Fluorescence Imaging And Biosensing Of Biological Species Related To Alzheimer's Disease

Alzheimer's disease?AD?,as one of the neurodegenerative diseases that cannot be prevented,cured or even slowed down,has become a common major disease that threatens human life and health.The metabolic balance of metal ions in the brain tissue of early AD patients is disturbed,triggering a large amount of reactive oxygen species?ROS?,which in turn causes the production of hyperphosphorylated tau?p-tau?protein,resulting in neuronal death;at the same time enhances?-secretase??-Secretase,BACE1?activity and expression level,BACE1 cleavage of amyloid precursor protein?APP?promotes the accumulation of?-amyloid protein?A??in brain tissue,causing brain tissue lesions.To achieve accurate monitoring of active substances in the early stage of AD,and to thoroughly study the development of AD from the molecular mechanism,it is of great significance for accurate prevention and treatment of AD.A variety of detection methods have been reported for the analysis of active substances related to AD.Among them,fluorescence analysis has many advantages,such as simple operation and dynamic visualization imaging of target analytes in biological samples.Therefore,it is widely used in biomedicine,biodetection and other fields.However,there are still great challenges to achieve high selective,high sensitive,high accurate and rapid fluorescence imaging method of important biological species related to AD.1?The composition of biological samples is complex,and the concentration of analyte is low.It is necessary to develop the probe with good selectivity and low detection limits.2?An in-depth understanding of the crosstalk between multiple components is beneficial for understanding the pathological process of AD.So the fluorescent probe for simultaneous detection of multiply biological species need to be developed,to provide effective methods for revealing important metabolic processes and signaling pathways.3?The fluorescence intensity of probe is easily disturbed,because the microenvironment of living tissue is dynamic and changeable.Therefore,the fluorescent lifetime probe and ratio fluorescent probe need to be developed to avoid the interference of environmental factors on the detection.Focous on the existing scientific problems,in order to accurately monitor the biological species in AD,this paper has carried out the following three research topics:1?A novel DNA nano fluorescent lifetime probe?-metal was designed and assembled,which can simultaneously detect Cu²⁺,Zn²⁺,and Ca²⁺in the brain of AD mouse.The main frame of the probe is a DNA tetrahedron with side length of about12 nm,and metal ion DNAzymes that specifically detect Cu²⁺,Zn²⁺,and Ca²⁺are assembled at the three vertices of the tetrahedron.The three DNAzymes contain three fluorophores with the same excitation wavelength and different emission wavelength,and corresponding quenchers.The target metal ion cut the substrate strand of DNAzyme,and then the DNA strand containing the quencher removed,so that the fluorescence lifetime of the?-metal probe changed,to realize the detection of the target metal ion.The?-metal probe was used for fluorescence lifetime imaging and biosensing of free Cu²⁺,Zn²⁺,and Ca²⁺in cytoplasm of neurons in cerebral cortex,striatum and hippocampus regions of mouse.2?The first fluorescent lifetime probe?-p-tau was designed and synthesized,which can be used to detect the p-tau protein in single neurons with high selectivity.The two Zn-Dpa units were designed as recognition groups and an improved cyanine with large Stokes-shift?160 nm?was synthesized as dye unit.The fluorescence lifetime of?-p-tau probe showed a good linearity with the concentration of p-tau protein from 1.0 to 5.0?M,and a response time less than 6.2 s.The?-p-tau probe can be used for fluorescence lifetime imaging and biosensing of p-tau protein in the cytoplasm of single neuron.3?The first two-photon ratiometric fluorescent probe AF633mCyd was designed and synthesized for detection of BACE1 in AD mouse brain,in which the fluorescence resonance energy transfer?FRET?system was designed and synthesized by a novel two-photon donor,merocyanine derivative?mCyd?,connected with an acceptor,Alexa Fluor 633?AF633?,through a peptide substrate?EVNL-DAEFRHDSGYK?with a length of less than 10 nm.With the probe being cleaved by BACE1,fluorescence intensity of mCyd is increased accompanied by the decrease of that of AF633.The probe AF633mCyd is the first reported two-photon ratiometric fluorescent probe for detecting BACE1 with the advantages of good tissue penetration,low biological background fluorescence,and resistance to environmental interference.The probe can be used for ratiometric fluorescence imaging and biosensor detection of BACE1 in cerebral cortex,striatum,thalamus and hippocampus regions of normal and AD mouse brain.The result demonstrated that compared with normal mouse brain,the level of BACE1 in the AD mouse brain was increased,and the increase in cortex and hippocampus was particularly obvious.In addition,ROS kit was also used to detect the ROS content in the cortex,striatum,thalamus and hippocampus of normal and AD mouse brain.The results showed that compared with normal mouse brain,the level of ROS in the AD mouse brain was also increased,and the increase in the cortex and hippocampus was particularly significant.Thus,it was found that both of AD and oxidative stress were closely associated with the level of BACE1.