Ou Li-Juan, Li Xiao-Yan, Li Li-Juan, Liu Hong-Wei, Sun Ai-Ming, Liu Kai-Jian
College of Material and Chemical Engineering, Hunan Institute of Technology, Hengyang 421002, P. R. China.
Analyst. 2015 Mar 21;140(6):1871-5. doi: 10.1039/c4an01994f.
A new, simple and sensitive fluorescence strategy was developed for the trypsin assay based on copper nanoparticles (CuNPs) and its different fluorescence response toward trypsin-catalyzed hydrolysis of cytochrome c (Cyt c). Polythymine (poly T)-templated CuNPs served as effective fluorescent probes. Cyt c is well-known to act as a quencher. However, herein, a low concentration of Cyt c was designed specially to act as the substrate of trypsin to avoid the quenching effects by electron transfer from Cyt c to CuNPs. In the presence of trypsin, Cyt c hydrolyzes to small peptides, releasing free cysteine residues. Nonfluorescent coordination complexes were formed upon exposure to free cysteine residues by a metal-ligand bond between Cu atoms and sulfur atoms, leading to a decreased fluorescence response to CuNPs. This novel method for the quantitative determination of trypsin has a linear detection range from 0.25 μg mL(-1) to 1000 μg mL(-1) and a relatively low detection limit of 42 ng mL(-1). To the best of our knowledge, this is the first application of the trypsin-catalyzed hydrolysis reaction of Cyt c to produce quenching effect in bioanalysis, which provided a novel approach for the biochemical sensing strategy.
基于铜纳米颗粒（CuNPs）及其对细胞色素c（Cyt c）胰蛋白酶催化水解的不同荧光响应，开发了一种用于胰蛋白酶检测的新型、简单且灵敏的荧光策略。聚胸腺嘧啶（poly T）模板化的CuNPs作为有效的荧光探针。众所周知，Cyt c可作为淬灭剂。然而，在此处，特意设计了低浓度的Cyt c作为胰蛋白酶的底物，以避免因电子从Cyt c转移至CuNPs而产生的淬灭效应。在胰蛋白酶存在的情况下，Cyt c水解为小肽，释放出游离的半胱氨酸残基。通过Cu原子与硫原子之间的金属 - 配体键，在暴露于游离半胱氨酸残基时形成非荧光配位络合物，导致对CuNPs的荧光响应降低。这种定量测定胰蛋白酶的新方法线性检测范围为0.25 μg mL(-1)至1000 μg mL(-1)，检测限相对较低，为42 ng mL(-1)。据我们所知，这是首次将Cyt c的胰蛋白酶催化水解反应产生淬灭效应应用于生物分析，为生化传感策略提供了一种新方法。
