State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Biosens Bioelectron. 2013 Mar 15;41:256-61. doi: 10.1016/j.bios.2012.08.030. Epub 2012 Aug 31.
Cystatin C (Cys C) is a significant cysteine protease inhibitor in human bodies, and is proposed as a fascinating novel marker of glomerular filtration rate for kidney injury detection. Almost all traditional methods for Cys C measurement are immunoassays. In this article, we report a simple, immune-independent (no need to rely on immunoassay) and label-free method for Cystatin C detection using BSA-stabilized Au nanoclusters (Au NCs) as a fluorescent probe. This method relies on the BSA scaffold degradation caused by the cysteine protease activity of papain and the specific inhibition of papain activity by Cys C. The fluorescence of BSA-Au NCs can be effectively quenched by papain, and restored by the coexistence of Cys C. Under optimized conditions, this method enables sensitive and selective measurement of Cys C concentration in the range of 25 ng/mL-2.0 μg/mL with the detection limit of 4.0 ng/mL, which is above 40 fold lower than that of commercial immune-based methods. SDS-PAGE, the absorption spectroscopy, transmission electron microscope, dynamic light scattering, and X-ray photoelectron spectroscopy were performed to discuss the quenching mechanism. In addition, percentage recoveries of Cys C in the spiked urine samples were ranged from 102.2% to 114.9% with the relative standard deviation ranging from 0.9-1.8%, demonstrating the applicability of the developed method in clinical samples. Furthermore, the present approach would be potentially extended to other proteases and their inhibitors detection with different protein-stabilized Au NCs.
半胱氨酸蛋白酶抑制剂 C（Cys C）是人体中一种重要的半胱氨酸蛋白酶抑制剂，被提议作为一种新颖的肾小球滤过率标志物，用于检测肾脏损伤。几乎所有传统的 Cys C 测量方法都是免疫测定法。在本文中，我们报告了一种简单、免疫独立（无需依赖免疫测定）且无需标记的方法，使用牛血清白蛋白（BSA）稳定的金纳米簇（Au NCs）作为荧光探针来检测 Cystatin C。该方法依赖于木瓜蛋白酶的半胱氨酸蛋白酶活性导致 BSA 支架降解，以及 Cys C 对木瓜蛋白酶活性的特异性抑制。BSA-Au NCs 的荧光可以被木瓜蛋白酶有效猝灭，并且可以通过 Cys C 的共存而恢复。在优化条件下，该方法可以在 25 ng/mL-2.0 μg/mL 的范围内对 Cys C 浓度进行灵敏和选择性的测量，检测限为 4.0 ng/mL，比商业免疫测定法低 40 多倍。进行了 SDS-PAGE、吸收光谱、透射电子显微镜、动态光散射和 X 射线光电子能谱分析，以讨论猝灭机制。此外，在添加尿液样本中 Cys C 的回收率在 102.2%-114.9%之间，相对标准偏差在 0.9-1.8%之间，表明该方法在临床样本中的适用性。此外，该方法有望扩展到使用不同的蛋白质稳定的 Au NCs 来检测其他蛋白酶及其抑制剂。
