研究目的
To develop a FRET-based mitochondria-targeted fluorescent probe for ratiometric detection of SO2 derivatives (specifically bisulfite) in biological systems, with fast response, high selectivity, and low detection limit, for real-time monitoring and cell imaging applications.
研究成果
The developed FRET-based mitochondria-targeted fluorescent probe CBP enables fast (<1 min), selective, and sensitive (detection limit 17.7 nM) ratiometric detection of HSO3- with visible color change. It successfully localizes in mitochondria and is applicable for imaging intracellular SO2 derivatives in living cells, demonstrating potential for real-time biological monitoring.
研究不足
The probe may have interference from HS- (sulfide) as it causes moderate spectral changes. The working pH range for optimal response is 7.0-11.0, which might not cover all physiological conditions. The study was conducted in vitro and in cell cultures; in vivo applications and long-term stability were not addressed.
1：Experimental Design and Method Selection:
The study designed a FRET-based fluorescent probe (CBP) using coumarin as energy donor and benzopyrylium as energy acceptor, with a mitochondria-targeted group. The sensing mechanism is based on Michael-addition reaction of bisulfite to the benzopyrylium moiety, blocking FRET and causing ratiometric fluorescence changes.
2：Sample Selection and Data Sources:
Synthetic compounds were prepared and characterized. Analytes including various ions, biothiols, and reactive oxygen species were tested in DMF/H2O solution. Cell lines (EC-109 and MCF-7) were cultured for imaging experiments.
3：List of Experimental Equipment and Materials:
UV-2102 spectrometer, F-4500 spectrophotometer, pHs-3C meter, Bruker DTX-400 NMR spectrometer, HPLC Q-Exactive HR-MS spectrometer, Nikon-80i inverted fluorescence microscope, Leica TCS SP8 confocal microscope. Chemicals from commercial sources, solvents purified, analytes prepared as 10 mM solutions.
4：Experimental Procedures and Operational Workflow:
Synthesis of probe CBP via multi-step reactions. Spectra measurements: stock solution of CBP in DMSO diluted to 10 μM in DMF/H2O/PBS buffer; selectivity tests with 10 equiv. analytes; titration with HSO3-; pH effect studies; kinetic experiments; cell culture and imaging with probe incubation and HSO3- addition.
5：Data Analysis Methods:
Fluorescence spectra analyzed for intensity ratios (F482/F630); detection limit calculated using 3SD/K formula; NMR and MS for mechanism confirmation; confocal microscopy for cell imaging and co-localization analysis.
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UV-2102 double-beam UV/VIS spectrometer
UV-2102
Perkin Elmer
Used for absorption spectra measurement.
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Bruker DTX-400 spectrometer
DTX-400
Bruker
Used for 1H and 13C NMR measurements with TMS as internal reference.
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HPLC Q-Exactive HR-MS spectrometer
Q-Exactive
Thermo
Used for ESI-MS measurements with methanol as mobile phase.
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Leica TCS SP8 confocal microscope
TCS SP8
Leica
Used for confocal microscopy in cell imaging.
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F-4500 spectrophotometer
F-4500
Not specified in paper
Used for fluorescence spectra measurement with excitation and emission wavelength band passes set at 5.0 nm.
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pHs-3C meter
pHs-3C
Shanghai, China (likely a manufacturer from Shanghai, but brand not explicitly stated)
Used for pH recording.
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Nikon-80i inverted fluorescence microscope
80i
Nikon
Used for fluorescent imaging experiments.
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Mito Tracker Green FM
Not specified
Not specified (commercially available mitochondrial dye)
Used as a co-localization reference for mitochondrial staining in cells.
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