An Enzyme Flow Immunoassay that Uses β-Galactosidase as the Label and a Cellobiose Dehydrogenase Biosensor as the Label Detector

Abstract

The aim was to develop a fast generic enzyme flow immunoassay (EFIA) using a beta-galactosidase (beta-GAL) label in combination with colorimetric detection as well as with a new amperometric biosensor as the label detector. The amperometric biosensor was previously developed within the group for the determination of diphenols in surface water samples. Antigen (Ag, analyte), tracer (Ag*, antigen labeled with beta-GAL), and antibody (Ab) were incubated off-line. After the equilibrium was reached, the sample was introduced into the flow system. The antibody complexes, AgAb and Ag*Ab, were trapped in a protein G column while the free unbound tracer was eluted and detected by an amperometric biosensor downstream after substrate reaction. The enzyme label beta-GAL converted the substrate 4-aminophenyl-beta-D-galactopyranoside (4-APG) into 4-aminophenol (4-AP), which subsequently was detected by a cellobiose dehydrogenase (CDH) modified solid graphite electrode. 4-AP was first oxidized at the electrode surface at +300 mV vs Ag/AgCl, and the formed 4-imino quinone (4-IQ) was reduced back to 4-AP by the CDH in the presence of cellobiose. By combining the EFIA with the CDH biosensor, the overall signal of one tracer molecule is amplified at two occasions, i.e., one enzyme label converts the substrate into many 4-AP molecules, and second these are further amplified by the CDH biosensor. The optimum conditions for the EFIA in terms of the molar ratio between tracer and beta-GAL, temperature, flow rate, etc., was investigated with colorimetric detection, using 2-nitrophenyl-beta-D-galactopyranoside (2-NPG) as the beta-GAL substrate. The performance of both the colorimetric and CDH biosensor detection was investigated and both methods were applied for determination of the model compound atrazine in spiked surface water samples. Detection limits of 0.056 +/- 0.008 and 0.038 +/- 0.007 microg L(-1) and IC50 values of 2.04 +/- 0.294 and 0.42 +/- 0.08 microg L(-1) were obtained for colorimetric and CDH detection, respectively. Matrix effects were less pronounced with the CDH biosensor than with colorimetric detection.

Direct Electron Transfer Between Ligninolytic Redox Enzymes and Electrodes

“…Because of its attractive and unique properties CDH has several technical applications, e.g., in biosensors for the sensitive and selective detection of toxic diphenols [179,180], of cellodextrins and lactose [181 ± 185], or as sensor in conjunction with flow immunoassays [186], in bioremediation for the degradation of recalcitrant pollutants including munitions and polyacrylate polymers [42,56,187], or in biocatalysis for the preparation of organic acids [187].…”

Sensors and Food Quality

“…With so much effort being focused on improving biosensor analysis, the applications of the technology are likely to expand throughout the coming decade, with food analysis at the forefront. Improvements in the performance of immunosensors have been achieved in two key areas; the increased use of molecular biology to produce rapidly highly specific antibodies, eg, phage display [73] and antibody cloning [74], and the improvement of the transduction process from the enzyme label, eg, osmium redox polymers [75] and amplification systems [76]. Chemical sensors have been pushed to improve their own performance, including new polymer deposition techniques [77], semiconductor oxide materials [78], and the continuous improvement in sensitivity of surface plasmon resonance systems [79].…”

Recent Advances in Electrochemical Enzyme Immunoassays

“…This choice has been successfully applied to detect aflatoxin M1 [82] in raw milk and atrazine [83], using in both cases a Protein G column. Aflatoxin M1 is determined amperometrically at a glassy carbon electrode, monitoring the activity of the enzyme HRP in presence of TMB, while the enzyme b-galactosidase and the substrate PAPG are used for the amperometric detection of atrazine with a cellobiose dehydrogenase (CDH) modified solid graphite electrode.…”