Department of Chemical & Biomolecular Engineering, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 04107, Republic of Korea.
Nano Lett. 2020 Oct 14;20(10):7100-7107. doi: 10.1021/acs.nanolett.0c02343. Epub 2020 Aug 24.
Although fluorescence-based analytical methods have been used in intracellular analyses, their sensitivity is low for the precise analysis of intracellular proteolytic enzymes to observe cell apoptosis related to cancer and neurodegenerative diseases. In this study, a metal-enhanced-fluorescence (MEF)-based highly sensitive biosensor for the detection of proteolytic enzymes is proposed for the first time by using a bifunctional Au nanoparticle (AuNP), which is connected to the fluorophore by both single-stranded DNA (ssDNA) and a peptide. Once caspase-3, a proteolytic enzyme, cuts the peptide specifically, the fluorescence signal is drastically increased because the ssDNA maintains an optimal distance for the MEF. The proposed sensing method shows the highly sensitive detection of caspase-3 based on just a simple enzymatic cleavage reaction within 1 h, and caspase-3-related preapoptotic cell detection was successfully carried out with high sensitivity. The proposed sensing method is a rapid, simple, and one-step technique for the real-time monitoring of intracellular proteolytic enzymes and can be applied to the early diagnosis of cancer and neurodegenerative diseases.
虽然基于荧光的分析方法已被用于细胞内分析，但它们的灵敏度较低，难以精确分析细胞内的蛋白水解酶，从而观察与癌症和神经退行性疾病相关的细胞凋亡。在这项研究中，首次提出了一种基于金属增强荧光（MEF）的高度灵敏生物传感器，用于检测蛋白水解酶，该传感器使用双功能金纳米粒子（AuNP），通过单链 DNA（ssDNA）和肽将荧光团连接起来。一旦半胱天冬酶-3（一种蛋白水解酶）特异性切割肽，由于 ssDNA 保持 MEF 的最佳距离，荧光信号会急剧增加。该传感方法仅通过 1 小时内的简单酶切反应即可实现对半胱天冬酶-3 的高灵敏度检测，并且可以成功地以高灵敏度进行 caspase-3 相关的预凋亡细胞检测。该传感方法是一种快速、简单的一步法技术，可用于实时监测细胞内蛋白水解酶，并可应用于癌症和神经退行性疾病的早期诊断。
