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

Bekhit, Michael, Gorski, Waldemar
Analytical chemistry 2019 v.91 no.4 pp. 3163-3169
antibodies, carbon nanotubes, electrochemistry, electrodes, enzyme-linked immunosorbent assay, hydrogen peroxide, microorganisms, monitoring, myeloperoxidase, rapid methods, saliva, signal transduction, thiocyanates
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).