Quantification of ATP in cell by fluorescence spectroscopy based on generalized ratio quantitative analysis model

Area of Science:

• Biochemistry
• Analytical Chemistry
• Spectroscopy

Background:

• Accurate quantification of adenosine triphosphate (ATP) is crucial for understanding cellular processes.
• Existing methods for ATP detection can be complex or require fluorescent labeling.
• G-quadruplex structures offer unique properties for molecular recognition and signal generation.

Purpose of the Study:

• To develop a simple, rapid, and label-free fluorescence spectroscopy method for intracellular ATP quantification.
• To utilize G-quadruplex formation for sensitive ATP detection.
• To validate the method's accuracy and reliability in real cell samples.

Main Methods:

• Development of a fluorescence assay based on thioflavin T (ThT) binding to an ATP aptamer G-quadruplex.
• ATP competes with ThT for binding to the G-quadruplex, leading to a change in fluorescence intensity.
• Application of a generalized ratio quantitative analysis model to correct for background interference in cell suspensions.

Main Results:

• The fluorescence intensity of the ThT-G-quadruplex complex decreased in the presence of ATP due to displacement.
• The method successfully quantified ATP in two different cell lines with accuracy comparable to a commercial ATP detection kit.
• Achieved a limit of detection of 0.22 μM and a limit of quantification of 0.66 μM for intracellular ATP.

Conclusions:

• The developed fluorescence spectroscopy method provides a highly simple, rapid, and label-free approach for cellular ATP quantification.
• The G-quadruplex-based assay demonstrates good sensitivity and accuracy for ATP detection in complex biological samples.
• This method offers a promising alternative for routine ATP analysis in cellular research.