Electrochemical Flow-ELISA for Rapid and Sensitive Determination of Microcystin-LR Using Automated Sequential Injection System

Abstract

An amperometric immunoanalysis system based on monoclonal antibodies immobilized on Sepharose beads and packed into a micro-immunocolumn was developed for the quantification of microcystin-LR. Microcystin-LR (MCLR) was used as a reference microcystin variant. Inside the immunocolumn, free microcystins and microcystin-horseradish peroxidase (tracer) were sequentially captured by the immobilized antibodies, and the detection was performed electrochemically using Super AquaBlue ELISA substrate 2,2′-azinobis(3-ethylbenzothiazoline-sulfonic acid) (ABTS). The ABTS●+ generated by enzymatic oxidation of ABTS was electrochemically determined at a carbon working electrode by applying a reduction potential set at 0.4 V versus Ag/AgCl reference electrode. The peak current intensity was inversely proportional to the amount of analyte bound to the immunocolumn. The amperometric flow-ELISA system, which was automatically controlled through the CapSenzeTM (Lund, Sweden) computer software, enabled determination of MCLR as low as 0.01 µg/L. The assay time was very short (20 min for one assay cycle). In addition, the electrochemical signals were not significantly affected by possible interferences which could be present in the real samples. Along with the simplicity of automation, this makes the developed method a promising tool for use in water quality assessment.

Electrochemical Biosensors for Tracing Cyanotoxins in Food and Environmental Matrices

“…In Lebogang’s work [ 43 ], another approach was used: in particular, to determine the presence of MC-LR in freshwater samples, an amperometric flow ELISA system, called VersAFlo, was produced, as shown in Figure 2 B. The system was fully automated, and the recognition was based on the presence of monoclonal antibodies linked to sepharose beads and packed into a micro-immunocolumn.…”

Development of an automated flow‐based spectrophotometric immunoassay for continuous detection of zearalenone

“…Prior to the flow-ELISA study, anti-ZEN was immobilized on CNBr activated Sepharose 4B beads reconditioned by adding 0.5 g of dry weight into a sintered glass filter where the beads were suspended and washed in 1.0 mM HCl (100 mL) for 15 min. The gel was washed with 50 mL of coupling buffer (0.2 M NaHCO 3 containing 0.5 M NaCl, pH 8.3) before removing the supernatant and the gel was kept at 4°C until use [24][25]. Anti-ZEN (5.0 mg L -1 ) in the coupling buffer and the gel were mixed in a centrifuge tube at 1:1 (v/v) ratio.…”

“…Since traditional ELISA cannot be directly applied to a process control on field, the combination of flow injection and traditional ELISA can offer fast, affordable, on-site detection, an approach which has been established by Nilsson et al [20][21] and further developed by others [22][23][24][25]. The Versatile Continuous Flow System (VersAFlo) based on sequential flow-injection created by Kumar et al (2012) [24] for bioanalytical application was designed to be reproducible, robust and easy to integrate to an online monitoring system.…”

Sequential Injection Analysis of Butyrylcholinesterase Using Butyrylcholine Ion-Selective Electrode Detector

“…On the other hand, sequential injection analysis (SIA) method is an analytical method in which a sample solution and a reagent solution are injected sequentially into a holding coil (HC) using a syringe pump (SP) and a multi-port selection valve (SV) controlled by a computer (PC) to induce mixing and chemical reactions through bidirectional flow, and the reaction products are led to a detector for measurement. [11][12][13][14][15][16][17][18][19][20] Unlike the flow injection analysis (FIA) method, the SIA method does not require continuous pumping of reagent solutions, and thus has the advantage of extremely low reagent consumption, making it an analysis method that conforms to the zero-emission concept.…”