Spray coated bentonite/MWCNT composite enzymatic biosensor for uric acid detection

Biosensors are widely used in healthcare and diagnostics, offering rapid and reliable detection of biological analytes. This study presents the development of an enzymatic uric acid (UA) biosensor based on a nanocomposite of bentonite (BT) and multi-walled carbon nanotubes (MWCNTs) fabricated via spray coating. The composite utilizes the high conductivity and surface area of MWCNTs along with the ion-exchange capacity and antifouling properties of BT to improve both electron transfer and long-term stability. Uricase (UOX) was immobilized on the BT/MWCNTs modified electrode using glutaraldehyde (GA) crosslinking, forming a stable enzyme matrix with strong adhesion and reduced leaching. The fabricated BT/MWCNT/GA/UOX biosensor was systematically characterized through morphological, chemical, and electrochemical analyses. Optimizing spray parameters enabled uniform film deposition and consistent surface roughness, thereby improving reproducibility across electrodes. The biosensor exhibited a broad linear detection range, high sensitivity, and a low detection limit of 5.31 µM in phosphate buffer, outperforming the control sensors prepared with BT-only and MWCNT-only films. Excellent selectivity was demonstrated against common interferents such as ascorbic acid and dopamine. The proposed sensor also exhibited good antifouling ability and was applicable to hydrodynamic conditions. Stability studies indicated that after 60 days of storage at 4 °C, the biosensor retained 92% of its initial response, showing minimal signal drift and structural degradation. Furthermore, real-sample validation using spiked human serum demonstrated reliable recovery (92.2–110.8%) and strong correlation (R² = 0.997) with the standard calibration, confirming the biosensor’s clinical applicability. These results confirm the mechanical, electrochemical, and operational robustness of the BT/MWCNT-based biosensor platform and suggest its strong potential for future integration into point-of-care (POC) diagnostic systems for UA and related metabolites.