samer Doughan Dr Peter Mitrakos Dr Sreekumair Nair Dr Thottackad

Radhakrishnan Anna Shahmuradyan Yi Han Phillip Rolo Hifza Najib Muhammad Shahrukh

David Hrovat Richard Fuku Hamna Fayyaz and Alex Escobar for their support

Lastly I would like to acknowledge my family and friends for their continued support I would

like to express my gratitude to my sister brother-in-law and baby niece for always making life

enjoyable I would also like to thank my girlfriend for her continued support throughout my time

in grad school Finally none of this would have been possible without the sacrifice and

encouragement from my parents I am truly blessed to have you as my role models

iv

Table of Contents

Acknowledgments iii

Table of Contents iv

List of Tables vi

List of Figures vii

Chapter 1 1

Introduction 1

11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein 1

111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508

mutation of CFTR Gene1

12 Nucleic Acids and Oligonucleotide Detection 3

121 Structure and Composition of DNA Hybridization 4

122 Thermodynamics of DNA Hybridization 5

123 Notes and Considerations for POC Application 7

13 Quantum dots 8

131 Quantum Confinement and The Particle in a Box 10

14 Fluorescence and Resonance Energy Transfer 11

141 Fluorescence Resonance Energy Transfer (FRET)11

15 Paper Based Analytical Devices 14

151 Paper Substrates for Sensing Technology Overview 15

152 Cellulose Modification and Smartphone-based Detection 15

16 Thesis Objectives and Contributions 17

Chapter 2 19

Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation

Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera 19

21 Experimental 19

211 Methods20

v

212 Instrumentation 24

22 Results and Discussion 25

221 FRET Pair Characterization (gQD ndash Cy3) 25

222 Oligonucleotide Hybridization in Solution 26

223 Oligonucleotide Hybridization in Paper Substrates 28

224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by

Smartphone Imaging 32

225 Analytical Figures of Merit 38

226 Selectivity for Mixtures of WT and MT Targets 40

227 Paper-based Assay Response for Complex Sample Matrices 42

228 Blind Assay for Detection and Quantification of CFTR Target Mixes 43

Chapter 3 45

Conclusion and Future Work 45

31 Future Directions 46

References 47

vi

List of Tables

Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF 2

Table 2 Oligonucleotide Sequences used in Hybridization Assays 20

Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids 34

Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids 34

Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids 34

Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids 34

Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids 36

Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids 36

Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids 36

Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids 37

Table 11 Analytical Performance Direct and Sandwich Bioassays 40

Table 12 Blind Assay for Direct and Sandwich Assays 44

vii

List of Figures

Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and Crick

Arrows on the ribbons represent the directionality bias for the single strands and dimensions for

the polymer are presented with one turn of the helix every 34 nm the distance between base pairs

every 034 nm and the distance between the phosphate backbone and the central axis every 1 nm

B shows the hydrogen bonding taking place between complementary pairs of nucleobases as

proposed by Chargaff with adenine (A) having two hydrogen bonds with thymine (T) and guanine

(G) having three hydrogen bonds with cytosine (C) Image was adapted with permission

Copyright Nature Education 201331 5

Figure 2A Representation of the core-shell model of quantum dots with corresponding high-

resolution TEM image Here core material is composed of CdSe and shell is composed of ZnS

B Quantum dots of different colors are presented with their corresponding core size image of

solution and photoluminescence spectra and color C Diagram representing the quantum

confinement and the change in band gap energy as material size decreases below the Bohr-exciton

radius Here CB and VB represent the conduction and valence band respectively and Eg represent

the band gap energies Image adapted with permission Copyright 2011 American Chemical

Society60 9

Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of colloidally

stable and spherical QD (green) with multiple FRET acceptors (yellow) (b) Change in FRET

efficiency based on changes in the distance between donor and acceptor (c) QD (green)

immobilized on a surface can interact with multiple FRET acceptors by interacting with adjacent

acceptors Image acquired with permission from Algar et al70 Copyright Elsevier 2010 12

Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in blue)

are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)

functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3 on

the proximal end and upon hybridization is brought to proximity with gQDs allowing for FRET

to take place (B) In sandwich assay format the probe strand hybridizes with the target strand (seen

in red) such that there is an overhang on the distal end Reporter strand (seen in green) hybridizes

with the overhang region of the target strand bringing to proximity the Cy3 label on the proximal

end of the reporter 14

viii

Figure 5 Chemical modification of cellulose via periodate mediated oxidation yields aldehyde

functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society of

Chemistry 2016 16

Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A) Reaction

zones consisted of chemically modified paper that were conjugated with gQD-oligonucleotide

probes Zones contained WT and MT controls and test zones where unknown samples were

spotted and imaged Detection was based on the principle of RET with gQDs used as donors and

Cy3 labels on oligonucleotide strands as acceptors (B) Imaging used a smartphone camera with

data processing by ImageJ to split the image to RGB color channels 18

Figure 7 Image of buffer solution leakage from hydrophilic paper zones 23

Figure 8 Normalized absorption and emission spectra for the gQD and Cy3 FRET pair The

spectral overlap is represented by the shaded region Absorption is shown as dashed lines and

emission is shown as solid lines 26

Figure 9 Hybridization of the gQD-probe strands was investigated in solution by fluorescence

spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii) CFTR single

DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT target strands The

concentration-response curves for the different gQD-probe conjugates are shown A WT Cy3

labelled target strands are seen in blue and MT Cy3 labelled target strands are seen in orange

Normalized PL spectra for the calibration curves are shown for B) CFTR WT Cy3 labelled target

strands and C) CFTR MT Cy3 labelled target strands ( indicates increasing target concentration)

27

Figure 10 Representations of the two different direct assay formats investigated in solution phase

gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA MT probe

and were mixed with complementary CFTR WT Cy3 target strands and CFTR MT Cy3 target

strands Hybridization resulted in proximity of gQDs and Cy3 which resulted in FRET 28

Figure 11 Digital smartphone image and the accompanying post-processing PL images (post

processing included R-G-B color splitting yielding pseudocolored images) gQD-WT probe

conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of Cy3

labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol (vii) 75

ix

pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of spots that

may not be visible otherwise 29

Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers to

WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target

(D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and Tm) were calculated

using the nearest neighbor method3839 30

Figure 13 The various probe-target conjugates explored for the sandwich assay format (A) refers

to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash MT target



Figure 14 Determination of optimal wash conditions for direct and sandwich assay considered

RG Ratios with variation of formamide concentration for wash times of 0 5 10 15 and 20 min

The optimal wash conditions for direct assay was found to be BB+10F for 5 minutes for (A)

gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal wash conditions for

sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-WT probe sequence and

BB+5F for 20 minutes for (D) gQD-MT probe sequence 38

Figure 15 Concentration-response curves showing the RG ratiometric response of the direct and

sandwich assay formats (Ai) gQD-WT probe conjugates were used for determination of Cy3

labelled WT targets and (Bi) gQD-MT probe conjugates were used for determination of Cy3

labelled MT targets (Ci) gQD-WT probe conjugates were used for determination of unlabelled

WT targets with Cy3 labelled reporters and (Di) gQD-MT probe conjugates were used for

determination of unlabelled MT targets with Cy3 labelled reporters The RG ratiometric response

of the direct assay at the low pmol concentration range was also determined (Aii) gQD-WT probe

conjugates and (Bii) gQD-MT probe conjugates The sandwich assay format (Cii) gQD-WT probe

conjugates and (Dii) gQD-MT probe conjugates Note that the scale for (A) and (B) is logarithmic

Each error bar represents one standard deviation for n=4 replicates 39

Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes and

(Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined using

background corrected RG ratio plots for hybridization of gQD-probe conjugates with Cy3 labelled

x

targets (for direct assay A and B) and gQD-probe conjugates with unlabeled targets and Cy3

labelled reporter sequences (for sandwich assay C and D) Response of the hybridization assay

was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-wash (Bi and Di) MT

probe conjugates Post-wash assays yielded signal response shown in Aii and Cii for WT probe

conjugates and in Bii and Dii for MT probe conjugates Error bars represent one standard deviation

for n = 4 replicates 41

Figure 17 Background corrected RG ratios for hybridization of gQD-probe conjugates in

complex matrices Direct assays used 24 pmol of CFTR Cy3 TGTs in either BBS GS SS or

BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates and

(B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to direct assay

and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was collected for (C)

gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars represent one standard

deviation for n = 4 replicates 42

1

Chapter 1

Introduction

11 Cystic Fibrosis and Genes Associated with Cystic Fibrosis Transmembrane Protein

Cystic fibrosis (CF) is a multi-system fatal autosomal recessive disorder that is

characterized by viscous secretions in the lungs of patients due to mutations in cystic fibrosis

transmembrane conductance regulator protein (CFTR) CF affects 1 in 3000 births with ~70000

people affected worldwide1ndash5 Over 1500 mutations for the CFTR protein have been found but few

are common and fewer result in the disease Of the few mutations responsible for the disease state

the deletion of phenylalanine at the 508 position (∆F508) is responsible for over two-thirds of the

cases while all other mutations account for no more than 5 of the cases individually256

Development of sensing technology for early detection of ∆F508 would serve to enable improved

screening by clinicians to identify the predominant gene carriers The strategies for diagnosing CF

are based on newborn screening (NBS) programs that work via screening for serum markers

including the immunoreactive trypsinogen (IRT) assay7ndash9 This assay is typically followed by

diagnosis of the genetic basis of disease including detection of ∆F508 and related mutations based

on determining the presence of specific oligonucleotide sequences Finally a sweat chloride test

is performed to diagnose patients with CF All of these techniques require skilled technicians to

process samples perform and analyse tests via resource-intensive technologies10 The aim of this

work is to contribute to the development of a low cost easy to use and portable method for sensing

CFTR ∆F508 gene mutations beginning with a focus on a suitable transduction strategy

111 Nucleic Acids based Detection of Genes Associated with CF and ∆F508 mutation of CFTR Gene

There are multiple strategies for transducing the presence of genes associated with CF and

some of the technologies that have been approved by the United Stated Food and Drug

Administration (FDA) for use as in-vitro medical devices are presented in Table 1 (accessed Feb

20th 2018)11

2

Table 1 Summary of FDA approved Nucleic Acid Based Tests for Diagnosis of CF

Manufacturer Trade Name Detection Strategy

Illumina Inc Illumina MiSeqDx Cystic

Fibrosis Clinical Sequencing

Assay

Next-gen sequencing by

synthesis

Illumina MiSeqDx Cystic

Fibrosis 139-Variant Assay

Luminex Molecular

Diagnostics Inc

xTAG Cystic Fibrosis 60 kit v2 Microbead-dye barcode

coupled microarray analysis xTAG Cystic Fibrosis 39 kit v2

Osmetech Molecular

Diagnostics

eSensor CF Genotyping Test Sandwich hybridization assay

with ferrocene tag for cyclic

voltammetry analysis

Nanosphere Inc Verigene CFTR and Verigene

CFTR PolyT Nucleic Acid Tests

Genomic amplification

followed by sandwich assay

with probes and gold

nanoparticle reporters for

analysis

Third Wave Technology Inc InPlex CF Molecular Test PCR coupled with FRET based

microwell plate

Celera Diagnostics Cystic Fibrosis Genotyping

Assay

PCR coupled with capillary

electrophoresis and

oligonucleotide ligation assay

Typically these technologies require the use of specialized facilities and dedicated

technicians for analysis of patient samples and confirmation of CF may take up to a few weeks79

The resources and time required to diagnose patients may be reduced through the development of

point-of-care (POC) devices In particular the use of paper-based test strips with smartphone

detection for on-site rapid screening of disease markers would serve to alleviate the burden placed

on the health care system by more expensive techniques12

At the core of POC technology is the transduction strategy and much effort has gone into

developing optical13 and electrochemical methods14 for generating and measuring signal Yet the

application of this technology has not been investigated for selective sensing of similar nucleic

acid sequences that are often found to be associated with the genetic basis of disease Thus to

further discuss the challenges in this field it is important to address some of the background

technology that has been developed for POC sensors In particular this chapter will discuss nucleic

acid detection and the thermodynamics associated with hybridization interactions the use of

3

formamide as a chaotrope for controlling hybridization the use of nanomaterials like quantum dots

as integrated components in the bioassays for fluorescence resonance energy transfer-based

sensing strategies and the application of paper as a platform and substrate for sensing

12 Nucleic Acids and Oligonucleotide Detection

Deoxyribonucleic acid (DNA) is a class of biopolymers that stores hereditary information

and ribonucleic acid (RNA) functions as the set of instructions for synthesis of proteins15 The two-

step process by which the DNA nucleobase sequence is transcribed for production of RNA and

subsequently RNA is used as a template for translation to produce proteins is referred to as the

central dogma of molecular biology16 Proteins carry out the function that is encoded in the genetic

regions of DNA by interacting with other molecules and biopolymers present within and on the

surface of cells The specific interactions that govern the proteinsrsquo function are due to the three-

dimensional structure of the amino acid sequence that composes proteins17 The order of amino

acids which composes proteins is based on the nucleobase sequence of transcribing RNA (and

therefore DNA) Thus hereditary information stored as the base sequence of DNA can govern the

sequence of amino acids and therefore the structure and function of proteins1617 There are

numerous types of diseases that have arisen due to nucleobase-pair mutations in the sequence of

gene coding regions of DNA18 Mutations of DNA bases influence the amino acid sequence that

compose proteins and a three-base pair deletion like the one found for phenylalanine at codon 508

significantly alters the function of the protein associated with the CFTR gene Other types of

genetic diseases also arise due to mutations of the base pair sequence associated with DNA and

strategies for detection of nucleic acid mutations offer a method to detect the presence of a disease

state

To determine the genetic basis of disease for guiding clinical treatment diagnostic

technology for sensing nucleic acids must be further developed The main goal of clinical

diagnostic technology is to determine the molecular basis of disease for guiding patient therapy

because knowledge obtained from diagnostics are paramount for programing treatment strategies

