Double-signal aptamer sensor for detecting GP73 based on MoS2-CeO2-Pd nano-enzyme

Abstract

A double-signal sandwich-type electrochemical aptamer sensor for detecting GP73 is constructed on the basis of a molybdenum disulfide-cerium oxide-palladium nano-enzyme material, a high-performance MoS2-CeO2-Pd nano-enzyme is prepared firstly, and GP73Apt is marked to form a signal probe; then, GP73Apt is incubated on the surface of the Au (at) PDA / SPE to form a capture probe, and a 3D sandwich type space structure of GP73Apt-GP73-GP73-GP73Apt is constructed through a specific recognition effect. Through the excellent electrochemical activity of the nano enzyme, an original is indicated as an electrochemical signal, the peroxidase-like property is utilized, H2O2 oxidation is catalyzed, OPD is accelerated to be catalyzed into OPDox, double-signal response current change of CeO2 and OPD is obtained by adopting DPV, high-specificity and high-sensitivity detection of GP73 is achieved, and the lowest detection limit reaches 0.12 pg / mL.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a dual-signal electrochemical aptamer sensor for detecting Golgi protein 73 based on nanoenzyme materials. Background Art

[0002] Primary hepatocellular carcinoma (HCC) is a malignant tumor with high incidence and high risk. Golgi transmembrane glycoprotein 73 (GP73) is a tumor marker for HCC. Common detection methods for GP73 include immunofluorescence technology, enzyme-linked immunosorbent assay technology, and western blotting.

[0003] The invention patent with publication number CN118259015A discloses a kit for determining the content of human GP73 based on latex immunoturbidimetry. The invention patent with publication number CN115754290A discloses a kit for detecting early liver cancer. The current detection methods have the disadvantages of high cost, poor specificity and narrow detection range. Therefore, there is an urgent need for a low-cost, high-specificity and wide-range detection scheme for GP73. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a dual-signal sandwich electrochemical aptamer sensor based on molybdenum disulfide-cerium oxide-palladium nanozyme (MoS2-CeO2-Pd) to detect GP73; the minimum detection limit reaches 0.12pg / mL.

[0005] The detection principle of the present invention is: using the high specific surface area and high conductivity of molybdenum disulfide (MoS2), combined with the excellent electrochemical activity and peroxidase-like properties of cerium oxide (CeO2), introducing precious metal palladium (Pd) nanoparticles to improve conductivity and biocompatibility, and preparing high-performance MoS2-CeO2-Pd nanozyme materials; MoS2-CeO2-Pd and GP73 aptamers (GP73 Apt ) to obtain MoS2-CeO2-Pd-GP73 Apt Signal probe; screen printed electrode (SPE) was activated by dilute sulfuric acid (H2SO4), chloroauric acid (HAuCl4) and dopamine (DA) were electropolymerized and deposited on the SPE electrode to obtain Au@PDA / SPE. Polydopamine (PDA) is a biomimetic material with strong adhesion, large specific surface area and high conductivity. Combined with gold nanoparticles, the conductivity of the electrode was further improved, which was GP73 Apt The Au-N bond was used to bind GP73 Apt Incubate on Au@PDA / SPE to obtain GP73 Apt / Au@PDA / SPE capture probe; GP73 and MoS2-CeO2-Pd-GP73 were sequentiallyApt Signal probe incubated in GP73 Apt / Au@PDA / SPE surface. Using GP73 and GP73 Apt Due to the advantage of specific binding, a stable "aptamer-protein-aptamer" sandwich structure is formed on the SPE surface. On the one hand, a redox reaction occurs between CeO2 (Ce(III) / Ce(IV)), providing an electrochemical signal. On the other hand, MoS2-CeO2-Pd has excellent peroxidase-like activity, catalyzing the oxidation of hydrogen peroxide H2O2 and o-phenylenediamine OPD to generate oxidized o-phenylenediamine OPDox, indicating another electrochemical signal. Differential pulse voltammetry (DPV) is used to obtain the oxidation response current changes of CeO2 and OPD, and the dual signals confirm each other, thereby realizing the detection of GP73.

