Method for detecting GP73 by constructing dual-signal aptamer sensor based on nano composite material and quantum dots

Abstract

A method for detecting GP73 by constructing a dual-signal aptamer sensor based on a nanocomposite and quantum dots comprises the following steps: modifying the surface of a screen-printed electrode with nanogold, and fixing a GP73apt2-BNCQD capture probe on the surface of the electrode through an Au-SH bond to increase the conductivity; the preparation method comprises the following steps: preparing an NSrGO-H-MoS2-GP73apt1 signal probe; therefore, the sandwich type electrochemical aptamer sensor is constructed. H2O2 is catalyzed by the peroxidase-like property of the NSrGO-H-MoS2 to oxidize TMB into oxTMB, and an electrochemical signal YTMB is obtained; meanwhile, Fe (II) in Hemin in the NSrGO-H-MoS2 nano composite material is oxidized into Fe (III), another electrochemical signal YHemin is obtained, and the two signals prove each other, so that double-signal accurate detection of GP73 is realized.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically relates to a method for detecting GP73 by constructing a dual-signal aptamer sensor based on nanocomposite materials and quantum dots. Background Art

[0002] Primary hepatocellular carcinoma (HCC) is one of the top five cancers in the world and poses a huge threat to life and health. Golgi protein 73 (GP73) is a new liver cancer marker, and the main detection methods include radioimmunoassay, fluorescent immunoassay, electrochemical immunosensor, piezoelectric immunosensor, etc. The invention patent with publication number CN 111413502A relates to a method for detecting GP73 serum using a commercial ELISA kit; the invention patent with publication number CN 113945713A relates to a method for detecting tumor markers such as GP73 using a new biochip, which meets the needs of large-scale sample testing. However, there are still disadvantages such as low sensitivity of GP73 detection, complex operation, and high cost. Therefore, a rapid, sensitive, and easy-to-operate GP73 detection method is needed. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a nanocomposite material based on nitrogen-sulfur doped reduced graphene oxide-hemin chloride-molybdenum disulfide (NSrGO-H-MoS2), combined with GP73 aptamer 1 (GP73 apt1 ) formed a signal probe (NSrGO-H-MoS2-GP73 apt1 ); Boron nitrogen doped quantum dots (BNCQD) combined with GP73 aptamer 2 (GP73 apt2 ) to form a capture probe (GP73 apt2 -BNCQD) was used to construct a highly sensitive sandwich-type dual-signal electrochemical sensor to detect GP73, with the lowest detection limits of 0.88 pg / mL and 1.09 pg / mL.

[0004] The detection principle of the present invention is to successfully synthesize a nanocomposite material with high specific surface area and high conductivity by utilizing dehydration condensation and π-π bond adsorption, and to bond the NSrGO-H-MoS2 nanocomposite material with GP73 through π-π bond action. apt1 Combined with GP73 to form NSrGO-H-MoS2-GP73 apt1 ; Gold nanoparticles (Au NPs) were modified on the surface of screen-printed electrode (SPCE) by electrodeposition technology; GP73 apt2 -BNCQD was loaded on the electrode surface modified with Au NPs; finally, GP73 and NSrGO-H-MoS2-GP73 apt1Incubated on the above electrode surfaces, GP73 can interact with GP73 apt1 GP73 apt2 Specific binding, orderly arrangement on the electrode surface, forming SrGO-H-MoS2-GP73 apt1 / GP73 / GP73 apt2 -BNCQD sandwich structure, thus constructing GP73 sandwich electrochemical aptamer sensor. BNCQD can enhance the conductivity of the electrode surface, NSrGO-H-MoS2 has excellent peroxidase-like properties and can catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to oxide TMB (oxTMB), and the square wave voltammetry (SWV) is used to record the change in the oxidation peak current of Hemin and TMB in NSrGO-H-MoS2, thereby realizing dual signal detection of GP73.

