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通过将活性酶共价连接到上转换荧光粉上来实现对底物的比率检测。

Covalent Attachment of Active Enzymes to Upconversion Phosphors Allows Ratiometric Detection of Substrates.

机构信息

Department of Chemistry, School of Natural Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PL, United Kingdom.

Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom.

出版信息

Chemistry. 2020 Nov 20;26(65):14817-14822. doi: 10.1002/chem.202001974. Epub 2020 Oct 16.

DOI:10.1002/chem.202001974
PMID:32476171
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7756657/
Abstract

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface-accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide-thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state.

摘要

上转换荧光粉（UCPs）通过光子上转换过程将多个低能量光子转换为更高能量的发射，为用作发光探针提供了一种有吸引力的有机荧光团替代物。在这里，UCPs 用功能化的硅烷进行了封端，以提供一个表面，用于通过带有表面可接近的半胱氨酸的马来酰亚胺-硫醇偶联将蛋白质共价连接。然后，通过马来酰亚胺-硫醇偶联将绿色荧光蛋白（GFP）和黄素酶五碳糖四硝酸还原酶（PETNR）的变体分别连接到 UCP 上，以分别允许能量从 UCP 转移到 GFP 或 PETNR 的黄素辅因子。当 PETNR 与 UCP 偶联时，它保留其活性，这允许通过酶氧化还原状态的比率检测来可逆地检测酶底物。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/f539a7ba6ded/CHEM-26-14817-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/834b8dcc4614/CHEM-26-14817-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/164bd6cd58e9/CHEM-26-14817-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/55f4762fbb82/CHEM-26-14817-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/c929cb34caaf/CHEM-26-14817-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/f539a7ba6ded/CHEM-26-14817-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/834b8dcc4614/CHEM-26-14817-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/164bd6cd58e9/CHEM-26-14817-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/55f4762fbb82/CHEM-26-14817-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/c929cb34caaf/CHEM-26-14817-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e345/7756657/f539a7ba6ded/CHEM-26-14817-g004.jpg

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