Study On Detection Methods Of Alkaline Phosphatase Activity Based On Nanozyme Catalysis

Considering that alkaline phosphatase?ALP?plays a vital role in disease early warning and dephosphorylation-related cellular regulation,it is widely considered as an important biomarker for clinical diagnosis.Therefore,there is an urgent need to develop effective and simple methods to monitor ALP activity.Based on this,this paper applies the emerging nanozyme catalysis to the detection of ALP activity,and the detailed contents are as follows:1.we propose a novel sensing strategy for ALP activity based on the hexametaphosphate ion?HMPi?-involved 3,3'-5,5'-tetramethylbenzidine?TMB?color reaction catalyzed by sulfuration-engineered Co O_xderived from ZIF-67.After sulfuration engineering,the Co O_xcomposite originating from the pyrolysis of ZIF-67 can exhibit enhanced peroxidase-mimicking catalytic activity to trigger the oxidation of TMB to its oxide TMBox,giving a remarkable color change from colorless to deep blue;with the presence of HMPi,the rapid electrostatic assembly of negatively charged HMPi and positively charged TMBox leads to the aggregation of the latter,resulting in a color fading phenomenon of the reaction solution after filtration;when ALP is added to hydrolyze the HMPi mediator,the aggregation procedure is significantly suppressed,and such that the solution color can be recovered.Based on this new principle,high-performance detection of ALP activity was gained,providing a wide linear range from 0.8 to 320 U·L^-1and a limit of detection as low as 0.38 U·L^-1.Reliable determination of the target in serum samples was also achieved,verifying the feasibility and practicability of our strategy in measuring ALP activity for clinical applications.2.we report a new colorimetric assay of ALP activity based on the enzyme-triggered in situ formation of Ag nanoparticles?NPs?with oxidase-mimicking activity.ALP can hydrolyze the ascorbic acid phosphate?AAP?substrate to produce ascorbic acid?AA?;the produced AA with strong reducing capacity can further transform Ag⁺into Ag NPs;in comparison with the Ag⁺precursor,the in-situ formed Ag NPs show much higher oxidase-like activity to catalyze the 3,3'-5,5'-tetramethylbenzidine?TMB?color reaction mediated by dissolved O₂at neutral p H.With the above principle,amplified colorimetric detection of ALP activity with high sensitivity was obtained,providing a linear scope of0.15?5 U·L^-1and a limit of detection down to 0.037 U·L^-1.In addition,our developed assay exhibited specific response toward ALP against other biological enzymes and species.Accurate and reliable determination of ALP activity in human plasma was also verified by our assay,suggesting its great potential as a facile and powerful tool for monitoring of ALP activity in clinical practice.3.we report a new strategy based on the target-induced valence state regulation of oxidase-mimicking Ce-based nanorods for ALP activity sensing.The mixed-valent Ce-based material?MVCM?with a relatively high Ce???/Ce???ratio can exhibit good oxidase-mimicking activity to trigger the catalytic oxidation of3,3'-5,5'-tetramethylbenzidine?TMB?to TMBox with the participation of O₂,resulting in a notable chromogenic reaction.When ALP catalyzes the hydrolysis of ascorbic acid phosphate to ascorbic acid?AA?,the formed AA induces the partial reduction of the MVCM to one with a low Ce???/Ce???ratio,which shows much less activity to trigger the chromogenic reaction.According to the above principle,a facile colorimetric assay was developed for ALP activity detection,providing a linear scope of 0.5?25 U·L^-1and a detection limit down to 0.1 U·L^-1.Besides,the proposed strategy could offer favorable selectivity for the determination of ALP activity.Accurate monitoring of the target in human serum samples was confirmed by our assay as well,indicating its potential as a reliable tool for clinical diagnosis.4.we proposed a novel and facile colorimetric assay based on phosphate anion-quenched oxidase-mimicking activity of Ce???ions for sensitive and selective detection of ALP activity.Free Ce???ions exhibited a strong oxidase-like capability to catalyze the oxidation of colorless 3,3'-5,5'-tetramethylbenzidine?TMB?into its blue product TMBox mediated by dissolved O₂at neutral p H,thus triggering a remarkable color reaction visually.When PO₄^3-was added,its strong affi nity to Ce???ions rapidly precipitated these free Ce???ions,resulting in the quenching of their enzymatic ability.Given ALP catalyzed the hydrolysis of adenosine triphosphate to produce PO₄^3-,determination of ALP activity could be achieved using the colorimetric assay,with no need of complicated instrumentation and protocol.As demonstrated,our assay offered a highly sensitive readout for ALP activity in linear scopes of 0?50 U·L^-1and 50?250 U·L^-1,providing a detection limit down to 2.3 U·L^-1.Furthermore,the developed assay was successfully applied to evaluate the ALP activity in human plasma accurately and reliably,indicating its great promise as a powerful and convenient tool for monitoring of ALP activity in clinical practice.