▎ 摘　　要

NOVELTY - Method for detecting glypican-3 (GPC3) with label-free aptamer sensor involves (I) preparing reduced graphene oxide solution, adding heme solution and hydrazine hydrate to obtain hydrogen-reduced graphene oxide, adding poly(diallyldimethylammonium chloride), sodium chloride and sodium hexachloroplatinate(IV) solution, obtaining hydrogen-reduced graphene oxide-platinum nano material, (II) placing electrode in sulfuric acid for voltammetric scanning, soaking in mixed solution containing chloroauric acid solution, reduced graphene oxide solution, glutaraldehyde and hydrogen-reduced graphene oxide-platinum nano particles solution, adding aminated GPC3 aptamer solution and bovine serum albumin solution, obtaining sensor interface, adding GPC3 solution, obtaining working electrode, taking normal human serum samples, adding mixed solution to sensor interface, and calculating the concentration of GPC3 in human serum sample. USE - Method for detecting glypican-3 with label-free aptamer sensor. ADVANTAGE - The method detects GPC3 with high load capacity, excellent electron transfer effect, high conductivity and high specific recognition effect. DETAILED DESCRIPTION - Method for detecting GPC3 with label-free aptamer sensor involves (I) (1) weighing 20 mg graphene oxide and 20 mL pure water to prepare, using ultrasonic breaker for crushing for 1 hour, adding 10 mg of ascorbic acid, stirring for 12 hours under a constant temperature magnetic stirrer, obtaining 1 mg/mL reduced graphene oxide solution, (2) taking 20 mg of heme into the beaker, adding 10 mu L ammonia and 20 mL pure water, stirring, obtaining 1 mg/mL heme solution, taking 2.0 mL heme solution and 2.0 mL reduced graphene oxide solution to mix, adding 8 mu L hydrazine hydrate, placing in a water bath at a constant temperature of 60 degrees C to react for 4 hours, centrifuging at a speed of 10000 rpm, removing the supernatant to obtain hydrogen-reduced graphene oxide, (3) taking 10 mL of hydrogen-reduced graphene oxide solution and pour it into a beaker, adding 2.0 mL of 0.2% poly(diallyldimethylammonium chloride) and 5.0 mL of 0.2 mol/L sodium chloride, stirring for 12 hours under constant temperature magnetic stirrer, adding 2.0 mL of 20 mmol/L sodium hexachloroplatinate(IV) solution, stirring for 12 hours, adding 10 mL of ethylene glycol, adjusting the pH value of the mixed solution to 12.0 with 1.0 mol/L sodium hydroxide, centrifuging at 12000 rpm to remove the supernatant, washing twice, obtaining hydrogen-reduced graphene oxide-platinum nano material, (II) (1) placing the electrode in 0.5 mol/L sulfuric acid for 20 cyclic voltammetric scanning at voltage of 0.4-1.0 V, soaking the activated screen-printed electrode in 5 mL of a mixed solution containing 0.01% chloroauric acid solution and 1.0 mg/mL reduced graphene oxide solution, placing under a magnetic stirrer for stirring, depositing at 0.4 V constant potential for 120 seconds, washing 3 times with pure water, blow drying to get reduced graphene oxide-gold nano material/screen-printed electrode, (2) soaking the reduced graphene oxide-sold nano material/screen-printed electrode with 2.5% glutaraldehyde for 15 minutes, washing with phosphate-buffered saline solution with a pH of 7.0, blow drying, adding 6 mu L of hydrogen-reduced graphene oxide-platinum nano particles solution in drops and heat-preserving for 30 minutes, washing 3 times with phosphate-buffered saline solution and pure water, drying, obtaining hydrogen-reduced graphene oxide-platinum nano particles/reduced graphene oxide-gold nano material/screen-printed electrode, (3) taking 2.0 mu L of 0.5 mu mol/L aminated GPC3 aptamer solution and adding in drops on the hydrogen-reduced graphene oxide-platinum nano particles/reduced graphene oxide-gold nano material/screen-printed electrode sensing interface, placing in a shaking incubator and heat-preserving for 2 hours, adding 6 mu L of 1% bovine serum albumin solution in drops to block, naturally drying, obtaining sensor interface, (III) (1) adding 2.0 mu L GPC3 solution of different concentrations on the sensor interface, heat-preserving for 20 minutes at 25 degrees C, washing twice with pH 7.0 phosphate-buffered saline solution and pure water, blow drying, obtaining working electrode, (2) placing the working electrode obtained above in a phosphate-buffered saline solution with a concentration of 0.2 mol/L and a pH of 7.0, using the differential pulse voltammetry scan of the CHI660E electrochemical workstation, recording its peak current, controlling the concentration of GPC3 at 0.001-10 mu g/mL, (IV) taking normal human serum samples,, adding 2.0 mu L of mixed solution in drops to the sensor interface constructed in step (II), heat-preserving for 20 minutes at 25 degrees C, obtaining working electrode of GPC3, placing the working electrode in the phosphate-buffered saline solution for differential pulse voltammetry scanning, recording current value, obtaining GPC3 working curve through step (3), and calculating the concentration of GPC3 in human serum samples. In step (III), the sensor current response value (Y') has a linear relationship with the GPC3 concentration (X), the working curve equation is given by the formula: Y = 0.4449X+0.1745, where the correlation coefficient is 0.9874.