Role of Cysteamine on Antibody Immobilization in Immunosensor design Faryal Kabir 08-arid-942 Ph.D (Biochemistry) 1

Immunosensor  Immunosensors are affinity ligand-based biosensor solid-state devices in which the immunochemical reaction is coupled to a transducer  It utilizes the very specific binding affinity of antibodies for a specific antigen to form a stable complex 2

Components of Immunosensor Two major components of immunosensor are:  Biological recognition element In order to recognize the cancer biomarker, antibodies are considered to be well suited recognition elements for immunosensors. The high specificity and affinity of an antibody for its antigen allows a selective binding of antigen which is present in low level  Signal transducer The transducer converts electrical, optical and mass changes of a solution into a measureable signal 3

Oxidative stress  The imbalance between oxidant producing systems and oxidative defense mechanisms resulting in an excessive production of reactive oxygen species  ROS are direct causes of DNA damage. They are produced either endogen- ously or exogenously can attack lipid, protein and nucleic acid simultaneously in the living cells  Oxygen molecule is used to accept electrons and to make water. If there is only one electron to give, a free radical is form. OFRs attack not only the bases but also the backbone of DNA 4

Cancer Biomarkers  Biomarker generally refers to a measured characteristic which may be used as an indicator of some biological state or condition  Level of biomarkers in biological fluid depends on different disease conditions and stages  Tumor associated antigens have been used as biomarkers for cancer diagnosis  These are cellular molecules that can be detected in tumor cells or other body fluids which are over expressed due to cancer onset and growth  There are a range of biomarkers which have been identified with different types of cancers. These can either be present inside the cancer cells or extracellular 5

8-hydroxy-2-deoxyguanosine (8OHdG) 8OHdG act as an indicator of DNA oxidation It is the most sensitive biomarker for oxidative stress and can be detected in urine, plasma or DNA isolated from cells and tissues. Upon DNA repair 8- OHdG is excreted in the urine It is frequently detected and extensively studied DNA lesion where mismatch repair plays a key role This lesion is important because it is relatively easily formed but also be a risk factor for cancer 6

Chemical structure of 8-OHdG (Lily, et al., 2004) 7

Limitations of Current Cancer Diagnostics Most of current diagnostics detect cancer after it has already spread to other parts of the body Early detection of cancer improves treatment options and survival rates Currently, methods used for cancer diagnosis involve complex, expensive and non- portable techniques Therefore, there is a need for simple and sensitive diagnostic method that can detect cancer biomarkers that exist at low concentration in biological fluids Biosensor can fulfill these requirements 8

Antibody Large protein composed of hundreds of amino acids Amino acids are arranged in a tridimensional order that is recognized as a Y shape Carboxyl (COOH) end of the peptide chain is positioned at the lower end this Y shape structure and is known as Fc region Each antibody binds to epitope region of an antigen through the two upper end parts of this Y shape that are amine terminated, called Paratope regions Because there are two paratope sites in a single structure, so each antibody is able to bind with two antigen species 9

Structure of an Antibody 10

Immunodetection Antigen: Any substance which stimulates the production of an antibody and than binds specifically to it Epitope: The portion of an antigen that is recognized and bound by an antibody Paratope: The site in the variable (V) domain of an antibody that binds to an epitope on an antigen Antibody based methods allowing the specific: Detection Quantification Localisation of antigens by means of antibody binding 11

Immunosensor design  Gold support  Creation of amine layer  Antibody immobilization  Antibody/antigen affinity reaction  Evaluation 12

Gold Support  Electrochemical studies used Au-screen printed electrodes (Au-SPEs)  Working and counter electrodes are made of gold  Reference electrode and electrical contacts made of silver  These are interfaced in a switch box to enable its galvanostat reading  The gold layer of electrode is washed with 70% alcohol and deionized water before any use 13

Creation of amine layer This is done by placing a solution of Cysteamine (50mM) on clean Au for 1hour Cysteamine is a two carbon chain carrying an amine group (NH2) at one and a thiol (SH) at the other end Au and Sulphur interact with each other and closely packed monolayer is formed Au surface becomes a stable amine layer 14

