Construction method and application of zwitter-ion antifouling and dual-signal-ratio electrochemical biosensor

Abstract

The invention discloses a construction method and application of a zwitter-ion antifouling and dual-signal-ratio electrochemical biosensor, and belongs to the technical field of biochemical sensing analysis and life science. According to the invention, 3-aminopropyl dimethylamine oxide (APDMAO) zwitterions are prepared to be used as an antifouling interface. Specific positive and negative charges (N < + >-O-) of APDMAO promote hydrogen bond combination with water molecules, and a firm hydration barrier is formed. Meanwhile, the development of APDMAO overcomes the defect that zwitterions are difficult to synthesize and modify, and the application of APDMAO in biosensing analysis is expanded. A methylene blue (MB) internal standard modified alpha fetoprotein (AFP) aptamer is coupled with APDMAO under a controllable potential, so that the detection precision of the sensor is improved. The constructed ratio type electrochemical antifouling biosensor realizes sensitive and accurate detection of AFP, and the detection limit is as low as 3.41 fg / mL.

Description

Technical Field

[0001] The invention relates to a method for constructing a zwitterion antifouling and dual-signal ratio electrochemical biosensor and its application in detecting disease marker alpha-fetoprotein, and belongs to the field of biochemical sensor analysis and life science technology. Background Art

[0002] According to statistics, more than 100,000 people lose their lives due to liver cancer in my country every year. At present, the diagnosis of liver cancer mainly relies on imaging examinations and biomarker detection. However, imaging examinations have certain limitations. Liver cancer can only be monitored when it develops to a certain extent, resulting in many patients being in the middle and late stages when diagnosed. In order to better achieve early screening of liver cancer, efficient and sensitive detection of liver cancer markers is crucial. Alpha-fetoprotein (AFP) is an acidic glycoprotein with low content in the normal human body. If its level exceeds 25ng / mL, there is a risk of primary liver cancer. Therefore, there is an urgent need to develop an efficient analytical method to detect AFP in human serum, which is of great significance for the clinical diagnosis and timely intervention treatment of liver cancer.

[0003] Electrochemical analysis technology is widely used in biomarker detection due to its high sensitivity, specificity and low cost. However, in addition to the target analytes, there are many interfering biomolecules in serum, which can undergo nonspecific adsorption and biooxidation, thereby affecting the electrochemical signal, which poses a huge challenge to the accurate detection of the target. Based on this, it is very important to develop efficient antifouling materials for sensor interface modification to resist the above-mentioned biofouling.

[0004] So far, polyethylene glycol, polypeptides and zwitterions have been reported to have anti-fouling properties. Among them, polyethylene glycol is easy to oxidize and degrade, polypeptides are relatively expensive, and zwitterions have excellent anti-fouling ability due to their strong hydration, and are regarded as the gold standard of anti-fouling materials. The strong hydration between the surface charge of zwitterions and water molecules can form an ordered strong hydrogen bond water layer to prevent the surface adsorption of non-specific biological molecules, which is the key to determining their anti-fouling properties. At the same time, relevant studies have shown that the shorter the distance between the positive and negative charge groups, the smaller the dipole of the zwitterion, and the stronger the anti-fouling ability given by hydration. However, the main disadvantage of zwitterions is that they are difficult to synthesize and modify, which limits their application in anti-fouling systems. In order to meet the above challenges, the present invention designs and synthesizes an amino-functionalized 3-aminopropyldimethylamine oxide (APDMAO) as an efficient zwitterion anti-fouling interface by a one-step oxidation method. The one-step synthesis strategy and inherent functional groups give it the characteristics of easy synthesis and modification, thereby solving the defects of zwitterions in the application of electrochemical anti-fouling systems. Summary of the invention

[0005] One of the technical tasks of the present invention is to make up for the deficiencies of the prior art and prepare an antifouling coating based on APDMAO zwitterions to give the sensing interface a highly efficient antifouling ability.

[0006] The second technical task of the present invention is to construct an electrochemical biosensor with zwitterion antifouling and dual signal ratio.

