Development of a Piezoelectric Immunosensor for

antibody titration for vaccinated fish

Ricardo Vieira Ribeiro

Thesis to obtain the Master of Science Degree in

Bioengineering and Nanosystems

Supervisors: Prof. Luís Joaquim Pina da Fonseca

Prof. Marília Clemente Velez Mateus

Examination Committee

Chairperson: Prof. Gabriel António Amaro Monteiro

Supervisor: Prof. Marília Clemente Velez Mateus

Members of the Committee: Dr. Cristina Maria Grade Couto da Silva Cordas

November 2017

iii

Acknowledgments

First of all I would like to express my sincere gratitude to Prof. Gabriel Monteiro, Prof. Luís Fonseca and

Prof. Marília Mateus, my supervisors. I would like to thank you for accepting me to develop this work

and for all the trust and freedom that was given to me to on the lab, for the encouragement to test new

idea and for every advice and knowledge given throughout this time. Thank you.

To all of my lab friends and colleagues, especially to Ana Rosa, Flávio, Rui Carvalho, Sara and Teresa,

for all the successes and failures we shared, for all the advices and help in the lab, for all of the support

and friendship, thank you.

To Raquel Agostinho and her big heart for always being there in the good and bad times. This journey

wouldn’t be completed without her by my side. Thank you.

A special note to Dr. Cristina Cordas and Rui Silva for their time spent answering my many questions

about this work with patience, kindness and altruism when they didn’t have to. Thank you.

Finally, to the most important people in my life, mom, dad, brother – thank you for everything, for all the

sacrifices, all the belief, trust, patience and love. I am forever grateful to you.

iv

v

Abstract

The antibody-antigen interaction in immunosensing is having huge interest in the last years.

Innumerous strategies have been successfully created and applied towards detection, quantification or,

simply, as a purification step of antibodies or antigens. Biosensor technology has been in constant

development towards the detection of biological or biochemical interactions where piezoelectric

immunosensors, based on quartz crystal microbalance (QCM), can perform label-free detection of

antibodies in a quick and sensitive way.

This work will focus on developing a QCM-based piezoelectric immunosensor. Taking advantage

of the gold and thiol group affinity, a self-assembled monolayer (SAM) was produced on the QCM

surface and, then, if the crystal surface is coated with an antigen, the binding of specific antibody to that

antigen can be detected by a change in the measured frequency and the mass in the surface calculated.

Bovine Serum Albumin (BSA) and antibody anti-BSA were used as antigen and antibody, respectively.

Later, fish serum was also studied. The layers packed and the immobilization process were achieved

with high success. The calibration curve results showed a good agreement and linear response between

the mass deposited and the frequency shift. A proof-of-concept specifically to this system was

demonstrated and can be performed in later studies.

In future works, the outer membrane protein K (OmpK) purified from Vibrio alginolyticus, a fish

pathogen infecting Portuguese aquacultures is the antigen to be immobilized at the surface for early

detection and quantitation of antibody production after immunization.

Keywords:

Quartz Crystal Microbalance; Gold surface functionalization; QCM development; Cyclic voltammetry.

vi

vii

Resumo

A interação antigénio-anticorpo na imuno-sensibilidade tem despertado muito interesse nos

últimos anos. Inúmeras estratégias foram criadas e aplicadas com êxito na detecção, quantificação ou,

mesmo como passo de purificação em anticorpos ou antigénios. A tecnologia de biossensores tem

apresentado desenvolvimentos ao longo do tempo na deteção tanto de interações biológicas como

bioquímicas onde os imunossensores piezoeléctricos derivados de microbalanças de cristal de quartzo

(QCM) podem efectuar deteções rápidas e sensíveis de anticorpo sem recorrer a marcadores.

Este estudo focar-se-á num QCM como imunossensor piezoeléctrico. Aproveitando a afinidade

entre ouro e grupos tiol, foi possível a produção de self-assembled monolayer (SAM) à superfície do

QCM e, depois, estando a superfície revestida com antigénio, foi possível detectar a ligação do

anticorpo específico a partir de uma variação da frequência medida para que, no final, a massa

depositada seja calculada. A albumina de soro bovino (BSA) e o anticorpo anti-BSA foram usados como

antigénio e anticorpo, respectivamente. Posteriormente, soro de peixe também foi estudado. A

deposição de camadas e o processo de imobilização foram obtidas com elevado sucesso. Os

resultados das rectas de calibração apresentaram uma boa concordância e linearidade entre a massa

depositada e variação frequência. Uma prova-de-conceito para este sistema específico foi

demonstrada.

Em trabalhos futuros, a proteína membranar externa K (OmpK) purificada do Vibrio alginolyticus,

um patogénico de peixe que infecta aquaculturas Portuguesas, é um possível antigénio capaz de serem

imobilizados à superfície metálica revestida do cristal de quartzo para a deteção precoce e

quantificação do anticorpo depois da imunização.

Palavras-chave:

Microbalança de cristal de quartzo; Funcionalização da superfície de ouro; Desenvolvimento da

microbalança; Voltametria cíclica.

viii

ix

Table of Contents

Acknowledgments ................................................................................................................... iii

Abstract ..................................................................................................................................... v

Resumo .................................................................................................................................... vii

List of Figures ......................................................................................................................... xii

List of Abbreviations ............................................................................................................ xvii

State of the Art ................................................................................................................ 1

Biosensors ................................................................................................................... 1

Alternative to biosensors ............................................................................................. 2

Quartz Crystal Microbalance – QCM ........................................................................... 2

Theory .......................................................................................................................... 4

QCM as a biosensor .................................................................................................... 5

Piezoelectric immunosensor based on QCM .............................................................. 7

Antibody-antigen complex quantification ................................................................... 10

Autolab EQCM module .............................................................................................. 11

Vibrio alginolyticus ..................................................................................................... 11

Immunization ............................................................................................................. 14

Material and Methods ................................................................................................... 16

Introduction ................................................................................................................ 16

Reagents ................................................................................................................... 16

Materials .................................................................................................................... 17

QCM Characterization ............................................................................................... 17

Gold electrode surface active area and Sensibility factor ............................ 17

Cyclic voltammetry........................................................................................ 20

QCM functionalization ............................................................................................... 21

Quantitative methods ................................................................................................. 25

Experimental Methods ............................................................................................... 26

Ionic strength behavior .............................................................................................. 29

QCM regeneration ..................................................................................................... 30

x

Results and Discussion ............................................................................................... 31

QCM Characterization ............................................................................................... 31

Gold electrode surface active area and Sensibility factor ............................ 31

Cyclic Voltammetry ....................................................................................... 35

QCM functionalization ............................................................................................... 36

Quantitative methods ................................................................................................. 39

Ionic strength behavior .............................................................................................. 44

Conclusions and Future Perspectives ....................................................................... 46

References .................................................................................................................... 48

Annexes ......................................................................................................................... 55

xi

xii

List of Figures

Figure 1 - Schematic illustration of a Biosensor. A biosensor contains a molecular sensing layer that

is directly attached to a physicochemical transduction element. The molecular sensing layer ensures

the recognition of specific targets, while the transduction element converts binding events into an

electrical signal, the intensity of which is correlated with the concentration or activity of the target

analytes. [23] ........................................................................................................................................... 1

Figure 2 - Schematics of QCM and QCM-D operation. Side view of the crystal. Application of

oscillatory voltage results in a cyclical deformation. The fundamental frequency (black waves at the

edges of the crystal) and the third overtone (blue wave in the middle) are illustrated. [22] .................... 4

Figure 3 - Crystallographic orientation of an AT-cut quartz substrate. The crystal is cut 35º 15’ to the

optical axis. [88] ....................................................................................................................................... 5

Figure 4 - Schematic representation of antibody immobilization main approaches and site-

directed antibody immobilization methods. A – Random antibody immobilization, B - site-directed

antibody immobilization, C - site-directed antibody immobilization via Fc binding proteins, D - via

antibody fragments and E - via oxidized oligosaccharide moieties [89]. ................................................. 9

Figure 5 – Schematic presentation of an amplification strategy using magnetic beads. [44] ................ 9

Figure 6 – Representation of two different quantification approaches. A – Calibration curve of a

HIV-1 antigen-QCM sensor enhanced by streptavidin-Au with different concentrations of the target. B -

Schematic illustration of binding targets and respective frequency shift uptake in order to determine the

mass change. [48] ................................................................................................................................. 10

Figure 7 – Representation of A - Autolab faraday cage and, B - Quartz Crystal Microbalance and C -

its holder. ............................................................................................................................................... 11

Figure 8 – 3D structures of 2 different Outer Membrane Proteins. A - Bacterial β-barrel membrane

protein A (OmpA) and B - Bacterial β-barrel membrane protein F (OmpF). [90] .................................. 12

Figure 9 - The OMP journey to the outer membrane. Nascent OMPs (maroon) are synthesized in the

bacterial cytoplasm and delivered by the SecA/SecB chaperones (purple) or by the ribosome (red) to

the Sec translocase (grey) for passage through the IM. Once in the periplasm, nascent OMPs can be

delivered to the BAM complex by the periplasmic chaperones SurA (slate) or Skp (blue). Any OMPs

xiii

which misfold during their journey will be degraded by the protease DegP (light blue) to prevent

aggregation. [51] .................................................................................................................................... 13

Figure 10 - Gram-negative bacterial double membrane envelope. Schematic representation of inner

membrane, periplasm and outer membrane and its constituents [57]. ................................................. 13

Figure 11 – Cyclic voltammetry in H2SO4. Characteristic iodine-adsorbed gold electrode

voltammogram [68]. ............................................................................................................................... 19

Figure 12 – Scheme of the different steps taking place during the self-assembly of alkanethiol

on Au: i) physisorption, (ii) lying down formation, (iii) nucleation of the standing phase, (iv) completion

of the standing up phase [73]. ............................................................................................................... 22

Figure 13 – Schematic representation of a SAM on Au. Final structure of a series of 11-

Mercaptoundecanoic acid molecules after reacting with QCM Au surface. .......................................... 22

Figure 14 – Representation of the reactions by a carbodiimide-catalyzed reaction using the EDC plus

NHS system to form an amide bond [76]. ............................................................................................. 24

Figure 15 – Schematic illustration of a reaction cell. a) before and b) after tightening [78]. ................ 26

Figure 16 - Schematic representation of a typical EQCM instrument. The quartz crystal has a

fundamental frequency of 7.995 MHz and is coated with thin gold films on both sides. The gold disk

deposited on the top side of the crystal is in contact with the electrolyte solution and used as the working

electrode. The top view of the gold-coated crystal is also shown. ........................................................ 28

Figure 17 – Schematic representation of 11-Mercaptundecanoic acid SAM formation on gold electrode

in conjugation with antigen and antibody immobilization [80]. .............................................................. 29

Figure 18 – Control voltammogram in H2SO4 solution. Cyclic voltammetry performed in a clean

QCM ranging 0 to 1.5 volt sweep with 2 peaks exhibited, peak number 2 and 3, the oxidation and

reduction process of Au substrate, respectively. ................................................................................... 31

Figure 19 – Test voltammogram. Cyclic voltammetry performed in an iodine-pretreated QCM ranging

0 to 1.5 volt sweep with 4 peaks exhibited. Peak number 1 and 4 are the charge of the oxidation and

process of adsorbed and desadsorbed iodine. Peak number 2 and 3, the oxidation and reduction

process of Au substrate, respectively. ................................................................................................... 32

Figure 20 – Frequency shift of a test QCM showing the desadsorption process from the Au substrate

after a sweep from 0 to 1.5 volts. .......................................................................................................... 33

xiv

Figure 21 – Cyclic voltammogram from -0.6 to 0.8V of Bare Au (Blue); 2mM 11-MUA (Orange);

EDC/NHS 50MM (Gray); BSA 1 mg/mL (Red) and Antibody anti-BSA (Black). ................................... 35

Figure 22 – Frequency shift of 2mM 11-MUA after addition on top of a clean QCM surface. Purple

background - Ethanol 70% solution; Yellow background – 2mM 11-MUA in ethanol 70% solution. 36

Figure 23 - Frequency shift of EDC/NHS 50 mM after addition on top of a thiolated-QCM surface. Brown

background – 100mM MES Buffer; Dark Green background – EDC/NHS 50mM in 100mM MES

buffer. ..................................................................................................................................................... 37

Figure 24 - Frequency shift of BSA 1 mg/mL after addition on top of a functionalized-QCM surface. Red

background – Milli-Q water; Light Green background – BSA 1mg/mL in Milli-Q water. .................. 37

Figure 25 - Frequency shift of antibody anti-BSA 10 µg/mL after addition on top of a BSA-QCM surface.

Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA in PBS pH7 buffer

solution. ................................................................................................................................................. 38

Figure 26 - Frequency shift of EDC/NHS 50 mM after addition on top of a thiolated-QCM surface. Red

background – Milli-Q water; Dark Green background – EDC/NHS in Milli-Q water. ........................ 40

Figure 27 – Illustration of a BSA in a horizontal position above a SAM. K-1 – Debye length; r – Sum of

the BSA radius and Debye length. ........................................................................................................ 42

Figure 28 – Calibration curve of a BSA-QCM sensor with different BSA concentrations. ................... 42

Figure 29 - Calibration curve of an anti-BSA immunosensor with different antibody anti-BSA

concentrations. ...................................................................................................................................... 43

Figure 30 - Calibration curve of a QCM with 5 different fish serum dilutions - 1:300; 1:250; 1:200; 1:150

and 1:100. .............................................................................................................................................. 44

Figure 31 – Abnormal frequency shift of BSA 1 mg/mL after BSA addition and removal on top of a

functionalized-QCM surface. Gray background – PBS pH 7 buffer; Light Green background – BSA

in PBS pH7 buffer solution. ................................................................................................................... 45

Figure 32 – Original frequency shift behavior for the sensibility factor calculation............................... 55

Figure 33 – Frequency shift behavior treated after using “moving average” with a period = 20. ......... 56

Figure 34 - Frequency shift behavior treated after using “moving average” with a period = 50. .......... 57

Figure 35 - Frequency shift behavior treated after using “moving average” with a period = 200. ........ 57

Figure 36 - Frequency shift of BSA 0.75 mg/mL after addition on top of a functionalized-QCM surface.

