Zheng, X. T., Choi, Y., Phua, D. G. G., & Tan, Y. N. (2020). Noncovalent Fluorescent Biodot–Protein Conjugates with Well-Preserved Native Functions for Improved Sweat Glucose Detection. Bioconjugate Chemistry, 31(3), 754–763. doi:10.1021/acs.bioconjchem.9b00856
Abstract:
To overcome the traditional issues of protein labelling, we report herein an effective approach to non-covalently conjugate the biomolecule-derived fluorescent nanodots (biodot) to functional proteins without the addition of chemical linkers for biosensor development. The as-prepared fluorescent biodot-protein conjugates are very stable near physiological pH, exhibiting excellent photostability and thermal stability. More importantly, the native functions of proteins, including drug binding and enzymatic activities, are well-preserved after conjugating with biodots. The optimized protein conjugation strategy is then applied to prepare glucose oxidase (GOx)-biodots fluorescent sensing probes for sweat glucose detection. Results show that the as-prepared sensing probes could achieve better assay performance than those covalent conjugates as demonstrated herein. Specifically, GOx in the non-covalently bound conjugates are able to catalyze the oxidation of glucose effectively which generates hydrogen peroxide as by-product. In the presence of Fe2+, Fenton reaction takes place to produce hydroxyl radicals and Fe3+, leading to significant fluorescence quenching of biodots on the conjugates. This simple one-step enzymatic assay in a single probe achieves a wide linear range of 25-1000 µM (R2 = 0.99) with a low detection limit of 25 µM. Furthermore, negligible interference is observed in the complex artificial sweat sample for accurate glucose quantification, achieving an excellent recovery rate of 100.5 ± 2.2%. This work provides a facile conjugation method that is generally applicable to a wide range of proteins, which will help to accelerate future development of multifunctional fluorescent probes to provide optical signals with unique protein functions (e.g., enzymatic, recognition, etc.) for biomedical sensing and imaging.
License type:
Attribution 4.0 International (CC BY 4.0)
Funding Info:
This research is supported by core funding from: IMRE
Grant Reference no. : IMRE/ 16-1P1401