An electrochemical biosensor and its application and detection method and device

Abstract

An electrochemical biosensor and its application and detection method and device, wherein zwitterion DMAPS provides strong antifouling performance for the sensing interface, effectively preventing the nonspecific adsorption of biological molecules such as proteins and cells, using click chemistry as a sensing strategy to maintain the structural integrity of biological molecules, effectively reducing the dissociation of aptamers / CA72-4, and improving the surface protein binding efficiency. The biosensor prepared by the present invention is used for real patient blood samples to achieve direct and rapid electrochemical detection of CA72-4.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to an electrochemical biosensor and its application, detection method and device. Background Art

[0002] Gastric cancer is an aggressive malignant tumor with a high incidence rate and has always been a global health concern. It is well known that the 5-year survival rate of gastric cancer is relatively high in the early development stage. Therefore, it is of great significance to establish a direct and effective biomarker detection platform for early gastric cancer. Since a variety of disease biomarkers circulate in the blood, blood is considered to be one of the most important biological fluids. Carbohydrate antigen 72-4 (CA72-4) is a mucinous glycoprotein in human blood. Many studies have shown that CA72-4 has a high sensitivity to gastric cancer. At present, enzyme-linked immunosorbent assay (ELISA) is considered to be the clinical gold standard for quantitative detection of protein biomarkers. However, in order to reduce the matrix effect, sample purification processes such as centrifugation are essential. Centrifugation is time-consuming and labor-intensive, and requires expensive equipment. Therefore, achieving direct and highly sensitive detection of protein biomarkers in whole blood is of great significance for shortening the detection time and reducing the detection cost.

[0003] Electrochemical biosensors can directly analyze complex clinical samples (such as urine, serum, and even whole blood) without sample pretreatment, showing great application prospects in clinical diagnosis. Unfortunately, electrochemical biosensors have only been commercialized for the detection of blood glucose. In terms of detecting other biomarkers, electrochemical biosensors still face serious biological contamination problems, which will lead to a significant decrease in electrical signals and signal-to-noise ratio (S / N), and even cause false positives. Integrating antifouling materials onto the electrode surface to form a hydration layer on the sensing interface can effectively alleviate the above problems. Inspired by the phospholipid bilayer of the cell membrane, zwitterions have received widespread attention in the field of electrochemical sensing due to their high biocompatibility and hydrophilicity.

[0004] Another obstacle for electrochemical biosensors to achieve high-sensitivity biomarker detection in whole blood is the low efficiency of surface protein binding due to matrix effects. Aptamers refer to nucleic acid sequences obtained from random nucleic acid libraries through SELEX screening technology, which have high affinity and strong specificity for specific target molecules. They bind to specific targets by forming secondary or tertiary structures, becoming a promising alternative in clinical diagnosis. Click chemistry is a common bioconjugation technology that has high conversion rates and can maintain the structural integrity and activity of biomolecules. Therefore, the click chemistry signal amplification strategy can effectively reduce the dissociation of aptamers from their binding proteins. To date, there are no published patents or literature reports on the direct detection of CA72-4 in whole blood samples. Summary of the invention

[0005] In order to solve the technical defects of the conventional detection methods in the prior art, such as complicated sample pretreatment and expensive instruments, the present invention provides an electrochemical biosensor and its application, detection method and device, which integrates the interface antifouling layer and sensing strategy of zwitterion DMAPS on the ITO electrode, and realizes the direct quantitative detection of CA72-4 in whole blood. The detection results of the sensor constructed by the present invention on clinical whole blood samples are true and reliable, so that the biosensor has great clinical application potential.

[0006] The technical solution adopted by the present invention is: an electrochemical biosensor, the electrochemical biosensor includes a zwitterionic biointerface constructed on the surface of an ITO electrode, and the zwitterionic biointerface is prepared by the following steps:

[0007] S1. Pretreatment of ITO electrode: immerse ITO in acetone, anhydrous ethanol and ultrapure water in turn, perform ultrasonic treatment respectively, and then use inert gas to dry the surface moisture of ITO electrode;

[0008] S2. Deposition of pDA: 20-30 mg of dopamine was added to 10 mL of Tris-HCl buffer, and the ITO electrode was vertically immersed in the above solution at room temperature for 0.5-6 h. The ITO surface was cleaned with ultrapure water and then dried with inert gas.

