Electrochemical sensor based on framework nucleic acid and carboxyethyl glucan as well as preparation method and application of electrochemical sensor

Abstract

The invention relates to an electrochemical sensor based on framework nucleic acid and carboxyethyl glucan as well as a preparation method and application of the electrochemical sensor. The surface of the silk-screen printing gold electrode is chemically modified to improve the sensitivity of the electrode, and then a double-antibody sandwich method is used for amplifying signals, so that the detection accuracy and sensitivity are further improved, the detection range is expanded, the detection limit is reduced, and the pancreatic cancer serum marker CA19-9 can be rapidly, accurately and sensitively detected. The CA19-9 sensor is combined with a portable electrochemical workstation, so that a patient can detect the change of CA19-9 at home, and the electrochemical sensor has the advantages of portability, low cost, high efficiency, simplicity in operation and the like, and can be used at home for detecting the CA19-9 at home.

Description

Technical Field

[0001] This invention relates to the fields of biomedical analysis and detection and electrochemical sensing technology, and in particular to an electrochemical sensor based on framework nucleic acid and carboxyethyl dextran, its preparation method and application. Background Technology

[0002] The latest cancer statistics released by the American Cancer Society in 2025 show that while the overall cancer mortality rate continues to decline, pancreatic cancer is bucking the trend and rising. [1] The incidence of pancreatic cancer in my country is also on the rise, with persistently high morbidity and mortality rates, drawing widespread attention. Pancreatic cancer is a common malignant tumor of the digestive tract, often referred to as the "king of cancers." Most pancreatic cancer patients are diagnosed at an advanced stage, at which point radical surgery is no longer possible, and many who undergo resection develop metastases within four years post-surgery. Therefore, highly sensitive and specific biomarkers are urgently needed for postoperative assessment of pancreatic cancer.

[0003] Carbohydrate antigen 19-9 (CA19-9) is a mucin-type carbohydrate antibody and is considered the most effective blood biomarker for diagnosing pancreatic malignancies. It is an important indicator for the auxiliary diagnosis of pancreatic cancer. The cutoff value for serum CA19-9 is 37 U / mL. [2] CA19-9 levels rise as pancreatic cancer progresses, and CA19-9 detection can be used for early auxiliary diagnosis of pancreatic cancer. Elevated serum CA19-9 levels after pancreatic cancer surgery are an important prognostic factor. Elevated CA19-9 levels can be detected 7 to 10 months before imaging diagnosis of pancreatic cancer recurrence. Accurate and rapid detection of serum CA19-9 can also be used for evaluating treatment efficacy and monitoring recurrence in pancreatic cancer patients.

[0004] The current industry standard method for detecting CA19-9 is chemiluminescence immunoassay. Subsequent methods, such as fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay, have also been developed. While these methods offer high accuracy and sensitivity, they are complex, time-consuming, and require bulky and expensive equipment. Furthermore, patients need to undergo frequent testing in hospitals.

[0005] Currently, electrochemical biosensors, with their advantages of high sensitivity, strong specificity, simple operation, and low cost, can achieve sensitive detection of a range of serological tumor markers. Existing technologies utilize framework nucleic acid probes constructed on gold electrode surfaces for the detection of various biomarkers. [3] However, because each binding site extending from the apex of a framework nucleic acid can only recognize one target molecule, it limits its ability to detect low concentrations of target analytes.

[0006] The references are as follows: [1] Cancer statistics, 2025. CA Cancer J Clin, 2025, 75(1): 10-45. [2] Prognostic Impact of Postoperative Serum CA19-9 Levels inPatients with Resectable Pancreatic Cancer. Ann Surg Oncol, 2010, 17, 2321–2329. [3] DNA Nanostructure-Based Universal Microarray Platform for High-Efficiency Multiplex Bioanalysis in Biofluids. ACS Applied Materials &Interfaces, 2014, 6: 17944-17953 Summary of the Invention

