A Diazonium-Based Colorimetric Approach for the Sensitive Detection of Creatinine in Serum Using Spectrophotometric and Microfluidic Techniques

Creatinine is an essential biomarker for assessing kidney function. This study presents a novel diazonium-based colorimetric method for measuring creatinine in human serum, utilizing both traditional spectrophotometry and a microfluidic detection platform. The technique involves the in situ formation of a diazonium salt (generated with acidified sodium nitrite), which reacts with p-phenylenediamine and α-naphthol to form a stable reddish-violet azo dye. The absorbance of this dye correlates directly with creatinine levels. The spectrophotometric method was optimized to detect maximum absorbance at 524 nm, while the microfluidic method was optimized with a smartphone-based colorimetric app. Calibration curves showed excellent linearity (R² = 0.9980 for 1.0–10.0 mg/dL, R² = 0.9983 for 2.0–14.0 mg/dL with both methods). The detection and quantification limits were 0.5 mg/dL (5 µg/mL) and 1.515 mg/dL (15.15 µg/mL) for spectrophotometry, and 1.2 mg/dL (12 µg/mL) and 2.62 mg/dL (26.2 µg/mL) for microfluidics. Both methods demonstrated high precision (RSDs 1.02% and 1.82%) and accuracy (recovery rates between 96.5% and 104.2%), comparable to the Cobas c311 analyzer. These results emphasize the method’s robustness, sensitivity, and suitability for both centralized lab testing and point-of-care diagnostics.