[0005] In order to solve this technical problem, the present invention is carried out according to the following steps:

[0006] Step 1: NSrGO-H-MoS2-GP73 Apt1 Preparation of signal probe

[0007] (1) Preparation of nitrogen-sulfur-doped reduced graphene oxide (NSrGO): Graphene oxide (GO) is added to pure water and ultrasonically crushed to obtain a GO solution; urea (CH4N2O) and thiourea (CH4N2S) are added to the GO solution to carry out a hydrothermal reaction; centrifugation and washing are performed to obtain a NSrGO solution.

[0008] (2) Preparation of nitrogen-sulfur doped reduced graphene oxide-hemin chloride (NSrGO-H): Dissolve hemin chloride (Hemin) with ammonia water and add it to the NSrGO solution and stir; centrifuge and wash; obtain NSrGO-H solution.

[0009] (3) Preparation of aminated molybdenum disulfide (MoS2-NH2): dissolve molybdenum disulfide (MoS2) and β-mercaptoethylamine in N,N-dimethylformamide (DMF), stir; centrifuge, wash; and obtain MoS2-NH2 solution.

[0010] (4) Preparation of nitrogen-sulfur doped reduced graphene oxide-hemin chloride-molybdenum disulfide (NSrGO-H-MoS2): MoS2-NH2 solution and NSrGO-H solution were mixed, centrifuged, and the supernatant was removed to obtain NSrGO-H-MoS2 solution.

[0011] (5) Nitrogen-sulfur doped reduced graphene oxide-hemin chloride-molybdenum disulfide-GP73 aptamer 1 (NSrGO-H-MoS2-GP73 apt1) Signal probe preparation: GP73 aptamer 1 (GP73 apt1 ) solution and NSrGO-H-MoS2 solution were ultrasonically uniformly mixed, incubated, centrifuged, and washed to obtain NSrGO-H-MoS2-GP73 apt1 Signal probe.

[0012] Step 2: GP73 apt2 -Preparation of BNCQD capture probes

[0013] (1) Preparation of boron-nitrogen-doped quantum dots (BNCQD): Add citric acid monohydrate (C6H8O7·H2O) and boric acid (H3BO3) to pure water and mix well; add ethylenediamine monohydrate (C2H8N2·H2O) to the solution, stir and transfer to a reactor for hydrothermal reaction; dialyze to obtain a BNCQD solution.

[0014] (2) GP73 aptamer 2-boron nitrogen doped quantum dots (GP73 apt2 Preparation of capture probe: aptamer 2 (GP73 apt2 ) solution and BNCQD solution were uniformly mixed by ultrasonication, incubated, centrifuged, washed, and GP73 was obtained. apt2 -BNCQD capture probe.

[0015] Step 3: Electrode modification and construction of sensing interface

[0016] (1) Preparation of Au NPs / SPCE: The screen-printed electrode (SPCE) was placed in a dilute H2SO4 solution and activated using cyclic voltammetry (CV). The activated SPCE was then placed in a chloroauric acid (HAuCl4) solution and subjected to constant potential deposition using chronoamperometry (IT) to obtain an AuNPs / SPCE electrode.

[0017] (2)GP73 apt2 -Preparation of BNCQD / Au NPs / SPCE sensing interface: Dropwise addition of GP73 apt2 -BNCQD solution was incubated on the AuNPs / SPCE surface, and then the active sites were blocked with bovine serum albumin (BSA) solution to obtain GP73 apt2 -BNCQD / AuNPs / SPCE electrochemical sensing interface.

[0018] Step 4: Drawing of GP73 working curve

[0019] (1) Add different concentrations of GP73 standard solution to the electrochemical biosensor interface obtained in step 3, incubate, wash, and blow dry to obtain GP73 / GP73 apt2 -BNCQD / Au NPs / SPCE;

[0020] (2) In GP73 / GP73 apt2 -NSrGO-H-MoS2-GP73 was added onto BNCQD / Au NPs / SPCE apt1 Solution, incubation, washing, and drying to obtain NSrGO-H-MoS2-GP73 apt1 / GP73 / GP73 apt2 -BNCQD / Au NPs / SPCE. The working electrode was then immersed in a phosphate buffered saline (PBS) solution containing H2O2 and TMB, and the response current of the dual-signal sensor was recorded using SWV scanning of an electrochemical workstation.