[0007] The purpose of this invention is to provide an electrochemical sensor based on framework nucleic acids and carboxyethyl dextran, its preparation method, and its applications. This invention improves the sensitivity of a screen-printed gold electrode by chemically modifying its surface. Then, a double-antibody sandwich method is used to amplify the signal, further improving the accuracy and sensitivity of detection, expanding the detection range, and lowering the detection limit. This enables rapid, accurate, and sensitive detection of the pancreatic cancer serum biomarker CA19-9. By connecting it to a portable electrochemical workstation, patients can monitor changes in CA19-9 at home. The electrochemical sensor has advantages such as portability, low cost, high efficiency, and simple operation, making it a suitable home-use sensor for CA19-9 detection.

[0008] The objective of this invention can be achieved through the following technical solutions: An electrochemical sensor based on framework nucleic acids and carboxyethyl dextran includes an electrode on which a capture probe interacting with the pancreatic cancer biomarker CA19-9 is immobilized. The capture probe is immobilized on the electrode surface using framework nucleic acid and carboxyethyl dextran.

[0009] Furthermore, the framework nucleic acid is assembled from four DNA strands into a tetrahedral structure. The bottom of the framework nucleic acid tetrahedron is connected to the electrode surface through groups such as thiol, amino, or aldehyde groups, and the amino group at the top interacts with the carboxyl group on the carboxyethyl dextran.

[0010] Furthermore, the sequences of the four DNA single strands are shown in SEQ ID NO.1 to SEQ ID NO.4.

[0011] Furthermore, the carboxyethyl dextran is a polymer with multiple carboxyethyl groups, wherein some carboxyl groups interact with amino groups on the framework nucleic acid through activation, and other carboxyl groups are linked by condensation with amino groups on the capture probe.

[0012] As a preferred technical solution, the electrode surface is immobilized with a capture probe that interacts with the pancreatic cancer biomarker CA19-9 through a scaffolding effect of framework nucleic acid and carboxyethyl dextran.

[0013] Furthermore, the electrodes include screen-printed gold electrodes, screen-printed carbon electrodes, and nanoparticle electrodes, etc.

[0014] As a preferred technical solution, the nanoparticle electrode is prepared by electrochemical deposition on the surface of other electrodes. The electrode can also be prepared by magnetic sputtering, vacuum evaporation or photolithography on other electrode materials.

[0015] Furthermore, the capture probe includes substances such as antibodies and DNA aptamers that can recognize proteins.

[0016] This invention also provides a method for preparing an electrochemical sensor based on framework nucleic acids and carboxyethyl dextran, the specific steps of which are as follows: S1. Dextran, acrylamide and sodium hydroxide are mixed and reacted to obtain carboxyethyl dextran; S2, Synthesizing framework nucleic acids; S3. Fix the framework nucleic acid obtained in step S2 on the electrode surface to obtain framework nucleic acid / electrode; S4. The carboxyethyl dextran obtained in step S1 is fixed on the surface of the framework nucleic acid / electrode obtained in step S3 to obtain carboxyethyl dextran / framework nucleic acid / electrode, i.e., electrochemical sensor.

[0017] Further, in step S1, the mass ratio of dextran, acrylamide and sodium hydroxide is 100:35~45:50~60, preferably 100:40:53.

[0018] Furthermore, in step S1, the reaction time is 20-30 hours and the reaction temperature is 40-50°C.

[0019] Further, in step S1, dextran, acrylamide and sodium hydroxide are mixed, reacted and then purified with ethanol to obtain carboxyethyl dextran.

[0020] Further, in step S2, the method for synthesizing the framework nucleic acid is as follows: Four DNA single strands were dissolved in TE buffer solution to obtain single-stranded mother liquor; four different single-stranded mother liquors were added to TM buffer solution, mixed well and heated for reaction, and annealed to obtain framework nucleic acid.

[0021] Furthermore, the sequences of the four DNA single strands are shown in SEQ ID NO.1 to SEQ ID NO.4.

[0022] Furthermore, the concentration of the single-chain mother liquor is 90~110 μmol / L.

