Design And Synthesis Of Water Soluble Polythiophene-based Ratiometric Probes And Their Sensing Property

ATP is an important biological small molecule in the body and it plays a crucial role in many physiological processes.ATP contents abnormalities,often lead to many diseases.Therefore,the establishment of simple,rapid,and high sensitivity detection methods have great significance not only for the investigating of life processes,but also for clinical diagnosis,drug analysis.Fluorescence technology possesses the advantages of simple operation,high sensitivity,good selectivity,easy in situ imaging and so on.It has been widely used in the detection of bioactive substances.The reported probes for ATP are usually based on a single emission intensity change,which is easily interfered by environmental factors and suffered from low signal to noise ratio.Ratiometric probes involve the simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio.After combined with substrate,probes exhibit significant shift in emission wavelength and change in two fluorescence emission intensity,which can be used for detection.The ratiometric method can improve the detection dynamic response range and has become a convenient and effective way for improving the sensitivity of fluorescent probe because it establish the internal standard for greatly weakening the interference from other factors.In recent years,the conjugate polymer-based fluorescent probes have attracted much attention because of their good photoelectric properties and signal amplification effect.Among those probes,the polythiophene derivatives have received increasing interest because of their utilities as a fluorescent and colorimetric dual-mode probe.When induced by analytes,the conformation and aggregation of polythiophene derivatives which are very sensitive to the external stimuli would be changed and induce the significant change in fluorescence spectra and the color of the solution.This paper reports a new FRET-based ratiometric probe,Cou-PT,for the detection of ATP.The introduction of coumarin fluorophores as FRET donors into the side-chain of polythiophene(PT)as the FRET acceptor provides the ratiometric probe.The detection principle is as follows: upon addition of ATP,the formation of Cou-PT /ATP complex leads to the conformational changes of the PT backbone from a random coil to the planar conformation and aggregation,which induce the system fluorescence quenching and the red shift of UV spectrum.As such,the ratiometric ATP detection can be achieved by taking advantage of spectral overlap changes to modulate the FRET efficiency from coumarin fluorophores to the PT backbone before and after the addition of ATP,coupled with ACQ.In this paper,the sensitivity of the ratio probe Cou-PT to ATP was studied by means of fluorescence spectroscopy,UV absorption spectroscopy,circular dichroism spectroscopy and dynamic light scattering.The results of Job’s-plot showed that the probe Cou-PT and ATP could form a 1: 1 complex with a binding constant of Kapp = 3.12 × 104 M-1.CD spectra and DLS showed that the addition of ATP resulted in the formation of polythiophene from non-planar to planar conformational transformation and aggregation.The probe exhibited a clear seesaw-type dual-emission signal change upon binding to ATP in aqueous solution.At the same time,the red shift of the absorption spectrum was accompanied by the change of the color of the solution.So the probe can also be utilized for naked-eye detection of ATP.In addition,the probe exhibited excellent selectivity for ATP and no obvious emission and UV spectral changes were observed with ADP,AMP or various other anions.The probe can also be utilized for real-time monitoring of the ATP hydrolysis reaction by apyrase under physiological pH conditions and initially achieved fluorescence imaging of living cells.