[0007] The third technical task of the present invention is to provide the use of the zwitterion antifouling and dual-signal ratio electrochemical biosensor constructed by the construction method, namely to achieve sensitive and accurate detection of the liver cancer marker AFP. The electrochemical biosensor has a wide linear range and a low detection limit, as well as excellent stability, specificity and reproducibility.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] 1. Construction method of a zwitterion antifouling and dual-signal ratio electrochemical biosensor

[0010] 1-3 mg / mL dopamine was mixed with 2-8 mg / mL APDMAO zwitterions, and then 6 μL of the mixed solution was dropped onto the electrode surface and incubated for 50 min to obtain an electrode modified with a polydopamine-3-aminopropyldimethylamine oxide (PDA-APDMAO) antifouling coating; a 5 mM Mg 2+ and 0.5 mM tri(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution; in the AFP aptamer-methylene blue solution, an electrode modified with a PDA-APDMAO antifouling coating is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum wire electrode is used as an auxiliary electrode, and the AFP aptamer-methylene blue is coupled to the surface of the PDA-APDMAO antifouling coating by a constant potential method; the obtained working electrode is placed in an AFP solution and incubated for 45 minutes, and then incubated in a concanavalin-silver nanoparticle (ConA-Ag NPs) solution for 1 hour, and a dual-signal ratio electrochemical biosensor with a PDA-APDMAO antifouling interface is constructed; that is, an electrochemical biosensor with zwitterion antifouling and dual-signal ratio;

[0011] The zwitterion antifouling and dual signal ratio electrochemical biosensor, after each modification step, the electrode surface is gently rinsed with ultrapure water to remove unbound reagents;

[0012] The APDMAO zwitterion is prepared by dissolving 10 g of 3-dimethylaminopyrrolamine in 100 mL of ultrapure water, stirring thoroughly, and then dropping 15 mL of 30% H2O2, followed by purging with oxygen at 60° C. for 6 h, and rotary evaporation at 80° C. to obtain the APDMAO zwitterion;

[0013] The mixture contained 5 mM Mg 2+ The AFP aptamer-methylene blue solution containing 0.5 mM tris(2-carboxyethyl)phosphine is prepared by dissolving 234 mg of NaCl, 41 mg of MgCl2·6H2O, 5 mg of tris(2-carboxyethyl)phosphine and 97 mg of tris(hydroxymethyl)aminomethane in 40 mL of ultrapure water, and adjusting the pH of the solution to 8.5 with 1 M dilute hydrochloric acid to prepare a buffer solution for standby use; subsequently, the AFP aptamer modified with methylene blue is dispersed in 15.4 μL of the buffer solution, heated to 95° C., maintained for 3 min, and then rapidly cooled to room temperature to obtain a 5 mM MgCl2·6H2O solution containing 0.5 mM tris(2-carboxyethyl)phosphine. 2+ and 0.5 mM tris(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution;

[0014] The constant potential coupling method has a potential of 0.55 V and a coupling time of 500 s;

[0015] The concentrations of the AFP solutions are 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, and 10 ng / mL, respectively;

[0016] The ConA-Ag NPs solution is prepared by dropping 0.1M AgNO3 into a mixed solution containing 10mM NaOH and 0.25mM sodium citrate; stirring is continued until the solution turns bright yellow, and glutathione is added; stirring is continued for 20 minutes, and 5.0μL of bis(N-hydroxysuccinimide) suberate is added; and 10μL of a 0.5mg / mL ConA solution is added, and after incubation for 15 minutes, a ConA-Ag NPs solution is obtained.

[0017] 2. Application of the zwitterion antifouling and dual signal ratio electrochemical biosensor constructed by the construction method for the detection of liver cancer marker AFP

[0018] A three-electrode system was constructed using the modified glassy carbon electrode as the working electrode, the calomel electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode; the three-electrode system was connected to an electrochemical workstation with a voltage range of -0.45 to 0.3 V, an amplitude of 50 mV, and a pulse width of 0.05 s; a phosphate buffered saline solution with a pH of 6.5 to 8.5 was used as the detection base solution, and the electrochemical signals under different concentrations of AFP were detected using the three-electrode system; a working curve was drawn based on the linear relationship between the obtained electrochemical signal and the logarithm of the AFP concentration; the constructed electrochemical antifouling biosensor has good stability, specificity and reproducibility, and achieves sensitive and accurate detection of AFP in a wide dynamic range of 10 fg / mL to 10 ng / mL, with a detection limit as low as 3.41 fg / mL.