[0009] S3. Co-modification of zwitterionic 3-(2-methacryloyloxyethyldimethylamino)propanesulfonate (DMAPS) with aptamers: The electrode with pDA deposited on the surface was immersed in a solution containing 3-5 mg / mL DMAPS and 1-3 μM aptamer at 50-55 °C. o C overnight, clean the ITO surface with ultrapure water, blow dry with inert gas, and store for future use.

[0010] The specific steps of co-modification of zwitterion 3-(2-methacryloyloxyethyldimethylamino)propanesulfonate (DMAPS) and aptamer in step S3 are as follows: triethylamine, ethanol and ultrapure water are uniformly mixed in a volume ratio of 3:160:800, 3-5 mg DMAPS and 1-3 μM aptamer are added to the above solution to prepare a DMAPS and aptamer co-modification reaction solution.

[0011] An application of the electrochemical biosensor in preparing electrochemical biosensing detection materials for gastric cancer antigen CA72-4.

[0012] The gastric cancer antigen CA72-4 electrochemical biosensor detection material is used by the following steps:

[0013] (a) co-incubating the electrode of the electrochemical biosensor with the surface modified zwitterion with CA72-4 in the whole blood sample, and capturing CA72-4 on the electrode surface through the specific recognition of the aptamer and the target molecule;

[0014] (b) Using the covalent cross-linking of the boronic acid group and the ortho-dihydroxyl group, the azide group of click chemistry was modified onto the surface of the ITO electrode through the chemical binding between the CA72-4 sugar chain and PBA-PEG-N3, so as to carry out the subsequent click chemistry signal amplification strategy;

[0015] (c) The probe current signal introduced by click chemistry was detected by square wave voltammetry to calculate the CA72-4 concentration in the whole blood sample.

[0016] The electrode with surface modified zwitterions in step (a) is co-incubated with CA72-4 in the whole blood sample, comprising the following steps: incubating the electrode with surface modified zwitterions and aptamers with whole blood samples with different concentrations of CA72-4 at 37°C for 20 min to form an aptamer / CA72-4 complex, then washing the ITO surface with ultrapure water and blowing it dry with inert gas.

[0017] The click chemistry signal amplification strategy in step (b) comprises the following steps: immersing the electrode with the aptamer / CA72-4 complex formed on the surface in 15~20 mM, 150 μL 4Arm-PEG-DBCO and 15~20 mM, 150 μL 4Arm-PEG-N3 solutions, reacting at 37°C for 1 h, and then immersing the electrode in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 8~10 mM Cu(II) / BPDS and 100 μL 8~10 mM ferrocene, reaction potential: -0.25 V, time: 1800 s, and repeating two steps until the square wave voltammetric current signal reaches a maximum value.

[0018] The square wave voltammetry detection in step (c) comprises the following steps:

[0019] I. The electrical signal of the electroactive probe ferrocene was monitored in 0.5-1.0 M LiClO4 by square wave voltammetry, and the standard solutions of CA72-4 with different concentrations of 10-1000 U / mL were measured. The scanning voltage was set to 0-0.6 V and the scanning rate was 0.1 V / s.

[0020] II. Use real patient blood samples instead of CA72-4 standard solution for testing.

[0021] An electrochemical biosensor detection method for gastric cancer antigen CA72-4 based on zwitterionic biointerface and click chemistry signal amplification strategy comprises the following steps:

[0022] (1) Using the adhesion effect of polydopamine (pDA) to construct a zwitterionic biointerface on the ITO surface;

[0023] (2) co-incubating the electrode with the surface modified with zwitterions with CA72-4 in the whole blood sample, and capturing CA72-4 on the electrode surface through the specific recognition between the aptamer and the target molecule;

[0024] (3) By utilizing the covalent cross-linking of the boronic acid group and the ortho-dihydroxyl group, the azide group of click chemistry was modified onto the surface of the ITO electrode through the chemical binding between the CA72-4 sugar chain and PBA-PEG-N3, so as to carry out the subsequent click chemistry signal amplification strategy;

[0025] (4) The probe current signal introduced by click chemistry was detected by square wave voltammetry to calculate the CA72-4 concentration in the whole blood sample.

