Construction Of Transition Metal Nanozyme Colorimetric Immunoassay System And Its Application In Carcinoembryonic Antigen Detection

Carcinoembryonic antigen（CEA）,as a common tumor marker,is expressed in many cancers,including gastric cancer,breast cancer,ovarian cancer,lung cancer and colorectal cancer.Therefore,the detection of CEA concentration becomes particularly important.Nowadays,colorimetric immunoassay based on nanozymes has received extensive attention due to its low cost,easy operation and visualization.In this paper,three kinds of transition metal nanomaterials with peroxidase-like activity were prepared by solvothermal method and instant synthesis method:Fe₃S₄,FeS₂@NiS,CuCo PBA,and a colorimetric immunoassay CEA system was constructed.The main research work is as follows:（1）Fe₃S₄nanomaterials were synthesized by one-step solvothermal method using ferric chloride and thiourea as raw materials.Its morphology is uniform flaky nanomaterials and has peroxidase-like activity.In the presence of H₂O₂,Fe₃S₄nanomaterials can catalyze the color change of 3,3’,5,5’-tetramethylbenzidine（TMB）to blue.Based on this principle,a sandwich colorimetric immunoassay for CEA concentration was constructed.In the range of 0.05-50 ng/mL,the concentration of CEA was linearly related to the absorbance of the solution,and the detection limit was82 pg/mL.（2）Transition metal FeS₂@NiS composite nanomaterials were synthesized by solvothermal method using iron nitrate,nickel nitrate and thioacetamide as raw materials.The antibody was coupled with the composite nanomaterials by carbodiimide method to construct a sandwich colorimetric immunoassay for CEA detection.Compared with Fe₃S₄nanomaterials,the synthesized FeS₂@NiS composite nanomaterials have better peroxidase-like activity.Under the optimal conditions,the colorimetric immunoassay was linear in the range of 0.005-40 ng/mL with a detection limit of 12 pg/mL.The system has a wide detection range and lower detection limit.（3）In order to reduce the influence of experimental operation and environment on the synthesis of nanomaterials,we propose a method for instant synthesis of nanozymes.When the cobalt potassium cyanide solution is mixed with the copper acetate solution,a transition metal nanomaterial copper-cobalt Prussian blue analogue（CuCo PBA）with peroxidase-like activity can be synthesized in real time.Therefore,glucose oxidase（GOD-Ab₂）labeled with CEA antibody was used to determine the CEA concentration on the microplate coated with CEA antibody.After the formation of sandwich immune complexes by glucose oxidase（GOD）,GOD catalyzes the reaction of H₂O₂generated by glucose with CuCo PBA nanomaterials and TMB mixed solution to turn blue,thereby constructing a highly sensitive colorimetric immunoassay detection method for CEA.The detection of CEA was linear in the range of 0.05-60 ng/mL with a detection limit of 22 pg/mL.