| Objective:Add an new new chromogen2,4,6-Tribromo-3-hydroxybenzoic acid (TBHBA) in the original creatinine, uric acid enzymatic Kit, to improve the sensitivity of creatinine, uric acid enzymatic detection methods; And with this result, develop a self-intellectual property, high sensitive, rapid creatinine and uric acid enzymatic strip, to provide a simple, quick, semi-quantitative or quantitative and reliable detection method, for chronic kidney disease (CKD) patients monitoring kidney function and end stage renal disease (ESRD) patients evaluating effect of dialysis, especially for home health monitoring.Methods:1. Highly sensitivity enzymatic detection method with a new chromogen:Add highly purity new chromogen TBHBA to the original R1reagent of creatinine, uric acid kit, be new chromogen reagent after mixing. Determine the best colorimetric wavelength of new chromogen reagent through the whole wavelength scanning test; According to concentration-absorbance curve, select the optimum concentration of TBHBA; By contrast experiment, to ascertain the best reagents proportion and sample size of the new testing method; then evaluating the performance of the new chromogen method under the optimal test parameters. Ensure TBHBA detection method can provide testing sensitivity.2. The production of strip:Firstly, conducting lots of wet chemical experiments on micro porous plate and exploring the optimum conditions for reaction system in strip; Then, compounding impregnated solutions for filter paper, handmade enzymatic creatinine and uric acid test strips3. The production of standard colorimetric card:Conducting lots of wet chemical experiments on micro porous plate, According to the reaction’s color gradient of series sample standard solution, making standard colorimetric cards of creatinine and uric acid strips.4. Evaluation of the test strip:Detect standard solution of known concentration and clinical serum samples, contrast to automatic biochemical analyzer, analyze the sensitivity and accuracy of test strip, evaluate the clinical value of creatinine and uric acid strips.Results:1. Parameters of highly sensitive new assays on creatinine and uric acid:the detection wavelength were520nm,510nm; the concentration of TBHBA were3g/L,5g/L; the maximum sample size were20μL,15μL; best reagent ratios were3:1,4:1.2. The linear regression equations of creatinine and uric acid new assays were y=0.0006x+0.0165, y=0.0009x-0.0209respectively. The linear range respectively were12.5μmol/L～1000μmol/L,40μmol/L～1600μmol/L, which cover the range of creatinine and uric acid levels of both healthy people and nephropathy patients, the linear ranges meet the needs of clinical testing. As the new assays, chromogenic reaction can be stable for2hours, the CV value of high or low standard solutions of creatinine and uric acid were2.02%,4.13%and3.25%,2.86%respectively. The new chromogenic reagent can be stored for2months in4℃. Compared with the original kits, creatinine and uric acid new detection method respectively were2times and1.5times of original method.3. Developed a creatinine, uric acid test strip and standard colorimetric card; For the strips, dipping time was10seconds, sample size was15uL, the best time of reading results was20～30minutes; From the detection of clinical serum samples, the mean and median concentrations of this samples, divided by strip test, were in the theory interval. And semi-quantitative results of the strip were basically consistent with the concentration of automatic biochemical analyzer.Conclusions:1. TBHBA selected as chromogen can effectively improve the methodological sensitivity for creatinine and uric acid testing, this method provide the basis for the research and development of creatinine and uric acid strip.2. The testing results of creatinine and uric acid dry chemistry strip were accurate and reliable, and repeatability was good. Be able semi-quantitatively or quantitatively detection sample with low level creatinine and uric acid, suitably for saliva sample.3. Creatinine and uric acid strip was portable, rapid for clinical monitoring of renal function, especially for CKD patients home monitoring and ESRD patients evaluating dialysis effect.4. The independent invention, enzymatic dry strip of creatinine and uric acid, has a good application prospect and market development value. |