Clinical diagnostics using a POC strategy offers improved opportunity for wide-scale screening

due to the advantages of low cost ease of manufacturing ease of transport ease of use and ease

of disposal19 One approach to the detection of genetic materials (deoxyribonucleic acid) is based

4

on hybridization and this process requires consideration of the chemical composition structure

and thermodynamics associated with hybridization

121 Structure and Composition of DNA Hybridization

Elucidation of DNArsquos structure and function has a long-storied history that has impacted

many fields of research including chemistry biology and medicine Much of the early work

related to DNA was focused on the structure of DNA with scientists focusing on the key details

related to the chemical composition of the monomers and the structural format of the polymeric

structure of DNA20ndash24 The key findings of the structure of DNA are summarized as follows

1 The structure for the DNA salt is composed of two helical polymer chains that are

coiled around one another and around a shared axis (see Figure 1A) The outside of the

chains is composed of phosphate-sugars groups and the chains are linked together on

the inside via hydrogen bonds between the nucleotide bases

2 The chains are anti-parallel in conformation with ie the 5rsquo end of one chain is bound

via the nucleobases to the 3rsquo end of the other chain

3 Both chains follow a right-handed helix (one type of DNA known as Z-DNA follows

a left-handed helix but this was discovered later)25 and base residues are present on the

chains every 34 Å with structural repeats every ten residues The distance from the

central shared axis to the phosphorous atom is 10 Å

4 The four bases composing DNA bond to a specific pair (see Figure 1B) ie adenine

(purine) binds with thymine (pyrimidine) and guanine (purine) binds with cytosine

(pyrimidine) The relationship of molar equivalency between pairs of bases ie A ndash T

and G ndash C was determined earlier by Chargaff in 195026

Details related to the structure and composition of DNA has formed the basis of our

understanding of the role of DNA in molecular and cell biology Through the structure of DNA

the mechanism for DNA replication27 transcription28 and translation29 for protein synthesis was

elucidated The confirmation of DNA as the storage for hereditary information paved the way for

initiatives like the Human Genome Project and insights from this undertaking have fueled research

regarding the genetic basis of disease30

5

Figure 1A shows the double helix structure for DNA polymer as proposed by Watson and

Crick Arrows on the ribbons represent the directionality bias for the single strands and

dimensions for the polymer are presented with one turn of the helix every 34 nm the

distance between base pairs every 034 nm and the distance between the phosphate

backbone and the central axis every 1 nm B shows the hydrogen bonding taking place

between complementary pairs of nucleobases as proposed by Chargaff with adenine (A)

having two hydrogen bonds with thymine (T) and guanine (G) having three hydrogen bonds

with cytosine (C) Image was adapted with permission Copyright Nature Education 201331

122 Thermodynamics of DNA Hybridization

Design and development of DNA-based technologies have been guided by the

thermodynamic modelling of hybridization Techniques like PCR3233 and isothermal

amplification34 rely on accurate control over the annealing of primers and DNA sensors often uses

temperature and chaotropic agents for achieving selectivity35 One of the useful tools for modelling

hybridization and mismatch-based interactions is the nearest neighbor method (NN)36 To explore

the strategy between the NN method and hybridization of DNA it is useful to understand some

details related to predicting the melting temperature (Tm)

First the system of interest will be defined at the equilibrium of dsDNA and ssDNA at the

point where both populations are equal ie half the strands of DNA are in the double helix state

and the other half are single-stranded and are often in various conformations Tm is the temperature

6

at which this equilibrium is found Next the equilibrium constant Keq is defined as being a ratio

of the concentration of dsDNA and ssDNA (as stated in Equation 1) A second expression can be

derived from the Vanrsquot Hoff equation (as stated in Equation 2) where ∆Ho and ∆So are the standard

enthalpy and entropy of hybridization and lnCT is the natural logarithm of the total strand

concentration This second equation can be used to calculate the thermodynamic parameters

related to Tm with the same being true vice versa37

Equation 1 = [][]

Equation 2 = ∆∆

With this foundation investigation into the NN method for modelling can be undertaken

The thermodynamics associated with a base pair are related to some degree with neighboring base

pairs Free energy values and other related parameters have been determined experimentally for

model oligonucleotide sequences This information is then used in conjunction with the nearest

neighbor algorithm (as presented in Equation 3) for obtaining the Tm for the strand of interest

Here base pair doublets are considered for sequence stability with ten unique combinations of

doublets (5rsquo-3rsquo) CG GC AT AA (also = TT) AG (also = CT) AC (also = GT) GA (also = TC)

GG (also = CC) TG (also = CA)38

Equation 3 ∆ = ∆ + ∆ + sum ∆

Equation 4 ∆ = ∆ minus ∆

In Equation 3 the ∆Gi(total) refers to the free energy of the strand of interest ∆G(init)

refers to the free energy of the strand of initiating base pair ∆G(sym) refers to the free energy of

symmetry Gj refers to the free energy associated with one of ten nearest neighbor stacking

interactions and nij is the appearance rate of the stacking interaction of interest Thermodynamic

parameters are also present for entropy and enthalpy allowing for the calculation of the Tm using

Equation 43638 Higher Tm values indicate greater stability than lower Tm values39ndash42 The NN

method can also be used along with a database of mismatch energetics to determine the

thermodynamic parameters related to those sequences

7

Tm values when used in conjunction with the free energies provide a theoretical basis for

designing probe ndash capture strand interactions This understanding can be useful when designing

wash conditions that control stringency for oligonucleotides composed of sequences with high

similarity Stringency control can be achieved using higher temperature (because increasing

temperature results in de-annealing of sequences and has greater effect on hybrids with partial

complementarity)43 by controlling the ionic strength of hybridization44 and via chaotropic agents

such as formamide45 and urea46 Although all strategies are valid for reducing false positive signals

(that arise from partially complementary strands of oligonucleotides) the use of washes containing

chaotropic agents may be more applicable for the POC given that temperature control requires a

temperature module

Chaotropic agents like formamide lower the melting temperature of duplex DNA by

engaging with the hydrogen bond network of DNA The degree by which temperature is lowered

depends on the GC content the conformations of single and duplex forms and the hydration state

of the strand (but typically can range 24 ndash 29 degC per mole of formamide)45 Chemically

formamide strongly associates with DNA is capable of four hydrogen bonds (same as water) and

is a stronger hydrogen bond acceptor than water Formamide ndash water bonds have been reported to

be 20 stronger than water ndash water bonds and it is accepted that formamide engages the hydration

network around DNA4547 Thus using formamide in washes for DNA hybridization can lower the

melting temperature favoring fully complementary hybrids over partially complementary hybrids

123 Notes and Considerations for POC Application

Developing a DNA screening device for the POC application requires consideration of the

many challenges faced by clinicians When screening genetic samples from blood it is important

to note that samples are often complex with proteins and other type of biomolecules (in addition

to cellular debris) and these materials may occlude the signal generated from target detection48

Another challenge to note for nucleic acid-based diagnostics is the low amount of target present in

clinical samples For example one milliliter of human blood contains approximately 107

leukocytes corresponding to femtomolar quantities (fM or attomoles 10-18 moles) of target nucleic

acid Thus detection strategies requiring hybridization-based assay require enzymatic

amplification of the target materials or improved analytical figures of merit for application in

POC49 Presently there are many different strategies for enzymatic amplification of nucleic acids

8

including polymerase chain reaction32 loop-mediated isothermal amplification34 helicase-

dependent isothermal amplification50 and recombinase polymerase amplification51 Post

amplification targets are often detected using hybridization-based assays using Watson-Crick base

pairing for detection of targets of interest Typically capture probes of complementary sequence

to targets are immobilized on a surface and the presence of target forms hybrids that are transduced

via electrochemical5253 or optical strategies54 Nano-surfaces can be integrated into this detection

strategy with oligonucleotides immobilized on the surface of nanoparticles allowing for

transduction via near-field phenomenon

13 Quantum dots

Nanomaterials based on gold and semiconductor composites have had a significant impact

across many different research fields including the chemical physical and biological sciences

Interest in nanoparticles has been driven due to the unique fundamental properties of these

materials as they approach and occupy size regions between bulk material and isolated atoms

Luminescent semiconductor-based quantum dots (QDs) in particular have attracted attention due

to their unique electro-optical properties arising from small size scales (typically ranging from

2 ndash 10 nm and consisting of 102 ndash 104 atoms per crystal) The key factors of interest for these

particles are material composition and size with a combination of the two giving rise to control of

physical properties such as the spectral profile and photon band gap energies55ndash59

There are many strategies for preparing and tuning the electro-optical properties of QDs

but some of the most studied from a synthetic perspective are based on binary composites of

elements from groups II-VI (like CdSe CdS or CdTe) and III-V (like InAs)55ndash58 For binary

composites luminescent properties can be controlled by choice of materials (selecting specific

regions of the UV-vis spectrum) and by control of size whereby smaller crystals are blue-shifted

and larger crystals are red-shifted5759 QDs used in most biological investigations are constructed

in a coreshell manner where the core is composed on a composite of materials previously

mentioned and the shell is composed of an inert coating (ie ZnS see Figure 2A) The QD shell

protects the nanoparticle from environmental degradation forming a protective layer and provides

a larger potential energy barrier for confining the exciton The shell material also provides a

synthetic strategy for controlling the core size and the type of shell allows for designing a class of

ligands for functionalizing the nanoparticle5556

9


resolution TEM image Here core material is composed of CdSe and shell is composed of

ZnS B Quantum dots of different colors are presented with their corresponding core size

image of solution and photoluminescence spectra and color C Diagram representing the

quantum confinement and the change in band gap energy as material size decreases below

the Bohr-exciton radius Here CB and VB represent the conduction and valence band

respectively and Eg represent the band gap energies Image adapted with permission

Copyright 2011 American Chemical Society60

The resulting particles have been incorporated into biological systems using surface ligands

with chemistry that allows the crystals to be stable in aqueous and physiological buffers6162

Further functionalization of these ligands has also allowed for the integration of biomolecules like

nucleic acids63 and proteins64 and polymers like polyethylene glycol (PEG) allowing applications

that extend from biological imaging65 to diagnostic device development and commercial

technologies566667 Optically quantum dots (QD) have broad absorption wavelengths (from the

UV into the visible) narrow and symmetrical emission photoluminescence (PL) profiles (25 nm

of full width at half maximum) high quantum yields and photochemical stability59 These

spectral properties in addition to the large surface area of QDs make them favourable donors for

RET processes

10

131 Quantum Confinement and The Particle in a Box

A brief overview of the quantum mechanics related to QDs will be discussed before

detailing the advantages and disadvantages of nanomaterials for optical detection of nucleic acids

As the semiconducting material that composes QDs transitions from the bulk scale to the nano-

scale the valence and conductance bands of the semiconductor material split into discrete

energetic states (see Figure 2A and B)60 The band gaps of semiconductors are fixed by the

composite of materials however for nanomaterials the band gap can also be tuned by modulating

the size of the nanomaterials58 Control of nanoparticle band gap energies occurs when the

dimensionality of the material reduces below the Bohr-exciton radius (~5 nm for materials like

CdSe)5960 The Bohr-exciton radius represents the minimal distance for the separation of an

electron-hole pair When an electron is excited by a photon of greater energy than the band gap

(the probability increases at higher energies yielding broad absorption spectra) the separation of

the electron-hole (exciton) is confined to the dimensionalities of the nanomaterial The term used

to describe this phenomenon is called quantum confinement and the model that best describes it is

the particle in a box575960

In this model a particle is said to be confined in a symmetrical box (of diameter a) where

the center of the box is denoted as = 0 and the edges of the box are denoted as = (

( Here

the potential energy inside the box +( le le

(- is said to be zero and the potential energy outside

the box + le ( ge

(- is said to be infinite The resulting probability of finding a particle outside

the confines of the box is zero 0 = 0 + le ( ge

(-1 and the discrete energy

eigenfunctions for the particle is 023 = radic2 sin9 where 9 = 123 etc In QDs the particle of

interest is the exciton and it is loosely confined to the crystal lattice of the semiconducting material

The surface of the material represents the impenetrable barrier (potential energy is infinity)

restricting the exciton to the interior of the QD and the oscillation energy to a few transitions6869

As size of the QDs decreases the energy required for excitation increases because the

exciton transitions within the nanoparticle becomes increasingly restricted59 Beyond the spectral

properties of QDs the conjugation of biomolecules to surfaces of QDs is also advantageous for a

RET based system because the surface area of QDs allows for loading of multiple biomolecules

Thus multiple pathways of RET can take place that can collectively improve energy transfer

11

efficiency and increases the optical signal Of note for signal reproducibility is that a ratiometric

data processing approach where acceptor and QD donor emission are tracked together thus greater

precision for biological interactions is achieved70

14 Fluorescence and Resonance Energy Transfer

The ideas related to fluorescence are important for building an understanding of the details

related to FRET Thus it is suggested to the reader that other resources such as Lakowiczrsquos