[0021] (3) Detect different concentrations of GP73, record the current peak value, draw the GP73 working curve based on the relationship between the two signal current peak values ​​at different concentrations and the GP73 concentration, and calculate the two minimum detection limits corresponding to this method.

[0022] Step 5: Detection of GP73 in actual serum samples

[0023] (1) First dilute the actual serum sample and then dilute the GP73 obtained in step 3. apt2 -BNCQD / AuNPs / SPCE sensing interface, add the actual sample to be tested, and then add NSrGO-H-MoS2-GP73 apt1 The signal probe was incubated, washed and dried to obtain a working electrode. The prepared working electrode was immersed in a PBS solution, and the SWV scanning of the electrochemical workstation was used to record the response current value of the dual signal sensor.

[0024] (2) According to the working curve obtained in step 4, the concentration of GP73 in the actual serum sample to be tested is calculated.

[0025] As a preference:

[0026] As described in step 1, the reaction temperature for preparing NSrGO by hydrothermal reaction is 180°C and the reaction time is 12h; GP73 apt1 The DNA sequence is 5′-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3′;

[0027] Step 2 GP73 Apt2 The DNA sequence is 5'-SH-ACGCTCGGATGCCACTACAG-TTGGTTTTTTTTTGTTATTTAGAGTAAA AACCTTGTGTGTAGTGA-CTCATGGACGTGCTGGTGAC-3';

[0028] In step 3, the concentration of the dilute H2SO4 solution is 0.5 mol / L, the CV scanning voltage is -1.2V to 1.2V, the concentration of the HAuCl4 solution is 0.01%, the constant voltage is -0.5V, and the scanning time is 2min;

[0029] The GP73 dropped on the electrode surface in step 3 and step 4 apt2 -BNCQD and NSrGO-H-MoS2-GP73 apt1 The dosage is 2.5 μL;

[0030] In step 4 and step 5, the SWV scan has a voltage range of -0.4 V to 0.8 V, the incubation temperature of the electrode is 25° C., the incubation time is 40 min, the pH value of the PBS buffer is 5.5, the volume ratio of TMB:H2O2 is 2:1, the concentration of the signal probe is 0.6 mg / mL, the concentration of the capture probe is 0.4 mg / mL, and the blank response current of the sensor is the largest.

[0031] Among them, step 1 synthesizes NSrGO-H-MoS2 with high electroactivity and large specific surface area, and GP73 apt1 NSrGO-H-MoS2-GP73 was formed by π-π bonding apt1 Signal probe. In step 2, BNCQD with high conductivity is synthesized and reacted with GP73 apt2 GP73 is formed by π-π binding apt2 -BNCQD capture probe. Step 3 Formation of GP73 apt2 -BNCQD / AuNPs / SPCE sensing interface. The signal probe in step 1 not only provides the oxidation signal of TMB in PBS for steps 4 and 5, but also provides the oxidation signal of Hemin in NSrGO-H-MoS2. The GP73 dual-signal working curve in step 4 provides a calculation basis for the determination of GP73 concentration in the actual serum sample in step 5. It can be seen that steps 1-5 support each other and work together to realize the detection of GP73 using the electrochemical aptamer dual-signal sensor based on NSrGO-H-MoS2 nanocomposite and BNCQD.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] 1. This method first prepared NSrGO-H-MoS2 nanocomposites with unique morphology, large specific surface area, high electroactivity, excellent electron transfer rate and peroxidase-like properties; and successfully prepared NSrGO-H-MoS2-GP73 apt1 Signal probe that can specifically recognize GP73 protein.

[0034] 2. Using GP73 apt2-BNCQD capture probe modified electrode surface can not only effectively fix GP73 apt2 , and improves the conductivity of the sensor, enhances the transfer efficiency between electrons, and effectively amplifies the detected current signal.

