Acetylcholine receptor fiber-optic evanescent fluorosensor.
An acetylcholine receptor-based optical biosensor was developed, which uses the evanescent wave to excite fluorescein isothiocyanate-labeled alpha-bungarotoxin (FITC-alpha-BGT), that is bound to a receptor protein immobilized on the surface of a quartz fiber. After excitation of the fluorophore just outside the waveguide boundary, the resultant fluorescence was trapped by and propagated back up the fiber. Pure nicotinic acetylcholine receptor (nAChR) protein, isolated from Torpedo electric organ, was immobilized noncovalently on quartz optic fibers and its density was quantitated by 125I-BGT binding. In the absence of nAChR, FITC-alpha-BGT alone bound to the quartz fiber. This nonspecific (i.e., nonreceptor) binding of FITC-alpha-BGT was totally eliminated by the addition of 0.1 mg/ml bovine serum albumin to the phosphate-buffered saline medium. This solution did not interfere with FITC-alpha-BGT binding to the nAChR that was immobilized on the fiber. Thus, only specific binding was observed under these conditions, resulting in a very high signal-to-noise ratio of greater than 99. Specific FITC-alpha-BGT binding to the nAChR protein on the optic fibers was inhibited by agonists (e.g., acetylcholine, nicotine, and carbamylcholine) and antagonists (e.g., pancuronium and d-tubocurarine) of the nAChR and was insensitive to high salt concentrations (e.g., 154 mM NaCl). The binding was most sensitive to the highly nAChR-specific alpha-BGT and alpha-cobratoxin. The optic-fiber sensor in its present form does not distinguish between receptor agonists or antagonist and cannot be regenerated for repeated use.(ABSTRACT TRUNCATED AT 250 WORDS)
Rogers
K R
KR
University of Maryland School of Medicine, Department of Pharmacology & Experimental Therapeutics, Baltimore 21201.
Valdes
J J
JJ
Eldefrawi
M E
ME
D000818:Animals; D002038:Bungarotoxins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002217:Carbachol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005069:Evaluation Studies as Topic; D005336:Fiber Optic Technology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005470:Fluorometry / Q000295:instrumentation* / Q000379:methods; D055100:Optical Fibers; D009994:Osmolar Concentration; D011791:Quartz; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism; D015398:Signal Transduction; D013861:Thiocyanates; D014403:Tubocurarine / Q000378:metabolism
D002038:Bungarotoxins; D005452:Fluoresceins; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D013861:Thiocyanates; D011791:Quartz; D002217:Carbachol; D016650:Fluorescein-5-isothiocyanate; D014403:Tubocurarine