Immunosensors

CNTs also have promising applications in the immunosensing field and many examples of such sensor devices can be found in the literature. For example, immunosensors with an electrochemiluminescence readout signal have been designed using CNTs as support of the immunorecognition systems [167]. 

Recently, some papers have started to highlight the performance of PSf-(bio) composites-CNT as electrode material for electrochemical immunosensing [112]. The authors have highlighted the attractive combination of PSf/CNTplus disposable screen-printed electrodes for monitoring the errzymatic activity of horseradish peroxidase and the RIgG inmunosensor response. In both cases an enhanced electroanalytical response was demonstrated in comparison with standard graph-ite/PSf composites. 

Sandwich-type sensors are applicable for measuring large antigens that are capable of binding two different antibodies. Such sensors utilize an antibody that binds the analyte-antigen, which then binds an enzyme-labeled second antibody. After removal of the nonspecifically adsorbed label, the probe is placed into the substrate-containing solution, and the extent of the enzymatic reaction is monitored 

FIGURE 6-13 Enzyme immunosensors based on the competitive or sandwich modes of operation. (Reproduced with pennission from reference 40.) 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (59). In this case, the content of the immunoassay reac- 

More recent trends aim in the direction of fabricating electrochemical protein array systems (for detecting multiple protein targets) and miniaturization of such immunoassays. These include an electrochemical protein chip with an array of 36 platinum electrodes on a glass substrate (64) and electrical immunoassays using microcavity formats down to the zmol antigen level (65). 

In addition to antibodies, it is possible to use artificial nucleic acids ligands, known as aptamers, for the selective detection of proteins. The tight binding properties make aptamers attractive candidates as molecular recognition elements in a wide range of bioassays and for the development of protein arrays. Electrochemistry has been shown useful for monitoring aptamer-protein interactions (66). 

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

Fig. 9. Immunosensor approaches where A is the analyte, is the labeled analyte, and Y is the antibody, (a) Direct immunosensors where the actual antigen—antibody interaction is measured (b) indirect immunosensors 1 and 2 which utilize formats similar to competitive and displacement...

Immunosensors promise to become principal players ia chemical, diagnostic, and environmental analyses by the latter 1990s. Given the practical limits of immunosensors (low ppb or ng/mL to mid-pptr or pg/mL) and their portabiUty, the primary appHcation is expected to be as rapid screening devices ia noncentralized clinical laboratories, ia iatensive care faciUties, and as bedside monitors, ia physicians offices, and ia environmental and iadustrial settings (49—52). Industrial appHcations for immunosensors will also include use as the basis for automated on-line or flow-injection analysis systems to analyze and control pharmaceutical, food, and chemical processing lines (53). Immunosensors are not expected to replace laboratory-based immunoassays, but to open up new appHcations for immunoassay-based technology. 

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). 

Enzyme immunosensors are employed for the determination of Hepatitis B surface antigen, IgG, alpha-fetoprotein, estradiol, theophylline, insulin [9004-10-8] and alburnin (69,70). However, these immunosensors generally have slow response times and slow reversibiUty (57). 

Enzyme immunosensors are used in flow injection systems and Hquid chromatography to provide automated on-line analyses (71—73). These systems are capable of continuously executing the steps involved in the immunoassays, including the binding reactions, washing, and the enzyme reaction, in about 10 minutes. 

AN AMPEROMETRIC ENZYME IMMUNOSENSOR BASED ON SCREEN-PRINTED ELECTRODE FOR THE DETERMINATION OF KLEBSIELLA PNEUMONIAE BACTERIAL ANTIGEN... 

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. 

The developed amperometric enzyme immunosensor was probed to determine the Klebsiella pneumoniae antigen in the human sera samples. The obtained results were juxtaposed with the data of the bacteriological analysis. 

Explain clearly how the use of enzymes can enhance the power of electrochemical immunosensors. 

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

Particularly attractive for numerous bioanalytical applications are colloidal metal (e.g., gold) and semiconductor quantum dot nanoparticles. The conductivity and catalytic properties of such systems have been employed for developing electrochemical gas sensors, electrochemical sensors based on molecular- or polymer-functionalized nanoparticle sensing interfaces, and for the construction of different biosensors including enzyme-based electrodes, immunosensors, and DNA sensors. Advances in the application of molecular and biomolecular functionalized metal, semiconductor, and magnetic particles for electroanalytical and bio-electroanalytical applications have been reviewed by Katz et al. [142]. 

Electrogenerated chemiluminescence (ECL) has proved to be useful for analytical applications including organic analysis, ECL-based immunosensors, DNA probe assays, and enzymatic biosensors. In the last few years, the electrochemistry and ECL of compound semiconductor nanocrystallites have attracted much attention due to their potential applications in analytical chemistry (ECL sensors). 

The current trend in analytical chemistry applied to evaluate food quality and safety leans toward user-friendly miniaturized instruments and laboratory-on-a-chip applications. The techniques applied to direct screening of colorants in a food matrix include chemical microscopy, a spatial representation of chemical information from complex aggregates inside tissue matrices, biosensor-based screening, and molec-ularly imprinted polymer-based methods that serve as chemical alternatives to the use of immunosensors. 

The development of immunosensors is one of the most active research areas in immun-odiagnostics. A large number of immunosensors, which combine the sensitivity and specificity of immunoassays with physical signal transduction, have been developed... 

A.M. Campbell, Monoclonal Antibody and Immunosensor Technology the Production and Application of Rodent and Human Monoclonal Antibodies, Elsevier, Amsterdam (1991). 

Thin films of functionalized amorphous silica for immunosensors application. Journal of Sol-Gel Science and Technology, 2, 823-826. 

Fiber Optic Affinity Sensors, Immunosensors and Gene Sensors... 

Bluenstein B., Walczak I., Chen S.Y., Fiber-optic evanescent-wave immunosensors for medical diagnostics, Trends Biotechnol 1990 161-168. 

Heideman R.G., H. Kooyman R. P., Greve J., Performance of a highly sensitive optical waveguide Mach-Zehnder interferometer immunosensor, Sensors and Actuators B 1993 10 209-217. 

Stemesjo A., Mellgren C., Bjorck L., Analysis of Sulfamethazine in Milk by an Immunosensor assay Based on Surface Plasmon Resonance, Immunoassays For Residue Analysis, ACS Symposium Series, 621 463-470 (1996). 

Ymeti A., Kanger J.S., Wijn R., Lambeck P.V., Greve J., Development of a multichannel integrated interferometer immunosensor, Sens. andActuat. B 2002 83 1-7. 

Figure 2 shows the most abundant class of antibodies found in blood serum and lymph - immunoglobulin G (IgG). IgG of molecular mass about 156 000, is most frequently used as a receptor in immunosensors. According to X-ray data6 8, IgG is a Y-shaped molecule consisting of two identical antigen binding Fab arms of dimensions 6.5 nm by 3.5 nm and an inactive Fc shank of dimensions 5 nm by 3.5 nm. 

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