[0035] 3. Using the peroxidase-like properties of NSrGO-H-MoS2 to catalyze H2O2 to release hydroxyl radicals to oxidize TMB to oxTMB, Hemin in the NSrGO-H-MoS2 nanocomposite was used as the electroactive substance. During SWV scanning, Fe(Ⅱ) in Hemin was oxidized to Fe(III), and a sandwich electrochemical aptamer sensor was constructed to detect GP73. The sandwich structure is more stable and has higher accuracy. The oxidation peak current values ​​of TMB and Hemin were recorded, and the relationship curve between the current value of the dual-signal sensor and the GP73 concentration was drawn. The two signals can corroborate each other, improve accuracy, and achieve quantitative detection of GP73. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :Schematic diagram of the sensor constructed based on NSrGO-H-MoS2 nanocomposite and BNCQD quantum dots for detecting GP73;

[0037] Figure 2 :Scanning electron microscopy (SEM) image of NSrGO-H-MoS2;

[0038] Figure 3 :Transmission electron microscopy (TEM) image of BNCQD;

[0039] Figure 4 : SWV working curves of electrochemical aptamer dual signal sensor based on NSrGO-H-MoS2 nanocomposite and BNCQD quantum dots for detecting different concentrations of GP73. DETAILED DESCRIPTION

[0040] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] A method for constructing a sensor based on NSrGO-H-MoS2 nanocomposite and BNCQD quantum dots for detecting GP73. The detection principle is shown in Figure 1 First, the SPCE was activated, and then AuNPs were electrodeposited on the SPCE to enhance the conductivity. apt2 -BNCQD capture probes were immobilized on Au NPs / SPCE to form GP73 apt2 -BNCQD / AuNPs / SPCE electrochemical sensing interface. The addition of BNCQD can enhance the conductivity of the sensing interface. The prepared NSrGO-H-MoS2 nanocomposite material combined with GP73 apt1Formation of NSrGO-H-MoS2-GP73 apt1 Signal probe, NSrGO-H-MoS2 has good peroxidase-like properties, NSrGO has excellent biocompatibility, Hemin is an electroactive substance that can act as an electronic medium to provide signals for the sensor. At the same time, the addition of Hemin can prevent NSrGO from agglomerating, and the addition of MoS2 enhances the conductivity of the nanocomposite material. GP73 is added to the electrochemical sensor interface, incubated, and then the signal probe is added. GP73 apt1 GP73 apt2 It specifically binds to GP73 protein, forming an aptamer-protein-aptamer sandwich complex and presenting a stable spatial structure and orderly arrangement. The more GP73 protein, the more sandwich complexes, and the more oxidized TMB and Hemin. The change in the oxidation peak current of TMB and Hemin is recorded. The relationship curve between the current value of the sensor and the GP73 concentration is drawn to achieve quantitative detection of GP73.

[0042] The specific implementation steps are as follows:

[0043] 1. NSrGO-H-MoS2-GP73 apt1 Preparation of signal probe

[0044] (1) Weigh 30.0 mg of GO and transfer it to a beaker containing 30.0 mL of ultrapure water. Ultrasonicate for 2 h to obtain a GO solution with a concentration of 1.0 mg / mL. Add 900.0 mg of urea and 900.0 mg of thiourea and stir at room temperature for 0.5 h. Then transfer it to a high-pressure reactor made of special polytetrafluoroethylene and hydrothermally react at 180 ° C for 12 h. Centrifuge and wash to obtain NSrGO solution.

[0045] (2) Weigh 30.0 mg of Hemin, add 60.0 μL of NH3·H2O, and then add 30.0 mL of ultrapure water, and stir to obtain a 1.0 mg / mL Hemin solution. Mix the Hemin solution and NSrGO solution in a volume ratio of 2:1 and stir for 24 h. Centrifuge and wash to obtain NSrGO-H solution.

[0046] (3) Weigh 20 mg of MoS2 and dissolve it in 20 mL of N,N-dimethylformamide (DMF), ultrasonically crush it for 1 hour, then add 40 mg of mercaptoethylamine to the MoS2 solution and stir it for 24 hours; add 20 mL of NSrGO-H to the above solution; centrifuge and wash; obtain NSrGO-H-MoS2 solution.

