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Electrochemical Assays and Immunoassays of the Myeloperoxidase/SCN–/H₂O₂ System

Author:
Bekhit, Michael, Gorski, Waldemar
Source:
Analytical chemistry 2019 v.91 no.4 pp. 3163-3169
ISSN:
1520-6882
Subject:
antibodies, carbon nanotubes, electrochemistry, electrodes, enzyme-linked immunosorbent assay, hydrogen peroxide, microorganisms, monitoring, myeloperoxidase, rapid methods, saliva, signal transduction, thiocyanates
Abstract:
Strategies to detect and characterize myeloperoxidase (MPO) are needed, given that this “split personality” enzyme kills harmful microorganisms but also damages a host tissue. Here, we describe electrochemical approaches to measure MPO by using the pseudohalogenation (MPO/SCN–/H₂O₂) and catalase-like (MPO/H₂O₂) cycles. Their kinetics were determined by monitoring the consumption of H₂O₂ with a nitrogen-doped carbon nanotubes (N-CNT) electrode, which could detect 0.50 μM H₂O₂ at −0.20 V. The unique design of internally calibrated electrochemical continuous enzyme assay (ICECEA) and electrode stability allowed use of one N-CNT electrode for over half a year to reliably determine MPO. The kinetic measurements showed that (a) SCN– did not affect the affinity of MPO for H₂O₂, (b) catalase-like cycle was slower, and (c) MPO retained enzymatically active conformation after complexation with its antibody Ab both in a solution and on the surface of an antibody dipstick (d/Ab). The homogeneous assays could detect 5.2 μg L–¹ MPO (35 pM) via a faster cycle. The heterogeneous immunoassays with the capture of MPO on d/Ab could detect 60 μg L–¹, which was suitable for the accurate detection of MPO in human saliva (101% recovery). Replacing a detection antibody of ELISA with ICECEA as a signal transducer for immunoassays offers a rapid method for the selective determination of enzymes; for example, time of MPO quantification was cut from 3–4 h (sandwich ELISA) to ∼20 min (ICECEA-dipstick).
Agid:
6311740