[0019] Beneficial technical effects of the present invention:

[0020] 1. The present invention prepares a high-efficiency antifouling coating based on APDMAO zwitterions. Zwitterions are dipole ions with both positive and negative charges on the same molecule. The strong hydration between the surface charge of the zwitterions and water molecules can form an ordered strong hydrogen bond water layer, which prevents the surface adhesion of non-specific biological molecules and greatly improves the anti-fouling ability of the interface. At the same time, APDMAO solves the defect that zwitterions are difficult to synthesize and modify, and promotes the application of zwitterionic antifouling materials in electrochemical biosensing analysis.

[0021] 2. The present invention constructs a zwitterion antifouling and dual-signal ratio electrochemical biosensor, in which the thiol-containing AFP aptamer modified with methylene blue as the internal standard is coupled with APDMAO under a controllable potential, which not only shortens the aptamer incubation time but also improves the detection accuracy of the sensor.

[0022] 3. The zwitterion antifouling and dual-signal ratio electrochemical biosensor constructed in the present invention exhibits excellent antifouling performance, as well as high stability, specificity, and reproducibility, and achieves sensitive and accurate detection of the liver cancer marker AFP in serum. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto, and all changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0024] Example 1 A method for constructing a zwitterion antifouling and dual signal ratio electrochemical biosensor 1 mg / mL of dopamine was mixed with 2 mg / mL of APDMAO zwitterion, and then 6 μL of the mixed solution was dropped onto the electrode surface and incubated for 50 min to obtain an electrode modified with a polydopamine-3-aminopropyldimethylamine oxide (PDA-APDMAO) antifouling coating; a 5 mM Mg 2+ and 0.5 mM tri(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution; in the AFP aptamer-methylene blue solution, an electrode modified with a PDA-APDMAO antifouling coating is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum wire electrode is used as an auxiliary electrode, and the AFP aptamer-methylene blue is coupled to the surface of the PDA-APDMAO antifouling coating by a constant potential method; the obtained working electrode is placed in an AFP solution and incubated for 45 minutes, and then incubated in a concanavalin-silver nanoparticle (ConA-Ag NPs) solution for 1 hour, and a dual-signal ratio electrochemical biosensor with a PDA-APDMAO antifouling interface is constructed; that is, an electrochemical biosensor with zwitterion antifouling and dual-signal ratio;

[0025] The zwitterion antifouling and dual signal ratio electrochemical biosensor, after each modification step, the electrode surface is gently rinsed with ultrapure water to remove unbound reagents;

[0026] The APDMAO zwitterion is prepared by dissolving 10 g of 3-dimethylaminopyrrolamine in 100 mL of ultrapure water, stirring thoroughly, and then dropping 15 mL of 30% H2O2, followed by purging with oxygen at 60° C. for 6 h, and rotary evaporation at 80° C. to obtain the APDMAO zwitterion;

[0027] The mixture contained 5 mM Mg 2+ The AFP aptamer-methylene blue solution containing 0.5 mM tris(2-carboxyethyl)phosphine is prepared by dissolving 234 mg of NaCl, 41 mg of MgCl2·6H2O, 5 mg of tris(2-carboxyethyl)phosphine and 97 mg of tris(hydroxymethyl)aminomethane in 40 mL of ultrapure water, and adjusting the pH of the solution to 8.5 with 1 M dilute hydrochloric acid to prepare a buffer solution for standby use; subsequently, the AFP aptamer modified with methylene blue is dispersed in 15.4 μL of the buffer solution, heated to 95° C., maintained for 3 min, and then rapidly cooled to room temperature to obtain a 5 mM MgCl2·6H2O solution containing 0.5 mM tris(2-carboxyethyl)phosphine. 2+ and 0.5 mM tris(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution;

[0028] The constant potential coupling method has a potential of 0.55 V and a coupling time of 500 s;