Antibody Immobilization A critical step in immunosensor design is the immobilization of Ab on the solid support Random Immobilization: Many approaches for immobilizing antibodies on solid support leads to a random orientation which would ultimately decrease the binding affinity Electrostatic non-covalent adsorption or coupling via amine terminals yielding inactive orientation of antibodies due to steric blocking of paratope sites 15

Oriented immobilization: Most common approach includes the attachment of protein A or protein G to the biosensing surface before antibody binding These proteins bind to Fc region of antibodies providing a suitable orientation of the antibodies Ordered organization will be decreased because the proteins are also randomly attached to the surface Orientation of antibodies may reduce the disulfide bonds between peptide chains leads to inactive antibody fragments So, there is a random binding of these fragments to the gold surface. 16

Oriented immobilization Random immobilization (Anke, et al., 2013) 17

Optimized Orientation  Methods for oriented Ab binding involve: Complex chemical procedures Chemical modification So, there is a need for the development of a simple method for site oriented immobilizaion Chemical modification Activation of Carboxylic residues via carbodiimide reaction Reaction with ethylenediamine Addition of BSA 18

Activation of Carboxylic residues via carbodiimide reaction Different and simpler immunosensor is designed by activation of carboxylic residues at Fc region of an antibody COOH group when activated, it forms highly reactive O-acylisourea intermediate O-acylisourea rapidly reacts with NHS to produce a more stable succinimydyl ester intermediate The resulting ester undertakes easy nucleophilic substitution with the amine groups on the Au/amine layer Carboxylic residues then covalently bind with amine layer, therefore preventing significant loss of antibody activity 19

Carbodiimide Reaction (Jiang, et al., 2004) 20

Reaction with Ethylenediamine  The activation of carboxylic group in the antibody is not carried out specifically at the Fc region  It may affect all carboxylic groups in the outer surface of 3D structure of antibody  Amine based compounds in a biological sample could bind to these activated positions and in this way decrease the selectivity of immunosensor  After antibody binding the activated carboxylic groups are deactivated by reaction with ethylenediamine 21

Addition of BSA  Bovine serum albumin, low cost protein is added to block the non-specific response of Ab  Ab has enormous dimensions, so it can interacts with other substances of protein nature which decreases the overall selectivity Antigen/antibody affinity reaction  The time for affinity reaction between the Ab-8OHdG immobilized in the gold layer and the antigen was 15 minutes  Antibody binds with 8OHdG by covalent bonding 22

The Ag-Ab interaction is due to lots of non-covalent interactions 23

Schematic representation of antibody immobilization by covalent linkage to a gold surface modified by cysteamine (Mendes, et al., 2009) 24

Immunosensor employing antibodies against 8OHdG 25

Design of an immunosensor (Nadia, et al., 2014) 26

Evaluation The immunosensor design is evaluated by following techniques:  Quartz-crystal microbalance with dissipation (QCM-D)  Atomic force microscopy (AFM)  Electrochemical impedance spectroscopy (EIS) 27

Quartz Crystal Microbalance with Dissipation  QCM consist of thin quartz disc sandwiched between the pair of electrodes and it is possible to excite the crystal to oscillation by applying AC voltage across its electrodes.  The principle is based on piezoelectric properties of quartz crystals  Quartz crystals are used as a transducer in immunological reactions because they allow the direct detection of antigen antibody reaction without the need of using labels  In QCM-D, two parameters frequency and dissipation are monitored simultaneously in real time  Is a real time analytical instrument which measures the mass and viscoelastic properties of molecular layers 28

 QCM relies on a voltage being applied to a quartz crystal causing it to oscillate at a specific frequency  When the mass changes the frequency of the oscillation changes  Changes in mass on the quartz surface are related to changes in frequency of the oscillating crystals through the Sauerbrey relation Δm = C. Δf  Dissipation occurs when the driving voltage to the crystal is shut off and the energy from the oscillating crystal dissipates from the system D=Elost/2 π Estored 29

Piezoelectric quartz crystal microbalance and scheme of vibration (Petr, S. 2003) 30

