Zwitterionic polymer, electrochemical sensor and preparation method and application thereof

Abstract

The application discloses a zwitterionic polymer, an electrochemical sensor and a preparation method and application thereof, the zwitterionic polymer is a sulfobetaine type zwitterionic polymer, has strong hydrophilicity and electric neutrality, and solventization of charged functional groups and hydrogen bond interaction can make a hydration layer formed on the surface of the zwitterionic polymer; the surface of the hydration layer can effectively resist non-specific adsorption, has high anti-fouling performance, and has great application potential in the field of anti-fouling electrochemical sensors. The application further provides an electrochemical sensor, and a fouling-resistant layer formed by the zwitterionic polymer on the electrochemical sensor can effectively resist non-specific adsorption, has high anti-fouling performance, can reduce and exclude signal interference in the detection of actual samples, thereby reducing sample pretreatment work, and has the characteristics of high anti-interference ability and good reproducibility in the detection of chloramphenicol.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection technology, specifically relating to an amphoteric polymer, an electrochemical sensor, its preparation method, and its application. Background Technology

[0002] Compared to traditional detection methods, electrochemical sensors offer advantages such as fast analysis speed, high sensitivity, ease of operation, and the ability to be miniaturized and portable. Therefore, they are commonly used in analytical detection in fields such as medicine and food safety. However, when applied to actual sample detection, interfering substances in the sample, such as proteins and carbohydrates, can easily interact with the sensor surface, causing non-specific adsorption and contaminating the sensor, severely affecting its sensitivity and accuracy. Researchers typically perform sample pretreatment, but this process is time-consuming, complex, and inefficient. Therefore, by modifying the electrode surface with antifouling materials, electrochemical sensors with inherent antifouling properties can be constructed. This allows for sensitive and accurate detection of samples requiring only simple pretreatment, significantly improving detection efficiency.

[0003] Commonly used antifouling materials for modifying sensor surfaces include polyethylene glycol (PEG) and its derivatives, zwitterionic materials, and peptides. All of these antifouling materials possess strong hydrophilicity and electroneutrality. The hydrophilic surfaces formed by their modification can bind with water molecules through electrostatic interactions and hydrogen bonds to form a dense hydration layer, thereby reducing non-specific adsorption. PEG and peptides are frequently used in the fabrication of antifouling electrochemical sensors; however, PEG is easily oxidized and broken down, and peptides are easily enzymatically hydrolyzed in biological media and are relatively expensive. Zwitterionic materials offer stable antifouling properties in electrochemical sensors, but due to their complex polymerization process, they are difficult to anchor onto the sensor surface after synthesis. Currently, there are relatively few reported cases of zwitterionic material-modified sensors. Summary of the Invention

[0004] To overcome the problems existing in the prior art, one objective of this invention is to provide a zwitterionic polymer. A second objective is to provide a method for preparing the aforementioned zwitterionic polymer. A third objective is to provide an electrochemical sensor. A fourth objective is to provide a method for preparing the aforementioned electrochemical sensor. A fifth objective is to provide applications of the aforementioned electrochemical sensor.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a zwitterionic polymer, the structural formula of which is shown in Formula 1 below: Formula 1.

[0006] The second aspect of the present invention provides a method for preparing the zwitterionic polymer of the first aspect, comprising the following steps: oxidizing N-[(3-(dimethylamino)propyl]methacrylamide to obtain a zwitterionic monomer, and photopolymerizing the zwitterionic monomer with methacrylate sulfobetaine to obtain the zwitterionic polymer.

[0007] Preferably, the oxidation reaction step includes: reacting N-[(3-(dimethylamino)propyl]methacrylamide with hydrogen peroxide in an oxygen atmosphere.

[0008] More preferably, the oxidation reaction also uses a stabilizer.

[0009] More preferably, the stabilizer is diethylenetriaminepentaacetic acid.

[0010] More preferably, the oxidation reaction is carried out at a temperature of 60-70°C.

[0011] More preferably, the oxidation reaction takes 4 to 7 hours.

[0012] More preferably, the oxidation reaction is carried out by reacting N-[(3-(dimethylamino)propyl]methacrylamide in an aqueous solution of hydrogen peroxide.

