研究目的
To design, synthesize, and characterize a novel fluorescent probe for the sensitive and selective detection of cysteine in biological systems, distinguishing it from other biothiols like homocysteine and glutathione, and to apply it for imaging in living cells and zebrafish.
研究成果
IPPA is a highly selective and sensitive fluorescent probe for cysteine, with low detection limits, good cell permeability, and low toxicity. It successfully imaged cysteine in living cells and zebrafish, demonstrating potential for bioassay applications. Future work should focus on structural optimizations for improved in vivo diagnostics.
研究不足
The probe may have limitations in distinguishing Cys from Hcy completely in all scenarios, and its application in vivo for mammals or rodents may require further optimization, such as developing red or near-infrared probes for better penetration and reduced background interference.
1：Experimental Design and Method Selection:
The study involved designing a small-molecule fluorescent probe (IPPA) based on an imidazo[1,2-a]pyridine fluorophore and an acrylate recognition group, utilizing Michael addition reactions for selectivity. Methods included synthesis, characterization (NMR, FT-IR, HRMS), fluorescence and UV-Vis spectroscopy, cytotoxicity assays, cell imaging, and zebrafish imaging.
2：Sample Selection and Data Sources:
Samples included synthesized IPPA, biothiols (Cys, Hcy, GSH), amino acids, metal ions, HepG2 cells, and zebrafish. Data were sourced from experimental measurements and standard reagents.
3：List of Experimental Equipment and Materials:
Equipment included fluorescence spectrophotometer (Cary Eclipse, Agilent), UV-Vis spectrophotometer (UV-1800, Shimadzu), NMR spectrometer (Agilent), pH meter (pH-25, Shanghai REX), fluorescence microscopes (EVOS FL Auto, Nikon Ti-S), microtiter plate reader (ELx808, BioTek), and LC-HRMS systems (Waters UPLC/XevoTQ, Agilent Q-TOF). Materials included chemicals from Sigma-Aldrich, Sinopharm, and J&K, and cell culture reagents.
4：Experimental Procedures and Operational Workflow:
Procedures involved synthesizing IPPA, optimizing detection conditions (pH, DMSO concentration), conducting selectivity tests with various analytes, performing fluorescence titrations, studying reaction mechanisms via NMR and HRMS, DFT calculations, cytotoxicity assays (CCK-8), cell imaging with HepG2 cells, and zebrafish imaging.
5：Data Analysis Methods:
Data were analyzed using fluorescence intensity measurements, pseudo-first-order kinetics, limit of detection calculations, statistical methods (mean ± SE, n=3), and DFT computations with Gaussian 09.
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fluorescence spectrophotometer
Cary Eclipse
Agilent Technologies
Recording fluorescence spectra for detection and analysis of fluorescent probes.
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UV-Vis spectrophotometer
UV-1800
Shimadzu
Measuring UV-Vis absorption spectra of samples.
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NMR spectrometer
600 MHz
Agilent Technologies
Collecting 1H and 13C NMR spectra for characterization of synthesized compounds.
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fluorescence microscope
EVOS FL Auto
Invitrogen (part of Thermo Fisher Scientific)
Performing fluorescence imaging of cells and tissues.
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LC-HRMS system
Agilent Accurate-Mass-Q-TOF MS 6520
Agilent
High-resolution mass spectrometry for characterization.
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pH meter
pH-25
Shanghai REX Instrument Factory
Measuring pH values of solutions.
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fluorescence microscope
Nikon Ti-S
Nikon
Performing epifluorescence microscopy for imaging applications.
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microtiter plate reader
ELx808
BioTek
Measuring absorbance in cytotoxicity assays.
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ultrapure water system
Milli-Q Plus
Millipore
Producing deionized water for experiments.
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LC-HRMS system
UPLC/XevoTQ MS/MS
Waters
Collecting mass spectra for compound analysis.
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