[0023] Furthermore, the heating reaction time is 5 to 15 minutes, and the heating reaction temperature is 90 to 100°C.

[0024] Further, in step S3, the electrode is subjected to cyclic voltammetric cleaning in sulfuric acid, and the framework nucleic acid obtained in step S2 is immobilized on the surface of the cleaned electrode, resulting in a framework nucleic acid / electrode.

[0025] Furthermore, in step S3, the concentration of the framework nucleic acid is 1~20 μmol.

[0026] Further, in step S4, the activator and the carboxyethyl dextran obtained in step S1 are mixed and added to the framework nucleic acid / electrode interface prepared in step S3 for reaction. After washing, the carboxyethyl dextran / framework nucleic acid / electrode is obtained, which is the electrochemical sensor.

[0027] Further, in step S4, the concentration of the carboxyethyl dextran is 10~100 mg / mL.

[0028] Furthermore, the activator includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0029] Furthermore, the volume ratio of the carboxyethyl dextran, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 50:0.5~1.5:0.5~1.5.

[0030] Furthermore, the concentration of the carboxyethyl dextran is 10-100 mg / mL; The concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.1~0.4 mol / L; The concentration of the N-hydroxysuccinimide is 0.1~0.4 mol / L.

[0031] In addition, the present invention also provides an application of an electrochemical sensor based on framework nucleic acid and carboxyethyl dextran in immobilizing biomolecules and detecting biomarkers.

[0032] Furthermore, the biomarker includes the pancreatic cancer serum biomarker CA19-9.

[0033] Furthermore, the method for detecting the pancreatic cancer serum biomarker CA19-9 using the electrochemical sensor is as follows: S1. Immobilize the CA19-9 monoclonal antibody on the surface of the above-mentioned electrochemical sensor based on framework nucleic acid and carboxyethyl dextran, and after the reaction, obtain CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode; S2. Add CA19-9 antigen, biotinylated CA19-9 antibody and horseradish peroxidase-labeled streptavidin to the surface of CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode obtained in step S1. After the reaction, streptavidin-modified HRP / biotinylated antibody / antigen / CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode is obtained. S3. Add enzyme substrate to the surface of the streptavidin-modified HRP / biotinylated antibody / antigen / CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode obtained in step S2, and perform quantitative analysis using an electrochemical workstation.

[0034] Further, in step S1, the concentration of the CA19-9 monoclonal antibody is 20~200 μg / mL.

[0035] Furthermore, in step S1, the reaction time is 2-20 hours and the reaction temperature is 4-40°C.

[0036] Further, in step S2, the volume ratio of the CA19-9 antigen, the biotinylated CA19-9 antibody, and the horseradish peroxidase-labeled streptavidin is 1:0.5~1.5.

[0037] Furthermore, in step S2, the reaction time is 10-20 minutes and the reaction temperature is 40-50°C.

[0038] Further, in step S3, the enzyme substrate includes 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and o-Phenylenediamine (OPD).

[0039] The principle of this invention is as follows: This invention involves modifying one strand of a bifunctional framework nucleic acid with amino groups. This modified strand is then mixed in equimolar proportions with three other DNA strands terminally modified with thiol groups, and rapidly assembled into a DNA tetrahedral nanostructure probe via a one-step annealing process. The amino-modified portion of the probe is maintained at the apex of the tetrahedral structure to form a complex with dextran.

[0040] Dextran is a polymer formed by α-D-glucose linked by α-1,6-glycosidic bonds. It possesses excellent biocompatibility, biodegradability, high stability, and non-immunogenicity, leading to its widespread application in biomedicine. Therefore, by attaching carboxyethyl dextran polymers to a framework nucleic acid, a composite interface between the framework nucleic acid and the carboxyethyl polymer can be constructed. The multiple binding sites on the carboxyethyl dextran can be used to immobilize multiple capture probes, thereby improving the detection sensitivity of target molecules.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention constructs a sensing interface using a framework nucleic acid and dextran composite material. The framework nucleic acid can directionally regulate molecular recognition, and the synthesized carboxyethyl dextran enhances antibody immobilization, synergistically improving the detection sensitivity of CA19-9. First, carboxyethyl dextran is synthesized. Then, framework nucleic acid is modified onto the surface of a gold electrode, and the synthesized carboxyethyl dextran is immobilized on it. Finally, through the specific recognition of antibody and antigen, CA19-9 in the sample is captured, and electrochemical detection is performed, providing a rapid and sensitive method for detecting CA19-9.