[0026] A gastric cancer antigen CA72-4 electrochemical biosensor detection device comprises the electrochemical biosensor.

[0027] The beneficial effects of the present invention are: an electrochemical biosensor and its application and detection method and device, wherein the zwitterion DMAPS provides a strong anti-fouling property for the sensing interface, effectively preventing the nonspecific adsorption of biological molecules such as proteins and cells, using click chemistry as a sensing strategy to maintain the structural integrity of biological molecules, effectively reducing the dissociation of aptamers / CA72-4, and improving the surface protein binding efficiency. The biosensor prepared by the present invention is used for real patient blood samples to achieve direct and rapid electrochemical detection of CA72-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a diagram showing the feasibility of the electrochemical biosensor for quantitative detection of CA72-4.

[0029] Figure 2 The electrochemical biosensor of the present invention is used to analyze the performance of quantitative detection of CA72-4 in whole blood. The concentrations of CA72-4 standards are 10 U / mL, 100 U / mL, 200 U / mL, 250 U / mL, 500 U / mL, and 1000 U / mL, respectively.

[0030] Figure 3 This is a diagram showing the detection results of the electrochemical biosensor of the present invention on a real sample. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources.

[0033] Example 1

[0034] (1) The construction of zwitterionic biointerface is as follows:

[0035] 1) Pretreatment of ITO electrode: ITO was immersed in acetone, anhydrous ethanol and ultrapure water in turn, and ultrasonicated for 30 min respectively, and then the surface moisture of the ITO electrode was dried with N2.

[0036] 2) Deposition of pDA: 20 mg of dopamine was added to 10 mL of Tris-HCl (pH=8.5) buffer. The ITO electrode was vertically immersed in the above solution at room temperature for 4 h. The ITO surface was cleaned with ultrapure water and then blown dry with N2.

[0037] 3) Co-modification of zwitterionic DMAPS and aptamer: The electrode with pDA deposited on the surface was immersed in a solution containing 3 mg / mL DMAPS and 1 μM aptamer at 50 °C. o The reaction was carried out overnight at a temperature of 4 °C, the ITO surface was cleaned with ultrapure water, dried with N2, and stored at 4 o Reserved under C conditions.

[0038] Preparation of co-modification solution of zwitterionic DMAPS and aptamer:

[0039] Triethylamine, ethanol and ultrapure water were evenly mixed at a volume ratio of 3:160:800, and 3 mg DMAPS and 1 μM aptamer were added to the above solution to prepare a DMAPS and aptamer co-modification reaction solution.

[0040] (2) The electrode modified with zwitterions on the surface is co-incubated with CA72-4 in the whole blood sample, the steps are as follows:

[0041] The electrodes modified with zwitterions and aptamers were incubated with 100 U / mL -1 The whole blood samples of CA72-4 were incubated for 20 min to form the aptamer / CA72-4 complex, and then the ITO surface was washed with ultrapure water and dried with N2.

[0042] (3) Click chemical signal amplification strategy, the steps are as follows:

[0043] The electrode with the aptamer / CA72-4 complex formed on the surface was immersed in 15 mM and 150 μL 4Arm-PEG-DBCO (a) and 15 mM and 150 μL 4Arm-PEG-N3 (a) solutions, respectively, and reacted at 37 °C for 1 h. The electrode was then immersed in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 8 mM Cu(II) / BPDS and 100 μL 10 mM ferrocene. The reaction potential was -0.25 V and the reaction time was 1800 s. The two steps (a) and (b) were repeated until the square wave voltammetric current signal reached the maximum value.

[0044] (4) Square wave voltammetry detection, the steps are as follows:

[0045] Square wave voltammetry was used to monitor the electrical signal of the electroactive probe ferrocene in 0.5 M LiClO4 and measure the standard solution of 100 U / mL CA72-4. The scanning voltage was set to 0~0.6 V and the scanning rate was 0.1 V / s.

