| Cancer is one of the biggest threats to human health.Due to the abnormal proliferation of cancer cells,the tumor microenvironment is in a weak acidic state(pH～6.5),where the concentrations of K+and H2O2 are significantly higher than normal physiological levels.These physiological characteristics are of great significance for the diagnosis and treatment of the related diseases.At present,a number of biosensors have been developed for monitoring the above physiological characteristics,including those with fluorescent probes,i-motif and DNA G-quadruplex(G4)that respond to H2O2,pH and K+,respectively.On this basis,we try to combine the pH-responsive fluorescent probe with the G4 structure to develop a fluorescence sensing system in response to multiple physiological characteristics of the cancer microenvironment.Although various fluorescent probes have been reported as pH switches,but most of their pKa does not match with pH of the cancer microenvironment.This problem,however,can be well solved by introducing a G4 structure that responds to the high level of environmental K+.With this in mind,we developed a fluorescent G4 ligand functioning in a given pH range,and accordingly established a H2O2 sensor logically controlled by K+and H+,which was then applied to the detection of H2O2 in cancer cell environment.Such a simultaneous specific analysis of three targets can improve the environmental recognition ability of the sensing platform.This research paper includes the following two parts:1.Design and synthesis of G4 fluorescent ligand for pH switch.Benzothiazolebased fluorescent dyes are a kind of commonly used ones,as its conjugate chain of carbon carbon double bond is easy to react with exogenous molecules.So they are chosen as the molecular skeleton for the design of G4 fluorescent ligands.By modifying the N atom in the thiazole ring with different substituents such as amino,carboxyl,hydroxyl groups,the corresponding fluorescent ligands are obtained,of which the amino group modified one,i.e.(E)-3-(3-aminopropyl)-2-(4-(pyrrolidin-lyl)styryl)benzo[d]thiazol-3-ium(PSNB),can combine with the G4 structure,accompanied by a strong fluorescence emission with a ca.160-fold fluorescence enhancement.Under acidic conditions,PSNB is in the lighting-up state,which can effectively bind the target G4 structure(Kd～0.67 μM).In this case,the pKa of PSNB/G4 complex increases by 1.8 units as compared to the pKa(～5.2)of free PSNB.This is consistent with the pH range of cancer cell microenvironment,laying a solid foundation for the related applications in biological analysis.2.Logically controlled H2O2 fluorescent sensor.On the basis of the above work,we further applied the designed PSNB fluorescent probe to target detection in cancer cell microenvironment.PSNB is subject to an addition reaction in the presence of HSO3-,and thus the fluorescence is quenched.After the addition of H2O2,HSO3-is oxidized and shed off from the molecule.As a result,the structure of PSNB is reset to the initial state,accompanied by a fluorescence increase.Based on the above principle,we constructed a K+and H+logically controlled H2O2 sensing platform using PSNB and G4.Under optimized conditions,the fluorescence recovery intensity(ΔF)shows a good linear relationship with H2O2 concentration in the range of 1～1000 μM,and the detection limit is 1 μM.The results shows that the H2O2 sensing platform almost not responds to other reactive oxygen species(ROS),showing a good selectivity over the target.Furthermore,we used this sensing platform to analyze the intracellular H2O2 in MCF-7 cell lysis solution,and evaluate that its endogenous level is about 0.3 mM,which is consistent with the previously reported range of 0.1～1 mM.Considering the weak acidic and high K+ environment on the surface of cancer cells,this sensing platform would have a good application potential in real-time monitoring of the flow of H2O2 signaling molecules between cells. |
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