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Mitigating dithiothreitol interference to gold/thiol interface in electrochemical detection of cathepsin B activity toward multiplex protease analysis

  • 0Department of Chemistry, Kansas State University, Manhattan, KS, 66502, USA.
Biosensors & Bioelectronics +

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January 26, 2025

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January 26, 2025

View abstract on PubMed

Summary

This summary is machine-generated.

This study developed a new electrochemical sensor to detect cathepsin B (CB) protease activity. By optimizing reagent use, the sensor accurately measures protease kinetics, overcoming interference issues for improved disease biomarker detection.

Area Of Science

  • Electrochemistry
  • Biosensing
  • Biochemistry

Background

  • Proteases are overexpressed in cancers and can serve as disease biomarkers.
  • Electrochemical techniques offer multiplexing capabilities for detecting extracellular protease activity.
  • Existing methods face interference from reagents, masking protease activity and reducing sensitivity.

Purpose Of The Study

  • To develop a robust electrochemical biosensor for monitoring cathepsin B (CB) protease activity.
  • To address and overcome interference issues caused by dithiothreitol (DTT) in protease detection.
  • To accurately determine CB proteolysis kinetics and substrate-specific cleavage.

Main Methods

  • Utilized a 3x3 gold microelectrode array (MEA) functionalized with (2-aminoethyl)ferrocene (AEF) tagged peptide substrates.
  • Employed alternating current voltammetry (ACV) to monitor changes in signal upon protease cleavage.
  • Implemented centrifugal filtration to remove DTT and incorporated EDTA to maintain enzyme activity, enabling accurate kinetic measurements.

Main Results

  • Demonstrated that protease cleavage causes an exponential decay in the ACV signal, inversely proportional to protease activity (1/τ).
  • Successfully mitigated interference from DTT by optimizing reagent removal and addition protocols.
  • Showcased substrate-dependent cleavage of three different peptide substrates by CB using the MEA chip.

Conclusions

  • The optimized electrochemical sensor accurately quantifies cathepsin B activity and kinetics.
  • Understanding and mitigating reagent interference at the thiol/gold interface is crucial for redox-tagged electrochemical biosensors.
  • The developed MEA chip has potential for rapid activity profiling of multiple proteases for disease diagnosis.

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