Preparation Of Zeolitic Imidazolate Frameworks Composites And Their Application In Chemiluminescence Imaging Immunoassay

Immunoassay plays an important role in biomedical research,clinical diagnosis,food safety,and environmental monitoring by utilizing specific binding between antigens and antibodies to quantitatively detect the content of antigenic substances,such as bacteria and proteins.Chemiluminescence imaging immunoassay combines the advantages of chemiluminescence immunoassay technology and imaging technology,with advantages such as high detection flux,wide linear range and fast analysis speed.Traditional chemiluminescence immunoassay usually uses natural enzyme labeled antibodies to catalyze hydrogen peroxide to trigger luminol luminescence.However,the shortcomings of natural enzymes such as easy deactivation,difficulty in extraction,and poor tolerance to extreme environments limit the development and application of chemiluminescence immunoassay.Metal organic frameworks(MOFs)have been proved to be an effective catalyst.Their high-density and dispersed metal active site ensure sufficient catalytic centers.The porous structure makes all active site easy to use,and open channels promote the related mass transfer and diffusion.MOFs have been found to have natural enzyme properties and have received widespread attention in the field of biosensing.Zeolitic imidazolate frameworks(ZIFs)are a subclass of MOFs formed by the coordination of metal ions with 2-methylimidazole organic ligands.They not only have rich topological structure and high porosity,but also have excellent thermal and chemical stability.In this work,we synthesized Au@CuZn-ZIF,Fe3O4@ZIF-67 and ZIF-67/NiCo-LDH/NC composite nanoenzymes based on the ZIFs,studied their simulated enzyme properties,developed nanoenzyme labeled detection probes and constructed a chemiluminescence imaging immunosensor to realize the high sensitivity and rapid detection of human IgG.The specific research work is as follows:(1)Chemiluminescence imaging immunoassay based on Au@CuZn-ZIF nanozymeFirstly,CuZn-ZIF was synthesized through a simple solvent method,and then HAuCl4 and NaBH4 were added to react to load Au nanoparticles on the surface of CuZn-ZIF,forming Au@CuZn-ZIF nanozyme,and then nanozyme signal probes were prepared by connecting Au@CuZn-ZIF nanozyme with secondary antibodies.The Au@CuZn-ZIF and nanozyme detection probes were characterized using scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray photoelectron spectroscopy(XPS)and ultraviolet spectroscopy(UV).Copper and zinc act as bimetallic centers to synergistically enhance the enzymatic catalytic activity of MOFs,and the introduction of Au nanoparticles enhances the biocompatibility of nanozyme.By using the sandwich method,the nanozyme and human immunoglobulin(HIgG)antigens were added to the surface of the carrier array modified with HIgG antibodies for incubation.Nanoenzymes on the complex can catalyze the oxidation of hydrogen peroxide to trigger luminol luminescence.By using an inductively coupled CCD camera to integrate and collect luminescence signals,high sensitivity detection of HIgG can be achieved.The experimental results indicated that the linear range of the chemiluminescence imaging immunosensor based on Au@CuZn-ZIF nanozyme for HIgG detection is 1.0 ng/mL to 10μg/mL,and the detection limit is 0.017 ng/mL when the signal-to-noise ratio is 3.This sensor has good specificity for HIgG detection.The sensor showed good specificity for HIgG detection,and its signal value decreased by only 1.6%after four weeks of storage in PBS solution,showing good storage stability.It can accurately detect actual serum samples and has important clinical application value and significance.(2)Chemiluminescence imaging immunoassay based on Fe3O4@ZIF-67 nanozymeBy using a simple preparation method,magnetic Fe3O4 was doped into ZIF-67,the construction of Fe3O4@ZIF-67 composite nanozyme can generate strong synergistic effects between Fe3O4 nanoparticles and ZIF-67,greatly improving the enzymatic catalytic performance of nanozyme,while also possessing good stability and dispersibility.Fe3O4@ZIF-67 nanozyme signal probes were prepared by connecting nanozyme with secondary antibodies,and then their characterization was analyzed by SEM,TEM,XPS,UV,etc.Using the sandwich method,the nanozyme labeled with secondary antibody and HIgG antigen were added to the surface of the carrier array modified with primary antibody for temperature incubation.The nanozyme on the complex catalyzed the oxidation of hydrogen peroxide to trigger luminol luminescence,and the emission signal was collected using an inductively coupled CCD camera to achieve highly sensitive detection of HIgG,The experimental results indicated that the linear range is 0.01 ng/mL-1μg/mL for the chemiluminescence imaging immunosensor based on Fe3O4@ZIF-67 nanozyme to detect HIgG,and its detection limit is 0.0023 ng/mL(the signal-to-noise ratio was 3).This sensor has good specificity for the detection of HIgG,and the operation is relatively simple with low sample consumption,which has great potential in clinical analysis.(3)Chemiluminescence imaging immunoassay based on ZIF-67/NiCo-LDH/NC nanozymeThe ZIF-67/NiCo-LDH/NC with a sheet-like structure was synthesized by using ZIF-67 as a template.After carboxylation,it was combined with secondary antibodies to prepare nanozyme signal probes.The characterization of ZIF-67/NiCo-LDH/NC and detection probes was carried out through SEM,TEM,XPS,UV,etc.Then,with the help of the good biocompatibility of chitosan,the primary antibody was embedded on the surface of the epoxy activated carrier,and then the antigen and signal probe were sequentially added.The nanoenzymes on the probe would catalyze oxidation of hydrogen peroxide to trigger the luminol chemiluminescence signal.The luminescence signal was collected by an inductively coupled CCD camera,and the luminescence signal will increase with increasing antigen concentration.The experimental results showed that the linear range is 0.001 ng/mL-10 ng/mL for the chemiluminescence imaging immunosensor based on ZIF-67/NiCo-LDH/NC nanozyme to detect HIgG.When the signal-to-noise ratio is 3,the detection limit is 0.00042 ng/mL.This sensor has good specificity for HIgG detection.After being stored in PBS solution for four weeks,its signal value only decreased by 1.5%,demonstrating good storage stability and accurately detecting actual human serum samples.