Dual-site IDA-based Nanoassemblies For Detection Of Some Vital Substances In Living Organisms

Simultaneous detection of multiple active substances in living organisms can uncover the processes of life.For example,nitric oxide and hydrogen peroxide must coexist in macrophages to effectively kill pathogens,and insulin must be co located with zinc in the pancreatic islets to promote its packaging and release.Multiple-site fluorescent probes for simultaneous detection of various analytes can provide an effective platform for exploring the mechanisms of interaction between active specie and improving the accuracy of disease diagnosis.At present,this fluorescent probe is mainly limited to the covalent bond linked fluorescent probe using direct method.However,there are few reports on the examples of multiple-site probes based on indicator displacement assay（IDA）through the non-covalent bond complexation of indicator and receptor.This paper focuses on some active substances in living organisms as research object,like reactive oxygen species（H₂O₂）,active sulfur species（GSH,H₂S）,and energy supply substance（ATP）.Several amphiphilic receptors with multiple reaction sites and positive charges were successfully designed and synthesized.We also investigated the ability of these receptors forming IDA nanoassemblies with different anionic fluorescent dyes,and studied the fluorescence recognition properties of the nanoassemblies towards multiple analytes.The details are as follows:1.An amphiphilic receptor Bp-SS-C14 containing disulfide bond and4,4-bipyridyl onium was designed and synthesized.The amphiphilic receptor can be assembled with different anionic dyes in aqueous solution into non-fluorescence Bp-SS-C14（?）dye（dye=UD,SR101,HPTS,EY,TPPS,RB）nanoassemblies.Based on the following two mechanisms:one is the indicator and analytes substitution competition,the other is analytes can guide disulfide bond cleavage to disrupt the assembly and release indicator;the Bp-SS-C14（?）dye nanoassemblies can specifically recognize ATP and GSH.Accordingly,ATP can only achieve the competitive replacement of UD and SR101,however,since GSH can effectively cleaved the disulfide bond to disrupt assembly,these indicators can all be released.Especially,UD and photosensitizer dye TPPS can more conveniently recognize ATP and GSH through dual channels due to their more matched excitation and emission wavelengths.The Bp-SS-C14（?）UD/TPPS nano assemblies can recognize ATP and GSH through dual channels,where the UD channel can distinguish these two analytes through dynamics control（ATP:rapid displacement,GSH:slow reaction）.We have achieved dual-channel fluorescence imaging of ATP and GSH in cells,and distinguished the concentration of ATP and GSH in cancer cells and normal cells.In addition,GSH can effectively induce the release of TPPS,achieving photodynamic therapy of cancer cells.2.An amphiphilic receptor Pe-SS-C14 containing disulfide bond and Phenylborate ester was designed and synthesized,which can be complexed with anionic indicator HPTS and assembled into non-fluorescence IDA nanoassemblies.The phenylborate ester group and disulfide bond serve as recognition sites for H₂O₂and H₂S,respectively.The departure of phenylborate ester groups caused by H₂O₂and the the cleavage of disulfide bonds by hydrogen sulfide can disrupt the assembly of the system,thereby promoting the release of the indicator.The Pe-SS-C14/HPTS nanoassembly has excellent selectivity and sensitivity for the detection of H₂O₂and H₂S,with detection limits as low as 24.5 n M and 18.2 n M,respectively.And it has been successfully applied to distinguish the levels of H₂O₂and H₂S in cancer cells and normal cells.