Antibody/Antigen binding  The ability of antibody to bind with antigen is checked by QCM-D studies  After the injection of Ab to the Au-NH2 layer the Δf values decreased which shows the physical adsorption of antibody. Increase in dissipation indicate that the layer of antibody has non-rigid structure showing viscoelastic properties  The observed frequency under equilibrium will increase when antigen 8OHdG bound to its Ab layer. This increase in frequency is due to the decrease in mass. The molar mass of antigen (283.2 g/mol) is very small as compared to Ab, which shows that this binding stage would not generate a significant mass increase  Ag binding to the immobilized antibody produce conformation changes in the antibody. Extraction of hydration molecules over the Ab/Au layer leads to a subsequent mass loss, so there is very small decrease in dissipation 31

QCM-D records reporting measurements of frequency and dissipation against time for the different stages of reaction 32

Atomic Force Microscopy  AFM is very high resolution type of microscopy which can be used to image the topography of soft biological materials in their native environment  Principle of AFM is simple, sharp tip is fixed at the end of a flexible cantilever is scanned over the surface of a sample  In most AFMs the sample is positioned on top of a four segments of piezoelectric tube and is scanned under a fixed tip  When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever, which is measured by using a laser deflection technique 33

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 Microcantilever based sensors are used for cancer biomarker detection  Affinity reactions are detected through the bending of a sensor due to mass and resonant frequency changes  AFM image of clean gold surface is not exact flat because the gold support had been reused from a previous experiment  AFM image of the presence of antibody on the Au/amine support shows that the surface is also quite uniform because of similar orientation through the Fc region  The binding of Ag to the Au/Amine/Ab support does not give rise to a significant change 36

AFM images in 2D (left) and 3D (right) views of different materials: Au layer (top), Au/amine/Ab (middle) and Au/amine/Ab with antigen (bottom). 37

Electrochemical Impedance Spectroscopy  EIS transduction is a direct and label free method. This technique allows the direct detection of affinity complex formation through the charge transfer resistance at the biofunctionalized electrode  Impedance is a measure of the ability of a circuit to resist the flow of electrical current. Electrochemical impedance is usually measured by applying an AC potential to an electrochemical cell and measuring the current through the cell. Electrochemical Impedance is normally measured by using a small excitation signal. 38

The presence of Ab attached to the amine layer is confirmed by a significant increase of the Rct observed in EIS spectra. Rct corresponds to the diameter of semicircle and measures the ability of the charge transfer between electrodes and surrounding electrolytes. Increase in diameter of semicircle shows an increase in resistance The binding of 8OHdG and anti-8OHdG lead to an increase in Rct. Due to the formation of affinity complexes insulating layer is formed. This layer cause resistance in electron transfer kinetics between the electrodes, thus increasing the electron transfer resistance. 39

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Summary  This is a novel approach for a suitable orientation of antibodies on gold modified platform  Ag binding to the immobilized Ab seems to produce conformation changes in the antibody  EIS studies suggest that a suitable orientation of the antibody is successfully achieved  The time and effort required for the devices seutup are significantly reduced, allowing it to be disposed off afterwards without significant costs 41

References Lily, L. B. C. Wua, C. Chiuan-Chian, C. Pi-Yueh, T. James and C. Wua Urinary 8- OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clinica Chimica Acta., 339: 1 –9. Anke, K. T. J. Beekwilder and H. Zuilhof Antibody orientation on biosensor surfaces: a minireview. 138: Jiang, K.,Schadler,L.S.,Siegel,R.W.,Zhang,X.,Zhang,H.,Terrone,M.,2004.Journal of MaterialsChemistry14,37–39. Mendes, R. K., D. C. M. Ferreira, R. F. Carvalhal, L. A. Peroni, D. R. Stach-Machado and L. T. Kubota Development of an electrochemical immunosensor for Phakopsora pachyrhizidetection in the early diagnosis of soybean rust. J. Braz. Chem. Soc., 20(4): 474. Nádia, S. F., M.Goreti and F.Sales Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases. Biosensors and Bioelectronics, 53: 193–199. Petr, S Piezoelectric quartz crystal sensors applied for bioanalytical assays and characterization of affinity interactions. J. Braz. Chem. Soc., 14(4):