[0029] The concentrations of the AFP solutions are 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, and 10 ng / mL, respectively;

[0030] The ConA-Ag NPs solution is prepared by dropping 0.1M AgNO3 into a mixed solution containing 10mM NaOH and 0.25mM sodium citrate; stirring continuously until the solution turns bright yellow, adding glutathione; stirring continuously for 20 minutes, adding 5.0μL of bis(N-hydroxysuccinimide) suberic acid; continuing to add 10μL of a 0.5mg / mL ConA solution, incubating for 15 minutes, and obtaining a ConA-Ag NPs solution. Example 2 A method for constructing an electrochemical biosensor with zwitterion antifouling and dual signal ratio 2mg / mL of dopamine is mixed with 5mg / mL of APDMAO zwitterion, and then 6μL of the mixed solution is dropped onto the electrode surface and incubated for 50 minutes to obtain an electrode modified with a polydopamine-3-aminopropyldimethylamine oxide (PDA-APDMAO) antifouling coating; preparing a solution containing 5mM Mg 2+and 0.5 mM tri(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution; in the AFP aptamer-methylene blue solution, an electrode modified with a PDA-APDMAO antifouling coating is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum wire electrode is used as an auxiliary electrode, and the AFP aptamer-methylene blue is coupled to the surface of the PDA-APDMAO antifouling coating by a constant potential method; the obtained working electrode is placed in an AFP solution and incubated for 45 minutes, and then incubated in a concanavalin-silver nanoparticle (ConA-Ag NPs) solution for 1 hour, and a dual-signal ratio electrochemical biosensor with a PDA-APDMAO antifouling interface is constructed; that is, an electrochemical biosensor with zwitterion antifouling and dual-signal ratio;

[0031] The zwitterion antifouling and dual signal ratio electrochemical biosensor, after each modification step, the electrode surface is gently rinsed with ultrapure water to remove unbound reagents;

[0032] The APDMAO zwitterion is prepared by dissolving 10 g of 3-dimethylaminopyrrolamine in 100 mL of ultrapure water, stirring thoroughly, and then dropping 15 mL of 30% H2O2, followed by purging with oxygen at 60° C. for 6 h, and rotary evaporation at 80° C. to obtain the APDMAO zwitterion;

[0033] The mixture contained 5 mM Mg 2+ The AFP aptamer-methylene blue solution containing 0.5 mM tris(2-carboxyethyl)phosphine is prepared by dissolving 234 mg of NaCl, 41 mg of MgCl2·6H2O, 5 mg of tris(2-carboxyethyl)phosphine and 97 mg of tris(hydroxymethyl)aminomethane in 40 mL of ultrapure water, and adjusting the pH of the solution to 8.5 with 1 M dilute hydrochloric acid to prepare a buffer solution for standby use; subsequently, the AFP aptamer modified with methylene blue is dispersed in 15.4 μL of the buffer solution, heated to 95° C., maintained for 3 min, and then rapidly cooled to room temperature to obtain a 5 mM MgCl2·6H2O solution containing 0.5 mM tris(2-carboxyethyl)phosphine. 2+ and 0.5 mM tris(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution;

[0034] The constant potential coupling method has a potential of 0.55 V and a coupling time of 500 s;

[0035] The concentrations of the AFP solutions are 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, and 10 ng / mL, respectively;

[0036] The ConA-Ag NPs solution is prepared by dropping 0.1M AgNO3 into a mixed solution containing 10mM NaOH and 0.25mM sodium citrate; stirring continuously until the solution turns bright yellow, adding glutathione; stirring continuously for 20 minutes, adding 5.0μL of bis(N-hydroxysuccinimide) suberic acid; continuing to add 10μL of a 0.5mg / mL ConA solution, incubating for 15 minutes, and obtaining a ConA-Ag NPs solution. Example 3 3mg / mL of dopamine is mixed with 8mg / mL of APDMAO zwitterions, and then 6μL of the mixed solution is dropped onto the electrode surface and incubated for 50 minutes to obtain an electrode modified with a polydopamine-3-aminopropyldimethylamine oxide (PDA-APDMAO) antifouling coating; preparing a solution containing 5mM Mg 2+ and 0.5 mM tri(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution; in the AFP aptamer-methylene blue solution, an electrode modified with a PDA-APDMAO antifouling coating is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum wire electrode is used as an auxiliary electrode, and the AFP aptamer-methylene blue is coupled to the surface of the PDA-APDMAO antifouling coating by a constant potential method; the obtained working electrode is placed in an AFP solution and incubated for 45 minutes, and then incubated in a concanavalin-silver nanoparticle (ConA-Ag NPs) solution for 1 hour, and a dual-signal ratio electrochemical biosensor with a PDA-APDMAO antifouling interface is constructed; that is, an electrochemical biosensor with zwitterion antifouling and dual-signal ratio;