[0013] More preferably, it further includes: extracting and collecting the aqueous phase after the oxidation reaction, dissolving it in anhydrous ethanol, removing the water by rotary evaporation, and freeze-drying to obtain the zwitterionic monomer.

[0014] Preferably, the photopolymerization reaction step includes: the zwitterionic monomer reacting with methacrylate sulfobetaine under the action of a photoinitiator.

[0015] More preferably, the photoinitiator is selected from at least one of 2-hydroxy-2-methylphenylacetone, 1-hydroxy-cyclohexylphenylacetone, and 2-hydroxy-4-(2-hydroxyethoxy)-2-methylphenylacetone.

[0016] More preferably, the photopolymerization reaction is carried out under ultraviolet light.

[0017] More preferably, the reaction time of the photopolymerization reaction is 20-40 min.

[0018] Preferably, the mass ratio of the zwitterionic monomer to methacrylate sulfobetaine is (1~9):1.

[0019] More preferably, the mass ratio is (3~6):1.

[0020] A third aspect of the present invention provides an electrochemical sensor comprising an electrode, the surface of which is an antifouling layer formed of the zwitterionic polymer described in the first aspect, the surface of which is loaded with silver-copper nanoparticles and thiol aptamers.

[0021] Preferably, the electrode is a glassy carbon electrode.

[0022] Preferably, the thiol aptamer is a thiol chloramphenicol aptamer.

[0023] More preferably, the sequence of the thiol chloramphenicol aptamer is 5'-SH-(CH2)6-ACT TCA GTG AGT TGTCCC ACG GTC GGC GAG TCG GTG GTAG-3'.

[0024] A fourth aspect of the present invention provides a method for preparing the electrochemical sensor described in the third aspect, comprising the following steps: S1. N-[(3-(dimethylamino)propyl]methacrylamide is oxidized to obtain a zwitterionic monomer. The zwitterionic monomer, methacrylate sulfobetaine and photoinitiator are mixed in a solvent to obtain a mixture. The mixture is then coated on the surface of the electrode and photopolymerization is carried out to obtain electrode 1 with an antifouling layer on the surface. S2. Coat the surface of the antifouling layer with a dispersion containing silver-copper nanoparticles, and react to modify the surface of the antifouling layer with silver-copper nanoparticles to obtain electrode 2; place electrode 2 in a mercapto aptamer solution for incubation to obtain the electrochemical sensor.

[0025] This invention coats the electrode surface with zwitterionic monomers and methacrylate sulfobetaine, and modifies the electrode surface with zwitterionic copolymers by photo-initiated in-situ polymerization to form an anti-fouling surface. The in-situ synthesis method solves the problem that zwitterionic materials are difficult to anchor onto the sensor surface after synthesis.

[0026] Preferably, in step S1, the coating method is drop coating.

[0027] Preferably, in step S1, the photopolymerization reaction is carried out under ultraviolet light.

[0028] Preferably, in step S1, the reaction time of the photopolymerization reaction is 20-40 min.

[0029] Preferably, step S1 further includes: after the photopolymerization reaction, immersing the electrode in PBS to remove excess monomers and photoinitiators.

[0030] Preferably, the silver-copper nanoparticles are synthesized using a seed growth method with silver nanoparticles as the substrate.

[0031] More preferably, the preparation steps of the silver nanoparticles include: reacting a reducing agent, a ligand, and a silver source to obtain silver nanoparticles.

[0032] More preferably, the reducing agent is glucose; the ligand is hexadecylamine; and the silver source is silver nitrate.

[0033] More preferably, the reaction time is 50-70 min; the reaction temperature is 90-120℃.

[0034] More preferably, the preparation steps of the silver-copper nanoparticles include: reacting trimethylamine, silver nanoparticles, and a copper source to obtain silver-copper nanoparticles.

[0035] Preferably, in step S2, a dispersion containing silver-copper nanoparticles is coated on the surface of the antifouling layer, and after standing for a period of time, a self-assembly reaction is carried out to modify the surface of the antifouling layer with silver-copper nanoparticles, thereby obtaining electrode 2.

[0036] Preferably, in step S2, the incubation time is 8-12 h; and the incubation temperature is 0-5℃.

[0037] Preferably, in step S2, the concentration of the thiol aptamer solution is 0.1~3 μmol / L. -1 .

[0038] The fifth aspect of the invention provides the application of the electrochemical sensor described in the third aspect in the detection of chloramphenicol.

