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 for E. coli and 35.14 nJ pixel 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.