▎ 摘　　要

NOVELTY - Unlabeled impedance type immunosensor is prepared by immobilizing monoclonal antibody PEDV-2C11 of porcine epidemic diarrhea virus on the surface of an electrode modified by a nano-gold/molybdenum disulfide/reduced graphene complex nano-material. USE - The unlabeled impedance type immunosensor is useful for detecting porcine epidemic diarrhea virus (claimed). ADVANTAGE - The sensor is prepared in a simple manner, and detects porcine epidemic diarrhea virus with high sensitivity. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the following: (1) method for preparing the unlabeled impedance type immunosensor involves (a) preparing gold nano solution, (b) synthesizing nano-gold/molybdenum disulfide/reduced graphene complex by forming molybdenum disulfide/reduced graphene complex by adding 0.08 g sodium molybdate dihydrate and 6 mg graphene to 10 ml deionized water, dispersing for 10 minutes under ultrasound, adding 0.126 g thiourea to dissolve, ultrasonicating the resulting mixture for 30 minutes, transferring to a 10 ml polytetrafluoroethylene-lined stainless steel autoclave, placing the autoclave in an oven, heating at 200 degrees C for 24 hours, cooling to room temperature, collecting the precipitate by performing centrifugation at 6000 revolutions per minute (rpm) for 5 minutes, washing with water and ethanol for several times, then drying at 60 degrees C, converting graphene oxide into reduced graphene oxide during the reaction, subsequently, adding 10 mg molybdenum disulfide/reduced graphene oxide (MoS2/rGO) to 10 ml the prepared gold nano solution, shaking at room temperature for 12 hours, washing the AuNPs/MoS2/rGO complex with water centrifuging at 6000 rpm for 5 minutes to obtain a precipitate, and collecting the final product by drying at 60 degrees C, (c) preparing immunosensor with a diameter of 0.15 mu m, polishing the glassy carbon electrode (GCE) surface to a mirror finish with aluminum oxide polishing solution, performing ultrasonic cleaning using ultra-pure water, drying in a nitrogen stream, completely drying the electrode, dripping 5 mu l AuNPs/MoS2/rGO aqueous solution onto the surface of the bare GCE, air-drying at room temperature, immersing the modified electrode in 200 mu l phosphate buffer solution of 0.15 mg/mL monoclonal antibody PEDV-2C11, placing at 4 degrees C for 12 hours, removing the electrode, placing in 10 mg/ml bovine serum protein for 1 hour to block non-specific sites, rinsing the bovine serum albumin on the surface with secondary water, immersing the prepared electrode in a phosphate buffer solution of pH 7, storing at 4 degrees C, immersing the antibody-immobilized electrode in phosphate buffered solution of porcine epidemic diarrhea virus of different concentrations having a pH of 7.4, placing in 37.5 degrees C water bath for 140 minutes, taking out the secondary water and phosphate buffered solution, and then subjecting to electrochemical impedance detection; and (2) method for detecting porcine epidemic diarrhea virus by using the immunosensor involves (a) establishing the relationship between the relative change rate of impedance (% delta Rct) and the concentration of porcine epidemic diarrhea virus by determining the initial impedance value Rct (BSA) of the impedance immunosensor, and the impedance of the immunosensor with the porcine epidemic diarrhea virus standard solution with different concentrations after the immune response for 140 minutes, (b) performing immunological reaction of the impedance immunosensor and the solution of the test object according to the method and conditions of step (a), measuring the impedance value, and calculating % delta Rct of the impedance caused by the reaction of the antibody to the porcine epidemic diarrhea virus, and (c) determining the content of porcine epidemic diarrhea virus in the test solution according to the relationship between % delta Rct of the impedance and the concentration of porcine epidemic diarrhea virus.