[0042] This invention modifies the electrode surface with carboxyethyl dextran. Since a single dextran molecule can carry many carboxyl groups, it increases the amount of antibody immobilized on the electrode surface, thereby solving the problem of insufficient antibody immobilization in existing methods. Furthermore, the hydroxyl groups on the dextran can reduce non-specific adsorption, thus enabling rapid and sensitive detection of CA19-9. Attached Figure Description

[0043] Figure 1 This is a schematic diagram illustrating the fabrication of an electrochemical sensor based on framework nucleic acids and carboxyethyl dextran and its principle for rapid detection of CA19-9.

[0044] Figure 2 A standard curve for rapid detection of CA19-9 using an electrochemical sensor based on framework nucleic acids and carboxyethyl dextran.

[0045] Figure 3 A standard curve for rapid detection of CA19-9 using a framework-based nucleic acid electrochemical sensor.

[0046] Figure 4 A bar graph showing the change in current on the electrode after immobilizing the same concentration of CA19-9 antibody in an electrochemical sensor for framework nucleic acids and an electrochemical sensor for framework nucleic acids and carboxyethyl dextran.

[0047] Figure 5 The standard curve for detecting CA19-9 using the purchased human sugar chain antigen 19-9 (CA19-9) enzyme-linked immunosorbent assay kit is shown.

[0048] Figure 6 This is a flowchart illustrating the preparation of an electrochemical sensor based on framework nucleic acids and carboxyethyl dextran and its rapid detection of CA19-9. Detailed Implementation

[0049] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0050] In the following embodiments, the sources of the reagents and materials are as follows: The DNA strands that self-assembled to form framework nucleic acids (specific sequences are shown in Table 1): DNA-1 (5' end thiol modified), DNA-2 (5' end thiol modified), DNA-3 (5' end thiol modified), DNA-4 (5' end amino modified), DNA-5 (5' end carboxyl modified) and streptavidin-modified HRP were all purchased from Shanghai Sangon Biotech Co., Ltd. Dextran, acrylamide, and casein blocking solution were purchased from Sigma-Aldrich. CA19-9 monoclonal antibody and CA19-9 antigen were purchased from Fitzgerald, Inc., USA. The biotinylated CA19-9 antibody was purchased from Shanghai Lingchao Biotechnology Co., Ltd. The screen-printed gold electrodes were purchased from Metrohm, Switzerland. Sodium hydroxide, ethanol, phosphate buffer solution, and sulfuric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; blank human serum was purchased from Beijing Huizhi Heyuan Biotechnology Co., Ltd. The human carbohydrate antigen 19-9 (CA19-9) enzyme-linked immunosorbent assay kit was purchased from Wuhan Elite Biotechnology Co., Ltd.

[0051] In the following embodiments, the sequences are as shown in Table 1: Table 1 Framework Nucleic Acid DNA Sequences The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0052] Example 1 This embodiment provides a method for preparing an electrochemical sensor based on framework nucleic acids and carboxyethyl dextran, the specific steps of which are as follows: S1. Synthesis of carboxyethyl dextran: S1-1. Weigh 10g of dextran T500 and dissolve it in 40mL of pure water to prepare solution A. S1-2. Take 20 mL of 10 mol / L NaOH solution as solution B; S1-3. Weigh 5.3g of acrylamide and dissolve it in 40mL of pure water to prepare solution C; S1-4. Take liquid A obtained in step S1-1, liquid B obtained in step S1-2, and liquid C obtained in step S1-3 in a volume ratio of 2:1:2 and stir them at room temperature until they are evenly dissolved. Then, place them in a 45°C water bath and magnetically stir for 24 hours to obtain a mixture. S1-5. Slowly add 3 times the volume of ethanol to the mixture obtained in step S1-4 for separation. Remove the supernatant, dissolve the white flocculent precipitate in 80 mL of water, and repeat the separation with ethanol once to obtain carboxyethyl dextran. S1-6. Adjust the pH of the carboxyethyl dextran obtained in step S1-5 to 7.3 with hydrochloric acid, and bring the concentration of carboxyethyl dextran to 100 mg / mL. Store it in a refrigerator at 4°C for later use.