Red background – Milli-Q water; Light Green background – BSA 0.75mg/mL in Milli-Q water. ...... 58

xv

Figure 37 - Frequency shift of BSA 0.5 mg/mL after addition on top of a functionalized-QCM surface.

Red background – Milli-Q water; Light Green background – BSA 0.5 mg/mL in Milli-Q water. ....... 59

Figure 38 - Frequency shift of BSA 0.25 mg/mL after addition on top of a functionalized-QCM surface.

Red background – Milli-Q water; Light Green background – BSA 0.25 mg/mL in Milli-Q water. ..... 59

Figure 39 - Frequency shift of BSA 0.15 mg/mL after addition on top of a functionalized-QCM surface.

Red background – Milli-Q water; Light Green background – BSA 0.15 mg/mL in Milli-Q water. ..... 60

Figure 40 - Frequency shift of BSA 0.125 mg/mL after addition on top of a functionalized-QCM surface.

Red background – Milli-Q water; Light Green background – BSA 0.125 mg/mL in Milli-Q water. ... 60

Figure 41 - Frequency shift of antibody anti-BSA 7.5 µg/mL after addition on top of a BSA-QCM surface.

Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 7.5 µg/mL in PBS

pH7 buffer solution. ............................................................................................................................... 61

Figure 42 - Frequency shift of antibody anti-BSA 5 µg/mL after addition on top of a BSA-QCM surface.

Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 5 µg/mL in PBS

pH7 buffer solution. ............................................................................................................................... 62

Figure 43 - Frequency shift of antibody anti-BSA 2.5 µg/mL after addition on top of a BSA-QCM surface.

Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 2.5 µg/mL in PBS

pH7 buffer solution. ............................................................................................................................... 62

Figure 44 - Frequency shift of antibody anti-BSA 1 µg/mL after addition on top of a BSA-QCM surface.

Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 1 µg/mL in PBS

pH7 buffer solution.Δf = 18 Hz .............................................................................................................. 63

xvi

xvii

List of Abbreviations

11-MUA 11-Mercaptoundecanoic acid

Au Gold

BSA Bovine Serum Albumin

DL Debye Length

DDL Diffusion Double layer

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

ELISA Enzyme-Linked Immunosorbent Assay

Fab Antigen-binding Fragment

FET Field-Effect Transistor

NHS N-Hidroxisuccinimide

IM Inner Membrane

OM Outer Membrane

OMP Outer Membrane Protein

OMPK Outer Membrane Proteins K

PBS Phosphate Buffer Saline

QCM Quartz Crystal Microbalance

RIA Radioimmunoassay

SAM Self-assembled monolayer

SPR Surface Plasmon Resonance

1

State of the Art

Biosensors

Biosensors are analytical apparatus that use a molecular sensing event to convert a biological

response into an electrical signal. So, it is combined a sensing element specific for a determined analyte

promoting a biological response which is detected and converted through a transducer [1]. Sensors are

classified through their physical-chemical transducers, i.e, electrochemical sensors can detect the

production or consumption of many chemical products in a redox reaction, calorimetric sensors exploits

a temperature change, optical sensors sense an optical diffraction or electrochemiluminescense and

resonant sensors measure mass variation [2] (Figure 1). In the end, the biosensor has the capacity to

process and display the signal by a detector. Sensors should have a good linearity, sensitivity and

selectivity along with good accuracy, recovery time and working lifetime.

Clark et al. [3] were the first to develop and build sensors, in this case, electrochemical sensors,

giving the first step towards the development of this technique. His investigation about the concept that

enzymes could be immobilized at the surface of electrochemical detectors to form enzyme electrodes

was supported by the creation of the Clark Electrode [4]. This electrode is the most basic and simpler

amperometric sensor where the oxygen concentration at the electrode surface will decrease

proportionally to the glucose concentration which is transform to gluconic acid by glucose oxidase. Since

then, numerous applications have appeared like glucose biosensors to measure glucose levels in the

blood in a fast and point-of-care usage for diabetic people and pregnancy tests.

In this project, a Quartz Crystal Microbalance is used as an acoustic biosensor for the uptake of

frequency change regarding a mass variation.

Figure 1 - Schematic illustration of a Sensor. A sensor contains a molecular sensing

layer that is directly attached to a physicochemical transduction element. The molecular sensing layer ensures the recognition of specific targets, while the transduction element converts binding events into an electrical signal, the intensity of which is correlated with the concentration or activity of the target analytes. [23]

2

Alternative to biosensors

Antibody-antigen interaction can be measured through immuno-biosensors but also by

immunoassays. Immunoassays are tests that use antibody and antigen complexes as a mean of

generating a measurable result and there are different kinds of immunoassays.

One of the most used is the Enzyme-Linked Immunosorbent Assay (ELISA) and uses along with

the antigen-antibody complex, an enzyme that will, by a chemical reaction, produce a color change. This

method has the antigen immobilized at the surface through which its specific antibody will bind after

been applied in the solution. The antibody has an enzyme linked to it, catalyzing a reaction when its

substrate is added in the solution. The reaction will produce a detectable signal in a form of color change

[5].

Radioimmunoassay (RIA) is a very sensitive and specific assay employing radioisotopes making

this assay dangerous. In here, a radioisotope is attached to the antigen of interest that will bind to its

specific antibody. After the complex formation, a sample with the antigen to be measured is added to

the solution and will compete with the radioactive antigen, replacing it. The radioactive signal is then

registered and is inversely proportional related to the antigen in the complex [6].

Fluoroimmunoassay and Chemiluminescence immunoassay are other techniques similar to ELISA

but with different labeling. All of this assays can quantify the antibody-antigen complex and serve as

alternative and comparative strategies for immunosensors [7].

Immunohistochemistry is a similar assay that stains the antigen-antibody complex in a cell [8].

Immunohistochemistry and ELISA are powerful molecular tools for monitoring and detecting specific

antigens. But for these analytical methods appropriate labeled antibodies are required.

Quartz Crystal Microbalance – QCM

Quartz crystal microbalance is an acoustic biosensor that has the ability of measuring and detecting

biological events in real time and label-free way [9]. The applicability in liquids has changed the use of

QCM in biosensors because it allowed not only the study of interface organization but also the study of

material coupled to the surface and the function played by the liquid in which the interfacial layer is

immersed [23,24].

Initially, QCM was developed to be a vapor phase sensor detecting gas molecules with the aid of

vapor species binding elements, such as polymers [25,26]. However, over the years QCM has been

applied in different molecular sensing event like DNA, proteins or cells. DNA biosensors were first

described by Fawcett et al. [14] where QCM was used to detect de hybridization of complementary

3

strands of synthetic RNAs. In the recent years, genetically modified organisms are being investigated

by hybridization of promoter sequences [15].

Protein adsorption for biosensor studies are difficult due to the different and complex structure of

proteins and their variety of structures among the protein population. This brings different frequency

responses that can’t be solved by Sauerbrey equation thus, the mass response concept can’t be applied

to forecast the amount of protein adsorbed. This is one of the QCM drawbacks and there are strategies

to overcome this situations. Nonetheless, it is important to know exactly if a protein adsorbs or not onto

a solid surface to determine if a material resists or promotes protein adsorption [16].

QCM can also evaluate the cell adhesion, spreading and proliferation to understand their

mechanisms in solid surfaces. For tissue engineering, this becomes of huge important to evaluate

material biocompability, like polymers. It is necessary to know the attachment differences in different

surfaces and the QCM has the ability to do that [17]

When QCM is used together with antibodies/antigen system it can be used as an immunosensor.

This strategy confers high specificity and detection limit which are important characteristics to measure

and quantify low antigen quantities. Highly pathogen avian influenza (H5N1) was detected in an early,

rapid and sensitive way in order to prevent and/or diagnose disease and mortality [18].

As it was previously stated, one of the QCM drawbacks is the difficulty to match the weight of

protein adsorbed with the Sauerbrey equation calculations due to the structural variety of proteins.

Although the QCM non-specificity nature emerges as a bigger problem, this technique has the ability to

measure mass changes but can’t translate that change with a specific analyte or other molecule

prevenient from a solution/medium due to its label-free characteristic. So, it’s necessary to mitigate the

undesired molecule to produce a frequency change. One approach is to purify the molecule of interest

being the only cause for the frequency shift but this is an expensive method. Another approach is in an

immunosensor coated with antigen, to block any free space of the surface with non-reactive species,

like ethanolamine, after coating the surface with self-assembled monolayer (SAM), for example. This

way there is an improvement of the binding efficiency due to a blocking of unreacted sites from non-

specific adsorption. [19]

It is possible to combine the QCM technique with other techniques to have a better understanding

of the system in different aspects. QCM technique can be combined with Atomic Force Microscopy

(AFM) to not only measure the relation of frequency with weight but also the surface coverage of the

molecule on top of the surface. Johannsmann et al. observed that even a 50% coverage film found a

good agreement with the acoustic terms [20].

In another analysis, it was combined three techniques: QCM, Surface Plasmon Resonance (SPR)

and Field Effect Transistor (FET). The FET measures the charge on the surface and the SPR the optical

refraction of the medium. These three different characteristics can be used to determine the protein-

material interactions both quantitatively and qualitatively, structural changes and biomolecular

rearrangements which is important to understand many biological functions, including structural

transformations, folding/unfolding and protein-protein interactions [21].

4

Theory

Quartz crystal has a piezoelectric effect which means that stress applied on the crystal causes

generation of an electric field, however the inverse piezoelectric effect is also observed. Quartz is

composed by two elements, silicon and oxygen (SiO2), and due to the non-linear nature of this bond (Si-

O) it is created a net dipole moment in the quartz. By applying a certain voltage on the quartz sensor,

which is covered with metal electrodes on the upper and lower sides, mechanical deformation is

generated. So, in theory, different voltages lead to different extents of mechanical deformation.

Therefore, the application of alternating electric field on the quartz sensor results in a cyclical

deformation, which is generated at the same frequency as the applied voltage (Figure 2). If this

deformation frequency matches the crystal’s inherent resonant frequency, an acoustic wave is

generated [22]. Thus, the surface event on the QCM sensor can be probed by its acoustic wave

propagation and properties variation, which can be converted into electrical signal through transducers

[23].

Quartz crystal can be cut in different angles that have different properties used in different

applications. The most widely cut used is the AT-cut obtained by an angle of 35º 15’ from the z-axis

(Figure 3) because of its frequency stability and high quality factor (Q) which means that quartz crystal

will be stable concerning time, temperature and other environmental changes, and have low energy loss

in oscillating systems thereby manifesting high accuracy.

Figure 2 - Schematics of QCM and QCM-D operation. Side view of the

crystal. Application of oscillatory voltage results in a cyclical deformation. The fundamental frequency (black waves at the edges of the crystal) and the third overtone (blue wave in the middle) are illustrated. [22]

5

As it will be later presented in the Sauerbrey equation, the frequency change is proportional to the

resonance frequency of the QCM and inversely proportional to the electrode area. So, an approach to

get higher mass sensitivity is to decrease the QCM thickness. This is one proposed physical strategy to

overcome low sensitivity.

QCM as a biosensor

The vibrational motion of the quartz crystal results in an acoustic wave that propagates back and

forth across the thickness of the crystal, between the crystal faces. When an alternating field of

appropriate frequency is applied on the electrodes of the QCM, it resonates at its resonance frequency,

which is given by:

𝑓0 =√𝜇𝑞

√𝜌𝑞×2𝑡𝑞 (1.1)

Where 𝑓0 is the resonance frequency of the resonating crystal, 𝜇𝑞 is the shear modulus of the

quartz crystal, 𝜌𝑞 is the quartz density and 𝑡𝑞 is the quartz thickness.

Considering 𝑀 as mass per unit area, 𝑚 the total quartz crystal mass and 𝐴 the area, then 𝑀 can

be defined as the product of the quartz density and thickness:

𝑀 =𝑚

𝐴= 𝑡𝑞 × 𝜌𝑞 (1.2)

Figure 3 - Crystallographic orientation of an AT-cut quartz

substrate. The crystal is cut 35º 15’ to the optical axis. [88]

6

Combining equation 1.1 and 1.2, it is possible to get:

𝑓0 =√𝜇𝑞×𝜌𝑞

2𝑀 (1.3)

The addition of mass on the top of the quartz crystal ∆𝑀𝑞 causes a change in the resonance

frequency of the oscillating crystal ∆𝑓:

𝑓0 + ∆𝑓 =√𝜇𝑞×𝜌𝑞

2(𝑀+∆M) (1.4)

Combining equation 1.3 with 1.4 and rearrange it:

𝑓0 = −𝑓0∆𝑀

𝑚(1+∆𝑀

𝑀) (1.5)

Now, considering a thin deposit wherein ∆𝑀 ≪ 𝑀 then equation 1.6 becomes:

∆𝑓 = −𝑓0∆𝑀

𝑀 (1.6)

Substituting 𝑀 and 𝑡𝑞 for 𝑡𝑞𝜌𝑞 and √𝜇𝑞

√𝜌𝑞×

1

2𝑓0, respectively, it is possible to get:

∆𝑓𝑠 = −2𝑓0

2

√𝜇𝑞×𝜌𝑞× ∆𝑀 = −𝐶𝑚 ×

∆𝑚

𝐴 (1.7)

Equation 1.7 is the so-called Sauerbrey equation. In 1959, Sauerbrey successfully demonstrated

the relationship between a change in frequency with adsorbed mass [24], where ∆𝑚 is the mass

deposited in the QCM, 𝐴 is the area of the resonator, ∆𝑓𝑠 is frequency shift and 𝐶𝑚, the Sauerbrey

constant, is known as the sensitivity factor dependent on the fundamental resonance frequency.

