Enzyme Activity Detection Based On Novel Fluorescent Materials And Screening Of Inhibitors

In recent years,the fluorescence method has been widely used for testing substances,molecular recognition and biological imaging and so on and has gotten the researchers' extensive concern.Simultaneously,various novel fluorescent materials have sprung up constantly.The novel fluorescent nanomaterial,gold nanoclusters and silicon quantum dots,which have good optical stability and biocompatibility have been applied in the field of biological and chemical analysis.Part one:IntroductionFirstly,the concept,classification,property and application of fluorescentnanomaterial were summarized.Then the synthesis and application of novel fluorescent nanomaterial gold nanoclusters were illustrated.Furthermore,the synthesis and application of the other novel fluorescent nanomaterial silicon quantum dots were introduced briefly.Finally,the purposes and significance of the research were put forwword.Part two:acetylcholinesterase activity detection and it's inhibitor screeningAcetylcholinesterase can catalyze the hydrolysis of acetylcholine to produce acetic acid and choline,regulating the content of acetylcholine in synaptic cleft.Acetylcholinesterase inhibitor can adjust the content of acetylcholine in synaptic cleft by suppressing the activity of acetylcholinesterase,meanwhile it can penetrate the Blood-Brain-Barrier,so it is a kind of effective drug for treating the diseases of above.The studies found that the content of acetylcholine is bound up with a variety of diseases,such as myasthenia gravis,glaucoma and Alzheimer's disease etc..A direct fluorescence turn-on method for rapid and sensitive acetylcholinesterase(AChE)activity assays and it's inhibitor screening has been developed by first using low-fluorescence glutathione-capped gold nanoclusters(GSH-AuNCs).The thiocholine produced by AChE-catalyzed hydrolysis of S-acetylthiocholine iodide could effectively enhance the fluorescence of GSH-AuNCs via Au-S bond formation.In the presence of inhibitors,AChE activity was suppressed and thus fluorescence enhancement decreased.Therefore,AChE activity assays and it's inhibitor screening could be performed by measuring the fluorescence intensity of the system.The linear range of the AChE activity assay was 0-30 mU mL-1 with a limit of detection(LOD)of 0.03 mU mL-1(S/N=3).The IC50 values of two inhibitors(tacrine and neostigmine bromide)were 42.92 nM and 37.04 nM,respectively.The developed protocol provides a simple and rapid platform for assaying AChE activity and screening its inhibitors and it was successfully applied to detecting the activity of AChE in the rat brain tissue.Part three:a-glucosidase activity assay and it's inhibitor screeningUp to now,diabetes is a incurable chronic diseases but it can be controlled.Its main characteristic is high blood sugar,while long-term high blood sugar will cause a series of complications,which substantially reduce the life quality of diabeticpatients,so it has become a health problem which has attaracted extensive attention worldwide.a-glucosidase inhibitor is a kind of important drug that can treat diabetes,which can slow the digestion rate of carbohydrates and effectively control the postprandial blood glucose level.So constantly establishing new method to realize the screen of a-glucosidase inhibitor is of great significance for the control of diabetes.In the study,we used silicon quantum dots(SiQDs)which is innocuous and unpoisonous and have good water solubility and biocompatibility as a fluorescence probe,and established a simple and sensitive a-glucosidase activity assay and it's inhibitor screening method.4-nitrophenyl-a-D-glucopyranoside can be catalyzed by a-glucosidase and producted p-nitrophenol which is a good electron acceptor.P-nitrophenol can cause the fluorescence quenching of SiQDs via electron transfer process.By monitoring the relationship between the fluorescence quenching rate of SiQDs and the content of p-nitrophenol,we can implement a-glucosidase activity assay and its inhibitor screening.The SiQDs is ease of synthesis and don't need to be modified,so this method is easy to operate.The linear range of the a-glucosidase activity assay was 1-60 mU mL-1 with a limit of detection(LOD)of 0.35 mU mL-1(S/N=3).The IC50 values of two common inhibitors(acarbose and 2,4,6-tribromophenol)were 564.8 ?M and 22.32 ?M,respectively.The experimental results show that we build a rapid,simple and sensitive method for detecting a-glucosidase activity and it was successfully applied to detecting the activity of a-glucosidase in human serum samples.Moreover,it was expected to be used for screening the inhibitors of a-glucosidase.