Development and Comparative Evaluation of Two Enzyme-Based Amperometric Biosensor Designs for Alanine Aminotransferase Determination in Biological Fluids

Alanine aminotransferase (ALT) is a key biomarker of liver function. Compared with conventional assays for ALT detection—which are expensive, time-consuming, labor-intensive, and require experienced personnel—biosensors represent a promising alternative, but it remains unclear which biorecognitive enzymatic configuration offers the best analytical performance for ALT detection. This study presents the development and comparative evaluation of two amperometric biosensors based on oxidase biorecognition elements: pyruvate oxidase (POx) and glutamate oxidase (GlOx). Enzymes were immobilized onto platinum electrodes under optimized conditions using entrapment for POx (pH 7.4, enzyme loading 1.62 U/µL, PVA-SbQ concentration 13.2%) and covalent crosslinking for GlOx (pH 6.5, enzyme loading 2.67%, glutaraldehyde concentration 0.3%). Analytical parameters were systematically assessed, including linear range (1–500 U/L for POx vs. 5–500 U/L for GlOx), limit of detection (1 U/L for both), and sensitivity (0.75 vs. 0.49 nA/min at 100 U/L). The POx-based biosensor demonstrated higher sensitivity and lower detection limits, whereas the GlOx-based biosensor exhibited greater stability in complex solutions and reduced assay costs due to a simpler working solution. Moreover, while the POx-based system is uniquely suited for ALT determination, the GlOx-based sensor can be affected by AST activity in samples but may also be adapted for targeted AST detection. Overall, the study highlights a trade-off between sensitivity, robustness, and versatility in ALT biosensor design, providing guidance for the rational development of clinically relevant devices.