▎ 摘　　要

NOVELTY - Detecting Golgi protein 73 (GP73) using electrochemical sensor based on nanocomposite material and aptamer by: (A) preparation of reduced graphene oxide-ferrocene-trimanganese tetraoxide (RGO-Fc-Mn3O4) nanocomposite; (B) modification of electrodes and construction of aptasensors; (C) drawing the working curve of GP73; (D) detection of GP73 in actual serum samples: (1) collecting human serum samples in the hospital (GP73 concentration has been obtained by enzyme-linked immunosorbent assay (ELISA)), adding dropwise to the GP73Apt/RGO-Fc-Mn3O4/Au-POPD/SPE sensing interface, reacting for a certain period of time, washing to obtain the working electrode, and drying it for later use; (2) placing the working electrode into 5 ml PBs solution, using the DPV scan of the electrochemical workstation, and recording its peak current; and (3) according to the working curve, calculating the concentration of GP73 in the actual sample to be tested. USE - Detecting Golgi protein 73 using electrochemical sensor based on nanocomposite material and aptamer. ADVANTAGE - GP73 aptamer is used as recognition molecule, using reductive graphene-trimanganese tetroxide (RGO-FcMn3O4) nano-composite material good electron transfer effect, excellent load capacity and excellent electrochemical activity. GP73 aptamer is capable of specifically recognizing and binding GP73 protein, constructing a suitable ligand sensor capable of specifically recognizing and quantitatively analyzing the GP73 protein, detecting the content of GP73 in serum. The method has simple operation, time-saving, low cost, and lowest detection limit of 0.01 ng/ml. DETAILED DESCRIPTION - Detecting Golgi protein 73 (GP73) using electrochemical sensor based on nanocomposite material and aptamer involves (A) preparation of reduced graphene oxide-ferrocene-trimanganese tetraoxide (RGO-Fc-Mn3O4) nanocomposite: (1) preparation of reduced graphene oxide (RGO): pouring graphene oxide into distilled water, crushing evenly, adding ascorbic acid for reduction, and obtaining RGO solution; (2) preparation of reduced graphene oxide-ferrocene (RGOFc): adding ferrocene formic acid solution and ethanol to the RGO solution, stirring and reacting to obtain RGO-Fc; (3) preparation of trimanganese tetraoxide (Mn3O4): adding manganese chloride tetrahydrate powder and polyvinylpyrrolidone (PVP) to pure water and mixing and stirring and heating; mixing sodium hydroxide solution and manganese chloride solution and stirring, centrifuging and washing to obtain Mn3O4 solution; and (4) preparation of reduced graphene oxide-ferrocene-trimanganese tetraoxide (RGO-Fc-Mn3O4) nanocomposite: adding RGO-Fc mixed solution and Mn3O4 solution to PVP, stirring for reaction, and centrifuging to obtain RGO-Fc-Mn3O4; (B) modification of electrodes and construction of aptasensors: (1) modification of gold-poly-o-phenylenediamine/screen-printed electrode (Au-POPD/SPE): placing the SPE in dilute sulfuric acid solution, performing cyclic voltammetry scanning to obtain activated screen-printed electrode, soaking the activated screen-printed electrode in a mixed solution of chloroauric acid and OPD for constant potential deposition, rinsing and drying to obtain Au-POPD/SPE; (2) preparation of reduced graphene oxide-trimanganese tetroxide-ferrocene/gold-poly-o-phenylenediamine/screen-printed electrode (RGO-FcMn3O4/Au-POPD/SPE) sensing interface: adding RGO-Fc-Mn3O4 solution dropwise to the obtained Au-POPD/SPE, incubating, rinsing, and drying to obtain RGO-FcMn3O4/Au-POPD/SPE; and (3) construction of GP73 aptamer/reduced graphene oxide-trimanganese tetroxide-ferrocene/gold-poly-o-phenylenediamine nanoparticles/screen printing electrode (GP73Apt/RGO-Fc-Mn3O4/Au@POPD/SPE) electrochemical aptasensor: dropping the GP73Apt solution on the RGOFc-Mn3O4/Au-POPD/SPE interface, placing it into the shaking incubator and incubating for a certain period of time, rinsing it with pure water, drying, and adding bovine serum albumin (BSA) solution on the sensing interface, and drying to obtain GP73Apt/RGO-FcMn3O4/Au@POPD/SPE electrochemical aptasensor; (C) drawing the working curve of GP73: (1) adding the standard GP73 solution dropwise to the GP73Apt/RGO-Fc-Mn3O4/Au-POPD/SPE sensing interface, reacting for a certain period of time, washing with pure water, and obtaining the working electrodes, and drying for later use, (2) placing the working electrode into phosphate buffer solution (PBS), using the differential pulse scanning method DPV scanning of the electrochemical workstation, and recording its peak current; and (3) detecting different concentrations of GP73 respectively, and drawing a working curve; and (D) detection of GP73 in actual serum samples: (1) collecting human serum samples in the hospital (GP73 concentration has been obtained by enzyme-linked immunosorbent assay (ELISA)), adding dropwise to the GP73Apt/RGO-Fc-Mn3O4/Au-POPD/SPE sensing interface, reacting for a certain period of time, washing to obtain the working electrode, and drying it for later use; (2) placing the working electrode into 5 ml PBs solution, using the DPV scan of the electrochemical workstation, and recording its peak current; and (3) according to the working curve, calculating the concentration of GP73 in the actual sample to be tested.