Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets

Mikaela Nichkova, Jun Feng, Francisco Sanchez-Baeza, M. Pilar Marco, Bruce D. Hammock, Ian M. Kennedy

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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 μm) produced by a piezoelectric generator system with 10-μm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 × 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 μg L-1 reaching a LOD of 0.04 μg L-1. The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 μg L-1 in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 μg L-1, with a dynamic range between 4 and 149.5 μg 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.

Original languageEnglish (US)
Pages (from-to)83-90
Number of pages8
JournalAnalytical Chemistry
Volume75
Issue number1
DOIs
StatePublished - Jan 1 2003

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Chlorophenols
Quenching
Fluorescence
Lasers
Buffers
Haptens
CCD cameras
Orifices
Fluorescein
Biosensors
Risk assessment
Dilution
Spectrometers
Assays
Ions
Antibodies

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets. / Nichkova, Mikaela; Feng, Jun; Sanchez-Baeza, Francisco; Marco, M. Pilar; Hammock, Bruce D.; Kennedy, Ian M.

In: Analytical Chemistry, Vol. 75, No. 1, 01.01.2003, p. 83-90.

Research output: Contribution to journalArticle

Nichkova, Mikaela ; Feng, Jun ; Sanchez-Baeza, Francisco ; Marco, M. Pilar ; Hammock, Bruce D. ; Kennedy, Ian M. / Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets. In: Analytical Chemistry. 2003 ; Vol. 75, No. 1. pp. 83-90.
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