[0037] The zwitterion antifouling and dual signal ratio electrochemical biosensor, after each modification step, the electrode surface is gently rinsed with ultrapure water to remove unbound reagents;

[0038] The APDMAO zwitterion is prepared by dissolving 10 g of 3-dimethylaminopyrrolamine in 100 mL of ultrapure water, stirring thoroughly, and then dropping 15 mL of 30% H2O2, followed by purging with oxygen at 60° C. for 6 h, and rotary evaporation at 80° C. to obtain the APDMAO zwitterion;

[0039] The mixture contained 5 mM Mg 2+ The AFP aptamer-methylene blue solution containing 0.5 mM tris(2-carboxyethyl)phosphine is prepared by dissolving 234 mg of NaCl, 41 mg of MgCl2·6H2O, 5 mg of tris(2-carboxyethyl)phosphine and 97 mg of tris(hydroxymethyl)aminomethane in 40 mL of ultrapure water, and adjusting the pH of the solution to 8.5 with 1 M dilute hydrochloric acid to prepare a buffer solution for standby use; subsequently, the AFP aptamer modified with methylene blue is dispersed in 15.4 μL of the buffer solution, heated to 95° C., maintained for 3 min, and then rapidly cooled to room temperature to obtain a 5 mM MgCl2·6H2O solution containing 0.5 mM tris(2-carboxyethyl)phosphine.2+ and 0.5 mM tris(2-carboxyethyl)phosphine in an AFP aptamer-methylene blue solution;

[0040] The constant potential coupling method has a potential of 0.55 V and a coupling time of 500 s;

[0041] The concentrations of the AFP solutions are 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, and 10 ng / mL, respectively;

[0042] The ConA-Ag NPs solution is prepared by dropping 0.1M AgNO3 into a mixed solution containing 10mM NaOH and 0.25mM sodium citrate; stirring is continued until the solution turns bright yellow, and glutathione is added; stirring is continued for 20 minutes, and 5.0μL of bis(N-hydroxysuccinimide) suberate is added; and 10μL of a 0.5mg / mL ConA solution is added, and after incubation for 15 minutes, a ConA-Ag NPs solution is obtained.

[0043] Example 4 Use of the zwitterionic antifouling and dual-signal ratio electrochemical biosensor constructed by the construction method described in Example 1, Example 2 and Example 3 for the detection of liver cancer marker AFP. A three-electrode system is constructed using a modified glassy carbon electrode as a working electrode, a calomel electrode as a reference electrode, and a platinum wire electrode as an auxiliary electrode; the three-electrode system is connected to an electrochemical workstation with a voltage range of -0.45 to 0.3 V, an amplitude of 50 mV, and a pulse width of 0.05 s; a phosphate buffered saline solution with a pH of 6.5 is used as a detection base solution, and the electrochemical signals under different concentrations of AFP are detected using the three-electrode system; a working curve is drawn based on the linear relationship between the obtained electrochemical signal and the logarithm of the AFP concentration; the constructed electrochemical antifouling biosensor has good stability, specificity and reproducibility, and achieves sensitive and accurate detection of AFP within a wide dynamic range of 10 fg / mL to 10 ng / mL, with a detection limit as low as 3.41 fg / mL.