Principles of Fluorescence Spectroscopy may provide a more detailed treatment on the topic71

The reader is also directed to Jaris-Erijman and Jovinrsquos review on FRET Imaging72 and Medintz

and Hildebrandtrsquos FRET ndash Foumlrster Resonance Energy Transfer From Theory to Applications73

for more details on theory of FRET

141 Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET sometimes referred to as Foumlrster resonance

energy transfer) is the near-field phenomenon where a chromophore in the excited state (donor)

undergoes a dipole-dipole through-space interaction with a ground state fluorophore (acceptor)

The result of this distance-dependent interaction forms the basis of bio-recognition based assays73

Although the theory of FRET has been discussed in detail elsewhere7273 the specific application

of FRET for QD-based sensors will be discussed further herein QDs have spectral properties that

make them excellent donors in FRET and two strong arguments for their advantage in FRET assays

involve the relationship between distance and FRET efficiency (see Equation 5) and the Foumlrster

distance (see Equation 6)7073

Equation 5 = = sum gt frasl ABsum gt frasl A

asymp gtAAgtA

Equation 6 gtA = DEF BgtGHIJBHKLMN = K PD Q BgtHKB sdot GHIN S TUVUUNU

S TUU

The efficiency of FRET details the degree to which energy transfer between the donor and

the acceptor is achieved This is primarily a function of the number of acceptors and the distances

related to the FRET pair For an individual QD of (near) spherical structure multiple FRET

acceptors are predicted to self-assemble on the surface of the crystal The specific location and

orientation of the acceptors are predicted to vary However the variations can be assumed to be

12

averaged In solution these acceptors are expected to self-assemble in all directions and the

resulting effect on FRET efficiency can be described using Equation 5 (see Figure 3(A)) From

Figure 3(b) it can be seen that an increase in the distance between FRET acceptors and donors

results in a decrease in FRET efficiency This again agrees with the theory of FRET efficiency as

described by Equation 5 When QDs are immobilized on a surface the number of acceptors

coordinating on the nanoparticle are expected to be less than in solution because a portion of the

QD is interfacing with the immobilizing surface (see Figure 3(c)) However this does not mean

that advantages of multiple FRET pathways are lost on the surface QDs when on a surface can

undergo FRET with acceptors on adjacent nanoparticles given that the proximity criteria are met

Thus multiple donors can interact with multiple acceptors In Equation 5 the efficiency of FRET

is represented by E the Foumlrster distance is represented by R0 the distance between the donor and

the acceptor is represented by r and the total number of acceptors is represented by a7073

Figure 3 Changes in FRET efficiency and QD-acceptor formats (a) Configuration of

colloidally stable and spherical QD (green) with multiple FRET acceptors (yellow) (b)

Change in FRET efficiency based on changes in the distance between donor and acceptor

(c) QD (green) immobilized on a surface can interact with multiple FRET acceptors by

interacting with adjacent acceptors Image acquired with permission from Algar et al70

Copyright Elsevier 2010

13

The Foumlrster distance is characteristic of the FRET pair (donor and acceptor) of interest and

represents the distance at which the efficiency of energy transfer is at 50 Parameters from both

the donor and the acceptor contribute to the Foumlrster distance In Equation 2 the orientation factor

is W the quantum yield of the donor is XY the refractive index of the medium is 9 the spectral

overlap is Z Avogadrorsquos number is [ the donor fluorescence is Y the wavelength is ] and the

molar absorption coefficient for the acceptor is ^_ Maximizing the Foumlrster distance can be

achieved with QDs because their spectral properties as FRET donors can be controlled affording

large donor-acceptor spectral overlap and donor quantum yield The emission of QDs is narrow

and the photoluminescence (PL) wavelength range is tunable based on control of the size of the

nanoparticle Thus QD emission can be designed to allow for large spectral overlap between QD

emission and the acceptorrsquos absorption profile QDs also have high quantum yields (XY asymp 02 ndash

09) with absorption profiles extending from the emission region to high energy UV Thus QDs

can be excited at higher energies avoiding excitation of the acceptor from QD light sources In

addition to excitation wavelength the excitation power required for QDs is lower than molecular

dyes because QDs have high molar absorptivity coefficients (^ asymp 104 ndash 106 M-1 cm-1) thus a lower

intensity excitation minimizes the rate of photobleaching These properties make QDs good donors

in FRET based processes and biosensors that integrate QD based FRET for sensing

biomolecules6070

Fluorescence is a high-sensitivity method among oligonucleotide-based detection

strategies74 Labelling of oligonucleotides can be accomplished during the amplification step via

the integration of fluorescently labelled deoxynucleotides but is not necessary or desired in some

applications74 The performance of fluorescence-based systems can be further improved by

integrating luminescent nanomaterials and adopting a fluorescence resonance energy transfer

(FRET) strategy for application in microPADs75 A representation of two analysis formats based on

labelled and unlabelled amplified oligonucleotide is presented in Figure 4 as the basis for the

methodology proposed in the work herein

14

Figure 4 Schematic of the ldquoDirectrdquo and ldquoSandwichrdquo FRET assays Probe strands (seen in

blue) are immobilized on the surface of green-emitting QDs (gQDs) via dithiolane (DTPA)

functionality (A) In direct assay format the target strand (seen in red) is modified with Cy3

on the proximal end and upon hybridization is brought to proximity with gQDs allowing for

FRET to take place (B) In sandwich assay format the probe strand hybridizes with the

target strand (seen in red) such that there is an overhang on the distal end Reporter strand

(seen in green) hybridizes with the overhang region of the target strand bringing to proximity

the Cy3 label on the proximal end of the reporter

15 Paper Based Analytical Devices

Advances in bioassays and sensing technologies for point-of-care (POC) or resource-

limited settings have been guided by recommendations of the World Health Organizationrsquos

ASSURED criteria that states devices must be affordable sensitive specific user-friendly rapid

and robust equipment free and deliverable to those who need them1976 Paper as a substrate has

been growing in popularity for device development primarily due to this criteria for POC devices

Paper based analytical devices (PADs) are affordable to manufacture with commercial options

offering reproducible pore size and flow rates19 PADs are also easy to fabricate with wax printing-

based technology77 and easy transport is possible via stacking sheets of devices19 The wicking

properties of paper allow for elimination of pumps and power supply modules often required for

microfluidic devices Paper also has well-defined chemistry allowing for bioconjugation and

15

modification of cellulose for developing sensing technology PADs can also be incinerated after

use allowing for safe disposal of biohazardous wastes commonly used on the device131978 With a

multitude of advantages PADs were chosen as a platform for developing sensing chemistry and

the following sections will introduce cellulose modification and fluorescence transduction

strategies used in conjunction with paper

151 Paper Substrates for Sensing Technology Overview

Paper is a suitable substrate for development of analytical devices with fluidic capabilities

(as stated earlier eg microfluidic paper-based analytical devices microPADs) It has been

implemented as a platform for screening and semi-quantitative assays of biomarkers offering

reliable performance at low cost with ease of use and disposal79 As an emerging technology for

POC application microPADs are uniquely poised to function as systems that can process raw samples

and then complete an analysis to yield information regarding the genetic basis of disease80

Research within the microPAD field has often focused on individual functional components of a

complete device including sample preparation81 (ie extraction of analytes from complex

samples) amplification of analytes of interest82ndash84 and detection commonly using

electrochemical8485 or optical (ie colorimetric or fluorimetric) techniques8687 For portable or in-

field applications the preference is isothermal enzymatic amplification yielding products that are

either labelled or unlabelled with dyes depending on the detection scheme and the desired

analytical figures of merit88ndash90 It is clear that sample processing and gene fragment amplification

can be achieved on paper substrates91 providing product for the transduction step which is the

focus of the work in this investigation

152 Cellulose Modification and Smartphone-based Detection

Whatman chromatography paper is one of the most common substrates for developing

PADs and is primarily composed of cellulose fibers manufactured from 100 cotton92 Cellulose

chemistry is well defined but only specific modifications that do not alter the spectroscopic quality

of paper are suitable for PAD development Incompatible chemistry may discolour the paper and

this would create challenges for reproducibility and accuracy of sensing One of the strategies for

modifying cellulose includes periodate-mediated oxidation1993 This reaction oxidizes the sugar

groups on cellulose yielding aldehyde functionality (see Figure 5) that can be modified further for

bioconjugation or nanoparticle coordination chemistry8794 Using this strategy reductive

16

amination has allowed for aldehyde activated papers to be modified with amine-modified nucleic

acids95 amine-functionalized nanoparticles96 and amine linkers that then coordinate to

nanoparticle surface for attachment87 The aldehyde chemistry coupled with amine-based linkers

was incorporated into this investigation due to the reproducibility and yield of the modification

reaction


functional groups for further bioconjugation Image from Ju et al74 Copyright Royal Society

of Chemistry 2016

Imaging of fluorescenceluminescence from PADs is typically accomplished using (epi-)

fluorescence microscopy however this technique is mainly calibrated for use in a laboratory and

is difficult to integrate into a portable system To overcome these challenges the camera (imaging)

technology in smartphones and personal electronic devices offer an effective compromise that is

readily accessible1297 Smartphone cameras lack the sensitivity of the high-end imagers used with

microscopes but these portable digital cameras have advanced processing systems and computing

power in these devices that rival most personal computers Integration of smartphone technology

for colourimetric and fluorescence-based assays has been demonstrated for many applications

providing figures of merit that are comparable to most other commercially available imaging

technologies1298 A FRET sandwich-based nucleic acid assay using green QDs and Cy3 dye

labelled DNA that uses i-Pad imaging has been reported by our group This format has reported a

limit of detection (LOD) of 450 fmol with a dynamic range spanning 2 orders of magnitude In

contrast epifluorescence microscopy provided a LOD of 30 fmol but the i-Pad and smartphone-

based cameras are also orders of magnitude lower in cost that the full microscopy system98 Thus

a smartphone-based sensor was chosen for spectroscopic detection of gene variations of CFTR

gene on paper

17

16 Thesis Objectives and Contributions

Investigations of the detection of oligonucleotides in a paper matrix have primarily focused

on fully complementary hybrids in the presence of non-complementary oligonucleotides8799ndash101

The results of these investigations suggest potential for distinction between mismatches and this

has been examined using a paper-based format to detect a three-base pair deletion associated with

CFTR ∆F508 The work described herein determined that a paper substrate can serve as a platform

for a ratiometric hybridization bioassay for detection of nucleic acids using QDs as RET donors

Green quantum dots (gQDs) and Cy3 dye labelled oligonucleotides were chosen as the RET pair

Hybridization of complementary strands of oligonucleotides resulted in proximity of the RET

donor and acceptor allowing for the near-field phenomenon to alter the PL of the FRET pair

Stringency was controlled by addition of formamide to tune selectivity for wild-type (WT) and

mutant-type (MT) targets Hybridization was conducted in both direct and sandwich formats with

the intention of comparison of analytical performance to guide the subsequent development of an

amplification format in the future Smartphone imaging was used to collect PL data A schematic

detailing the operation of the paper-based assay is presented as Figure 6

18

Figure 6 Schematics detailing the bioassay of CFTR related genetic mutations (A)

Reaction zones consisted of chemically modified paper that were conjugated with gQD-

oligonucleotide probes Zones contained WT and MT controls and test zones where

unknown samples were spotted and imaged Detection was based on the principle of RET

with gQDs used as donors and Cy3 labels on oligonucleotide strands as acceptors (B)

Imaging used a smartphone camera with data processing by ImageJ to split the image to

RGB color channels

19

Chapter 2

Detection of Cystic Fibrosis Transmembrane Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization Assay and a Smartphone Camera

Author Contribution Statement

All experimental work was done by K Malhotra All authors contributed to the

experimental design data analysis and preparation of the manuscript This chapter is based on the

following manuscript

Malhotra K Noor M O Krull U J Detection of Cystic Fibrosis Transmembrane

Conductance Regulator ∆F508 Gene Mutation Using a Paper-Based Nucleic Acid Hybridization

Assay and a Smartphone Camera Manuscript submitted

21 Experimental

Reagents and Oligonucleotides

Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQD with peak

photoluminescence (PL) of 525 nm) capped with oleic acid in toluene were obtained from

Cytodiagnostics (Burlington ON Canada) Whatmanreg cellulose chromatography papers (Grade

1 20 cm x 20 cm) tetramethylammonium hydroxide solution (TMAH 25 ww in methanol) L-

glutathione (GSH reduced ge98) sodium (meta)periodate (NaIO4 ge99) formamide (F

ge995) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) chloroform (CH3Cl anhydrous

ge99) granular lithium chloride (LiCl) sodium cyanoborohydride (NaCNBH3 95) 1-(3-

aminopropyl)imidazole (API 98) sodium dodecyl sulfate (SDS ge985) and 4-(2-

hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES ge995) were obtained from Sigma-

Aldrich (Oakville ON Canada) Buffer solutions were prepared using a water purification system

(Milli-Q 18 M`cm-1) and were autoclaved prior to use

20

The oligonucleotide sequences used in the hybridization assays are listed below in (ACGT

Corporation Toronto ON Canada Integrated DNA Technologies IDT Coralville Iowa USA)

The oligonucleotides were dissolved in autoclaved deionized water and stored at -20degC

