Ultra-Bright and pH-Stable DBCO-Capped Gold Clusters as NIR-II Gram-Typing Nanoprobes for Deciphering Microgravity-Related Gut Bacterial Restructuring in Rats

Microgravity-induced alterations of gut microbiota are implicated in gastrointestinal pathology during spaceflight, yet conventional 16S rRNA sequencing lacks the resolution to remotely track real-time spatial dynamics of in situ bacteria. Here, we develop ultrabright, pH-stable DBCO-capped gold nanoclusters (AuNCs@DBCO) as NIR-II nanoprobes for selective imaging of Gram-positive (G⁺) and Gram-negative (G⁻) bacteria. The DBCO modification not only enhances the quantum yield by 5.6-fold through restricted intramolecular vibrations of water-soluble Au₂₅ clusters but also enables bioorthogonal labeling with azido-precursors metabolically incorporated into bacterial cell walls. Using D-Ala-N₃ for G⁺ and Kdo-N₃ for G⁻ labeling, we achieve in vivo and ex vivo imaging of gut bacteria in rats under simulated microgravity (SMG). Our approach reveals position-specific gut bacterial restructuring that SMG increases ileal G⁺ bacteria while suppressing G⁻ populations, such a spatial imbalance is difficult to be detected by sequencing-based phylum analysis. The gut dysbiosis coincides with intestinal barrier disruption and inflammation, driven by enrichment of opportunistic pathogens and depletion of beneficial commensals. The utilization of this nanoprobe enables precise deciphering of SMG-induced gut microbiogeography and Gram-typing imbalance associated pathological consequences, providing a powerful tool for studying spaceflight-associated gastrointestinal disorders.