[0006] The present invention is carried out according to the following steps:

[0007] Step 1: Preparation of MoS2-CeO2-Pd-GP73 Apt Signal Probe

[0008] (1) Preparation of molybdenum disulfide (MoS2): MoS2 is dissolved in water and ultrasonically crushed to obtain a MoS2 solution; sodium hydroxide (NaOH) is added to the MoS2 solution, stirred, hydrothermally stripped, and dried to obtain a single layer of MoS2.

[0009] (2) Preparation of molybdenum disulfide-cerium oxide (MoS2-CeO2) nanomaterials: adding cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and hydrogen peroxide (H2O2) to a mixed solution of urea (CO(NH2)2) and sodium citrate (C6H5Na3O7), stirring, and performing a hydrothermal reaction to obtain a CeO2 solution; centrifuging, washing, and drying to obtain CeO2 nanocrystals; mixing a monolayer MoS2 solution with the CeO2 solution, stirring, and drying to obtain a MoS2-CeO2 nanomaterial.

[0010] (3) Preparation of molybdenum disulfide-cerium oxide-palladium (MoS2-CeO2-Pd) nanozyme: Take MoS2-CeO2 solution, add sodium hexachloropalladate (Na2PdCl6) solution and hydrazine hydrate (N2H4) solution, stir, centrifuge, wash, and dry to obtain MoS2-CeO2-Pd nanozyme.

[0011] (4) Construction of MoS2-CeO2-Pd-GP73 aptamer Apt )Signal probe: MoS2-CeO2-Pd nanozyme and amino-modified GP73 Apt Shake and mix, incubate; centrifuge, wash; remove free GP73 in the upper layer Apt, MoS2-CeO2-Pd-GP73 was obtained Apt Solution.

[0012] Step 2: Electrode modification and construction of biosensor working electrode

[0013] (1) Activation of SPE: The SPE was placed in H2SO4, and the electrode was activated by cyclic voltammetry using an electrochemical workstation, then cleaned and dried to obtain an activated SPE.

[0014] (2) Preparation of Au@PDA / SPE: The activated SPE was immersed in a mixed solution of HAuCl4 and DA, and electropolymerization deposition was performed using an electrochemical workstation, followed by washing and drying to obtain Au@PDA / SPE.

[0015] (3) Preparation of GP73 Apt / Au@PDA / SPE capture probe: GP73 Apt Add Au@PDA / SPE surface, incubate, wash, and dry; add bovine serum albumin BSA solution, dry, and obtain GP73 Apt / Au@PDA / SPE capture probe.

[0016] (4) Preparation of GP73 / GP73 Apt / Au@PDA / SPE: Take the standard GP73 protein and add it dropwise to GP73 Apt / Au@PDA / SPE surface, incubation, washing and drying; GP73 / GP73 Apt / Au@PDA / SPE.

[0017] (5) Preparation of MoS2-CeO2-Pd-GP73 Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode: MoS2-CeO2-Pd-GP73 Apt Add dropwise to GP73 / GP73 Apt / Au@PDA / SPE surface, incubation, cleaning and drying; MoS2-CeO2-Pd-GP73 Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode.

[0018] Step 3: Draw the working curve of GP73

[0019] (1) Construction of MoS2-CeO2-Pd-GP73 using different concentrations of standard GP73 protein Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode.

[0020] (2) The working electrode was immersed in a phosphate buffered saline (PBS) solution containing hydrogen peroxide (H2O2) and o-phenylenediamine (OPD), and differential pulse voltammetry (DPV) was used to scan the electrode using an electrochemical workstation to record the oxidation current values ​​of CeO2 and OPD.

[0021] (3) GP73 solutions of different concentrations were tested and the current peaks of the dual signals were recorded respectively; based on the relationship between the current response value of the sensor and the GP73 concentration, a working curve was drawn and the minimum detection limit of the method was calculated.

[0022] Step 4: Detection of GP73 in actual serum samples

[0023] (1) Using the actual serum sample to be tested to construct the sandwich electrochemical aptamer sensor in step 2, incubate, wash, and dry to obtain a working electrode.

