Ratiometric Fluorescent Detection Of Intracellular Singlet Oxygen By Semiconducting Polymer Dots

Singlet oxygen?¹O₂?is one of reactive oxygen species.It plays important roles in many physical,chamical,and biological processes,and it is closely related to the generation of many diseases,the aging of organisms and many physiological processes.Singlet oxygen has been widely used in wastewater treatment,chemosynthesis,and photodynamic therapy to kill cancer cells.However,the molecular mechanism underlying the effects of ¹O₂ are not completely understood.The ¹O₂ concentration in living cells is essential for biological processes,thus,the detection and quantification of ¹O₂ are extremely important for understanding its roles in different processes.In the traditional direct detection method,the near-infrared luminescence of ¹O₂ at 1270 nm is measured to detect it.However,owing to the low quantum yield and signal-to-noise ratio as well as the short lifetime in aqueous solutions of ¹O₂,the efficiency of this method is limited.Various probes that are sensitive to ¹O₂ have been used for detection and quantification.One method bases on absorbance.The absorbance intensity of this kind of probes will change in the presence of ¹O₂.¹O₂ can be quantified according to the rate of change.Despite the convenience of probes based on absorbance,the low sensitivity limits their applications in live cells.Chemiluminescent probes are reported to be highly sensitive to ¹O₂,which detect ¹O₂ according to the chemiluminescent change of probes.But they have various shortcomings,such as a low selectivity and high toxicity.In the past few years,fluorescence probes have attracted a great deal of attention for singlet oxygen sensing.They generally detect ¹O₂ by monitoring changes in various properties,such as wavelength and emission intensity.The high sensitivity,quick response,and excellent spatial resolution in microscopic imaging make them promising probes.Although the background fluorescence of samples sometimes has interference,the high sensitivity and low detection line still make them become the most potential probe.Semiconducting polymer dots?Pdots?as fluorescence probes exhibit bright fluorescence,good photostability and biocompatibility for optical imaging and detection.Pdots are widely applied in fluorescence imaging,sensing and detection,photoacoustic imaging,and photodynamic and photothermal therapy.The superior light-harvesting capability of semiconducting polymers provide an ideal system to engineer new functions by molecular dye doping.In this paper,we designed a Pdot probe to detect ¹O₂ in living cells.We doped a dye,i.e.,singlet oxygen sensor green?SOSG?in Pdots.The fluorescence of SOSG will enhance gradually in the presence of¹O₂,however,SOSG is supplied as a cell-impermeant probe,and thus does not allow for intracellular ¹O₂ detection.We used blue light as the excitation light source,and made use of the fluorescence resonance energy transfer between the polymer and the dye,to monitor the response of the probe to ¹O₂ produced by photosensitizer.At the same time,because of the fluorescence of polymer unchanged while that of dye changed a lot,a ratio fluorescence detection was carried out.The main work are as follows:?1?We used PFO as a polymer donor and doped SOSG,and prepared Pdots by the method of reprecipitation.Chlorin e6?Ce6?served as the source of ¹O₂.Under the irradiation of corresponding light,PFO transferred energy to SOSG.With the increasing of ¹O₂ produced by photosensitizer,the green fluorescence intensity of Pdot probe also increased.The Pdots were characterized,such as size,morphology,zeta potential,absorbance and emission spectra.By analyzing the emission spectra of different doping ratios,it is confirmed that there is energy transfer between polymer PFO and dye SOSG.Then,we evaluated the detection of ¹O₂ in vitro,and the results proved that the emission intensity of Pdots did change with the increase of ¹O₂ concentration,which proved that it was promising to conduct further detection in living cells.?2?We detect intracellular ¹O₂ by using the Pdot probe.After Pdots entered cells,Ce6 was added to the system.Ce6 was stimulated at 650 nm to generate ¹O₂,and the fluorescence intensity of Pdots in cells was monitored by confocal fluorescence microscope.The collected fluorescence images showed that the green fluorescence of Pdots became brighter and brighter as the time of illumination increased.The results showed that our Pdots has a good application prospect in the detection of ¹O₂ in living cells.