[0047] (4) Add 20.0 μL of 100.0 μM GP73 apt1Mix with 100.0 μL 2.0 mg / mL NSrGO-H-MoS2 solution, incubate at 4 °C for 12 h, centrifuge, wash, and obtain NSrGO-H-MoS2-GP73 apt1 Signal probe.

[0048] 2. GP73 apt2 -Preparation of BNCQD capture probes

[0049] (1) Weigh 2.5 g C6H8O7·H2O and 1.0 g H3BO3, dissolve in 15 mL ultrapure water and stir, add 1.0 mL C2H8N2·H2O, pour the above solution into a polytetrafluoroethylene-lined autoclave, react at 200°C for 8 h, cool and centrifuge, dialyze the supernatant, and filter to obtain a BNCQD solution.

[0050] (2) Add 20.0 μL of 100.0 μM GP73 apt2 Mix with 100.0 μL 2.0 mg / mL BNCQD solution, incubate at 4°C for 12 h, centrifuge, wash, and obtain GP73 apt2 -BNCQD capture probe.

[0051] The SU8020 scanning electron microscope (SEM) produced by Hitachi, Japan, was used to characterize NSrGO-H-MoS2. Figure 2 shown. Figure 2 A is the SEM scan of NSrGO, where an obvious wrinkled spatial structure can be seen. Figure 2 B is the SEM scan of NSrGO-H. It can be seen that the wrinkle morphology of the material surface changes to become film-like, which is due to the presence of Hemin with a film-like structure. Figure 2 C is the SEM scan of NSrGO-H-MoS2. Figure 2 D is a local scan magnified image of NSrGO-H-MoS2, where small fragments can be seen attached to the surface of the wrinkled membrane, indicating that MoS2 is successfully attached to the NSrGO-H material.

[0052] The nanomaterials were characterized using a JEM-1200EX transmission electron microscope (TEM). Figure 3 shown. Figure 3 A and 3B are TEM images of BNCQDs, where the spherical structure of BNCQDs can be clearly observed, and the BNCQD quantum dots show lattice fringes at a scale of 5 nm.

[0053] 3. Electrode modification and construction of biosensing interface

[0054] (1) The bare SPCE was activated in a 0.5 mol / L H2SO4 solution, and CV scanning was performed at -1.2 V to 1.2 V, with 20 cycles at 0.5 V / s. After activation, a small beaker containing 0.01% HAuCl4 solution was placed on a magnetic stirrer, and it was scanned at a constant voltage of -0.5 V for 2 min, washed three times with ultrapure water, and dried to obtain Au NPs / SPCE.

[0055] (2) Take 2.5 μL of 0.4 mg / mL GP73 apt2 -BNCQD solution was dropped on the electrode surface of Au NPs / SPCE, incubated at 25°C for 45 min, washed, and dried; then 4.0 μL 0.5% BSA was used to block the active sites and incubated for 45 min to obtain the working electrode GP73 apt2 -BNCQD / Au NPs / SPCE.

[0056] 4. Drawing of GP73 working curve

[0057] (1) Add 0.01 ng / mL to 100.0 ng / mL of GP73 standard solution to the GP73 electrochemical sensing interface constructed in step 3, incubate at 25°C for 45 min, and obtain GP73 / GP73 apt2 -BNCQD / Au NPs / SPCE sensing interface.

[0058] (2) Add 2.5 μL 0.6 mg / mL NSrGO-H-MoS2-GP73 onto the sensing interface prepared above. apt1 The solution was incubated at 25 °C for 45 min, washed and dried to obtain NSrGO-H-MoS2-GP73 apt1 / GP73 / GP73 apt2 -BNCQD / Au NPs / SPCE.

