CN114965637B - A method for detecting GPC3 by constructing a sandwich-type aptasensor based on nanocomposite materials - Google Patents

Abstract

Method for detecting GPC3 by constructing sandwich type aptamer sensor based on nanocomposite, modifying gold-reduced graphene oxide (Au NPs@rGO) on surface of screen printing electrode, and fixing GPC3 aptamer (GPC 3) by physical adsorption _Apt ) Preparing H-rGO-Pt@Pd NPs-GPC3 by taking heme-reduced graphene oxide-Pt@Pd (H-rGO-Pt@Pd NPs) as a carrier _Apt And (3) a signal probe, and constructing the sandwich type electrochemical nano aptamer sensor. The effective current amplification is carried out by utilizing the peroxidase property of the H-rGO-Pt@Pd NPs nanocomposite material to catalyze Ag deposition, the DPV method is adopted to scan, the peak current is recorded, the detection of GPC3 is realized, and the minimum detection limit is 0.4801 mug/mL.

Description

A sandwich-type aptasensor based on nanocomposite materials to detect GPC3 method

technical field

The invention belongs to the field of biological detection, and in particular relates to a method for detecting GPC3 by constructing a sandwich-type aptamer sensor based on nanocomposite materials.

Background technique

Glypican-3 (glypican-3, GPC3) detection methods include ELISA, radioimmunosorbent assay, fluorescent immunoassay, chemiluminescence immunoassay, electrochemical immunosensor, etc. The invention patent with the publication number CN 112014577A discloses an immune kit, which uses magnetic bead microparticles coupled with reagent M diluted with mouse anti-human GPC3 monoclonal antibody and reagent M diluted with alkaline phosphatase-labeled rabbit anti-human GPC3 polyclonal antibody R is coupled to detect GPC3 in serum. This method has high sensitivity, but the preparation process is cumbersome and the cost is high. The invention patent with the publication number CN 105717104 A uses a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with liver cancer, uses cell wax block technology to make thin-layer sections, and then detects GPC3. This method has good stability and specificity, but The instruments used are expensive, the operation is complicated, and professional technicians are required to detect. Therefore, it is necessary to establish a rapid, sensitive and portable method for detecting GPC3.

Contents of the invention

The technical problem to be solved by the present invention is to provide a sandwich-type aptamer sensor based on heme-reduced graphene oxide-platinum@palladium nanocomposite (H-rGO-Pt@Pd NPs) to realize GPC3 detection, with the lowest The detection limit was 0.4801 μg/mL.

In order to solve this technical problem, H-rGO-Pt@Pd NPs nanocomposites with high specific surface area, high conductivity and peroxidase-like properties were synthesized by π-π bond adsorption, and the NPs combined by π-π bonds The method combines the nanocomposite material with the GPC3 aptamer (GPC3 _Apt ) to prepare the H-rGO-Pt@Pd NPs-GPC3 _Apt signal probe that can specifically bind to GPC3; the Au NPs@rGO is modified by electrodeposition technology On the surface of the screen-printed electrode; the GPC3 aptamer was modified on the surface of the Au NPs@rGO electrode by electrostatic adsorption. Since GPC3 can specifically bind to the GPC3 aptamer to form a stable chemical structure, the H-rGO-Pt @Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt / Au NPs@rGO/SPCE sandwich type electrochemical aptamer sensor. Dropping hydrogen peroxide (H ₂ O ₂ ) and silver nitrate (AgNO ₃ ) on the electrode surface, H-rGO-Pt@Pd NPs can effectively catalyze the reaction of H ₂ O ₂ and AgNO ₃ due to their peroxidase-like properties. Ag ⁺ gets electrons and is reduced to metal Ag and deposited on the electrode surface. The current response of the deposited Ag sensor is detected by differential pulse voltammetry (DPV). The current response of Ag is positively correlated with the concentration of GPC3, and the detection of GPC3 is realized.