7

Equation 1.7 dictates that a change in the mass deposited on the active area of the exposed electrode

will decrease the resonant frequency and can be used as mass tool sensor. However, the Sauerbrey

equation only modulates thin, rigid and uniform films where it is assumed that the adsorbed mass follows

the vibration of the crystal, hence it behaves as if it were thicker. A different approach has to be taken

when the resonator becomes immersed in a fluid or the acoustic properties of the viscoelastic probe

layer are significantly different from those of the crystal sensor because if the mass is elastic it will not

follow rigidly the vibration and its viscoelasticity will contribute to a mass shift.

Since these situations are characteristic for biosensor applications, Kanazawa and Gordon

developed and expanded the Sauerbrey equation to include the liquid density and viscosity dependence

[25].

∆𝑓𝑙 = −𝑓03/2

× √𝜂𝐿𝜌𝐿

𝜋𝜇𝑞𝜌𝑞 (1.8)

Where 𝑓0 is the oscillation frequency of the free (dry) crystal, 𝜂𝐿and 𝜌𝐿 are the absolute viscosity

and density of the liquid, respectively. This relation is obtained from a simple physical model which

couples the shear wave in the quartz to a damped shear wave in the fluid.

So, whenever there is a two layer system combining a biological sample with a SAM thin film, then

it is necessary to add Equation 1.7 and 1.8 in order to have an overall frequency shift:

∆𝑓 = ∆𝑓𝑠 + ∆𝑓𝑙 = −𝑓02 × (√

𝜂𝐿𝜌𝐿

𝜋𝜇𝑞𝜌𝑞𝑓0+

2∆𝑚

𝐴√𝜇𝑞×𝜌𝑞) = −𝐶𝑚𝑠

× 𝐶𝑚𝑙×

∆𝑚

𝐴 (1.9)

Piezoelectric immunosensor based on QCM

The use of antibodies to coat the surface of the QCM gives an immunosensor character to this

technique. It is possible to introduce specificity and selectivity due to the antigen-antibody interaction

and add sensitivity from QCM properties. This relation has been studied and improved over the last

decades in order to quantitatively measure these biomolecules. Vibrio alginolyticus, a fish pathogen, is

infecting Portuguese aquacultures causing high mortality in fish species promoting an adequate

procedure treatment. The piezoelectric immunosensor is able to collect and detect the antibodies from

fish serum to develop effective vaccination protocols.

8

The detection of proteins with high sensitivity and selectivity is crucial for early diagnosis of

diseases and is the key factor for monitoring disease recurrence and therapeutic treatment efficacy. A

wide variety of analytes has been investigated where the first use of QCM as an immunosensor was

given by Shons et al. [26] where Bovine Serum Albumin (BSA) antibodies were detected. Since then,

there were studies using QCM immunosensor to detect deathly diseases at the early stages. A QCM

immunosensor was investigated targeting celiac disease which is an autoimmune disorder and the

autoantibodies, produced by an autoimmune reaction from people who suffers from this disease when

eat gluten, were found [27]. An immunoassay using antibodies against carcinoembryonic antigen (CEA)

was fabricated for early diagnosis and differentiating, monitoring curative effect, and follow-up on the

patients with tumour or carcinoma [28]. Diabetic nephropathy is a common cause of renal failure and is

a major complication of both type 1 and type 2 diabetes and the quantification of its markers are of huge

importance towards early treatment [29]. An immunosensor array for the rapid detection of HIV was

developed [30]. Cardiovascular disease was also studied were a competitive assay to detect C-reactive

protein (CRP), a cardiovascular biomarker, was developed [31].

Surface functionalization have a continuous and important role towards the immobilization of either

antigen or antibody. QCM immunosensors integrated with site-directed antibody immobilization

techniques improved the immunosensor sensitivity and stability as the antibodies started to be in a non-

random orientation (Figure 4A, B and C). In the beginning, one of the methods to aid the antibody

immobilization was using protein A or protein G, which are immunoglobulin-binding proteins with

different specificities. Protein A was first reported in 1987 by Muramutsu et al. [32] as binding proteins

in immunosensing applications. It has also been used to help the detection of pesticides [8,9], bacteria

[10,11], and viruses [37] while protein G was used in BSA, bacteria [38] and other analytes [39]. It is

also possible to immobilize recombinant Fab’ fragments to produce self-assembled monolayers directly

on the gold surface in order to create oriented antibody (Figure 4D) [40].

Nowadays, chemically functionalized surface provides an antibody/antigen immobilization without

altering the biological activity in a cheaper and easy way. The formation of self-assembled monolayers

(SAMs) with terminal active functional groups (i.e., COOH, OH and NH2) has been recognized as one

of the most effective approached for immobilizing probes. QCM gold surface can be treated with thiol

followed by EDC-NHS to form an active NHS ester which is highly specific to amine groups present in

antibodies [41]. Other SAMs were develop and optimized for each specific study. A mixture of

mercaptopropionic acid and mercaptoethanol acid was formed to control the concentration of biological

molecules on the surface of the probe sensor fixing the immunecompetence and reducing steric

hindrance [17,18]. Different 16-mercaptohexadecanoc acid and 11-mercapto-1-undecanol ratios were

constructed to investigate the best result in terms of SAM orientation with protein immobilization [42].

9

Figure 5 – Schematic presentation of an amplification strategy using magnetic beads. [44]

In some cases QCM immunosensors suffer from low sensitivity because the mass change by

antigen binding to the probe is small. So, there is a need to enhance the assay sensibility using different

strategies like increasing the deposited mass with nanoparticles, enzymes, liposomes, etc. Gold

nanoparticles are currently being used due to a good biocompatibility, large specific surface area and

high surface free energy [43]. With the same purpose magnetic beads were also used because it

provides easy and simple purification by magnetic separation (Figure 5) [44].

Figure 4 - Schematic representation of antibody immobilization main approaches and site-directed antibody immobilization methods. A – Random antibody immobilization, B - site-directed antibody

immobilization, C - site-directed antibody immobilization via Fc binding proteins, D - via antibody fragments and E - via oxidized oligosaccharide moieties [89].

A

C D

B

E

10

Antibody-antigen complex quantification

For QCM immunosensors there are two different approaches to quantify the antibody-antigen

complex. One way is in taking advantage of the QCM physical properties and applying the Sauerbrey

equation in the study to determinate the mass variation uptake. This is a direct and simple approach

however, sometimes the method is not the best suit given some limitations aforementioned like the non-

specific binding of undesirable molecules [16,21,45-48].

Another approach is to do a calibration curve using different concentration of the molecule of

interest and measuring the respective signal, in this case the frequency shift. It is possible to produce a

linear regression of frequency shift versus concentration in a given detection limit range. After that, it is

only necessary to add the sample with the unknown concentration of molecule of interest and read the

frequency shift value in order to take the concentration by the calibration curve [17,18,47].

There is also the possibility to join these two approaches to take advantage of their characteristics

and also to give confidence in the final results (Figure 6). The calibration curve used with the Sauerbrey

equation has the purposed of determine the detection limits. When this point is passed there will be a

non-linear response of the frequency shift over the concentration meaning that the results feasibility are

lost [48].

Figure 6 – Representation of two different quantification approaches. A – Calibration curve of a HIV-1 antigen-

QCM sensor enhanced by streptavidin-Au with different concentrations of the target. B - Schematic illustration of binding targets and respective frequency shift uptake in order to determine the mass change. [48]

A B

11

Autolab EQCM module

After a mass change in the QCM surface and, therefore, a change in the quartz crystal resonance

frequency it is necessary to detect, process and display the signal to the user. This is done by a

potentiostat using the module for QCM measurements. The resonant frequency will change as a linear

function of the mass of material deposited on the crystal surface.

To perform this assay the front-surface QCM (Figure 7B) has to be immersed in an electrolyte

inside of a crystal holder (Figure 7C) and the measurements are done inside a faraday cage to mitigate

vibrations caused by the exterior environment (Figure 7A). An oscillator is in contact with the cell which

receives the frequency output and digitalizes to voltage by a proper software, displaying in the end. This

device has a detection limit of the order of micrograms range. Finally, the quantification of the mass

change can be performed using the Sauerbrey equation because the frequency change and its constant

are known [49].

Vibrio alginolyticus

Vibrio alginolyticus is a halophilic gram-negative bacteria that is a natural host in aquifers

environment. It is a pathogen in humans which is responsible for skin, eye and ear infections. The

species carry the trh virulence gene which has the potential to infect not only humans but also fish. [50].

This subject is important due to economic losses because V. alginolitycus has been associated to

disease outbreaks in cultured fish. In this project there is an interest to study new vaccination protocols

towards the fish treatment in aquaculture environments.

Figure 7 – Representation of A - Autolab faraday cage and, B - Quartz Crystal Microbalance and C - its holder.

A

B

C

12

Gram-negative bacteria, such as vibrio alginolyticus, have double membranes that provide nutrient

uptake for viability and external protection. It is composed by an Inner Membrane (IM) and an Outer

Membrane (OM). Between these two membranes is located the periplasm. Regarding the OM, it

presents a composition between lipids, lipopolysaccharide, lipoproteins and integral membrane proteins.

These proteins located in the OM are called Outer Membrane Proteins (OMP) [51]. They have a

characteristic β-barrel structure that helps in a gain of stability to overcome harsh and variable

environment (Figure 8) [52]. Moreover, because of their external exposition OMPs are integral proteins

capable of nutrient uptake, waste export and cell adhesion and signaling. However, they serve as a

virulence factor for nutrient scavenging and evasion of host defense mechanism in pathogenic strains.

Their biogenesis begins with OMPs being synthesized in the cytoplasm by ribosomes [53]. After

that, they pass through a small pore in the IM barrier, still unfolded. As they enter in the periplasm

aqueous environment, the unfolded OMPs interact to chaperones to guarantee a correct fold and to

avoid misfold due to a prone aggregation in the periplasm. Finally, OMPs are set in the OM by the β-

barrel assembly machinery complex (

Figure 9 and Figure 10).

The vibrio alginolyticus OmpW was investigated to be used has a vaccine candidate in fish [54]. In

a related study, vibrio alginolyticus OmpK was also investigated for the same purposes [55]. In fact,

these proteins showed high similarities with other vibrio species in the C-terminal, where a phenylalanine

is located, which translates as a high conserved locus that can serve as an epitope [56].

Figure 8 – 3D structures of 2 different Outer Membrane Proteins. A - Bacterial

β-barrel membrane protein A (OmpA) and B - Bacterial β-barrel membrane protein F (OmpF). [90]

A

B

13

As it was stated, OMPs have external exposition and these fractions can be recognized as foreign

antigens by hosts, triggering an immune response. Over the years, these fractions, or epitopes, have

been studied and used to develop vaccines by immunization technique.

Figure 9 - The OMP journey to the outer membrane. Nascent OMPs (maroon) are synthesized in the bacterial

cytoplasm and delivered by the SecA/SecB chaperones (purple) or by the ribosome (red) to the Sec translocase (grey) for passage through the IM. Once in the periplasm, nascent OMPs can be delivered to the BAM complex by the periplasmic chaperones SurA (slate) or Skp (blue). Any OMPs which misfold during their journey will be degraded by the protease DegP (light blue) to prevent aggregation. [51]

Figure 10 - Gram-negative bacterial double membrane envelope. Schematic representation of inner membrane,

periplasm and outer membrane and its constituents [57].

14

Immunization

Vaccination is a type of immunization that stimulates an immune response to develop an adaptive

immunity to a pathogen after an antigen material is administered. The antigen, presenting no

pathogenicity, is exposed to the host who considers it as foreign and develops the ability to quickly

respond to a subsequent encounter due to the immunological memory, creating specific antibodies

towards the antigen. Thus, the host specific antibody acquisition can be further quantified in different

regimes. In the 21st century, an array of microbiological and molecular allow antigens for new vaccines

to be specifically identified, designed, produced and delivered with the aim of optimizing the induction

of a protective immune response against a well-defined immunogen [58].

Vaccine delivery can be performed by different approaches like oral, immersed or injection. Oral

vaccination with antigen is added in the feed. However, the antigen is destroyed in the gut making this

approach poor and inconsistent [59]. Injecting in the body cavity, typically an intraperitoneal injection,

has the advantage of administer precisely the right vaccine concentration but it makes a labor and

intensive method. To overcome these drawbacks, it is possible to immerse the vaccine in the solution

making easy to administer and cheap. The first commercial available bacterial vaccines in fish were

against vibrosis. The inactivated whole-cell formulations were added in the solution so the administration

was done by immersion. [60].

After immunization, serum is collected and processed to titrate the specific antibodies.

15

16

Material and Methods

Introduction

This chapter will describe the methodology used in this thesis. All steps from the calibration process

to the work electrode characterization, through quartz crystal microbalance functionalization and

quantitative and experimental methods, until the quartz crystal microbalance regeneration are

discriminated and justified.

The strategy followed in this thesis was a simply and common one. To achieve the final goal, the

antibody quantification, it was first produced a thiolated-SAM on top of the QCM Au surface. Following

that, build up a suitable reactive group, by the EDC plus NHS conjugation, to manage antigen biding.

Prior to antibody quantification, a calibration curve related to the antigen was performed to evaluate and

study a film coverage around 80% of the surface. Another calibration curve was realized, only this time

using the antibody, after setting an antigen concentration and work through that value. Fish serum was

also evaluated

A cyclic voltammetry was realized between each experiment to guarantee and confirm the molecule

binding. Finally, to regenerate the QCMs used, they were immersed in a piranha solution.

Reagents

Sulphuric acid (H2SO4, 98%) and Sodium iodide (NaI, 99,5%) were purchased from Merck

Company (Germany). Hydrogen peroxide (H2O2, 30% v/v), 11-Mercaptoundecanoic acid (11-MUA,

95%), N-Hidroxisuccinimide (NHS, 98%), Bovine Serum Albumine (BSA, 96%), Ethanolamine (98%)

were supplied from Sigma-Aldrich (USA), respectively. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride) (EDC) and Anti-Bovine Serum Albumin Rabbit Polyclonal Antibody was purchased from

Thermo Fisher Scientific (USA). Potassium hexacyanoferrate (II) trihydrate (99,5%) was purchased from

Fluka (USA).

