Enzyme–Coupled Fluorescence Assays for the Detection and Quantification of Amino Acids

Amino acids are fundamental to cellular physiology, and deviations in their concentrations are closely linked with metabolic and pathological disorders. Accurate and rapid quantification of amino acids is therefore essential for clinical diagnostics and biomedical research. However, conventional enzymatic assays based on absorbance detection typically lack the sensitivity required for clinical amino acid analysis. In this study, we developed enzyme-based assays for the detection and quantification of four clinically relevant amino acids, L-valine, L-phenylalanine, L-proline, and L-lysine, representing branched-chain, aromatic, cyclic, and basic amino acids, respectively. Recombinant dehydrogenases specific to each amino acid were coupled with a resazurin-based fluorescence readout, enabling highly sensitive detection of NADH generated during amino acid oxidation. The assays demonstrated high specificity toward their respective amino acids and enabled quantification in the submillimolar range using minimal sample volumes. Moreover, application to serum samples from sepsis patients revealed accurate amino acid concentrations, which were further validated by NMR spectroscopy, showing a strong correlation (R2 = 0.97-0.99). The fluorescence-based assay required only 15-20 µL of serum and delivered results within 30-40 min, highlighting its potential for point-of-care diagnostics.