[0024] (2) The working electrode was placed in a PBS solution containing H2O2 and OPD, and the oxidation current values ​​of CeO2 and OPD were recorded using a DPV scan of an electrochemical workstation.

[0025] (3) According to the working curve described in step 3, the concentration of GP73 in the actual serum sample is calculated.

[0026] Furthermore, in step 1, the molar concentration ratio of MoS2 and NaOH is 1:5.

[0027] Furthermore, in step 1, a single-layer MoS2 solution is prepared, the reaction temperature is 220°C, and the time is 8 hours.

[0028] Furthermore, in step 1, a CeO2 suspension is prepared, the reaction temperature is 180°C, and the reaction time is 20 hours.

[0029] Furthermore, in step 2, the volume ratio of DA to HAuCl4 is 3:1.

[0030] Furthermore, in step 2, the concentration of the MoS2-CeO2-Pd solution is 1.0 mg / mL.

[0031] Furthermore, in step 2, the mass fraction of the BSA solution is 1.0%.

[0032] Preferably, in step 1, GP73 Apt The base sequence is 5'-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3';

[0033] Preferably, in step 3, the optimal incubation temperature of GP73 is 35° C., and the optimal incubation time is 35 min.

[0034] Preferably, in steps 3 and 4, the concentration of PBS solution is 0.2 M, the pH value is 5.5, the concentration of H2O2 is 50 mM, and the concentration of OPD is 25 mM. The DPV linear scanning range is -0.1 V to 0.9 V, and the scanning rate is 0.1 V / s.

[0035] Among them, step 1 provides a MoS2-CeO2-Pd nanozyme with high specific surface area, excellent conductivity and high electrochemical activity, and combines with GP73 Apt Combined to form MoS2-CeO2-Pd-GP73 Apt Signal probe, laying the foundation for steps 2, 3, and 4; Step 2 constructed MoS2-CeO2-Pd-GP73 Apt / GP73 / GP73 Apt / Au@PDA / SPE electrochemical aptamer sensor; Steps 3 and 4 verify the performance of the sensor in detecting GP73; Steps 1-4 confirm each other and prove that GP73 Apt Combining MoS2-CeO2-Pd nanozyme can realize dual-signal electrochemical detection of GP73.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The present invention utilizes the high specific surface area and high conductivity of molybdenum disulfide (MoS2), combines the excellent electrochemical activity and peroxidase-like properties of cerium oxide (CeO2), introduces precious metal palladium (Pd) nanoparticles to improve conductivity and biocompatibility, and prepares high-performance MoS2-CeO2-Pd nanozyme materials; GP73 Apt As recognition molecules, MoS2-CeO2-Pd-GP73 was prepared by chemical bonding Apt Signal probe; GP73 is bonded by Au-N bond Apt Incubate with Au@PDA / SPE to construct GP73 Apt / Au@PDA / SPE capture probe; using GP73 and GP73 Apt The specific binding advantage forms a stable "aptamer-protein-aptamer" sandwich 3D structure on the SPE surface. On the one hand, the excellent electrochemical activity of CeO2 (Ce(III) / Ce(IV)) is utilized to generate a redox reaction and obtain the electrochemical oxidation signal of CeO2; the peroxidase-like properties of MoS2-CeO2-Pd are utilized to catalyze the oxidation of hydrogen peroxide H2O2 and o-phenylenediamine OPD to generate oxidized o-phenylenediamine OPDox, and the electrochemical oxidation signal of OPD is obtained, thereby constructing a dual-signal sandwich electrochemical aptamer sensor to detect GP73. The sensor has strong specificity, high stability, good reproducibility, and the minimum detection limit can reach 0.16pg / mL (I CeO2) and 0.12 pg / mL(I OPD ), providing new ideas for HCC diagnosis and prognosis detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of dual-signal electrochemical aptasensor for detecting GP73 based on MoS2-CeO2-Pd;

[0039] Figure 2 Scanning electron microscopy (SEM) images of MoS2-CeO2-Pd, (A) CeO2, (B) MoS2-CeO2, (C) MoS2-CeO2-Pd;

[0040] Figure 3 Scanning electron microscopy (SEM) characterization images of different modification processes on the electrode surface;