[0039] Preferably, the method for detecting chloramphenicol includes the following steps: Chloramphenicol standard solutions of different concentrations were prepared and detected using the electrochemical sensor described in the third aspect. Differential pulse voltammetry (DPV) was used for scanning, and the signal inhibition rate was calculated by recording the current signals before and after detection. A standard curve was plotted based on the obtained signal inhibition rates of different concentrations and the concentrations of the chloramphenicol standard solutions. The electrochemical sensor described above was used to collect and detect the sample liquid to be tested. The presence of chloramphenicol in the sample liquid was determined by analyzing the current signals before and after detection. The obtained signal inhibition rate was compared with the standard curve to obtain the concentration of chloramphenicol in the sample liquid to be tested.

[0040] Preferably, the electrochemical sensor is used in the detection of chloramphenicol residues in food.

[0041] More preferably, the method for detecting chloramphenicol residues in food includes the following steps: The food sample after simple processing is diluted to a certain factor to obtain the liquid sample to be tested. The liquid sample to be tested is collected and detected by the electrochemical sensor described above. The current signal before and after detection is analyzed to determine whether the liquid sample to be tested contains chloramphenicol. The obtained signal inhibition rate is compared with the standard curve to obtain the concentration of chloramphenicol in the liquid sample to be tested.

[0042] The beneficial effects of this invention are: This invention provides an amphoteric polymer, which is a sulfonate betaine-type amphoteric polymer with strong hydrophilicity and electroneutrality. The solvation and hydrogen bonding of charged functional groups can form a hydration layer on the surface of the amphoteric polymer. This hydration layer can effectively resist nonspecific adsorption and has high antifouling performance, showing great application potential in the field of antifouling electrochemical sensors.

[0043] Specifically, compared with the prior art, the present invention has the following advantages: (1) The present invention also provides an electrochemical sensor. The antifouling layer formed by zwitterionic polymer on the electrochemical sensor can effectively resist non-specific adsorption and has high antifouling performance. It can reduce and eliminate signal interference in the detection of actual samples, thereby reducing the sample pretreatment work. In addition, the silver-copper nano-ions (Ag-CuNDs) on the electrochemical sensor can not only be used for the connection of thiol aptamers, but also enhance the electrochemical signal. Moreover, the loaded silver-copper nano-ions do not affect the antifouling performance of zwitterionic polymer.

[0044] (2) The electrochemical sensor of the present invention can be used as a chloramphenicol electrochemical aptamer sensor. Due to the high selectivity of the mercaptochloramphenicol aptamer, the chloramphenicol electrochemical aptamer sensor has specific selectivity and high sensitivity to chloramphenicol within a certain range. In addition, the zwitterionic polymer can make the electrochemical sensor have high anti-interference ability and good reproducibility in detecting chloramphenicol residues in food. Attached Figure Description

[0045] Figure 1 The hydrogen nuclear magnetic resonance spectra characterized by DMAPMA (a) and TMAOMA (b) are shown. Figure 2 Characterization of Ag-Cu NDs, including transmission electron microscopy (a), energy-dispersive X-ray spectroscopy (b), and ultraviolet absorption spectrum (c). Figure 3 Scanning electron microscope (SEM) images of different modified electrodes; GCE (a), PTMAO-SBMA / GCE (b), and Ag-Cu NDs / PTMAO-SBMA / GCE (c). Figure 4To optimize the ratio of zwitterionic monomers, (a) bovine serum albumin (BSA), (b) lysozyme (Lys), and (c) glycogen (Gn). Figure 5 Concentration optimization of Ag-Cu NDs (a) and aptamer concentration optimization (b); Figure 6 A comparison of the signal inhibition rates of GCE, PTMAO-SBMA / GCE, and Ag-Cu NDs / PTMAO-SBMA / GCE at different concentrations of (a) glucose, (b) fructose, and (c) glycogen. Figure 7 The DPV signal response of GCE in (a) glucose, (b) fructose and (c) glycogen, respectively; Figure 8 The DPV signal response of PTMAO-SBMA / GCE in (a) glucose, (b) fructose and (c) glycogen, respectively; Figure 9 The DPV signal responses of Ag-Cu NDs / PTMAO-SBMA / GCE in (a) glucose, (b) fructose and (c) glycogen, respectively; Figure 10 For GCE, PTMAO-SBMA / GCE, Ag-Cu NDs / PTMAO-SBMA / GCE at 10 mg mL -1 Signal inhibition rates of glucose, fructose, and glycogen solutions; Figure 11 To incorporate the DPV response signals of different concentrations of chloramphenicol into the antifouling electrochemical sensor; Figure 12 The curve showing the relationship between chloramphenicol concentration and signal inhibition rate is shown; the inset represents the corresponding standard curve. Figure 13 DPV response during the fabrication process of an antifouling electrochemical sensor; Figure 14 The results are (a) specificity test results, (b) repeatability test results, and (c) stability test results for the antifouling electrochemical sensor. Detailed Implementation

