Metal Nanomaterials For Enzyme Activity Detection And Screening Of Enzyme Inhibitor

Research have shown that abnormal activity of some enzymes leads to dysfunction and disease.Regulation of enzyme activity can treat and cure disease.Therefore,searching for new molecules inhibited the activity of enzyme and establishment of new method to detect enzyme activity have significant scientific values.Metal nanoclusters,as a new type of nanomaterials,are widely used in the field of biomedicine because of low toxicity,good light stability,and long Storck shift.Based on these excellent properties,a series of fluorescent nanosensors have been constructed for monitoring of three hydrolase enzymes and screening of inhibitors.The main research is briefly summarized as follows:（1）α-Glucosidase（α-Glu）is closely related to the occurrence of diabetes,and it has been applied as biomarker and therapeutic target in clinical diagnosis.4-nitrophenyl-α-D-glucopyranoside（NGP）is selected as the substrate,which is further hydrolyzed to yield 4-nitrophenol（4-NP）through the catalysis ofα-Glu.The quenching efficiency is positively correlated to the concentration ofα-Glu.Furthermore,the inhibitory effects of the extracts from four Chinese herbal medicines（i.e.,the rind of Punica granatum L.,Momordica grosvenorii Swingle.,Crataegus pinnatifida Bge.and Lycium barbarum L.）on theα-Glu activity have been studied.Last but not the least,by immobilizing DT-Cu NCs into agarose hydrogels in polyethylene tubes,a visual device is fabricated to screenα-Glu inhibitors with high throughput and sensitivity.（2）The biochemical indicators of trypsin are used in the clinical diagnosis of acute pancreatitis and other diseases.At present,detection methods that use macromolecular proteins as substrates has potential problems,such as poor selectivity.The reason is thatα-helices andβ-sheets in the secondary structures of proteins can prevent trypsin to efficiently hydrolyze proteins,resulting in poor sensitivity.Herein,a novel peptidyl probe is first synthesized by the amide reaction between glutathione-capped gold nanoclusters（GSH-Au NCs）and peptide RRRRRRRRR（Arg₉）,and it is called Arg₉-GSH-Au NCs.Polyuridylic acid（poly U）,as a new trigger,is firstly used to induce the high aggregation-induced emission enhancement（AEE）of positive charged Arg₉-GSH-Au NCs through electrostatic attraction.Due to the hydrolysis of Arg₉ by trypsin,the peptide bond on the carboxyl terminal of the arginine residues is cleaved.Hence,poly U is hard to induce the aggregation of peptidyl probe again,and the AEE from GSH-Au NCs becomes weak.It is an ideal platform to detect trypsin and screen trypsin inhibitors.A good linear range of trypsin is obtained from 1 to 50 ng·m L^-1 with the limit of detection（LOD）0.31 ng·m L^-1。Finally,the proposed method is applied to screen out the best trypsin inhibitors from various types of soybeans,indicating that the biosensor has great potential in bioanalysis and new drug development.（3）Alkaline phosphatase（ALP）plays an important role in cell signal transmission and protein activity regulation.In this paper,the fluorescent copper nanoclusters（Cu NCs）templated by G-quadruplex（G4）were first synthesized,and the label-free fluorescent probe（G4-Cu NCs）is utilized to determine ALP.When adenosine triphosphate（ATP）is added,the interaction between Cu²⁺and ATP results in the fluorescence quenching of G4-Cu NCs.The addition of ALP triggers the hydrolysis of ATP,so the fluorescence intensity of G4-Cu NCs recovers gradually.This novel system enables the specific detection of ALP with the wide linear range of 10-500U/L,respectively,and the corresponding limits of detection as low as 2.8 U/L for ALP.Moreover,this present system has been successfully applied for the detection of ALP in human serum samples with satisfactory recoveries.