Preparation,Characterization Of Functional Nanoenzymes With Multi-enzyme Mimetic Activities And Their Application In Point-of-care Testing

Recently,by virtue of their excellent enzyme mimetic activities,high catalytic activity,good stability and low cost,nanozymes have attracted extensive attention,and were applied in biosensing,nanomedicine,tumor therapy,biocatalysis and environmental remediation.Although nanoenzymes have entered a stage of diverse preparation methods,diverse mimetic enzyme types,and diverse applications.However,there are still problems such as single mimetic enzyme activity,complex application processes and low catalytic efficiency,which have prompted hundreds of research groups worldwide to investigate nanoenzymes in greater depth.Compared with single enzyme-like nanoenzymes,nanoenzymes with multi-enzyme mimetic activities have higher catalytic efficiency and environmental response selectivity.Furthermore,the synergistic effect and cascade reaction between enzymes and enzymes enable nanoenzymes with multi-enzyme mimetic activities more suitable for application in complex living systems.Moreover,in analytical sensing,nanoenzymes with multi-enzyme mimetic activities can not only simplify the process but also shorten the sensing time.As we know that ASSURED is now the standard for the development of biosensing systems,the Point-of-care testing（POCT）technology is favored for its simple and rapid application in routine large-scale high-throughput assays.Moreover,testing equipment with simple operation,economy and miniaturization,especially in resource-poor areas to deal with large-scale testing is extremely effective.In recent years,studies have shown that the combination of nanoenzymes and POCT is a comprehensive solution.Since nanoenzymes have similar chemical properties to natural enzymes,they are able to catalyze a series of specific chemical or biochemical reactions that produce specific output signals.Moreover,the clear robustness,low cost and high stability for field applications make nanozymes more suitable as transduction components in POCT devices,demonstrating great potential in bionanotechnology,biosensing and biomedical applications.Based on the above,this thesis is dedicated to constructing nanoenzymes with multi-enzyme mimetic activities and combining them with POCT devices（portable digital thermometer and smartphone）for application in rapid,portable and ultra-sensitive biomolecules detection.（1）Glutathione（GSH）is an important endogenous antioxidant that maintains the proper functioning of the immune system at all times.Although there have been various ways to assay GSH to help us solve the problem,it is still not a sustainable solution because of the high cost of unportable equipment and facilities,complicated assay process and the need for professional operators.Inspired by this,this work prepared lyrium-like Cu₂O/CuO hierarchical microspheres（Cu₂O/CuO HMSs）with four enzyme mimetic activities by a simple green solvothermal strategy.The Cu₂O/CuO HMSs exhibited the mimetic properties of four enzymes,including peroxidase-like（POD-like）,laccase（Laccase-like）,superoxide dismutase（SOD-like）,and catalase-like（CAT-like）activities.Based on the POD-like activity of Cu₂O/CuO HMSs,a multi-signal response GSH sensing platform was designed.Since GSH can compete with 3,3′,5,5′-tetramethylbenzidine（TMB）to consume H₂O₂,which affects the absorbance,color and temperature signals of the detection system.Therefore,a three-mode sensing strategy was designed for the quantitative portable detection of GSH by combining a UV-spectrophotometer,a smartphone and a portable digital thermometer.The experimental results showed that the proposed sensing strategy exhibited good selectivity and reusability,and could be successfully applied to the detection of GSH in human serum samples.This work provides a new solution for the preparation of multi-enzyme mimics and also demonstrates that multi-enzyme mimics can serve as a paradigm for bio-portable sensing candidates.（2）The potential anticoagulant properties of heparin have led to its widespread use in clinical treatment.However,the imbalance in the amount of heparin application also causes great damage to the vital system.In order to protect the normal operation of living systems at all times,it is important to develop POCT biosensing technology for sensitive and specific detection of heparin.In this work,firstly,a double transition metal-based coordination polymer nanoflowers（CoNi CPNFs）were synthesized by a one-pot method.Au/CoNi CPNFs were formed by anchoring gold nanoparticles（Au NPs）with CoNi CPNFs as the support matrix.Surprisingly,Au/CoNi CPNFs exhibited four-fold independent enzymatic activities:oxidase-like（OXD-like）,ascorbate oxidase like（AAO-like）,laccase like（Laccase-like）,and superoxide dismutase like（SOD-like）activities.In addition,a three-mode portable sensing platform（colorimetric,photothermal,colorimetric）was constructed based on the oxidase-like activity of Au/CoNi CPNFs coupled with a UV-spectrophotometer,a portable digital thermometer,and a smartphone for the rapid on-site quantitative detection of heparin.The mechanism of the study showed that heparin could cause the color shift of the solution by inhibiting the oxidase-like activity of Au/CoNi CPNFs,and a blue-purple product was generated.More importantly,the constructed sensing platform achieved high sensitive and specific recognition of heparin.In conclusion,this work provides a good guide for the development and application of multifunctional nanoenzymes with multiple enzymatic activities.