Resurfaced Fluorescent Protein as a Sensing Platform for Label-Free Detection of Copper(II) Ion and Acetylcholinesterase Activity

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• 作者：Chunyang Lei ; Zhen Wang ; Zhou Nie ; Honghua Deng ; Huiping Hu ; Yan Huang ; Shouzhuo Yao
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：February 3, 2015
• 年：2015
• 卷：87
• 期：3
• 页码：1974-1980
• 全文大小：376K
• ISSN：1520-6882

文摘

Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H₃₉GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H₃₉GFP as the signal reporter, a label-free fluorometric sensor for Cu²⁺ sensing was developed based on the unique multivalent metal ion-binding property of H₃₉GFP and fluorescence quenching effect of Cu²⁺ by electron transfer. The high affinity of H₃₉GFP with Cu²⁺ (K_d, 16.2 nM) leads to rapid detection of Cu²⁺ in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H₃₉GFP/Cu²⁺ complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu²⁺ and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL^鈥?) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H₃₉GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.