[0053] S2, Synthesizing framework nucleic acids: DNA-1, DNA-2, DNA-3, and DNA-4 (sequences of the four strands are shown in Table 1) purchased from Shanghai Sangon Biotech were dissolved in TE buffer to a final concentration of 100 μmol / L. 1 μL of each of the four strands was added to 96 μL of TM buffer and mixed well. The mixture was heated at 95°C for 10 min, then rapidly cooled to 4°C and held for at least 30 s. The temperature control was performed using a PCR instrument to obtain a framework nucleic acid with a final concentration of 1 μmol / L.

[0054] S3. Fix the framework nucleic acid obtained in step S2 on the electrode surface: S3-1. Electrode cleaning: The screen-printed gold electrode (purchased from Metrohm, Switzerland) was cleaned with 0.1M H2SO4 solution. Electrochemical cleaning was performed using cyclic voltammetry to remove surface impurities. Then, it was rinsed with ultrapure water and finally dried with nitrogen to obtain the pretreated electrode. S3-2. Add 20 μL of the 1 μmol / L framework nucleic acid prepared in step S2 to the cleaned electrode surface obtained in step S3-1, and react at 37℃ for 2 h. After the reaction is completed, wash twice with phosphate buffered saline with 0.05% Tween-20 (PBST) and twice with phosphate buffered saline (PBS), each time for 5 min. Dry with nitrogen to obtain the framework nucleic acid / electrode.

[0055] S4. Add the carboxyethyl dextran obtained in step S1 to the surface of the framework nucleic acid / electrode obtained in step S3 for fixation: S4-1. Prepare a mixed solution by mixing the carboxyethyl dextran obtained in step S1 (50 mg / ml), 0.4 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 0.1 mol / L N-hydroxysuccinimide (NHS) at a volume ratio of 50:1:1. S4-2. Take 20 μL of the mixed solution obtained in step S4-1 and add it to the framework nucleic acid / electrode interface prepared in step S3. React at 25℃ for 2 h. After the reaction is complete, wash twice with PBST and twice with PBS, each time for 5 min. Dry with nitrogen to obtain carboxyethyl dextran / framework nucleic acid / electrode, i.e., electrochemical sensor.

[0056] Example 2 This embodiment provides a method for rapid detection of the pancreatic cancer serum biomarker CA19-9 using an electrochemical sensor based on framework nucleic acids and carboxyethyl dextran. The specific steps are as follows: S1, Immobilized CA19-9 antibody: S1-1. The carboxyethyl dextran / framework nucleic acid / electrode prepared in Example 1 was activated for 30 min with 40 μL of EDC / NHS mixed solution (where the concentration of EDC was 0.2 mol / L and the concentration of NHS was 0.05 mol / L). S1-2. Add 20 μL of CA19-9 monoclonal antibody with a concentration of 100 μg / mL (purchased from Fitzgerald, USA) to the membrane surface, react at 37°C for 2 h, or at 4°C for 18 h, and wash and dry after the reaction is complete. S1-2. Take another 50 μL of 5% casein blocking solution and add it to the surface of carboxyethyl dextran / framework nucleic acid / electrode and react for 30 min to block the remaining carboxyl groups on the electrode surface. S1-3. After each reaction step, wash twice with PBST and twice with PBS, 5 min each time, to obtain CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode, and store at 4℃. Other antibodies are fixed using a similar method.

