A capillary-based amperometric flow immunoassay for 2,4,6-trichlorophenol

Abstract

This paper describes the development of two different capillary-based heterogeneous competitive flow immunoassay formats (capillary flow injection immunoassay (CFIIA) and capillary sequential injection immunoassay (CSIIA)) for the determination of 2,4,6-trichlorophenol (2,4,6-TCP). The assays are based on the competition between the analyte and an analyte derivative labelled with the enzyme beta-galactosidase, for an anti-TCP antibody, followed by the injection of the mixture at equilibrium into a flow stream, where separation between the fractions bound and unbound to the antibody is performed in a glass capillary containing immobilised protein A. The antibody-tracer fraction retained inside the protein A capillary was measured by injection of 4-aminophenyl- beta- D-galactoside (4-APG), followed by amperometric detection of the enzymatically generated 4-aminophenol (4-AP), leading to a negative correlation between the signal and the analyte concentration. The two immunoassay formats were compared in terms of sensitivity and speed, giving IC(50) values of 1.41+/-0.03 and 1.64+/-0.07 micro g L(-1), detection limits of 0.2 and 0.4 micro g L(-1), and sample throughputs of 6 and 4 h(-1) for the CFIIA and CSIIA system, respectively. The influence of different interfering chlorophenolic compounds in the assay was minor, with only one exception (i.e. 2,4-dichlorophenol). In addition, different water matrices were tested (surface, tap, and rain water), showing that the matrix influence was negligible, except for rainwater, which resulted in a 30% increase in sensitivity. As a conclusion, the assay is suitable for the fast screening of TCP present at low concentration levels in water samples.

Recent Advances in Electrochemical Enzyme Immunoassays

“…The first choice has been followed by Nistor et al to detect 4-nitrophenol [80] and 2,4,6-trichlorophenol [81], using bgalactosidase as label and PAPG as enzyme substrate. The system designed for 4-nitrophenol incorporates a Protein G column and the product of the enzymatic reaction is amperometrically detected at a glucose dehydrogenase (GDH) screen-printed biosensor, while a Protein A capillary is used in combination with an Au based screen-printed electrode, for the 2,4,6-trichlorophenol determination.…”

Recent developments in electrochemical flow detections—A review

“…It was found, however, that the response obtained with non-conducting beads was significantly larger, which was attributed to different capacity of beads for enzyme immobilization. A special type of a flow-through reactor was used in FIA and SIA systems for immunochemical determinations of 2,4,6-trichlorophenol with amperometric detection [116]. In determination based on enzyme labeling of the antibody, the immunochemical reaction was carried out off-line, and after an appropriate incubation time the mixture was injected into a flow system, where the capillary reactor with immobilized protein A was used for separation between the fractions of bound and unbound to the antibody.…”

Biochemical analysis with microfluidic systems

“…The automation of heterogeneous immunoassays is wellknown from the development of flow-through systems comprising immunoreactors as packed-bed columns [80] or as open-tubular capillaries [81] with specific capture antibodies immobilized on glass beads or on the walls of the capillary. Those capillaries were fabricated also from PDMS resulting in the first element of an heterogeneous immunoassay microchip [82].…”