▎ 摘　　要

NOVELTY - A sandwich-type electrochemical immune sensor for detecting cervical cancer marker comprises working electrode, reference electrode, and base electrode. The working electrode contains glassy carbon electrode, surface modified sequence hatched anti-reduced graphene oxide-tetraethylene pentamine, anti-bovine serum albumin, cervical cancer tumor marker antigens and gold-mesoporus carbon, is respectively connected with the neutral red, thionine secondary antibody, the reference electrode is saturated calomel electrode, and the base electrode is molybdenum electrode. USE - As sandwich-type electrochemical immune sensor for detecting cervical cancer marker (claimed). ADVANTAGE - The sandwich-type electrochemical immunosensor allows simultaneous detection of tumor markers in cancer applications. The electrochemical immune sensor can be simultaneously detect two samples, make up the limitations of single indicators, improves the early diagnosis of cervical cancer, and can be detect other types of cancer tumor markers simultaneously. DETAILED DESCRIPTION - A sandwich-type electrochemical immune sensor for detecting cervical cancer marker comprises a working electrode, a reference electrode, and a base electrode as counter electrode, the working electrode contains glassy carbon electrode, surface modified sequence hatched anti-reduced graphene oxide-tetraethylene pentamine (anti-rGO-TEPA), an anti-bovine serum albumin, cervical cancer tumor marker antigens and Au CMK-3 (gold mesoporus carbon), is respectively connected with the neutral red, thionine secondary antibody, the reference electrode is saturated calomel electrode, and the base electrode is molybdenum electrode. The preparation method of sandwich-type electrochemical immune sensor involves: a) preparing Au CMK-3 and electron mediator secondary antibody incubation (Ab2) solution; b) preparing an electrochemical immune sensor working electrode; c) preparation of electrochemical immune sensor working curve, where the step (a), involves: Au (CMK-3) synthesis: taking a solution (100 ml) containing chloroauric acid (0.01 g) into flask, and heating to boil, then dropwise adding sodium citrate (1%) and maintain the temperature for 40 minutes, cooling to room temperature, adding the solution with mesoporous CMK-3 for 4 hours at 9000 revolutions per minute (rpm) speed for 20 minutes, centrifuging for three times, vacuum drying to obtain the Au CMK-3; preparation of Au CMK-3 and electronic medium secondary antibody incubation solution: weighing Au CMK-3, adding phosphate buffer solution (PBS) ultrasonic having pH value of 7.4, adding ant-1-carcinoembryonic antigen (ant-1-CEA), squamous cell carcinoma antigen (ant-1-SCCA), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-N-hydroxysuccinimide imide (NHS) solution, at 4 degrees C for 12 hours, then respectively adding neutral red solution (2 mg/ml), thionine solution (2 mg/ml), and incubating for 10 hours at 4 degrees C, centrifuging for 20 minutes at speed of 6000 revolutions per minute (rpm), redispersing after washing in a PBS having pH of 7.4 to obtain different secondary antibody solution, mixing two secondary antibody solution, such as to obtain Au CMK-3-ant1-CEA-neutral red and AuOCMK-3-ant-1-SCCA-thionine secondary antibody mixed solution. The step (b) involves: polishing the surface of glassy carbon electrode to make the surface smooth; adding EDC and NHS (50 mmol/l) to the antibody solution SCCA (10 Pg/ml) or CEA (10 Pg/ml), and then adding amino graphene (2 mg), shaken for 4 hours by centrifugation, washing with PBS and diluted to the original volume, to give the corresponding solution rGO-TPEA-Ab1, final volume of the solution obtained by mixing the mixture dropped on the electrode, drying at room temperature, modifying using solution (mass fraction of 1%) of bovine serum albumin (BSA) on the electrode surface to eliminate non-specific active sites of the electrode, cleaning with PBS having pH of 7.4, cleaning electrodes, taking both cancer tumor marker antigen solution, after drying, followed by the dropwise addition of different concentrations of antigen and antibody incubation at room temperature for 30 minutes, and to ensure an anti-antigen immune complexes formed half; taking the AuO CMK-3-ant1-CEA neutral red and AuO CMK-3-ant1-SCCA-thionine secondary antibody modified mixed solution to the electrodes; storing at 4 degrees C, drying to obtain the electrochemical immunosensor working electrode; the step (c) for manufacture of electrochemical immune sensor involves: connecting the reference electrode, saturated calomel electrode, the working electrode of a molybdenum wire electrode and the electrochemical workstation properly; adding PBS buffer solution having pH 7.4 (containing 0.1 mol/l potassium chloride (KCl) as supporting electrolyte) as a base solution, by differential pulse voltammetry (DPV) modified working electrode, neutral red (-0.62V) and thionine (-0.17V) at different potentials response; according to different electronic medium obtained current response corresponding to its different tumor markers antigen standard solution relation, drawing the working curve.