Interference-Free Duplex Detection of Total and Active Enzyme Concentrations at a Single Working Electrode

Area of Science:

• Electrochemistry
• Biomedical Engineering
• Analytical Chemistry

Background:

• Simultaneous detection of total and active enzyme concentrations at a single electrode is challenging.
• Achieving two distinct redox-cycling reactions without interference is difficult.
• Existing methods often struggle with interferences when combining different assays.

Purpose of the Study:

• To develop a simple, sensitive, and interference-free combined assay for total prostate-specific antigen (tPSA) and free prostate-specific antigen (fPSA).
• To utilize two redox-cycling reactions at a single electrode for duplex detection.
• To integrate an immunoassay for tPSA with a protease assay for fPSA.

Main Methods:

• A single working electrode was used with two distinct incubation periods and applied potentials.
• Electrochemical-enzymatic (EN) redox cycling involving ferrocenemethanol for tPSA immunoassay.
• EN redox cycling involving 4-amino-1-naphthol for fPSA protease assay.
• The assay combines immunoassay label and fPSA affinity-bound to the electrode for enhanced sensitivity and specificity.

Main Results:

• The developed assay demonstrated highly interference-free, specific, and sensitive duplex detection in both buffer and serum.
• Detection limits were achieved at approximately 10 pg/mL for tPSA and 1 pg/mL for fPSA.
• The method successfully combined two different assays and redox-cycling reactions without mutual interference.

Conclusions:

• The presented combined assay offers a robust solution for simultaneous tPSA and fPSA quantification.
• This approach simplifies duplex detection by employing a single electrode and distinct electrochemical conditions.
• The assay shows significant potential for clinical diagnostics requiring accurate measurement of both total and active enzyme forms.