[0046] Example 2

[0047] (1) The construction of zwitterionic biointerface is as follows:

[0048] 1) Pretreatment of ITO electrode: ITO was immersed in acetone, anhydrous ethanol and ultrapure water in turn, and ultrasonicated for 30 min respectively, and then the surface moisture of the ITO electrode was dried with N2.

[0049] 2) Deposition of pDA: 25 mg of dopamine was added to 10 mL of Tris-HCl (pH=8.5) buffer. The ITO electrode was vertically immersed in the above solution at room temperature for 4 h. The ITO surface was cleaned with ultrapure water and then blown dry with N2.

[0050] 3) Co-modification of zwitterionic DMAPS and aptamer: The electrode with pDA deposited on the surface was immersed in a solution containing 3 mg / mL DMAPS and 1 μM aptamer at 50 °C. o The reaction was carried out overnight at a temperature of 4 °C, the ITO surface was cleaned with ultrapure water, dried with N2, and stored at 4 o Reserved under C conditions.

[0051] Preparation of co-modification solution of zwitterionic DMAPS and aptamer:

[0052] Triethylamine, ethanol and ultrapure water were evenly mixed at a volume ratio of 3:160:800, and 3 mg DMAPS and 1 μM aptamer were added to the above solution to prepare a DMAPS and aptamer co-modification reaction solution.

[0053] (2) The electrode modified with zwitterions on the surface is incubated with CA72-4 in the whole blood sample, as follows:

[0054] The electrodes modified with zwitterions and aptamers were incubated with 100 U / mL -1 The whole blood samples of CA72-4 were incubated for 20 min to form the aptamer / CA72-4 complex, and then the ITO surface was washed with ultrapure water and dried with N2.

[0055] (3) Click chemical signal amplification strategy, the steps are as follows:

[0056] The electrode with the aptamer / CA72-4 complex formed on the surface was immersed in 15 mM, 150 μL 4Arm-PEG-DBCO (a) and 15 mM, 150 μL 4Arm-PEG-N3 (a) solutions, respectively, and reacted at 37 °C for 1 h. The electrode was then immersed in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 8 mM Cu(II) / BPDS and 100 μL 10 mM ferrocene. The reaction potential was -0.25 V and the reaction time was 1800 s. The two steps (a) and (b) were repeated until the square wave voltammetric current signal reached the maximum value.

[0057] (4) Square wave voltammetry detection, the steps are as follows:

[0058] Square wave voltammetry was used to monitor the electrical signal of the electroactive probe ferrocene in 0.5 M LiClO4 and measure the standard solution of 100 U / mL CA72-4. The scanning voltage was set to 0~0.6 V and the scanning rate was 0.1 V / s.

[0059] Example 3

[0060] (1) The construction of zwitterionic biointerface is as follows:

[0061] 1) Pretreatment of ITO electrode: ITO was immersed in acetone, anhydrous ethanol and ultrapure water in turn, and ultrasonicated for 30 min respectively, and then the surface moisture of the ITO electrode was dried with N2.

[0062] 2) Deposition of pDA: 20 mg of dopamine was added to 10 mL of Tris-HCl (pH=8.5) buffer. The ITO electrode was vertically immersed in the above solution at room temperature for 2 h. The ITO surface was cleaned with ultrapure water and then blown dry with N2.

[0063] 3) Co-modification of zwitterionic DMAPS and aptamer: The electrode with pDA deposited on the surface was immersed in a solution containing 4 mg / mL DMAPS and 1 μM aptamer at 50 °C. o The reaction was carried out overnight at a temperature of 4 °C, the ITO surface was cleaned with ultrapure water, dried with N2, and stored at 4 o Reserved under C conditions.

[0064] Preparation of co-modification solution of zwitterionic DMAPS and aptamer:

[0065] Triethylamine, ethanol and ultrapure water were evenly mixed at a volume ratio of 3:160:800, and 3 mg DMAPS and 1 μM aptamer were added to the above solution to prepare a DMAPS and aptamer co-modification reaction solution.

[0066] (2) The electrode modified with zwitterions on the surface is incubated with CA72-4 in the whole blood sample, as follows:

[0067] The electrodes modified with zwitterions and aptamers were incubated with 100 U / mL -1 The whole blood sample of CA72-4 was incubated for 20 min to form the aptamer / CA72-4 complex, and then the ITO surface was washed with ultrapure water and dried with N2.

