Rapid Modification Of Protein To Endow It With Peroxidase Activity And Construction Of New Biosensors

In this study,new strategy for protein modification has been proposed.Firstly,hemoglobin was selectively modified with tartaric acid to form tartaric acid hemoglobin complex,which significantly enhances the peroxidase mimetic activity of hemoglobin,the unique property of TA-Hb was employed to develop a rapid colorimetric assay for detecting Hb in human serum samples.Secondly,based on the coordination between carboxyl group and iron porphyrin(TPPFe),target proteins could be modified with TPPFe,to form a stable protein complexs with peroxidase-like properties.A new biosensor platform was thereby constructed by using the catalytic activity of the complex,which can realize simple,fast and effective protein detection.In order to verify the universality of this method,we detected the proteins related to diabetes and got reliable results.Finally,TPPFe was used to modify the glucose oxidase to form a complex with dual enzyme activities.The one-step colorimetric detection of glucose concentration in blood was achieved,and the dual enzyme was applied to kill cancer cells and inhibit Escherichia coli.The main research contents are as follows:(1)In this study,we developed a facile method to rapid and selectively modify hemoglobin(Hb)with tartaric acid to form complex(TA-Hb),which exhibits strong peroxidase-like activity comparable to the native horseradish peroxidase(HRP).The structure change of Hb accompanied by the formation of TA-Hb complex was validated by UV-vis absorption,fluorescence,fluorescence lifetime and circular dichroism data.The oxygen atom of the carboxyl group of tartaric acid can easily coordinate with heme and become the axial ligand of porphyrin,and this specific structure facilitates to form stable intermediate,significantly enhancing Hb peroxidase-like activity.This study is expected to provide important insight into the interactions of the physiologically important protein Hb with various ligands.In addition,the unique property of TA-Hb was employed to develop a rapid colorimetric assay for detecting Hb in human serum samples,regardless of whether the sample is in the form of fresh or long-term storage,liquid or dried blood.This colorimetric assay can detect Hb over the concentration range of 0.01 ? 0.1 mg/m L and the limit of detection(LOD)is 0.1 ?g/m L.(2)By rapid,direct treatment of target proteins with iron porphyrin(TPPFe)in situ,carboxyl group of amino acid conjugates with Fe atom of the TPPFe molecule,forming a stable protein complex.We have shown that this complex not only maintains the integrity and functions of original proteins,but also acquires peroxidase activity.This study is unique since such modification is difficult to achieve with standard chemical modification or molecular biology methods.An innovative biosensing assay was developed for simplified,cost-effective and sensitive detection based on this protein modification.In addition,the proposed immunoassay is superior to traditional ELISA as it eliminates expensive and complicated cross-linking process of enzyme-labeled antibody.From a practical point of view,we extended this assay to rapid detection of clinically relevant proteins.The results show that this simple immunoassay provides clinical diagnose,food safety,and environmental monitoring in an easy-to-implement manner.(3)The glucose oxidase was modified by iron porphyrin to endow it with new peroxidase activity and double enzyme activity.The cascade reaction could be realized in a short time.Based on the double enzyme activity,we designed a one-step colorimetric method for the detection of glucose in blood,which can quantitatively detect the glucose in the range of 0.01-0.4 m M,and the limit of detection(LOD)is 0.0002 m M.The application of the double enzyme was extended to implement cytotoxicity and antibacterial experiments to verify the versatility of the modified method.