Study On Novel Visual Detection Methods For Disease-associated Proteins

Protein is the main bearer of human life activities and has a variety of physiological functions.The abnormal expression of certain proteins can reflect a variety of diseases in the human body.Thus,accurate detection of disease-associated proteins in the human body is of great significance for disease prevention.At present,traditional protein detection methods are more accurate,but rely heavily on large instruments and professional operators,and cannot meet the requirements for convenient detection.The visual detection method based on nanozyme catalysis has many advantages such as low cost,simple operation and high sensitivity,and has broad development potential in the field of biochemical analysis.Thus,this study constructed three new visual detection methods to detect disease-associated proteins.The main research contents are as follows:（1）A visual and quantitative detection platform for alkaline phosphatase was constructed based on the colorimetric reaction of Fe³⁺and 2-phosphate ascorbate.The research in this chapter found that Fe³⁺can specifically coordinate with the colorless substrate 2-ascorbate phosphate（AAP）to form a brown Fe3⁺-AAP complex with a specific UV absorption peak（472 nm）.When alkaline phosphatase（ALP）is added,ALP can hydrolyze the AAP substrate to form ascorbic acid（AA）and phosphate ion（Pi）,and cannot react with Fe³⁺in colorimetric.Based on this principle,this chapter realizes the colorimetric detection of ALP with high sensitivity and selectivity.The linear range is 0.8～80 U/L,and the detection limit is as low as 0.66 U/L.It has been successfully applied to the quantitative detection of ALP in serum.（2）A visual and quantitative detection platform for alkaline phosphatase was constructed based on Mn O₂ microsphere oxidase.In this chapter,the detection sensitivity of ALP is improved by introducing the nanozyme catalytic system to amplify the detection signal.First,synthesized Mn O₂ microspheres with good oxidase-like activity,which can catalyze the oxidation of colorless 3,3’,5,5’-tetramethylbenzidine（TMB）to form blue 3,3’,5,5’-Tetramethylbenzidine oxide（TMBox）.When ALP is added,it can hydrolyze AAP to generate AA and reduce Mn O₂ microspheres to Mn²⁺.At this time,Mn²⁺has no oxidase-like activity and cannot catalyze the TMB colorimetric reaction.Based on this strategy,highly sensitive and selective colorimetric detection of ALP can be achieved,with a detection range of 0.5-120 U/L and a detection limit as low as 0.42 U/L,and it has been successfully applied to the quantitative detection of ALP in serum.（3）Based on Zr-MOF peroxidase to construct phosphorylated protein quantitative detection and discrimination colorimetric sensor array.This chapter introduces Principal Component Analysis（PCA）to distinguish phosphorylated proteins associated with multiple diseases.Firstly,the Zr-MOF with good peroxidase-like activity was prepared,which can catalyze the TMB colorimetric reaction in the presence of H₂O₂.At the same time,the Zr in Zr-MOF can specifically recognize phosphate groups in proteins.When phosphorylated protein is added,phosphorylated protein can bind Zr-MOF and inhibit its peroxidase-like activity.Based on this principle,a high-efficiency quantitative detection ofα-casein（α-CS）is realized,with the detection range of 0.17～0.5μg/m L,and the detection limit as low as 0.16μg/m L.In addition,this chapter also uses PCA to accurately distinguish between phosphorylated and non-phosphorylated proteins.