Fast ratiometric colorimetric sensing of total protein content in human serum based on classic biuret method

A ratiometric colorimetric assay based on the classical biuret reaction was developed and systematically optimized for the quantification of total protein (TP) in human serum. By exploiting absorbance ratios rather than single-wavelength measurements, the method compensates for fluctuations associated with reagent blank instability, matrix effects, and instrumental drift, particularly under finely tuned alkaline conditions. The assay was analytically validated using the Bradford method as reference and benchmarked against the conventional absorbance at 550 nm, showing improved robustness, linearity, and reproducibility. The assay was successfully applied to both commercial pooled serum and clinical specimens, confirming reliable performance across real and heterogeneous biological matrices. Owing to its simplicity, low cost, and enhanced stability, the proposed ratiometric biuret assay represents a practical alternative for routine TP quantification, including use in decentralized or resource-limited analytical settings.