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Covalent Attachment of Active Enzymes to Upconversion Phosphors Allows Ratiometric Detection of Substrates |
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| Authors: | Dr. Letitia Burgess Hannah Wilson Dr. Alex R. Jones Dr. Peter Harvey Dr. Louise S. Natrajan Dr. Sam Hay |
| Affiliation: | 1. 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;2. Department of Chemistry, School of Natural Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PL United Kingdom |
| 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. |
| Keywords: | biosensors energy transfer enzymes lanthanides upconversion |