[0046] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0047] The information on the raw materials and reagents used in the embodiments and experimental analyses of this invention is as follows: 2-Hydroxy-2-methylphenylacetone, copper acetate (I) (CuOAc), diethylenetriaminepentaacetic acid, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), glycogen (Gn) were purchased from Ron Reagents; bovine serum albumin (BSA) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; hydrogen peroxide (H2O2, 30) Dichloromethane (DCM), anhydrous ethanol, and sodium chloride were sourced from Tianjin Damao Chemical Reagent Co., Ltd.; lysozyme (Lys) was purchased from Boster Biologics; fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) was sourced from Beijing Zhongke Chenyu Technology Co., Ltd.; potassium ferrocyanide trihydrate (K4Fe(CN)6·3H2O), potassium ferricyanide (K3Fe(CN)6), potassium chloride (KCl), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), L-(+) ascorbic acid (99%), and sodium chloride were also used. (NaCl) was purchased from Sinopharm Group; chloramphenicol aptamers were purchased from Shanghai Sangon Biotech; D-(+)-glucose (Glu), D-fructose (Fru), and α-lactose (Lac) were purchased from Shanghai Lanji Technology Co., Ltd.; chloramphenicol (CAP), tetracycline hydrochloride (TET), ofloxacin (OFLX), kanamycin sulfate (KANA), amoxicillin (AMX), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; ultrapure water (18.2 MΩ cm⁻¹) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. -1 (Made in the laboratory)

[0048] Example 1 This embodiment provides an electrochemical sensor, the specific preparation process of which is as follows: (1) The synthesis equation for the zwitterionic monomer (TMAOMA) is as follows:

[0049] The preparation process was as follows: In a three-necked round-bottom flask, diethylenetriaminepentaacetic acid (80 mg) was dissolved in 30 mL of deionized water under vigorous stirring. Then, hydrogen peroxide (30% solution, 4.78 g) was slowly added to the flask, and the mixture was heated to 60 °C and purged with oxygen. DMAP (15 g) was dissolved in 10 mL of deionized water and slowly added to the flask. The temperature was raised to 65 °C, and the reaction time was 5 h. After the solution cooled naturally to room temperature, it was extracted multiple times with DCM, and the aqueous phase was collected. The collected aqueous phase was dissolved in anhydrous ethanol, and most of the water was removed by rotary evaporation. The resulting product was lyophilized for 24 h to obtain a yellowish-white waxy substance.

[0050] The proton nuclear magnetic resonance spectra of TMAOMA and DMAPMA are as follows: Figure 1 As shown, the synthesis of the target product can be demonstrated.

[0051] (2) Preparation of zwitterionic copolymer antifouling interface on electrode surface: Glassy carbon electrode (GCE) was polished with alumina powder, then ultrasonically cleaned with ultrapure water and anhydrous ethanol, and dried with high-purity nitrogen to obtain a clean glassy carbon electrode. The TMAOMA from step (1) was prepared to a concentration of 200 mg / mL. -1 TMAOMA aqueous solution; sulfonyl betaine (SBMA) was prepared to a concentration of 200 mg / mL. -1 SBMA aqueous solution was prepared; TMAOMA solution and SBMA solution were mixed at volume ratios of 10:0, 9:1, 8:2, and 7:3, and 0.1% (w / w) of photoinitiator (2-hydroxy-2-methylphenylacetone) was added to obtain a mixed solution. 5 μL of the mixed solution was dropped onto the electrode surface, and an in-situ reaction was initiated using 365 nm photoinitiation for 30 min, resulting in the polymerization synthesis of a series of copolymers of PTMAO-SBMA, named PTMAO-SBMA0, PTMAO-SBMA1, PTMAO-SBMA2, and PTMAO-SBMA3, respectively. The copolymers were then immersed in PBS (10 mmol / L). -1 In the solution, the excess monomer is fully swollen and removed to obtain an electrode with a stable modified antifouling sensing interface, denoted as PTMAO-SBMA / GCE.

