Enhancer effect of fluorescein on the luminol-H2O2-horseradish peroxidase chemiluminescence: energy transfer process.

The chemiluminescence of the luminol-H2O2-horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intensity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects.

Variable-angle synchronous fluorescence spectrometry and rank annihilation methods for mixture resolution.

The potential of variable angle synchronous spectroscopy (VASS) for fluorescent mixtures resolution was assessed and compared with the rank annihilation method (RAM). For this purpose, a set of excitation-emission matrices from three standard cyclodextrin fluorescence-enhanced solutions of the pesticides aminocarb, carbendazim and coumatetralyl and a mixture of them was obtained. Careful selection of the spectral routes to be scanned provides analyte signals that are free of interferences. Application of the rank annihilation method to excitation-emission matrices (EEMs) obtained by conventional scanning spectrofluorimetry gives quantitative results that show poor precision and accuracy when compared to those of VASS. The recoveries from ternary mixtures by VASS are within 99-104% and by RAM within 84-130%.

Enzyme-linked immunosorbent assay by image analysis using a charge-coupled device array detector.

This paper describes a fluorescence enzyme-linked immunosorbent assay (ELISA) for the quantification of (+/-)-2-(2, 4-dichlorophen-oxy)propionic methyl ester (dichlorprop methyl ester). Antibodies for dichlorprop methyl ester were produced by immunizing rabbits with a conjugate of dichlorprop methyl ester with bovine serum albumin. Data acquisition on microtiter wells is performed by a spectrofluorometer through a fiber optic and by a charge-coupled device camera. A correlation was obtained between the image analysis data on ELISA and the data acquired by the spectrofluorometer. The results demonstrate that the fluorescence image analysis performed by the charge-coupled device detector is applicable to ELISA, and the analysis time, sensitivity, and precision of the ELISA procedure are compared to conventional fluorescence ELISA performed by the spectrofluorometer. The ELISA procedure was selective for structurally similar compounds or usually found in formulation pesticides. Concentrations for 50% displacement curves were dichlorprop, 83.59 microg/ml, and 2,4,5-T, 388.23 microg/ml; triclopyr, ioxynil, bentazone, and MCPA had no response.

Phase-modulation fluorescence lifetime immunoassay of dichlorprop.

A homogeneous immunofluorimetric technique to determine dichlorprop herbicide is described. Simultaneous determinations of both free and antibody-bound fluoresceinamine-labeled dichlorprop were carried out by measuring phase-resolved fluorescence or modulation-resolved fluorescence. The difference between the recorded values of the fluorescence lifetimes of the two species quantifies selectivity. Multifrequency heterogeneity analyses of free and bound labeled dichlorprop gave 3.68 +/- 0.03 and 4.28 +/- 0.02 ns, respectively. The differences between the phase-angles of the two species (117.5 degrees and 127.2 degrees) correspond to a difference between fluorescence lifetime values of 600 ps. This lifetime difference permits the quantitative determination of the two species simultaneously. The mean relative standard deviation of the set of three calibration curves when fitted to radioimmunoassay (chi 2 5 x 10(-4)) was 6%. The practical dynamic range was 0.1 to 100 micrograms.ml-1. On the other hand, the fractional contributions of both species, free and bound, to the total lifetime measured by demodulation, permit quantitative calculation of the two components. Both methods gave similar analytical specifications.