New Strategy For In Vivo Ananlysis Of Ischemia And Alzheimer's Disease Related Molecules

Alzheimer's disease?AD?and ischemia are the two most common brain diseases,and there is a potential link between them.The study found that,on the one hand,the possibility of subsequent AD in patients with cerebral ischemia is greatly enhanced;on the other hand,the occurrence of cerebral ischemia that patients with previous AD further aggravates the decline in cognitive ability.However,the differences and associations between the pathological molecular mechanisms between the two diseases are still unclear.Therefore,the studies of ischemic and AD-associated molecules is important for exploring the link between the two diseases,early diagnosis and treatment of the diseases.However,due to the complex micro-environment of the brain,large differences in the environment between in vivo and in vitro,and the interrelationship between molecules in the brain,it is still a huge challenge to establish an analysis method with high selectivity,high accuracy and simultaneous detection of multiple substances!This topic focuses on the key scientific issues of selectivity and accuracy in the detection of chloride ions and active sulfur of ischemia and AD.A series of new methods for in vivo analysis are designed and constructed,and a new method for in vivo analysis is established.In vivo analysis of chloride and active sulphur in the brains of ischemia and AD provides an effective method for studying the molecular mechanisms in the brain of rats under ischemia and AD disease models.The details are as follows:1.Based on the principle that AgNPs are first oxidized to form Ag⁺under electrochemical conditions,and further reacted with Cl^-.Here,We choosed the oxidation potential of AgNPs as the measurement signal,and methyl blue as the internal reference signal.Thus,a label-free selective and accurate electrochemical biosensor was first developed for real-time monitoring of Cl^-levels in mouse brain model of AD and rat brain upon global cerebral ischemia.The prepared sensor has a good Nernst response to Cl^-with the concentrations ranged from 1 to 300 mM,and the sensitivity is 58.3 mV/pCl^-.Moreover,MB was selected as an inner reference molecule for a built-in correction to avoid the effects from the complicated brain and improve the accuracy.In addition,the electrochemical biosensor showed remarkable selectivity for determination of Cl^-over other anions,metal ions,amino acids,and other biomolecules.Finally,the developed biosensor with high analytical performance,as well as unique characteristic of CFME itself including easy to inert in live brain and good biocompatibility,was successfully applied to in vivo determination of Cl^-levels in three brain regions:striatum,hippocampus,and cortex of live mouse and rat brains,and the levels of Cl^-in normal striatum,hippocampus,and cortex of normal mouse brains and those in the mouse model brains of AD were reported.In addition,the results in the rat brains followed by cerebral ischemia demonstrated that the concentrations of Cl^-decreased by 19.8?0.5%?n=5?in the striatum and 27.2?0.3%?n=5?in hippocampus of rat brains after cerebral ischemia for 30 min,but negligible change in Cl^-concentration was observed in cortex.2.Herein,an efficient biosensor was created for the simultaneous monitoring of endogenous H₂S_n and H₂S in mouse brains.Integrating with density functional theory?DFT?calculation,the novel electrochemical probe,3,4-bis??2-fluoro-5-nitrobenzoyl?-oxy?benzoic acid(M_PS-1)was rationally designed and synthesized for specific recognition of H₂S_n.At the same time,N-?4-?2,5-dinitrophenoxy?phenyl?-5-?1,2-dithiolan-3-yl?pentanamide(M_HS-1)was prepared and co-assembled at the mesoporous gold film?mAu?as selective element for H₂S.This single biosensor can simultaneously determine H₂S_n concentrations from 0.2 to 50?M,as well as H₂S concentrations from 0.2 to 40?M.More importantly,negligible crosstalk was observed between Na₂S₂ and Na₂S determination channels..In addition,both of M_PS-1and M_HS-1S-1 exhibited fast response time for Na₂S₂ and Na₂S of within 2 min,and high selectivity for Na₂S₂ and Na₂S against the other sulfur-containing species,reactive oxygen species and metal ions.Finally,the novel biosensor was successfully applied in the quantification of H₂S_n and H₂S in the living mouse brains and brain slices.The averaged levels of H₂S_n and H₂S were reported for the first time in cortex,hippocampus and striatum in mouse brains with cerebral ischemia and mouse brains model of Alzheimer's diseases?AD?,respectively.In the brain model of ischemia and AD mice,the level of H₂S decreased and the level of H₂S_n increased.In addition,the expression of TRPA1 protein was positively correlated with changes in H₂S_n levels.More importantly,the brain slice suggested that H₂S_n is H₂S-derived signal molecules and exhibit⁴.4 folds more potent activity of TRPA1 channels than H₂S under stimulation with the same amount.This study not only demonstrates the unprecedented roles of H₂S_n,but also provides a new insight on the reactive sulfur species playing in brain diseases.3.We designed a high-accuracy and sensitive single-molecule ratio Raman probe?AuNRs/DEP?for the simultaneous monitoring of multiple active sulfurs in cerebrospinal fluid of mouse brains model of AD,based on the surface-enhanced Raman?SERS?and PCA techniques.First,the gold nanorods?AuNRs?was synthesized and charactized by UV-Vis and TEM.Then,the organic molecule 2-?3,5-dinitrophenoxy?-4-?2-?4-ethynylbenzamido?ethyl?phenyl 2-fluoro-5-nitrobenz-oate?DEP?was synthesized and further are linked to AuNRss by acetylene bond.The molecular mainly design consists of an internal reference group and a reactive group,and has multiple recognition functions:the first part is acetylene group,which can be bonded to AuNRs by covalent bonding,and the recognition molecule DEP is assembled into the nano-substrate in one step.At the same time,the alkynyl group is located in the Raman silent region of the organism,and can be used as an internal reference to construct a ratiometric probe to improve the accuracy of the assay.The second partial response group consists of an ether bond,an ester group and a C-F bond,awhich are the H₂S,H₂S_n,and both H₂S_n and Cys recognition sites,respectively.Next,a multi-functional Raman probe of AuNRs/DEP was prepared in one step by linking the DEP molecule to the AuNRs via a galvanic bond.Finally,the multifunctional Raman probe AuNRs/DEP exhibits a different SERS response at different wavelengths for the reactive sulfur species.According to the PCA analysis,PC1 has a good linear relationship with the concentration logarithm of H₂S,H₂S_n and Cys,respectively.In addition,the Raman probe shows good selectivity for other biologically relevant species.More importantly,the probe has high accuracy and excellent reproducibility with an internal reference.Finally,combined with the above good analytical performance,the AuNRs/DEP Raman probe was successfully used for simultaneous monitoring of H₂S,H₂S_n and Cys in microdialysis fluid of mouse brains model of AD...