The present invention carries out according to the following steps:

Step 1: Preparation of H-rGO-Pt@Pd NPs-GPC3 _Apt signaling probe

(1) Preparation of reduced graphene oxide (rGO): Dissolve graphene oxide (GO) in pure water and ultrasonically crush it; add ascorbic acid (AA) and stir to obtain rGO suspension;

(2) Preparation of heme-reduced graphene oxide (H-rGO): take heme (Hemin) and dissolve it in ammonia water (NH ₃ ·H ₂ O), add rGO solution and hydrazine hydrate (N ₂ H ₄ ·H ₂ O) solution, stirred in a water bath, centrifuged, and washed to obtain a H-rGO suspension;

As an improvement, the following steps are also included:

(3) Preparation of H-rGO-Pt@Pd NPs: Add polydiallyldimethylammonium chloride (PDDA) and sodium chloride (NaCl) to the H-rGO suspension, stir, and add tetrachloropalladium sodium chloride (Na ₂ PdCl ₄ ) and sodium tetrachloroplatinate (Na ₂ PtCl ₄ ) and continue to stir, then add hydrazine hydrate (N ₂ H ₄ ·H ₂ O) solution and stir, store, centrifuge, wash, dry, Obtain H-rGO-Pd@Pd NPs solid;

(4) Preparation of H-rGO-Pt@Pd NPs-GPC3 _Apt signal probe: GPC3 aptamer (GPC3 _Apt ) solution and H-rGO-Pt@Pd NPs solution were uniformly mixed by ultrasonic, incubated, centrifuged and washed to obtain H-rGO-Pt@Pd NPs-GPC3 _Apt signaling probe.

Step 2: Modification of electrodes and construction of biosensing interface

(1) Activate the screen-printed electrode (SPCE) in dilute sulfuric acid (H ₂ SO ₄ ) solution;

(2) The activated SPCE was placed in a mixture containing auric acid (HAuCl ₄ ) and GO for electrodeposition to obtain an AuNPs@rGO/SPCE electrode;

(3) Add GPC3 _Apt dropwise on the surface of Au NPs@rGO/SPCE, incubate, wash, and dry to obtain GPC3 _Apt /AuNPs@rGO/SPCE.

Step 3: Drawing of GPC3 working curve

(1) Add different concentrations of GPC3 standard solutions onto the GPC3 electrochemical biosensing interface, incubate, wash, and dry to obtain GPC3/GPC3 _Apt /Au NPs@rGO/SPCE;

(2) Add H-rGO- _Pt @Pd NPs-GPC3 _Apt solution dropwise on GPC3/GPC3 Apt /Au NPs@rGO/SPCE, incubate, wash and dry to obtain H-rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt /Au NPs@rGO/SPCE;

(3) Add the H ₂ O ₂ and AgNO ₃ solution dropwise onto the H-rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/ GPC3 _Apt /Au NPs@rGO/SPCE, react in the dark, and wash to obtain the working electrode Ag /H-rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt /AuNPs@rGO/SPCE, spare;

(4) Immerse the working electrode in the glycine-sodium hydroxide (NaOH) buffer solution of nitric acid (HNO ₃ ) and potassium nitrate (KNO ₃ ), scan with the DPV of the electrochemical workstation, and record the response current value of the sensor;

(5) Test the GPC3 working electrodes with different concentrations, record the response current of the sensor, and draw the standard working curve according to the relationship between the current response value of the sensor and the GPC3 concentration; calculate the minimum detection limit of the method.

Step 4: Detection of GPC3 in actual serum samples

(1) Immerse the working electrode prepared by the actual serum sample to be tested into the glycine-NaOH buffer solution containing HNO ₃ and KNO ₃ , scan with the DPV of the electrochemical workstation, and record the response current value of the sensor;

(2) Calculate the concentration of GPC3 in the actual sample to be tested according to the working curve obtained in step 3.

As preferred:

The DNA sequence of the GPC3 _Apt described in step 1 is 5'-TAA CGC TGA CCT TAG CTG CAT GGC TTTACA TGT TCC A-NH ₂ -3';

The concentration of the _HAuCl4 solution used to deposit gold in step 2 is 0.1%, the concentration of the rGO suspension is 1.0 mg/mL, and when the mixing volume ratio is 1:8, the conductivity is the best, and the current value reaches 63.9 μA;

The incubation temperature of the electrode described in steps 3 and 4 was 25 °C, the incubation time was 30 min, and the response current of the sensor was maximum when the pH of the PBS buffer was 7.0.

