Fiber optics, immunoassay

An example of an evanescent wave fiber optic immunoassay and the associated optics has been described in detail for measurement of anti-rabbit IgG.(130) Rabbit antibody is immobilized on the distal face of an optical fiber. Unlabeled anti-rabbit IgG competes with fluorescein-labeled anti-rabbit IgG for rabbit antibody binding sites... 

Fluorescence-based fiber optic immunoassay (a) 100 pg/mL (b) 1 ng/mL (a) Buffer solution (b) River water... 

King K.D., Vanniere J.M., LeBlanc J.L., Bullock K.E., Anderson G.P., Automated fiber optic biosensor for multiplexed immunoassays, Environ. Sci. Technol. 2000 34 2845-2850. 

Fluorometers designed for research purposes(31) are typically equipped with a xenon arc lamp, monochromators, one or more photomultiplier tubes, cuvet holders, and a computer interface. Some research level fluorometers, such as the Perkin-Elmer LS50, have optional microtiter plate reading accessories with fiber optic bundles. This is convenient since 96-well microtiter plates are commonly used for immunoassay development, and many commercial immunoassays are based on the use of microtiter plates. Fluorometers designed for commercial immunoassay purposes are generally dedicated instruments with few, if any, data acquisition and reduction parameters that can be manipulated by the user. 

R. D. Petrea, M. J. Sepaniak, and T. Vo-Dinh, Fiber-optic time-resolved fluorimetry for immunoassays, Talanta 35, 139-144 (1988). 

J. D. Andrade, W. M. Reichert, D. E. Gregonis, and R. A. VanWagenen, Remote fiber-optic biosensors based on evanescent-excited fluoro-immunoassay Concept and progress, IEEE Trans. Electron Devices ED-32, 1175-1179 (1985). 

Fiber-optic sensors based on controlled-release polymers provide sustained release of indicating reagents over long periods. This technique allows irreversible chemistries to be used in the design of sensors for continuous measurements. The sensor reported in this paper is based on a fluorescence energy transfer immunoassay. The sensor was cycled through different concentrations of antigen continuously for 30 hours. 

For example, several strategies have been used for immunoassay techniques with fiber-optic biosensors. In the sandwich format, the receptor is immobilized on the stu"face of the fiber waveguide and a secondary or tracer antibody (which is labelled with a fluorescent dye) is added to the solution. In the absence of the analyte, the tracer remains in solution and little fluorescence is observed. However, after addition of the analyte, a molecular sandwich is formed on the sensor smface within the evanescent excitation volume. The sandwich assay is usually more sensitive than a competitive-binding assay because the fluorescence intensity increases with analyte concentration. 

Immunochromatography, colloidal gold, silver enhancement 10 minutes Evanescent wave fiber-optic immunosensor Colorimetric and chemiluminescence ELISA Immunochromatography, colloidal gold, silver enhancement 10 minutes Sandwich hybrid receptor-immunoassay,... 

Boyer and coworkers were the first to develop instrumentation for near-infrared fluorescence immunoassays [117]. WiUiams and coworkers also developed instrumentation for detection of near-infrared fluorescence in sohd-phase immunoassays [118]. The instrument consists of a semiconductor laser coupled with a fiber-optic cable, a silicon photodiode for detection, a sample stage coupled to a motor drive, and a data acquisition device. The instrument could detect 500 pM concentrations of human immunoglobulin G (IgG) on a nitrocellulose matrix. The assay was performed in roughly two hours. The detection limits obtained on this instrument were comparable to that obtainable with ELISA. The assay developed by WiUiams suffers from excessive scatter generated from the membrane, nonspecific binding, and incompatibility with conventional microtiter plate immunoassay formats [140]. Patonay and coworkers developed a NIR fluorescence immunoassay apparatus that overcame many of these limitations. Baars and Patonay have evaluated a novel NIR dye NN382 (Fig. 14.25) for the ultrasensitive detection of peptides with capilary electrophoresis [141]. A solid-phase, NIR fluorescence immunoassay system was... 

A fiber-optic electrode was fabricated for the simultaneous generation and transmission of electrochemical luminescence by preparing a transparent electrode on the optical and surface of a fiber-optic. The opto-electrochemical properties of the micro-optical device were characterized in solutions containing the compounds required for luminol luminescence. The validity of sensitive measurement of electrochemiluminescence to be employed in a homogeneous immunoassay was evaluated by using potential step excitation of luminol in the presence and in the absence of hydrogen peroxide. 

We report here the performance characteristics of the fiber-optic electrode by app iqg the device to the electrochemiluminescence of luminol to clarify the feasibility of luminol as an electrochemiluminescent label for a sensitive homogeneous immunoassay. 

Lubbers and Optlz (9) and Andrade et al (10 have employed fiber optic sensors for the continuous measurement of chemical reactions in biological systems. High sensitivity may be achieved using these sensors to measure fluorescent-tagged antigens or antibodies in competitive binding immunoassay reactions In solution. 

This chapter describes an evanescent wave fiber optic biosensor and its application to immunoassays for rapid detection of bacterial cells and pollutants. Whole cells of Burkholderia cepacia G4 5223-PRl (G4) are of interest for their ability to degrade trichloroethylene (TCE) which is one of the most prevalent contaminants of ground water in the United States. The lower limit of detection of the G4 with this system is 10 - 10 cells/ml. In addition to TCE, the explosive trinitrotoluene (TNT) is a known contaminant of ground water. Limits of detection of TNT with this system is 10 ng/ml. 

What is possible, however, is a technique which weights each of these properties in accordance to its importance in a given application. The use of immunoassays in conjunction with an evanescent wave fiber optic biosensor is a unique way of balancing the demands for specificity, sensitivity, rapidity, adaptability, and simplicity. 

Figure 5. TNT competitive immunoassay on fiber optic biosensor. A single fiber optic probe was exposed to various solutions containing Cy5-TNB(7.5 ng/ml) + TNT (0-50 ng/ml). Concentrations of TNT (ng/ml) were A=0, B=l, C=5,D=10, E=50.

Figure 6. Standard response curve for the TNT competitive immunoassay in buffer. TNT (1-860 ng/ml) solutions were assayed using the fiber optic biosensor. The percent inhibition of the reference signal for Cy5-TNB only for each concentration is shown. The 95% confidence intervals are shown. A minimum of 3 assays were performed for each concentration with the exception of 200 ng/ml TNT.

FIGURE 16 Schematic principles of bioaffinity fiber-optic biosensors. (a) Detection of intrinsically fluorescent molecule using immobilized antibody, (b) Competition assay using a fluorescent-labeled antigen, (c) Sandwich immunoassay using an immobilized antibody and a fluorescent-labeled antibody. 

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