[0041] Figure 4 DPV curves of the MoS2-CeO2-Pd based electrochemical sensor detecting different GP73 concentrations. DETAILED DESCRIPTION

[0042] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] A dual-signal electrochemical aptasensor for detecting GP73 based on MoS2-CeO2-Pd nanozyme. The detection principle is shown in Figure 1 The high-performance MoS2-CeO2-Pd nanozyme material was prepared by hydrothermal method and urea precipitation method; the amino-modified GP73 Apt Through π-π stacking on MoS2-CeO2-Pd, MoS2-CeO2-Pd-GP73 is formed Apt As a signal probe; Au@PDA / SPE was prepared by electropolymerization deposition to further enhance the conductivity of SPE; GP73 was bonded by Au-N bond Apt Incubated on the Au@PDA / SPE electrode surface to form GP73 Apt / Au@PDA / SPE as capture probe. Apt GP73 solution was added to the surface of / Au@PDA / SPE to form GP73 / GP73 Apt / Au@PDA / SPE interface; MoS2-CeO2-Pd-GP73 Apt Fixed on GP73 / GP73 Apt / Au@PDA / SPE surface, forming a stable "aptamer-protein-aptamer" sandwich structure. As the concentration of GP73 increases, more GP73 is captured on the electrode surface, resulting in more MoS2-CeO2-Pd-GP73 AptThe probe is fixed, thus generating a stronger CeO2 oxidation current. At the same time, due to the peroxidase-like activity of MoS2-CeO2-Pd, H2O2 catalyzes the oxidation of OPD, causing the oxidation current peak of OPD to increase. The oxidation current values ​​of CeO2 and OPD are positively correlated with the concentration of GP73 and can be used for accurate quantitative detection of GP73. The specific steps are as follows:

[0044] 1. MoS2-CeO2-Pd-GP73 Apt Preparation of signal probe

[0045] (1) 32.04 mg of MoS2 was dissolved in 10.0 mL of ultrapure water and ultrasonically crushed for 2 h; 400 mg of NaOH was dissolved in 10.0 mL of ultrapure water, uniformly mixed with the MoS2 solution, and stirred for 12 h. The mixed solution was transferred into a high-pressure reactor and reacted at 220° C. for 8 h. After cooling, a single-layer MoS2 solution was obtained, which was centrifuged, washed, and dried to obtain a single-layer MoS2 solid.

[0046] (2) Take 0.105g Ce(NO3)3·6H2O and 0.09mL 30% H2O2 solution and add it to 17mL 0.1MCO(NH2)2 and 4mL 0.01M C6H5Na3O7 solution, stir for 45min; transfer the mixed solution into a high pressure reactor, react at 180℃ for 20h, and obtain CeO2 colloidal suspension after cooling; centrifuge, wash and dry to obtain CeO2 nanocrystals. Take 10.0mL MoS2 solution and 5.0mL CeO2 colloidal suspension, mix, add 50mg PVP-K30, stir for 12h, obtain MoS2-CeO2 nano solution, centrifuge, wash and dry to obtain MoS2-CeO2 nano material.

[0047] (3) Take 10.0 mL of the MoS2-CeO2 solution obtained above, add 2.0 mL of 20 mM Na2PtCl6 and 5.0 μL of hydrazine hydrate N2H4, stir for 12 h, centrifuge, wash, and dry to obtain MoS2-CeO2-Pd nanozyme.

[0048] The MoS2-CeO2-Pd nanozyme was characterized and analyzed using a SU8020 scanning electron microscope (SEM) produced by Hitachi, Japan. Figure 2 As shown, CeO2( Figure 2 A) The SEM image shows a black crystalline granular structure; Figure 2 B is the SEM image of MoS2-CeO2, where it can be clearly seen that crystal particles are adsorbed on the surface of the flaky structure, indicating that MoS2-CeO2 was successfully constructed. Figure 2C is the SEM image of MoS2-CeO2-Pd. Crystal particles and bright small particles of nanomaterials are adsorbed on the surface of the flaky structure, proving that Pd nanoparticles have been successfully attached to the surface of MoS2-CeO2, indicating that MoS2-CeO2-Pd was successfully prepared.

