Enzyme and Metal Particles labeled Electrochemical Immunosensors
In this mini review paper, the experimental aspects electrochemical immunosensors and their application in detection of Human Epidermal growth factor Receptor 2 (HER2) in breast cancer patients are briefly discussed. Generally, electrochemical immunosensors fall in to two different groups; they are either labeled or label free. Here, the enzyme and metal nano particles labeled electrochemical immunosensors are briefly explained.
According to the introduced definition by IUPAC, Biosensors are analytical devices that produce quantitative analytical information using biochemical receptor such as antibodies and enzymes that is in direct contact with the transduction element 1. So far, many kinds of biosensors like immunosensors, enzyme-based and DNA biosensors, have been made 2. The very first biosensor was glucose biosensor made by Clark and Lyons. Immunosensors are group of biosensors which work based on the recognition of antibody and antigen. In an electrochemical immunosensor, the analyte or the antigen binds to a specific region of antibody or bioreceptor on the surface of the electrode and form a complex 3. In this kind of biosensor, the concentration of antigen which acts as an analyte can be determined by different methods such as amperometric, impedimetric, conductometric or potentiometric signals 3. Fig. 1 exhibits Schematic representation of electrochemical immunosensor.
Figure 1schematic presentation of an electrochemical immunosensor 3
Recently, Electrochemical immunsesnors, due to some advantages such as portability, low cost, high sensitivity and selectivity has attracted great attention in different fields including bioprocess control, food quality and military and medical specially for cancer diagnosis 4.
Basic principles of immunosensors
In Immunosensors, an antigen which can be viruses, tissue cells, fungi and proteins (with a molecular weight larger than 1.5 kDa) form bind with antibody to make a complex. The magnitude of affinity between antibody and antigen is shown by Ka or association constant which is the strength of binding forces in the formed complex (Fig 2) 4.
Immunosensors are divided in to several different groups such as: Radioactive labels (radioimmunoassays), enzymes (enzyme-linked immunosorbent assay) ELISA, ?uorophore compounds (?uoroimmunoassays) and chemiluminescent compounds (chemiluminescent immunoassays) 4. Among the above-mentioned techniques, ELISA, due to the lower limit of detection, is the most widely used technique so far. ELISA has two different formats: sandwich and competitive formats 5.
In the figure 3A, which is a schematic representation for ELISA sandwich format, the antigen should have at least two active binding sites or epitopes. In this format, the first antibody is immobilized on a solid surface, after adding antigen, it interacts with the immobilized antibody and forms binds. However, the interaction between antibody and antigen cannot be determined directly, so a second antibody which is labelled with an enzyme is added to the solution. The role of this enzyme labelled antibody is to generate a detectable signal5. In the competitive format, in figure 3B, there is a competition between the antigens (analyt) and the enzyme-labelled antigens added to make binds with immobilized antibody. While, in figure 3C, the competition is between the antigens (analyt) and the immobilized antigens for binding with the enzyme-labelled reporting antibodies 5. Based on the type of applied transducer, the immunosensors fall in to 3 categories: electrochemical, piezoelectric and optical. Among these techniques electrochemical immunosensors, due to some advantages such as short time of response, small sample volume, high sensitivity and low cost have attracted great attention 5.
Figure 2 KA =a?nity or association constant, KD = dissociation constant, Ab = concentration of the unoccupied antibody binding sites, Ag = concentration of the unoccupied antigen binding sites and Ab-Ag = concentration of the antibody-antigen complex 4
Figure 3 different ELISA formats: (A) sandwich, (B) competitive antibody coating and (C) competitive antigen coating 5.
Electrochemical immunosensors classification:
The electrochemical detection of immunosensors classified as interfacial methods which can occur in both static and dynamic mode (potentiometry and voltammetry) and bulk methods (conductometry). In the static mode, the detection is done under equilibrium condition (no net current) and in the dynamic mode the measurements are carried out under current or potential control. In voltammetry, the focus is on current responses Fig 4. 4.
Components of an electrochemical immunosensor:
Depending on the formats of immunosensors (sandwich or competitive), the recognition element which is either antibody or antigen is immobilized on the surface of an electrode. The measurement is done by injection of the enzyme labelled antibody which produce a detectable signal. Fig.5 illustrates the main components of an electrochemical immunosensor 6.
