Affinity EnzymoMetric Assay (AEMA) as the Highly Sensitive and Disposable Test for Cholinesterase Inhibitors

Usually, bioanalytical methods for the detection of organophosphorous (OP) compounds and other cholinesterase inhibitors (ChEI) are based on the measurement of inhibition of the cholinesterase activity in the presence of such compounds. In this case the analytical response is inversely related to inhibitor concentration. Thus, the accuracy of detection of low inhibitor concentrations is insufficient because the final analytical signal is represented by a small change of a large basic signal. To overcome this limitation the new sensitive method for the analysis of ChEI has been developed. Our approach is called Affinity EnzymoMetric Assay (AEMA) and consists of three steps (Fig.): first, the recognition, where ChEI binds to horseradish peroxidase (HRP) labelled cholinesterase (bi-enzyme HRP-ChE conjugate). The second, the excess HRP-ChE conjugate is removed after incubation the conjugate with paraoxon-modified affinity support. Finally, the inhibitor-HRP-ChE complex is detected in supernatant (or collected eluant) fluid by measure HRP activity with 3,3'5,5'-tetramethylbenzidin in the presence of hydrogen peroxide at 450 nm.

First, the developed approach was realized using Langmuir-Blodgett films as affinity support for the binding of inhibitor-free bi-enzyme conjugate HRP-ChE. The capacity of affinity support to cholinesterase (8,5.10-12 mol/cm2) was enough to perform the disposable ChEI assay. However the disposable micro column containing paraoxon modified porous carrier was more suitable format for fast flow-injection assay of highly toxic substances. The disposable micro columns (about 10 ml volume) containing porous affinity support can be used. Only two minutes is required to separate of excess HRP-ChE conjugate on affinity micro column at the analysis of ChEI. The lower limit of detection is about 1 pM for OP compounds such as diisipropylfluorophosphate (DFP), paraoxon and trichlorfon. The test allows to detect carbamates (carbofuran, carbetamide and carbaryl) in the concentration 0.1-1.0 nM.

Figure 1: The principle of Affinity EnzymoMetric Assay (AEMA) of ChEI.


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