[0057] S2, Antigen-antibody interaction: S2-1. Prepare CA19-9 antigen solutions of 0, 6.25, 12.5, 25, 50, 100, 200, and 400 U / mL using blank human serum. Then prepare a mixture of biotinylated CA19-9 antibody and streptavidin-modified HRP (where the concentration of biotinylated CA19-9 antibody is 10 μg / mL and the concentration of streptavidin-modified HRP is 8 μg / mL). Mix the antigens of different concentrations with the biotinylated CA19-9 antibody and streptavidin-modified HRP solutions at a volume ratio of 1:1 to obtain the mixture. S2-2. The mixture obtained in step S2-1 is dropped onto the surface of CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode, which was immobilized with CA19-9 antibody in step S1. The reaction is carried out at 42℃ for 15 min. After the reaction is completed, the mixture is washed 3 times with PBST and 2 times with PBS, and dried with nitrogen to obtain streptavidin-modified HRP / biotinylated antibody / antigen / CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode.

[0058] S3, Electrochemical detection of CA19-9: 30 μL of HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) was dropped onto the surface of streptavidin-modified HRP / biotinylated antibody / antigen / CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode at different CA19-9 antigen concentrations. The reaction was allowed to proceed for 15 s, and the reaction was detected by chronoamperometry using an electrochemical workstation.

[0059] Experimental results are as follows Figure 2 As shown, an electrochemical sensor using framework nucleic acids and carboxyethyl dextran was used to detect CA19-9 antigen in blank human serum as low as 6.25 U / mL.

[0060] Comparative Example 1 This comparative example provides a method for rapid detection of CA19-9 using an electrochemical sensor based on a framework nucleic acid. The specific steps are as follows: S1. Synthesizing framework nucleic acids: DNA-1, DNA-2, DNA-3, and DNA-5 (sequences of the four strands are shown in Table 1) purchased from Shanghai Sangon Biotech were dissolved in TE buffer to a final concentration of 100 μmol / L. 1 μL of each of the four strands was added to 96 μL of TM buffer and mixed well. The mixture was heated at 95°C for 10 min, then rapidly cooled to 4°C and held for at least 30 s. The temperature control was performed using a PCR instrument to obtain a framework nucleic acid with a final concentration of 1 μmol / L.

[0061] S2. Immobilize the framework nucleic acid obtained in step S2 on the electrode surface: S2-1. Electrode cleaning: The screen-printed electrode (purchased from Metrohm, Switzerland) was cleaned with 0.1M H2SO4 solution. Electrochemical cleaning was performed using cyclic voltammetry to remove surface impurities. Then, it was rinsed with ultrapure water and finally dried with nitrogen to obtain the pretreated electrode. S2-2. Add 20 μL of the 1 μmol / L framework nucleic acid prepared in step S1 to the cleaned electrode surface obtained in step S3-1, and react at 37℃ for 2 h. After the reaction is completed, wash twice with phosphate buffered saline with 0.05% Tween-20 (PBST) and twice with phosphate buffered saline (PBS), 5 min each time, and blow dry with nitrogen to obtain the framework nucleic acid / electrode.

[0062] Then, following Example 2, 100 μg / ml of CA19-9 antibody was fixed, and CA19-9 in blank human serum was detected.

[0063] The experimental results of detecting different concentrations of CA19-9 in blank human serum are as follows: Figure 3 As shown in the curves, the framework nucleic acid sensor can detect 25 U / mL of CA19-9 antigen in blank human serum, while the electrochemical sensor using framework nucleic acid and carboxyethyl dextran (…) Figure 2It can detect CA19-9 antigen in blank human serum as low as 6.25 U / mL. Compared with the two methods, the electrochemical sensor of framework nucleic acid and carboxyethyl dextran is more sensitive.

