Engineering a Reversible Fluorescent Probe for Real-Time Live-Cell Imaging and Quantification of Mitochondrial ATP

Abstract

Real-time imaging and quantification of adenosine triphosphate (ATP) fluctuation in cells are significant for understanding the relationship between energy metabolism and cell functions. However, few synthetic fluorescent probes have been reported to tackle this challenge due to lack of accurate fluorescence readout and suitable response concentration. Herein we designed and synthesized a ratiometric fluorescent probe (Rh6G–ACFPN) for quantitatively detecting the fluctuation of mitochondrial ATP in living cells. Rh6G–ACFPN selectively and reversibly responds to ATP with an ideal dissociation constant (K d) of 4.65 mM (3–10 mM: the range of mitochondrial ATP concentrations). Live-cell imaging allows us to directly monitor the dynamic changes of mitochondrial ATP in high temporal resolution. Moreover, for the first time, mitochondrial ATP in normal and cancer cells lines was successfully quantified and discriminated. These results demonstrate the versatility of Rh6G–ACFPN as a useful imaging tool to elucidate the function of mitochondrial ATP in living cells.

A General Strategy for Development of Activatable NIR‐II Fluorescent Probes for In Vivo High‐Contrast Bioimaging

“…In vitro and in vivo studies in the clinical pathology environment had proved remarkable stability of NIRII‐RT4 (Figure 1 H, Figure 4 and Figure S21), thus the emission change in liver of living mice reflected the actual ATP fluctuation. From the cells imaging experiments with an NIR ATP probe ACF‐ATP4, [38] we further demonstrated the increase of intracellular ATP in hepatocytes during hepatotoxicity (Figure S26,S27). Moreover, real‐time experiments showed that NIRII‐RT‐ATP also can image hepatotoxicity even within 1 h APAP (300 mg kg −1 ) treated (Figure 6 B and D).…”

“…The fluorescence OFF‐ON switching of NIRII‐RT4 amide derivative NIRII‐RT‐pH is very similar to that of the classic CS dyes (NIR rhodamine derivatives) and their amide derivatives, [26] indicating that, like the classic CSs, the novel NIR‐II dye NIRII‐RT4 (or NIRII‐RT3) could be used as platforms to design NIR‐II fluorescent probes. To further verify this, we then constructed NIRII‐RT‐ATP and NIRII‐RT‐Hg (Figure 5 D) as novel NIR‐II fluorescent probes for detecting adenosine triphosphate (ATP) [38] and Hg 2+ , [39] respectively. As shown in Figure 5 E, like probe NIRII‐RT‐pH, NIRII‐RT‐ATP itself had no fluorescence.…”

“…By analyzing the emission intensity (925 nm) changes of NIRII-RT-pH with the Henderson-Hasselbalch-type mass action equation, we acquired apKavalue of 2.93 for NIRII-RT-pH (Figure 5C). Thef luorescence OFF-ON switching of NIRII-RT4amide derivative NIRII-RT-pH is very similar to that of the classic CS dyes (NIR rhodamine derivatives) and their amide derivatives, [26] indicating that, like the classic CSs,t he novel NIR-II dye NIRII-RT4 (or NIRII-RT3) could be used as platforms to design NIR-II fluorescent probes.T of urther verify this,w et hen constructed NIRII-RT-ATPa nd NIRII-RT-Hg (Figure 5D)a sn ovel NIR-II fluorescent probes for detecting adenosine triphosphate (ATP) [38] and Hg 2+ , [39] respectively.A sshown in Figure 5E,l ike probe NIRII-RT-pH, NIRII-RT-ATPitself had no fluorescence.However,with the addition of ATPi nH EPES buffer (pH 7.4, containing 20 %D MSO &1%t ween-80), an intense emission (absorption) peak at 918 nm (854 nm) was observed (Figure 5E and Figure S23a) and the fluorescence intensity of F 918 nm kept linearly increasing (Figure S23b). Additionally,t he probe NIRII-RT-ATPe xhibited high selectivity to ATPover other biological species such as ADP,A MP,G TP,G SH etc.…”

A General Strategy for Development of Activatable NIR‐II Fluorescent Probes for In Vivo High‐Contrast Bioimaging

“…[43] In vitro and in vivo studies in the clinical pathology environment had proved remarkable stability of NIRII-RT4 (Figure 1H,F igure 4a nd Figure S21), thus the emission change in liver of living mice reflected the actual ATP fluctuation. From the cells imaging experiments with an NIR ATPp robe ACF-ATP4, [38] we further demonstrated the increase of intracellular ATPi nh epatocytes during hepatotoxicity (Figure S26,S27). Moreover,r eal-time experiments showed that NIRII-RT-ATPa lso can image hepatotoxicity even within 1hAPAP (300 mg kg À1 )t reated (Figure 6B and D).…”

DNA Logic Nanodevices for Real-Time Monitoring of ATP in Lysosomes

“…Fluorescence (Aptamer) 0.125−2 mM 19 μM [1] Fluorescence (Split Aptamer) 0.03−2 mM 30 μM [2] Fluorescence (Small organic molecules) None (Resonpse range 2−9 mM) None [3] Fluorescence (Aptamer) 0−1.6 mM 78 μM [4] Fluorescence (Aptamer) None (Resonpse range 2−8 mM) None [5] Fluorescence (Aptamer) 0.1−2 mM 18 μM This work S-8…”