研究目的
To develop an intracellular signal amplification-based MCE-LIF method for the quantification of ATP present inside single cells.
研究成果
The proposed method provides a useful tool for studying the biomedical functions of ATP at the single cell level and for early diagnosis of related diseases.
研究不足
The method requires specific conditions for signal amplification and may not be applicable to all cell types without optimization.
1：Experimental Design and Method Selection:
Developed an intracellular signal amplification strategy using carboxyl luciferin-labeled ATP aptamer as target probe (TP), liposomes as the carrier for transporting the TP and exonuclease III into cells, and a TP labeled with a fluorophore (FAM) for selective identification of ATP and generation of fluorescent signals.
2：Sample Selection and Data Sources:
Used Hela, HepG2, and HL-7702 cell lines.
3：List of Experimental Equipment and Materials:
Microchip electrophoresis with laser induced fluorescence detection, liposomes, exonuclease III, ATP aptamer.
4：Experimental Procedures and Operational Workflow:
Cells were incubated with liposomes encapsulated TP and exonuclease III, followed by lysis and MCE-LIF analysis.
5：Data Analysis Methods:
Analyzed using MCE-LIF conditions including pH of electrophoresis buffer solution, concentration of borax and SDS, and separation voltage.
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microchip electrophoresis
Used for the separation and analysis of ATP in single cells.
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laser induced fluorescence detection
Used for the sensitive detection of fluorescent signals generated from ATP quantification.
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liposomes
Used as carriers for transporting the target probe and exonuclease III into cells.
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exonuclease III
Used for catalyzing the hydrolysis of the target probe to release ATP and fluorescent FAM.
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ATP aptamer
Used as the target probe for selective identification of ATP and generation of fluorescent signals.
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