[0068] (3) Click chemical signal amplification strategy, the steps are as follows:

[0069] The electrode with the aptamer / CA72-4 complex formed on the surface was immersed in 15 mM, 150 μL 4Arm-PEG-DBCO (a) and 15 mM, 150 μL 4Arm-PEG-N3 (a) solutions, respectively, and reacted at 37 °C for 1 h. The electrode was then immersed in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 8 mM Cu(II) / BPDS and 100 μL 10 mM ferrocene. The reaction potential was -0.25 V and the reaction time was 1800 s. The two steps (a) and (b) were repeated until the square wave voltammetric current signal reached the maximum value.

[0070] (4) Square wave voltammetry detection, the steps are as follows:

[0071] Square wave voltammetry was used to monitor the electrical signal of the electroactive probe ferrocene in 0.5 M LiClO4 and measure the standard solution of 100 U / mL CA72-4. The scanning voltage was set to 0~0.6 V and the scanning rate was 0.1 V / s.

[0072] Example 4 Feasibility Analysis

[0073] The anti-fouling electrochemical biosensor prepared in Examples 1 to 3 was used for feasibility analysis of CA72-4 detection, and the steps were as follows:

[0074] (1) The construction of zwitterionic biointerface is as follows:

[0075] 1) Pretreatment of ITO electrode: ITO was immersed in acetone, anhydrous ethanol and ultrapure water in turn, and ultrasonicated for 30 min respectively, and then the surface moisture of the ITO electrode was dried with N2.

[0076] 2) Deposition of pDA: 25 mg of dopamine was added to 10 mL of Tris-HCl (pH=8.5) buffer. The ITO electrode was vertically immersed in the above solution at room temperature for 3 h. The ITO surface was cleaned with ultrapure water and then blown dry with N2.

[0077] 3) Co-modification of zwitterionic DMAPS and aptamer: The electrode with pDA deposited on the surface was immersed in a solution containing 3 mg / mL DMAPS and 1 μM aptamer at 50 °C. o The reaction was carried out overnight at a temperature of 4 °C, the ITO surface was cleaned with ultrapure water, dried with N2, and stored at 4 o Reserved under C conditions.

[0078] Preparation of co-modification solution of zwitterionic DMAPS and aptamer:

[0079] Triethylamine, ethanol and ultrapure water were evenly mixed at a volume ratio of 3:160:800, and 3 mg DMAPS and 1 μM aptamer were added to the above solution to prepare a DMAPS and aptamer co-modification reaction solution.

[0080] (2) The electrode modified with zwitterions on the surface is incubated with CA72-4 in a whole blood sample and a blank buffer solution without CA72-4, as follows:

[0081] The electrodes modified with zwitterions and aptamers were incubated with 100 U / mL -1 The whole blood sample containing CA72-4 and the blank buffer without CA72-4 were incubated for 20 min, and then the ITO surface was washed with ultrapure water and dried with N2.

[0082] (3) Click chemical signal amplification strategy, the steps are as follows:

[0083] The above electrodes were immersed in 15 mM, 150 μL 4Arm-PEG-DBCO and 15 mM, 150 μL 4Arm-PEG-N3 solutions, respectively, and reacted at 37°C for 1 h. Then, the electrodes were immersed in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 10 mM Cu(II) / BPDS and 100 μL 10 mM ferrocene. The reaction potential was -0.25 V and the reaction time was 1800 s.

[0084] (4) Square wave voltammetry detection, the steps are as follows:

[0085] The electrical signal of the electroactive probe ferrocene was monitored by square wave voltammetry in 0.5 M LiClO4. The standard solution of 100 U / mL CA72-4 was measured with a scan voltage of 0-0.6 V and a scan rate of 0.1 V / s. -1 The final signals of the electrodes after incubation with CA72-4 and blank buffer without CA72-4 were compared. Figure 1 From the comparison results, it can be seen that only when the test solution contains the target molecule CA72-4, will a strong current signal be shown at a potential of 0.42 V, indicating that this method is feasible for detecting CA72-4.