[0052] The polymerization equations for TMAOMA and SBMA are as follows:

[0053] (3) Preparation of silver-copper nanoparticles and their loading on electrodes: Preparation of Ag-Cu NDs: 0.045 g hexadecylamine, 3.43 mL water, and 1.2 mL silver nitrate (100 mmol / L) were added at room temperature. -1 0.37 mL of glucose (1 M) and 0.37 mL of 0.37 mL of glucose (1 M) were added sequentially to a 40 mL pressure-resistant bottle. After magnetic stirring for 5 hours, the mixture was heated in a 100 °C oil bath for 1 hour. The pressure-resistant bottle was then removed and allowed to cool naturally. The mixture was washed with ultrapure water and centrifuged 2-3 times (13300 rpm, 10 min), and then directly redispersed in 9 mL of ultrapure water for inoculation and growth of Ag-Cu NDs.

[0054] Add 0.3 g trimethylamine and 0.3 mL anhydrous copper acetate (100 mmol / L) -18.9 mL of ultrapure water and 200 μL of an aqueous solution of silver nanoparticles (Ag NPs) were sequentially added to a 40 mL pressure-resistant bottle. The resulting mixture was stirred for 5 hours, followed by the addition of 0.6 mL of ascorbic acid (1 mol L⁻¹) while stirring. -1 Stir for 10 minutes. Place the pressure-resistant bottle in an oil bath at 150 °C and heat for 12.5 min. Wash with ultrapure water and centrifuge 3-5 times (13300 rpm, 10 min), and concentrate to 10 mL for later use (the concentration of Ag-Cu NDs is defined as 1×).

[0055] Figure 2 Characterization of Ag-Cu NDs, where Figure 2 In this context, 'a' stands for transmission electron microscope (TEM). Figure 2 In this context, b represents the energy-dispersive X-ray spectrum. Figure 2 In the diagram, c represents the ultraviolet absorption spectrum.

[0056] Figure 2 The synthesized Ag-Cu NDs were characterized by TEM, EDX, UV-Vis spectroscopy, and a complete characterization, as shown by TEM images of Ag-Cu NDs. Figure 2 In (a), a gourd-like nanostructure was observed, consisting of two hemispherical regions. Each domain ranged in size from 25 to 35 nm. (EDX elemental map) Figure 2 (b) Confirmed the existence of separated Ag and Cu domains in the nanocrystals, with the Ag domains being yellow and the Cu domains being red. Furthermore, the unique structural features of Ag-Cu NDs are also reflected in their optical properties, as shown in the UV-Vis spectrum (…). Figure 2 c) Two peaks appear at ~409 nm and ~585.5 nm, a blue shift of ~6.5 nm compared to the peak of Ag NPs (~415.5 nm). Since the size and shape of Ag NPs remain unchanged during the seed growth of Ag-CuNDs, the blue shift of the Ag NPs peaks may indicate charge transfer between the Cu and Ag domains. In summary, the characterization results confirm the successful synthesis of Ag-CuNDs nanocrystals in this embodiment.

[0057] Ag-Cu NDs solutions with concentrations of 1×, 2×, 3×, 4×, and 5× were drop-coated onto the surface of the prepared antifouling sensing interface PTMAO-SBMA / GCE. After being left at room temperature for 30 min, the poorly bonded Ag-Cu NDs were rinsed with ultrapure water to obtain an electrode with a stable modified antifouling sensing interface and silver-copper nanoparticles, denoted as Ag-Cu NDs / PTMAO-SBMA / GCE.

[0058] (4) At 4 °C, Ag-Cu NDs / PTMAO-SBMA / GCE were placed in solutions of concentrations of 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.1 μmol L, respectively. -1 Incubate the chloramphenicol aptamer in 200 μL of chloramphenicol aptamer solution for 10 h. The chloramphenicol aptamer has the sequence 5'-SH-(CH2)6-ACT TCA GTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTAG-3' to obtain a working electrode modified with the aptamer, denoted as Apt-Ag-Cu NDs / PTMAO-SBMA / GCE.

