Label-free Electrochemical Immunoassay Based On CuFe-MOF And Its Complexes Nanozymes

Label-free electrochemical immunosensors taking advantage of the rapidity and simplicity of label-free immunoassays and the high sensitivity of electrochemical detection have been widely used in clinical diagnosis and food safety test.Metal-organic frameworks（MOFs）are the class of materials composed of organic ligands and metal nodes,with highly diverse structures,large specific surface area,tunable pore size,and customizable chemical properties.It has been found that some MOF materials exhibit functionally similar catalytic activity to natural enzymes.Therefore,spindle-shaped bimetallic CuFe-MOF,corn-like ZIF-8（Zn）@CuFe-MOF and sheet-like ZIF-67 modified ZIF-67（Co）@CuFe-MOF were synthesized in this paper.It was found that these MOFs materials have high peroxidase activity,and can be used as mimetic enzymes to construct label-free electrochemical immunosensors,which realize low-cost,high-sensitivity,fast and accurate label-free human immunoglobulin G（HIgG）detection.This thesis proposes a new idea and a new method for label-free electrochemical immunoassay based on MOF nanozymes,which integrates high stability,low cost and simple preparation of novel MOF nanozymes,high sensitivity of electrochemical assay,and high specificity of immunoassays.Metal-organic frameworks used as electrode modification materials,can greatly improve the immobilization capacity,biological activity and stability of bioactive molecules,thus improving the analytical performance of biosensors.The detailed research work is as follows:（1）In this study,a new method of label-free electrochemical immunoassay based on spindle-shaped bimetallic CuFe-MOF nanozyme was proposed for the highly sensitive detection of IgG.The CuFe-MOF material was successfully prepared by a one-pot hydrothermal method.Its morphology and property were characterised by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,X-ray diffraction（XRD）and ultraviolet-visible absorption spectroscopy（UV-vis）,which showed a spindle-shaped structure and good peroxidase-like activity.A label-free electrochemical immunosensor was constructed using CuFe-MOF nanozyme as an electrode material for modification of antiobodies.In the presence of the CuFe-MOF nanozyme,the mimetic-catalyzed activity of H2O2 toward the oxidation of the reaction substrate of o-phenylenediamine（o-PD）was greatly enhanced,generating a strong electrochemical signal.The immune complex formed by the antigen-antibody specific reaction inhibits this catalytic oxidation reaction,thus causing a decrease in the electrochemical signal intensity.The linear range of the label-free electrochemical immunosensor for IgG detection is 0.001-50 ng/mL,with a detection limit as low as 0.00045 ng/mL（S/N=3）.The CuFe-MOF-based label-free electrochemical immunosensor enables highly sensitive detection of HIgG and has been successfully applied to measure real samples with important clinical applications.（2）This study proposed a new method for the label-free electrochemical immunoassay based on the corn-like ZIF-8（Zn）@CuFe-MOF double MOF nanozyme.The structure and peroxidase-like activity of ZIF-8（Zn）@CuFe-MOF was characterised using SEM,TEM,XRD and UV-vis.The ZIF-8（Zn）@CuFe-MOF material shows corn-cob shaped morphology and superior peroxidase activity compared to pristine CuFe-MOF.The label-free electrochemical immunosensor proposed in this work can be easily fabricated in a simple step by capturing biotinylated antibodies with the aid of streptavidin.After incubation with the corresponding antigen at different concentrations,the immune complex formed by the specific binding of the antibody antigen prevents the contact between the ZIF-8（Zn）@CuFe-MOF nanozyme and the catalytic substrate,resulting in a reduction of the electrochemical signal intensity,which in turn leads to a quantitative label-free immuoassay.Using HIgG as the analytical model,the label-free electrochemical method based on the ZIF-8（Zn）@CuFe-MOF double MOF nanozyme showed good linearity over the concentration range of 0.001-80 ng/mL of HIgG,with detection limits as low as 0.00032 ng/mL（S/N=3）,resulting in high sensitivity and a wide linear range of IgG detection.（3）In this study,sheet-like ZIF-67（C o）-modified spindle-shaped CuFe-MOF（ZIF-67（Co）@CuFe-MOF）was fabricated for the first time using a bimetallic CuFe-MOF material as a template.The structure and peroxidase-like activity of ZIF-67（Zn）@CuFe-MOF were characterized using SEM,TEM,XRD and UV-vis.A ZIF-67（Co）@CuFe-based MOF-based label-free electrochemical immunoassay strategy was proposed for the highly sensitive detection of HIgG.The ZIF-67（Co）@CuFe-MOF nanozyme was modified to the surface of glassy carbon electrode,and then biotinylated antibody was immobilized by means of streptavidin-biotin interaction.The immune complex formed by the specific reaction between antigen and antibody hindered the catalytic oxidation of o-phenylenediamine by ZIF-67（Co）@CuFe-MOF nanozyme,resulting in the decrease of electrochemical signal value.The label-free immunosensor has a linear range of 0.0005-100 ng/mL for HIgG detection with a detection limit as low as 0.00013 ng/mL（S/N=3）.Moreover,the immunosensor has been successfully applied to the detection of practical samples.