Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Dong Chuan Road 500, Shanghai 200241, P.R. China.
Analyst. 2017 Nov 6;142(22):4215-4220. doi: 10.1039/c7an01295k.
Developing a sensitive and accurate method for Furin activity is still the bottleneck for understanding the role played by Furin in cell-surface systems and even in Alzheimer's disease. In this work, a ratiometric electrochemical biosensor was developed for sensitive and accurate determination of Furin activity in the cell based on dual signal amplification stemming from a peptide with multiple response sites and the antifouling gold nano-bellflowers (GBFs). A new peptide, HS-CMRVRR↓YKDFDFG (P3), was designed for the first time to be selectively cleaved by Furin at site↓. More importantly, this peptide P3 constitutes three amino acid residues with the -COOH group subsequently used to bind with the response molecule of ferrocene, and can remarkably improve the determination sensitivity by about 2.3 fold. Meanwhile, GBFs stabilized by PEG were taken as a second element to magnify the signal of the ferrocene group via a large ratio surface area and good conductivity, as well as an antibiofouling nanosurface to reduce the biofouling of the electrode surface in cells. This double amplification strategy can greatly enhance the sensitivity of Furin detection by 6.5-fold, which is favorable for detection of low amounts of Furin. In addition, 5'-MB-GGCGCGA(T)-SH-3' was co-assembled as an inner reference to provide a built-in element to correct the determination error resulting from a complicated analysis environment. Finally, this sensitive and accurate Furin biosensor was successfully applied to detect Furin activity in Furin overexpressed U251 and MDA-MB-468 cells. As far as we know, this is the first report to mention an electrochemical strategy to detect Furin activity in cells.
开发一种灵敏准确的 Furin 活性检测方法仍然是理解 Furin 在细胞表面系统中作用的瓶颈，甚至是在阿尔茨海默病中的作用。在这项工作中，基于具有多个响应位点的肽和抗污金纳米钟（GBF）的双重信号放大，开发了一种灵敏准确的用于细胞内 Furin 活性检测的比率型电化学生物传感器。首次设计了一种新的肽 HS-CMRVRR↓YKDFDFG（P3），使其能够在位点↓处被 Furin 特异性切割。更重要的是，该肽 P3 由三个具有 -COOH 基团的氨基酸残基组成，随后与二茂铁的响应分子结合，可以显著提高测定灵敏度约 2.3 倍。同时，PEG 稳定的 GBF 被用作第二个元素，通过大的比表面积和良好的导电性放大二茂铁基团的信号，以及作为一个抗生物污染的纳米表面来减少细胞中电极表面的生物污染。这种双重放大策略可以使 Furin 的检测灵敏度提高 6.5 倍，有利于检测低丰度的 Furin。此外，还共同组装了 5'-MB-GGCGCGA(T)-SH-3'作为内参，提供了一个内置元件来校正由于复杂分析环境而导致的测定误差。最后，成功地将这种灵敏准确的 Furin 生物传感器应用于检测过表达 Furin 的 U251 和 MDA-MB-468 细胞中的 Furin 活性。据我们所知，这是第一个报道用于检测细胞中 Furin 活性的电化学策略。
