▎ 摘　　要

NOVELTY - Detecting glypican 3 (GPC3) by light-addressable potentiometric sensor (LAPS) based on nanocomposite materials comprises (a) taking 30 mg of graphene oxide (GO), adding 30 ml ultrapure water, ultrasonically breaking, then adding 10 mg ascorbic acid (AA), and stirring for 12 hours to obtain reduced graphene oxide (rGO), placing the mixed solution in a 60 degrees C water bath for 4 hours, centrifuging, washing, and drying to obtain hemin-rGO composite nanomaterials, (b) placing LAPS chip in an ethanol solution for 10 minutes, placing it in an acetone solution for ultrasonic washing for 10 minutes, then placing it in ultrapure water for ultrasonic washing for 10 minutes, and drying, (c) soaking the above-mentioned LAPS sensor into a phosphate-buffered saline buffer with pH of 6.5 and concentration of 2 mol/l, under the action of the applied bias voltage, and (d) calculating the concentration of GPC3 in the sample to be tested according to the working curve of GPC3. USE - The method is useful for detecting GPC3 by LAPS based on nanocomposite materials. ADVANTAGE - The method utilizes specific recognition effect between GPC3Apt and GPC3 to cause the change between the potentials in the LAPS sensitive unit to realize the detection of GPC3 with minimum detection limit of 0.212 ng/ml. DETAILED DESCRIPTION - Detecting glypican 3 (GPC3) by light-addressable potentiometric sensor (LAPS) based on nanocomposite materials comprises (a) (1) taking 30 mg of graphene oxide (GO), adding 30 ml ultrapure water, ultrasonically breaking, then adding 10 mg ascorbic acid (AA), and stirring for 12 hours to obtain reduced graphene oxide (rGO), (2) dissolving 30 mg hemin in 10 mu l aqueous ammonia and adding 30 ml ultrapure water, mixing hemin solution and rGO solution, then adding 8 mu l hydrazine hydrate solution, vortexing for 10 minutes, placing the mixed solution in a 60 degrees C water bath for 4 hours, then centrifuging at 12000 revolutions/minute for 10 minutes, removing supernatant, washing, and drying to obtain hemin-rGO composite nanomaterials, and (3) adding 2 ml 0.2% PDD solution and 5 ml 0.2 mol/l sodium chloride solution to 10 ml 0.5 mg/ml hemin-rGO solution, stirring and reacting for 12 hours, centrifuging and washing to obtain the phthalate diethylene glycol diacrylate (PDDA)-modified hemin-rGO solution, adding 2 ml 20 mmol/l sodium hexachloroplatinate solution and 2 ml 20 mmol/l sodium tetrachloropalladate solution to the PDDA modified 0.5 mg/ml hemin-rGO solution, stirring for 12 hours, then adding 10 ml ethylene glycol (EG) solution to the solution for mixing, adjusting the pH value of the mixed solution to 12 using with 1 mol/l sodium hydroxide solution, refluxing at 140 degrees C for 4 hours, centrifuging and washing to obtain heme-reduced graphene oxide-platinum-palladium (H-rGO-Pt-Pd) nanoparticles composite nanomaterials, (b) (1) placing LAPS chip in an ethanol solution for 10 minutes, placing it in an acetone solution for ultrasonic washing for 10 minutes, then placing it in ultrapure water for ultrasonic washing for 10 minutes, and drying, (2) adding 6 mu l 1 mol/l sodium hydroxide solution dropwise to the surface of the chip after pretreatment, rinsing using pure water after 30 minutes, then adding dropwise 6 mu l amino propyl triethoxy silane (APTES) solution with a mass fraction of 1%, placing in a refrigerator at 4 degrees C for 12-24 hours, washing using pure water for three times, then dropping 6 mu l 2.5% glutaraldehyde solution on the surface of the chip, and coupling for 30 minutes, (3) adding 6 mu l H-rGO-Pt-Pd nanoparticles solution dropwise to the chip prepared under the above conditions, incubating at 25 degrees C for 3 hours, and rinsing using pure water, and (4) dropping 6 mu l GPC3Apt solution with concentration of 5 mu mol/l on the above chip, incubating at 25 degrees C for 2 hours, then washing, then adding dropwise 6 mu l 1% bovine serum albumin solution, washing and drying after 30 minutes, and obtaining the LAPS chip sensitive unit interface, (c) (1) adding 6 mu l GPC3 solution dropwise to the interface of the LAPS chip sensitive unit, incubating at 25 degrees C for 1 hour and taking it out to make LAPS sensor, (2) soaking the above-mentioned LAPS sensor into a phosphate-buffered saline buffer with pH of 6.5 and concentration of 2 mol/l, under the action of the applied bias voltage, due to the specific recognition between GPC3Apt and GPC3, the potential of the LAPS sensitive unit changes, making the I-V curve of the LAPS sensor produce a corresponding offset, using the LabVIEW (RTM: System-design platform and development environment for a visual programming language) platform to record the voltage offset of the LAPS sensor with different GPC3 concentrations, and drawing the working curve, and (d) (1) adding 6 mu l sample solution to be tested to the LAPS chip sensitive unit obtained in step (b), incubating at 25 degrees C for 1 hour, then taking out to make the LAPS sensor, soaking the LAPS sensor in a phosphate-buffered saline buffer with a pH of 6.5 and a concentration of 2 mol/l, under the action of the applied bias voltage, the I-V curve of the LAPS sensor produces a corresponding offset, and using the LabVIEW (RTM: System-design platform and development environment for a visual programming language) platform to record the voltage offset value of the LAPS sensor, and (2) calculating the concentration of GPC3 in the sample to be tested according to the working curve of GPC3 obtained in step (c).