[0064] The results of the electrode surface current change experiment after immobilizing the same concentration of CA19-9 antibody in the electrochemical sensor of framework nucleic acid and the electrochemical sensor of framework nucleic acid and carboxyethyl dextran are as follows: Figure 4 As shown, from Figure 4 As can be seen, the current change value of the electrochemical sensor with framework nucleic acid and carboxyethyl dextran is significantly higher than that of the electrochemical sensor with framework nucleic acid, indicating that the electrochemical sensor with framework nucleic acid and carboxyethyl dextran immobilizes more CA19-9 antibody.

[0065] Comparative Example 2 This comparative example provides a double-antibody sandwich ELISA method for detecting CA19-9, used for comparison with Example 2. The method of use follows the instructions for the Elabscience® Human Glycan Antigen 19-9 Enzyme-Linked Immunosorbent Assay Kit (purchased from Wuhan Elabscience® Biotechnology Co., Ltd.). The specific steps are as follows: S1. Remove the kit from the refrigerator 20 minutes in advance and allow it to equilibrate to room temperature (18-25℃).

[0066] S2, Antigen-antibody interaction: S2-1. Prepare CA19-9 antigen solutions of 0, 6.25, 12.5, 25, 50, 100, 200, and 400 U / mL using blank human serum; S2-2. Add 100 μL of antigen solution of different concentrations from step S2-1 to the wells of the ELISA plate immobilized with CA19-9 antibody, and react at 37°C for 90 min; after the reaction is complete, discard the liquid in the wells. S2-3. Add 100 μL of biotinylated antibody to each well and react at 37°C for 60 min. After the reaction is complete, wash with the washing solution provided in the kit. S2-4. Add 100 μL of HRP conjugate to each well and react at 37°C for 60 min. After the reaction is complete, wash with the washing solution provided in the kit.

[0067] S3, enzyme-linked immunosorbent assay (ELISA) detection S3-1. Add 90 μL of substrate solution (TMB) to each well and react at 37°C in the dark for 15 min. S3-2. Add 50 μL of stop solution to each well to terminate the reaction. Immediately measure the optical density (OD value) of each well using a microplate reader at a wavelength of 450 nm.

[0068] The detection results of CA19-9 at different concentrations are as follows: Figure 5As shown, the ELISA method can detect CA19-9 antigen as low as 6.25 U / mL. However, compared to Example 2, in step S2, i.e., antigen-antibody interaction, the ELISA takes 210 min, while the method provided by this invention takes 15 min. In step S3, substrate addition reaction, the ELISA takes 15 min, while the method of this invention takes 15 s. Therefore, the method of this invention is faster. Furthermore, the method provided by this invention requires a sample volume of 20 μL, less than the 100 μL required by the ELISA method; the method of this invention requires even less sample volume.

[0069] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An electrochemical sensor based on a framework nucleic acid and carboxyethyl dextran, characterized by, The device includes electrodes on which capture probes interacting with the pancreatic cancer biomarker CA19-9 are immobilized. The capture probe is immobilized on the electrode surface using framework nucleic acid and carboxyethyl dextran.

2. The electrochemical sensor based on framework nucleic acid and carboxyethyl dextran according to claim 1, characterized in that, The framework nucleic acid is assembled into a tetrahedral structure by four DNA strands. The bottom of the framework nucleic acid tetrahedron is connected to the electrode surface through thiol, amino or aldehyde groups, and the amino group at the top of the tetrahedron interacts with the carboxyl group on the carboxyethyl dextran. The carboxyethyl dextran is a polymer with multiple carboxyethyl groups, some of which interact with amino groups on the framework nucleic acid through activation, while other carboxyl groups are linked by condensation with amino groups on the capture probe. The electrodes include screen-printed gold electrodes, screen-printed carbon electrodes, and nanoparticle electrodes. The capture probe includes an antibody and a DNA aptamer.

3. The electrochemical sensor based on framework nucleic acid and carboxyethyl dextran according to claim 2, characterized in that, The sequences of the four DNA single strands described are shown in SEQ ID NO.1 to SEQ ID NO.

4.