[0049] (4) 40.0 μL of amino-modified GP73 Apt (10.0 μM) and 20.0 μL MoS2-CeO2-Pd were mixed, ultrasonicated for 10 min, incubated at 4 °C for 12 h, centrifuged at 8000 rpm for 10 min, and washed to obtain MoS2-CeO2-Pd-GP73 Apt Signal probe.

[0050] 2. Electrode modification and construction of dual-signal sandwich electrochemical aptamer sensor

[0051] (1) The SPE electrode was immersed in a 0.5 M H2SO4 solution and scanned by cyclic voltammetry. The scanning voltage range was 0.4 V-1.0 V. The scanning was performed for 20 sections. The electrode was then cleaned and dried to obtain an activated SPE.

[0052] (2) 0.25 M dopamine DA and 0.01% HAuCl4 were mixed in a volume ratio of 3:1, stirred, and deposited at a potential of -0.5 V for 120 s to obtain Au@PDA / SPE;

[0053] (3) Take 2.5 μL of 10.0 μM GP73 Apt Add dropwise onto the Au@PDA / SPE surface, incubate at 25°C for 1 h, wash, and dry; then add dropwise 3.0 μL (0.5%) BSA solution, and dry to obtain GP73. Apt / Au@PDA / SPE.

[0054] (4) Take 2.5 μL of GP73 standard solution of different concentrations and add it dropwise to GP73 Apt / Au@PDA / SPE surface, incubated at 35°C for 35 min, washed, and dried to obtain GP73 / GP73 Apt / Au@PDA / SPE.

[0055] (5) Take 2.5 μL MoS2-CeO2-Pd-GP73 Apt Add the solution dropwise to GP73 / GP73 Apt / Au@PDA / SPE, incubated at 25 °C for 1 h, washed, and dried to obtain MoS2-CeO2-Pd-GP73 Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode.

[0056] The electrode modification process was characterized and analyzed using a SU8020 scanning electron microscope produced by Hitachi, Japan. Figure 3 As shown. The surface of the SPE electrode is rough and gray, with no particle distribution ( Figure 3 A); As Au@PDA is electrodeposited onto the SPE surface, many evenly distributed bright particles can be clearly seen on the SPE surface ( Figure 3 B); Incubate GP73 on the surface of Au@PDA / SPE electrode Apt , the surface shows obvious wrapped spherical bright particles ( Figure 3 C) Prove GP73 Apt It has been successfully incubated on the Au@PDA / SPE surface; further to GP73 Apt / Au@PDA / SPE surface incubated with GP73 protein, the surface gaps were reduced, and it was clearly visible that large and small bright-colored granular substances were connected together ( Figure 3 D); finally, MoS2-CeO2-Pd-GP73 Apt Probe incubation to GP73 / GP73 Apt / Au@PDA / SPE surface, a large number of chain complexes can be clearly seen attached to the surface ( Figure 3 E), demonstrating the formation of a stable “aptamer-protein-aptamer” structure on the SPE surface.

[0057] 3. Drawing of GP73 working curve

[0058] (1) The working electrode was placed in a PBS (0.2 M, pH = 5.5) solution containing H2O2 (50 mM) and OPD (25 mM). The DPV method was used in a CHI660E electrochemical workstation with a scan range of -0.1 V to 0.9 V and a scan rate of 0.1 V / s to record the peak oxidation currents of CeO2 and OPD. The DPV curves of GP73 at different concentrations are shown in Figure 2. Figure 4 As shown in the figure, as the concentration of GP73 protein is added, more conductive signal probes are fixed, and the oxidation current values ​​of CeO2 and OPD increase. When the concentration of GP73 is within 0.001ng / mL-100.0ng / mL, there is a logarithmic linear relationship between the oxidation current value of CeO2 (or the oxidation current value of OPD) (Y) and the concentration of GP73 (X), and the working curves are respectively and I OPD =2.24218lgC+18.09797(R 2 =0.99202). According to formula C LOD =3S d / b(S d The minimum detection limit is calculated as follows: and 0.12 pg / mL (I OPD Signal).