Among them, step 1 provides a H-rGO-Pt@Pd NPs nanocomposite with high specific surface area, high conductivity and peroxidase-like properties, which is combined with GPC3 _Apt through π-π bonds to form H-rGO -Pt@Pd NPs-GPC3 _Apt nanosignal probe, providing a detection signal for step 2; step 2 constitutes a sandwich-type biosensing interface that specifically recognizes GPC3, using the specific binding of GPC3 aptamer and GPC3 protein and H -rGO-Pt@Pd NPs nanocomposites have both peroxidase-like properties, which are beneficial for electron transfer and catalysis. The construction of the biosensing interface in step 2 provides a feasible condition for the determination of GPC3, which is an essential prerequisite step in the electrochemical detection of GPC3 in step 3 and step 4. It can be seen that steps 1-4 are progressive, and each step is indispensable, so H-rGO-Pt@Pd NPs nanocomposites, GPC3 aptamers (GPC3 _Apt ) and Au NPs@rGO nanohybrids can be used to achieve sandwich type Detection of GPC3 by aptasensor.

Compared with the prior art, the present invention has the following advantages:

1. The H-rGO-Pt@Pd NPs nanocomposite material constructed in this patent has the characteristics of large specific surface area, excellent peroxidase-like catalytic activity and good biocompatibility. The material has conductive substance Pt@Pd NPs, enhance the electron transfer efficiency. In addition, the efficient peroxidase-like properties of H-rGO-Pt@Pd NPs nanocomposites can effectively catalyze the redox reaction between H ₂ O ₂ and AgNO ₃ , and reduce Ag ⁺ in AgNO ₃ to metal Ag deposited on On the electrode surface, the effective current amplification is performed, and the current response of the deposited Ag sensor is detected by the DPV method to realize the sensitive detection of the GPC3 protein.

2. Using Au NPs@rGO and H-rGO-Pt@Pd NPs nanocomposites to modify the electrode surface can not only effectively immobilize GPC3 _Apt , but also have excellent electrical conductivity, enhance electron transfer, and effectively amplify the detection signal.

3. Using Hemin in the H-rGO-Pt@Pd NPs nanocomposite as the electroactive substance, construct H-rGO-Pt@Pd NPs-GPC3 _Apt as the recognition probe, and design a sandwich of GPC3 _Apt as the capture probe A method for detecting GPC3 with a type electrochemical sensor. The sandwich structure is more stable, with high precision and good stability, which is conducive to the detection of target molecules. The minimum detection limit of this method is 0.4801 μg/mL.

Description of drawings

Fig. 1 Schematic diagram of the sandwich-type electrochemical aptasensor based on H-rGO-Pt@Pd NPs nanomaterials to detect GPC3;

Fig. 2 SEM image and XRD image of H-rGO-Pt@Pd NPs nanocomposite;

Fig. 3 Verification diagram of peroxidase-like properties of H-rGO-Pt@Pd NPs nanocomposites;

Figure 4 H-rGO-Pt@Pd NPs-GPC3 _Apt signal probe verification map;

Fig.5 SEM image of electrode modification process;

Fig. 6 DPV profiles of different GPC3 concentrations.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