[0044] Example 5 Use of the zwitterionic antifouling and dual-signal ratio electrochemical biosensor constructed by the construction method described in Example 1, Example 2 and Example 3 for the detection of liver cancer marker AFP. A three-electrode system is constructed using a modified glassy carbon electrode as a working electrode, a calomel electrode as a reference electrode, and a platinum wire electrode as an auxiliary electrode; the three-electrode system is connected to an electrochemical workstation with a voltage range of -0.45 to 0.3 V, an amplitude of 50 mV, and a pulse width of 0.05 s; a phosphate buffered saline solution with a pH of 7.4 is used as a detection base solution, and the electrochemical signals under different concentrations of AFP are detected using the three-electrode system; a working curve is drawn based on the linear relationship between the obtained electrochemical signal and the logarithm of the AFP concentration; the constructed electrochemical antifouling biosensor has good stability, specificity and reproducibility, and achieves sensitive and accurate detection of AFP within a wide dynamic range of 10 fg / mL to 10 ng / mL, with a detection limit as low as 3.41 fg / mL.

[0045] Example 6 Use of the zwitterionic antifouling and dual-signal ratio electrochemical biosensor constructed by the construction method described in Example 1, Example 2 and Example 3 for the detection of liver cancer marker AFP. A three-electrode system is constructed using a modified glassy carbon electrode as a working electrode, a calomel electrode as a reference electrode, and a platinum wire electrode as an auxiliary electrode; the three-electrode system is connected to an electrochemical workstation with a voltage range of -0.45 to 0.3 V, an amplitude of 50 mV, and a pulse width of 0.05 s; a phosphate buffered saline solution with a pH of 8.5 is used as the detection base solution, and the electrochemical signals under different concentrations of AFP are detected using the three-electrode system; a working curve is drawn based on the linear relationship between the obtained electrochemical signal and the logarithm of the AFP concentration; the constructed electrochemical antifouling biosensor has good stability, specificity and reproducibility, and achieves sensitive and accurate detection of AFP within a wide dynamic range of 10 fg / mL to 10 ng / mL, with a detection limit as low as 3.41 fg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Flowchart of the construction method for zwitterion antifouling and dual-signal ratiometric electrochemical biosensor.

[0047] Figure 2 This is the H NMR spectrum of APDMAO zwitterion.

[0048] Figure 3 Figure 2 is the synthetic mechanism diagram of PDA-APDMAO.

[0049] Figure 4 This is the attenuated total reflection infrared spectrum of PDA-APDMAO.

[0050] Figure 5Figure 2 is the X-ray photoelectron spectrum of PDA-APDMAO, where (A) is the full-area X-ray photoelectron spectrum of PDA-APDMAO; (B) and (C) are high-resolution X-ray photoelectron spectra of the O1s region and the N1s region, respectively.

[0051] Figure 6 Static water contact angle diagrams of (A) bare electrode and (B) PDA-APDMAO modified electrode.

[0052] Figure 7 Differential pulse voltammetry curves of (A) bare electrode and (B) PDA-APDMAO modified electrode incubated in different concentrations of human serum.

[0053] Figure 8 Fluorescence imaging of (A) bare electrode and (B) PDA-APDMAO modified electrode after incubation in fluorescein-conjugated bovine serum albumin solution for 2 h.

[0054] Fig. 9 Schematic representation of the process for constructing an electrochemical biosensor based on (A) cyclic voltammetry and (B) electrochemical impedance spectroscopy.

[0055] Fig.10 The differential pulse voltammetry curves of different modified electrodes based on the dual-signal ratio sensing strategy.

[0056] Fig.11 (A) DPV responses of the zwitterionic antifouling and dual signal ratio electrochemical biosensor after incubation with different concentrations of AFP and (B) the corresponding calibration curve, where a to g represent concentrations of 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, and 10 ng / mL, respectively.

[0057] Fig.12 (A) Specificity, (B) long-term stability, (C) reproducibility of the zwitterion antifouling and dual-signal ratio electrochemical biosensor constructed by the construction method, and (D) comparison of sample detection results with hospital methods.

