Work

Electrochemical bienzymatic biosensor for pyruvate kinase activity evaluation and inhibitor screening

Year: 2025

Type: article

Abstract: This study describes the development of a pyruvate kinase (PyK)-biosensor for the evaluation of PyK activity, as a diagnostic tool for early cancer screening and detection of kinase inhibitors used in cancer treatment, with the evaluation of the inhibition mechanism. The biosensor was constructed by co-immobilizing the enzymes PyK and pyruvate oxidase (PyOx) on Au film electrodes by crosslinking with glutaraldehyde (GA) and evaluated electrochemically by cyclic voltammetry (CV) and fixed potential amperometry (CA). First, the experimental conditions were optimized in terms of applied potential, enzyme ratio PyK:PyOx and enzyme substrate concentration: phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP). The biosensor sensitivity towards PEP detection was 2.11 ± 0.08 μA mM −1 cm −2 , with very high reproducibility and repeatability, which made it suitable for inhibition studies of PyK inhibitor. The inhibition mechanism of shikonin was determined in relation to both PEP and ADP, with the calculation of IC 50 values and binding constants ( K i ). Detection of shikonin was possible at very low concentrations in the linear range of 0.1 – 4.0 pM. The electrochemical results were validated by UV-Vis spectrophotometry. The developed biosensor is a valuable tool for drug screening by enabling enzyme catalytic function examination with applicability to identify inhibitors, estimate their affinity, inhibition mechanism linked to their molecular mechanisms of action and evaluate selectivity, of great interest in both pharmaceutical and medical domains. • Electrochemical assay using a bienzymatic biosensor for pyruvate kinase activity evaluation; • Pyruvate kinase activity can be used as a diagnostic tool for early cancer diagnose; • Fixed potential amperometry for detection of pyruvate kinase inhibitor; • Electrochemical screening of PyK inhibitors based on PyK inhibition studies.

Source: Talanta

DOI: 10.1016/j.talanta.2025.127886

Authors: Ricardo Jose Branco Leote, Madalina Maria Barsan, Caroline G. Sanz, Victor C. Diculescu

Institutions: University of Bucharest, National Institute of Materials Physics

Language: English

Topic: Electrochemical sensors and biosensors

Subfield: Electrical and Electronic Engineering

Field: Engineering

Domain: Physical Sciences

SDG: Good health and well-being

Open Access status: hybrid

Estimated APC paid: $4,610