The Preparation Of Sensors Based On Gold Nanorods,Polymer Dots And Their Application In Enzyme Activity Detection

Biosensor is a highly selective analytical system composed of biological recognition element and coupled to a chemical or physical transducer for biological,chemical analysis detection.Compared with the traditional complex and time-consuming detection methods,the new biosensor has the advantages of low cost,high selectivity,high sensitivity,speediness and miniaturization.It is widely used in environmental monitoring,food safety,disease diagnosis and other fields.Recent advancements of nanotechnology have greatly improve the tremendous achievements of biosensing.Due to the excellent optical,electrical and magnetic properties of nanomaterials,some biosensors based on the nanomaterials is more sensitive.Therefore,in this thesis,several biosensors were developed for the detection of enzyme activities and enzyme inhibitors screening based on gold nanorods（AuNRs）and polymer dots（PDs）,respectively.The specific work is as follows:1.In chapter 2,an ultrasensitive colorimetric biosensor forα-glucosidase sensing based on iodine-mediated etching of AuNRs was developed.In this method,2-O-α-D-glucopyranosyl-L-ascorbic acid（AA-2G）was used as an enzymatic substrate.The hydrolysate ascorbic acid in situ reduced KIO₃ to I₂,which subsequently etched the AuNRs.A blue-shift of the localized surface plasmon resonance（LSPR）absorption of AuNRs was clearly observed by UV-vis spectroscopy and reflected with visual color changes.The shift showed a good linear relationship with the concentration ofα-glucosidase during 2.5 U/L to 45 U/L and a lowα-glucosidase detection limit of 0.5U/L（S/N=3）was provided.This strategy was featured with low cost,simplicity,excellent sensitivity,high selectivity and successfully applied in theα-glucosidase inhibitor screening with Chinese Wolfberries as candidates.2.In chapter 3,the PDs with high stability,hypotoxicity,high quantum yield of53.6%were synthesized by a mild one-step method.The PDs were characterized by TEM,FT-IR spectroscopy,UV-Vis spectroscopy,fluorescent spectroscopy,XPS and XRD.The new type PDs fluorescent sensor was built for the detection of alkaline phosphatase（ALP）activity based on inner filter effect.P-Nitrophenylphosphate（PNPP）was chosen as the ALP substrate and could be hydrolyzed by ALP to give p-nitrophenol（PNP）,whose absorption overlapped the fluorescence excitation spectrum of the PDs.It resulted in a turn-off fluorescence signal due to inner filter effect.A detection limit of 0.01 U/L and a good linear ralationship with the ALP activity from 0.1 U/L to 20 U/L（R²=0.999）was obtained.The analysis of serum samples and inhibitors screening were also tested and a satisfactory result was achieved.The results suggested that our proposed sensing approach could be used for ALP activity detection in biochemical applications and for the screening of ALP inhibitors in drug discovery.3.In chapter 4,a novel,sensitive and selective PDs fluorescent sensing strategy based on inner filter effect was firstly developed for AFu activity determination.The detection mechanism of this system was based on the inner filter effect between PDs and p-nitrophenol（PNP）which was the hydrolysate of 4-nitrophenyl-α-L-fucopyranoside（PNPF）catalyzed by AFu.The absorption of PNP overlapped the fluorescence excitation spectrum of the PDs,which resulted in a fluorescence quenching or weakening of PDs.The sensing system showed a good linear relationship in 0.01 U/L to 0.9 U/L and provided a low AFu detection limit of 0.001 U/L（S/N=3）.It was successfully applied in the determination of AFu in human serum samples.Furthermore,a new chemical compound（AA-2βFuc）was successfully synthesized and showed good inhibitory effect to AFu activity.4.In chapter 5,a highly sensitive and selective PDs-based fluorescence sensor was firstly developed for sensitive detection of horseradish peroxidase（HRP）activity and glucose content by employing p-phenylenediamine（PPD）as the reaction substrate.HRP/hydrogen peroxide（H₂O₂）-catalyzed oxidation of PPD could result in the formation of colored Bandrowski’s base（PPDox）with a maximum absorption at 510 nm.The overlapping between the absorption of PPDox and the fluorescence excitation/emission spectra of the PDs resulted in a turn-off fluorescence signal due to inner filter effect.On the basis of converting glucose into H₂O₂ catalyzed by glucose oxidase（Gox）,the sensing system was further exploited for glucose assay.The sensing system was sensitive and demonstrated to give a detection limit of 0.001 ng/mL for HRP and 0.1μmol/L for glucose.Meanwhile,the sensing assay was successfully extended into the HRP-based fluorescent ELISA for Cry1Ab/Ac protein detection and rice leaves analysis.