[0086] Example 5 Analysis of the quantitative detection performance of CA72-4 in whole blood

[0087] The anti-fouling electrochemical biosensor prepared in Examples 1 to 3 is used for quantitative detection performance analysis of CA72-4 in whole blood, and the steps are as follows:

[0088] (1) The construction of zwitterionic biointerface is as follows:

[0089] 1) Pretreatment of ITO electrode: ITO was immersed in acetone, anhydrous ethanol and ultrapure water in turn, and ultrasonicated for 30 min respectively, and then the surface moisture of the ITO electrode was dried with N2.

[0090] 2) Deposition of pDA: 25 mg of dopamine was added to 10 mL of Tris-HCl (pH=8.5) buffer. The ITO electrode was vertically immersed in the above solution at room temperature for 3 h. The ITO surface was cleaned with ultrapure water and then blown dry with N2.

[0091] 3) Co-modification of zwitterionic DMAPS and aptamer: The electrode with pDA deposited on the surface was immersed in a solution containing 3 mg / mL DMAPS and 1 μM aptamer at 50 °C. o The reaction was carried out overnight at a temperature of 4 °C, the ITO surface was cleaned with ultrapure water, dried with N2, and stored at 4 o Reserved under C conditions.

[0092] Preparation of co-modification solution of zwitterionic DMAPS and aptamer:

[0093] Triethylamine, ethanol and ultrapure water were evenly mixed at a volume ratio of 3:160:800, and 3 mg DMAPS and 1 μM aptamer were added to the above solution to prepare a DMAPS and aptamer co-modification reaction solution.

[0094] (2) The electrode modified with zwitterions on the surface was incubated with different concentrations of CA72-4 in whole blood samples, as follows:

[0095] The electrodes modified with zwitterions and aptamers were incubated with whole blood standard samples of different concentrations of CA72-4 (10 U / mL, 100 U / mL, 200 U / mL, 250 U / mL, 500 U / mL, 1000 U / mL) at 37°C for 20 min. The ITO surface was then washed with ultrapure water and dried with N2.

[0096] (3) Click chemical signal amplification strategy, the steps are as follows:

[0097] The above electrodes were immersed in 15 mM, 150 μL 4Arm-PEG-DBCO and 15 mM, 150 μL 4Arm-PEG-N3 solutions, respectively, and reacted at 37°C for 1 h. Then, the electrodes were immersed in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 10 mM Cu(II) / BPDS and 100 μL 10 mM ferrocene. The reaction potential was -0.25 V and the reaction time was 1800 s.

[0098] (4) Square wave voltammetry detection, the steps are as follows:

[0099] Square wave voltammetry was used to monitor the electrical signal of the electroactive probe ferrocene in 0.5 M LiClO4. Different concentrations of CA72-4 were measured with the scan voltage set at 0-0.6 V and the scan rate at 0.1 V / s. Figure 2 The standard curve of different concentrations of CA72-4 and the response electrical signal is shown in Figure 2. The linear regression equation between the logarithm of CA72-4 concentration and the response current signal is i(10 -6A)=2.56lg(CA72-4 / U / mL)-1.83 ( R 2 =0.997), and the detection limit was 7.11 U / mL ( S / N =3).

[0100] Example 6 Biosensor Real Sample Analysis

[0101] The anti-fouling electrochemical biosensor prepared in Examples 1 to 3 is used for quantitative detection performance analysis of CA72-4 in whole blood, and the steps are as follows:

[0102] (1) The construction of zwitterionic biointerface is as follows:

[0103] 1) Pretreatment of ITO electrode: ITO was immersed in acetone, anhydrous ethanol and ultrapure water in turn, and ultrasonicated for 30 min respectively, and then the surface moisture of the ITO electrode was dried with N2.

[0104] 2) Deposition of pDA: 25 mg of dopamine was added to 10 mL of Tris-HCl (pH=8.5) buffer. The ITO electrode was vertically immersed in the above solution at room temperature for 3 h. The ITO surface was cleaned with ultrapure water and then blown dry with N2.

[0105] 3) Co-modification of zwitterionic DMAPS and aptamer: The electrode with pDA deposited on the surface was immersed in a solution containing 3 mg / mL DMAPS and 1 μM aptamer at 50 °C. o The reaction was carried out overnight at a temperature of 4 °C, the ITO surface was cleaned with ultrapure water, dried with N2, and stored at 4 o Reserved under C conditions.

