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Electrochemical Assays and Immunoassays of the Myeloperoxidase/SCN-/H2O2 System
Michael Bekhit1, Waldemar Gorski1
1Department of Chemistry , University of Texas at San Antonio , One UTSA Circle , San Antonio , Texas 78249 , United States.
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).
Insights
New electrochemical methods accurately detect myeloperoxidase (MPO), an enzyme involved in both host defense and damage. These assays are faster and more sensitive than traditional methods, enabling rapid MPO quantification in biological samples like saliva.
Area of Science:
- Electrochemistry
- Biomedical Engineering
- Enzyme Kinetics
Background:
- Myeloperoxidase (MPO) is a critical enzyme with dual roles in host defense and tissue damage.
- Accurate and rapid detection of MPO is essential for understanding its physiological and pathological functions.
- Existing methods for MPO detection can be time-consuming and lack sensitivity.
Purpose of the Study:
- To develop novel electrochemical strategies for sensitive and rapid detection of myeloperoxidase (MPO).
- To characterize the kinetics of MPO using pseudohalogenation and catalase-like cycles.
- To establish an internally calibrated electrochemical continuous enzyme assay (ICECEA) for reliable MPO quantification.
Main Methods:
- Utilized nitrogen-doped carbon nanotubes (N-CNT) electrodes to monitor hydrogen peroxide (H2O2) consumption.
- Employed pseudohalogenation (MPO/SCN-/H2O2) and catalase-like (MPO/H2O2) enzymatic cycles for MPO detection.
- Developed homogeneous and heterogeneous (antibody dipstick) immunoassay formats incorporating ICECEA.
Main Results:
- Achieved sensitive detection of H2O2 (0.50 μM) and MPO (35 pM in homogeneous assays, 60 μg L-1 in heterogeneous assays).
- Demonstrated that SCN- does not affect MPO's affinity for H2O2, and the catalase-like cycle is slower.
- Showed MPO retains activity after antibody complexation, with high recovery (101%) in human saliva samples.
- Reduced MPO quantification time from 3-4 hours (ELISA) to approximately 20 minutes (ICECEA-dipstick).
Conclusions:
- Electrochemical assays using ICECEA offer a rapid, selective, and sensitive method for MPO determination.
- The developed method significantly reduces assay time compared to traditional ELISA.
- This approach is suitable for accurate MPO quantification in complex biological matrices like saliva.
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