A fiber-optic cocaine biosensor.
A fiber-optic biosensor was developed for detection of cocaine, its metabolites, and other coca alkaloids, using a monoclonal antibody (mAb) against a derivatized benzoylecgonine (BE). The mAb was immobilized noncovalently on quartz fibers and a flow fluorometer was used to detect changes in evanescent wave fluorescence. A fluorescein (FL) conjugate of BE bound to the mAb specifically in a saturable manner and with high affinity (Kd = 7.6 nM). Cocaine or other test compounds competed with FL-BE for binding to the mAb in a concentration-dependent manner, thereby reducing the initial rate or steady-state fluorescence. Addition of cocaine to the flow buffer after reaching steady-state fluorescence enhanced the dissociation of bound FL-BE, and cocaine removal allowed fiber regeneration for multiple measurements. The detection limits for cocaine, cocaethylene, norcocaine, and BE were 5, 5, 29, and 30 ng/ml, respectively, but for ecgonine it was 4600 ng/ml and for methylecgonine it was 2000 ng/ml. Tropacocaine was detected at 10 ng/ml, but atropine was detected at 2900 ng/ml. The biosensor discriminated by 833-fold between cocaine and its stereoisomer pseudococaine. Structural features necessary for high-affinity recognition by this mAb are benzoate and 3 beta configuration, both of which are found in BE, cocaine, norcocaine, and cocaethylene.
Devine
P J
PJ
Department of Pharmacology and Experimental Therapeutics, School of Medicine, University of Maryland at Baltimore, 21201, USA.
Anis
N A
NA
Wright
J
J
Kim
S
S
Eldefrawi
A T
AT
Eldefrawi
M E
ME
D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002138:Calibration; D003042:Cocaine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D018796:Immunoconjugates; C005618:benzoylecgonine; D003042:Cocaine; D019793:Fluorescein