The Study Of Preparation And Sensing About Metal/Carbon Nanomaterial Composite Nano-enzyme

Biological enzymes play a vital catalytic role in people’s lives.Scientists have been committed to applying enzymes to production.However,natural enzymes are not only expensive,but also cannot be produced in large quantities,and the reuse rate is low.Nanoenzymes have gradually become substitutes for natural enzymes in scientific research due to their advantages of stability,economy,high efficiency and controllability.Among them,carbon-based nanozymes have become a research hotspot in recent years due to their excellent geometry,good meterial compatibility and tunable enzyme activity.But the active sites of pure carbon-based nanozymes are not clear,and the enzyme-like activity is not high.Most of the preparation requires the participation of high temperature and high pressure environment,which limits the further production and application of carbon-based nanozymes.In order to solve this difficulty,in this paper,we prepared a series of metal-C₃N₄novel carbon-based nanozymes by growing metal nanoparticles on the surface of graphite carbon nitride by self-deposition method without high temperature and high pressure environment.On the basis of low noble metal usage,the catalytic activity of C₃N₄nanozyme was greatly improved,and it was successfully applied to biological analysis.The sensing systems for sensitive detection of ascorbic acid concentrationand alkaline phosphatase activity in human serum,evaluation of total antioxidant activity in human saliva,and concentration of thiol amino acids were designed.The details are as follows:1.Ruthenium nanoparticles-carbon nitride nanozyme（Ru-C₃N₄）prepared by self-deposition of ultra-small ruthenium nanoparticles on carbon nitride has high peroxidase mimic enzyme activity and can be used for sensitive colorimetric detection of ascorbic acid and alkaline phosphatase.Alkaline phosphatase plays an important role in phosphate metabolism.The content of alkaline phosphatase in human serum is related to the diagnosis of many diseases.Therefore,it is necessary to develop a flexible,convenient,efficient and low-cost alkaline phosphatase detection method.The mechanism of detecting alkaline phosphatase activity using Ru-C₃N₄as a peroxidase mimetic enzyme is as follows:Ru-C₃N₄can produce strong oxidizing singlet oxygen by decomposing H₂O₂,oxidize colorless TMB to blue oxidized TMB（ox TMB）and produce a strong ultraviolet absorption peak at 652 nm to record the ultraviolet absorption peak at 652 nm by ultraviolet spectrophotometer.Alkaline phosphatase can incubate L-ascorbic acid-2-phosphate（AAP）to produce ascorbic acid.Ascorbic acid has the reducibility of reducing blue ox TMB to colorless TMB.On this basis,the concentration of AAP is fixed,and the activity of alkaline phosphatase can be changed to obtain different concentrations of ascorbic acid.The obtained ascorbic acid is added to change the absorbance of the system.Based on this,the activity of alkaline phosphatase is detected by ultraviolet-visible absorption method.2.To verify whether carbon nitride can reduce other similar metal ions to prepare nanozymes by similar principles,we prepared Pd-C₃N₄nanozymes with peroxidase-like activity by self-deposition of palladium nanoparticles on carbon nitride for the detection of total saliva resistance.Oxidative stress in the human body can cause changes in the redox balance in the human body,which is reflected in the total antioxidant capacity（TAC）in the human body.Total antioxidant capacity,the sum of enzymatic and non-enzymatic antioxidants in the body,is an important biological analysis parameter.The level of TAC in human saliva also reflects the level of TAC in other body fluids.In this work,palladium nanoparticles-carbon nitride（Pd-C₃N₄）was prepared by self-deposition for the first time.Compared with other synthesis methods of carbon nitride-palladium composites,this method does not require the participation of high temperature and high pressure environment,and the synthesis steps are simple and convenient.The detection method is similar to the previous work.After studying the optimal reaction conditions and kinetic tests,the peroxidase-like activity of Pd-C₃N₄was evaluated by Pd-C₃N₄-H₂O₂-TMB system with UV absorbance as the standard.The free radical scavenging experiment proved that the free radicals obtained by the reaction of Pd-C₃N₄with H₂O₂were superoxide radicals and singlet oxygen.Then,the common antioxidant substances in saliva such as glutathione,cysteine,ascorbic acid,uric acid and typical sulfhydryl antioxidant potassium thiocyanate were detected,and a good linear range was obtained respectively.On this basis,based on the total saliva resistance of normal people,the visual evaluation of total saliva resistance level was realized by using mobile phone APP.3.Pt-C₃N₄ nanozyme with peroxidase-like activity was prepared by self-deposition of platinum nanoparticles on graphite carbon nitride to detect the concentration of mercaptoaminoacid in organisms.In the above work,glutathione,cysteine and other sulfhydryl antioxidants in the human body were detected.Unlike reducing substances such as ascorbic acid,which rely on electron transfer mechanisms,the antioxidant capacity of antioxidants such as cysteine is derived from the hydrogen atom exchange mechanism,which inhibits oxidation by limiting the ability of the active site of the material to produce free radicals.When the concentration of thiol compounds in the system gradually increases,the ability to inhibit the active site does not increase linearly.However,in some of the previously reported detection processes,the absorbance and the concentration of the compound are often inaccurate due to excessive errors.The approximate linear relationship has played a misleading role in the study of nanozymes.Therefore,it is necessary to update a detection method for thiol compounds.In this work,for the first time,we prepared Pt-C₃N₄nanomaterials with high peroxidase mimic enzyme activity by combining Pt nanoparticles with graphite phase carbon nitride by self-deposition at room temperature.We studied the optimal reaction conditions,kinetic tests,and used them to detect mercaptoaminoacid.A linear system based on the correspondence between the UV absorbance and the reciprocal of the concentration of thiol compounds was established.It provides a new idea for the detection and calculation of thiol amino acids in organisms.