Table 2 Oligonucleotide Sequences used in Hybridization Assays

Name Sequence

CFTR WT Probe (WT DTPA) DTPA-5rsquo-AAT ATC ATC TTT GGT GTT-3rsquo

CFTR MT Probe (MT DTPA) DTPA-5rsquo-AAT ATC ATT GGT GTT TCC-3rsquo

CFTR WT Cy3 TGT Cy3-3rsquo-TTA TAG TAG AAA CCA CAA-5rsquo

CFTR MT Cy3 TGT Cy3-3rsquo-TTA TAG TAA CCA CAA AGG-5rsquo

CFTR NC Cy3 TGT Cy3-3rsquo-GAA TGA AGG TAC TAA AGA-5rsquo

CFTR WT TGT 3rsquo-TTA TAG TAG AAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo

CFTR MT TGT 3rsquo-TTA TAG TAA CCA CAA AGG ATA CTA CTT ATA TCT-5rsquo

CFTR NC TGT 3rsquo- GAA TGA AGG TAC TAA AGA AAT TGA TAC GGC CCT AGG

TAG

CFTR Reporter (REP Cy3) Cy3-5rsquo-TAT GAT GAA TAT AGA-3rsquo

TGT = target WT = wild type MT = mutant type NC = non-complementary Cy3 =

Cyanine 3 DTPA = dithiol phosphoramidite REP = reporter

211 Methods

2111 Preparation of QD-Probe Oligonucleotide Conjugates

In general oleic acid capped CdSxSe1-xZnS quantum dots with green PL (gQDs peak PL

at 525 nm) in toluene were made water soluble by a ligand exchange reaction using glutathione

(GSH) The resulting glutathione capped QDs (GSH-QDs) were conjugated with DTPA modified

CFTR oligonucleotide probes A detailed protocol for preparing water-soluble and

oligonucleotide conjugated QDs is presented as follows

Green-emitting CdSxSe1-xZnS (coreshell) quantum dots (gQDs peak PL = 525 nm)

capped with oleic acid in toluene were made water-soluble via a ligand exchange reaction with

glutathione (GSH) In a typical reaction 200 mg of L-glutathione was dissolved in 600 microL of

tetramethylammonium hydroxide solution (TMAH) The solution of L-glutathione in TMAH was

added dropwise to a solution of 035 microM organic gQDs dissolved in 2 mL of chloroform The

resulting solution was agitated for 5 minutes using a vortex mixer and stored overnight in darkness

at room temperature Glutathione modified gQDs (GSH-QDs) were extracted into aqueous

solution using borate buffer saline solution 100 microL of 50 mM borate buffer saline (BBS pH 925

21

100 mM NaCl) solution was added to the GSH-QD solution and agitated for 1 minute using a

vortex mixer The organic (bottom) layer was discarded and ethanol was added to the aqueous

(top) layer at a 11 ratio Ethanol-buffer precipitations were performed as follows the mixture was

centrifuged at 8000 rpm for 5 minutes to obtain a pellet of GSH-QDs The resulting supernatant

was discarded and the pellet was dissolved in 150 microL of BBS solution These ethanol-buffer

precipitations were done two additional times and the resulting QD pellet was dissolved in 200 microL

of 50 mM borate buffer (BB pH 925)98 The concentration of GSH-QDs was determined using

UV-vis absorption spectroscopy and GSH-gQDs (V = 21x105) were stored at 4 degC102

GSH-QDs were conjugated with wild type or mutant type (WT or MT respectively)

oligonucleotide probe strands via self-assembly of oligonucleotide probes terminated with a single

or double disulfide (DTPA) moiety at the 5rsquo terminus Self-assembly was accomplished via in-situ

reduction of the disulfide moiety of probe strands to dithiol using Tris(2-carboxyethyl)phosphine

hydrochloride (TCEP) In a typical reaction GSH-QDs (400 nM) were incubated with 40 times

molar excess of CFTR probes (165 microM) and 500 times molar excess of TCEP (83 mM) in 50 mM

borate buffer saline (BBS pH 925 100 mM NaCl) The mixture was agitated overnight via an

orbital shaker After overnight incubation the QD-probe conjugates were subjected to ldquosalt agingrdquo

The concentrations of NaCl and TCEP were increased in small increments over a period of 2 hours

to 400 mM and 97 mM respectively The mixture was subsequently shaken overnight using an

orbital shaker The solution containing QD-probe conjugates was used without further purification

(unless otherwise stated) and stored at 4 degC98

2112 Solution-Phase Hybridization Assays

Solution-phase hybridization assays were conducted in triplicate and direct assay format

For a typical FRET assay gQD-probe conjugates were mixed with 3rsquo Cy3 labelled oligonucleotide

targets (CFTR WT Cy3 TGT and CFTR MT Cy3 TGT) at various concentrations (625 ndash 75 pmol)

in 50 mM borate buffered saline solution (BBS pH 925 100 mM NaCl) with a reaction time of

15 minutes prior to sample measurements

22

2113 Surface Modification of Paper with Imidazole Groups

Enclosed reaction zones on Whatman cellulose chromatography paper (Grade 1) paper

substrates were prepared by wax printing circular boundaries with a Xerox ColorQube 8570DN

solid ink printer The paper in the zones was subsequently oxidized to obtain aldehyde

functionalities that were further reacted via reductive amination to obtain imidazole groups on the

paper A detailed protocol for preparing paper substrates is presented as follows

Paper reaction zones were created on 20 cm x 20 cm sheets of Whatman cellulose

chromatography paper (Grade 1) using an array pattern designed with AutoCAD 2012 software

The design was an array pattern consisting of 32 circular zones (diameter ca 3 mm) in a 4 by 8

format The dimensions of the paper sheets containing the reaction zones were 25 mm by 60 mm

Wax printing was done using a Xerox ColorQube 8570DN solid ink printer Paper devices were

printed with one pass at the high resolution using black wax (product number = 108R00930

although other wax colors could theoretically be used for printing without any impact on the

chemistry)9498 After fabrication wax was melted into the paper by placing paper sheets in an oven

at 120 degC for 2 minutes

Modification of paper was based on a two-step reaction First cellulose was oxidized to

yield aldehyde groups and then an imidazole functionality was added via reductive amination87

Oxidation of paper with aldehyde functionality was based on periodate oxidation of cellulose In

a typical procedure 018 g of LiCl and 006 g of NaIO4 was dissolved in 6 mL of Milli-Q water

and vortexed Next the solution was spotted onto paper zones in 5 μL aliquots Papers were then

placed in an oven at 50 degC until dry Spotting and drying was repeated two more times after which

the papers were washed Washing was accomplished by placing the papers in Milli-Q water and

agitating for 2 minutes after which the papers were dried in a desiccator overnight

Imidazole functionality was added to the aldehyde modified paper via reductive amination

with sodium cyanoborohydride (NaCNBH3) Briefly a solution of 200 mM NaCNBH3 and 160

mM 1-(3-aminopropyl)imidazole (API) in 100 mM 4-(2-Hydroxyethyl)piperazine-1-

ethanesulfonic acid (HEPES pH 80) buffer was prepared Next 2 μL aliquots of the solution were

spotted onto the aldehyde modified paper zones and allowed to react at room temperature over an

hour

23

21131 Note on Troubleshooting Leaking of Paper Zones

A challenge that arose was leakage from hydrophilic zones defined by wax barriers in the

paper (see Figure 7) To minimize this issue papers were heated for no more that 2 min at 120degC

In addition to this previous protocols for paper modification have reported the use of a 10 min

wash in BB buffer solution containing 01 sodium dodecyl sulfate (SDS) after spotting with

imidazole solution This step was modified to a BB wash for 10 min because it is believed that

addition of SDS was resulting in erosion of wax from paper substrates

Figure 7 Image of buffer solution leakage from hydrophilic paper zones

2114 Immobilization of QD-Probe Oligonucleotide Conjugates and Solid-Phase Hybridization Assays

Hybridization assays on paper substrates were conducted using two formats direct assay and

sandwich assay (see Figure 4) For direct assay first QD-probe conjugates were immobilized on

imidazole modified paper zones dried in darkness and washed with 50 mM borate buffer (BB pH

925) for 5 minutes Next 3rsquo Cy3 labelled oligonucleotide targets (CFTR WT TGT Cy3 or CFTR

MT TGT Cy3) were spotted onto the paper zones and dried at room temperature before washing

with BBS for 30 sec Papers were then dried for an hour under vacuum before imaging using a

smartphone camera Depending on the desired investigation (ie wash conditions for stringency)

a further wash step was done followed by drying under vacuum for an hour before imaging with a

smartphone For sandwich-based assays first QD-probe conjugates were immobilized on paper

zones dried at room temperature and then washed with BB for 5 minutes Next oligonucleotide

targets (CFTR WT TGT or CFTR MT TGT) were spotted onto the paper zones dried at room

temperature and then 3rsquo Cy3 labelled reporter sequences were spotted and dried at room

24

temperature before being washed with BBS for 30 sec Papers were then dried for an hour under

vacuum before imaging with a smartphone Depending on the desired investigation (ie wash

conditions for stringency) a further wash step was done followed by drying under vacuum for an

hour before imaging with a smartphone camera

212 Instrumentation

2121 PL Spectra and Digital Image Acquisition

PL spectra for hybridization assays done in solution-phase were acquired using a

QuantaMaster Photon Technology International spectrofluorimeter (London ON Canada) The

excitation source was a xenon arc lamp (Ushio Cypress CA) and the detector was a red-sensitive

R928P photomultiplier tube (PMT Hamamatsu Bridgewater NJ) The FRET ratios for the PL

spectra were calculated using Equation 7

Equation 7 bc = sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt

1

minus sum bcUMDgtUdMAgtsum bcUMNgtUdMBgt

1

Digital color images for paper substrates were acquired using an iPhone SE with the built-

in camera software (Apple Cupertino CA USA) A ND16 filter (Nikon Mississauga Canada)

was placed in front of the camera to prevent saturation of the detector and the imaging was done

in a dark room Default settings were used for all images with no alterations to exposure time or

detector sensitivity A hand-held ultraviolet (UV) lamp (UVGL-58 LWSW 6W The Science

Company Denver CO USA) operated at the long wavelength (365 nm) setting was used to

illuminate paper substrates at some distance 10 cm The power of the UV lamp was measured

using a power meter at λ = 405 nm when the attenuator was in the OFF position (Newport power

meter model 1918-C Irvine California U S A) The measured power from the UV lamp was

44 microW at a distance of 10 cm from the UV lamp (sensing area was A cong 0785 cm2 radius = 05

cm) Paper substrates were dried in a desiccator before data collection (dry format) The RG ratios

(ratiometric response) from the digital images were calculated using Equation 8

Equation 8 bc = + =e=e

-

minus + =e=e

-

25

2122 Digital Imaging for Ratiometric Detection of Oligonucleotide Hybridization

Data for a ratiometric format of signal transduction requires simultaneous measurement of

intensity from two wavelength bands associated with the PL of the RET donor and acceptor

Imaging was done to make use of the R-G-B colour palette of a smartphone camera with donor

PL associated with the green color channel and acceptor PL was associated with the red color

channel and dividing the average signal intensity of the red color channel with the green color

channel Images were processed using ImageJ software (version 149v National Institutes of

Health Bethesda MB USA) to obtain the average signal intensities of the R-G-B color channels

in the reaction zones on the paper substrates with the average signal obtained via measurement of

n = 4 test spots see Paper zones containing only gQD-probe conjugates (no added targets) were

used as the brightest spots and served as background control Imaging was conducted in a dark

room using dried paper which has previously been reported to offer greater fluorescence

intensity98

22 Results and Discussion

221 FRET Pair Characterization (gQD ndash Cy3)

The optical signal from the bioassay explored in this investigation was based on the near-

field interaction between gQDs as FRET donors and Cy3 as FRET acceptors Foumlrster formalism

was used to characterize the FRET pair and the Foumlrster distance was determined to be 47 nm

Detection of target sequences of interest was observed as a decrease in the PL of the RET donor

and an increase in the PL of the RET acceptor With increasing concentration of dye labelled target

the fluorescence from the paper zones were observed to change from green to yellow indicating

that RET was occurring (see Figure 8)

26


spectral overlap is represented by the shaded region Absorption is shown as dashed lines

and emission is shown as solid lines

Solution based measurements were done to determine the Foumlrster distance (Ro) using

where 9 refers to the refractive index of the surrounding medium (in this investigation a value of

133 was used) W( refers to the orientation factor (in this investigation a random orientation was

assumed and a value of 23 was assigned) The quantum yield (QY ΦY) of glutathione modified

green quantum dots (GSH-gQDs) was taken to be 65 (previously reported)87 Finally the spectral

overlap interval (Z) was determined using

Equation 9 A = K PD Q BgtHK Q NGHgJ

In Y is the fluorescence intensity associated with the donor as a function of wavelength (]) ^_

is the molar extinction coefficient associated with the FRET acceptor as a function of ]

Equation 10 J = S TUVUUNUS TUU

222 Oligonucleotide Hybridization in Solution

Solution-phase assays were conducted to characterize the interaction between probe and

target oligonucleotide strands Investigation of the FRET pair in solution was accomplished via

spectral analysis to obtain a ratiometric value for the interaction Normalized and background

corrected spectra were mathematically processed via Equation 7 to obtain a ratio corresponding to

the energy transfer process Background correction used the Cy3 dye emission spectra

corresponding to the wavelength range of 560 nm to 590 nm and the gQD emission spectra

corresponding to the wavelength range of 510 nm to 540 nm A ratio of the Cy3 emission to gQD

