Impact of Biotinylation Strategy on the Luminescent Properties and Quantum Yield of Carbon Dots

Impact of biotinylation strategy on the luminescent properties and quantum yield of carbon dots (CDs) is systematically investigated for four types of solvothermally synthesized CDs from citric acid and urea in dimethylformamide (CD-CU), citric acid in formamide (CD-CF), citric acid and ethylene diamine in formamide (CD-CEF), and glutathione in formamide (CD-GF) emitting in the 470-690 nm range. We compare direct carbodiimide coupling (EDC/NHS) with a two-step method employing presynthesized biotin NHS ester, followed by conjugation to CDs in aqueous media. Direct coupling (Method 1) leads to substantial quenching of surface-related emissive centers and formation of urea by-products, which strongly reduce photoluminescence quantum yield (PLQY), while careful purification partially restores the initial optical responses. In contrast, the NHS-ester strategy (Method 2) efficiently attaches biotin with minimal perturbation of the CD optical responses, preserving or even enhancing PLQY. In particular, glutathione/formamide-derived CDs (CD-GF) demonstrate the highest robustness: their deep-red emission is retained, and PLQY increases from 10.6% for pristine CDs to 15.5% after biotin NHS-ester functionalization. Cytotoxicity assays on A549 and HeLa cells confirm low toxicity at relevant concentrations, supporting the applicability of biotinylated CD-GF as red-emitting nanoprobes for targeted fluorescence bioimaging and theranostic applications.