Immunosensors immobilization

On the basis of an enzyme thermistor, Mattiasson et al. (1977) developed one of the first immunosensors. Immobilized antibodies against albumin are placed in a column and set into an ET. After injection of an albumin-sample and a known amount of enzyme-labeled albumin, both are separated from the sample matrix by antibody-antigen-interaction. After injection of a substrate, the change in heat is a measure of analyte concentration. The less heat produced means that more albumin has been bound. An elution step regenerates the ELISA. Due to its thermal detection principle, the procedure is called TELISA (thermometric enzyme-linked immunosorbent assay). Figure 3 shows the principle of the TELISA procedure in its sandwich configuration. 

Fig. 8. Basic components of a biosensor. In the case of an immunosensor, the antibody (or antigen) would be immobilized onto the transducer.

The aim of our investigation was the development of the amperometric enzyme immunosensor for the determination of Klebsiella pneumoniae bacterial antigen (Ag), causes the different inflammatory diseases. The biosensing pail of the sensors consisted of the enzyme (cholinesterase) and antibodies (Ab) immobilized on the working surface of the screen-printed electrode. Bovine seiaim albumin was used as a matrix component. 

The working conditions of the immunosensor (enzyme and antigen concentrations, dilutions of the antibodies, pH of the buffer solution) were found. The cholinesterase immobilized demonstrated the maximum catalytic activity in phosphate buffer solution with pH 8.0. The analytical chai acteristics of the sensor - the interval of the working concentrations and detection limit - have been obtained. The proposed approach of immunoassay made possible to detect 5T0 mg/ml of the bacterial antigen. 

Ilkovic equation, 62 Immobilization,, enzyme, 172 Immunoassays, 185 Immunosensors, 183 Infrared spectroelectrochemistiy, 44 Instrumentation, 104 Insulin release, 178... 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

FIGURE 5.6 Schematic representation of the immunosensor based on a Protein A-GEB biocomposite as a transducer, (a) Immobilization of RlgG on the surface via interaction with Protein A, (b) competitive immunoassay using anti-RIgG and biotinylated anti-RIgG, (c) enzyme labeling using HRP-streptavidin and (d) electrochemical enzyme activity determination. (Reprinted from [31] with permission from Elsevier.)... 

The development of electrochemical immunosensors generally involves the immobilization of an immunocomplex on a single electrode, followed by detection via the... 

O. Ouerghi, A. Touhami, N. Jaffrezic-Renault, C. Martelet, H. Ben Ouada, and S. Cosnier, Impedimetric immunosensor using avidin-biotin for antibody immobilization. Bioelectrochemistry 56, 131—133... 

Since immunosensors usually measure the signals resulting from the specific immu-noreactions between the analytes and the antibodies or antigens immobilized, it is clear that the immobilization procedures of the antibodies (antigens) on the surfaces of base transducers should play an important role in the construction of immunosensors. Numerous immobilization procedures have been employed for diverse immunosensors, such as electrostatic adsorption, entrapment, cross-linking, and covalent bonding procedures. They may be appropriately divided into two kinds of non-covalent interaction-based and covalent interaction-based immobilization procedures. 

Lee et al. utilized the self-assembled layer of thiol group-modified protein A for the oriented immobilization of antibodies [64], An increased binding capacity was further observed. As another illustrative instance, a protein A-based orientation-controlled immobilization strategy for antibodies was proposed for the fabrication of a QCM immunosensor using nanometer-sized gold particles and amine-terminated PPF [65], Moreover, in recent years, there has emerged another oriented immobilization... 

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