Research On Sensing Of Microorganisms And Their Metabolites Based On Functionalized Nanozyme

Diseases caused by microorganisms is a great burden of public health,seriously threatening people’s health and life,and hindering economic development worldwide.The traditional detection method is time-consuming,requires complex and expensive equipment,has low sensitivity and is not suitable for field detection.In order to meet the challenge posed by harmful microorganisms and reduce the impact on human health,it is essential to effectively and rapidly identify pathogens.The detection of pathogens and metabolites is constructive to develop better diagnostics or treatments,so the biosensor emerges as The Times require.Its advantages are short detection time,fast analysis speed,portability,and no need for complex equipment and operation.In addition,it can combine a variety of technologies to obtain broad prospects in the detection of bacteria and its metabolites.Nanozyme is a kind of artificial enzyme,which is a kind of nano-material with enzyme simulation activity.It makes up for the physical and chemical instability,low durability and high cost of natural enzyme,which has aroused great interest of people.Combined with its physical and chemical properties,it can show advantages in many fields such as clinical diagnosis,environmental monitoring and wastewater treatment.Nanozymes with catalase activity can catalyze hydrogen peroxide（H₂O₂）to produce hydroxyl radical（·OH）,which can oxidize 5,5’-tetramethylbenzidine（TMB）and the product shows blue color.Accordingly,nano-materials with catalase activity can be used as colorimetric probes in the field of biosensing,showing great application value.Colorimetric method has attracted much attention due to its portability,simplicity and rapidity.The problem of colorimetric detection is poor specificity and low sensitivity.The regulation of enzyme activity of nanozymes provides the possibility to explore a more efficient colorimetric probe.In this paper,a functionalized iron trioxide（Fe₂O₃）nanozyme was prepared for selective detection of endotoxin.In order to further detect bacteria more sensitively,iron molybdenum bimetallic nanozyme（Fe₂Mo O₄）was prepared and was functionalized by nucleic acid to construct a sensing platform to detect Pseudomonas aeruginosa.1.In order to develop nanomaterials with good catalytic activity and strong specificity,we use a simple one-pot hydrothermal method to prepare 3-aminophenylboric acid functionalized nanomaterials with high peroxidase-like catalytic activity.The prepared nanomaterial has rough surface providing more substrate binding sites;the addition of 3-aminophenylboric acid makes the material doped with N and B elements,which is conducive to the enhancement of catalytic activity,and makes it binding to endotoxin specifically at the same time.The enzymatic kinetics study results showed that functionalized Fe₂O₃ had stronger affinity for substrates and higher catalytic efficiency compared with HRP and other iron-based peroxide mimics.The selective binding of endotoxin and functionalized Fe₂O₃ makes catalytic sites of nanozyme covered,resulting in less TMB oxidation and lighter solution color.Signal changes based on absorbance extraction can be used for selective and sensitive detection of endotoxin.Functionalized Fe₂O₃ with peroxidase-like activity can be used as the colorimetric probe.This sensor has good performance in the simulation analysis of beer,5%glucose injection and human health serum has practical application value.2.In order to further detect bacteria more sensitively,it is necessary to prepare more efficient colorimetric probes and combine signal amplification strategies to build biosensor.Bimetal nanozyme can improve the catalytic activity of enzyme like greatly;hybridization chain reaction（HCR）has the advantages of high amplification efficiency,isothermal conditions,simple procedure and no enzyme reaction.It is a better method to replace the traditional polymerase chain reaction（PCR）.We prepared a series of Fe-Mo bimetallic nanozymes by hydrothermal method by changing the addition amount of sodium citrate.Meanwhile,it was found that with the increase of Fe/Mo,the enzyme-like activity decreased by the analysis of the results of their catalytic activity and element content.Then the mechanism of the difference in enzyme activity was explored,sodium citrate,as a reducing agent and stabilizer,could affect the catalytic activity by adjusting the composition and morphology.The bimetallic Fe₂Mo O₄ nanozyme with excellent peroxidase-like activity was selected as colorimetric probe combined with HCR to build biosensor to detect Pseudomonas aeruginosa gene.The activity of the nanozyme modified by nucleic acid decreased,which was caused by catalytic active site of Fe₂Mo O₄ nanozyme being covered and the competitive binding of HCR products and Fe₂Mo O₄ to TMB.Those lead to the shallow solution and reduced absorbance after triggering HCR.HCR can achieve selective and amplify the change of absorbance,which can be used as a signal to detect Pseudomonas aeruginosa gene specifically and sensitively.In addition,the proposed method was tested in the simulation analysis of serum samples,and the good recovery rate proved that the strategy has a broad prospect in biochemical analysis and clinical application.