High-performance liquid chromatography enables purification of red-emitting carbon dots for cellular imaging.

BACKGROUND: The hydrothermal method is widely employed for the synthesis of carbon dots (CDs); however, the resulting products often contain complex compositions, leading to inaccurate morphological and photophysical characterization and reducing their reliability in applications. In this study, reversed-phase high-performance liquid chromatography (RP-HPLC) was employed to fractionate the heterogeneous products generated from the hydrothermal treatment of resorcinol. RESULTS: Ten distinct fractions were well-separated by RP-HPLC, followed by comprehensive characterization through spectroscopic and electron microscopy-related techniques. Among these fractions, the CDs in fraction 7 (designated F7-CDs) exhibit the highest quantum yield (50%), excellent photostability, and abundant hydroxyl and carboxylic acid groups. Transmission electron microscopy further reveals, the F7-CDs behave as crosslinked polymer-encapsulated CDs. To enable the F7-CDs to image specific organelles, they were functionalized with cysteine via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-sulfohydroxysuccinimide coupling, followed by conjugation with a maleimide-poly(ethylene glycol)-succinimidyl carbonate linker, and subsequently attached to amine-terminated triphenylphosphonium (TPP) or morpholine (MP). The TPP- and MP-conjugated F7-CDs exhibited strong colocalization with MitoView and LysoView 488, respectively, confirming their ability to target mitochondrial and lysosomal compartments. In contrast, their negligible overlap with nuclear stains and a relatively poor colocalization with non-targeting organic dyes demonstrate that the high organelle-targeting selectivity is highly connected with the surface ligands of the F7-CDs. SIGNIFICANCE: Collectively, RP-HPLC is a powerful purification method for producing high-quality CDs, well-suited for surface ligand engineering in cellular imaging. Notably, this work reports the very first example of red-emitting CDs that retain strong luminescence and biocompatibility after chromatographic purification, demonstrating their potential for cellular imaging.