▎ 摘　　要

NOVELTY - Preparing electrochemical immunosensor based on nickel-cobalt double metal hydroxide combined with graphene oxide reinforced luminol involves preparing nickel-cobalt double metal hydroxide NiCo-LDH by dissolving 0.2-0.5g of cobaltous nitrate hexahydrate in 25mL of methanol as solution A, dissolving 0.3-0.8 g of 2-methylimidazole in 25mL of methanol as solution B. The solution B is poured into solution A under stirring condition, aged at room temperature for 24 hours and centrifuged to obtain product which is placed in oven and dried to obtain ZIF-67, 20-50mg of ZIF-67 is dispersed in 25mL of ethanol, sonicated for 30 minutes, 50-100mg of nickel carbonate hexahydrate is dissolved in 30mL of ethanol to obtain dispersion liquid and stirred for 10 minutes to obtain mixed solution. The solution is transferred to 100mL reaction kettle, reacted at 90degrees Celsius for 2 hours, centrifuged to obtain centrifuged product. USE - Method for preparing an electrochemical immunosensor based on nickel-cobalt double metal hydroxide combined with graphene oxide reinforced luminol for detecting neuron-specific enolase. ADVANTAGE - The method prepares electrochemical immunosensor which improves provides strong and stable electrochemiluminescence signal and achieves ultra-sensitive detection of neuron-specific enolase with a detection limit of 3.14fg per ml. DETAILED DESCRIPTION - Preparing electrochemical immunosensor involves preparing nickel-cobalt double metal hydroxide NiCo-LDH by dissolving 0.2-0.5g cobaltous nitrate hexahydrate in 25mL methanol as solution A, dissolving 0.3-0.8g 2-methylimidazole in 25mL methanol as solution B. The solution B is poured into solution A, aged at room temperature for 24 hours and centrifuged to obtain product which is dried in oven to obtain ZIF-67, 20-50mg of ZIF-67 is dispersed in 25mL of ethanol, sonicated for 30 minutes, 50-100mg of nickel carbonate hexahydrate is dissolved in 30mL ethanol to obtain dispersion liquid and stirred for 10 minutes to obtain mixed solution. The solution is transferred to 100mL kettle, reacted at 90degrees Celsius for 2 hours, centrifuged and dried to obtain NiCo-LDH. The graphene oxide is prepared by dispersing 0.2-0.5g graphite in 40mL concentrated sulfuric acid and phosphoric acid in ratio of 9:1, adding 1.5-3.0g potassium permanganate, reacting it in oil bath at 50degrees Celsius for 12 hours to obtain reacted solution and 40mL of water is prepared to freeze into ice. The solution is poured into ice, stirred, 200-500microL hydrogen peroxide is added, stirred and reacted for 30 minutes, solution is washed with 0.2mol/L hydrochloric acid, ethanol, and ether for three times and dried in oven to obtain graphene oxide. The GO@NiCoLDH compound of nickel and cobalt double metal hydroxide combined with graphene oxide is prepared by dispersing 10-30mg graphene oxide in 20mL water for 2 hours, adding 10-50mg nickel-cobalt double metal hydroxide, stirring it for 12 hours, washing it with water, centrifuging and drying it to obtain GO@NiCoLDH composite. The composite of nickel and cobalt double hydroxide combined with graphene oxide loaded luminol GO@NiCo-LDH-luminol is prepared by dispersing 10-30mg of nickel and cobalt double hydroxide combined with graphene oxide in 20mL water, stirring it for 30 minutes, adding 5-15mL of 5mmol/L luminol solution, stirring it for 12 hours, centrifuging and dispersing it in aqueous solution to obtain final product, which is stored at 4degrees Celsius. The CeO2@MnO2 composite is prepared by dissolving 1-2g cerium nitrate in 10mL water, adding 20-50mL ethylene glycol, 1-3mL acetic acid and 0.5-2g polyvinylpyrrolidone K30, transferring solution to 50mL reactor and reacting it at 150-200degrees Celsius for 22 hours to obtain reacted product. The product is centrifuged and washed with ethanol and methanol to obtain cerium dioxide precursor, which is calcined in air at 500degrees Celsius for 2 hours to obtain cerium dioxide and 20-50mg of cerium dioxide is dispersed in 35mL of 0.01-0.05mol/L potassium permanganate solution to obtain dispersed solution, heated at 140degrees Celsius for 12 hours, centrifuged, washed and dried to obtain final composite. The PBS buffer solution is prepared by taking 11.94g disodium hydrogen phosphate dodecahydrate in 500mL flask, configuring it with concentration of 1/15mol/L as solution A, taking 4.54g potassium dihydrogen phosphate in flask and configuring it as solution B, and mixing solution A and B to form series of PBS buffer solutions with pH of 6.0-8.5. The secondary antibody marker CeO2@MnO2-Ab2 of cerium dioxide and manganese dioxide complex combined with neuron-specific enolase recognition antibody is prepared by dissolving 0.1-0.5g of cerium dioxide and manganese dioxide complex in 10mL ethanol, adding 0.1-0.5mL 3-aminopropyltriethoxysilane, refluxing, centrifuging, washing and drying it to obtain complex. The 100-300microL of 10microg/mL neuron-specific enolase secondary antibody Ab2 is added in 10mL of PBS, 0.5-1mL of 400mmol/L of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100mmol/L of N-hydroxysuccinimide mixed solution, stirred for 4 hours, 20-50mg of aminated cerium dioxide and manganese oxide complex are added and stirred for 6 hours, centrifuged and washed with PBS to obtain secondary antibody marker CeO2@MnO2-Ab2 of cerium dioxide and manganese dioxide complex combined with neuron-specific enolase recognition antibody. The product is dispersed in 5mL of PBS and stored in a refrigerator at 4degrees Celsius. The immunosensor is prepared by polishing glassy carbon electrode with aluminum oxide polishing powder, cleaning it with water, dropping 6microL of 1.0-3.0mg/mL of GO@NiCo-LDH-luminol solution onto surface of electrode and drying it at room temperature. The 5micro-20uL of 400mmol/L of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100mmol/L of N- hydroxysuccinimide mixed solution activates the carboxyl group on GO@NiCo-LDH-luminol luminescent material on surface of modified electrode. The 6microL of 10microg/mL of neuron-specific enolase primary antibody Ab1 is added and washed with PBS buffer to remove excess Ab1. The 3microL of 0.1 wt.% bovine serum albumin solution is dropped onto surface to block non-specific binding sites, washed with PBS buffer to remove unbound BSA, and 6microL of 0.00001-300ng/ml of neuron-specific enolase antigens are dropped onto surface, incubated for 2 hours and washed with PBS buffer. The 6microL of CeO2@MnO2-Ab2 solution is added, washed with PBS buffer, dried at 4degrees Celsius to prepare immunosensor.