Figure 4 schematic representation of electrochemical classification 4
Figure 5 schematic representation of the main components of an electrochemical immunosensor 6
the generated signal from biological event, produce either measurable potential (potentiometry), a change in the conductive characteristics of the environment between electrode (conductometric), measurable current (voltammetry/amperometry) or a change in the impedance (impedimetric). The electrochemical part of an immunosensor, usually is composed of a working electrode which work as transducer, a reference electrode which is located outside the reaction in order to maintain a stable potential and finally an auxiliary or counter electrode connect the solution to the working electrode 7.
In electrochemical approaches, the measurements are done at the solution/electrode interface. The closer the analyte to the surface of the working electrode, the greater the sensitivity to the enzymatic product. Therefore, for higher sensitivity the analyte concentration should be high in the diffusion layer of working electrode. An efficient immobilization technique not only increase the antibody/antigen interaction, but also improve the sensing surface of the electrode 6.
Amperometric and voltammetric immunosensor:
In amperiometry, the generated current from the oxidation or reduction of an electroactive species is measured at constant potential. The amperometric method divides in to two groups; direct and indirect transduction. In indirect method, oxidizing reagents or mediators are used for the electrochemical reaction of the analyte at the working electrode. In the direct amperometric biosensors, the redox enzyme is directly bound to the surface of the electrode and no more mediators are needed. It is important to mention that the redox reaction between the enzyme and analyte should occur as close as possible to the surface of electrode 8. When the generated current is measured during variations of potential it is called voltammetry. Because we can vary the potential in many ways, there are different types of voltammetry such as: linear sweep, polarography, reverse pulse, normal pulse and so on8. Among the mentioned voltammetric techniques, cyclic voltammetry is one of the widely used. The voltage, at a fixed rate is swept between two values. When it gets to V2 the scan reverse and back to V1. It provides useful information about the electrochemical reaction rates and redox potential 8.
Enzyme-labelled-based amperometric immunosensors
As it was said earlier, we have two different types of amperometric immunsensors: direct and indirect transduction. In the indirect immunsensor type an electron transfer mediator is introduced in to the system. For example, the hydroquinone mediator in presence hydrogen peroxide oxidized by horseradish peroxidase (HRP) enzyme which serve as a label, then reduced at the surface of the electrode. Fig.6 5.
Figure 6 HRP-based amperometric detection in the presence (mediated) and in the absence (direct electron transfer) of a mediator. 5
The second type of amperometric immunsensors is s direct electron transfer (DET). In this type, the surface of the electrode is modified by nanomaterials which act as electron wire between the surface of the electrod and redox center of the enzyme 5. Therefore, in this case, there is no mediator Fig. 7.
Figure 7 Indirect (A) and Direct (B) enzyme-labelled electrochemical immunosensors 5
Metal particle labelled immunosensors
In this type of electrochemical immunsesors, the metal particle are used instead of enyme for labelling the AB-2. When the immobilized antibody, captured the analyte, the antibody2-nano particles are added to the solution to bind with analyte. Then, considerable amount of electroactive metal ions generates by nanoparticle dissolving in acid. Finally the released electroactive metal ions detect by an electrochemical device such amperemeter or anodic stripping voltammetry 9. The Au nanoparticles are commonly used as labels. they first electrooxidize of to AuCl4? ions and then detection of generated Au ions is done by either the Au nanoparticles oxidized in an acidic solution and then the ions detect by SV or they directly reduced to Au metal and the reduction current measured by DPV or SWV 5.
Figure 8 Au nanoparticles as label 5.
Cancer biomarker detection:
Shamsipour and coworker 10 designed and reported a sandwich type electrochemical immunosensor for detection of Human Epidermal growth factor Receptor 2 (HER2) in breast cancer patients. The reported immunosensor was consist of: glassy carbon electrode modified by antiHER2-Fe3O4 NPs as the working
electrode, a platinum wire as the counter electrode and Ag/AgCl (Saturated KCl) electrode as the reference electrode. They also employed antiHER2/[email protected] NPs as label. The calculated limit of detection toward HER2 biomarker was about 2.0 × 10?5 ng mL?1.
Figure 9 Preparation of (A) antiHER2/APTMS-Fe3O4 as platform bioconjugate (PB) and (B) antiHER2/[email protected] as label bioconjugate (LB)10
Compared to chromatography and other methods, electrochemical immunosensors offers many advantages such as portability, potential of automation, low cost, fast response time, selectivity and good sensitivity. However, due to a lack of validated protocols there are few commercially available electrochemical immunosensors 11. Therefore, further studies and efforts in the development of immunosensors need to be done.
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