4. A method for preparing an electrochemical sensor based on framework nucleic acid and carboxyethyl dextran as described in any one of claims 1-3, characterized in that, The specific steps are as follows: S1. Dextran, acrylamide and sodium hydroxide are mixed and reacted to obtain carboxyethyl dextran; S2, Synthesizing framework nucleic acids; S3. Fix the framework nucleic acid obtained in step S2 on the electrode surface to obtain framework nucleic acid / electrode; S4. The carboxyethyl dextran obtained in step S1 is fixed on the surface of the framework nucleic acid / electrode obtained in step S3 to obtain carboxyethyl dextran / framework nucleic acid / electrode, i.e., electrochemical sensor.

5. The method for preparing the electrochemical sensor based on framework nucleic acid and carboxyethyl dextran according to claim 4, characterized in that, In step S1, the mass ratio of dextran, acrylamide, and sodium hydroxide is 100:35~45:50~60; In step S2, the method for synthesizing the framework nucleic acid is as follows: Four DNA single strands were dissolved in TE buffer to obtain single-stranded mother liquor; four different single-stranded mother liquors were added to TM buffer, mixed well, heated and reacted, and annealed to obtain framework nucleic acid; In step S3, the electrode is cyclically voltammetrically cleaned in sulfuric acid, and the framework nucleic acid obtained in step S2 is immobilized on the cleaned electrode surface. After cleaning, the framework nucleic acid / electrode is obtained. In step S3, the concentration of the framework nucleic acid is 1~20 μmol; In step S4, the activator and the carboxyethyl dextran obtained in step S1 are mixed and added to the framework nucleic acid / electrode interface prepared in step S3 for reaction. After washing, the carboxyethyl dextran / framework nucleic acid / electrode is obtained, which is the electrochemical sensor. In step S4, the concentration of the carboxyethyl dextran is 10~100 mg / mL.

6. The method for preparing the electrochemical sensor based on framework nucleic acid and carboxyethyl dextran according to claim 5, characterized in that, In step S2, the sequences of the four DNA single strands are shown in SEQ ID NO.1 to SEQ ID NO.4; the concentration of the single-strand mother liquor is 90 to 110 μmol / L; In step S4, the activator includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The volume ratio of the carboxyethyl dextran, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 50:0.5~1.5:0.5~1.

5. The concentration of the carboxyethyl dextran is 10~100 mg / mL; The concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.1~0.4 mol / L; The concentration of the N-hydroxysuccinimide is 0.1~0.4 mol / L.

7. The application of an electrochemical sensor based on framework nucleic acid and carboxyethyl dextran as described in any one of claims 1-3 in the immobilization of biomolecules and the detection of biomarkers.

8. The application according to claim 7, characterized in that, The biomarkers include CA19-9, a serum biomarker for pancreatic cancer.

9. The application according to claim 8, characterized in that, The method for detecting the serum biomarker CA19-9 in pancreatic cancer using the electrochemical sensor is as follows: S1. Immobilize the CA19-9 monoclonal antibody on the surface of the above-mentioned electrochemical sensor based on framework nucleic acid and carboxyethyl dextran, and after the reaction, obtain CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode; S2. Add CA19-9 antigen, biotinylated CA19-9 antibody and horseradish peroxidase-labeled streptavidin to the surface of CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode obtained in step S1. After the reaction, streptavidin-modified HRP / biotinylated antibody / antigen / CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode is obtained. S3. Add enzyme substrate to the surface of the streptavidin-modified HRP / biotinylated antibody / antigen / CA19-9 antibody / carboxyethyl dextran / framework nucleic acid / electrode obtained in step S2, and perform quantitative analysis using an electrochemical workstation.

10. The application according to claim 9, characterized in that, In step S1, the concentration of the CA19-9 monoclonal antibody is 20~200 μg / mL; In step S2, the volume ratio of the CA19-9 antigen, the biotinylated CA19-9 antibody, and the horseradish peroxidase-labeled streptavidin is 1:0.5~1.

5. In step S3, the enzyme substrate includes 3,3',5,5'-tetramethylbenzidine, 3,3'-diaminobenzidine, 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid), and o-phenylenediamine.

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