Spectral Analysis Of Substances Related To Neurodegenerative Diseases

Alzheimer’s disease（AD）is a typical neurodegenerative disease,and its pathological features are mainly manifested by the massive formation of senile plaques and neurofibrillary tangles in patients’brains.Among them,senile plaques are mainly composed of amyloidβ-protein（Aβ）and metal ions such as Zn²⁺,Fe³⁺and Cu²⁺.With the increasing aging of the world,the harm of AD to human health is also intensifying.So far,the pathogenesis of AD is still not clearly resolved,and there is also no effective treatment to prevent or reverse the disease.Therefore,the study of metal ions and Aβplaques related to neurodegenerative diseases is the key to the analysis and treatment of AD pathological features.Spectral analysis is an important research method for metal ions and Aβplaques related to neurodegenerative diseases.On the one hand,for the important metal ions,the two-photon ratiometric fluorescent probe has the advantages of strong penetration ability,low background signal and small tissue damage,which can realize the biological sensing of metal ions.Metal ions can affect the folding and aggregation of Aβprotein,which in turn affects the generation,growth and structure of senile plaques.Therefore,on the other hand,polarized light analysis was used to further analyze the structure and growth mechanism of Aβplaques and the effects of metal ions and inhibitors on their growth process.However,it is still challenging to realize the fluorescence analysis of metal ions and the polarization analysis related to Aβplaques.The current main challenges are mainly in the following three aspects:（1）To achieve biosensing of metal ions in brain slices and zebrafish,it is necessary to construct probes with high two-photon absorption cross sections（>100 GM）;（2）Most of the reported two-photon ratiometric metal ion fluorescent probes need to add a large amount of organic solvents.To better use the probe for biosensing,it is necessary to increase the water solubility of the probe;（3）In the study of amyloid accumulation spherulites,the huge potential of polarized light analysis is far from being realized,and it is urgent to establish a complete and systematic Aβplaque polarized analysis.This article has carried out the following three tasks on the key scientific issues raised above:（1）To improve the two-photon absorption cross section of the probe,we synthesized a two-photon ratiometric probe,5,5-bis（（E））-4-（（2,5,8,11-tetraoxatridecan-13-yl）oxy）-3,5 dimethylstyryl)-2,2’-bipyridine（P-Zn）,with high selectivity and spatiotemporal resolution.After P-Zn probe specifically interacted with Zn²⁺,the maximum value of the two-photon absorption cross section increased from 516±77 GM to 958±144 GM.P-Zn probe also have the advantages of high stability,low toxicity,and good biocompatibility.By comparing the fluorescence imaging of AD mouse brain slices with normal mouse brain slices,it was found that there were obvious plaques in AD mouse brain slices,and the content of Zn²⁺in this plaque was higher than that of normal mouse brain.（2）In order to overcome the shortcomings of poor water solubility of probes,we synthesized a two-photon ratiometric fluorescence probe,（6-（4-（2-（2-（2-methoxyethoxy）ethoxy）eth-oxy）-3,5-dimethylstyryl）quinolin-2-yl）-N,N-bis（pyridin-2-ylmethyl）methanamine（TEO-MPVQ）,with higher water solubility and selectivity,by using the derivative of Dipyridylmethylamine（DPA）as Zn²⁺complexing agent.TEO-MPVQ probe has the characteristics of strong tissue penetration ability,small biological damage and short response time.Neuronal imaging showed that endogenous Zn²⁺was rapidly released from metallothionein in neurons under NO stimulation,reducing the intracellular Zn²⁺concentration（3）Metal ions are closely related to the formation of Aβplaques.We systematically studied the structure and growth mechanism of Aβplaques and the effects of key biomolecule ions such as metal ions on their growth process.Studies have shown that Aβplaques exist in the form of spherulites in the brains of patients.Lots of Aβ_1-16 spherulites were prepared by the solvent evaporation method,and it was confirmed that the Aβ_1-16 spherulites were accumulated of Aβfiber.Muller matrix polarized imaging studies have shown that the linear birefringence angle of Aβ_1-16spherulites is parallel to the rapid growth direction of the fibers in the spherulites.Zn²⁺and Cu²⁺affect the growth of Aβ_1-16 spherulites through nucleation and coordination,so that the growth rate of Aβ_1-16 spherulites changes with the concentration of Zn²⁺and Cu²⁺,which increases first and then decreases.To further explore the interaction between foreign substances and fibers,the arrangement of some dye molecules in the spherulite was investigated,and it was confirmed that the dyes such as rhodamine B were arranged radially along the spherulite.It is further speculated that the inhibitor mainly interacts with the radial direction of the Aβfiber,thereby slowing the growth of the Aβfiber in the radial direction and reducing the growth rate of spherulites.