Design of a Piezoelectric Immunosensor for antibody ... estudo focar-se-á num QCM como imunossensor...
Transcript of Design of a Piezoelectric Immunosensor for antibody ... estudo focar-se-á num QCM como imunossensor...
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
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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.
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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]
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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
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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].
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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]
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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]
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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.
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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
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6, no. 2, pp. 153–159, 2016.
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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.