Near-Infrared Hybrid Rhodol Dyes with Spiropyran Switches for Sensitive Ratiometric Sensing of pH Changes in Mitochondria and Drosophila melanogaster First-Instar Larvae

Abstract

Near-infrared hybrid rhodol dyes (probes A and B) for sensitive ratiometric visualization of pH changes were prepared by incorporating hemicyanine dyes into traditional rhodol dyes. This approach was based on π-conjugation changes involving a rhodol hydroxyl group as a spiropyran switch upon pH changes. Probes A-2 and B-2 consist of a rhodol fluorophore and an N,O-disubstituted hemiaminal ether residue linked through a cyclic carbon–carbon double-bonded connection. Electronic spectra of probes A-2 and B-2 contain sharp absorption peaks at 535 nm and fluorescence peaks at 558 nm with similar π-conjugation and a closed spiropyran form at a basic pH of 10.2. However, acidic pH conditions break down the hemiaminal ether groups, leading to indolenium moieties and significantly extending the π-conjugation within the rhodol fluorophores, resulting in additional near-infrared emissions for probes A-1 and B-1. As a result, probes A and B exhibit gradual decreases of the absorption peaks at 535 nm and gradual increases in absorption peaks at 609 and 622 nm upon transition from basic to acidic pH, respectively. Both probes display ratiometric fluorescence sensing responses to pH downgrades from 10.2 to 3.6 with visible fluorescence decreases at 558 nm, as well as corresponding increases of the near-infrared fluorescence peaks at 688 and 698 nm, respectively. They exhibit fast, sensitive, and selective fluorescence responses with clearly defined ratiometric features to pH changes and show low cytotoxicity and excellent cell permeability. Our probes were successfully applied to ratiometrically detect pH changes in mitochondria, D. melanogaster first-instar larvae, and to visualize the mitophagy process caused by either cell nutrient starvation or drug treatment.

A near-infrared fluorescent probe reveals decreased mitochondrial polarity during mitophagy

“…In this respect, small molecular fluorescent probes have attracted much attention due to their high sensitivity, good biocompatibility and incomparable spatiotemporal resolution. 9 Several small molecular fluorescent probes have been designed for the detection of mitochondrial pH and viscosity changes, revealing a decreased pH and an increased viscosity during mitophagy. 10 However, the polarity changes in this process are still unclear.…”

De novo strategy with engineering anti-Kasha/Kasha fluorophores enables reliable ratiometric quantification of biomolecules

“…It has been established that the preferential ratiometric patterns of a unimolecular fluorophore can overcome some inevitable side effects between donor and acceptor fluorophores in FRET-based methods [20][21][22][23] . Unimolecular ratiometric probes can react with the analyte to form a new fluorophore.…”

Ratiometric Near-Infrared Fluorescent Probes Based on Hemicyanine Dyes Bearing Dithioacetal and Formal Residues for pH Detection in Mitochondria

“…In order to demonstrate that our probes can respond to intracellular pH changes, we incubated HeLa cells with AH + and BH + , adjusted intracellular pH to 9.0, 8.0, 7.5, 6.5, 6.0, and 5.0 by nigericin (H + /K + ionophore) [ 16 , 18 , 23 , 24 , 40 , 41 , 42 , 43 , 44 ], and collected cellular fluorescence in two channels from 650 nm to 657 nm, and from 700 nm to 750 nm under excitation of 635 nm. With the confocal fluorescence imaging ( Figure 16 ), upon gradual decrease of intracellular pH from 9.0 to 5.0, we observed an expected gradual decrease in fluorescence in the channel II and gradual increase in fluorescence in the channel I ( Figure 16 and Figure 17 ).…”

“…HeLa cells were cultured in the high glucose DMEM media supplemented with 10% FBS, 1% penicillin streptomycin in an incubator (37 °C, 5% CO 2 ) [ 16 , 18 , 23 , 24 , 40 , 41 , 42 , 43 , 44 ]. Cells were replenished with fresh medium every 2 days.…”

“…Ratiometric near-infrared fluorescent probes have desirable advantages such as near-infrared imaging to achieve deep tissue penetration, low photodamage to cells, and less biological sample autofluorescence. Our probes also possess self-calibration capability with two emissions to overcome systematic errors of intensity-based fluorescent probes produced by excitation light fluctuation, probe concentration changes, uneven distribution, and compartmental localization [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. Both AH + and BH + probes ( Scheme 1 ) show ratiometric fluorescent probes to pH changes with the largest bathochromic shifts of 38 nm and 59 nm based on the protonation and deprotonation of a hydroxy group attached to the hemicyanine dyes, respectively.…”