All chemicals used to produce buffer solutions were of analytical pure grade or better quality such

as ultrapure and low conductivity Milli-Q water. 2-(N-morpholino)ethanesulfonic acid (MES, >99,0%),

sodium chloride (NaCl, ≥ 99,5%), potassium chloride (KCl, ≥ 99,5%), were purchased from Sigma-

Aldrich (USA). Potassium dihydrogen phosphate (KH2PO4, 99%) and were purchased from Panreac

(ES).

17

Materials

Quartz Crystal Microbalance – 7.995 MHz AT-cut quartz crystals, coated with optically flat polished

gold electrodes on both sides was purchased from IJ Cambria Scientific (Carmarthenshire, UK). The

sensing gold electrode area is 0.205 cm2.

Autolab faraday cage and CHI440B 220V potentiostat were supplied from CH Instruments, Inc.

(Texas, USA)

All data points registered were recorded by CH Instruments Software version 11.04 © and all

graphics were produced by Wolfram Mathematica 11 version 11.0.1.0 ©.

QCM Characterization

Gold electrode surface active area and Sensibility factor

It is important to assess if there are defects and deviations between experimental and theoretical

approaches through the Sauerbrey equation before biomolecule quantification. Therefore, a calibration

of the microbalance is necessary to have confidence in the subsequent results.

The microbalance sensitivity factor can vary due to differences in the resonant frequency, materials

and shear modulus and it may occur a difference between theoretical and experimental values of 20%

[61]. One can neglect deviation as far as 10% where the optimum value should be 1%.

Sensibility factor lies on the verification of the Sauerbrey law and is described as the relation

between the frequency variation of a crystal quartz and its mass change. Its value can be calculated

through two distinct electrochemical methods, the metal electrodeposition and halide ions adsorbption

in metal surfaces [64, 65, 66]. Metal electrodeposition calibration method, as the silver

potentiodynamical deposition and dissolution, has been used widely [67,68]. However, after silver

deposition it becomes difficult to remove it entirely from the electrode surface leading to interferences in

further experiments. In this work halide ion adsorbption method was accomplished using iodine as

covering neutral monolayer [69,70]. In terms of QCM characterization the Au electrode surface area and

sensibility factor will be calculated.

The Au electrode active surface area (𝐴′) can be calculated after treating the Au electrode with

iodine and scanning by cyclic voltammetry. Since the iodine chemisorbs on Au surface to form a neutral

monolayer then the area can be obtained by the relevant electrolytic charge (𝑄) [69]:

18

𝐴′ =𝑄

𝑛×𝐹×𝛤𝐼 (2.1)

Where 𝑛 is the number of electrons for the specific electrode reaction associated with chemisorbed

iodine, 𝐹 is the Faraday’s constant and 𝛤𝐼 is the calculated iodine packing density, based on the premise

that the chemisorption of iodine is space-limited. This means that it is assumed an iodine chemisorbed

hexagonal close-packing with unassociated atoms. This premise allows the calculus of the average area

occupied by the spherical iodine atom (𝜎𝐼):

𝜎𝐼 = 2√3 × 𝑟𝑣𝑑𝑤2 (2.2)

Here, 𝑟𝑣𝑑𝑤 is the Van der Waals radius of the iodine atom and can be related to 𝛤𝐼 by:

𝛤𝐼 =1

𝑁𝐴×𝜎𝐼 (2.3)

Where 𝑁𝐴 is the Avogadro’s constant. Finally, the number of electrons is given by the iodine

oxidation reaction:

𝐼(𝑎𝑑𝑠) + 3𝐻2𝑂 = 𝐼𝑂3(𝑎𝑞)− + 6𝐻+ + 5𝑒− (2.4)

And 𝑄 is obtained as the net oxidation charge of iodine between peak 1 and 3, in Figure 11. The

peak 1 consists not only by the charge of the oxidation process of adsorbed iodine but also the Au

substrate. Meanwhile, peak number 3 represents the charge of the reduction process of the Au substrate

and since they are symmetric it is possible to substrate peak 3 from peak 1 to have the net oxidation

charge for the adsorbed iodine.

19

Figure 11 – Cyclic voltammetry in H2SO4. Characteristic iodine-adsorbed gold

electrode voltammogram [68].

The sensibility factor (C) can be obtained rearranging the Sauerbrey equation (1.7):

∆𝑓 =−2∆𝑚×𝑓0

2

𝐴′×√𝜇𝑞×𝜌𝑞 (2.5)

Where ∆𝑓 is the frequency change caused by ∆𝑚, ∆𝑚 is the mass change of the quartz crystal

surface, 𝑓0 is the resonant frequency of the quartz crystal in the absence of the deposited mass, 𝐴′ is

the geometric area of the electrode, 𝜇𝑞 and 𝜌𝑞 are the shear modulus and density of quartz, respectively

[66].

𝐶 =∆𝑓

∆𝑚 (2.6)

Plotting the ∆𝑓 vs. 𝑃𝑜𝑡𝑒𝑛𝑡𝑖𝑎𝑙 assay, one can assess the frequency change due to the iodine

desorbed from the Au electrode. The mass of this desorbed iodine can be calculated by the following

equation:

𝑚𝑎𝑑𝑠 =𝑄×𝑀𝐼2

𝑛×𝐹 (2.7)

20

Where 𝑀𝐼2 is the molecular weight of the adsorbed iodine on the Au surface. It is then possible to

correct these variables in the Sauerbrey equation to fit in the real conditions worked.

Cyclic voltammetry

In order to fully guarantee that the frequency changes are indeed a direct cause of the mass

retained at the Au surface a cyclic voltammetry was realized after every assay.

Cyclic voltammetry is an electroanalytical technique that measures the variation of the potential

difference between the work electrode and counter electrode and the resulting current between working

and counter electrode. This technique translates the redox potential behavior of electroactive species

by understanding how the current changes according to the voltage applied. It is then possible to plot a

voltage versus current to obtain a voltammogram [70]. The voltage is swept at two fixed potential values

where a scan is done at a certain rate, so in a positive scan as the voltage increase the current will also

increase because it becomes closer to the redox potential. Past this point, and the voltage continue

increasing, the current will start to decrease, thus forming a peak. This happens because all the reduced

form was oxidized near the electrode surface when a potential is applied higher than the redox potential.

The negative scan will also produce a peak because there will be a regeneration of the reduced

species originating a current with inverse polarity, completing the cycle. This peak is important because

indicates if the reaction is reversible. Moreover, the shape of voltammograms are not only dependent of

the analyzed species but also of their concentrations.

In this work a three-electrode cell was used in junction with potassium ferrocyanide as electrolyte.

The reference electrode was Ag/AgCl, the counter electrode was a platinum wire and the gold QCM

surface as a working electrode. When the positive scan starts the current will gradually increase as the

voltage applied begins to approach the potassium ferrocyanide redox potential, where it will be oxidized

at the gold surface releasing electrons:

𝐹𝑒(𝐶𝑁)6−4 → 𝐹𝑒(𝐶𝑁)6

−3 + 𝑒− (2.8)

After reaching this potential, the reduced species starts to deplete and the current decays until the

negative scan starts and the potential is reversed. A negative peak is formed as the voltage applied is

strong enough to regenerate the reduced species.

𝐹𝑒(𝐶𝑁)6−3 + 𝑒− → 𝐹𝑒(𝐶𝑁)6

−4 (2.9)

21

The current value will be proportional to the barrier resistance between the electroactive species

and the Au surface. So, one can study if there are adsorbed species after each assay blocking the

electrons transfer to the working electrode. Therefore, it is expected a decrease in the current as layers

begin to pack above the Au surface [71].

QCM functionalization

The strategy followed in this work consisted of two key steps for QCM functionalization. Taking

advantage of the gold selectivity for thiols, the first step was covering the gold electrode by an alkylthiol

to form a SAM and, then, conjugate the SAM with a zero-length crosslinker presenting a suitable active

group for bioconjugation.

The self-assembled method presents a simple, oriented, ultra-thin and well-ordered packed

monolayer strategy. The gold selectivity for thiols comes with a strong chemical bond between gold and

sulphur [72]. This spontaneously and reproducible adsorption is dependent of the thiol chain size. It is

expected that long-chain thiols need more time to stabilize due their adsorbtion kinetics, both physisorb

and chemisorb, as they undergo different configurations [73]. For example, in the initial adsorption there

is physisorption through Wan der Waals interaction where the alkane chain interacts with the surface

producing a “lying down” configuration. Afterwards, the molecule evolves to a “standing up” mode where

thiol molecules chemisorb through the sulphur headgroup (Figure 12) [74].

The activation of the quartz crystal surface was performed by immersing it in a solution of 11-

Mercaptoundecanoic acid. As the result of the self-assembled monolayer deposition it was generated

an oriented monolayer with carboxylic groups in the outer end, demonstrated in Figure 13.

22

Figure 12 – Scheme of the different steps taking place during the self-assembly of alkanethiol on Au: i) physisorption, (ii) lying down formation, (iii) nucleation of the standing

phase, (iv) completion of the standing up phase [73].

Figure 13 – Schematic representation of a SAM on Au. Final structure of a series of 11-Mercaptoundecanoic

acid molecules after reacting with QCM Au surface.

23

The last step is generating a zero-length crosslinker bonded covalently to the thiol molecule, as

can be seen in Figure 14. Zero-length crosslinkers are small molecules that are widely used in

bioconjugation due to their water-solubility and low cross-reactivity. Carbodiimides are one of that group

capable of overcome the absence of high reactive chemical groups at the surface of thiol molecules,

thus they can mediate the condensation of primary amine with a carboxylic acid originating an amide

linkage [75].

EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) plus NHS (N-

hydroxysuccinimide) are very popular water-soluble carbodiimides. Although its advantages EDC is very

unstable in the presence of water so there are some precautions to be taken when using this reagent

as a crosslinker. First of all, it should be stored at -20ºC and warmed to room temperature before its use

to avoid condensation and decomposition over time.

Moreover, EDC can react with other nucleophiles, such as sulfhydryl groups and oxygen atoms,

and for that reason water molecules can hydrolyze o-acylisurea, a byproduct, which can lead to the

formation of isourea and, consequently, the regeneration of the original carboxylate group [76].

One way to increase the yield’s reaction is to perform it in a pH near or above 6.5 where the

carbodiimide stability reaches best results [77]. A suitable buffer is important like MES (2-(N-morpholino)

ethanesulfonic acid) because exhibits a pKa around 6.15 at 25ºC. In this work the yield differences

between MES buffer and 70% ethanol are studied.

It goes without saying that every experiment was performed under room temperature, 25ºC, to

mitigate error and deviations that temperature may cause to the QCM.

24

Figure 14 – Representation of the reactions by a carbodiimide-catalyzed reaction using the EDC

plus NHS system to form an amide bond [76].

25

Quantitative methods

Using the Sauerbrey equation it is possible to quantify the adsorbed species on the QCM surface.

So, rearranging the Sauerbrey equation to obtain a mass variation we get:

∆𝑚 =𝐴×𝛥𝑓×√𝜇×𝜌

−2×𝑓02 (2.10)

The next step is to take the mass variation and translate it to number of molecules (N) adsorbed

by the following equation:

𝑁 =∆𝑚×𝑁𝐴

𝑀𝑊 (2.11)

Where 𝑁𝐴 is the Avogadro’s number and 𝑀𝑊 is the molecular weight. Having the number of

molecules adsorbed it is also possible to calculate the overlay area cause by them and calculate the

bind ratio between two different molecules. In the first case, the final result can give an indication of how

many layers of a given molecule exists after an assay [78]. The overlay area (AT) can be calculated

simply by multiplying the molecule area (Amol) and its number of molecules:

𝐴𝑇 = 𝐴𝑚𝑜𝑙 × 𝑁 (2.12)

A calibration curve was performed varying the antigen concentration, in this work BSA was used,

and the consequent frequency change was registered. If it appears to be in the linear zone, there is a

need to find the precise BSA concentration that traduces in a QCM surface overlay of 80%,

corresponding to a BSA monolayer.

After setting the corresponding BSA concentration another calibration curve varying the antibody

anti-BSA was done. The quantification is based in the same procedure done so far, using the same line

of thought and equations. The goal is to achieve a proof-of-concept about this methodology for

determination and antibody quantification through QCM analysis in this particular system.

26

Experimental Methods

QCM cleansing

The QCM were cleaned before use by rinsing with 70% ethanol and Milli-Q water before immersion

in Piranha solution (30%, v/v, H2O2:H2SO4 = 1:3) for a few minutes, to obtain a clean gold surface.

Cleaned QCM were then rinsed with water and dried in an air stream. This is an important step because

it can remove impurities, such as oxides, from the gold surface.

After cleaning, the QCM was mounted in the cell holder composed by three round Teflon pieces.

Each Teflon piece has different purposes, for instance, the bottom piece holds the QCM tight with their

four screws against the middle piece, the middle piece is the reaction chamber where the solution is

dropped on and the top piece aids the counter and reference electrode balance as exhibited in Figure

15 [78].

Figure 15 – Schematic illustration of a reaction cell. a) before and b) after tightening [78].

QCM characterization

The first experimental method done was the calibration and to get the Au surface area, a control

(clean) and a test (iodine-pretreated) QCM were examined. For the control experiment, a clean QCM

Au surface was immersed in 3mL of 1M H2SO4 solution and, for the test experiment, a clean QCM Au

surface was also used but, this time, it was immersed on a 3mL solution of 100mM NaI/1M H2SO4. In

both situations, a cyclic voltammetry was performed ranging a sweep between 0 and 1.5 volts and their

respective voltammograms were obtained. Afterwards, the voltammograms were compared and

analyzed.

a b

27

At the same time the test QCM cyclic voltammetry was underway, a plot of Δf vs. Potential was

executed. The goal was to find the sensibility factor due to a frequency change caused by desorption of

iodine from 1.5 to 0 volts.

