Graphene hybrid nanoprobes for targeted microbial sensing and ultralow-energy, deep-tissue, noninvasive multiphoton imaging in the NIR-I/II region

Nitrogen (N) doping and amino functionalization markedly enhance the electron-donating capacity of graphene quantum dots (GQDs), thereby improving charge-transfer efficiency in amino-N-GQDs and yielding substantially superior photophysical performance compared with amino-free N-GQDs and N-free amino-GQDs. Further optimization was achieved through conjugation of amino-N-GQDs with sulfur- and nitrogen-rich polymers, polystyrene sulfonate and polyethylenimine, resulting in amino-N-GQD-polymer nanohybrids with significantly improved optical behavior. These hybrid nanostructures exhibited high quantum yields, excellent photostability, negligible reactive oxygen species generation, and strong two-photon luminescence, positioning them as promising contrast agents for nonlinear bioimaging. To enable molecular specificity, antibody functionalization was incorporated. When conjugated with anti-lipopolysaccharide or anti-TasA antibodies, the nanohybrids selectively targeted Escherichia coli ( E. coli ) and Bacillus subtilis ( B. subtilis ), generating bright fluorescence, strong signal intensity, and high signal-to-noise ratios under two-photon excitation. Using a custom-built Ti:sapphire laser system operating at 970 nm (near-infrared-II region), imaging depths of up to 270 μm were achieved with ultralow excitation energies, 42.96 nJ pixel⁻¹ for E. coli and 35.14 nJ pixel⁻¹ for B. subtilis , acquired over 100 scans (total exposure = 0.666 s). The nanohybrids produced two-photon luminescence using only 1/49 and 1/36 of the energy required for cellular autofluorescence, corresponding to ∼2401- and ∼1296-fold signal enhancements, respectively. This remarkable efficiency supports deep, noninvasive imaging and underscores the potential of amino-N-GQD-polymer nanohybrids as versatile near-infrared-I/II-responsive probes for next-generation biomedical imaging applications.