0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27

emission was taken for samples containing both donor and acceptor (ie subscript DA for donor-

acceptor) and the background donor emission was subtracted (ie subscript D for donor) The

ratios obtained from this processing were further averaged using three measurements in total

A range of stoichiometric concentrations for gQDs-probe conjugates and targets were

investigated to obtain concentration-response curves for the different gQD-probe conjugates In

total two different types of conjugates were investigated in solution including gQD-WT probe

conjugates and gQD-MT probe conjugates (shown visually as Figure 10i and ii respectively) The

response curves generated for the two conjugates are shown in Figure 9Ai to Figure 9Aii For each

of the conjugates hybridization of two different types of targets were investigated Data points

shown in orange correspond to CFTR MT Cy3 TGTs and data points shown in blue correspond to

CFTR WT Cy3 TGTs For gQD-probe conjugates with WT probes the FRET signals for CFTR

WT Cy3 TGTs (fully complementary FC) were expected to be greater than that for CFTR MT

Cy3 TGTs (partially complementary PC) due to formation of more stable oligonucleotide hybrids

Similar results were also expected for gQD-probe conjugates with MT probes (ie greater FRET

signals from samples of FC hybrids vs PC hybrids)

Figure 9 Hybridization of the gQD-probe strands was investigated in solution by

fluorescence spectroscopy gQD-probe conjugates with i) CFTR single DTPA WT probe ii)

CFTR single DTPA MT probe were hybridized with CFTR Cy3 WT and CFTR Cy3 MT

target strands The concentration-response curves for the different gQD-probe conjugates

are shown A WT Cy3 labelled target strands are seen in blue and MT Cy3 labelled target

strands are seen in orange Normalized PL spectra for the calibration curves are shown for

28

B) CFTR WT Cy3 labelled target strands and C) CFTR MT Cy3 labelled target strands (

indicates increasing target concentration)

It was found that the fully complementary (FC) hybrids were more stable

thermodynamically than the partially complementary (PC) hybrids PC hybrids for gQD-WT probe

conjugates yielded approximately 25 of the absolute FRET intensity of FC hybrids and PC

hybrids for gQD-MT probe conjugates yielded approximately 50 of the absolute FRET intensity

of the FC hybrids This data led us to believe that with wash stringency control sufficient

discrimination for FC vs PC hybrids would be possible and distinguishing FC and PC on solid-

substrates may be accomplished

Figure 10 Representations of the two different direct assay formats investigated in solution

phase gQDs were modified with i) CFTR Single DTPA WT probe ii) CFTR Single DTPA

MT probe and were mixed with complementary CFTR WT Cy3 target strands and CFTR

MT Cy3 target strands Hybridization resulted in proximity of gQDs and Cy3 which

resulted in FRET

223 Oligonucleotide Hybridization in Paper Substrates

Selectivity of base pair hybridization of DNA strands can be controlled by environmental

manipulation for detection of unique oligonucleotide strands of interest Stringency can be adjusted

by control of the ionic strength the pH of the hybridization solution and by altering the

thermodynamic stability of the probe-target conjugate via addition of chaotropes like formamide

Formamide (HCONH2) can be added to hybridization buffers for lowering oligonucleotide

stability Chemically it is a stronger hydrogen bond acceptor than water and can engage with the

hydrogen bond network of oligonucleotides to displace hydration of DNA The extent of melt

29

temperature depression caused by addition of formamide is dependent on factors including GC

composition of the oligonucleotide strand the helical conformation and the state of hydration

Studies on the denaturing effects from formamide find sensitivity of AT containing duplexes to be

lower than those containing GC perhaps due to the different hydration pattern of AT containing

oligonucleotides3545 Control of the melting temperature of hybrids on paper substrates can be

achieved by adjusting the ratio (vv ) of formamide added to washes and the amount of time that

the paper undergoes the wash A preliminary investigation of the thermodynamic parameters

associated with FC and PC oligonucleotide hybrids was conducted The nearest-neighbor method

was used to determine the thermodynamic parameters associated with the expected probe ndash target

hybrids used in the design of this experiment42 The resulting data was used to interpret the

information produced from the FRET-based system undergoing wash conditions of various

stringencies

Investigation of the fluorescence response caused by hybridization within paper substrates

was accomplished by image analysis to obtain a ratiometric value for the FRET process

Background correction was accomplished using Equation 8 where the intensity of signal in the

paper zone for the red color channel (ie EMRed) corresponded to emission of Cy3 and the intensity

of signal for the green color channel (ie EMGreen) corresponded to emission of gQD A ratio of

the Cy3 emission to gQD emission was taken for samples containing both donor and acceptor

(subscript DA for donor-acceptor) and the background donor emission was subtracted (subscript

D for donor) for each sample spot The data was further processed by obtaining an average value

of four background corrected paper zones for each sample concentration (example of images used

for data processing provided as Figure 11)



conjugates with green channel (gQDs) and red channel (Cy3) for varying concentrations of

30

Cy3 labelled target (i) 0 pmol (ii) 24 pmol (iii) 3 pmol (iv) 39 pmol (v) 48 pmol (vi) 6 pmol

(vii) 75 pmol (viii) 9 pmol of CFTR Cy3 TGT The white dashed circle indicates locations of

spots that may not be visible otherwise

2231 Direct Assay Format

The direct assay made use of hybridization of probe strands with fluorescently labelled targets

Imidazole modified paper substrates were used to immobilize either gQD-WT probe conjugates or

gQD-MT probe conjugates Real samples are expected to be composed of CFTR WT TGT strands

CFTR MT TGT strands or a combination of the CFTR TGT strands Thus four different

variations of probe and target oligonucleotide conjugates were investigated as presented in Figure

12 Hybrids (A) and (B) were fully complementary (FC) and have ∆Gmax = -30 kcal mol-1 and -31

kcal mol-1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and

(D) were partially complementary (PC) and have ∆Gmax = -14 kcal mol-1 and -14 kcal mol-1 for

WT probe ndash MT Target and MT Probe ndash WT Target respectively3639 These differences in

stabilities indicate that careful control of formamide concentration may be sufficient to distinguish

between WT and MT gene fragments at room temperature

A WT Probe ndash WT Target

(18 Complementary Base Pairs with Probe)

B MT Probe ndash MT Target


∆Gmax = -30 kcal mol-1 ∆Gmax = -31 kcal mol-1

Theoretical Tm = 43 degC (316 K) Theoretical Tm = 44 degC (317 K)

C WT Probe ndash MT Target


D MT Probe ndash WT Target




Figure 12 The various probe-target hybrids explored for the direct assay format (A) refers

to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe ndash

gQD gQD

gQD gQD

31

MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and

Tm) were calculated using the nearest neighbor method3839

2232 Sandwich Assay Format

A sandwich assay strategy was based on the step-wise hybridization of probe strands with

unlabeled targets and the subsequent hybridization with a fluorescently labelled reporter sequence

Imidazole modified paper substrates were conjugated with either gQD-WT or gQD-MT probe

systems Clinical samples are expected to be composed of CFTR WT TGT strands CFTR MT

TGT strands or a combination of the CFTR TGT strands Thus four different variations of probe

and target oligonucleotide conjugates were investigated as summarized in Figure 13 In contrast

to direct assay the sandwich assay consists of two hybridization events Of the two hybridization

events only the first event was expected to yield partially complementary (PC) structures while

the second event was expected to always yield fully complementary (FC) structures For the first

hybridization event hybrids (A) and (B) are FC and have ∆Gmax = -30 kcal mol-1 and -31 kcal mol-

1 for WT probe ndash WT Target and MT Probe ndash MT Target respectively Hybrids (C) and (D) are

PC and have ∆Gmax = -14 kcal mol-1 and -20 kcal mol-1 for WT probe ndash MT Target and MT Probe

ndash WT Target respectively The thermodynamic parameters for hybrids (A) and (B) agreed with

those determined for the direct assay and as expected were higher than the values for hybrids (C)

and (D)36 However the parameters for hybrid (D) suggest that it was more stable in the sandwich

assay format than the direct assay format Hybrid (D) in sandwich assay was predicted to form a

PC conjugate with fourteen base pairs which was six more base pairs that all other PC conjugates

(eight) Thus discrimination of MT Probe ndash WT Target and MT Probe ndash MT Target was predicted

to require wash conditions of greater stringency than other PC conjugates For the second

hybridization event (target strands and reporter strands) ∆Gmax = -21 kcal mol-1 and Tm = 30degC

(303 K) were expected The ∆Gmax for the first hybridization events were higher than the second

hybridization event in FC conjugates The result was that wash conditions required to achieve the

mismatch discrimination would also result in signal loss for FC conjugates because for a single

paper system FC hybrids were washed in the same conditions as PC hybrids

32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)



Figure 13 The various probe-target conjugates explored for the sandwich assay format (A)

refers to WT probe ndash WT target (B) refers to MT probe ndash MT target (C) refers to WT probe

ndash MT target (D) refers to MT probe ndash WT target Thermodynamic parameters (∆Gmax and


224 Optimization of Wash Conditions for Direct and Sandwich Based Assay by Smartphone Imaging

To determine the optimized conditions of stringency required to achieve selectivity for the

fully complementary oligonucleotide hybrids wash conditions were explored where selectivity

was controlled as a function of time and added formamide (vv) Paper substrates were washed

with borate buffer containing formamide with increments from 0 to 10 vv ratios for 0 5 and

10 min for direct assays (up to 20 minutes for sandwich assays) Following the washes and after

drying the paper substrates were imaged and the average intensity from reaction zones was

measured to calculate a quantitative ratiometric signal A wider range of wash conditions were

investigated for the sandwich assays because the energy associated with the PC hybrid MT probe

gQD gQD

gQD gQD

33

ndash WT Target was larger than other PC hybrids and could significantly shift conditions for

discrimination between FC and PC hybrids

Data from these wash condition experiments were summarized as heat map tables (see

Table 3 toTable 10) For each of the probe sequences WT or MT in both direct and sandwich

formats wash conditions were explored with FC or PC targets For each probe sequence pairs of

heat maps (FC and PC) were prepared to determine optimal wash conditions Data for FC targets

were presented as green heat maps and PC targets were presented in red heat maps Wash

conditions suitable for assay development would have high signal from FC heat maps and very

low to zero signal from PC heat maps ie large signal (dark green) for FC and small signal (white

ndash light red) for PC Wash conditions chosen for further investigations were then summarized in

Figure 14

2241 Labelled Target (Direct Format)

The resulting ratiometric values were summarized in Table 3 for gQD-WT probe ndash WT

Target hybrids Table 4 for gQD-WT probe ndash MT Target hybrids Table 5 for gQD-MT probe ndash

MT Target hybrids and Table 6 for gQD-MT Probe ndash WT Target hybrids Based on the predicted

energies of hybridization (Figure 12 and Figure 13) FC hybrids were expected to be more stable

and to retain more signal under stringent wash conditions than PC hybrids

For WT probe the wash condition that offered the greatest signal for FC hybrids (Table 3)

and the least signal for PC hybrids (Table 4 ie within noise) was chosen as the wash condition to

continue further investigations Similarly for MT probe the wash conditions offering the greatest

signal for FC hybrids (Table 5) and the least signal for PC hybrids (Table 6 ie within noise) was

chosen as the wash condition to continue further investigations For WT probe the wash conditions

meeting the criteria for mismatch discrimination were BB+5 formamide at 10 min and BB+10

formamide at 5 and 10 min For MT probe the wash conditions meeting the criteria for mismatch

discrimination were BB+5 formamide at 10 min BB+75 formamide at 10 min and BB+10

formamide at 5 and 10 min

34

Table 3 Summary of RG Ratiometric Signals for gQD-WT probe ndash WT Target hybrids

WT Probe - WT Target

RG Ratio Signal

BB+X Wash Times (minutes)

0 5 10

Amount of

Formamide

Added ( vv)

0 108 plusmn 003 101 plusmn 003 094 plusmn 002




Table 4 Summary of RG Ratiometric Signals for gQD-WT probe ndash MT Target hybrids

WT Probe - MT Target

RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)

0 098plusmn 002 020 plusmn 004 010 plusmn 001




Table 5 Summary of RG Ratiometric Signals for gQD-MT probe ndash MT Target hybrids

MT Probe - MT Target

RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





Table 6 Summary of RG Ratiometric Signals for gQD-MT probe ndash WT Target hybrids

MT Probe - WT Target

RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35

2242 Target Determination by Sandwich Assay

The process for determining the optimal wash conditions for sandwich assays was similar

to that used for direct assays The relevant ratiometric values of signals are summarized Table 7 in

for gQD-WT probe ndash WT Target hybrids Table 8 for gQD-WT probe ndash MT Target hybrids

Table 9 for gQD-MT probe ndash MT Target hybrids and Table 10 for gQD-MT Probe ndash WT Target

hybrids FC hybrids were expected to be more stable and to retain more signal under stringent

wash conditions than PC hybrids It is important to note that the gQD-MT probe ndash WT Target

hybrid had a much larger ∆Gmax than the other PC hybrids Thus it was expected to require more

stringent wash conditions to achieve discrimination of FC from PC sequences As with direct

assay discrimination of the FC hybrids from the PC hybrids required wash conditions where

ratiometric signal from FC hybrids was present and signal from PC hybrids was within the noise

of the detector Thus for WT probe the wash condition offering the greatest signal for FC hybrids

