▎ 摘　　要

NOVELTY - Preparing a graphene-based calprotectin biosensor comprises e.g. (i) dispersing the graphene oxide prepared by chemical oxidation in a phosphate buffer solution to form a stable dispersion, applying the dispersion to the cleaned glassy carbon electrode and drying the modified electrode at room temperature to prepare graphene modified electrode; (ii) improving the stability of the modified electrode and coating with chitosan acetic acid solution; (iii) using the modified electrode prepared in step (ii) as a working electrode, placing in a phosphate buffer solution, forming a three-electrode system with the saturated calomel reference electrode and the platinum plate counter electrode, performing the cyclic voltammetry scan in the voltage range of 1.5-0V to reduce the graphene oxide to graphene and improving the conductivity of the electrode and the adsorption performance of the calprotectin antibody and the adsorption amount of the calprotectin antibody on the electrode. USE - The graphene-based calprotectin biosensor is useful for detecting calprotectin in feces, serum or urine (claimed). ADVANTAGE - The method: can enrich and fix nature of calprotectin antibody on the electrode surface; blocks the active site, and finally fixes the calprotectin on the electrode through the immune response of the antigen and antibody; and takes graphene with the excellent conductivity which increases the peak current of the electrode in the electrochemical probe potassium ferricyanide solution and sensitivity of the immunosensor thus increases the amount of calprotectin antibody modification on the electrode surface and the electrode's detection range of calprotectin concentration. DETAILED DESCRIPTION - Preparing a graphene-based calprotectin biosensor comprises (i) dispersing the graphene oxide prepared by chemical oxidation in a phosphate buffer solution to form a stable dispersion, applying the dispersion to the cleaned glassy carbon electrode and drying the modified electrode at room temperature to prepare graphene modified electrode; (ii) improving the stability of the modified electrode and coating the modified electrode prepared in step (i) continuously with chitosan acetic acid solution; (iii) using the modified electrode prepared in step (ii) as a working electrode, placing in a phosphate buffer solution with a molar concentration of 0.05-0.2 M, forming a three-electrode system with the saturated calomel reference electrode and the platinum plate counter electrode, performing the cyclic voltammetry scan in the voltage range of 1.5-0V to reduce the graphene oxide to graphene and improving the conductivity of the electrode and the adsorption performance of the calprotectin antibody and the adsorption amount of the calprotectin antibody on the electrode; (iv) activating the carboxy group on the electrode surface with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to add 4-6 mu l (50-600 mM)1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dripping a phosphate buffer solution of N-hydroxysuccinimide onto the modified electrode prepared in step (iii), and activating at room temperature for 1-3 hours; (v) modifying the calprotectin antibody, adding 5-7 mu l calprotectin antibody solution with a concentration of 25-300 mu g/ml to the surface of the electrode prepared in step (iv), incubating in a thermostat at 25-35 degrees C for 0.5-2 hours and washing away the calprotectin antibody that is not firmly bound to the electrode with 8-12 mM phosphate buffer solution; (vi) sealing the non-specific active sites of the electrode, using 4-6 mu l bovine serum albumin solution with a concentration of 0.5-3 wt.% to block the non-specific active sites on the surface of the electrode prepared, incubating in a thermostat at 25-35 degrees C for 0.5-2 hours and using 8-12 mM phosphate buffer solution to wash off bovine serum albumin that is not firmly bound to the electrode; and (vii) taking the modified calprotectin which uses 5-7 mu l calprotectin with a concentration of 10-200 ng/ml in a series of different concentrations for specific recognition with the antibody on the electrode, incubating in a thermostat at 25-35 degrees C for 0.5-2 hours, washing off the calprotectin that is not firmly bound to the antibody on the electrode with 8-12 mM phosphate buffer solution, and storing it in a refrigerator at 4 degrees C for later use. An INDEPENDENT CLAIM is also included for detecting the concentration of calprotectin solution, comprising (a) forming the electrode modified with calprotectin, silver/silver chloride electrode and platinum electrode as three-electrode system; and (b) using a potassium ferricyanide solution with a molar concentration of 1-3 mM dissolved in a phosphate buffer solution as the electrolyte, carrying out cyclic voltammetry scan or differential pulse voltammetry scan in the voltage range of -0.2 to 0.6V on the electrochemical workstation, where the peak current on the cyclic voltammetry scan or differential pulse voltammetry scan curve changes regularly with the concentration of calprotectin thus realizing the test of the concentration of calprotectin