Membrane Immunosensors

Enzyme immunoelectrodes involve the spatial coupling of the sensor, the immunocomplex, and the catalytic amplification by indicator enzymes. Like the sensor systems described above, enzyme immunoelectrodes are based on common principles of EIA. The choice of enzymes for EIA is rather restricted and is further diminished when electrodes are to be used for detection. So far only GOD, catalase, and HRP have been combined with oxygen-sensing polarographic sensors. An overview of enzyme immunoelectrodes is given in Table 21. 

In one of the first reports concerning amperometric immunosensors, Mattiasson and Nilsson (1977) proposed an electrode system for determining insulin and albumin. An oxygen electrode was covered by a nylon 

Antigen Electrode Principle Marker enzyme Sensitivity References 

AFP = a-fetoprotein HCG = human chorionic gonadotrophine IgG = immunoglobulin G HBs = hepatitis B surface antigen H2Q/BQ = hydroquinone/benzoquinone 

Recently, Boitieux et al. (1987) proposed a novel method for separating labeled immunocomplexes from free unlabeled antigen. A membrane capable of specifically binding (5-galactosidase was fixed to an oxygen electrode. The antigen, IgG, competed with GOD-labeled IgG for 3-ga-lactosidase-labeled antibody. As soon as the complex was formed in the solution it was reversibly bound to the sensor surface. The GOD activity was inversely proportional to the concentration of IgG. 

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Figure 3.31 — (A) Continuous monitoring of analytes in a fermentation medium. In the absence of analyte, the analyte enzyme conjugate binds predominantly to sensor 1 (left). At high analyte concentrations in the medium, the signal shifts to sensor 2 (right). (B) Principle of the membrane immunosensor. (Reproduced from [225] and [226] with permission of VCH Publishers).

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Y.V. Plekhanova, A.N. Reshetilov, E.V. Yazynina, A.V. Zherdev, and B.B. Dzantiev, A new assay format for electrochemical immunosensors polyelectrolyte-based separation on membrane carriers combined with detection of peroxidase activity by pH-sensitive field-effect transistor. Biosens. Bioelectron. 19, 109-114(2003). 

Starodub et al. [98] studied different constructions and biomedical applications of immunosensors based on fiberoptic and enhanced CL. They discussed three different approaches of immobilization of one of the immunocomponents on the fiberoptic surface. Good results could be achieved by the use of a special membrane closely connected to the fiberoptic, with sensitivities compared to that obtained by the ELISA method but with a faster rate of analysis. The sensor was... 

An optical immunosensor for continuous T4 measurement has been described, in which the fluorescent indicator protein is separated from the sample flow chamber by a dialysis membrane.024) The indicator is T4-binding globulin (TBG), the intrinsic fluorescence (ex. 290 nm) of which is quenched by T4binding. Due to the high affinity of the TBG for thyroxine, the immunosensor is not reversible, but multiple measurements can be made until the TBG is saturated. Sensitivity is inadequate for clinically useful concentrations of T4, but suggestions for improvement of the method are made. 

Non-labelled immunosensors rely on various principles (Fig. 3.27.A). Either the antibody or the antigen is immobilized on the solid matrix to form a sensing device. The solid matrix should be sensitive enough at the surface to detect immunocomplex formation. Electrode, membrane, piezoelectric and optically active surfaces may in principle be used to construct non-labelled immunosensors. The antigen or antibody to be determined is dissolved in a solution and reacted with the complementary matrix-bound antibody or antigen to form an immunocomplex that alters the physical e.g. the electrode potential or intrinsic piezofrequency) or optical properties of the... 

Figure 3.28 — Sensing terminal for antibody-based FOCS (A) direct covalent attachment of immunochemical, (B) membrane entrapment of immunochemical, and (C) a regenerable immunosensor. (Reproduced from [216] with permission of the American Chemical Society).

Wilson et al. developed a flow-through immunosensor using an immunosorbent membrane holding an antigen (bovine IgG) for the determination of... 

Several amperometric immunosensors have been developed for monoclonal antibodies (IgG), a-interferon and the pesticide 2,4-dichlorophenoxy-acetic acid (2,4-D) by using a flow-cell with the catching antibody covalently bound to a cellulose acetate or activated nylon membrane as shown in Fig. 3.31. B. With multiepitope antigens (e.g. a protein), after the antigen is bound and washed, a second enzyme-labelled antibody is used to form a sandwich... 

Aizawa et al. developed immunosensors of the first type for syphilis and blood typing [228-230] based on measurements of the transmembrane potential across an immunoresponsive membrane. The potentiometric immunosensor using an antibody against human chorionic gonadotropin (hCG)... 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors require multiple steps for analyte determination, and either sandwich assays or competitive binding assays may be used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. 

Enantioselective, potentiometric membrane electrodes (EPMEs) are proposed for the potentiometric detection of the enantiomers [2,10]. The advantages of utilization of these electrodes over amperometric biosensors and immunosensors are a longer lifetime, a large working concentration range, no dilution required for the samples and possibility of decreasing of limit of detection by utilization of KC1 0.1 mol/L as internal solution [2],... 

H. Yu, F. Yan, Z. Dai and H.X. Ju, A disposable amperometric immunosensor for alpha-1-fetoprotein based on enzyme-labeled antibody/chito-san-membrane-modified screen-printed carbon electrodes, Anal. Biochem., 331 (2004) 98-105. 

Haga et al. developed another type of immunosensor by combining an enzyme membrane immunoassay and an enzyme sensor using oxygen electrodes (HI). In this assay antigen molecules (theophylline) are attached on the surface of the liposomes and an enzyme (horseradish peroxidase) is encapsulated in the sensitized liposome. When antibody (antitheophylline antibody) and complement are added, the enzyme is released by the liposome lysis. The enzyme activity with the NADH-NAD reaction can be determined by the oxygen electrode. When antigen is added, it competitively binds to antibodies, then liposome lysis and enzyme activity are decreased. The sensitivity of this method for theophylline determination was reported as 0.7 ng/ml. 

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