Target-Activated Modulation of Dual-Color and Two-Photon Fluorescence of Graphene Quantum Dots for in Vivo Imaging of Hydrogen Peroxide

The development of nanoprobes suitable for two-photon microscopy techniques is highly desirable for mapping biological species in living systems. However, at the current stage, the nanoprobes are restricted to single-color fluorescence changes, making it unsuitable for quantitative detection. To circumvent this problem, we report here a rational design of a dual-emission and two-photon (TP) graphene quantum dot (GQD⁴²⁰) probe for imaging of hydrogen peroxide (H₂O₂). For specific recognition of H₂O₂ and lighting the fluorescence of TPGQD⁴²⁰, a boronate ester-functionalized merocyanine (BMC) fluorophore was used as both target-activated trigger and the dual-emission fluorescence modulator. Upon two-photon excitation at 740 nm, TPGQD⁴²⁰–BMC displays a green-to-blue resolved emission band in response to H₂O₂ with an emission shift of 110 nm, and the H₂O₂ can be determined from 0.2 to 40 μM with a detection limit of 0.05 μM. Moreover, the fluorescence response of the TPGQD⁴²⁰–BMC toward H₂O₂ is rapid and extremely specific. The feasibility of the proposed method is demonstrated by two-photon ratiometrically mapping the production of endogenous H₂O₂ in living cells as well as in deep tissues of murine mode at 0–600 μm. To the best of our knowledge, this is the first paradigm to rationally design a dual-emission and two-photon nanoprobe via fluorescence modulation of GQDs with switchable molecules, which will extend new possibility to design powerful molecular tools for in vivo bioimaging applications.