Tumor-specific imaging is a major challenge in clinical tumor resection. To overcome this problem, several activatable probes have been developed for use in tumor imaging. However, most of these probes are activated based on a single-factor stimulation and are irreversible. Therefore, false signals that make tumor-specific imaging difficult are easily generated. We have developed a new dual-stimulus responsive near-infrared (NIR) reversible adenosine 5′-triphosphate (ATP)-pH probe for fluorescence and photoacoustic ratiometric imaging of tumors. Since the H+ and ATP content is significantly higher in the tumor microenvironment than that in normal tissues, the Förster resonance energy transfer-based probe ATP-pH was constructed with silicon rhodamine as the donor, CS dye as the acceptor, and ATP/H+ recognition units that could only be activated when both H+ and ATP were connected to the acceptor. The ATP-pH probe is reversibly activated by both the H+ and ATP, which effectively reduces the cumulative response of the probe in circulation after intravenous injection. Further, the NIR ratiometric property of the probe makes it suitable for in vivo imaging. Finally, our probe was successfully utilized in ratiometric photoacoustic and fluorescence tumor imaging and ratiometric fluorescence imaging-guided tumor resection.

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