Claims

1. A method for constructing a zwitterion antifouling and dual signal ratio electrochemical biosensor, characterized in that: 1-3 mg / mL dopamine was mixed with 2-8 mg / mL 3-aminopropyl dimethylamine oxide zwitterion, and then 6 μL of the mixed solution was dropped onto the electrode surface and incubated for 50 min to obtain an electrode modified with polydopamine-3-aminopropyl dimethylamine oxide antifouling coating; 5 mM Mg 2+ and 0.5 mM tris(2-carboxyethyl)phosphine; in the alpha-fetoprotein aptamer-methylene blue solution, an electrode modified with a polydopamine-3-aminopropyldimethylamine oxide antifouling coating is used as a working electrode, a calomel electrode is used as a reference electrode, and a platinum wire electrode is used as an auxiliary electrode, and the alpha-fetoprotein aptamer-methylene blue is coupled to the surface of the polydopamine-3-aminopropyldimethylamine oxide antifouling coating by a constant potential method; the obtained working electrode is placed in an alpha-fetoprotein solution and incubated for 45 minutes, and then incubated in a concanavalin-silver nanoparticle solution for 1 hour, to construct a dual-signal ratio electrochemical biosensor with a polydopamine-3-aminopropyldimethylamine oxide antifouling interface; that is, an electrochemical biosensor with zwitterion antifouling and dual-signal ratio; After each modification step, the electrode surface of the zwitterion antifouling and dual signal ratio electrochemical biosensor was gently rinsed with ultrapure water to remove unbound reagents.

2. The method for constructing the zwitterion antifouling and dual signal ratio electrochemical biosensor according to claim 1, characterized in that: The 3-aminopropyldimethylamine oxide zwitter ion is prepared by dissolving 10 g of 3-dimethylaminopyrrolamine in 100 mL of ultrapure water, stirring thoroughly, adding 15 mL of 30% H2O2 by mass, then purging with oxygen at 60° C. for 6 hours, and rotary evaporating at 80° C. to obtain the 3-aminopropyldimethylamine oxide zwitter ion.

3. The method for constructing the zwitterion antifouling and dual signal ratio electrochemical biosensor according to claim 1, characterized in that: The mixture contained 5 mM Mg 2+ The alpha-fetoprotein aptamer-methylene blue solution containing 0.5 mM tris(2-carboxyethyl)phosphine is prepared by dissolving 234 mg of NaCl, 41 mg of MgCl2·6H2O, 5 mg of tris(2-carboxyethyl)phosphine and 97 mg of tris(hydroxymethyl)aminomethane in 40 mL of ultrapure water, and adjusting the pH of the solution to 8.5 with 1 M dilute hydrochloric acid to prepare a buffer solution for standby use; subsequently, the alpha-fetoprotein aptamer modified with methylene blue is dispersed in 15.4 μL of the buffer solution, heated to 95° C., maintained for 3 min, and then rapidly cooled to room temperature to obtain a 5 mM MgCl2·6H2O solution containing 0.5 mM tris(2-carboxyethyl)phosphine. 2+ and 0.5 mM tris(2-carboxyethyl)phosphine in an alpha-fetoprotein aptamer-methylene blue solution.

4. The method for constructing the zwitterion antifouling and dual signal ratio electrochemical biosensor according to claim 1, characterized in that: The constant potential coupling was performed at a potential of 0.55 V and a coupling time of 500 s.

5. The method for constructing the zwitterion antifouling and dual signal ratio electrochemical biosensor according to claim 1, characterized in that: The concentrations of the alpha-fetoprotein solutions are 10fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, and 10ng / mL, respectively.

6. The method for constructing the zwitterion antifouling and dual signal ratio electrochemical biosensor according to claim 1, characterized in that: The concanavalin-silver nanoparticle solution is prepared by dropping 0.1M AgNO3 into a mixed solution containing 10mM NaOH and 0.25mM sodium citrate; continuously stirring until the solution turns bright yellow, adding glutathione; continuing stirring for 20 minutes, adding 5.0μL of bis(N-hydroxysuccinimide) suberate; continuing to add 10μL of a 0.5mg / mL concanavalin solution, incubating for 15 minutes, and obtaining a concanavalin-silver nanoparticle solution.

7. Use of the zwitterion antifouling and dual signal ratio electrochemical biosensor constructed by the construction method of claim 1 for detecting liver cancer marker AFP.

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