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This paper presents the development of near-infrared (NIR) fluorescent probes, A and B, engineered from hemicyanine dyes with 1,8-naphthalic and rhodamine derivatives for optimized photophysical properties and precise mitochondrial targeting. Probes A and B exhibit absorption peaks at 737 nm and low fluorescence in phosphate-buffered saline (PBS) buffer. Notably, their fluorescence intensities, peaking at 684 (A) and 702 nm (B), increase significantly with viscosity, as demonstrated through glycerol-to-PBS ratio experiments. This increase is attributed to restricted rotational freedom in the fluorophore and its linkages to rhodamine or 1,8-naphthalic groups. Theoretical modeling suggests nonplanar configurations for both probes, with primary absorptions in the rhodamine and hemicyanine cores (A: 543; B: 536 nm), and additional transitions to 1,8-naphthalic (A: 478 nm) and rhodamine (B: 626 nm) groups. Probe A is also responsive to human serum albumin (HSA), a key biomarker, with fluorescence increasing in HeLa cells as HSA concentrations rise. In contrast, probe B shows no response to HSA, likely due to steric hindrance from its bulky rhodamine group, illustrating a selectivity difference between the probes. Probe B, however, excels in mitochondrial imaging, confirmed through cellular and in vivo studies. In HeLa cells, it tracked viscosity changes following treatment with monensin, nystatin, and lipopolysaccharide (LPS), with fluorescence increasing in a dose-dependent manner. In fruit flies, probe B effectively detected monensin-induced viscosity changes, demonstrating its stability and in vivo applicability. These findings highlight the versatility and sensitivity of probes A and B as tools in biological research, with potential applications in monitoring mitochondrial health, detecting biomarkers like HSA, and investigating mitochondrial dynamics in disease.
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Near-Infrared Ratiometric Hemicyanine Fluorescent Probes for Monitoring Mitochondrial pH Dynamics in Live Cells during Oxidative Stress and Hypoxia
Novel near-infrared ratiometric molecules (probes A and B) produced by linking formyl-functionalized xanthene and methoxybenzene moieties, respectively, onto a xanthene-hemicyanine framework are detailed. Probe A exhibited a primary absorption peak at 780 nm and a shoulder peak at 730 nm and exhibited fluorescence at 740 nm↓ (signifies a downward shift in intensity upon acidification) in a pH 9.3 buffer and 780 nm↑ at pH 2.8 under excitation at 700 nm. Probe B featured absorptions at 618 and 668 nm at pH 3.2 and at 717 nm at pH 8.6, and fluorescence at 693 nm↑ at pH 3.2 and at 739 nm↓ at pH 8.6, in mostly the red to near-IR region. The ratiometric changes in the intensity of the fluorescent absorptions were reversed between A and B upon acidification as indicated by the arrows. Theoretical calculations confirmed that there were slight changes in conformation between probes and the protonated molecules, suggesting that the changes in emission spectra were due mostly to conjugation effects. Calculations at the APFD/6-311+g(d,p) level with a solvent described by the polarizable continuum model resulted in pKa values for A at 6.33 and B at 6.41, in good agreement with the experimentally determined value of 6.97 and an average of 6.40, respectively. The versatilities of the probes were demonstrated in various experimental contexts, including the effective detection of mitochondrial pH fluctuations. Live cell experiments involving exposure to different pH buffers in the presence of H+ ionophores, monitoring mitophagy processes during cell starvation, studying hypoxia induced by CoCl2 treatment, and investigating responses to various oxidative stresses are detailed. Our findings highlight the potential of attaching xanthene and methoxybenzaldehyde groups onto xanthene-hemicyanine structures as versatile tools for monitoring pH changes in a variety of cellular environments and processes.
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- Award ID(s):
- 2117318
- PAR ID:
- 10565777
- Publisher / Repository:
- ACS
- Date Published:
- Journal Name:
- ACS Omega
- Volume:
- 9
- Issue:
- 40
- ISSN:
- 2470-1343
- Page Range / eLocation ID:
- 42049 to 42060
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acsomega.4c07303