Cyclic Voltammetry

This technique was performed after every functionalization step. For the next step of experiments

clean and unused QCMs were chosen, and then, bare Au cyclic voltammetry was the experimental

method performed after the calibration. The goal was to obtain a control voltammogram of the system

where the electrolyte of this assay was a solution containing 5mM Potassium hexacyanoferrate(II)

trihydrate and 100mM KCl in deionized water. 3mL was dropped in the reaction chamber contacting the

Au surface, or working electrode, and both counter and reference electrode. It was performed a 10 cyclic

voltammetry ranging between -0.6V and 0.8V traducing in a bare Au voltammogram.

QCM functionalization - 2mM 11-Mercaptoundecanoic acid

Between each experiment the QCM was dismounted and the surface dried with an air stream. The

frequency change measurements started with a dried Au surface and, about 1 minute later, it was

dropped 200μL of the solvent solution, which in that case was the 70% ethanol, and was left to stabilize

for 30min. That strategy enables the user to see the frequency changes caused exclusively by the

solution molecules desired. The volume corresponding of the 70% ethanol in the reaction chamber is

pulled out entirely and only after that began the functionalization method where a 200μL of 2mM 11-

Mercaptoundecanoic acid in 70% ethanol was deposited on top of the Au surface. During 20h the

frequency change caused by the binding of the thiol molecule with gold was registered. It was also

removed the unbound molecules to the surface that were causing an unwanted frequency change by a

washing step.

To assess the functionalization process in a qualitatively way, another cyclic voltammetry was

realized in the same conditions used before. Therefore, it was added 3mL of the electrolyte on top of

the functionalized surface and obtained its voltammogram.

28

Figure 16 - Schematic representation of a typical EQCM instrument. The quartz crystal has a fundamental

frequency of 7.995 MHz and is coated with thin gold films on both sides. The gold disk deposited on the top side of the crystal is in contact with the electrolyte solution and used as the working electrode. The top view of the gold-coated crystal is also shown.

EDC/NHS complex

Continuing the functionalization process, a zero-length crosslinker solution was made by joining

50mM EDC and 50mM NHS in 50mM KCL MES buffer. Frequency change measurements started again

with dried QCM functionalized and, one minute after recording, 200 μL of MES buffer solvent was laid

for 30 min to stabilize. Continuing the functionalization process it was added 200μL of a solution

containing 50mM EDC/NHS in 50mM KCL MES buffer and, after 2h, the QCM surface was washed to

remove molecules in excess. Another cyclic voltammetry to evaluate the reaction occurred was

performed.

BSA and antibody anti-BSA

Afterwards, BSA was diluted in Milli-Q water at different concentrations, from 1 mg/mL to 0.125

mg/mL. In order to register the frequency variation through BSA bound to the crosslinker, 200 μL of Milli-

Q water was added for 30min to a dried QCM surface and, then, 200 μL of BSA solution were added to

the reaction chamber. Again, non-specific BSA ligated was removed by washing the QCM surface.

Finally, after the BSA-evaluation cyclic voltammetry, antibody anti-BSA assay was performed. It is

assumed an 80% BSA surface coverage so it is of extreme importance to block non-specific sites.

Ethanolamine is a molecule capable to react with the EDC/NHS reactive group and, therefore, prevent

the non-specific binding of antibody anti-BSA to other molecules than BSA [79]. 200 μL of 100mM

Ethanolamine was applied in the QCM BSA-surface before adding the antibody anti-BSA.

The starting procedure was then replicated as usual where the solvent solution was PBS buffer,

pH 6.5. 200 μL of PBS was dropped on top of the Au surface for 30min and, after PBS removal, 200 μL

of 10 μg/mL of anti-BSA antibody in PBS was added.

29

As always, the washing step was done to remove unbound antibodies and a cyclic voltammetry

was done. In the end it is expected to have a molecular structure as it is demonstrated in Figure 17.

Figure 17 – Schematic representation of 11-Mercaptundecanoic acid SAM formation on gold electrode in

conjugation with antigen and antibody immobilization [80].

Fish serum

Fish serum was also evaluated in terms of proteins in solution. The procedure used was based

as described in the beginning of this chapter, from the QCM treatment with piranha solution until the

EDC/NHS binding. From this point, a 200 μL of Milli-Q water was added for a few minutes and,

afterwards, removed. The final step consisted in dropping 200 μL of a fish serum on the QCM surface

and the frequency shift registered for 1h.

To make the calibration curve the fish serum stock solution was diluted in 5 samples in Milli-Q

water. The dilutions made from the stock solution were 1:100; 1:150; 1:200; 1:250 and 1:300.

Ionic strength behavior

In the present work BSA was diluted in Milli-Q water for measurement purposes. Although a PBS

medium is much more suitable for proteins, in this QCM analysis and, specifically in an electrolyte-liquid

30

system, ionic strength can produce an erratic behaviour through measurements involving the diffusion

double layer in the interface between the bulk phase and the molecule charged surface.

In biological systems, liquid state is present in many assays where solutions can include electrolyte

and biomolecules. For QCM measurements, the Kanazawa equation (1.8) describe linearly viscoelastic

films for non-electrolyte solutions. However, for electrolyte solutions this equation cannot predict the

behaviour because it causes strong interferences, which are mainly due to the elastic contribution of the

diffuse double layer (DDL) near the metal covered quartz [81].

When the DDL is formed, counter ions will be attracted to the surface of the charged molecule

creating a plane with a high density of this counter ions attached, the inner Helmholtz plane. At the other

end is present the outer Helmholtz plane with ions with the same charge as the molecule surface.

Between these 2 planes stands the diffuse part of the double layer [82].

This DDL thickness is called Debye length (DL) and can be influenced by the concentration and

valence of the electrolytes. If the concentration increases, or using ions with high valences, the tendency

is to shorten the Debye length where the entire double layer draws near the surface. Therefore, there

will be an accumulation of charges at the surface of the electrodes. Electronically, the DDL can be

compared as a capacitor-like system [83]. The DDL viscoelasticity is, then, strongly dependent on the

surface charge, the bulk electrolyte concentration and the dielectric constant of the solvent [84].

Interferences can be due to an ionic atmosphere relaxation. It is possible that the surface movement

at 8 MHz cannot be followed by the ionic atmosphere leading to transients mimicking desorption with

frequency variations less accurate [85,86]. A similar study to what is being done here, came to a

conclusion that these anomalies can cause an error more than 70% for a specific electrolyte

concentration [81].

QCM regeneration

In the end, the quartz crystals were regenerated before reuse by immersing in Piranha solution

(30%, v/v, H2O2:H2SO4 = 1:3) for a few minutes, to obtain a clean gold surface. Regenerated QCMs

were then rinsed with water and 70% ethanol and dried in an air stream and stored.

31

Results and Discussion

QCM Characterization

Gold electrode surface active area and Sensibility factor

To fully characterize the QCM parameters, hence calculating the gold electrode surface active area

and the sensibility factor, it is necessary to do a calibration assay. As it was stated in the previous

chapter, an iodine monolayer was formed in the QCM Au surface to assess the apparent active area

and, after this treatment, it was realized a cyclic voltammetry and, at the same time, a ∆𝑓 vs. 𝑃𝑜𝑡𝑒𝑛𝑡𝑖𝑎𝑙

was plotted. The cyclic voltammetry parameters were set to cover the key peaks which means

performing a cyclic voltammetry ranging a sweep between 0 and 1.5 volt in two voltammograms, one

control treated without iodine and one treated with iodine.

The control voltammogram is presented in Figure 18 and it has two main peaks, peak number 2

and 3. These peaks are related to the oxidation and reduction process of Au substrate, respectively. It

is important to know that the control QCM exhibits only Au substrate response as it is obsereved.

Figure 18 – Control voltammogram in H2SO4 solution. Cyclic voltammetry performed in a clean

QCM ranging 0 to 1.5 volt sweep with 2 peaks exhibited, peak number 2 and 3, the oxidation and reduction process of Au substrate, respectively.

2

3

32

The test voltammogram obtained shows 4 peaks, as indicated in Figure 19. This voltammogram is

similar as the one obtained by Dong et al. [68] and it is possible to correlate the peaks nature. Besides

peaks number 2 and 3 that are also present in control voltammogram, peaks number 1 and 4 are also

present. Peak number 1 represents, as said, by the charge of the oxidation process of adsorbed iodine

and corresponds to reaction of equation 2.4. Furthermore, peak number 4 represents the inverse

reaction of equation 2.4 and the desadsorbed iodine forms I2. Differences between voltammograms are

evident which give confidence in the further calculations.

Figure 19 – Test voltammogram. Cyclic voltammetry performed in an iodine-pretreated QCM ranging 0 to 1.5

volt sweep with 4 peaks exhibited. Peak number 1 and 4 are the charge of the oxidation and process of adsorbed and desadsorbed iodine. Peak number 2 and 3, the oxidation and reduction process of Au substrate, respectively.

The iodine packed density is calculated by equation 2.3 which is dependent of the average area

occupied by the spherical iodine atom, equation 2.2. The van der Waals radius of the iodine atom is

0.215 nm and the Avogadro’s constant is 6.022x1023.

𝜎𝐼 = 2√3 × 𝑟𝑣𝑑𝑤2 = 0.160 𝑛𝑚2

𝛤𝐼 =1

𝑁𝐴 × 𝜎𝐼=

1

6.022 × 1023 × 0.160= 1.04 𝑛𝑚𝑜𝑙/𝑐𝑚2

1

3

4

2

33

From equation 2.5, the number of electrons that take part in this reaction is 5, the Faraday constant

is 9.64x104 C/mol and the net charge (𝑄) is given by the difference between peak 1 and 3 is 1.2x10-4 C

and, thus, the Au surface active area (𝐴′) calculated by equation 2.0, is:

𝐴′ =𝑄

𝑛 × 𝐹 × 𝛤𝐼=

1.2 × 10−4

5 × 9.65 × 104 × 1.04 × 10−9= 0.23 𝑐𝑚2

After this, one can calculate the sensibility factor (C) by equation 2.6. But first, it is necessary to

achieve the mass adsorbed on the Au surface, using equation 2.7:

𝑚𝑎𝑑𝑠 =𝑄 × 𝑀𝐼2

𝑛 × 𝐹=

1.2 × 10−4 × 256.8

5 × 9.65 × 104= 54.18 𝑛𝑔

Figure 20 shows the frequency shift behavior caused by the desadsorbed iodine and its value is

34.80 Hz.

Figure 20 – Frequency shift of a test QCM showing the desadsorption process from the Au substrate after a

sweep from 0 to 1.5 volts.

Δf = 34.80 Hz

34

And, then, the sensibility factor calculated is:

𝐶 =∆𝑓

∆𝑚=

34.80

54.18= 0.64 𝐻𝑧/𝑛𝑔

To rectify the theoretical Sauerbrey equation it is important to assess the deviation from the

experimental values. For an error up to 10% it is possible to modify it and have confidence in the results

given by the QCM. Knowing the sensibility factor and Au active surface area it is possible to get the

resonance frequency and compare it with the batch fix values, which in this case the resonance

frequency is set at 7.995 MHz.

Now, assuming a resonance frequency of 7.995 MHz, substituting 𝐴′ for the newly found value

and knowing the crystal shear modulus (𝜇) and density (𝜌), 2.648 g/cm3 and 2.947x1011 g/cm.s2,

respectively, the theoretic sensibility factor is:

𝐶 =∆𝑓

∆𝑚= −

2 × (𝑓0)2

𝐴′ × √𝜇𝑞 × 𝜌𝑞

𝐶 = −2 × (7.995 × 106)2

0.23 × √2.648 × 2.947 × 1011= 0.63 𝐻𝑧/𝑛𝑔

So, there is a deviation of about 3% between theoretical and experimental values which is

acceptable and can be used in further results ahead.

Moreover, the software used is capable of giving the exact resonance frequency for each QCM

operated in a giving assay. This method is more accurate due to errors and variance in the sensibility

factor and Au active surface area experiments. Nonetheless, a calibration assay was proved to work in

the future.

35

Cyclic Voltammetry

Another type of characterization is cyclic voltammetry analysis but, instead of QCM parameter

assessment, this will evaluate biomolecule binding. This technique was applied before, between and

after the full assay, which means the biomolecule binding can be assessed in all steps.

A clean QCM was analyzed (Bare Au) before the coating started. From there, after every assay, a

cyclic voltammetry was performed and the results were compared. In the end, there will be a

voltammogram from Bare Au, 11-MUA, EDC/NHS, BSA and Antibody anti-BSA, as can be seen in

Figure 21.

Figure 21 – Cyclic voltammogram from -0.6 to 0.8V of Bare Au (Blue); 2mM 11-MUA (Orange);

EDC/NHS 50MM (Gray); BSA 1 mg/mL (Red) and Antibody anti-BSA (Black). Hexacyanoferrate (III) trihydrate was the electrolyte.

The results were as expected meaning that, in every step the current measured was gradually less

resulted by a resistance increase from the layered molecules. The electrons from the electrolyte found

an increased blocked pathway to the work electrode from bigger molecules. In this case, 11-MUA, BSA

and the antibody presented an incremented shift demonstrated in Figure 21 where for EDC/NHS, a

small molecule, produced little change.

This technique allowed to take conclusions about every step to the end and constitutes an aid in a

non-selective technique.

36

QCM functionalization

The first part of the functionalization is coating a clean QCM surface, previously treated with piranha

solution, with 2mM 11-MUA and, in the process, register the frequency shift along time. Figure 22 shows

this variation over 3h. Initially, ethanol was added to stabilize for 30 min (purple background) and, right

after ethanol was removed, the same volume of 11-MUA in ethanol was dropped on top of the QCM

surface to register the thiol binding on Au substrate for over 3h (yellow background).

Figure 22 – Frequency shift of 2mM 11-MUA after addition on top of a clean QCM surface. Purple background - Ethanol 70% solution; Yellow background – 2mM 11-MUA in ethanol 70% solution.

