Synthesis And Biological Applications Of Multifunctional Fluorescent Probes For Adenosine Triphosphate Detection

Adenosine 5’-triphosphate(ATP),as the "energy currency",is not only involved in all physiological metabolic activities but also participates in various pathological processes in living cells.ATP is also an important indicator for evaluating the cleanliness and safety of the environment.Consequently,there is an urgent need for sensitive and selective detection of ATP in live cells,tissues,and environmental samples.Fluorescent probes as an emerging tool for ATP detection,have aroused great attention from researchers due to their simple and convenient operation,economical cost,high selectivity,excellent biocompatibility,and low cytotoxicity.Moreover,the concentrations of ATP and some other biologically active substances(GSH、H2S、H2O2、ONOO-,etc)are abnormal in diseases such as inflammation,liver damage,Alzheimer’s disease,etc.Developing fluorescent probes that can simultaneously monitor the concentration fluctuations of ATP and these biologically active substances is of great significance for diagnosing related diseases.In addition,since ATP is over-expressed in cancer cells and tumor tissues,the design of multifunctional fluorescent probes that can simultaneously monitor ATP in cancer cells and have therapeutic effects is also imminent.For these reasons,we have designed and synthesized a series of multi-functional fluorescent probes to detect ATP and conducted research on their sensing performances and biological applications,the details are as follows:1.Synthesis and applications of rhodamine-anchored poly(norbomene)fluorescent probes for ATP detection.We have synthesized two rhodamine-anchored poly(norbomene)fluorescent probes named P1 and P2.P1 is composed of rhodamine B with spirolactam-ring contained and PEG long chains.The structure of P2 contains spironolactam ring-closed rhodamine B,1,8-naphthalimide and PEG fragments.Owing to the amphiphilic property,copolymers P1 and P2 self-assemble nanostructure aggregations in the aqueous solution.Upon the addition of ATP,the spirolactam ring of rhodamine B in P1 will open,accompanied by a visual color changing from colorless to red and a great fluorescence enhancement.P2 initially shows 1,8-naphthalimide-based green emission,upon interaction with ATP,a FRET process occurs from 1,8-naphthalimide donor and rhodamine B acceptor with a change in fluorescence color from green to red,which exhibits fluorescence ratiometric detection of ATP.Probes P1 and P2 have been successfully applied to detect ATP levels in living cells due to their outstanding photostability and biocompatibility.2.Synthesis and applications of an ATP-activated tetraphenylethene photosensitizer.In this work,we have synthesized and obtained a tetraphenylethylene-based fluorescent probe C14-Azo-Py-TPE with a D-π-A structure.Due to the introduction of amide/pyridinium-imidazolium recognition sites and TPE fluorophore,C14-Azo-Py-TPE can form hydrogen bonding,electrostatic binding and π-π stacking multiple interactions with ATP,resulting in greater aggregation behavior Based on the AIE effect,the fluorescence of the probe will be significantly enhanced.Probe C14-Azo-Py-TPE shows a highly sensitive and selective detectability for ATP and can be applied to monitor ATP levels in living cells.Meanwhile,the increased ATP concentration in the probe could promote its production of reactive oxygen species(·OH and O2·-),leading to a greater ability to kill cancer cells and more effective photodynamic therapy.C14-Azo-Py-TPE can be successfully applied in ATP imaging and activation-induced cancer cell death.3.Synthesis and applications of an ATP-activated triphenylamine dimer photosensitizer In this work,we have constructed and synthesized a triphenylamine dimer fluorescent probe TPA-Azo-TPA with bisimidazolium contained.TPA-Azo-TPA can form electrostatic binding and π-π stacking interactions with ATP,which leads to greater aggregation and fluorescence enhancement,and thus achieves highly sensitive and selective detection of ATP.Probe TPA-Azo-TPA can successfully image intracellular ATP due to its excellent photostability and biocompatibility.Meanwhile,the reactive oxygen species production capacity of the probe will be enhanced under ATP induction(mainly reflected in type Ⅰ reactive oxygen species ·OH and O2·-),which corresponds to a enhanced cancer cell killing ability,thus achieving a better photodynamic therapy effect.Compared to C14-Azo-Py-TPE,TPA-Azo-TPA has a more red-shifted absorption and emission,responds better to ATP and has a lower detection limit.In particular,TPA-Azo-TPA has more type Ⅰ reactive oxygen species production upon the addition of ATP.4.Synthesis and applications of a coumarin-linked tetraphenylethene fluorescent probe for detection of ATP and GSH.A bifunctional coumarin-linked tetraphenylethene probe Cou-SS-Py-TPE was successfully developed for the simultaneous detection of ATP and GSH.Due to the effective FRET process between the energy donor coumarin and the energy receptor TPE,probe Cou-SS-Py-TPE initially only shows red emission of the TPE segment,while the blue fluorescence of coumarin moiety is quenched.Cou-SS-Py-TPE can detect ATP with a fluorescence turn-on response in the red channel by the aggregation-induced fluorescence enhancement mechanism and showed a ratiometric detection toward GSH through the disruption of the FRET process by cleaving the disulfide bond,accompanied by a fluorescence enhancement in the blue channel and a fluorescence decrease in the red channel.Cou-SS-Py-TPE also displays a superior sensitivity and high selectivity to ATP and GSH,with a detection limit of 20.80 nM for ATP and 3.47 μM for GSH.Significantly,Cou-SS-Py-TPE can discriminate GSH from ATP and other analytes due to the different response times.Furthermore,Cou-SS-Py-TPE has been discovered to be capable of imaging ATP and GSH in living cells and zebrafish.