(Table 7) and the least signal for PC hybrids (Table 8 ie within noise) was chosen as the optimal

wash condition to continue further investigations The wash conditions offering the greatest signal

for FC hybrids (Table 9) and the least signal for PC hybrids (Table 10 ie within noise) was chosen

as the optimal wash condition to continue further investigations

For MT probe the wash conditions meeting the criteria for mismatch discrimination are

more limited than those for WT probe due to the stability of the PC hybrid (see the thermodynamic

treatment of the hybrids in the main article) Of the various wash conditions BB+5 formamide

at 20 min wash BB+75 formamide at 20 min and BB+10 Formamide at 5 10 15 and 20 min

meet the criteria for the assays Of the different wash conditions for MT probe only BB+5

formamide at 20 min met all the criteria because the BB+75 formamide and BB+10

formamide washes were rejected for WT Probe Thus BB+5 formamide at 5 min for gQD-WT

probes and 20 min wash for gQD-MT probes was chosen for further characterization of the figures

of merit for the assays

36

Table 7 Summary of RG Ratiometric Signal for gQD-WT probe ndash WT Target hybrids

WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)

0 062 plusmn 005 046 plusmn 002 040 plusmn 001 040 plusmn 002 037 plusmn 004

125 0697plusmn0006 062 plusmn 002 060 plusmn 001 053 plusmn 003 042 plusmn 006






Table 8 Summary of RG Ratiometric Signal for gQD-WT probe ndash MT Target hybrids

WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)


125 059 plusmn 002 007 plusmn 004 000 plusmn 002 001 plusmn 001 -001 plusmn 004



5 048 plusmn 002 -002 plusmn 004 000 plusmn 002 000 plusmn 003 -001 plusmn 006


10 037 plusmn 005 -005 plusmn 003 -004 plusmn 004 -004plusmn 003 -002 plusmn 004

Table 9 Summary of RG Ratiometric Signal for gQD-MT probe ndash MT Target hybrids

MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37

Table 10 Summary of RG Ratiometric Signal for gQD-MT probe ndash WT Target hybrids

MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








2243 Optimizing Wash Conditions for Selectivity

Of the various conditions investigated many provided for full discrimination of FC and

PC sequences including all washes done for 10 minutes (see Table 3 to Table 10) The optimal

wash conditions for direct assays that provided the best resolution between FC and PC while

minimizing loss of FC hybrid for both WT and MT sequences was BB + 5 vv formamide

(BB+5F) for both WT and MT sequences (presented as Figure 14A for WT and Figure 14B for

MT) The optimal wash conditions for the sandwich assay reflected the stability of the PC hybrids

for gQD-MT probe which was greater than that of all the other sequences (Figure 13) At

BB+10F complete signal loss for FC gQD-WT probes was noted The wash condition for

sandwich assays was determined to be BB+5F with WT sequences being washed for 5 minutes

while MT sequences were washed for 20 minutes This is schematically presented in Figure 14C

for gQD-WT probe and Figure 14D for gQD-MT probe These washing conditions were then

further investigated for the analytical figures of merit and performance in complex sample

matrices

38

Figure 14 Determination of optimal wash conditions for direct and sandwich assay

considered RG Ratios with variation of formamide concentration for wash times of 0 5 10

15 and 20 min The optimal wash conditions for direct assay was found to be BB+10F for

5 minutes for (A) gQD-WT probe sequence and (B) gQD-MT probe sequence The optimal

wash conditions for sandwich assay was found to be BB+5F for 5 minutes for (C) gQD-

WT probe sequence and BB+5F for 20 minutes for (D) gQD-MT probe sequence

225 Analytical Figures of Merit

The performance of the bioassay was investigated in both direct and sandwich assay

formats and concentration-response curves are presented in Figure 15 Paper substrates were

washed with BB+10F or BB+5F for direct and sandwich assays respectively with wash times

of 20 minutes for MT sandwich assays and 5 minutes for direct phase assays and WT sandwich

assays Performance of the bioassays in the low pmol range is presented as insets for each of the

respective curves Regression analysis for the dataset was done to obtain the analytical figures of

merit which are presented in Table 11

0 5 10 1500

05

10

15

Formamide in BB Wash (vv)

RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD

Optimized Condition (Direct Assay) BB+10F for 5 mins

Optimized Condition (Sandwich Assay) BB+5F for 5 mins (WT) and 20 mins (MT)

C D

A B

39

Figure 15 Concentration-response curves showing the RG ratiometric response of the

direct and sandwich assay formats (Ai) gQD-WT probe conjugates were used for

determination of Cy3 labelled WT targets and (Bi) gQD-MT probe conjugates were used

for determination of Cy3 labelled MT targets (Ci) gQD-WT probe conjugates were used for

determination of unlabelled WT targets with Cy3 labelled reporters and (Di) gQD-MT

probe conjugates were used for determination of unlabelled MT targets with Cy3 labelled

reporters The RG ratiometric response of the direct assay at the low pmol concentration

range was also determined (Aii) gQD-WT probe conjugates and (Bii) gQD-MT probe

conjugates The sandwich assay format (Cii) gQD-WT probe conjugates and (Dii) gQD-MT

probe conjugates Note that the scale for (A) and (B) is logarithmic Each error bar

represents one standard deviation for n=4 replicates

The response of the WT and MT direct assays was similar with sensitivity (slope of

response line) LOD LOQ and dynamic range being in close agreement A dynamic range of two

orders of magnitude (03 ndash 15 pmol) was observed with an LOD (at 3 sigma) of 215 and 285 fmol

for WT and MT probes respectively This consistency in analytical performance reflects the

similar ∆G and Tm for the two FC and PC hybrids

40

Table 11 Analytical Performance Direct and Sandwich Bioassays

Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay

WT 03145 09857 215 fmol 650 fmol 03 ndash 15

MT 03147 09680 285 fmol 865 fmol 03 ndash 15

Sandwich

Assay

WT 00486 09934 422 fmol 128 pmol 04 ndash 20

MT 00285 09779 145 pmol 438 pmol 15 ndash 20

The sandwich assay response of WT and MT was found to vary with WT probes having

double the sensitivity in the region of linearity a lower LOD and LOQ (by three times) and a

larger dynamic range (04 ndash 20 as compared to 15 ndash 20) These differences in analytical

performance are also consistent with the thermodynamic stabilities of the various hybrids MT

probes were required to undergo washes of higher stringency and thus a larger proportion of the

FC was lost Quantification of the analytical parameters was accomplished using only WT or MT

targets However the discrimination of targets in mixtures is also of importance

226 Selectivity for Mixtures of WT and MT Targets

Clinical samples of oligonucleotides are expected to be composed of gene sequences of

WT only MT only (∆F508) or a mixture of WT and MT The cross-reactivity of WT and MT

sequences must therefore be evaluated Selectivity assays were determined in direct assay format

and signal from digital images was measured pre- and post- formamide washing Samples of 24

pmol of CFTR Cy3 FC targets were spiked with 0 ndash 24 pmol samples of CFTR Cy3 PC targets

(eg for WT probe WT targets were spiked with MT targets) Selectivity assays were also done

using the sandwich assay format and samples of 48 pmol of CFTR FC targets were spiked with

0 ndash 48 pmol samples of CFTR PC targets

41

Figure 16 Pre- and post-wash assays for determining the selectivity of (Ai) gQD-WT probes

and (Bi) gQD-MT probes in mixtures of WT and MT targets Selectivity was determined

using background corrected RG ratio plots for hybridization of gQD-probe conjugates with

Cy3 labelled targets (for direct assay A and B) and gQD-probe conjugates with unlabeled

targets and Cy3 labelled reporter sequences (for sandwich assay C and D) Response of the

hybridization assay was determined for pre-wash (Ai and Ci) WT probe conjugates and pre-

wash (Bi and Di) MT probe conjugates Post-wash assays yielded signal response shown in

Aii and Cii for WT probe conjugates and in Bii and Dii for MT probe conjugates Error

bars represent one standard deviation for n = 4 replicates

It was found that for both direct and sandwich assays in pre-wash WT and MT signals

showed linear response with increasing amount of PC targets (Figure 16(Ai) and (Bi) for direct

assay and (Ci) and (Di) for sandwich assay respectively) The signal showed a 25 increase from

0 to the highest pmol added This indicates substantial contribution of signal from PC hybrids

Post-wash it was found that there was no contribution of signal from the addition of PC targets to

either WT or MT assays in both direct and sandwich assays (Figure 16(Aii) and (Bii) for direct

42

assay and (Cii) and (Dii) for sandwich assay respectively) The results indicate that suitable

stringency control can obviate false positives in mixtures of WT and MT probes

227 Paper-based Assay Response for Complex Sample Matrices

The performances of the assays were investigated for samples that contained bovine serum

albumin (BSA 40 mg mL-1) goat serum (GS 85 vv) and salmon sperm DNA (SS 2000 bp

fragments 08 mg mL-1) These matrices were used to dilute either CFTR NC Cy3 TGT CFTR

WT Cy3 TGT or CFTR MT Cy3 TGT at 24 pmol concentration for direct assay and at 6 pmol

concentration for sandwich assay The resulting RG ratios from direct hybridization assays

(including BBS as a control) are shown in Figure 17(A) and (B) for WT and MT probe conjugates

respectively RG ratios from sandwich hybridization assays are shown in Figure 17 (C) and (D)

for WT and MT probes respectively



BSA Response of the hybridization assay was collected for (A) gQD-WT probe conjugates

and (B) gQD-MT probe conjugates Sandwich assays were conducted in similar manner to

direct assay and using 6 pmol of CFTR Cy3 TGTs Response of the hybridization assay was

43

collected for (C) gQD-WT probe conjugates (D) gQD-MT probe conjugates Error bars

represent one standard deviation for n = 4 replicates

High selectivity was retained for all hybridization assays in both direct and sandwich

format with the signal from NC and PC hybrids being within the experimental error Thus the

interfering effects of these sample matrices did not compromise the performance of either direct

or sandwich assays

228 Blind Assay for Detection and Quantification of CFTR Target Mixes

The performances of the direct and sandwich assays were investigated with a blind assay

experiment to confirm that the specific wash conditions in this thesis could be used for

determination of unknown mixtures of oligonucleotides associated with CFTR gene sequence

Mixtures of samples that contained WT only MT only and a mix of WT and MT targets were

used in particular because these are the expected combinations of oligonucleotides from clinical

samples The blind assays were prepared with external assistance such that sample identities and

concentration were unknown to the assayer Samples were prepared in BBS buffer with a final

concentration of 30 pmol for direct assay and 75 pmol for sandwich assay The resulting solutions

were then spotted on paper devices and ratiometric signal was measured pre-and post-wash for

sample identification Signal from the assays and subsequent identification of samples were found

to be in agreement and within experimental error supporting applicability of this technology for

clinical application (see Table 12) All spiked samples were correctly identified by the assayer

and signals generated from assays were within the dynamic range of the assay

44

Table 12 Blind Assay for Direct and Sandwich Assays

Assay Format Blind

Sample

Spiked

Samples

Signal Sample

Identification WT assay MT assay

Direct Assay 1 WT only 054 plusmn 003 002 plusmn 002 WT

2 WT and MT 049 plusmn 001 058 plusmn 004 Mix

3 MT only 000 plusmn 002 065 plusmn 006 MT


Sandwich Assay 1 MT only 002 plusmn 001 055 plusmn 003 MT




45

Chapter 3

Conclusion and Future Work

Fluorescence determination in a paper substrate of a predominant genetic marker for cystic

fibrosis has been explored This involves distinction between a mutant form and wild type

oligonucleotide sequence either of which could be present individually or in mixture in clinical

samples A FRET-based hybridization assay using QDs with green emission as donors and a Cy3

molecular fluorophore as an acceptor has provided for two assays methods One method relied on

labelled oligonucleotide target as commonly produced during enzyme amplification Another

method used a sandwich assay format to avoid the need for labelling of oligonucleotide targets