From Figure 22, it is observed a frequency shift of about 289 Hz demonstrating a mass deposition

in the QCM surface from 11-MUA. Past this functionalization, it was time to enhance selectivity by using

an EDC/NHS solution in MES buffer. Figure 23 exhibits the behavior of this molecule with the same

previously strategy applied.

Δf = 289 Hz

37

Figure 23 - Frequency shift of EDC/NHS 50 mM after addition on top of a thiolated-QCM surface. Brown background – 100mM MES Buffer; Dark Green background – EDC/NHS 50mM in 100mM

MES buffer.

This time, MES buffer (brown background) was added initially for 40min following the EDC/NHS

solution (dark green background) for 3h, ending with a MES washing step to remove unbound

molecules. This addition caused a frequency shift of 95 Hz. Taking advantage of this molecular structure

at this point, BSA 1 mg/mL was joined in the system as an antigen, Figure 24.

Figure 24 - Frequency shift of BSA 1 mg/mL after addition on top of a functionalized-QCM surface. Red background – Milli-Q water; Light Green background – BSA 1mg/mL in Milli-Q water.

Δf = 95 Hz

Δf = 126 Hz

38

This experiment started by dropping Milli-Q water (red background) for 30 min and, then, BSA 1

mg/mL in Milli-Q water (light green background) during 3h until a Milli-Q water washing step was

accopmlished. The washing step aims the removal of non-specific BSA binding which would cause

unwanted frequency shift, misleading to an unprecise read. The frequency variation registered was 126

Hz.

Between these two steps, ethanolamine was added to block non-specific sites that could result in

a false response when dropping the antibodies. This way, those sites are out of reach and the frequency

shift of the antibody is due to the specific binding antigen-antibody.

Finally, antibody anti-BSA 10 µg/mL was added and its frequency shift registered, as seen in Figure

25. The introduction of PBS solution (grey background) for 30 min was followed by an addiction of this

antibody (light blue background) during 2h. Once again, a PBS washing step was performed to remove

the excess of antibody. A 113 Hz frequency shift was recorded, as the final step.

Figure 25 - Frequency shift of antibody anti-BSA 10 µg/mL after addition on top of a BSA-QCM surface. Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA in PBS pH7 buffer solution.

Δf = 113 Hz

39

Quantitative methods

Now that one has the Sauerbrey equation reformed and frequency shift values registered, it is

important to understand the extent of them. This chapter will translate the frequency shift to adsorbed

mass, then, to number of molecules adsorbed and, finally, to QCM surface coverage due to those

molecules.

2mM 11-Mercaptoundecanoic acid

By the sensibility factor, C = 0.64 Hz/ng, the mass deposited on the Au surface for a frequency

shift of 289 Hz, is 451.56 ng. Since the 11-MUA molecular weight is 218.36 g/mol, the number of

molecules (N) on top of the surface, by equation 2.11, is:

𝑁 =∆𝑚 × 𝑁𝐴

𝑀𝑊

=451.56 × 10−9 × 6.02 × 1023

218.36= 1.24 × 1015 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠

To get the coverage done by this quantity of 11-MUA, it is simply multiply the number of molecules

by the area of the 11-MUA polar head (carboxylic group area = 37.3 Å2):

𝐴𝑇 = 𝐴𝑚𝑜𝑙 × 𝑁 = 3.73 × 10−15 × 1.24 × 1015 = 4.62 𝑐𝑚2

This means that, for a 2mM concentration of 11-MUA, 20 layers of thiol molecules are packed in

this assay.

EDC/NHS 50mM

The same strategy can be applied in the EDC/NHS reaction. A 95 Hz frequency shift translates a

148.43 ng of mass adsorbed. The molecular weight is 115.09 g/mol and the number of molecules (N)

is:

𝑁 =∆𝑚 × 𝑁𝐴

𝑀𝑊

=148.43 × 10−9 × 6.02 × 1023

115.09= 7.76 × 1014 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠

40

Now, it is interesting to know the efficiency of this reaction. Dividing both 11-MUA and EDC/NHS

number of molecules present, it gives:

𝑁𝑁𝐻𝑆

𝑁11−𝑀𝑈𝐴

× 100 = 7.76 × 1014

1.24 × 1015× 100 = 62.62%

It is shown that about 2 out of 3 11-MUA molecules can be activated by NHS molecules. This

efficiency can be improved using strategies stated in chapter 2.4. Moreover, the buffer effect in the

EDC/NHS reaction can be evaluated through the same calculations as above when using different buffer

solutions. When using a less suitable buffer this reaction drop efficiency and, in worst cases, there are

almost no 11-MUA molecule activated. Such example is demonstrated in Figure 26 where a dilution of

the EDC/NHS in Milli-Q water won’t activate almost none 11-MUA molecule.

The EDC/NHS concentration should be high enough to cover the thiol molecules bound at the Au

surface. In this case, 50mM of EDC/NHS should be enough for the 2mM 11-MUA, even taking into

account the efficiency problems that this particular reaction has.

Figure 26 - Frequency shift of EDC/NHS 50 mM after addition on top of a thiolated-QCM surface. Red background – Milli-Q water; Dark Green background – EDC/NHS in Milli-Q water.

Δf = 20 Hz

41

BSA 1 mg/mL

The strategy from this point on diverged slightly. The goal was to have a BSA surface coverage

around 80% (1 layer) allowing to study the antibody binding in an accurate and precise way. Hence, a

calibration curve was made for BSA concentration range from 0.125 to 1 mg/mL. Using as an example

the 1 mg/mL concentration, the mass deposited was 196.87 ng due to a 126 Hz frequency shift. Since

the BSA molecular weight is 66430 g/mol, then the number of BSA molecules deposited is:

𝑁 =∆𝑚 × 𝑁𝐴

𝑀𝑊

=196.87 × 10−9 × 6.02 × 1023

66430= 1.78 × 1012 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠

The total BSA area was calculated considering the BSA as an oblate ellipsoid with 2 radii, a or

“side-on” and b or “top-on”, Figure 27, where its values are set as a = 17.5 Å and b = 47.4 Å [85]. Besides

that, BSA Debye length (DL) has to be taken into account in these calculations. In this thesis, BSA was

diluted in Milli-Q water to mitigate anomalies in high ionic strength environments due to this precise

parameter but, even in this controlled condition, there will always be some charges in solution. For that

reason, a DL of 43.1 Å at 5mM ionic strength was set [86] and all these values were accounted for.

For lack of additional information, the BSA shape on top of the layer will be set as mostly on

horizontal position, as represented in Figure 27. This extrapolation is based on the number of possible

peptide bond formation between BSA and NHS being bigger than on the vertical position. Generally, it

will present more stability to the BSA and will be more likely to be in this position. Determination of the

BSA surface coverage will underway as follow:

Horizontal Position → 𝐴𝐵𝑆𝐴 = (𝜋 × (𝑟𝐵+𝐷𝐿)2) = (𝜋 × (47.4 + 43.1)2) = 24717.08 Å2 = 2.47 × 10−12 𝑐𝑚2

𝐴𝑡 = 2.47 × 10−12 × 1.78 × 1012 = 4.40 𝑐𝑚2

To find a suitable BSA concentration, a series of measurements were performed at 0.125; 0.15;

0.25; 0.75 and 1 mg/mL. The calibration curve is represented in Figure 28 along with its equation.

Forcing a 80% surface coverage, which is around 0.184 cm2, the number of molecules necessary to

achieve it would be 1.60 × 1011 and, consequently, a mass deposition of around 17.65 ng leading to a

frequency shift of 11.10 Hz. Substituting from the calibration curve equation, the optimum BSA

concentration is:

𝑦 = 122𝑥 + 4 ↔ 𝑥 =11.10 − 4

122= 0.05 𝑚𝑔/𝑚𝐿

42

Figure 27 – Illustration of a BSA in a horizontal position above a SAM. K-1 – Debye length; r – Sum of the BSA

radius and Debye length.

Since this is, not only really low frequency shift value, but also BSA concentration and, most likely,

will enter in the non-linear region of the technique or won’t be sensitive enough, a new value of BSA

concentration, 0.15 mg/mL, was set and used on further antibody experiments.

Nonetheless, the calibration produced a good linear and proportional interaction between frequency

shift and BSA concentration, achieving a R2=0.9932.

Figure 28 – Calibration curve of a BSA-QCM sensor with different BSA concentrations.

r

“Side-on”

“Top-on”

43

Antibody anti-BSA 10 µg/mL

After coating the QCM with 0.15 mg/mL of BSA, another calibration and its equation, Figure 29,

was performed using antibody anti-BSA ranging 1 to 10 µg/mL with an increment of 2.5 µg/mL. This had

the objective of proof-of-concept to, ultimately, knowing if another antigen-antibody system could be

used.

The molecular weight of a rabbit IgG is around 150 000 g/mol causing a frequency shift of 113 Hz.

The corresponding deposited mass was 176.56 ng and the number of molecules is:

𝑁 =∆𝑚 × 𝑁𝐴

𝑀𝑊

=176.56 × 10−9 × 6.02 × 1023

150000= 7.08 × 1011 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠

The calibration curve presented good results, showing a good relation between frequency shift and

antibody concentration, resulting in a R2 = 0.9923 and the respective equation is 𝑦 = 10.9𝑥 + 4

Figure 29 - Calibration curve of an anti-BSA immunosensor with different antibody anti-BSA concentrations.

44

Fish Serum

The 5 dilution samples produced a calibration curve represented in Figure 30 with an 𝑦 =

16915𝑥 − 0.9 equation and a R2 = 0.9961. Again, it was achieved a good linearity meaning it is possible

to measure the deposition of proteins with confidence in this system.

In the future, a QCM-based immunosensor could be applied to immobilize OmpK from Vibrio

Alginolitycus and quantify its specific antibody from a fish serum.

Figure 30 - Calibration curve of a QCM with 5 different fish serum dilutions - 1:300; 1:250; 1:200; 1:150

and 1:100.

Ionic strength behavior

Throughout this work, BSA was diluted both in PBS pH 7 and in Milli-Q water and the differences

in behavior were conclusive. The main difference between these two solutions is the ionic strength

where the formation of DDL between the SAM surface and the protein causes an erratic behavior when

measuring frequency variations. Moreover, BSA is negatively charged at pH = 7 [87].

Figure 31 shows this behavior where it is seen multiple random “ups-and-downs” without any

pattern. This kind of effect makes it practically impossible to analyze, evaluate and study the real

frequency shift caused by the protein. These variations can be due to a protein rearrangement in order

45

to be more stable and an ionic movement. A change to an almost zero ionic strength solution was proven

essential to the development and conclusion of this work.

Figure 31 – Abnormal frequency shift of BSA 1 mg/mL after BSA addition and removal on top of a functionalized-QCM surface. Gray background – PBS pH 7 buffer; Light Green background – BSA in PBS pH7 buffer

solution.

46

Conclusions and Future Perspectives

This work aims the development of a piezoelectric immunosensor to quantify antibodies from fish

serum. Ultimately, the final goal was not accomplished because neither the antibodies nor their specific

antigens were used. However, a piezoelectric immunosensor was successfully obtained from the

beginning using a well-known strategy. The fish antigen and antibody were substituted by BSA and

antibody anti-BSA in the process. Nevertheless, a proof-of-concept for this system was demonstrated

using these reagents along with fish serum showing good results.

It is important to know if the QCM is in order so a calibration is the starting step. By chapter 3.1.1,

the Au electrode surface active area and the sensibility factor were determined and compared with the

theoretical values. Although having deviations, both stood in the margin of error and were used to correct

further equations. The Au electrode active surface area and sensibility factor calculated were 0.23 cm2

and 0.64 Hz/ng, respectively. This is a good, simple and fast way to understand and have certainty of

following experiments.

After the calibration technique, cyclic voltammetry is also capable of characterizing the QCM,

mainly by the binding biomolecules. It is another simple technique able to assess this type of binding by

measuring the current flowing through the electrolyte and the working electrode. The resistance or

blocking increased by packing biomolecules led to an increase difficulty in current flowing. In chapter

3.1.2, it is observable that as the immunosensor number of layers increases, then, the current measured

decreases. The evaluation of the molecule binding is confirmed.

The immunosensor development strategy started with coating with 11-MUA responsible by a

frequency shift of 289 Hz due to 1.24 x 1015 molecules on top of the surface. Afterwards, the active

group was enhanced introducing the EDC/NHS reaction in the medium facing a frequency variation of

95 Hz from 7.76 x 1014 molecules. The reaction efficiency was calculated and 2 out of 3 11-MUA

molecules were activated by the EDC/NHS reaction. This particular step of the fabrication process is

important because EDC/NHS reaction can lose effectiveness from the buffer solution, hence, a MES

buffer is ideal as described in chapter 2.4, regarding Milli-Q water, per example. The differences are

incisive where a frequency shift of 20 Hz from Milli-Q water solution are way less than the 95 Hz

registered for MES buffer. Another key point in using this reaction fresh. A great loss in efficiency is

observed as time passes after being made.

The next step was introducing BSA molecules and perform a calibration curve to get the precise

BSA concentration needed to cover 80% of the QCM surface. The calibration curve was successfully

done. Due to the BSA shape it was difficult to set the optimum concentration and, because of that, a

value of 0.15 mg/mL was chosen. The antibody anti-BSA and fish serum calibration curve were also

successfully performed and because of this good agreement it is possible to quantify antibodies using

this technique and proteins from fish serum, respectively.

47

In the future, fabricating a different immunosensor using this present strategy is achievable. Per

example, Vibrio Alginolyticus OmpK could be immobilized in the QCM surface to early detection and

quantitation of antibodies, optimizing vaccination protocols. However, good washing steps and blocking

non-specific sites are key to minimize the errors because of the non-selectivity characteristic of this

technique combining with a several proteins and other biomolecules present in the serum that can be

misleading the final results.

