Zhang Lufeng, Qin Haiyan, Cui Wanwan, Zhou Yang, Du Jianxiu
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.
Talanta. 2016 Dec 1;161:535-540. doi: 10.1016/j.talanta.2016.09.011. Epub 2016 Sep 4.
The development of new detection methods for proteases activity assay is important in clinical diagnostics and drug development. In this work, a simple, label-free, and turn-on fluorescent sensor was fabricated for trypsin, a protease produced in the pancreas. Cytochrome c, a natural substance of trypsin, could be selectively cleaved by trypsin into heme-peptide fragment. The produced heme-peptide fragment exhibited an intensive catalytic role on the HO-mediated the oxidation of thiamine to form strong fluorescent thiochrome. The fluorescence intensity was closely dependent on the amount of trypsin presented. The procedure allowed the measurement of trypsin over the range of 0.5-20.0μg/mL with a detection limit of 0.125μg/mL. The sensor showed better precision with a relative standard deviation of 1.6% for the measurement of 1.0μg/mL trypsin solution (n=11). This sensing system was applied to screen the inhibitor of trypsin, the IC values were calculated to be 12.71ng/mL for the trypsin inhibitor from soybean and 2.0μg/mL for benzamidine hydrochloride, respectively, demonstrating its potential application in drug development and related diseases treatment.
开发用于蛋白酶活性测定的新检测方法在临床诊断和药物开发中具有重要意义。在这项工作中，制备了一种简单、无标记且开启型的荧光传感器，用于检测胰蛋白酶（一种在胰腺中产生的蛋白酶）。细胞色素c是胰蛋白酶的天然底物，可被胰蛋白酶选择性切割成血红素 - 肽片段。产生的血红素 - 肽片段对HO介导的硫胺素氧化形成强荧光硫色素具有强烈的催化作用。荧光强度与存在的胰蛋白酶量密切相关。该方法可在0.5 - 20.0μg/mL范围内测定胰蛋白酶，检测限为0.125μg/mL。对于1.0μg/mL胰蛋白酶溶液的测量，该传感器显示出较好的精密度，相对标准偏差为1.6%（n = 11）。该传感系统用于筛选胰蛋白酶抑制剂，大豆胰蛋白酶抑制剂的IC值计算为12.71ng/mL，盐酸苯甲脒的IC值为2.0μg/mL，证明了其在药物开发和相关疾病治疗中的潜在应用。