[0059] (3) The working electrode obtained above was placed in a PBS solution (0.2 M, pH 5.5) containing 2 mM H2O2 and 5 mM TMB, and SWV scanning was performed using a CHI660E electrochemical workstation to record the peak current. The SWV curves of different GP73 concentrations are shown in Figure 2. Figure 4 As shown in Figure 2, the TMB current response value (Y TMB ) and GP73 concentration (C1) have a logarithmic linear relationship, and the working curve is Y TMB =21.1651+7.5876*lgC1, the correlation coefficient is 0.9983; the sensor Hemin current response value (Y Hemin) and GP73 concentration (C2) have a linear relationship Y Hemin =5.9085+0.1171*C2, the correlation coefficient is 0.9832. LOD =3S d / b(S d represents the standard deviation of the six blank control groups, b represents the slope of the standard curve) and the minimum detection limits are calculated as C LOD(TMB) =0.88pg / mL, C LOD(Hemin) =1.09pg / mL.

[0060] 5. Detection of GP73 in serum samples

[0061] (1) The GP73 level in human serum samples was detected under optimal conditions by spiked measurement. Sample 1 was from a healthy person, sample 2 was from a patient with liver cirrhosis, and samples 3-6 were from patients with liver cancer. The ELISA method was first used to detect the concentration of GP73 in each serum sample. Then, the six serum samples with different concentrations were diluted 10 times and then added with 0 ng / mL, 1.0 ng / mL, and 10.0 ng / mL GP73 protein solutions at a ratio of 1:1. apt2 -BNCQD / Au NPs / SPCE sensing interface was dripped with 2.5 μL of the above mixed solution and incubated at 25 °C for 45 min, then the signal probe was washed and dripped and incubated again for 45 min to prepare NSrGO-H-MoS2-GP73 apt1 / GP73 / GP73 apt2 -BNCQD / Au NPs / SPCE sensor. Three groups of serum samples were measured for each concentration, and the working curve Y obtained in step 4 was TMB =21.1651+7.5876*lgC1 and Y Hemin =5.9085+0.1171*C2, the corresponding concentration of GP73 in the actual serum sample can be calculated, and the test results are shown in Table 1 and Table 2. TMB As the response value, the recovery rate of GP73 level in serum samples was 96.85-101.60%, and the RSD was between 0.63% and 4.09%. Hemin The response value was 91.53-102.61% and the RSD was 0.59-6.31%. Therefore, the sensor has a good application prospect, and the two signals can corroborate each other to improve the accuracy of detection.

[0062] Table 1 Detection results of GP73 in actual serum samples (Y TMB is the response value)

[0063]

[0064] Table 1

[0065]

[0066] Table 2 Detection results of GP73 in actual serum samples (Y Hemin is the response value)

[0067]

[0068] Table 2

[0069]

