Fluorescence Sensing And Quantitative Study Of ATP In Mitochondria Of Cells

Adenosine triphosphate(ATP),the most important bioorganic molecule containing phosphoric acid groups in living cells,is a direct energy substance of organisms and participated in a variety of physiological processes,and its content is closely related to the occurrence and development of many diseases.Therefore,quantitative and real-time monitoring of ATP changes in cells has important physiological and pathological significance.Although some fluorescent probes have been used for the monitoring of ATP content in organisms,these fluorescent probes mainly have the following problems:First,poor selectivity and narrow response range,it is difficult to achieve intracellular ATP imaging;Second,Many fluorescent probes has been reported to detect ATP based on the changes of emission intensity,which were difficult to realized quantitative detection of intracellular ATP,owing to the influence of the instrument and the cellular microenvironment.These problems limit the use of ATP fluorescent probes in biosensing and imaging.In response to the above problems,based on the combination of rational design and screening,this paper developed a FRET based ratiometric fluorescent probe for ATP detection with appropriate binding constants to achieve dynamic monitoring and quantitative analysis of mitochondrial ATP.The specific work is as follows:1)For the problems of the poor selectivity and narrow response range of traditional ATP fluorescent probes,we developed four compounds(ACF-ATP1-4)using a long-wavelength xanthene derivative(ACF27)as a fluorescent reporter unit and four amino compounds as recognition units.Under physiological conditions,we tested the response of four compounds to different biomolecules containing phosphate groups.The results showed that the compounds ACF-ATP1,ACF-ATP3,and ACF-ATP4 had good selectivity for ATP,while other biomolecules do not affect the fluorescent properties of three compounds.The compound ACF-ATP4 containing the piperazine recognition group has a fast response(within 2 min),good selectivity and a suitable response range for ATP.2)Ratio fluorescence imaging can effectively reduce the interference of the biological microenvironment and experimental instruments.Based on the previous work,we synthesized the mitochondrial-targeting ratio fluorescence probes(Rh6G-ACFPN,R6G-ACF-2)with ACF-ATP4 as the ATP response unit and energy acceptor,and a positively charged rhodamine dye as the energy donor and mitochondria targeting unit.At the same time,in order to study the response mechanism,we synthesized the compounds Rh6G-ACFTN and Rh6G-ACFDN as control probes.Spectroscopic experiments showed that the probe Rh6G-ACFPN not only had high selectivity and sensitivity to ATP,but also had fast response(within 3 minutes)and reversible detection.In addition,cell imaging experiments showed that Rh6G-ACFPN could not only be used for endogenous ATP detection under different conditions,but also successfully realized reversible real-time monitoring of ATP dynamic changes and quantification of different intracellular ATP content.