Objective To investigate differences in the metabolic activity of oral microbiota at the single-cell level between children with dental caries and healthy children using D₂O-labeled single-cell Raman microspectroscopy.

Methods After determining the optimal D₂O volume fraction, the correlations of the deuterium (C-D) Raman band with microbial metabolic activity and glucose consumption were evaluated. Saliva samples were collected from healthy and caries-affected children and labeled with 40% D₂O. Under different culture conditions (aerobic, hypoxia, neutral pH7, and acidic pH5.5), Raman spectra were acquired at multiple time points to monitor and quantify metabolic activity in real time.

Results 1) The C-D ratio was positively correlated with the D₂O volume fraction (Pearson r = 0.968, 95% CI: 0.962-0.972), and a D₂O volume fraction of 40% provided stable metabolic readouts without measurably affecting microbial growth. 2) The C-D ratio was positively correlated with glucose consumption (Pearson r = 0.95, 95% CI: 0.776-0.990) and changes earlier than OD₆₀₀ did, indicating that it might serve as a sensitive indicator of metabolic activity and glucose consumption of the oral microbiota. 3) The caries group showed faster growth, while growth was relatively slower in the healthy group, and 3 h, 6 h, and 9 h were identified as representative time points for subsequent Raman spectral acquisition. 4) Metabolic activity of the oral microbiota did not differ significantly under varying oxygen conditions (P > 0.05). Compared with the neutral condition, the metabolic activity index was significantly reduced under acidic conditions (P < 0.05). The healthy group exhibited higher inter-individual variability in microbial metabolic activity, whereas the caries group showed a more concentrated distribution pattern, with this trend being more pronounced under acidic conditions.

Conclusion D₂O-labeled single-cell Raman microspectroscopy rapidly and sensitively profiles metabolic activity and glucose consumption of children’s oral microbiota. Caries-associated microbial communities exhibit higher metabolic activity than healthy controls, providing a methodological basis for further elucidating caries-related metabolic features.