[0059] Figure 3 Scanning electron microscope (SEM) images of electrodes with different modifications.

[0060] Figure 3 As shown in Figure a, the polished GCE surface is smooth and flat. When an in-situ zwitterionic copolymer (PTMAO-SBMA) is formed on its surface... Figure 3 b). It can be seen that the GCE surface becomes rough, and the zwitterionic copolymer is uniformly distributed on its surface in a dotted pattern. The strong adhesion between polyacrylic acid and GCE is the result of a large number of synergistic interactions, including cation-π interactions, electrostatic attraction, and mechanical interlocking structures. When Ag-Cu NDs are further modified on the PTMAO-SBMA / GCE surface... Figure 3 c) You can see that even smaller spherical particles are evenly distributed.

[0061] Comparative Example 1 This comparative example provides an electrochemical sensor, the specific preparation process of which is as follows: The preparation method of Ag-Cu NDs solution is the same as in Example 1; A 4× Ag-Cu NDs solution was drop-coated onto the surface of the prepared antifouling sensing interface PTMAO-SBMA / GCE. After being left at room temperature for 30 min, the poorly bonded Ag-Cu NDs were rinsed with ultrapure water to obtain a stable electrode modified with silver-copper nanoparticles, denoted as Ag-Cu NDs / GCE.

[0062] At 4 °C, Ag-Cu NDs / PTMAO-SBMA / GCE were placed in a solution with a concentration of 0.5 μmol / L. -1Incubate the chloramphenicol aptamer in 200 μL of chloramphenicol aptamer solution for 10 h. The chloramphenicol aptamer has the sequence 5'-SH-(CH2)6-ACT TCAGTG AGT TGT CCC ACG GTC GGC GAG TCG GTG GTAG-3' to obtain a working electrode modified with the aptamer, denoted as Apt-Ag-Cu NDsGCE.

[0063] Detection experiment 1. The electrode system used in this invention is based on the traditional three-electrode system: a glassy carbon electrode as the working electrode; a platinum electrode as the counter electrode; and a saturated calomel electrode as the reference electrode.

[0064] All electrochemical experiments were conducted on an IVIUM electrochemical workstation.

[0065] All electrochemical tests were performed in an electrochemical probe solution. The electrochemical probe solution contained 5 mmol / L -1 K3[Fe(CN)6] / K4[Fe(CN)6] and 0.2 mol L -1 KCl in PBS. Preparation method of PBS: Accurately weigh 35.8 g of disodium hydrogen phosphate (Na₂HPO₄·12H₂O) and 15.6 g of sodium dihydrogen phosphate (NaH₂PO₄·2H₂O), place them in a 500 mL volumetric flask, and dilute to volume with ultrapure water to obtain 0.2 mol / L PBS. -1 Na2HPO4 solution and 0.2 mol L -1 NaH₂PO₄ solution. Measure 324 mL of 0.2 M Na₂HPO₄ solution and 0.2 mol L⁻¹ of NaH₂HPO₄ solution using a graduated cylinder, respectively. -1 76 mL of NaH₂PO₄ solution was mixed with 3.6 g of solid sodium chloride (NaCl) to obtain a solution with pH 7.4 in 0.2 mol / L solution. -1 The PBS solution was incubated at room temperature. Preparation of the electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6]): Accurately weigh 164.62 mg of potassium ferrocyanide (K3[Fe(CN)6]), 184.17 mg of potassium ferrocyanide (K4[Fe(CN)6]), and 1491.02 mg of potassium chloride (KCl). Add 0.2 mol / L... -1 Dilute the pH 7.4 PBS solution to 0.01 mol / L. -1 Then use 0.01 mol L -1 The above-mentioned drugs were dissolved in PBS solution at pH 7.4, and finally diluted to volume in a 100 mL volumetric flask to obtain a bright yellow solution, which yields 5 mmol L. -1The electrochemical probe K3[Fe(CN)6] / K4[Fe(CN)6], containing a 0.2 mol / L KCl solution, was wrapped in aluminum foil and stored at 4 ℃ for later use.

