Application Of Cupric Oxide Based Nano Enzyme In Chemiluminescence Imaging Immunoassay

In recent years,cancer is still a disease that is difficult to cure,and the outbreak of COVID-19 in 2019 has also dealt a great blow to people’s production and life.In view of the current large-scale screening and clinical diagnosis requirements of cancer and COVID-19,a sensitive and label-free chemiluminescence imaging immunosensor(CLIIS)based on nanoenzyme was constructed.And has achieving rapid and sensitive detection of tumor markers and novel coronavirus protein and antibodies.Compared with natural enzymes,nano-enzymes have the advantages of low cost,simple production,stable properties and controllable catalytic activity.Among non-precious metals,copper is highly productive and cost-effective.In addition,the multiple oxidation states of the metal components can provide interesting catalytic properties for the redox reactions and are suitable for the preparation of peroxidase.As a famous p-type semiconductor material,copper oxide has been widely used in various kinds of sensors because of its low energy band,high catalytic activity,non-toxic and reasonable price.It has been widely used in various kinds of sensors,however,there are few reports on chemiluminescent immune sensors.Three kinds of nanomaterials,CuO hollow cube,Au/CuO nanorods and defective ZIF-67@CuO,have been synthesized in this paper.The nano-enzymes all have excellent peroxidase activities and were applied to construct the labelfree CLIIS,so as to realize the low-cost,rapid and sensitive simultaneous detection of multiple markers.The specific research work is as follows:(1)In order to improve the accuracy of clinical diagnosis of liver cancer,a chemiluminescence immunosensor based on CuO hollow cube nano-enzyme was developed.Copper oxide hollow cubes were prepared by polyalcohol reduction and ammonia-air oxidation etching.The morphology,composition and enzyme-like activity of the prepared materials were characterized by scanning electron microscope(SEM),transmission electron microscope(TEM),ultraviolet-visible absorption spectrum(UV-vis),X-ray diffraction(XRD)and so on.The nanomaterial is a hollow cube with excellent peroxidase properties and excellent kinetic characteristics,which can enhance and prolong the luminescence of oxidized luminol.The nano-enzyme was embedded in the micropore of the immunosensor by chitosan,and the antibodies of liver cancer markers(AFP,CEA)were immobilized by chitosan-rich functional groups.When there is a target antigen in the sample,the formation of immune complex hinders the catalysis of nano-enzyme to the substrate and weakens the chemiluminescence signal,thus realizing the detection of the target analyte.Combined with the analysis of supporting software,the quantitative detection of tumor markers of liver cancer can be realized.The constructed CuO-based immunosensor showed a good linear relationship in the concentration range of 0.1-100 ng/mL(AFP)and 0.1-200 ng/mL(CEA),and the detection limit of AFP and CEA were 0.01 ng/mL.In addition,the prepared immunosensor shows good specificity and stability,and has important practical application value.(2)Aiming at the current home diagnosis of COVID-19,a chemiluminescence imaging immunosensor based on Au/CuO nanorods and nanorods was developed in this study.Au/CuO nanorods were prepared by reduction of chloroauric acid by sodium borohydride and oxidation of copper hydroxide by chlorauric acid at room temperature.The morphology,composition and enzyme-like activity of the prepared materials were characterized by SEM,TEM,UV-vis,XRD and X-ray photoelectron spectroscopy(XPS).The nanomaterial is rod-shaped and has excellent peroxidase-like properties.Through the film-forming property of chitosan,the rodshaped Au/CuO nano-enzyme was embedded in the micropore of the immune chip.The nanomaterial is rod-shaped and has excellent peroxidase-like properties.Through the filmforming property of chitosan,the Au/CuO nano-enzyme was embedded in the micropore of the immune chip.Chitosan has rich functional groups on its surface and good biocompatibility.It is used to connect a segment of peptide P4 of the spike protein,which is one of four main structural proteins of SARS-CoV-2,and successfully constructed an immunesensor for detecting novel coronavirus antibodies.When there is a target antibody in the sample,the formation of immune complex hinders the contact between the substrate and the nano-enzyme,reduces the chemiluminescence intensity,and realizes the highly sensitive detection of SARSCoV-2.Immunosensor constructed based on Au/CuO nanoenzyme shows a good linear relationship in the concentration range of 0.0001 to 50 ng/mL(G5),with a detection limit of 0.03 pg/mL.In addition,the prepared immunesensor showed good specificity,repeatability and has a good application prospect.(3)Zeolite imidazole skeleton(ZIF-67)has been proved to be an effective catalyst.Its high-density dispersion of metal active sites ensures sufficient catalytic centers,porous structure makes all active sites easy to use,and open channels promote related mass transfer and diffusion.In this work,a chemiluminescence imaging immunosensor based on defective ZIF-67@CuO nano-enzyme was developed.Firstly,sea urchin-like copper oxide was synthesized and modified with polyvinylpyrrolidone,then ZIF-67 was grown at room temperature by seed-mediated method.Finally,ZIF-67 was etched with polyoxometalates to expose CuO and more active sites.The morphology,composition and enzyme-like activity of the prepared materials were characterized by TEM,UV-vis,XRD and XPS.The prepared composites have excellent peroxidase-like activity and have the catalytic kinetic characteristics of both ZIF-67 and CuO,which can shorten the chemiluminescence intensity detection time.When there is a target analyte,the formation of immune complex hinders the contact between the substrate and the nano-enzyme.The higher the concentration of the target antigen/antibody,the lower the chemiluminescence intensity.The constructed CLIIS based on the defective ZIF-67@CuO mimic enzyme showed good linear relationship in the concentration range of 0.01 pg/mL-200 ng/mL(P4)and 0.01 pg/mL-100 ng/mL(G5),and the detection limit were 0.003 pg/mL.In addition,the prepared immunesensor showed good specificity and repeatability,providing a new strategy for the diagnosis of the novel coronavirus.