Last but not least, it is important to highlight once again the possibility of DL formation when using

proteins in an ionic strength solution. Comparing results of two situations (BSA in PBS and Milli-Q water)

like in chapter 3.4 is clearly visible those differences and the impossibilities of evaluate behaviors in a

PBS solution.

48

References

[1] P. Mehrotra, “Biosensors and their applications - A review,” J. Oral Biol. Craniofacial Res., vol.

6, no. 2, pp. 153–159, 2016.

[2] S. P. Mohanty and E. Koucianos, “Biosensors: A tutorial review,” IEEE Potentials, vol. 25, no. 2,

pp. 35–40, 2006.

[3] L. C. Clark and C. Lyons, “Electrode systems for continuous monitoring in cardiovascular

surgery,” Ann. N. Y. Acad. Sci., vol. 102, no. 1, pp. 29–45, 1962.

[4] “Clark, L. C. Monitor and control of blood and tissue oxygen tensions. Transactions American

Society for Artificial Internal Organs 1956, 2, 41–&.” .

[5] WHO, “The enzyme-linked immunosorbent assay (ELISA).,” Bull. World Health Organ., vol. 54,

no. 2, pp. 129–139, 1976.

[6] S. J. Goldsmith, “Radioimmunoassay: Review of basic principles,” Semin. Nucl. Med., vol. 5, no.

2, pp. 125–152, 1975.

[7] S. Wang, T. Yin, S. Zeng, H. Che, F. Yang, X. Chen, G. Shen, and Z. Wu, “A piezoelectric

immunosensor using hybrid self-assembled monolayers for detection of Schistosoma

japonicum,” PLoS One, vol. 7, no. 1, pp. 1–7, 2012.

[8] L. L. de Matos, D. C. Trufelli, M. G. L. de Matos, and M. A. da S. Pinhal, “Immunohistochemistry

as an important tool in biomarkers detection and clinical practice,” Biomark. Insights, vol. 2010,

no. 5, pp. 9–20, 2010.

[9] P. Bingen, G. Wang, N. F. Steinmetz, M. Rodahl, and R. P. Richter, “Solvation effects in the

QCM-D response to biomolecular adsorption - a phenomenological approach,” Anal. Chem., vol.

80, no. 23, pp. 8880–8890, 2008.

[10] E. Rojas, M. Gallego, and I. Reviakine, “Effect of sample heterogeneity on the interpretation of

quartz crystal microbalance data: Impurity effects,” Anal. Chem., vol. 80, no. 23, pp. 8982–8990,

2008.

[11] I. Carton, A. R. Brisson, and R. P. Richter, “Label-free detection of clustering of membrane-bound

proteins,” Anal. Chem., vol. 82, no. 22, pp. 9275–9281, 2010.

[12] N. C. Speller, N. Siraj, B. P. Regmi, H. Marzoughi, C. Neal, and I. M. Warner, “Rational design

of QCM-D virtual sensor arrays based on film thickness, viscoelasticity, and harmonics for vapor

discrimination,” Anal. Chem., vol. 87, no. 10, pp. 5156–5166, 2015.

[13] J. S. Sharp, J. A. Forrest, and R. A. L. Jones, “Swelling of Poly( dl -lactide) and Polylactide- co -

glycolide in Humid Environments,” Macromolecules, vol. 34, no. 25, pp. 8752–8760, 2001.

49

[14] N. C. Fawcett, J. A. Evans, L. C. Chien, and N. Flowers, “Nucleic Acid Hybridization Detected by

Piezoelectric Resonance,” Anal. Lett., vol. 21, no. 7, pp. 1099–1114, 1988.

[15] İ. Karamollaoğlu, H. A. Öktem, and M. Mutlu, “QCM-based DNA biosensor for detection of

genetically modified organisms (GMOs),” Biochem. Eng. J., vol. 44, pp. 142–150, 2009.

[16] M. C. Dixon, “Quartz crystal microbalance with dissipation monitoring: Enabling real-time

characterization of biological materials and their interactions,” J. Biomol. Tech., vol. 19, no. 3,

pp. 151–158, 2008.

[17] A. C. Da-Silva, S. S. Soares, and G. N. M. Ferreira, “Acoustic detection of cell adhesion to a

coated quartz crystal microbalance - implications for studying the biocompatibility of polymers,”

Biotechnol. J., vol. 8, no. 6, pp. 690–698, 2013.

[18] D. Li, J. Wang, R. Wang, Y. Li, D. Abi-Ghanem, L. Berghman, B. Hargis, and H. Lu, “A nanobeads

amplified QCM immunosensor for the detection of avian influenza virus H5N1,” Biosens.

Bioelectron., vol. 26, no. 10, pp. 4146–4154, 2011.

[19] S. Buchatip, C. Ananthanawat, P. Sithigorngul, P. Sangvanich, S. Rengpipat, and V. P. Hoven,

“Detection of the shrimp pathogenic bacteria, Vibrio harveyi, by a quartz crystal microbalance-

specific antibody based sensor,” Sensors Actuators, B Chem., vol. 145, no. 1, pp. 259–264,

2010.

[20] D. Johannsmann, I. Reviakine, E. Rojas, and M. Gallego, “Effect of sample heterogeneity on the

interpretation of QCM(-D) data: Comparison of combined quartz crystal microbalance/atomic

force microscopy measurements with finite element method modeling,” Anal. Chem., vol. 80, no.

23, pp. 8891–8899, 2008.

[21] T. Goda and Y. Miyahara, “Interpretation of protein adsorption through its intrinsic electric

charges: A comparative study using a field-effect transistor, surface plasmon resonance, and

quartz crystal microbalance,” Langmuir, vol. 28, no. 41, pp. 14730–14738, 2012.

[22] I. Reviakine, D. Johannsmann, and R. P. Richter, “Hearing what you cannot see and visualizing

what you hear: Interpreting quartz crystal microbalance data from solvated interfaces,” Anal.

Chem., vol. 83, no. 23, pp. 8838–8848, 2011.

[23] G. N. M. Ferreira, A.-C. da-Silva, and B. Tomé, “Acoustic wave biosensors: physical models and

biological applications of quartz crystal microbalance.,” Trends Biotechnol., vol. 27, no. 12, pp.

689–697, 2009.

[24] G. Z. Sauerbrey, “Use of quartz vibrator for weighing thin films on a microbalance,” Zeitschrift für

Phys., vol. 155, pp. 206–222, 1959.

[25] K. Keiji Kanazawa and J. G. Gordon, “The oscillation frequency of a quartz resonator in contact

with liquid,” Anal. Chim. Acta, vol. 175, no. C, pp. 99–105, 1985.

[26] A. Shons, F. Dorman, and J. Najarian, “An immunospecific microbalance,” J. Biomed. Mater.

50

Res., vol. 6, no. 6, pp. 565–570, 1972.

[27] T. Balkenhohl and F. Lisdat, “An impedimetric immunosensor for the detection of autoantibodies

directed against gliadins.,” Analyst, vol. 132, no. 4, pp. 314–22, 2007.

[28] B. Zhang, X. Zhang, H. hui Yan, S. jun Xu, D. hua Tang, and W. ling Fu, “A novel multi-array

immunoassay device for tumor markers based on insert-plug model of piezoelectric

immunosensor,” Biosens. Bioelectron., vol. 23, no. 1, pp. 19–25, 2007.

[29] Y. Luo, M. Chen, Q. Wen, M. Zhao, B. Zhang, X. Li, F. Wang, Q. Huang, C. Yao, T. Jiang, G.

Cai, and W. Fu, “Rapid and simultaneous quantification of 4 urinary proteins by piezoelectric

quartz crystal microbalance immunosensor array,” Clin. Chem., vol. 52, no. 12, pp. 2273–2280,

2006.

[30] L. Z. Wen Y, Shan G, Pu J, “Research on piezoelectric protein sensor array for rapid detecting

HIV (1+2) antibody.,” J. Biomed. Eng., vol. 52, pp. 2273–2280, 2009.

[31] N. Kim, D. K. Kim, and Y. J. Cho, “Development of indirect-competitive quartz crystal

microbalance immunosensor for C-reactive protein,” Sensors Actuators, B Chem., vol. 143, no.

1, pp. 444–448, 2009.

[32] H. Muramatsu, J. M. Dicks, E. Tamiya, and I. Karube, “Piezoelectric crystal biosensor modified

with protein A for determination of immunoglobulins.,” Anal. Chem., vol. 59, no. 23, pp. 2760–

2763, 1987.

[33] G. G. Guilbault, B. Hock, and R. Schimid, “A piezoelectric immunobiosensor for atrazine in

drinking water,” Biosens. Bioelectron., vol. 7, no. 6, pp. 411–419, 1992.

[34] M. Minunni, M. Mascini, R. M. Carter, M. B. Jacobs, G. J. Lubrano, and G. G. Guilbault, “A quartz

crystal microbalance displacement assay for Listeria monocytogenes,” Anal. Chim. Acta, vol.

325, no. 3, pp. 169–174, 1996.

[35] L. Pastorino, F. Caneva Soumetz, M. Giacomini, and C. Ruggiero, “Development of a

piezoelectric immunosensor for the measurement of paclitaxel,” J. Immunol. Methods, vol. 313,

no. 1–2, pp. 191–198, 2006.

[36] J. S. Bovenizer, M. B. Jacobs, C. K. O’Sullivan, and G. G. Guilbault, “The Detection of

Pseudomonas aeruginosa Using the Quartz Crystal Microbalance,” Anal. Lett., vol. 31, no. 8, pp.

1287–1295, 1998.

[37] B. König and M. Grätzel, “A novel immunosensor for herpes viruses.,” Anal. Chem., vol. 66, no.

3, pp. 341–344, 1994.

[38] B. N. Johnson and R. Mutharasan, “PH effect on protein G orientation on gold surfaces and

characterization of adsorption thermodynamics,” Langmuir, vol. 28, no. 17, pp. 6928–6934,

2012.

51

[39] B. Attili and A. Suleiman, “A piezoelectric immunosensor for the detection of cocaine,”

Microchem. J., vol. 179, no. 54, pp. 174–179, 1996.

[40] Z. Shen, G. A. Stryker, R. L. Mernaugh, L. Yu, H. Yan, and X. Zeng, “Single-chain fragment

variable antibody piezoimmunosensors,” Anal. Chem., vol. 77, no. 3, pp. 797–805, 2005.

[41] J. Chen and S. Sadhasivam, “A Simple and Sensitive Detection of OmpA Protein from

Escherichia Coli by Quartz Crystal Microbalance,” vol. 2, pp. 47–50, 2011.

[42] Y. J. Yuan, Q. Chen, and J. Li, “QCM-based immunosensor for determining immunoglobulin G,”

RSC Adv., vol. 6, no. 46, pp. 40336–40342, 2016.

[43] Z. Yan, M. Yang, Z. Wang, F. Zhang, J. Xia, G. Shi, L. Xia, Y. Li, Y. Xia, and L. Xia, “A label-free

immunosensor for detecting common acute lymphoblastic leukemia antigen (CD10) based on

gold nanoparticles by quartz crystal microbalance,” Sensors Actuators, B Chem., vol. 210, pp.

248–253, 2015.

[44] R. Akter, C. K. Rhee, and M. A. Rahman, “A highly sensitive quartz crystal microbalance

immunosensor based on magnetic bead-supported bienzymes catalyzed mass enhancement

strategy,” Biosens. Bioelectron., vol. 66, pp. 539–546, 2015.

[45] Y. Chen, X. Huang, H. Shi, and B. Mu, “RESEARCH COMMUNICATION A Novel and Cost-

Effective Method for Early Lung Cancer Detection in Immunized Serum,” Asian Pacific J. Cancer

Prev., vol. 12, pp. 3009–3012, 2011.

[46] M. Muratsugu, F. Ohta, Y. Miya, T. Hosokawa, S. Kurosawa, N. Kamo, and H. Ikeda, “Quartz

crystal microbalance for the detection of microgram quantities of human serum albumin:

relationship between the frequency change and the mass of protein adsorbed.,” Anal. Chem.,

vol. 65, no. 9, pp. 2933–7, 1993.

[47] S. Xiulan, Z. Yinzhi, S. Jingdong, S. Liyan, Q. He, and Z. Weijuan, “A quartz crystal microbalance-

based Immunosensor for Shrimp Allergen Determination in Food,” Eur. Food Res. Technol., vol.

231, no. 4, pp. 563–570, 2010.

[48] T. Nhiem, S. Park, and S. Joon, “Sensors and Actuators B : Chemical Detection of HIV-1 antigen

by quartz crystal microbalance using gold nanoparticles,” Sensors Actuators B. Chem., vol. 237,

pp. 452–458, 2016.

[49] Metrohm Autolab B.V., “EQCM Study of Underpotentially Deposited (UPD) Lead Adlayer on Gold

(Autolab Application Note EC05),” None, pp. 1–3, 2011.

[50] S. Mustapha, E. M. Mustapha, and C. Nozha, “Vibrio Alginolyticus : An Emerging Pathogen of

Foodborne Diseases,” vol. 2, no. 4, pp. 302–309, 2013.

[51] S. E. Rollauer, M. A. Sooreshjani, N. Noinaj, and S. K. Buchanan, “Outer membrane protein

biogenesis in Gram-negative bacteria,” Philos. Trans. R. Soc. London B Biol. Sci., vol. 370, no.

1679, 2015.

52

[52] J. W. Fairman, N. Noinaj, and S. K. Buchanan, “The structural biology of ??-barrel membrane

proteins: A summary of recent reports,” Curr. Opin. Struct. Biol., vol. 21, no. 4, pp. 523–531,

2011.

[53] J. H. Weiner and L. Li, “Proteome of the Escherichia coli envelope and technological challenges

in membrane proteome analysis,” Biochim. Biophys. Acta - Biomembr., vol. 1778, no. 9, pp.

1698–1713, 2008.

[54] R. Qian, W. Chu, Z. Mao, C. Zhang, Y. Wei, and L. Yu, “Expression, characterization and

immunogenicity of a major outer membrane protein from vibrio alginolyticus,” Acta Biochim.