[0059] 4. Detection of GP73 in actual serum samples:

[0060] (1) Collect three serum samples with known GP73 concentrations (normal human serum: sample 1, GP73 concentration of 15.26 ng / mL, serum of patients with liver disease: sample 2, GP73 concentration of 28.49 ng / mL, serum of patients with liver cancer: sample 3, GP73 concentration of 93.76 ng / mL). The standard addition method was used to add 0 ng / mL, 1.0 ng / mL and 10.0 ng / mL GP73 standard solutions to the serum samples for detection, and three experiments were performed on each serum sample. All human serum samples in this experiment were obtained from the Laboratory Department of the 924th Hospital of the Joint Logistics Support Force of the People's Liberation Army of China, and were approved by the Ethics Committee of the 924th Hospital of the Joint Logistics Support Force of the People's Liberation Army of China (No. Guiyi 2022-38). The concentration of GP73 in clinical serum was obtained by enzyme-linked immunosorbent assay (ELISA).

[0061] (2) Take 2.5 μL of serum sample and drip it on GP73 Apt / Au@PDA / SPE electrode surface, incubated at 35°C for 35 min, washed and dried.

[0062] (3) Take 2.5 μL MoS2-CeO2-Pd-GP73 Apt Add the signal probe to GP73 / GP73 Apt / Au@PDA / SPE, incubated at 25 °C for 1 h, washed, and dried to obtain MoS2-CeO2-Pd-GP73 Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode.

[0063] (4) The working electrode was placed in a PBS (0.2 M, pH = 5.5) solution containing H2O2 (50 mM) and OPD (25 mM). The DPV method was used in a CHI660E electrochemical workstation with a scan range of -0.1 V to 0.9 V and a scan rate of 0.1 V / s to record the peak oxidation currents of CeO2 and OPD.

[0064] (5) Using the GP73 working curve obtained in step 3 and I OPD=2.24218lgC+18.09797, the concentration of GP73 in each serum sample was calculated, and the results are shown in Table 1. As can be seen from Table 1, the relative standard deviations of the GP73 concentration measured by the constructed aptamer sensor and the GP73 concentration in the actual serum sample are between 2.04%-7.44% and 1.78%-7.92%, respectively, and the recovery rates are between 99.45%-127.83% and 96.25%-127.15%. This indicates that the dual-signal aptamer electrochemical sensor can be used to detect the GP73 concentration in actual serum samples.

[0065] Table 1 Results of GP73 detection in human serum samples (n=3)

[0066]

