Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
Biosens Bioelectron. 2016 May 15;79:347-52. doi: 10.1016/j.bios.2015.12.070. Epub 2015 Dec 21.
The development of simple and sensitive methods for protease sensing plays important roles in clinical diagnostics and drug development. Here a simple, rapid, label-free, and sensitive trypsin colorimetric sensor was developed by employing cytochrome c (cyt c) as an enzyme substrate and 3,3´,5,5´-tetramethylbenzidine (TMB) as a chromogenic reagent. It was found that cyt c hardly catalyzes H2O2-mediated TMB oxidation to produce a blue solution. But the hydrolysate of cyt c by trypsin displays an intense catalytic effect on the aforementioned reaction, resulting in the formation of a blue solution immediately. The detection process allows visually perceiving as low as 50 ng/mL trypsin with the naked eyes. With the aid of a spectrophotometer, the absorbance at 652 nm was proportional to the concentration of trypsin in the range from 5.0 ng/mL to 2.0 μg/mL with a detection limit of 4.5 ng/mL. The sensor showed better precision with relative standard deviation of 2.5% and 1.7% for eleven repetitive measurements of 50.0 ng/mL and 1.0 μg/mL trypsin solution, respectively. The procedure has been successfully applied to the determination of trypsin in human urines and for inhibitor screening, demonstrating its potential application in clinic diagnosis and drug development.
开发用于蛋白酶检测的简单而灵敏的方法在临床诊断和药物开发中起着重要作用。本研究采用细胞色素 c（cyt c）作为酶底物和 3,3´,5,5´-四甲基联苯胺（TMB）作为显色试剂，开发了一种简单、快速、无需标记且灵敏的胰蛋白酶比色传感器。研究发现，cyt c 几乎不能催化 H2O2 介导的 TMB 氧化生成蓝色溶液。但是，胰蛋白酶水解 cyt c 后对上述反应显示出强烈的催化作用，立即形成蓝色溶液。检测过程可以用肉眼检测低至 50 ng/mL 的胰蛋白酶。借助分光光度计，在 5.0 ng/mL 至 2.0 μg/mL 的范围内，652nm 处的吸光度与胰蛋白酶的浓度呈正比，检测限为 4.5 ng/mL。该传感器对 50.0 ng/mL 和 1.0 μg/mL 的胰蛋白酶溶液进行 11 次重复测量，其相对标准偏差分别为 2.5%和 1.7%，显示出较好的精密度。该方法已成功应用于人尿中胰蛋白酶的测定和抑制剂筛选，表明其在临床诊断和药物开发中有潜在的应用。
