Green-emissive carbon dots for multitarget staining of cells, fungi, and bacteria: Insights into mechanisms and clinical applications

Biological staining is essential for imaging, functional investigation, and disease diagnostics. In this research, innovative green-emissive carbon dots (G-CDs) were hydrothermally prepared from 1,2,4-triaminobenzene and ascorbic acid (AA) and further explored for versatile imaging of mammalian cells, plant cells, and microorganisms. The G-CDs, exhibiting fluorescence enhancement, outperform conventional dyes in terms of staining efficiency and specificity across various biological systems. For mammalian cells (SH-SY5Y, Hep3B, and HEK293), the G-CDs can stain both the nuclei and nucleoli in 10 min via a micropinocytic endocytosis approach, with the latter brighter. Fluorescence enhancement was greater for RNA than for DNA. Interestingly, for plant cells, only cell walls were stained, because positive charges on G-CDs enable strong adsorption onto the negatively charged cell walls, thereby hindering their further entry. G-CDs can stain cell walls and nuclei for fungi (including Aspergillus flavus and Aspergillus niger ), and cell walls and nucleoids for bacteria ( Bacillus subtilis and Escherichia coli ). The targeted staining arises from both the binding of G-CDs to DNA, RNA, pectin, peptidoglycan, and cellulose, and fluorescence enhancement due to surface passivation inhibiting rotation and vibration of functional groups. Electrostatic interactions, coordination, hydrogen bonding, and amidation collectively account for the interactions between G-CDs and these biomolecules . Finally, their practical applications in leucorrhea examinations demonstrated their capability to differentiate between bacterial and fungal vaginosis, highlighting their potential in clinical diagnostics.