研究目的
Developing a ratiometric fluorescent probe for lysosomal hypochlorous acid (HOCl) based on a through-bond energy transfer (TBET) strategy for selective and sensitive detection in living cells.
研究成果
The developed ratiometric fluorescent probe IRh-Ly, based on a TBET platform, exhibits excellent selectivity, sensitivity, and rapid response to HOCl, making it suitable for detecting lysosomal HOCl in living cells. The integration of a morpholine moiety enables lysosome-targeting, enhancing its applicability in bio-imaging.
研究不足
The study does not mention the probe's stability over time or its performance in complex biological matrices beyond the tested conditions. The specificity towards HOCl in the presence of other highly reactive species in vivo needs further validation.
1：Experimental Design and Method Selection:
The study involved the synthesis of a ratiometric fluorescence probe (IRh-Ly) using a TBET strategy on a rhodamine-imidazo[1,5-a]pyridine platform. The probe's response to HOCl was evaluated through UV-vis absorption and fluorescence spectroscopy.
2：Sample Selection and Data Sources:
RAW264.7 cells were used as the cell model for fluorescence imaging. The probe's selectivity was tested against various reactive oxygen and nitrogen species (ROS/RNS) and ions.
3：7 cells were used as the cell model for fluorescence imaging. The probe's selectivity was tested against various reactive oxygen and nitrogen species (ROS/RNS) and ions. List of Experimental Equipment and Materials:
3. List of Experimental Equipment and Materials: Instruments included a Bruker Avance 400 spectrometer for NMR, a Q-TOF6510 spectrograph for HRMS, a PHSJ-3F pH meter, a Hitachi U-3900 spectrophotometer for UV-vis absorption, and an F-4600 fluorescence spectrophotometer. Chemicals were sourced from Sigma-Aldrich and J & K Scientific LTD.
4：Experimental Procedures and Operational Workflow:
The probe's synthesis involved refluxing compound 1 in thionyl chloride, followed by purification. The probe's response to HOCl was measured in EtOH-PBS buffer. Cell imaging involved incubating RAW264.7 cells with the probe and stimuli (LPS and PMA) before fluorescence imaging.
5：7 cells with the probe and stimuli (LPS and PMA) before fluorescence imaging. Data Analysis Methods:
5. Data Analysis Methods: Fluorescence intensity ratios (I589/I462) were used to quantify HOCl concentration. The probe's cytotoxicity was assessed using SRB assay.
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Confocal fluorescence microscopy
Leica SP8
Leica
Used for obtaining the fluorescence images of RAW264.7 cells.
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Bruker Avance 400 spectrometer
Avance 400
Bruker
Used for obtaining 1H NMR and 13C NMR spectra.
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Q-TOF6510 spectrograph
Q-TOF6510
Agilent
Employed to collect the high-resolution mass spectroscopy.
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Hitachi U-3900 spectrophotometer
U-3900
Hitachi
Used for obtaining UV-vis absorption spectra.
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F-4600 fluorescence spectrophotometer
F-4600
HITACHI
Used for recording the fluorescent spectra.
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PHSJ-3F pH meter
PHSJ-3F
LeiCi
Used for determining the pH value.
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EPED laboratory water purification system
EPED-X3A-10T
EPED
Used for obtaining deionized water for preparing metal ions solution.
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