Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets.
An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 microm) produced by a piezoelectric generator system with 10-microm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 x 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A-fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the IC50 of the LIF-microdroplet QFIA is 0.45 microg L(-1) reaching a LOD of 0.04 microg L(-1). The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 microg L(-1) in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 ,g L(-1), with a dynamic range between 4 and 149.5 microg L(-1) in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment.
Nichkova
Mikaela
M
Department of Biological Organic Chemistry. IIQAB-CSIC, Jorge Girona, 18-26, 08034-Barcelona, Spain.
Feng
Jun
J
Sanchez-Baeza
Francisco
F
Marco
M Pilar
MP
Hammock
Bruce D
BD
Kennedy
Ian M
IM
D001667:Binding, Competitive; D002138:Calibration; D002733:Chlorophenols / Q000652:urine*; D005453:Fluorescence; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D007700:Kinetics; D007834:Lasers; D015394:Molecular Structure; D016273:Occupational Exposure; D012680:Sensitivity and Specificity
D002733:Chlorophenols; C024564:2,4,6-trichlorophenol