Optical fiber biosensors binding

Fig. 4 - An optic fiber biosensor for cocaine using a mAb against benzoylecgonine as the biological sensing element, (left) The time course of binding of FL-BE to the mAb-coated fiber expressed by the fluorescent signal transmitted via the fiber. After reaching steady state, FL-BE was withdrawn from the flow buffer (indicated by the arrow). The bound FL-BE dissociated and fluorescence decreased exponentially, (right) Reusability of the biosensor for multiple assays of cocaine introduced into the flow buffer after steady-state fluorescence (200 mV) was achieved. Cocaine at the indicated concentrations was added to the flow buffer for only the time intervals indicated by the bars. The downward deflection resulted from displacement of FL-BE by cocaine, but upon removal of cocaine from the flow buffer FL-BE displaced the bound cocaine. Reproduced with permission from reference 4, Copyright 1995 American Chemical Society.

This biosensor employs a quartz optical fiber as a transducer, and the chemical recognition element is the nicotinic acetylcholine receptor (AcChR). The receptor is a membrane protein that spans a lipid bilayer it binds acetylcholine rapidly and reversibly, and changes shape upon binding, to allow the transport of ions through a... 

The AcCh biosensor uses competitive binding between FITC labeled or unlabeled bungarotoxin and receptor immobilized by adsorption onto the surface of a quartz optical fiber. Once bound to the protein BTX, FITC exhibits absorption and emission maxima of 495 nm and 520 nm, respectively. In the absence of analyte (BTX), a maximal quantity of FITC-BTX binds to the immobilized receptor protein. 

Fig. 2 - The specificity of binding of the fluorescent probe to the Ab-coated optic fibers of the biosensor, (left) The fluorescent signal transmitted by quartz fibers coated with mAbs raised against BE, compared to that of untreated fibers and fibers coated with human IgG or casein. The flow buffer contained 10 nM FL-BE. The lines are offset from each other by 10 mV. Reproduced with permission from reference 4 Copyright 1995 American Chemical Society, (right) The fluorescence transmitted by TCPB-FL, bound to the fiber coated with rabbit anti-PCB IgGs, control rabbit IgG, human IgG or casein compared to a bare fiber. It is clear that the non-specific TCPB-FL binding to the quartz fibers, non-target IgG can be significant and must be corrected for. Reproduced with permission from references 4 and 27, Copyright 1995, American Chemical Society.

Biosensor Probes. For the fiber optic biosensor used here, a portion of protective cladding on the exterior of the optical fiber is removed from the distal 10 cm of the fiber to expose a core of fused silica. This exposed region becomes the probe. Antibodies are covalently attached to the exposed core. When the probe is in contact with a sample containing an analyte, the immobilized antibody specifically binds the analyte from the bulk solution and concentrates it on the surface of the fiber within the evanescent zone. Any fluorophore associated with the analyte is also immobilized within the evanescent wave. Excitation of the fluorophore by light in the evanescent wave leads to fluorescent emission which generates a detectable signal. Two different methods of associating a fluorophore with the analyte are described below. 

There were five steps involved in the preparation of the optical fibers tapering, cleaning, silanization, crosslinker attachment, and covalent binding of antibodies. The procedures used were based on methods outlined previously (Bhatia et al., 1989 Ogert et al., 1992). The goal was to immobilize affinity purified antibodies on the surface of the fibers for the capturing of complex molecules in solution and ultimately detection using the biosensor. 

Seo et al. (1999) used a planar optic biosensor that measures the phase shift variation in refractive index due to antigen binding to antibody. In this method, they were able to detect S. enterica serovar T) himurium with a detection limit of 1 x 10 cfu/ml. When chicken carcass fluid was inoculated with 20 cfu/ml, the sensor was able to detect this pathogen after 12 h of nonselective enrichment. A compact fiber optic sensor was also used for detection of S. T) himurium at a detection limit of 1 X 10" cfu/ml (Zhou et al., 1997, 1998) however, its efficacy with food samples is unproven. Later, Kramer and Lim (2004) used the fiber optic sensor, RAPTOR , to detect this pathogen from spent irrigation water for alfalfa sprouts. They showed that the system can be used to detect Salmonella spiked at 50 cfu/g seeds. An evanescent wave-based multianalyte array biosensor (MAAB) was also employed for successful testing of chicken excreta and various food samples (sausage, cantaloupe, egg, sprout, and chicken carcass) for S. T) himurium (Taitt et ah, 2004). While some samples exhibited interference with the assay, overall, the detection limit for this system was reported to be 8 x 10 cfu/g. 

Fluorescence Detection Fiber-optic Biosensor on Biomolecules Binding Kinetics Measurement... 

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. 

Lee and Walt used a related strategy to build a thrombin biosensor by covalent attachment of the TEA sequence to silica microspheres. They then used a fiber optic device to detect the binding of fluorescein-labeled thrombin to... 

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