Construction And Applications Of Phosphatase Activity Detection Methods Based On Nanozyme Catalysis

As an important functional enzyme in human body,phosphatase is involved in many biological processes from metabolism to signal transduction.Monitoring the physiological activity of phosphatase is of great significance for the prevention,diagnosis and treatment of diseases.Although traditional instrumental methods are accurate,they rely on expensive equipment and require professional operation,which cannot meet the need of convenient detection.In recent years,nanozyme-based analytical methods have drawn great attention in the field of clinical diagnosis due to their low cost,easy operation and high stability.They have a broad application prospect in clinical diagnosis,biochemical analysis,environmental monitoring,food safety and other areas.In this thesis,we have realized the effective analysis of phosphatase activity by designing and constructing several nanozyme catalyzed systems and developing a variety of convenient detection methods.Specific research contents are listed as follows:(1)Construction and application of a fluorescence detection method based on bifunctional MIL-53(Fe)peroxidase for alkaline phosphatase(ALP)activity.The developed MIL-53(Fe)exhibits good peroxidase-like activity to catalytically decompose H₂O₂ into hydroxyl radicals that have strong oxidizing capacity,stimulating the fluorescence of MIL-53(Fe)itself;because PPi has a strong coordination with Fe³⁺in MIL-53(Fe),the former with a relatively large molecular structure can inhibit the nanozyme's activity via capping active Fe³⁺sites,thus leading to the self-fluorescence suppression of MIL-53(Fe);when ALP is added,it hydrolyzes PPi and restores the double functions of MIL-53(Fe),offering the self-fluorescence readout again.Based on this strategy,the detection range of ALP activity was 2?80 U/L,and the detection limit was 0.7 U/L.This proves that the MIL-53(Fe)nanozyme can be used for the detection of ALP activity in serum.(2)Construction and application of a colorimetric assay based on CeVO₄ oxidase for alkaline phosphatase(ALP)activity.The developed CeVO₄ nanoparticles exhibit good oxidase-mimicking catalytic ability to promote the oxidation of 3,3?,5,5?-tetramethylbenzidine(TMB)to TMBox,triggering a prominent change of the solution color from colorless to blue;when HMPi as an ALP enzymatic substrate is added into the solution,the strong electrostatic interaction between the positively charged product TMBox and the negatively charged HMPi can result in the rapid aggregation of the former,forming a deep purple HMPi-TMBox agglomerate.After the agglomerate is filtered out,the reaction solution turns to be almost colorless;if ALP is applied to hydrolyze HMPi into phosphate ions(Pi)in advance,the aggregation process is restrained significantly,and such that the blue color of the oxidase-like CeVO₄catalyzed TMB system is well retained.On the basis of this new sensing strategy,colorimetric analysis of ALP activity was realized with the detection range of 1?210U/L and the detection limit as low as 0.68 U/L.This protocol can also accurately and rapidly detect ALP activity in serum samples,which proves that it has great potential in the clinical diagnosis of ALP activity.(3)Construction and application of a one-pot double-signal amplification electrochemical detection method based on Mn₃O₄ oxidase for acid phosphatase(ACP)activity.Mn₃O₄ microspheres are prepared and exhibit good oxidase-mimicking catalytic activity to trigger the oxidation of TMB to its corresponding product TMBox,which gives two remarkable peaks when it is re-reduced to TMB recorded by an integrated electrochemical system composed of screen-printed electrode(SPE)and 3D-printed holder.When HMPi as an ACP enzymatic substrate is added,the strong electrostatic interaction between positively charged TMBox and negatively charged HMPi leads to the rapid formation of the flocculent precipitate TMBox-HMPi,which will automatically sedimentate onto SPE surface.In this case,both the decreased amount of free TMBox in solution and the increased electrode surface passivation induced by TMBox-HMPi sedimentation significantly reduce the re-reduction signals of TMBox.If ACP exists,it can hydrolyze the HMPi substrate to Pi,and such that both the formation of TMBox-HMPi and its sedimentation onto electrode surface are dramatically relieved.As a consequence,the re-reduction peaks of TMBox become remarkable again.Based on this strategy,we constructed a one-pot double-signal amplification electrochemical detection method for ACP activity analysis.The detection range of ACP activity was 0.05?50 U/L,and the detection limit was as low as0.024 U/L,which proves the reliability of this method for ACP activity detection in clinical serum samples.