Chang Hui-Fang, Sun Yu-Ling, Yeh Fang-Yuan, Tseng I-Hua, Chang Chia-Chu, Lin Chih-Sheng
Department of Biological Science and Technology, National Chiao Tung University Hsinchu Taiwan
Division of Endocrinology, Department of Internal Medicine, Hsinchu Mackay Memorial Hospital Hsinchu Taiwan.
RSC Adv. 2018 Aug 14;8(51):29013-29021. doi: 10.1039/c8ra04322a.
Gold nanoparticles (AuNPs) can be applied in biosensors using fluorescence resonance energy transfer (FRET) technique. Based on this technique, we have established a sensitive and efficient biosensing method by modifying a peptide-probe onto AuNPs to detect proteinase enzyme activity in this study. This biosensing method was designed for chymase activity detection and applied in kidney disease diagnosis. In this study, 16 nm-AuNPs were used to construct the AuNPs-based fluorescence peptide probe (named AuNPs-peptide probe) for chymase activity determination. The peptide sequence is FITC-Acp-DRVYIHPFHLDDDDDC, which comprises a fluorophore at the N-terminal end, an enzyme (chymase) substrate (DRVYIHPFHL), a spacer (DDDDD) and cysteine (C) to conjugate to AuNPs surface. When the enzyme catalyzes the substrate sequence, the fluorophore drifts away from AuNPs and the fluorescence emitting signal can be excited at 495 nm and detected at 515 nm. The results indicate that the time required for the AuNPs-peptide probe for activity detection of chymase was only 15 min, and a linear correlation from 10 to 100 ng mL of chymase was acquired. The chymase reaction would be significantly inhibited by addition of specific chymase inhibitor chymostatin. The AuNPs-peptide probe was tested for the detection of high concentrations of trypsin and chymotrypsin, but only minor emitted fluorescence intensity was detected. According to these results, sensitivity and specificity of the AuNPs-peptide probe for chymase detection have been confirmed. AuNPs-peptide probe was successfully used for the detection of renal chymase activity; and the results indicate the pathogenically increased chymase activity in kidney tissue of nephropathic mice from aristolochic acid I treatment.
金纳米颗粒（AuNPs）可通过荧光共振能量转移（FRET）技术应用于生物传感器。基于该技术，在本研究中，我们通过在AuNPs上修饰肽探针建立了一种灵敏且高效的生物传感方法，用于检测蛋白酶的酶活性。这种生物传感方法是设计用于检测糜酶活性，并应用于肾脏疾病诊断。在本研究中，使用16纳米的AuNPs构建基于AuNPs的荧光肽探针（命名为AuNPs - 肽探针）来测定糜酶活性。肽序列为FITC - Acp - DRVYIHPFHLDDDDDC，其在N末端包含一个荧光团、一种酶（糜酶）底物（DRVYIHPFHL）、一个间隔区（DDDDD）和用于与AuNPs表面共轭的半胱氨酸（C）。当酶催化底物序列时，荧光团从AuNPs上漂移开，并且荧光发射信号可在495纳米处激发并在515纳米处检测到。结果表明，AuNPs - 肽探针检测糜酶活性所需时间仅为15分钟，并且获得了糜酶浓度在10至100纳克/毫升之间的线性相关性。添加特异性糜酶抑制剂抑糜酶素会显著抑制糜酶反应。测试了AuNPs - 肽探针用于检测高浓度的胰蛋白酶和胰凝乳蛋白酶，但仅检测到微弱的发射荧光强度。根据这些结果，已证实AuNPs - 肽探针检测糜酶的灵敏度和特异性。AuNPs - 肽探针成功用于检测肾脏糜酶活性；结果表明，来自马兜铃酸I处理的肾病小鼠肾脏组织中糜酶活性病理性增加。
