Transcript brązy 1

Role of Cysteamine on Antibody Immobilization in
Immunosensor design
Faryal Kabir
08-arid-942
Ph.D (Biochemistry)
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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
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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
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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
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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
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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 8OHdG 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
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Chemical structure of 8-OHdG
(Lily, et al., 2004)
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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
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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
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Structure of an Antibody
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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
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Immunosensor design
 Gold support
 Creation of amine layer
 Antibody immobilization
 Antibody/antigen affinity reaction
 Evaluation
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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
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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
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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
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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 .
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Oriented immobilization
Random immobilization
(Anke, et al., 2013)
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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
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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
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Carbodiimide Reaction (Jiang, et al., 2004)
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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
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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
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The Ag-Ab interaction is due to lots of non-covalent interactions
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Schematic representation of antibody immobilization by covalent linkage to a
gold surface modified by cysteamine (Mendes, et al., 2009)
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Immunosensor employing antibodies against 8OHdG
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Design of an immunosensor
(Nadia, et al., 2014)
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Evaluation
The immunosensor design is evaluated by following techniques:
 Quartz-crystal microbalance with dissipation (QCM-D)
 Atomic force microscopy (AFM)
 Electrochemical impedance spectroscopy (EIS)
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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
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 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/2πEstored
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Piezoelectric quartz crystal microbalance and scheme of vibration (Petr, S. 2003)
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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
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QCM-D records reporting measurements of frequency and dissipation against time for the different
stages of reaction
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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
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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
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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).
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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.
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 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.
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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
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References
 Lily, L. B. C. Wua, C. Chiuan-Chian , C. Pi-Yueh, T. James and C. Wua. 2004. Urinary 8OHdG: 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. 2013. Antibody orientation on biosensor surfaces: a
minireview. 138: 1619-1627.
 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. 2009. Development of an electrochemical immunosensor for Phakopsora
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 Nádia, S. F., M.Goreti and F.Sales. 2014. 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. 2003. Piezoelectric quartz crystal sensors applied for bioanalytical assays and
characterization of affinity interactions. J. Braz. Chem. Soc., 14(4): 441-445.
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