[0106] Preparation of co-modification solution of zwitterionic DMAPS and aptamer:

[0107] Triethylamine, ethanol and ultrapure water were evenly mixed at a volume ratio of 3:160:800, and 3 mg DMAPS and 1 μM aptamer were added to the above solution to prepare a DMAPS and aptamer co-modification reaction solution.

[0108] (2) The electrode modified with zwitterions on the surface is incubated with a real blood sample, as follows:

[0109] The electrodes modified with zwitterions and aptamers were incubated with real blood samples at 37°C for 20 min, and then the ITO surface was washed with ultrapure water and dried with N2.

[0110] (3) Click chemical signal amplification strategy, the steps are as follows:

[0111] The above electrodes were immersed in 15 mM, 150 μL 4Arm-PEG-DBCO and 15 mM, 150 μL 4Arm-PEG-N3 solutions, respectively, and reacted at 37°C for 1 h. Then, the electrodes were immersed in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 10 mM Cu(II) / BPDS and 100 μL 10 mM ferrocene. The reaction potential was -0.25 V and the reaction time was 1800 s.

[0112] Square wave voltammetry detection, the steps are as follows:

[0113] Square wave voltammetry was used to monitor the electrical signal of the electroactive probe ferrocene in 0.5 M LiClO4 to measure the CA72-4 content in real blood samples. The scanning voltage was set to 0-0.6 V and the scanning rate was 0.1 V / s. Figure 3 The figure is a comparison of the results of CA72-4 detection by the biosensor of the present invention and the results of detection by the hospital method. The accuracy of the sensor of the present invention in clinical application was explored by analyzing 5 clinical blood samples. The concentration of CA72-4 was measured by the conventional electrochemiluminescence method (ECL) in the hospital and the sensor of the present invention. Figure 3 As shown in Figure 2, the concentrations of CA72-4 in S1 and S2 were both >250 U mL-1, which were beyond the detection range of the hospital method, while the concentrations measured by the antifouling aptamer sensor were 370 U mL-1 and 370 U mL-1, respectively. -1 and 283.1 UmL -1 , indicating that the biosensor has a wider detection range. The test results of S3, S4, and S5 are basically consistent with the hospital results. These results show that the biosensor in the present invention has reliable analytical performance in clinical blood samples.

[0114] Technical personnel should note that although the present invention has been explained through the above specific implementation methods, the inventive concept of the present invention is not limited thereto. Any improvement or variation based on the inventive concept belongs to the protection scope of this patent right.

[0115] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments and experimental examples. Any technical solution that follows the concept of the present invention is included in the protection scope of the present invention. It should be emphasized that for ordinary technicians in this technical field, without departing from the purpose and scope of the present invention, any modification or equivalent replacement should be regarded as part of the protection scope of the present invention.

Claims

1. An electrochemical biosensor, characterized in that: The electrochemical biosensor comprises a zwitterionic biointerface constructed on the surface of an ITO electrode, and the zwitterionic biointerface is prepared by the following steps: S1. Pretreatment of ITO electrode: immerse ITO in acetone, anhydrous ethanol and ultrapure water in turn, perform ultrasonic treatment respectively, and then use inert gas to blow dry the moisture on the surface of ITO electrode; S2. Deposition of pDA: 20-30 mg of dopamine was added to 10 mL of Tris-HCl buffer, and the ITO electrode was vertically immersed in the above solution at room temperature for 0.5-6 h. The ITO surface was cleaned with ultrapure water and then dried with inert gas. S3. Co-modification of zwitterionic 3-(2-methacryloyloxyethyldimethylamino)propanesulfonate (DMAPS) with nucleic acid aptamers: The electrode with pDA deposited on the surface was immersed in a solution containing 3-5 mg / mL DMAPS and 1-3 μM nucleic acid aptamer at 50-55 °C. o C overnight, clean the ITO surface with ultrapure water, blow dry with inert gas, and store for future use.

