Amino-functionalized nitrogen-doped graphene-quantum-dot-based nanomaterials with nitrogen and amino-functionalized group content dependence for highly efficient two-photon bioimaging

We fabricated nanomaterials comprising amino-functionalized and nitrogen-doped graphene quantum dots (amino-N-GQDs) and investigated their photostability and intrinsic luminescence in the near-infrared spectrum to determine their suitability as contrast agents in two-photon imaging (TPI). We observed that amino-N-GQDs with a higher amount of bonded nitrogen and amino-functionalized groups (6.2%) exhibited superior two-photon properties to those with a lower amount of such nitrogen and groups (4.9%). These materials were conjugated with polymers containing sulfur (polystyrene sulfonate, PSS) and nitrogen atoms (polyethylenimine, PEI), forming amino-N-GQD–PSS–PEI specimens (amino-N-GQD-polymers). The polymers exhibited a high quantum yield, remarkable stability, and notable two-photon properties and generated no reactive oxygen species, rendering them excellent two-photon contrast agents for bioimaging. An antiepidermal growth factor receptor (Ab_EGFR) was used for labeling to increase specificity. Two-photon imaging (TPI) of amino-N-GQD (6.2%)-polymer-Ab_EGFR-treated A431 cancer cells revealed remarkable brightness, intensity, and signal-to-noise ratios for each observation at a two-photon excitation power of 16.9 nJ pixel⁻¹ under 30 scans and a three-dimensional (3D) depth of 105 µm, indicating that amino-N-GQD (6.2%)-polymer-Ab_EGFR-treated cells can achieve two-photon luminescence with 71 times less power required for two-photon autofluorescence (1322.8 nJ pixel⁻¹ with 500 scans) of similar intensity. This economy can minimize photodamage to cells, rendering amino-N-GQD-polymers suitable for noninvasive 3D bioimaging.