The principle of a sandwich-type electrochemical aptasensor based on H-rGO-Pt@Pd NPs nanomaterials to detect GPC3 is shown in Figure 1. First prepare the H-rGO-Pt@Pd NPs nanocomposite material, and then use the material to immobilize GPC3 _Apt to form H-rGO-Pt@Pd NPs-GPC3 _Apt signal probe; then activate the SPCE electrode and pass the Au NPs@rGO through Electrodeposition technology modified the surface of the activated electrode to enhance the conductivity of the electrode; GPC3 specifically combined with H-rGO-Pt@Pd NPs-GPC3 _Apt and GPC3 _Apt to form a stable chemical structure and construct H-rGO-Pt @Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt /AuNPs@rGO/ SPCE Sandwich Electrochemical Aptamer Sensor; Catalysis of H ₂ O ₂ via Peroxidase-like Properties of H-rGO-Pt@Pd NPs Nanocomposites React with AgNO ₃ , H ₂ O ₂ can reduce Ag ⁺ in AgNO ₃ to metal Ag and deposit on the electrode surface, use the electrochemical workstation DPV method, record the current signal before and after the detection of GPC3, and then draw according to the relationship between the GPC3 concentration and the response current The working curve is used to obtain the level of GPC3 in the serum to achieve the purpose of detecting GPC3.

The implementation steps are as follows:

1. Preparation of H-rGO-Pt@Pd NPs-GPC3 _Apt signaling probe

(1) Weigh 30.0 mg GO and disperse it in 30.0 mL pure water, break it up, add 30.0 mg ascorbic acid (AA), and stir it magnetically for 3 h to prepare a 1.0 mg/mL rGO suspension; then add the rGO suspension to 30.0 mL of Hemin solution, stirred for 30 min, added 5.0 μL of hydrazine hydrate solution, vortexed, 50 °C water bath for 2 h; centrifuged and washed to obtain H-rGO suspension.

(2) Add 2.0 mL of 2% PDDA solution and 2.0 mL of 2% NaCl solution to 30.0 mL of 1.0 mg/mL H-rGO suspension, and stir for 18 h; mix 1.0 mL of Na ₂ PdCl ₄ and 1.0 mL of Na ₂ PtCl ₄ was added to the H-rGO mixed solution, and then 10.0 μL of hydrazine hydrate solution was added, and stirred for 18 h to obtain the H-rGO-Pt@Pd NPs nanocomposite.

The H-rGO-Pt@Pd NPs nanocomposite was characterized by the SU8020 scanning electron microscope (SEM) produced by Hitachi, Japan, as shown in Figure 2A, it can be seen from the figure that small particles are attached to the surface of the wrinkled film, indicating that Pt and Pd particles were successfully attached on the H-rGO material. The H-rGO-Pt@Pd NPs nanocomposite was characterized by the D8 ADVANCE X-ray diffractometer produced by Bruker, USA. As shown in Figure 2B, the C(002) crystal plane of rGO is in phase with the diffraction peak at 2θ=23.51° Correspondingly, (111), (200) and (220) of Pt correspond to the diffraction peaks of 2θ=39.76°, 46.24°, 67.45°, and (111), (200) and (220) of Pd correspond to 2θ=40.12° , 46.66°, 68.12° diffraction peaks correspond to each other, indicating that the H-rGO-Pt@Pd NPs nanocomposite was successfully prepared.

The CHI660E electrochemical workstation produced by China Shanghai Chenhua Instrument Co., Ltd. was used to verify the peroxidase-like properties of H-rGO-Pt@Pd NPs nanocomposites with Shanghai Hitachi UH5300 UV-Vis spectrophotometer, as shown in Figure 3 . Figure 3A is the verification of the color reaction of peroxidase-like properties of H-rGO-Pt@Pd NPs, alone 3,3',5,5'-tetramethylbenzidine (TMB) (a) and alone H ₂ O ₂ (b) is colorless and transparent. When TMB and H ₂ O ₂ (c) are mixed, a color reaction occurs, showing a light blue color. Adding H-rGO-Pt@Pd NPs to TMB (d) and H Natural color when ₂ O ₂ (e), dark blue when H-rGO-Pt@Pd NPs are mixed with TMB and H ₂ O ₂ (f), light blue tube c compared with dark blue f tube , indicating that H-rGO-Pt@Pd NPs have peroxidase-like properties and can catalyze the reaction of TMB and H2O2. Figure 3B is the UV verification of the peroxidase-like properties of H-rGO-Pt@Pd NPs, a, b, d, e have no absorption peaks, c has a small peak, and f has a strong absorption peak, indicating that TMB and H ₂ O ₂ A color reaction occurs, and the nanocomposite H-rGO-Pt@Pd NPs has a catalytic effect, further indicating that the nanocomposite has a strong catalase-like property.