Analytical performance was primarily based on selective melting of undesired hybrids and

sufficient stringency control was possible to provide reliable detection of targets even in samples

that contained substantial quantities of protein and nucleic acid as interferents Despite the

performance differences due to thermodynamic stabilities of hybrids formed from two

oligonucleotides compared to sandwich hybrids using 3 oligonucleotides the data indicates that

both direct and sandwich assays could be implemented to distinguish between wild type and

mutant type samples

Of the two hybridization formats direct assay was observed to have better analytical

figures of merit including a lower LOD and greater sensitivity to target than sandwich assay which

had comparable LOL and dynamic range Wash times for both direct and sandwich assay were on

the order of five minutes with direct assay using more stringent wash conditions than sandwich

assay However the MT variant for sandwich assay was found to have a higher LOD and smaller

dynamic range than other sequences Wash times for the MT sandwich assay was four times as

long as WT and direct assays limiting the throughput of this assay in sandwich format Taking

these facts into account sandwich assay is still better suited for further development of this

technology than direct assay Sandwich assays can be incorporated with ease to different types of

amplification techniques when compared with direct assay which requires labelled nucleotides

limiting the options available for amplification

46

31 Future Directions

There are many requirements that need to be addressed for the application of this screening

technologies for the point-of-care The work in this thesis focused primarily on the detection of

targets related to Cystic Fibrosis but the sample processing target extraction target amplification

and clinical validation still need to be addressed Samples for POC genetic testing will need to be

processed without the use of large laboratory instruments because the technology for a device must

be portable and low cost Extraction and amplification of targets will also be required due to the

low number of targets present in samples

The two most likely applications for this technology are the incorporation of paper-based

test strips for new born screening of infants7-10 and general screening for CF genes of adult

patients The implementation of multi-level NBS programs is relatively new and is based firstly

on a heel prick blood test followed by a larger volume blood and sweat test The small volume of

blood that is collected in a heel prick (a few hundred L) would require an enzymatic technique

to amplify zeptomole (10-21 moles) quantities of genetic material to produce enough biomarker

for analysis with the paper-based test strip49 Amplification techniques like PCR and

tHDA7482 have been shown to detect these levels of genetic material and would be required for

further application of the proposed paper-based technology Blood tests for adults could include

screening for heterozygous carriers of CF Typical blood tests of adults provide larger volumes of

blood (a few mL) Such samples offer attomole (10-18 moles) quantities of genetic

material49 These larger amounts of nucleic acids can be amplified using simpler technology

associated with isothermal enzymatic methods given that exponential amplification may not be

essential to achieve sufficient signal from hybridization assays

Isothermal enzymatic amplification of nucleic acids is a promising strategy for overcoming

low target numbers because it eliminates the need for temperature control modules currently

required for enzyme-based amplification The lack of specialized equipment makes isothermal

techniques field portable and POC available Two popular isothermal techniques that are being

translated for paper substrates are loop mediated isothermal amplification (LAMP)34 and

recombinase polymerase amplification (RPA)51 These technologies will also require clinical

validation with real patient samples at the POC for further application

47

References

(1) Cutting G R Cystic Fibrosis Genetics From Molecular Understanding to Clinical

Application Nat Rev Genet 2015 16 (1) 45ndash56

(2) Rommens J M et al Identification of the Cystic Fibrosis Gene Chromosome Walking

and Jumping Science 1989 245 (4922) 1059ndash1065

(3) OrsquoSullivan B P Freedman S D Cystic Fibrosis The Lancet 2009 373 (9678) 1891ndash

1904

(4) Hodson M Bush A Geddes D Cystic Fibrosis Third Edition CRC Press 2012

(5) Kerem B et al Identification of the Cystic Fibrosis Gene Genetic Analysis Science

1989 245 (4922) 1073ndash1080

(6) Riordan J R et al Identification of the Cystic Fibrosis Gene Cloning and

Characterization of Complementary DNA Science 1989 245 (4922) 1066ndash1073

(7) Castellani C Massie J Sontag M Southern K W Newborn Screening for Cystic

Fibrosis Lancet Respir Med 2016 4 (8) 653ndash661

(8) Kharrazi M et al Newborn Screening for Cystic Fibrosis in California Pediatrics 2015

136 (6) 1062ndash1072

(9) Ross L F Newborn Screening for Cystic Fibrosis A Lesson in Public Health Disparities

J Pediatr 2008 153 (3) 308ndash313

(10) Dequeker E et al Best Practice Guidelines for Molecular Genetic Diagnosis of Cystic

Fibrosis and CFTR-Related Disorders ndash Updated European Recommendations Eur J

Hum Genet 2009 17 (1) 51ndash65

(11) Health C for D and R In Vitro Diagnostics - Nucleic Acid Based Tests

httpswwwfdagovMedicalDevicesProductsandMedicalProceduresInVitroDiagnostics

ucm330711htm (accessed Feb 22 2018)

(12) Martinez A W et al Simple Telemedicine for Developing Regions Camera Phones and

Paper-Based Microfluidic Devices for Real-Time Off-Site Diagnosis Anal Chem 2008

80 (10) 3699ndash3707

(13) Martinez A W Phillips S T Whitesides G M Three-Dimensional Microfluidic

Devices Fabricated in Layered Paper and Tape Proc Natl Acad Sci 2008 105 (50)

19606ndash19611

(14) Noiphung J et al Electrochemical Detection of Glucose from Whole Blood Using Paper-

Based Microfluidic Devices Anal Chim Acta 2013 788 39ndash45

(15) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Structure and

Function of DNA In Molecular Biology of the Cell 5th Edition Garland Science New York

2002

(16) Crick F Central Dogma of Molecular Biology Nature 1970 227 561ndash563

(17) Alberts B Johnson A Lewis J Raff M Roberts K Walter P The Shape and

Structure of Proteins In Molecular Biology of the Cell 5th Edition Garland Science New

York 2002

48

(18) Pierce B A Gene Mutations and DNA Repair In Genetics A Conceptual Approach W

H Freeman and Company 2014 pp 493ndash525

(19) Martinez A W Phillips S T Whitesides G M Carrilho E Diagnostics for the

Developing World Microfluidic Paper-Based Analytical Devices Anal Chem 2010 82

(1) 3ndash10

(20) Dahm R Friedrich Miescher and the Discovery of DNA Dev Biol 2005 278 (2) 274ndash

288

(21) Levene P A The Structure of Yeast Nucleic Acid IV Ammonia Hydrolysis J Biol

Chem 1919 40 415ndash424

(22) Franklin R E Gosling R G Molecular Configuration in Sodium Thymonucleate Nature

1953 171 (4356) 740ndash741

(23) Wilkins M H F Stokes A R Wilson H R Molecular Structure of Nucleic Acids

Molecular Structure of Deoxypentose Nucleic Acids Nature 1953 171 738ndash740

(24) Watson J D Crick F H C Molecular Structure of Nucleic Acids A Structure for

Deoxyribose Nucleic Acid Nature 1953 171 737ndash738

(25) Mitsui Y et al Physical and Enzymatic Studies on Poly d(IndashC)Poly d(IndashC) an Unusual

Double-Helical DNA Nature 1970 228 (5277) 1166ndash1169

(26) Chargaff E Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic

Degradation Experientia 1950 6 201ndash209

(27) Meselson M Stahl F W The Replication of DNA in Escherichia Coli Proc Natl Acad

Sci 1958 44 (7) 671ndash682

(28) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From DNA to RNA

In Molecular Biology of the Cell 5th Edition Garland Science New York 2002

(29) Alberts B Johnson A Lewis J Raff M Roberts K Walter P From RNA to Protein


(30) International Human Genome Sequencing Consortium Initial Sequencing and Analysis of

the Human Genome Nature 2001 409 (6822) 860ndash921

(31) Pray L Discovery of DNA Structure and Function Watson and Crick Nat Educ 1 (1)

100

(32) Saiki R K et al Primer-Directed Enzymatic Amplification of DNA with a Thermostable

DNA Polymerase Science 1988 239 (4839) 487ndash491

(33) Mullis K B Faloona F A [21] Specific Synthesis of DNA in Vitro via a Polymerase-

Catalyzed Chain Reaction In Methods in Enzymology Recombinant DNA Part F

Academic Press 1987 Vol 155 pp 335ndash350

(34) Notomi T et al Loop-Mediated Isothermal Amplification of DNA Nucleic Acids Res

2000 28 (12) e63

(35) Fuchs J et al Effects of Formamide on the Thermal Stability of DNA Duplexes on

Biochips Anal Biochem 2010 397 (1) 132ndash134

(36) SantaLucia J Hicks D The Thermodynamics of DNA Structural Motifs Annu Rev

Biophys Biomol Struct 2004 33 415ndash440

49

(37) Breslauer K J Frank R Bloumlcker H Marky L A Predicting DNA Duplex Stability

from the Base Sequence Proc Natl Acad Sci U S A 1986 83 (11) 3746ndash3750

(38) SantaLucia John Allawi H T Seneviratne P A Improved Nearest-Neighbor

Parameters for Predicting DNA Duplex Stability Biochemistry (Mosc) 1996 35 (11)

3555ndash3562

(39) Allawi H T SantaLucia J Thermodynamics and NMR of Internal GmiddotT Mismatches in

DNA Biochemistry (Mosc) 1997 36 (34) 10581ndash10594

(40) Peyret N Seneviratne P A Allawi H T SantaLucia J Nearest-Neighbor

Thermodynamics and NMR of DNA Sequences with Internal AmiddotA CmiddotC GmiddotG and TmiddotT

Mismatches Biochemistry (Mosc) 1999 38 (12) 3468ndash3477

(41) Allawi H T SantaLucia J Nearest-Neighbor Thermodynamics of Internal AmiddotC

Mismatches in DNAthinsp Sequence Dependence and pH Effects Biochemistry (Mosc) 1998

37 (26) 9435ndash9444



(43) Petersen J Poulsen L Petronis S Birgens H Dufva M Use of a Multi-Thermal

Washer for DNA Microarrays Simplifies Probe Design and Gives Robust Genotyping

Assays Nucleic Acids Res 2008 36 (2) e10

(44) Matsishin M J Ushenin I V Rachkov A E Solatkin A P SPR Detection and

Discrimination of the Oligonucleotides Related to the Normal and the Hybrid Bcr-Abl

Genes by Two Stringency Control Strategies Nanoscale Res Lett 2016 11

(45) Blake R D Delcourt S G Thermodynamic Effects of Formamide on DNA Stability

Nucleic Acids Res 1996 24 (11) 2095ndash2103

(46) Kourilsky P Leidner J Tremblay G Y DNA-DNA Hybridization on Filters at Low

Temperature in the Presence of Formamide or Urea Biochimie 1971 53 (10) 1111ndash1114

(47) Chang Y J Castner E W Femtosecond Dynamics of Hydrogen‐bonding Solvents

Formamide and N ‐methylformamide in Acetonitrile DMF and Water J Chem Phys

1993 99 (1) 113ndash125

(48) Innis M A Gelfand D H Sninsky J J White T J PCR Protocols A Guide to

Methods and Applications Academic Press 2012

(49) Mikkelsen S R Electrochecmical Biosensors for DNA Sequence Detection

Electroanalysis 1996 8 (1) 15ndash19

(50) Vincent M Xu Y Kong H Helicase-Dependent Isothermal DNA Amplification

EMBO Rep 2004 5 (8) 795ndash800

(51) Piepenburg O Williams C H Stemple D L Armes N A DNA Detection Using

Recombination Proteins PLoS Biol 2006 4 (7) e204

(52) Drummond T G Hill M G Barton J K Electrochemical DNA Sensors Nat

Biotechnol 2003 21 (10) 1192ndash1199

(53) Hahm J Lieber C M Direct Ultrasensitive Electrical Detection of DNA and DNA

Sequence Variations Using Nanowire Nanosensors Nano Lett 2004 4 (1) 51ndash54

50

(54) Fan X White I M Shopova S I Zhu H Suter J D Sun Y Sensitive Optical

Biosensors for Unlabeled Targets A Review Anal Chim Acta 2008 620 (1ndash2) 8ndash26

(55) Dabbousi B O et al (CdSe)ZnS CoreminusShell Quantum Dots Synthesis and

Characterization of a Size Series of Highly Luminescent Nanocrystallites J Phys Chem

B 1997 101 (46) 9463ndash9475

(56) Chan W C W Maxwell D J Gao X Bailey R E Han M Nie S Luminescent

Quantum Dots for Multiplexed Biological Detection and Imaging Curr Opin Biotechnol

2002 13 (1) 40ndash46

(57) M G Bawendi et al The Quantum Mechanics of Larger Semiconductor Clusters

(ldquoQuantum Dotsrdquo) Annu Rev Phys Chem 1990 41 (1) 477ndash496

(58) Murray C B Norris D J Bawendi M G Synthesis and Characterization of Nearly

Monodisperse CdE (E = Sulfur Selenium Tellurium) Semiconductor Nanocrystallites J

Am Chem Soc 1993 115 (19) 8706ndash8715

(59) Alivisatos A P Semiconductor Clusters Nanocrystals and Quantum Dots Science 1996

271 (5251) 933ndash937

(60) Algar W R Susumu K Delehanty J B Medintz I L Semiconductor Quantum Dots

in Bioanalysis Crossing the Valley of Death Anal Chem 2011 83 (23) 8826ndash8837

(61) Gerion D et al Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated

CdSeZnS Semiconductor Quantum Dots J Phys Chem B 2001 105 (37) 8861ndash8871

(62) Chan W C W Nie S Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic

Detection Science 1998 281 (5385) 2016ndash2018

(63) Mitchell G P Mirkin C A Letsinger R L Programmed Assembly of DNA

Functionalized Quantum Dots J Am Chem Soc 1999 121 (35) 8122ndash8123

(64) Willard D M Carillo L L Jung J Van Orden A CdSeminusZnS Quantum Dots as

Resonance Energy Transfer Donors in a Model ProteinminusProtein Binding Assay Nano Lett

2001 1 (9) 469ndash474

(65) Ballou B Lagerholm B C Ernst L A Bruchez M P Waggoner A S Noninvasive

Imaging of Quantum Dots in Mice Bioconjug Chem 2004 15 (1) 79ndash86

(66) Hill H D Mirkin C A The Bio-Barcode Assay for the Detection of Protein and Nucleic

Acid Targets Using DTT-Induced Ligand Exchange Nat Protoc 2006 1 (1) 324ndash336

(67) Nam J-M Thaxton C S Mirkin C A Nanoparticle-Based Bio-Bar Codes for the

Ultrasensitive Detection of Proteins Science 2003 301 (5641) 1884ndash1886

(68) El-Issa H D The Particle in a Box Revisited J Chem Educ 1986 63 (9) 761

(69) Manae M A Hazra A Helping Students Understand the Role of Symmetry in Chemistry

