Construction of near infrared ratiometric fluorescent probes with emission crosstalk-free for reversible ATP tracking in mitochondria and tumors

A reliable tool for real-time monitoring and quantifying the fluctuation of adenosine triphosphate (ATP) in living organisms is highly desired for further understanding its biological function. However, there is no probe available to trace ATP in vivo for the challenges of developing reversible responsive ratiometric probes in the near-infrared region with spectrum crosstalk-free. Here, the strategies to overcome above problems by elongating the π-conjugation system of the rhodamine skeleton and rational connecting the internal reference dyes are proposed to obtain the two proof-of-concept probes M838 and M746. M838 displays the NIR ratio emission signal (I749 nm/I535 nm) as well as over 200 nm wavelength differences between the two peaks after rapidly responding to ATP, which will greatly improve the imaging resolution and reliability. The confocal imaging experiments enables us to directly monitor the dynamic changes of mitochondrial ATP. Importantly, M838 fills the gap in recognizing ATP in tumor tissues in a reversible way, and could distinguish and quantify ATP content in tumor tissue and normal tissue. Thus, we believe that M838 has the potential as a useful molecular imaging tool to study of ATP physiological and pathological functions at the clinical level.