Fluorescent Probes Based On Cyclodextrin Polymers For Pesticide Discrimination And Intracellular PH Imaging

The fluorescence properties of dyes depend sensitively on their microenvironment.Changes in the microenvironment of dyes may lead to largely changes in one or more of their fluorescence parameters,such as maximum excitation/emission wavelength,fluorescence intensity,fluorescence lifetime and anisotropy.Especially the fluorescence properties of dyes have changed significantly in the presence of some macrocyclic molecules due to the formation of their host-guest complexes with fluorescent dyes.Based on this property,a variety of fluorescent molecular probes have been constructed,which have been widely used for the detection of ions and small molecules,the recognition and interaction of biological macromolecules,and even the staining of organelles in living cells.In the previous studies,we found that the β-cyclodextrin(β-CD)polymer has a significant enhancement to the fluorescence of pyrene.In addition,nanoparticles could be formed based on β-CD polymerc and enter cells without cytotoxicity.On this basis,the effects of β-cyclodextrin polymer(β-CDP)on the fluorescence properties of various dyes were investigated.Then,fluorescent probe array and fluorescent nanoprobes were constructed based on β-CDP,which respectively discriminated different pesticides and targeted mitochondrial pH imaging.(1)Fluorescent probe array based on β-cyclodextrin polymer for pesticides discriminationThe β-CDP has not only the host-guest interaction with dyes,but also has macromolecular effects,so the effect on the fluorescent properties of dyes is m ore significant than that of the cyclodextrin monomer.In order to make full use of this feature,we investigated the effects of β-CDP on the fluorescence properties of 13 common dyes and determined the binding constants.The results showed that the fluore scence intensity of 8 dyes did not change significantly after adding β-CDP,while the fluorescence intensity of the other 5 dyes(rhodamine B isothiocyanate,pyrene,5(6)-carboxynaphthalene fluorescein,curcumin and danamide)increased or decreased significantly after binding with β-CDP.Based on the above results,we selected four dyes with significant changes in fluorescence intensity after binding to β-CDP,and constructed fluorescence probe array for pesticides discrimination by using the competition be tween guest molecules.The degree of change in the fluorescence intensity of the β-CDP/dyes complex were different in the presence of different pesticides.The distinction of competitiveness between pesticides and dyes may be attributed to the difference o f hydrophobic properties of each pesticides.The data were anaylized by principal component analysis(PCA),three-dimensional maps showed that the five pesticides were divided into five regions without obvious overlap,indicating that the fluorescent probe array could be used for discriminating different pesticides.(2)Mitochondria targeted self-assembled ratiometric fluorescent nanoprobes based on β-cyclodextrin polymer for pH imaging in living cellsIn the previous study,we found that β-CDP could enhance the fluorescence of rhodamine B isothiocyanate.Moreover,rhodamine B and fluorescein were often used as signal units and reference units for pH detection.In addition,our group had constructed ratiometric probes for pH imaging in cells using β-CDP and two dyes.However,the distribution of pH in cells is actually uneven,the pH values in various organelles are different.On these foundations,we designed ratiometric fluorescent nanoprobes based on selfassembly for imaging in mitochondrial pH of living cells.The nanoprobes consisted of three parts:(1)a β-CDP acted as the backbone of the nanoprobes;(2)two lipophilic dyes acted as the pH sensitive unit and reference unit,i.e.adamantane-labeled fluorescein(Ad-F)and adamantane-labeled rhodamine B(Ad-R);(3)adamantane-labeled triphenylphosphonium(Ad-TPP)acted as the mitochondrial targeting unit.Confocal microscopy images proved that the nanoprobes could be applied to monitor intracellular pH in the range of pH 4.0-8.0.Moreover,as evidenced by co-localization imaging,the nanoprobes also showed good ability to target mitochondria(Pearson correlation coefficient was 0.88).Additionally,the nanoprobes showed low toxicity,excellent pH reversibility,and flexibility of construction,and have the potential to monitor pH of other organelles if different targeting units are conjugated.This strategy is expected to provide new tools for pH monitoring in specific organelles and diagnosis of pH related diseases.