(3) Mix 50.0 μL of GPC3 _Apt with a concentration of 5.0 μmol/L and 100.0 μL of a H-rGO-Pt@Pd NPs solution with a concentration of 1.0 mg/mL, incubate at 4°C for 12 h, and wash to obtain H-rGO- Pt@Pd NPs-GPC3 _Apt signaling probe.

The H-rGO-Pt@Pd NPs-GPC3 _Apt signal probe was verified by using a UH5300 UV-Vis spectrophotometer from Shanghai Hitachi, China. As shown in Figure 4, the wavelength of the amino GPC3 aptamer (NH ₂ -GPC3 _Apt ) was at 260 nm There are obvious absorption peaks at 260 nm and 397.5 nm for H-rGO-Pt@Pd NPs, and the probe and the centrifuged probe supernatant have absorption peaks at around 260 nm and 400 nm, and the probe absorbs The peak intensity was greater than that of the probe supernatant, indicating that H-rGO-Pt@Pd NPs were successfully combined with NH ₂ -GPC3 _Apt . The formula for calculating the binding rate is as follows:

K=(X ₀ -X)/X ₀ ×100%

Among them, X ₀ and X are the absorbance intensity of the signal probe (H-rGO-Pt@Pd NPs- GPC3 _Apt ) and the supernatant of the signal probe at the same concentration and the same amount, respectively, and the binding rate K=82.06% is obtained by calculation.

, Modification of electrodes and construction of biosensing interface

(1) Immerse the electrode (SPCE) in dilute H ₂ SO ₄ , and scan 20 laps of CV at a speed of 0.5 V/s at a scanning voltage of 0.4-1.2 V; put the pretreated electrode into 1 mL In the solution containing 0.01% HAuCl ₄ and 1.0 mL of rGO solution with a concentration of 1.0 mg/mL, using the it technology in the electrochemical workstation, deposited at a scanning voltage of 0-1.2 V for 120 s, washed and dried to obtain Au@ rGO/SPCE.

(2) Add 2.0 μL of GPC3 _Apt with a concentration of 5.0 μM onto the surface of Au NPs@rGO/SPCE electrode, incubate at 25 °C for 30 min, wash and dry; add 2.0 μL of 1% BSA solution on the electrode to block Active sites were incubated at 25°C for 30 min, washed and dried to obtain GPC3 _Apt /Au NPs@rGO/SPCE.

, Drawing of GPC3 working curve

(1) Add GPC3 standard solution in the concentration range of 1.0 μg/mL~70.0 μg/mL to the GPC3 electrochemical biosensing interface constructed in step 2, and incubate at 25 °C for 1 h to obtain GPC3/GPC3 _Apt /Au NPs@rGO / SPCE.

(2) Add 3.0 μL of 0.63 mg/mL H-rGO-Pt@Pd NPs-GPC3 _Apt solution dropwise on the sensing interface prepared in step (1), incubate at 25 °C for 1 h, wash and dry to obtain H- rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt /AuNPs@rGO/SPCE.

(3) Add 2.0 µL of 100 mM H ₂ O ₂ and 1.0 µL of 50.0 mM AgNO ₃ solution dropwise on the above electrode, react in the dark at 25 °C for 30 min, wash, dry, and set aside to get working Electrode Ag/H-rGO-Pt@PdNPs-GPC3 _Apt /GPC3/GPC3 _Apt /Au NPs@rGO/SPCE.

(4) Put the above electrode into the glycine-NaOH buffer solution of HNO ₃ and KNO ₃ , use the DPV of the electrochemical workstation to detect the concentration of GPC3 in the range of 1.0 μg/mL~70.0 μg/mL, and record the peak current. The DPV curve of GPC3 concentration is shown in Fig. 6 . Its linear equation is Y=0.282183X+22.64276 (Y is the response current, X is the concentration of GPC3), the correlation coefficient R ² =0.99564, the standard deviation is calculated to be 0.045 by measuring the blank sample many times, and through the formula, LOD=3S _b /b, where S _b is the standard deviation measured in six blank groups, b is the slope of the standard curve, and the lowest detection limit of sensitivity calculated by this method is 0.4801 μg/mL.

