Ratiometric Zinc Biosensor Based on Bioluminescence Resonance Energy Transfer: Trace Metal Ion Determination with Tunable Response

Abstract

Determination of metal ions such as zinc in solution remains an important task in analytical and biological chemistry. We describe a novel zinc ion biosensing approach using a carbonic anhydrase–Oplophorus luciferase fusion protein that employs bioluminescence resonance energy transfer (BRET) to transduce the level of free zinc as a ratio of emission intensities in the blue and orange portions of the spectrum. In addition to high sensitivity (below nanomolar levels) and selectivity, this approach allows both quantitative determination of “free” zinc ion (also termed “mobile” or “labile”) using bioluminescence ratios and determination of the presence of the ion above a threshold simply by the change in color of bioluminescence, without an instrument. The carbonic anhydrase metal ion sensing platform offers well-established flexibility in sensitivity, selectivity, and response kinetics. Finally, bioluminescence labeling has proven an effective approach for molecular imaging in vivo since no exciting light is required; the expressible nature of this sensor offers the prospect of imaging zinc fluxes in vivo.

Progress of Section “Biochemistry” in 2022

“…A relevant number of papers dealing with this enzyme, its inhibitors, activators and involvement in various diseases have been published in 2022 in this journal [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. The first group of contributed materials dealt with the use of this protein for investigations of basic biochemical approaches, such as protein folding [ 7 ], thermodynamic parameters assessment for protein–ligand interactions [ 8 ], bioluminescence resonance energy transfer connected to the binding of the metal ion to apoenzymes [ 9 ], the possibility to evidence chalcogen bonds in the X-ray crystal structures of CA–lig and adduct [ 10 ]. In two other papers, the role of CAs in CO 2 sequestration processes for alleviating global warming [ 11 ] or in photosynthesis in bacteria and plants (only the α-class CAs were considered in a very informative review by De Simone’s group [ 12 ]) were thoroughly discussed, proving again the potential environmental applications of these enzymes.…”

Ratiometric Detection of Zn2+ Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors

“…However, due to the toxicity and strong oxidative ability of H 2 O 2 , the CRET sensor can be difficult to apply in various biological samples. To address these issues, we resort to bioluminescence resonance energy transfer (BRET), which relies on a light-emitting donor enzyme to excite an adjacent fluorophore [86][87][88][89]. Like CRET, BRET does not require an external excitation source, thus could reduce the autofluorescence and photobleaching tendency.…”

“…Chemistry 2023, 5, FOR PEER REVIEW emitting donor enzyme to excite an adjacent fluorophore [86][87][88][89]. Like CRET, BRE not require an external excitation source, thus could reduce the autofluorescence an tobleaching tendency.…”