Multi-Enzyme Activities Exploration Of Copper(?)-Aspartate Nanozymes And Their Application In Juice Analysis

Nanomaterials are nanomaterials that can mimic the catalytic activity of natural enzymes,which have the advantages of low cost,high stability and mass production compared to natural enzymes.Nanomaterials targeting a single enzyme-like activity have been reported extensively,but nanomaterials with multiple enzymatic activities are less studied and limited to mimic peroxidase,superoxide dismutase(SOD)and oxidase.Nanomaterials with multiple enzymatic functions that can catalyze different kinds of substrates are beneficial to expand their applications or further realize cascade catalytic reactions.Considering the abundance and low price of copper,which has excellent properties comparable to those of precious metals,and many natural metalloenzymes contain copper in their catalytically active centers.Therefore,in this paper,we expect to explore a simple preparation method of copper-based nanomaterials with multi-enzyme activity and apply them to juice analysis.In this paper,a colorimetric/fluorescence sensing platform was constructed for the detection of target analytes in fruit juices by combining the multi-enzyme activities of copper(II)-aspartate nanozymes with a colorimetric/fluorescence method.And the fruit juices were successfully distinguished and identified by combining sensor array techniques.The main work is as follows:(1)A fibrous nanomaterial(CuAsp nanofibers)was formed based on the ligand selfassembly of copper and L-aspartate,and it was demonstrated for the first time that CuAsp nanofibers can mimic the catalytic activities of peroxidase,laccase,catalase,ascorbate oxidase(AAO),and SOD.XPS analysis showed that CuAsp nanofibers contain both Cu2+ and Cu+,and the simultaneous presence of mixed oxidation valence copper may be the main reason for the multi-enzyme activity of CuAsp.The peroxidase-like activity of CuAsp nanofibers is mainly derived from their catalytic generation of hydroxyl radicals from H2O2.In addition,it was found that the simulated activities of peroxidase and catalase of CuAsp nanofibers could be modulated by p H without interference.Kinetic studies showed that the synthesized CuAsp nanofibers have good substrate affinity and catalytic reaction rates.(2)A colorimetric sensor based on the peroxidase-like activity of CuAsp nanofibers was developed for the detection of total antioxidant capacity of fruit juices.Using 3,3',5,5'-tetramethylbenzidine(TMB)as the substrate,CuAsp nanofibers oxidized colorless TMB to blue ox TMB in the presence of H2O2.The introduction of antioxidants such as reduced glutathione(GSH),ascorbic acid(AA)and L-cysteine(Cys)will inhibit the above-mentioned color reactions to achieve the quantitative detection of antioxidants.This method could be applied successfully to determine the total antioxidant capacity of different commercial juices with high accuracy of results.(3)Based on CuAsp nanofibers mimicking peroxidase activity,three typical peroxidase chromogenic substrates TMB,2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)and o-phenylenediamine(OPD)were catalyzed to produce ox TMB,ox ABTS and ox OPD,respectively.A three-channel colorimetric sensor array was constructed for the differentiation of antioxidants using these three different colors of oxidation products as sensing probes.The type and amount of antioxidants contained in fruit juices vary depending on the taste,origin of raw materials and processing methods.Therefore,the colorimetric sensing array is used to further differentiate and identify different flavors,brands and types of juices.(4)A ratiometric fluorescence sensing platform was designed for the detection of AA based on the AAO-like and laccase-like activities of CuAsp nanofibers.OPD can be oxidized by the laccase-like activity of CuAsp to generate ox OPD with yellow fluorescence,when AA is added it is oxidized by the AAO-like activity of CuAsp to generate dehydroascorbic acid(DHAA),DHAA will couple with OPD to generate a blue fluorescent product,thus hindering the formation of ox OPD.The method can selectively detect AA with a linear detection range of 1-220 ?M and a detection limit of 0.32 ?M with good sensitivity.In addition,it is possible to visualize and detect AA based on smartphones and changes in fluorescence color under UV lights,which in turn enables on-site analysis.This ratiometric fluorescence sensing platform can also be used for AA detection in real fruit juices and has potential in practical applications.(5)Detection and removal of phenolic compounds from fruit juices based on the laccaselike activity of CuAsp nanofibers.A colorimetric sensor was developed for the detection of catechin using the principle that CuAsp can oxidize catechin to yellow products and generate colorimetric signals.The linear range of the colorimetric sensor was 20-1200 ?M,and the detection limit was 5.88 ?M with good anti-interference property.By investigating the catalytic performance of CuAsp nanofibers,the results showed that the stability of the nanozyme in terms of p H,temperature and storage time was better than that of natural laccase.In addition,CuAsp nanofibers could be recovered by centrifugation and could maintain above80% of the original activity after 10 consecutive cycles.The nanozyme can be used to remove phenolic compounds from actual juice samples with better removal results than natural laccase.The CuAsp nanofibers were still effective in removing phenolic compounds from fruit juices under high temperature conditions.