Fiber-Optic Fluoroimmunoassay System with a Flow-Through Cell for Rapid On-Site Determination of Escherichia coli O157:H7 by Monitoring Fluorescence Dynamics.

Dynamic fluoroimmunoassay with a flow-through system using optical fiber probes consisting of polystyrene was developed and applied to a quantitative detection of E. coli O157:H7. The system measures E. coli as fluorescence of sandwich-type immune complexes formed by capture antibodies immobilized on the surface of the probe, E. coli cells, and fluorescently labeled detection antibodies. Excitation was carried out using an evanescent wave from the probe. Resulting fluorescence recoupled into the probe was detected by a photodiode. The assay system was constructed with a flow cell which was available for sequential injection of experimental reagents. In vitro characterization was performed using the flow cell, and the calibration range of E. coli O157:H7 was from 10(3) to 10(7) cells/mL. The measurement for each sample was completed within 12 min. Furthermore, it was also possible to estimate the concentrations of E. coli O157:H7 by the increasing rate of fluorescence during binding reaction of detection antibodies to antigens. This minimized the time for measurement down to 6 min. The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.

Fluorescence immunoassay using an optical fiber for determination of Dermatophagoides farinae (Der f1).

Immunoassay methods are generally used for measuring of allergenic substances. However, they need special facilities, skilled handling, and time-consuming procedure. In this work, a fiber-optic immunoassay system which could measure allergen by fluorescent intensities of immune complexes formed by allergens and fluorescently labeled antibodies was established. Immune complexes absorbed on the optical fiber probe surface, and excitation light was injected into the probe, then evanescent field is created in the proximity of the probe. The fluorophores were excited by the evanescent light, and fluorescence was detected by a photo diode. The target allergen detected by our system was Der f1 derived from Dermatophagoides farinae that is one of the house dust mite and major source of inhaled allergens. The fluorophore used labeling on detecting antibody was cyanine 5. The system enabled to detect and quantitatively determine of Der f1. The measurement range was from 0.24 to 250 ng/ml, and the result competes with ELISA. The measurement time was 16 min/sample. The immunoassay system was applied to measurement of Der f1 from actual dust samples. Calculated values of Der f1 showed good correlations between the fiber-optic fluoroimmunoassay and ELISA.