Application Of Nanoenzymes In Removal Of Pollutants And Rapid Detection Of Biomolecules

As environmentally friendly catalysts,natural enzymes are greatly limited in practical application due to the disadvantages of high price,complicated preparation and purification,easy deactivation,poor recovery and difficult to reuse.Nanoenzymes can simulate the biocatalytic effect of natural enzymes and overcome the shortcomings of natural enzymes.Therefore,nanoenzymes are widely used in many fields,such as bacteriostatic,anti-infection,anti-cancer,early diagnosis of diseases and development of new drugs.Although there are a variety of functional materials prepared for nanoenzymes but with the disadvantages such as cumbersome steps,high energy consumption and consumption of a large amount of organic solvents.For improving the synthesis method,this project started with the concept of green,environmental protection,simple and efficient,to prepare a variety of nanoenzymes,to evaluate and expand their application.The details are as follows:(1)A kind of core-shell material with regular shape and size,and uniform composition,is prepared in aqueous phase and used for laccase immobilization.Magnetic Fe₃O₄nanoparticle is coated with MoS₂ by self-assembly,and it is further coated by polyethyleneimine,which acts as a spacer arm and is beneficial to enzyme immobilization.The nanocarrier affords the high loading capability of 120.0 mg/g and activity recovery of 90.02%for laccase.The immobilized laccase is stable in a wider range of p H(3.0-10.0),temperature(20-70°C)and storage time(1-14 days),as well as having the recyclability(1-10 cycles).The carcinogenic persistent organic pollutants(malachite green,bisphenol A,bisphenol F)in the water are degraded by the immobilized laccase with a higher efficiency(82.7%,87.6%,70.6%)than free laccase(14.4%,85.0%,48.0%),respectively.It is prospected that the constructed material is qualified as green nanoreactor for biological macromolecules immobilization.(2)A kind of supramolecular metal-organic coordination complex(SMOCC)functionalized Fe₃O₄@MoS₂ was prepared and used for enzyme immobilization.As possessing a protective nanocoating of PDA/PEI/Cu²⁺(polydopamine:PDA,polyethyleneimine:PEI),the proposed material can provide biocompatible microenvironment and flexible adhesion force on particle interface,which is conductive to loading enzyme(the upload capacity of laccase is 170.3 mg/g)with high activity(93.10%).Compared to the free laccase,the immobilized laccase has stability in a broader range of p H(3.0-10.0),temperature(20-80°C),storage time(1-18 days)and reusability(1-16 cycles).The removal of carcinogenic persistent organic pollutants(malachite green,bisphenol A,bisphenol F)in the water with the immobilized laccase shows a higher efficiency(89.4%,92.0%,76.0%,respectively)than free laccase(16.2%,88.5%,54.0%,respectively)under same conditions.The Fe₃O₄@MoS₂@(PDA/PEI/Cu²⁺)nanocomposites can also be used to immobilize lipase,trypsin and catalase successfully,showing a satisfactory enzyme loading(157.0 mg/g,151.6 mg/g,162.6 mg/g,respectively)and activity retention(95.1%,90.2%,91.2%,respectively).In view of the potential utilization as biocompatible materials,the fabricated SMOCC nanomaterial is a new strategy for preparing biocatalysts with high performance.(3)The peroxide-like catalytic activity of gold nanoclusters(Au-NCs)is very low under physiological conditions(p H 7.4),which greatly limits its biological detection applications.A new nanoenzyme platform was constructed by self-assembly of Au-NCs and ZIF-8/CQDs.It was found that heparin can significantly promote the peroxidase-like activity of Au-NCs on the nanoenzyme platform at p H 7.4.In the presence of H₂O₂,the catalytic activity of Au-NCs on the nanoenzyme platform for TMB increased nearly 50 times.Based on this phenomenon,a colorimetric method was developed to determine heparin in the range of 0.1-50?g/m L,with a detection limit of 0.012?M.When protamine is introduced,heparin and protamine take the lead in specific binding due to antagonism,which makes heparin unable to adsorb on the surface of ZIF-8/CQDS,thus inhibiting the enhancement of the catalytic activity of Au-NCs.Based on this phenomenon,a colorimetric method was developed to determine protamine in the range of 0.01-0.5?g/m L,with a detection limit of 0.01?g/m L.Therefore,this method provides a new idea for the visual detection of heparin and protamine under physiological conditions.(4)We successfully constructed a colorimetric and fluorescent dual-channel detection method for acid phosphatase(ACP)activity and its inhibitor screening based on the internal filtering effect between oxidized TMB(ox TMB)and rhodamine B(RB).Inspired by phenomenon of Au³⁺precursor in situ formed gold nanoparticles(Au NPs)can quickly and efficiently catalyze the oxidation from colorless3,3',5,5'-tetramethylbenzidine(TMB)to blue product(ox TMB)in the absence of H₂O₂,and the overlapping between emission of RB and absorption of ox TMB can lead to effectively quench the fluorescence emission of RB,the determination of ACP can be performed owing to that ACP can promote the hydrolysis of2-phospho-L-ascorbic acid trisodium salt(AAP)to generate ascorbic acid(AA),which can inhibit the internal filtering effect between RB and ox TMB by the reduction with ox TMB.Before the dual-channel detection for acid phosphatase activity,no material preparation was needed.The colorimetry and fluorescence methods can quantify ACP in the range of 0.06-5 m U/m L and 0.03-5 m U/m L,respectively.Furthermore,we constructed a smartphone-assisted sensing platform for on-site monitoring of ACP in the range of 0.75-50 m U/m L,and the detection limits can be 0.3 m U/m L.This study not only successfully established a simple method for the detection of ACP and the screening of ACP inhibitors,but also demonstrated a control strategy that converts color-based signal readout into colorimetric and fluorescence dual signal readout.