Using the Particle-in-a-Box Model J Chem Educ 2016 93 (6) 1056ndash1060

(70) Algar W R Tavares A J Krull U J Beyond Labels A Review of the Application of

Quantum Dots as Integrated Components of Assays Bioprobes and Biosensors Utilizing

Optical Transduction Anal Chim Acta 2010 673 (1) 1ndash25

(71) Lakowicz J R Principles of Fluorescence Spectroscopy 3rd ed Springer New York

2006

51

(72) Jares-Erijman E A Jovin T M FRET Imaging Nat Biotechnol 2003 21 (11) 1387ndash

1395

(73) FRET - Foumlrster Resonance Energy Transfer Medintz I Hildebrandt N Eds Wiley-

VCH Verlag GmbH amp Co KGaA Weinheim Germany 2013

(74) Ju Q Noor M O Krull U J Paper-Based Biodetection Using Luminescent

Nanoparticles Analyst 2016 141 (10) 2838ndash2860

(75) Hildebrandt N et al Energy Transfer with Semiconductor Quantum Dot Bioconjugates

A Versatile Platform for Biosensing Energy Harvesting and Other Developing

Applications Chem Rev 2017 117 (2) 536ndash711

(76) Peeling R W Holmes K K Mabey D Rapid Tests for Sexually Transmitted Infections

(STIs) The Way Forward Sex Transm Infect 2006 82 (Suppl 5) v1ndashv6

(77) Carrilho E Martinez A W Whitesides G M Understanding Wax Printing A Simple

Micropatterning Process for Paper-Based Microfluidics Anal Chem 2009 81 (16) 7091ndash

7095

(78) Carrilho E Phillips S T Vella S J Martinez A W Whitesides G M Paper

Microzone Plates Anal Chem 2009 81 (15) 5990ndash5998

(79) Cheng C-M et al Paper-Based ELISA Angew Chem Int Ed 2010 49 (28) 4771ndash

4774

(80) Yang Y et al Paper-Based Microfluidic Devices Emerging Themes and Applications

Anal Chem 2016

(81) Moghadam B Y Connelly K T Posner J D Isotachophoretic Preconcenetration on

Paper-Based Microfluidic Devices Anal Chem 2014 86 (12) 5829ndash5837

(82) Noor M O Hrovat D Moazami-Goudarzi M Espie G S Krull U J Ratiometric

Fluorescence Transduction by Hybridization after Isothermal Amplification for

Determination of Zeptomole Quantities of Oligonucleotide Biomarkers with a Paper-Based

Platform and Camera-Based Detection Anal Chim Acta 2015 885 156ndash165

(83) Connelly J T Rolland J P Whitesides G M ldquoPaper Machinerdquo for Molecular

Diagnostics Anal Chem 2015 87 (15) 7595ndash7601

(84) Tsaloglou M-N et al Handheld Isothermal Amplification and Electrochemical Detection

of DNA in Resource-Limited Settings Anal Biochem 2018 543 116ndash121

(85) Dungchai W Chailapakul O Henry C S Electrochemical Detection for Paper-Based

Microfluidics Anal Chem 2009 81 (14) 5821ndash5826

(86) He M Liu Z Paper-Based Microfluidic Device with Upconversion Fluorescence Assay

Anal Chem 2013 85 (24) 11691ndash11694

(87) Noor M O Shahmuradyan A Krull U J Paper-Based Solid-Phase Nucleic Acid

Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence

Resonance Energy Transfer Anal Chem 2013 85 (3) 1860ndash1867

(88) Connolly A R Trau M Isothermal Detection of DNA by Beacon-Assisted Detection

Amplification Angew Chem Int Ed 2010 49 (15) 2720ndash2723

52

(89) Liu H Li L Duan L Wang X Xie Y Tong L Wang Q Tang B High Specific

and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based

Exponential Rolling Circle Amplification Anal Chem 2013 85 (16) 7941ndash7947

(90) Hsieh K Patterson A S Ferguson B S Plaxco K W Soh H T Rapid Sensitive

and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic

Electrochemical Quantitative Loop-Mediated Isothermal Amplification Angew Chem

Int Ed 2012 51 (20) 4896ndash4900

(91) Liu M et al Target-Induced and Equipment-Free DNA Amplification with a Simple

Paper Device Angew Chem Int Ed 2016 55 (8) 2709ndash2713

(92) Yetisen A K Akram M S Lowe C R Paper-Based Microfluidic Point-of-Care

Diagnostic Devices Lab Chip 2013 13 (12) 2210ndash2251

(93) Arauacutejo A C Song Y Lundeberg J Staringhl P L Brumer H Activated Paper Surfaces

for the Rapid Hybridization of DNA through Capillary Transport Anal Chem 2012 84

(7) 3311ndash3317

(94) Noor M O Krull U J Paper-Based Solid-Phase Multiplexed Nucleic Acid

Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As

Donors in Fluorescence Resonance Energy Transfer Anal Chem 2013 85 (15) 7502ndash

7511

(95) Ju Q Uddayasankar U Krull U Paper-Based DNA Detection Using Lanthanide-Doped

LiYF4 Upconversion Nanocrystals As Bioprobe Small 2014 10 (19) 3912ndash3917

(96) Doughan S Uddayasankar U Krull U J A Paper-Based Resonance Energy Transfer

Nucleic Acid Hybridization Assay Using Upconversion Nanoparticles as Donors and

Quantum Dots as Acceptors Anal Chim Acta 2015 878 1ndash8

(97) de Villiers C A Lapsley M C Hall E A H A Step towards Mobile Arsenic

Measurement for Surface Waters The Analyst 2015 140 (8) 2644ndash2655

(98) Noor M O Krull U J Camera-Based Ratiometric Fluorescence Transduction of Nucleic

Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform

Using Immobilized Quantum Dots as Donors Anal Chem 2014 86 (20) 10331ndash10339

(99) Allen P B Arshad S A Li B Chen X Ellington A D DNA Circuits as Amplifiers

for the Detection of Nucleic Acids on a Paperfluidic Platform Lab Chip 2012 12 (16)

2951ndash2958

(100) Wang Y et al Photoelectrochemical Lab-on-Paper Device Equipped with a Porous Au-

Paper Electrode and Fluidic Delay-Switch for Sensitive Detection of DNA Hybridization

Lab Chip 2013 13 (19) 3945ndash3955

(101) Song Y et al Visual Detection of DNA on Paper Chips Anal Chem 2014 86 (3) 1575ndash

1582

(102) Uddayasankar U Shergill R T Krull U J Evaluation of NanoparticlendashLigand

Distributions To Determine Nanoparticle Concentration Anal Chem 2015 87 (2) 1297ndash

1305

53

Copyright Acknowledgements

Copyright permissions have been reported in the caption for relevant figures

ii



Karan Malhotra

Master of Science

Department of Chemistry

University of Toronto

2018

Abstract

Diagnostic technology that utilizes paper substrates and device cameras offers opportunities for

development of cost effective point-of-care technologies The translation of assays operating in

aqueous solution require further development for implementation in paper substrates This report

presents and compares two methods for determination of oligonucleotides that serve as indicators

of Cystic Fibrosis differentiating wild type and mutant type sequences containing a 3-base

deletion The transduction strategy operates by selective hybridization of dye-labelled

oligonucleotides (target or reporters) to capture probes immobilized on quantum dots and

hybridization results in emission of dyes via resonance energy transfer Detection is based on

hybridization of fluorophore labelled target or hybridization of unlabelled target and labelled

reporter in a sandwich assay format Selectivity to determine mismatched sequences required

control of stringency conditions using formamide as a chaotrope It was determined that both

formats can distinguish between wild type and mutant type samples on paper substrates

iii

Acknowledgments

























iv

Table of Contents

Acknowledgments iii


List of Tables vi

List of Figures vii

Chapter 1 1

Introduction 1








13 Quantum dots 8








Chapter 2 19



21 Experimental 19

211 Methods20

v












Chapter 3 45



References 47

vi

List of Tables













vii

List of Figures
















Society60 9














viii



Chemistry 2016 16


















27









ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



iii

Acknowledgments

























iv

Table of Contents

Acknowledgments iii


List of Tables vi

List of Figures vii

Chapter 1 1

Introduction 1








13 Quantum dots 8








Chapter 2 19



21 Experimental 19

211 Methods20

v












Chapter 3 45



References 47

vi

List of Tables













vii

List of Figures
















Society60 9














viii



Chemistry 2016 16


















27









ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



iv

Table of Contents

Acknowledgments iii


List of Tables vi

List of Figures vii

Chapter 1 1

Introduction 1








13 Quantum dots 8








Chapter 2 19



21 Experimental 19

211 Methods20

v












Chapter 3 45



References 47

vi

List of Tables













vii

List of Figures
















Society60 9














viii



Chemistry 2016 16


















27









ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



v












Chapter 3 45



References 47

vi

List of Tables













vii

List of Figures
















Society60 9














viii



Chemistry 2016 16


















27









ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



vi

List of Tables













vii

List of Figures
















Society60 9














viii



Chemistry 2016 16


















27









ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



vii

List of Figures
















Society60 9














viii



Chemistry 2016 16


















27









ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



viii



Chemistry 2016 16


















27









ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



ix






























x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



x














1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



1

Chapter 1

Introduction























20th 2018)11

2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



2





Assay


synthesis



Luminex Molecular

Diagnostics Inc



Osmetech Molecular

Diagnostics










analysis


microwell plate


Assay


electrophoresis and















3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



3






















state








4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



4





























5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



5




















6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



6







Equation 1 = [][]

Equation 2 = ∆∆









Equation 3 ∆ = ∆ + ∆ + sum ∆










7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



7










temperature module





















8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



8









13 Quantum dots






















9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



9


















RET processes

10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



10


















( Here



the box + le ( ge













11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



11






















asymp gtAAgtA


S TUU






12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



12



















13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



13


















biomolecules6070









14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



14




















15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



15































16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



16





reaction



of Chemistry 2016
















gene on paper

17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



17
















18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



18







RGB color channels

19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



19

Chapter 2









21 Experimental













20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



20





Name Sequence









TAG




211 Methods















21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



21



























22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



22





























hour

23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



23






















24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



24





212 Instrumentation








1


1














-

minus + =e=e

-

25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



25













intensity98










26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



26






















0

05

1

15

2

25

3

400 450 500 550 600 650 700

No

rma

lize

d A

BS

PL

Sp

ect

ra

Wavelength (nm)

gQD ABS

Cy3 ABS

gQD EM

Cy3 EM

gQD Cy3

27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



27






















28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



28














resulted in FRET









29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



29












stringencies














30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



30






























gQD gQD

gQD gQD

31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



31




























32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



32



(FC with REP)



(FC with REP)





(FC with REP)



(FC with REP)
















gQD gQD

gQD gQD

33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



33











Figure 14
















34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



34



RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)







RG Ratio Signal


0 5 10

Amount of

Formamide

Added ( vv)





35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



35


























36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



36


WT Probe - WT

Targt

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









WT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)









MT Probe - MT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)








37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



37


MT Probe - WT

Target

RG Ratio Signal


0 5 10 15 20

Amount of

Formamide

Added (

vv)





















matrices

38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



38















0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 15-02

00

02

04

06

08


RG

Rati

oWT Target

MT Target

0 5 10 1500

05

10

15


RG

Ra

tio

WT Target

MT Target

0 5 10 1500

02

04

06

08


RG

Ra

tio

WT Target

MT Target

gQD

gQD

gQD

gQD

gQD

gQD

gQD

gQD



C D

A B

39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



39

















40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



40


Assay

Format

Probe Slope of

Calibration

Curve

r2 LOD LOQ Linear

Range

(pmol)

Direct

Assay



Sandwich

Assay



















41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



41
















42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



42

















43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



43






or sandwich assays















44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



44


Assay Format Blind

Sample

Spiked

Samples

Signal Sample










45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



45

Chapter 3















mutant type samples












46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



46






























47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



47

References






1904



1989 245 (4922) 1073ndash1080






136 (6) 1062ndash1072


J Pediatr 2008 153 (3) 308ndash313









80 (10) 3699ndash3707



19606ndash19611





2002




York 2002

48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



48





(1) 3ndash10


288


Chem 1919 40 415ndash424


1953 171 (4356) 740ndash741










Sci 1958 44 (7) 671ndash682








100







2000 28 (12) e63





49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



49





3555ndash3562








37 (26) 9435ndash9444















1993 99 (1) 113ndash125






EMBO Rep 2004 5 (8) 795ndash800







50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



50





B 1997 101 (46) 9463ndash9475



2002 13 (1) 40ndash46





Am Chem Soc 1993 115 (19) 8706ndash8715


271 (5251) 933ndash937











2001 1 (9) 469ndash474














2006

51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



51


1395












7095




4774


Anal Chem 2016














Anal Chem 2013 85 (24) 11691ndash11694






52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



52







Int Ed 2012 51 (20) 4896ndash4900







(7) 3311ndash3317




7511













2951ndash2958



Lab Chip 2013 13 (19) 3945ndash3955


1582



1305

53



53



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Detection of Cystic Fibrosis Transmembrane Conductance ......Karan Malhotra Master of Science Department of Chemistry University of Toronto 2018 Abstract Diagnostic technology that