[0066] In this invention, differential pulse voltammetry (DPV) is used to record the current response values ​​of the modified electrode before and after incubation (the electrical signal before incubation is...). I 0, the electrical signal after incubation is I The differential pulse voltammetry method has a potential range of -0.2 V to 0.6 V, an amplitude of 10 mV, and a scan rate of 50 mV / s. -1 The change in electrical signal is calculated from these values. △I = I 0– I ) and signal suppression ratio (%) = [( I 0– I ) / I 0]×100).

[0067] 2. The prepared zwitterionic copolymer antifouling interface electrodes PTMAO-SBMA0 / GCE, PTMAO-SBMA1 / GCE, PTMAO-SBMA2 / GCE, PTMAO-SBMA3 / GCE, and GCE were immersed in 1.0, 10.0 mg / mL solutions, respectively. -1 Bovine serum albumin (BSA), lysozyme (Lys), and 10.0 and 50.0 mg / mL -1 The electrode was incubated in a lactose (Lac) solution for 30 min. The working electrode to be detected was immersed in the electrochemical probe solution, and DPV scanning was performed within a potential range of -0.2 V to 0.6 V. The current response values ​​before and after incubation were recorded to derive the signal suppression rate, thus providing a more intuitive comparison of the antifouling ability of zwitterionic copolymer antifouling sensing interfaces with different proportions. Figure 4 From a, b, and c, we can conclude that the signal suppression rate of PTMAO-SBMA2 / GCE is consistently the lowest, indicating that it has the best antifouling ability, and it can be used for the subsequent construction of an antifouling electrochemical sensor.

[0068] 3. Working electrodes Ag-Cu NDs / PTMAO-SBMA / GCE modified with different concentrations of Ag-Cu NDs, and working electrodes Apt-Ag-Cu NDs / PTMAO-SBMA / GCE modified with different concentrations of aptamer, were immersed in an electrochemical probe solution. DPV scanning was performed within a potential range of -0.2 V to 0.6 V, and the current response values ​​before and after incubation were recorded to derive the signal suppression rate. Figure 5 A 4× concentration Ag-Cu NDs solution and a 0.5 μmol L⁻¹ solution can be obtained.-1 Electrodes prepared using aptamer solutions exhibit the best performance.

[0069] 4. Based on the fact that the actual samples for subsequent testing were honey and simply pretreated pig liver sample solution, the main components that easily produce non-specific adsorption are glucose (Glu), fructose (Fru), and glycogen (Gn). Here, concentrations of 10, 20, and 50 mg / mL were used. -1 Glucose and fructose, and 0.1, 1, 10 mg mL -1 Glycogen solution was a contaminant; notably, the glycogen concentration in the pig liver sample was far below 0.1 mg / mL after simple pretreatment. -1 The bare GCE, zwitterionic copolymer antifouling sensing interface, and subsequently modified Ag-Cu NDs antifouling sensing interface were immersed in glucose, fructose, and glycogen solutions of the above concentrations and incubated for 30 min. The working electrode to be detected was immersed in the electrochemical probe solution, and DPV scanning was performed in the potential range of -0.2 V to 0.6 V. The DPV signal responses of different modified electrodes GCE, PTMAO-SBMA / GCE, and Ag-Cu NDs / PTMAO-SBMA / GCE in different pollutants are as follows: Figure 7-9 As shown. The signal suppression rate is obtained by recording the current response values ​​before and after incubation. Figure 6 It can be concluded that even at 50 mg / mL -1 glucose, fructose, 10 mg / mL -1 In glycogen solutions, the signal inhibition rates of PTMAO-SBMA / GCE were as low as 1.61%, 1.91%, and 7.08%, significantly lower than those of GCE (42.08%, 52.81%, and 65.58%). Furthermore, it is evident that loading Ag-Cu NDs onto the antifouling surface (Ag-Cu NDs / PTMAO-SBMA / GCE) did not negatively impact its antifouling effect.

[0070] Dilute GCE, Ag-Cu NDs / GCE, PTMAO-SBMA / GCE, Ag-Cu NDs / PTMAO-SBMA / GCE at 10 mgmL -1 After incubation for 30 min in glucose, fructose, and glycogen solutions, the results showed that the signal inhibition rate of Ag-Cu NDs / GCE was not significantly different from that of GCE, indicating that it is easily affected by interfering substances, leading to inaccurate test results. Therefore, Ag-Cu NDs / GCE cannot be used for detection in samples with simple pretreatment. The final experimental results show that ( Figure 10 Modification with Ag-Cu NDs does not enhance the antifouling properties of GCE, but it does not affect the antifouling effect of the antifouling interface.