2. The electrochemical biosensor according to claim 1, characterized in that The specific steps of co-modification of zwitterion 3-(2-methacryloyloxyethyldimethylamino)propanesulfonate (DMAPS) and nucleic acid aptamer in step S3 are as follows: triethylamine, ethanol and ultrapure water are uniformly mixed in a volume ratio of 3:160:800, 3-5 mg DMAPS and 1-3 μM nucleic acid aptamer are added to the above solution to prepare a DMAPS and nucleic acid aptamer co-modification reaction solution.

3. Use of the electrochemical biosensor according to claim 1 in preparing electrochemical biosensor detection materials for gastric cancer antigen CA72-4.

4. The use according to claim 3, characterized in that: The gastric cancer antigen CA72-4 electrochemical biosensor detection material is used by the following steps: (a) co-incubating the electrode of the electrochemical biosensor with the surface modified zwitterion with CA72-4 in the whole blood sample, and capturing CA72-4 on the electrode surface through the specific recognition of the nucleic acid aptamer and the target molecule; (b) Using the covalent cross-linking of the boronic acid group and the ortho-dihydroxyl group, the azide group of click chemistry was modified onto the surface of the ITO electrode through the chemical binding between the CA72-4 sugar chain and PBA-PEG-N3, so as to carry out the subsequent click chemistry signal amplification strategy; (c) The probe current signal introduced by click chemistry was detected by square wave voltammetry to calculate the CA72-4 concentration in the whole blood sample.

5. The use according to claim 4, characterized in that The electrode with surface modified zwitterions in step (a) is co-incubated with CA72-4 in the whole blood sample, comprising the following steps: incubating the electrode with surface modified zwitterions and nucleic acid aptamers with whole blood samples with different concentrations of CA72-4 at 37°C for 20 min to form a nucleic acid aptamer / CA72-4 complex, then washing the ITO surface with ultrapure water and blowing it dry with inert gas.

6. The use according to claim 4, characterized in that: The click chemistry signal amplification strategy in step (b) comprises the following steps: immersing the electrode with the nucleic acid aptamer / CA72-4 complex formed on the surface in 15~20mM, 150 μL of 4Arm-PEG-DBCO and 15~20 mM, 150 μL of 4Arm-PEG-N3 solutions, reacting at 37°C for 1 h, and then immersing the electrode in a freshly prepared click chemistry reaction solution containing 8.9 mL 100 mM KPF6, 900 μL DMSO, 100 μL 8~10 mM Cu(II) / BPDS and 100 μL 8~10 mM ferrocene, reaction potential: -0.25 V, time: 1800 s, and repeating two steps until the square wave voltammetric current signal reaches a maximum value.

7. The use according to claim 4, characterized in that: The square wave voltammetry detection in step (c) comprises the following steps: I. The electrical signal of the electroactive probe ferrocene was monitored in 0.5-1.0 M LiClO4 by square wave voltammetry, and the standard solutions of CA72-4 with different concentrations of 10-1000 U / mL were measured. The scanning voltage was set to 0-0.6 V and the scanning rate was 0.1 V / s. II. Use real patient blood samples instead of CA72-4 standard solution for testing.

8. A method for detecting gastric cancer antigen CA72-4 by electrochemical biosensing based on zwitterionic biointerface and click chemistry signal amplification strategy using the electrochemical biosensor according to claim 1, characterized in that: The following steps are involved: (1) constructing the zwitterionic biointerface of the electrochemical biosensor according to claim 1 on the ITO surface by utilizing the adhesion effect of polydopamine (pDA); (2) co-incubating the electrode with the surface modified with zwitterions with CA72-4 in the whole blood sample, and capturing CA72-4 on the electrode surface through the specific recognition between the aptamer and the target molecule; (3) By utilizing the covalent cross-linking of the boronic acid group and the ortho-dihydroxyl group, the azide group of click chemistry was modified onto the surface of the ITO electrode through the chemical binding between the CA72-4 sugar chain and PBA-PEG-N3, so as to carry out the subsequent click chemistry signal amplification strategy; (4) The probe current signal introduced by click chemistry was detected by square wave voltammetry to calculate the CA72-4 concentration in the whole blood sample.

9. An electrochemical biosensor detection device for gastric cancer antigen CA72-4, characterized in that: The invention comprises the electrochemical biosensor according to claim 1.

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