Claims

1. A dual-signal electrochemical aptasensor for detecting GP73 based on molybdenum disulfide-cerium oxide-palladium nanozyme MoS2-CeO2-Pd is carried out in the following steps: Step 1: Preparation of MoS2-CeO2-Pd-GP73 aptamer Apt Signal Probe (1) Preparation of molybdenum disulfide MoS2: MoS2 is dissolved in water and ultrasonically crushed to obtain a MoS2 solution; sodium hydroxide NaOH is added to the MoS2 solution, stirred, hydrothermally stripped, and dried to obtain a single-layer MoS2 solution; (2) Preparation of MoS2-CeO2 nanomaterials: adding urea CO(NH2)2 and sodium citrate C6H5Na3O7 to a mixed solution of cerium nitrate hexahydrate Ce(NO3)3·6H2O and hydrogen peroxide H2O2, stirring, and performing a hydrothermal reaction to obtain a CeO2 solution; centrifuging, washing, and drying to obtain CeO2 nanocrystals; mixing a monolayer MoS2 solution with a CeO2 solution and polyvinyl pyrrolidone, stirring, and drying to obtain a MoS2-CeO2 nanomaterial; (3) Preparation of molybdenum disulfide-cerium oxide-palladium MoS2-CeO2-Pd nanozyme: adding sodium hexachloropalladate Na2PdCl6 solution and hydrazine hydrate N2H4 solution to MoS2-CeO2 solution, stirring, centrifuging, washing, and drying to obtain MoS2-CeO2-Pd nanozyme; (4) Construction of MoS2-CeO2-Pd-GP73 aptamer Apt Signal probe: MoS2-CeO2-Pd nanozyme and amino-modified GP73 Apt Vortex to mix and incubate overnight; Centrifugation, washing; Removal of free GP73 in the upper layer Apt , MoS2-CeO2-Pd-GP73 was obtained Apt Solution; Step 2: Electrode modification and construction of biosensor working electrode (1) Activation of SPE: Place SPE in H2SO4, perform electrode activation using cyclic voltammetry on an electrochemical workstation, clean, and dry to obtain activated SPE; (2) Preparation of Au@PDA / SPE: The activated SPE was immersed in a mixed solution of HAuCl4 and DA, and electropolymerization deposition was performed using an electrochemical workstation, followed by washing and drying to obtain Au@PDA / SPE; (3) Preparation of GP73 Apt / Au@PDA / SPE: GP73 Apt Add to SPE surface, incubate, wash, and dry; add BSA solution, dry to obtain GP73 Apt / Au@PDA / SPE; (4) Preparation of GP73 / GP73 Apt / Au@PDA / SPE: Take the standard GP73 protein and add it dropwise to GP73 Apt / Au@PDA / SPE surface, incubation, washing and drying; GP73 / GP73 Apt / Au@PDA / SPE; (5) Preparation of MoS2-CeO2-Pd-GP73 Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode: MoS2-CeO2-Pd-GP73 Apt Add dropwise to GP73 / GP73 Apt / Au@PDA / SPE surface, incubation, cleaning and drying; MoS2-CeO2-Pd-GP73 Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode; Step 3: Draw the working curve of GP73 (1) Construction of MoS2-CeO2-Pd-GP73 using different concentrations of standard GP73 protein Apt / GP73 / GP73 Apt / Au@PDA / SPE working electrode; (2) The working electrode was immersed in a phosphate buffered saline (PBS) solution containing hydrogen peroxide H2O2 and o-phenylenediamine (OPD), and differential pulse voltammetry (DPV) was used to scan the working electrode using an electrochemical workstation to record the peak oxidation currents of CeO2 and OPD. (3) Detecting GP73 solutions of different concentrations, recording the corresponding oxidation current peaks of CeO2 and OPD; drawing a working curve based on the relationship between the current response value of the sensor and the GP73 concentration, and calculating the minimum detection limit of the method; Step 4: Detection of GP73 in actual serum samples (1) using the actual serum sample to be tested instead of the standard GP73 protein solution, and preparing a dual-signal sandwich electrochemical aptamer sensor according to step 2 to obtain a working electrode; (2) placing the working electrode in a phosphate buffered saline (PBS) solution containing hydrogen peroxide (H2O2) and o-phenylenediamine (OPD), scanning using the DPV method of an electrochemical workstation, and recording the peak current of the oxidation current of CeO2 and OPD; (3) According to the working curve described in step 3, the concentration of GP73 in the serum sample to be tested is calculated.

2. The sensor according to claim 1, characterized in that: In step 1, a monolayer MoS2 solution is prepared, the molar concentration ratio of MoS2 and NaOH is 1:5, the reaction temperature is 220°C, and the time is 8h.

3. The sensor according to claim 1, characterized in that: In step 1, a CeO2 suspension is prepared, the reaction temperature is 180°C, and the reaction time is 20 hours.

4. The sensor according to claim 1, characterized in that: In step 2, the volume ratio of DA and HAuCl4 is 3:1, the concentration of MoS2-CeO2-Pd solution is 1.0 mg / mL, and the mass fraction of BSA solution is 1.0%.

5. The sensor according to claim 1, characterized in that: GP73 Apt The base sequence is 5'-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3'.

6. The sensor according to claim 1, characterized in that: In step 3 and step 4, the incubation temperature of GP73 is 35°C, and the incubation time is 35 min; the concentration of PBS solution is 0.2 M, and the pH value is 5.5; the concentration of H2O2 is 50 mM; the concentration of OPD is 25 mM; the DPV linear scanning range is -0.1 V to 0.9 V, and the scanning rate is 0.1 V / s.

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