Construction Of Novel Colorimetric Methods For ALP And TYR Detection In Serm

Natural enzymes,important catalysts in organisms,control many enzymatic reactions in organisms and maintain the vital activities of organisms,and their content can be used as an important reference for the diagnosis of many diseases.Therefore,it is of great interest to develop methods that can efficiently and accurately detect natural enzyme content in real samples.On the basis of simple and green preparation of sensing materials,two new colorimetric analysis methods are constructed,which are successfully applied for the detection of alkaline phosphatase(ALP)and tyrosinase(TYR)in real samples,and the results are satisfactory.The main research contents are as follows:1.Photometric analysis method is widely used in practical clinical trials because of its simple operation,low cost and convenience.So far,the reported ALP colorimetric analysis is mostly a non-ratiometric analysis established based on changes in absorbance and concentration at a single wavelength,lacking an internal reference and often limited by the problem of changes in the surrounding microenvironment.The development of new colortric analysis is important for high precision quantitative determination.The ratio-type colorimetric analysis uses the ratio to provide internal self-calibration to correct for the effects of various interference factors and to increase the measured dynamic range of the colorimetric analysis.However,the difficulty of constructing the ratio color analysis method is whether the absorbance of the target changes significantly.On the basis of the previous work of the laboratory,an AA ratio color analysis method is constructed by using the color change of S(alizarin Red S,ARS)and Cu²⁺and the redox reaction between ascorbic Acid,AA)and Cu²⁺.based on AA hydrolysis of the ALP catalytic substrate2-phosphate-2-phospho-L-ascorbic acid acid(AAP),this method can be further used to detect ALP.Compared with most of the reported ALP colorimetric analysis methods,this method has the advantages of low detection limit,simplicity,fast and green.This method has been successfully applied to detect the amount of ALP in serum with satisfactory results.The establishment of this method provides a new method for the accurate detection of ALP in serum and for other ratio methods.2.Analysis methods based on enzymatic reaction construction have been widely used,but natural enzymes have the disadvantages of complex preparation and purification,high cost,and difficult recovery.To overcome the above shortcomings,artificial enzymes have been developed.Nanoenzymes refer to nanomaterials with natural enzyme activity,which have the unique advantages of high activity,good stability,low cost,easy amplification and modification.But at present,nanoenzymes still have disadvantages such as unclear catalytic mechanism,low activity and poor selectivity.In view of the above shortcomings,this paper successfully designed and prepared a series of layered double hydroxide(LDH),examined the enzyme activity and influencing factors,found that the synthetic LDH has horseradish peroxidase(HRP)activity,and through the Ni doping and chemical modification significantly enhanced its HRP enzyme activity.Cu Ni/Fe LDH has enhanced HRP activity,and dopamine(DA)can significantly enhance the HRP activity of Cu Ni/Fe LDH.based on the phenomenon that DA and Ni doping can significantly enhance Cu/Fe LDH class HRP activity,a new method for DA colorimetric analysis is constructed and successfully applied to detect the content of DA in artificial CSF with satisfactory results.In addition,based on DA can be catalyzed by TYR into dopaquinone,inhibiting the HRP activity of Cu Ni/Fe LDH,this method was successfully applied to detect TYR in serum.Compared with previous reported analysis methods,this method has the advantages of low cost,high sensitivity and simple operation,which provides new experience for the preparation of LDH-based nanoenzyme and the regulation of its enzyme activity,makes certain contributions to the better understanding and application of nanoenzymes,and provides reference for the development of other enzyme materials.The establishment of this method provides a new method for the highly sensitive detection of TYR in serum,and also provides some meaningful references for constructing other new colorimetric analysis methods based on nanoenzymes.