Claims

1. A method for detecting GP73 by constructing a dual-signal aptamer sensor based on nanocomposites and quantum dots for non-diagnostic purposes, comprising the following steps: Step 1: NSrGO-H-MoS2-GP73 Apt1 Preparation of signal probe (1) Preparation of nitrogen-sulfur doped reduced graphene oxide NSrGO: graphene oxide GO is added to pure water and ultrasonically crushed to obtain a GO solution; urea CH4N2O and thiourea CH4N2S are added to the GO solution, stirred and transferred to a reactor, and the temperature and pressure are controlled to perform a hydrothermal reaction; Centrifugation, washing; Obtaining NSrGO solution; (2) Preparation of nitrogen-sulfur doped reduced graphene oxide-hemin chloride NrGO-H: dissolve hemin in ammonia water and then add NSrGO solution and stir; Centrifugation, washing; Obtaining NSrGO-H solution; (3) Preparation of aminated molybdenum disulfide MoS2-NH2: dissolving molybdenum disulfide MoS2 and β-mercaptoethylamine ammonia in N,N-dimethylformamide DMF to obtain a MoS2-NH2 solution; (4) Preparation of nitrogen-sulfur doped reduced graphene oxide-hemin chloride-molybdenum disulfide NSrGO-H-MoS2: MoS2-NH2 solution and NSrGO-H solution were mixed, centrifuged and the supernatant was removed to obtain NSrGO-H-MoS2 solution; (5) Nitrogen-sulfur doped reduced graphene oxide-hemin chloride-molybdenum disulfide-aptamer 1NSrGO-H-MoS2-GP73 apt1 Preparation of signal probe: Adaptamer 1GP73 apt1 The solution and NSrGO-H-MoS2 solution were uniformly mixed by ultrasonication, incubated, centrifuged, and washed to obtain NSrGO-H-MoS2-GP73 apt1 Signal probe; Step 2: GP73 apt2 -Preparation of BNCQD capture probes (1) Preparation of boron-nitrogen doped quantum dots BNCQD: add citric acid monohydrate C6H8O7·H2O and boric acid H3BO3 into pure water, mix, add ethylenediamine monohydrate C2H8N2·H2O to the solution, stir and transfer to a reactor, perform hydrothermal reaction, and then dialyze to obtain a BNCQD solution; (2) Aptamer 2-BN-doped quantum dots GP73 apt2 - Preparation of BNCQD capture probe: aptamer 2GP73 apt2 The solution and the BNCQD solution were uniformly mixed by ultrasonication, incubated, centrifuged, and washed; GP73 apt2 -BNCQD capture probe; Step 3: Electrode modification and biosensing interface construction (1) Preparation of Au NPs / SPCE: The screen-printed electrode SPCE was placed in a 0.5 mol / L H2SO4 solution and activated by cyclic voltammetry; the activated SPCE was placed in a solution containing 0.01% HAuCl4 and deposited at a constant potential of minus 0.5 V using it for 2 min to obtain an AuNPs / SPCE electrode; (2) GP73 apt2 -Preparation of BNCQD / Au NPs / SPCE sensing interface: Dropwise addition of GP73 apt2 -BNCQD solution was incubated on Au NPs / SPCE, and then the active sites were blocked with bovine serum albumin BSA solution to obtain GP73 apt2 -BNCQD / AuNPs / SPCE electrochemical sensing interface; Step 4: Drawing of GP73 working curve (1) Add GP73 standard solution to GP73 apt2 -BNCQD / Au NPs / SPCE surface, incubation, washing, and drying; GP73 / GP73 apt2 -BNCQD / Au NPs / SPCE; (2) In GP73 / GP73 apt2 -NSrGO-H-MoS2-GP73 was added onto BNCQD / Au NPs / SPCE apt1 Solution, incubation; washing, drying; obtaining electrode NSrGO-H-MoS2-GP73 apt1 / GP73 / GP73 apt2 -BNCQD / AuNPs / SPCE; (3) The working electrode was immersed in PBS solution, and the double peak current was recorded by SWV scanning of the electrochemical workstation; (4) Detecting different concentrations of GP73 respectively and recording the double peak current; drawing the working curve of GP73 respectively according to the relationship between the current response value of the sensor and the concentration of GP73, and calculating the minimum detection limit; Step 5: Detection of GP73 in actual serum samples (1) The working electrode prepared with the actual serum sample to be tested is immersed in the PBS solution, and the SWV of the electrochemical workstation is used for scanning to record the response current value of the sensor; (2) According to the working curve obtained in step 4, the concentration of GP73 in the actual sample to be tested is calculated.

2. The method according to claim 1, characterized in that: The hydrothermal reaction in step 1 is carried out at a controlled temperature of 180° C. and a reaction time of 12 h.

3. The method according to claim 1, characterized in that: The concentration of the NSrGO-H-MoS2 solution described in step 1 is 1.0 mg / mL.

4. The method according to claim 1, characterized in that: NSrGO-H-MoS2-GP73 as described in step 1 apt1 The concentration of the signal probe was 0.6 mg / mL.

5. The method according to claim 1, characterized in that: Step 2 GP73 apt2 -BNCQD capture probe concentration was 0.4 mg / mL.

6. The method according to claim 1, characterized in that: The PBS solution in step 4 and step 5 has a concentration of 0.2 M and a pH of 5.5; it contains 2 mM H2O2 and 5 mM TMB.

7. The method according to claim 1, characterized in that: GP73 dropped on the electrode surface in step 3 apt2 -BNCQD and NSrGO-H-MoS2-GP73 dropped on the electrode surface in step 4 apt1 The dosage was 2.5 μL.

8. The method according to claim 1, characterized in that: In step 4 and step 5, the SWV scan has a voltage range of -0.4V to 0.8V.

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