Biophys. Sin. (Shanghai)., vol. 39, no. 3, pp. 194–200, 2007.

[55] R. H. Qian, Z. H. Xiao, C. W. Zhang, W. Y. Chu, L. S. Wang, H. H. Zhou, Y. wei Wei, and L. Yu,

“A conserved outer membrane protein as an effective vaccine candidate from Vibrio

alginolyticus,” Aquaculture, vol. 278, no. 1–4, pp. 5–9, 2008.

[56] M. Struyvé, M. Moons, and J. Tommassen, “Carboxy-terminal phenylalanine is essential for the

correct assembly of a bacterial outer membrane protein,” J. Mol. Biol., vol. 218, no. 1, pp. 141–

148, 1991.

[57] T. N. Ellis and M. J. Kuehn, “Virulence and Immunomodulatory Roles of Bacterial Outer

Membrane Vesicles,” Microbiol. Mol. Biol. Rev., vol. 74, no. 1, pp. 81–94, 2010.

[58] A. L. Cunningham, N. Garçon, O. Leo, L. R. Friedland, R. Strugnell, B. Laupèze, M. Doherty,

and P. Stern, “Vaccine development: From concept to early clinical testing,” Vaccine, vol. 34, no.

52, pp. 6655–6664, 2016.

[59] S. Hart, A. B. Wrathmell, J. E. Harris, and T. H. Grayson, “Gut immunology in fish: A review,”

Dev. Comp. Immunol., vol. 12, no. 3, pp. 453–480, 1988.

[60] I. Sommerset, B. Krossøy, E. Biering, and P. Frost,

“Vaccines_for_fish_in_aquaculture_review_2005,” pp. 89–101, 2005.

[61] M. P. Soriaga, J. A. Schimpf, A. Carrasquillo, and J. B. Abreu, “Electrochemistry of the I-on-Pd

single-crystal interface: studies by UHV-EC and in situ STM,” vol. 335, pp. 273–280, 1995.

[62] S. Tanaka, S. Yau, and K. Itaya, “In-situ scanning tunneling microscopy of bromine adlayers on

Pt ( 111 ),” no. 111, pp. 1–6, 1995.

[63] H. A. Gasteiger, N. M. Markovic, and P. N. Ross, “Bromide Adsorption on Pt ( 111 ): Adsorption

Isotherm and Electrosorption Valency Deduced from RRD Pt ( 111 ) E Measurements,” no. 111,

pp. 1414–1418, 1996.

[64] Z. Shi and J. Lipkowski, “Chloride adsorption at the Au ( 111 ) electrode surface,” 1996.

[65] M. Bruckenstein, S; Shay, “Experimental aspects of use of the quartz crystal microbalance in

solution,” Electrochem. Acta, vol. 30, no. 10, pp. 1295–1300, 1985.

53

[66] Y. Ratieuville, P. Viers, and J. Alexandre, “A new electrochemical cell adapted to quartz crystal

microbalance measurements,” Elsevier, vol. 2, pp. 839–844, 2000.

[67] H. Lei, H. Uchida, and M. Watanabe, “Electrochemical quartz crystal microbalance study of

adsorption of iodide on highly ordered Au ( 111 ),” vol. 413, no. 11 l, 1995.

[68] N. Gu, L. Niu, and S. Dong, “Simultaneous determination of both the calibration constant in an

electrochemical quartz crystal microbalance and the active surface area of a polycrystalline gold

electrode,” vol. 2, pp. 48–50, 2000.

[69] P. Soriaga, “Determination of the surface area of gold electrodes by iodine chemisorption,” J.

Electroanal. Chem., vol. 233, pp. 283–289, 1987.

[70] D. Grieshaber, R. Mackenzie, J. Vörös, and E. Reimhult, “Electrochemical Biosensors -Sensor

Principles and Architectures,” Sensors, vol. 8, no. March, pp. 1400–1458, 2008.

[71] “Cyclic Voltammetric Study of ferrocyanide / ferricyanide Redox Couple,” vol. 4, no. Cv, pp. 4–6.

[72] D. S. Karpovich and G. J. Blanchard, “Direct Measurement of the Adsorption Kinetics of

Alkanethiolate Self-Assembled Monolayers on a Microcrystalline Gold Surface,” no. 27, pp.

3315–3322, 1994.

[73] C. Vericat, M. E. Vela, G. Benitez, P. Carro, and R. C. Salvarezza, “Self-assembled monolayers

of thiols and dithiols on gold : new challenges for a well-known system,” pp. 1805–1834, 2010.

[74] L. L. Rouhana, M. D. Moussallem, and J. B. Schlenoff, “Adsorption of Short-Chain Thiols and

Disulfides onto Gold under Defined Mass Transport Conditions : Coverage , Kinetics , and

Mechanism,” pp. 16080–16091, 2011.

[75] M. Fuentes, J. M. Palomo, C. Mateo, A. Venteo, A. Sanz, and R. Ferna, “Optimization of the

modification of carrier proteins with aminated haptens,” vol. 307, pp. 144–149, 2005.

[76] J. Pinto, R. Oliveira-silva, A. L. Daniel-da-silva, and R. Vitorino, “Bionanoconjugation for

Proteomics applications — An overview,” Biotechnol. Adv., 2014.

[77] N. Nakajima, Y. Ikada, B. Engineering, R. June, I. Carbodiimides, and D.- Zadeh, “Mechanism

of Amide Formation by Carbodiimide for Bioconjugation in Aqueous Media,” pp. 123–130, 1995.

[78] L. Wang, C. Wu, Z. Hu, Y. Zhang, R. Li, and P. Wang, “Sensing Escherichia coli O157 : H7 via

frequency shift through a self-assembled monolayer based QCM immunosensor *,” vol. 9, no. 2,

pp. 121–131, 2008.

[79] Ö. Ertekin, S. Öztürk, and Z. Öztürk, “Label Free QCM Immunobiosensor for AFB1 Detection

Using Monoclonal IgA Antibody as Recognition Element,” Sensors, vol. 16, p. 1274, 2016.

[80] A. Ahmad and E. Moore, “Electrochemical immunosensor modified with self-assembled

monolayer of 11-mercaptoundecanoic acid on gold electrodes for detection of benzo [ a ] pyrene

in water,” pp. 5839–5844, 2012.

54

[81] R. Etchenique and T. Buhse, “Anomalous behaviour of the quartz crystal microbalance in the

presence of electrolytes presence of electrolytes,” no. August 2014, 2000.

[82] T. Mattke and H. Kecke, “Molecular Dynamic Simulations of Single , Interacting , and Sheared

Double Layers,” vol. 561, pp. 555–561, 1998.

[83] M. Encarnação, P. Stallinga, and G. Ferreira, “Influence of electrolytes in the QCM response :

Discrimination and quantification of the interference to correct ... Influence of electrolytes in the

QCM response : Discrimination and quantification of the interference to correct microgravimetric

data,” Elvesier, vol. 22, pp. 1351–1358, 2006.

[84] R. Etchenique and T. Buhse, “ANALYST Viscoelasticity in the diffuse electric double layer †,”

pp. 1347–1352, 2002.

[85] M. Heinen, F. Zanini, F. Roosen-Runge, D. Fedunová, F. Zhang, M. Hennig, T. Seydel, R.

Schweins, M. Sztucki, M. Antalík, F. Schreiber, and G. Nägele, “Viscosity and Diffusion:

Crowding and Salt Effects in Protein Solutions,” pp. 1–17, 2011.

[86] P. Mereghetti, M. Martinez, and R. C. Wade, “Long range Debye-Hückel correction for

computation of grid-based electrostatic forces between biomacromolecules,” BMC Biophys., vol.

7, no. 1, p. 4, 2014.

[87] F. Zhang, M. W. A. Skoda, R. M. J. Jacobs, R. A. Martin, C. M. Martin, and F. Schreiber, “Protein

interactions studied by SAXS: Effect of ionic strength and protein concentration for BSA in

aqueous solutions,” J. Phys. Chem. B, vol. 111, no. 1, pp. 251–259, 2007.

[88] E. Stava, M. Yu, H. C. Shin, H. Shin, J. Rodriguez, and R. H. Blick, “Mechanical actuation of ion

channels using a piezoelectric planar patch clamp system.,” Lab Chip, vol. 12, no. 1, pp. 80–7,

2012.

[89] A. Makaraviciute and A. Ramanaviciene, “Site-directed antibody immobilization techniques for

immunosensors,” Biosens. Bioelectron., vol. 50, pp. 460–471, 2013.

[90] R. Koebnik, K. P. Locher, and P. Van Gelder, “Structure and function of bacterial outer membrane

proteins: barrels in a nutshell.,” Mol. Microbiol., vol. 37, no. 2, pp. 239–253, 2000.

55

Annexes

This section will serve as to place graphics used to determine the points for calibration curve and

to explain the methodology used in the sensibility factor graphic.

Sensibility factor

The sensibility factor was determined by knowing the frequency shift of the desadsorbed iodine

from the Au QCM surface in the presence of potential sweep from 1.5 to 0 volts. However, the assay

was performed in a “wet” surface which means the QCM surface was immersed in the 100mM KI/1M

H2SO4 solution. There are differences in starting an assay in a “wet” or “dry” surface because the

referential will be different in those two situations. On a “wet” surface the frequency shift becomes more

“sensitive”, showing a lot of variance and error. Besides, the working scale is so low that this error are

even more pronounced.

The respective graphic is exhibited in Figure 32 and it shows, clearly, a lot of error and variation

with a slightly tendency or pattern, making it impossible to define precisely the frequency variation.

Figure 32 – Original frequency shift behavior for the sensibility factor calculation

56

One way to smooth the frequency shift is applying the “moving average” method. This method wilt

take the data points and the arithmetic mean of them in a certain period. The goal is removing noise

represented by the hefty oscillations and get a better trend line. For instance, choosing a “moving

average” with a period of 20, the graphic improves considerably, as can be seen in Figure 33, and now

a more precise value can be taken. Figure 34 was obtained using a period = 50 and served as

demonstration in the iodine adsorption strategy in chapter 3.1.1. Finally, Figure 35, represents a

frequency shift with almost hefty oscillation-free by using a period = 200.

Figure 33 – Frequency shift behavior treated after using “moving average” with a period = 20.

57

Figure 34 - Frequency shift behavior treated after using “moving average” with a period = 50.

Figure 35 - Frequency shift behavior treated after using “moving average” with a period = 200.

58

BSA Calibration curve

To create the BSA calibration curve it were necessary 6 different points of BSA concentrations. The

points used were: [0.125; 0.15; 0.25; 0.50; 0.75; 1] mg/mL. The point 1 mg/mL was already showed in

chapter 3.2 and the respective frequency shift was 126 Hz.

The 0.75 mg/mL point is represented in the Figure 366 and a frequency shift of 97 Hz:

Figure 36 - Frequency shift of BSA 0.75 mg/mL after addition on top of a functionalized-QCM surface. Red background – Milli-Q water; Light Green background – BSA 0.75mg/mL in Milli-Q water.

Δf = 97 Hz

59

Point 0.5 mg/mL in Figure 377 with a frequency shift of 60 Hz:

Figure 37 - Frequency shift of BSA 0.5 mg/mL after addition on top of a functionalized-QCM surface. Red background – Milli-Q water; Light Green background – BSA 0.5 mg/mL in Milli-Q water.

Point 0.25 mg/mL in Figure 388 with a frequency shift of 40 Hz:

Figure 38 - Frequency shift of BSA 0.25 mg/mL after addition on top of a functionalized-QCM surface. Red background – Milli-Q water; Light Green background – BSA 0.25 mg/mL in Milli-Q water.

Δf = 60 Hz

Δf = 40 Hz

60

Point 0.15 mg/mL in Figure 399 with a frequency shift of 25 Hz:

Figure 39 - Frequency shift of BSA 0.15 mg/mL after addition on top of a functionalized-QCM surface. Red background – Milli-Q water; Light Green background – BSA 0.15 mg/mL in Milli-Q water.

Point 0.125 mg/mL in Figure 40 with a frequency shift of 18 Hz:

Figure 40 - Frequency shift of BSA 0.125 mg/mL after addition on top of a functionalized-QCM surface. Red background – Milli-Q water; Light Green background – BSA 0.125 mg/mL in Milli-Q water.

Δf = 25 Hz

Δf = 18 Hz

61

Antibody anti-BSA calibration curve

To create the Antibody anti-BSA calibration curve it were necessary 5 different points of antibody

anti-BSA concentrations. The points used were: [1; 2.5; 5; 7.5; 10] µg/mL. The point 10 µg/mL was

already showed in chapter 3.2 and the respective frequency shift was 113 Hz.

The 7.5 µg/mL point is represented in the Figure 41 and a frequency shift of 81 Hz:

Figure 41 - Frequency shift of antibody anti-BSA 7.5 µg/mL after addition on top of a BSA-QCM surface. Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 7.5 µg/mL in PBS pH7 buffer

solution.

Δf = 81 Hz

62

The 5 µg/mL point is represented in the Figure 42 and a frequency shift of 63 Hz:

Figure 42 - Frequency shift of antibody anti-BSA 5 µg/mL after addition on top of a BSA-QCM surface. Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 5 µg/mL in PBS pH7 buffer solution.

The 2.5 µg/mL point is represented in the Figure 43 with a frequency shift of 32 Hz:

Δf = 63 Hz

Δf = 32 Hz

Figure 47 - Frequency shift of antibody anti-BSA 2.5 µg/mL after addition on top of a BSA-QCM surface. Gray background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 2.5 µg/mL in PBS pH7 buffer solution.

63

The 1 µg/mL point is represented in the Figure 48 with a frequency shift of 18 Hz:

Δf = 18 Hz

Figure 52 - Frequency shift of antibody anti-BSA 1 µg/mL after addition on top of a BSA-QCM surface. Gray

background – PBS pH 7 buffer; Light Blue background – Antibody anti-BSA 1 µg/mL in PBS pH7 buffer solution.