The electrode construction process was characterized by scanning electron microscopy (SEM), as shown in Figure 5. Figure 5A is the SEM image of SPCE, the surface of the electrode shows uniformly arranged particles due to its inherent carbon particles; Figure 5B is the SEM image of Au NPs@rGO/SPCE, it can be seen that the electrode is covered with a black film and uniformly distributed Many bright white spherical particles indicate that AuNPs@rGO has been successfully deposited on the electrode surface; Figure 5C is the SEM image of GPC3 _Apt /Au NPs@rGO/SPCE, it can be seen that the surface is covered with a thin film, which shows that GPC3 _Apt has been successfully immobilized on the electrode surface. It can be seen in Figure 5D that the surface of the film is relatively smooth, which is attributed to the reaction between GPC3 and GPC3 _Apt to form a stable structure, which shows that GPC3 is successfully adsorbed on the electrode surface. Figure 5E shows H-rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt /Au NPs@rGO/SPCE, the surface presents a typical wrinkled structure, and many spherical nanoparticles are wrapped, proving that H-rGO-Pt@Pd NPs-GPC3 _Apt has been uniformly modified on the electrode surface. Figure 5F shows Ag/H-rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/ GPC3 _Apt /Au NPs@rGO/SPCE, with bright particles on the surface, indicating that Ag has been effectively deposited on the surface.

, Detection of GPC3 in actual serum samples

(1) The level of GPC3 in human serum samples was detected under optimal conditions by the addition method. Mix normal human serum samples with 10.0 μg/mL, 20.0 μg/mL, and 40.0 μg/mL GPC3 standard solutions at a ratio of 1:1 to make a mixed solution.

(2) Add 2.0 μL of the mixed solution dropwise to the GPC3 electrochemical biosensing interface constructed in step 2, and incubate at 25 °C for 30 min, GPC3/GPC3 _Apt /Au NPs@rGO/SPCE.

(3) Add 3.0 μL of 0.63 mg/mL H-rGO-Pt@PdNPs-GPC3 _Apt solution dropwise on GPC3/GPC3 _Apt /Au NPs@rGO/SPCE, incubate at 25 °C for 1 h, wash and dry to obtain H -rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt /Au NPs@rGO/SPCE.

(4) Add 2.0 µL of 100 mM H ₂ O ₂ and 1.0 µL of 50.0 mM AgNO ₃ solution dropwise on the above electrode, react in the dark at 25 ℃ for 30 min, wash, and dry to obtain the working electrode Ag/H- rGO-Pt@Pd NPs-GPC3 _Apt /GPC3/GPC3 _Apt /Au NPs@rGO/SPCE.

(5) As described in step 3, place the working electrode in the glycine-NaOH buffer solution of HNO ₃ and KNO ₃ for DPV scanning, and record the current value. Through the GPC3 working curve obtained in step 3, the concentration of GPC3 in the human serum sample was calculated, and the results are shown in Table 1. The recovery rate was in the range of 100.96% to 121.15%, and the RSD value was 0.05% to 2.24%. The results show that the electrochemical aptasensor can be used to detect the concentration of GPC3 in actual serum samples.

Table 1 Detection results of GPC3 in actual serum samples

.

Sample 1: Normal Serum, AFP = 5.14 ng/mL

Sample 2: liver cancer serum, AFP = 88.27 ng/mL

Sample 3: liver cancer serum, AFP = 223.88 ng/mL

(Note: Serum samples were obtained from the Guangxi Key Laboratory of Metabolic Disease Research, the 924th Hospital of the Chinese People's Liberation Army (Guilin, China), and followed the requirements of the Ethics Committee of the Guangxi Metabolic Disease Research Key Laboratory of the 924th Hospital of the Chinese People's Liberation Army.).

