Sensors immunoassay systems

In contrast with sensors, sensing systems are ideal for exploitation of immunochemical selectivity. This accounts for various highly sensitive and successful competitive immunoassays. Incorporation of the manipulative step(s) opens the door for regeneration of the antibody or even for operation under virtually reversible conditions. 

We recently coupled nanoparticle-based sensors on an 8-biosensor array with off-line protein capture into a simple microfluidic system (Fig. 1.12) [112]. This microfluidic immunoassay system features AuNP sensor electrodes built on a screen-printed carbon platform inserted into a molded 70 pL PDMS channel... 

Surface plasmon resonance has been used to produce a wide variety of optical sensors, eg, systems which are able to change their color in presence of specific analytes. These devices can be used as sensors for immunoassay, gas, and liquid (see Fig. 11) (48). 

Figure 44. The cell-based hybrid biosensor immunoassay system. The inset depicts the arrangement of the key hybrid sensor components. The details of the system A to E are in the text (Reproduced fi-om Reference [12] with permission).

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 3.29.A shows a flow-cell of 20 iL inner volume used to hold immobilized anti-mouse IgG bound to a rigid beaded support (activated Pierce trisacryl GF-2000). The cell was used to develop a two-site immunoassay for mouse IgG by consecutive injection of the sample, acridinium ester-labelled antibody and alkaline hydrogen peroxide to initiate the chemiluminescence, which started the reaction sequence shown in Fig. 3.29.B. Regenerating the sensor entailed subsequent injection of an acid solution, which resulted in a determination time of ca. 12 min (this varied as a fimction of the flow-rate used, which also determined the detection limit achieved, viz. 50 amol for an overall analysis time of 18 min) [218]. The sensor was used for at least one week with an inter-assay RSD of 5.9%. Attempts at automating the hydrodynamic system for use in routine analyses are currently under way.

Further progress of ECL probes immobilization methods should result in new robust, stable, reproducible ECL sensors. Especially, the use of electrochemilumi-nescent polymers may prove to be useful in this respect. There are also good prospects for ECL to be used as detection in miniaturized analytical systems particularly with a large increase in the applications of ECL immunoassay because high sensitivity, low detection limit, and good selectivity. One can believe that miniaturized biosensors based on ECL technology will induce a revolution in clinical analysis because of short analysis time, low consumption of reactants, and ease of automation. 

Chlorophenoxy acid herbicides are also widely used to control broadleaf weeds and grass plants. Several immunoassays have been reported for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).246 247 Several immunosensors have been described using a transducing principle similar to the RIANA system already described in this chapter. Thus, Meusel et al.248 reported the use of monoclonal antibodies in a sensor chip to analyze river and lake water samples, obtaining detection limits of 0.1 ig L 1. Moreover, monoclonal antibodies, produced by Cuong et al.,249 were used in a dipstick immunoassay format to analyze pond water samples. When applied to the 2,4-D compound, this semiquantitative method yielded for an IC50 of 6 ug I. and an LOD of 0.5 pg L-1. 

The properties of high specificity and a wide applicability with many analytes have led to the widespread use of immunoanalytical techniques. The benefits of electrochemical sensors include technical simplicity, speed, and convenience via direct transduction to electronic equipment. Combining these two systems offers the possibility of a convenient assay technique with high selectivity. Because of the complexity of immunoassay methods, such devices have not yet found widespread use. Nevertheless, electrochemical immuno-sensors offer the potential for fast, simple, cost-effective analysis of many... 

Abdel-Hamid et al. [122] used a flow-injection amperometric immunofll-tration assay system for the rapid detection of total E. coli and Salmonella. Disposable porous nylon membranes served as a support for the immobilization of anti- ]. coli or anti-Salmonella antibodies. The assay system consists of a flow-injection system, a disposable filter-membrane, and an amperometric sensor. A sandwich immunoassay specifically and directly detected 50 cells ml total E. coli or 50 cells ml Salmonella. The immunosensor can be used as a highly sensitive and automated bioanalytical device for the rapid quantitative detection of bacteria in food and water. 

The capacitance measurement technique applied to the antibody-coated sensor in our model system yielded a sensitivity to atrazine concentrations over the ppb to sub-ppb range. The immunoassay standard curve showed that the sensor is capable of the resolution needed for a semiquantitative assay [39]. Scatter in the data is sufficiently small to allow for a positive or negative result within... 

The future use of coated piezoelectric devices as immunochemical sensors, even directly in the liquid phase, is very promising and could be considered a very competitive alternative to other types of immunoassay. Only this technique and that of surface plasmon resonance provide labelless methods for the direct study of antigen-antibody reactions, and their analytical possibilities. The devices can be easily automated or combined with flow injection systems, extending their capability of continuous and repeated assays. This raises an exciting possibility of using crystal arrays to assay for different analytes in complex samples with an on-line display of the results. 

This definition also clarifies what is not a chemical sensor or biosensor for purposes of market projections and commercialization. Passive assays such as, for example, colorimetric chemical reactions, immunoassays, and nucleic acid probes are not sensors. While such assays do result in a quantifiable entity (such as color production, fluorescence, etc), the assay itself does not provide the means to quantify the response. Rather, a separate quantification system is required. 

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