Interaction Analysis And Detection Of Biomolecules In Alzheimer’s Disease

Alzheimer’s disease(AD)is a neurodegenerative disease whose main features include progressive cognitive,functional and behavioural impairment and is the most prevalent cause of dementia.AD,as an age-related disease,has gradually evolved into a major social issue against the growing background of an ageing global population.To date,there is still much controversy in the study of the pathogenesis of AD.The prevailing view is that AD is a multifactorial disease,and the two mainstream hypotheses,the cholinergic hypothesis and the amyloid cascade hypothesis,have been proposed.The amyloid hypothesis suggests that the accumulation of β-amyloid(Aβ)leads to the neurotoxicity in central nervous system(CNS)and is the main pathogenic cause of AD.While the aggregation and clearance of Aβ are influenced by multiple factors,the study of the interaction of Aβ with other biomolecules can help to better understand the pathogenesis of AD and develop therapy options.In conteast,the cholinergic hypothesis suggests that reduced acetylcholine(ACh)levels lead to reduced nerve function,which causes the development of AD.And acetylcholinesterase(AChE),a key enzyme in biological neurotransmission,is responsible for degrading ACh and can regulate ACh levels and plays a key role in maintaining normal physiological function of the cholinergic nervous system.At the same time,the hydrolysis of ACh in the brain by AChE accelerates the aggregation of Aβ into amyloid plaques,and the two hypotheses are interrelated.Currently,AChE inhibitor drugs are one of the most important clinical treatments for AD,and AChE activity assay is an essential tool for high-throughput screening of AChE inhibitors and the evaluation of their inhibitory effects.Therefore,the development of simple,sensitive and reliable assays for AChE activity and its inhibitors is of great significance for the study of pathogenic mechanisms and the diagnostic treatment of AD.Based on the above research background,this thesis focused on the interaction analysis and detection needs of AD-related biomolecules,carried out real-time monitoring and analysis of the interaction between Aβ and related biomolecules,and constructed two novel methods for AChE activity detection and its inhibitor screening based on nanocatalytic materials,which includes the following three parts:1.The specific binding of three apolipoprotein E(Apo E)isoforms(Apo E2,Apo E3 and Apo E4)to Aβ was analyzed in real time by using dual polarization interferometry(DPI).Aβ was covalently coupled to the chip surface with the assistance of glutaraldehyde.When different Apo E isoforms flowed through the Aβ-modified chip surface,DPI can acquire the structure information of the chip surface,including mass,thickness and density,in real time.We have calculated the kinetic constants according to the mass change and analyzed the binding ability of Apo E isoforms and Aβ.The order of affinity was Apo E4>Apo E3>Apo E2,and their dissociation constants were 251 ± 37,40 ± 0.65 and 24.6± 2.42 nM,respectively.These results contribute to the understanding of the fact that Apo E4 has a higher risk of causing AD.Furthermore,this analytical approach to study the dynamics of biomolecular interactions is expected to contribute to the development of AD drugs and therapies targeting Apo E with Aβ.2.Based on the two-dimensional Zn-TCPP(Fe)nanosheets and the complexation of thiocholine(TCh)with metal ions,a label-free analytical method was established to detect the activity of AChE and screen its potential inhibitors.Ultra-thin twodimensional Zn-TCPP(Fe)nanosheets exhibit high peroxidase-mimicking activity,and can catalyze the oxidation of colorless 3,3’,5,5’-tetramethylbenzidine(TMB)to the blue product ox-TMB by hydrogen peroxide(H2O2),while AChE catalyzes the substrate acetylthiocholine(ATCh)to produce the reducing substance TCh,which was able to inhibit the catalytic activity of Zn-TCPP(Fe)nanosheets by complexing Zn2+.Meanwhile,TCh could reduce the blue ox-TMB to colorless TMB,and both reactions above would lead to the discoloration of the solution,so the AChE activity can be measured by testing the color change of the solution.The sensing system exhibited good selectivity and sensitivity(detection limit of 0.029 mU/mL)and has excellent potential for assessing AChE activity in human serum samples.Moreover,in the presence of AChE inhibitors,the activity of AChE is inhibited,thus hindering the production of TCh and indirectly restoring the formation of ox-TMB,and the semiinhibitory concentration(IC50)for AChE was 18.4 nM when tested with tacrine as a typical AChE inhibitor model.These results indicated that the analytical method successfully enables the detection of AChE activity,which can provide new avenues for early diagnosis and drug screening of AD and promote the development of AD therapy.3.BSA-Cu3(PO4)2 hybrid nanoflowers were prepared by a one-step coprecipitation method,and a simple and sensitive new method for the detection of AChE activity and its inhibitors was established.BSA-Cu3(PO4)2 hybrid nanoflowers,which possess laccase-like activity,can catalyze the coloration reaction of 2,4-dichlorophenol and 4-aminoantipyrine.When AChE and ATCh exist simultaneously,the substrate ATCh would be immediately hydrolyzed to TCh.And TCh could be able to reduce Cu2+to Cu+in BSA-Cu3(PO4)2 hybrid nanoflowers,thus enhancing the laccase-mimicking activity of the nanoflowers,and finally deepening the red color of the system solution.The enhanced activity of BSA-Cu3(PO4)2 hybrid nanoflowers and the degree of solution color deepening were directly related to the activity of AChE,showing a good linearity in the range of 0.25-5 mU/mL,and the limit of detection was 0.038 mU/mL.The selection of carbaryl and tacrine as inhibitor models confirmed that the method could be used for inhibitor screening.The method can accurately and reliably perform AChE activity and inhibitor assays,and has great potential for application in AD drug design and screening.