Claims (3)

1. A method for non-diagnostic purposes based on nanocomposite materials to construct a sandwich-type aptamer sensor to detect GPC3, carried out according to the following steps: Step 1: Preparation of heme-reduced graphene oxide-platinum@palladium nanocomposite H-rGO-Pt@Pd NPs-GPC3Apt signaling probe Preparation of rGO: Pour graphene oxide GO into ultrapure water, break it evenly, add ascorbic acid AA for reduction, and obtain rGO suspension; Preparation of H-rGO: Dissolve hemin Hemin in ammonia water, add rGO suspension and hydrazine hydrate N2H4·H2O, react in water bath, and centrifuge to obtain H-rGO suspension; Preparation of H-rGO-Pt@Pd NPs: Add polydiallyldimethylammonium chloride PDDA, NaCl to H-rGO suspension, stir, then add sodium tetrachloropalladate Na2PdCl4, sodium tetrachloroplatinate Na2PtCl4 and N2H4·H2O, stirred to get H-rGO-Pt@Pd solution; Preparation of H-rGO-Pt@Pd NPs-GPC3Apt signal probe: Mix GPC3 aptamer GPC3Apt and H-rGO-Pt@Pd solution, incubate, centrifuge, remove supernatant to obtain H-rGO-Pt@Pd NPs-GPC3Apt solution; Step 2: Modification of electrodes and construction of biosensing interface (1) Activate the screen-printed electrode SPCE in dilute sulfuric acid solution; (2) Put the activated SPCE into the mixture containing auric acid HAuCl4 and GO solution for constant potential deposition to obtain Au NPs@rGO/SPCE; (3) Add GPC3Apt dropwise on the surface of Au NPs@rGO/SPCE, incubate, wash and dry to obtain GPC3Apt/AuNPs@rGO/SPCE; (4) Add H2O2 and AgNO3 solutions dropwise on the above electrode, react in the dark, wash, and dry to obtain the working electrode Ag/HrGO-Pt@Pd NPs-GPC3Apt/GPC3 /GPC3Apt/Au NPs@rGO/SPCE; Step 3: Drawing of GPC3 working curve (1) Add GPC3 standard solution dropwise to the surface of GPC3Apt/Au NPs@rGO/SPCE, incubate, wash and dry to obtain GPC3/GPC3Apt/Au NPs@rGO/SPCE; (2) Add the H-rGO-Pt@Pd NPs-GPC3Apt solution dropwise on GPC3/GPC3Apt/Au NPs@rGO/SPCE, incubate, wash, and dry to obtain the electrode H-rGO-Pt@Pd NPs-GPC3Apt/GPC3/ GPC3Apt/Au NPs@rGO/SPCE; (3) Add H2O2 and AgNO3 solutions dropwise on the obtained electrode, react in the dark, wash, and dry to obtain the working electrode Ag/HrGO-Pt@Pd NPs-GPC3Apt/GPC3 /GPC3Apt/Au NPs@rGO/SPCE; (4) Immerse the working electrode in the glycine-sodium hydroxide buffer solution containing HNO3 and KNO3, scan with DPV, and record the response current value of the sensor; (5) Detect different concentrations of GPC3 and record the peak current; draw the working curve of GPC3 according to the relationship between the current response value of the sensor and the concentration of GPC3, and calculate the minimum detection limit of the method; Step 4: Detection of GPC3 in actual serum samples (1) Immerse the working electrode prepared by the actual serum sample to be tested into the glycine-sodium hydroxide buffer solution containing HNO3 and KNO3, scan with the DPV of the electrochemical workstation, and record the response current value of the sensor; (2) Calculate the concentration of GPC3 in the actual serum sample according to the working curve obtained in step 3. 2. The method according to claim 1, characterized in that: the concentration of the H-rGO-Pt@Pd NPs nanozyme in step 1 is 1.0 mg/mL, and the H-rGO-Pt@Pd NPs - GPC3Apt signaling probe concentration is 0.63 mg/mL. 3. The method according to claim 1, characterized in that: the ratio of HAuCl4 and rGO in step 2 is 1:8; the volume ratio of H2O2 and AgNO3 is 2:1.