Noncovalent Fluorescent Biodot-Protein Conjugates with Well-Preserved Native Functions for Improved Sweat Glucose...

To overcome the traditional issues of protein labeling, we report herein an effective approach for noncovalent conjugation of 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 biodot-glucose oxidase (GOx) 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 noncovalently bound conjugates are able to catalyze the oxidation of glucose effectively, which generates hydrogen peroxide as a byproduct. 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.