▎ 摘　　要

NOVELTY - Non-diagnostic target detection of glypican-3 using ratiometric electrochemical sensor of reduced graphene oxide (RGO)-cuprous oxide nanocomposite material and molybdenum disulfide-ferrocene composite material involves (i) preparing RGO-cuprous oxide nanocomposite solution, (ii) preparing molybdenum disulfide-ferrocene-aptamer detection probe, (iii) activating screen-printed electrode (SPE), depositing the activated SPE into chloroauric acid solution, washing to obtain gold nanoparticles/SPE, dripping RGO-cuprous oxide solution on gold nanoparticles/SPE, obtaining RGO-cuprous oxide/gold nanoparticles/SPE, adding bovine serum albumin (BSA) solution to the surface of prepared molybdenum disulfide-ferrocene-aptamer/RGO-cuprous oxide/gold nanoparticles/SPE, washing, and drying to obtain an electrochemical sensing interface, (iv) drawing working curve, and (v) calculating the concentration of glypican-3 in the actual serum sample to be tested based on the obtained working curve. USE - Non-diagnostic target detection of glypican-3 (GPC3) using ratiometric electrochemical sensor of reduced graphene oxide (RGO)-cuprous oxide nanocomposite (NC) material and molybdenum disulfide-ferrocene composite material. ADVANTAGE - The method provides high specific surface area and high electron transfer efficiency of reducing oxidation graphene (RGO) and excellent catalytic performance of nano cuprous oxide particles, synthesizing RGO-cuprous oxide composite material with stable differential pulse voltammetry (DPV) electric signal, and the redox electric signal of cuprous oxide can be used as a reference signal. The method improves the detection sensitivity and enhances specificity. The electrochemical aptamer sensor requires simple preparation, and ensures simple structure and wide detection range. DETAILED DESCRIPTION - Non-diagnostic target detection of glypican-3 (GPC3) using ratiometric electrochemical sensor of reduced graphene oxide (RGO)-cuprous oxide nanocomposite (NC) material and molybdenum disulfide-ferrocene composite material involves (i) dissolving graphene oxide in ultrapure water, adding copper nitrate and sodium hydroxide, adding hydrazine, and stirring for 2 hours under nitrogen-filled environment to obtain RGO-cuprous oxide nanocomposite solution, (ii) dissolving molybdenum disulfide in dimethylformamide (DMF), ultrasonically crushing for 2 hours, adding mercaptoethylamine to obtain molybdenum disulfide solution, stirring completely, centrifuging the mixed solution, separating the supernatant, and washing twice to obtain molybdenum disulfide-amine product, adding N-hydroxy succinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) into ultrapure water, uniformly stirring to prepare EDC/NHS solution, adding ferrocenoic acid and molybdenum disulfide-amine product into EDC/NHS solution, uniformly stirring, centrifuging at room temperature, separating the supernatant, washing thrice to obtain molybdenum disulfide-ferrocene composite solution, ultrasonically mixing aminated glypican-3 aptamer, molybdenum disulfide-ferrocene composite solution and EDC/NHS solution in a ratio of 1:2:1, heat-preserving at room temperature, slowly adding sodium chloride solution, aging, centrifuging the solution to remove free aptamer, and dispersing the residue in tris-EDTA buffer solution to obtain molybdenum disulfide-ferrocene-aptamer detection probe, (iii) placing screen-printed electrode (SPE) in sulfuric acid for activation by circulating voltammetry scanning, depositing the activated SPE into chloroauric acid solution by current-time (i-t) technology for constant potential deposition, washing with ultrapure water to obtain gold nanoparticles/SPE, dripping RGO-cuprous oxide solution on gold nanoparticles/SPE, heat-preserving for 30 minutes at 37℃, washing with phosphate-buffered saline (PBS) to obtain RGO-cuprous oxide/gold nanoparticles/SPE, dripping molybdenum disulfide-ferrocene-aptamer solution on RGO-cuprous oxide/gold nanoparticles/SPE, heat-preserving for 30 minutes at 37℃, washing with PBS to obtain molybdenum disulfide-ferrocene-aptamer/RGO-cuprous oxide/gold nanoparticles/SPE, adding bovine serum albumin (BSA) solution to the surface of molybdenum disulfide-ferrocene-aptamer/RGO-cuprous oxide/gold nanoparticles/SPE, heat-preserving at 37℃ to block non-specific binding site, washing, and drying to obtain an electrochemical sensing interface, (iv) dripping standard glypican-3 sample on the electrochemical sensing interface, heat-preserving at constant temperature, washing and drying to obtain glypican-3/molybdenum disulfide-ferrocene-aptamer/RGO-cuprous oxide/gold nanoparticles/SPE working electrode, placing the working electrode into 5 mL PBS buffer solution, using differential pulse voltammetry (DPV) for scanning, recording the detection signal and the reference signal of the sensor, detecting glypican-3 with different concentrations, recording the detection current (IFc) and reference current (ICu), calculating the ratio of the detection current (IFc) and reference current (ICu), drawing the working curve, and calculating the minimum detection limit, and (v) dripping an actual human serum sample solution at the electrochemical sensing interface, heat-preserving for a period of time, washing to obtain working electrode, placing the working electrode into 5 mL PBS buffer solution, using DPV for scanning, recording the detection current (IFc) and the reference signal current (ICu), calculating the ratio of the detection current (IFc) to the reference current (ICu), and calculating the concentration of glypican-3 in the actual serum sample to be tested based on the obtained glypican-3 working curve.