研究目的
To develop a rapid, single-point method for colorimetric creatinine determination in urine samples as an alternative to the Jaffé assay, using 3,5-dinitrobenzoic acid and an optoelectronic detector to improve specificity and reduce costs.
研究成果
The DNBA method for creatinine determination in urine is more specific and accurate than the single-point Jaffé assay, with reduced interferences from compounds like glucose and urea. The use of a PEDD detector lowers instrumentation costs without compromising analytical performance. Validation with real urine samples showed good agreement with reference methods, but applicability to serum is limited due to interference issues. Future work could focus on overcoming these interferences for broader clinical use.
研究不足
The method shows high interference from bilirubin and protein, which may limit its application to urine and dialysate samples, not serum. The reaction product is unstable over time, requiring precise control of reaction timing. The PEDD detector has slightly higher LOD and LOQ compared to the Jaffé assay, but this is not critical for urine analysis due to high creatinine content.
1：Experimental Design and Method Selection:
The study designed a colorimetric assay using 3,5-dinitrobenzoic acid (DNBA) in alkaline conditions as a chromophore for creatinine detection, comparing it to the Jaffé method. An optoelectronic detector based on paired emitter detector diodes (PEDD) was developed for low-cost detection.
2：Sample Selection and Data Sources:
Human urine samples with known creatinine levels were obtained from the Central Clinical Laboratory of Medical University of Warsaw, analyzed using the Jaffé protocol on a Cobas 6000 analyzer as a reference.
3：List of Experimental Equipment and Materials:
Equipment includes a Shimadzu UV-2401 PC spectrophotometer, disposable polystyrene cuvettes, LEDs from various manufacturers, a 3D-printed cuvette holder, an adjustable laboratory power circuit with L272 operational amplifier, and a Keysight Technologies U1231A multimeter. Materials include creatinine, 3,5-dinitrobenzoic acid, bilirubin, bovine serum albumin, and other analytical-grade chemicals from Sigma Aldrich and Avantor Performance.
4：Experimental Procedures and Operational Workflow:
Reaction conditions were optimized by testing different NaOH concentrations and volume ratios. For PEDD, 130 LED pairs were tested; the optimal pair was selected based on current and creatinine concentration tests. Measurements were taken at specific times after reaction initiation. Interference studies involved adding potential interferents to creatinine solutions. Real sample analysis involved diluting urine samples and measuring with spectrophotometry and PEDD.
5：Data Analysis Methods:
Data were analyzed using calibration curves, linear ranges, LOD, LOQ, and statistical tests including two-tail paired Student's t-tests and Bland-Altman plots for method validation.
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