[0071] 5. Take Apt-Ag-Cu NDs / PTMAO-SBMA / GCE and soak them in 200 μL of solutions with concentrations of 0.01, 0.05, 0.1, 1, 10, 50, and 100 ng / mL. -1 In a chloramphenicol standard solution, after incubation for 60 min, the loosely bound chloramphenicol was rinsed with ultrapure water to form the working electrode to be detected. Then, the working electrode was immersed in an electrochemical probe solution, and DPV scanning was performed within a potential range of -0.2 V to 0.6 V. The corresponding DPV images are shown below. Figure 11 As shown. The signal inhibition rate was calculated by recording the current signals before and after detection; the standard curve was obtained based on the signal inhibition rate and the logarithm of chloramphenicol concentration: y = 3.29x + 13.14, (R 2 = 0.997, S / N=3), LOD is 0.33 pg mL -1 ( Figure 12 ).

[0072] 6. Analyze the changes in DPV signal during the stepwise construction of the antifouling electrochemical sensor using different electrodes from Example 1 and the electrode from Comparative Example 1. Figure 13 As shown, the DPV current signal decreased (125.3 μA-120.3 μA) after GCE modification of the zwitterionic copolymer (PTMAO-SBMA). This is due to the poor conductivity of the zwitterionic copolymer and the thin modification thickness. Coating with Ag-Cu NDs, which connect the chloramphenicol aptamer via Ag-S bonds, resulted in a significant decrease in the DPV current signal to 88.1 μA after aptamer modification. This was attributed to the impaired charge transfer at the electrode surface. Notably, the DPV current response increased from 120.3 μA to 127.1 μA after Ag-Cu NDs coating. This demonstrates that Ag-Cu NDs can not only be used for aptamer connection but also enhance the electrochemical signal. The Apt / Ag-Cu NDs / PTMAO-SBMA / GCE mixture was prepared at 100.0 ng / mL... -1 After incubation in CAP solution for 60 min, the DPV current signal decreased again. This is due to the weak conductivity of CAP molecules and the conformational change that occurs when chloramphenicol aptamers bind to CAP molecules, resulting in a more compact accumulation on the electrode surface, which hinders electron transfer on the electrode surface.

[0073] 7. With 100 ng mL -1 Chloramphenicol and 1 μg mL -1 Amoxicillin, ofloxacin, kanamycin, doxytetracycline, and mixtures thereof were used as detection targets, and the prepared antifouling electrochemical aptamer sensor was used for detection. The results are as follows: Figure 14As shown in Figure a, the electrochemical signal changes for amoxicillin, ofloxacin, kanamycin, and doxytetracycline were very low, at 2.7%, 1.3%, 3.5%, and 4.1%, respectively; while the signal inhibition rates for detecting chloramphenicol alone and mixtures thereof were 18.2% and 18.9%, respectively, indicating that the sensor has excellent selectivity. Furthermore, the constructed antifouling electrochemical sensor was stored in PBS (10.0 mmol / L). -1 In a solution, used to detect 100.0 ng / mL -1 Five days later, the TC still maintained 97.1% of the initial signal, indicating that the antifouling electrochemical sensor has good stability. Figure 14 b). Repeatability was evaluated using seven different working electrodes. Following step 2.4, each electrode was used to prepare an antifouling electrochemical sensor three times for chloramphenicol detection. The results showed that the proposed antifouling electrochemical aptamer sensor exhibited excellent repeatability. Figure 14 c).

[0074] 8. Honey, pig liver, and physiological saline were selected as test samples, and the spiked recovery experiment was used to verify the practical application performance of the constructed sensor in the test samples.

[0075] Using pH 7.4 0.01 mol L -1 The honey was diluted 1% with PBS buffer. 3 ± 0.05 g of homogenized pig liver sample was weighed into a 50 ml centrifuge tube. 3 ml of deionized water was added and the mixture was shaken to mix. Then, 6 ml of ethyl acetate was added and shaken for 2 minutes. The mixture was centrifuged at 4000 rpm for 10 minutes at room temperature. 2 ml of the supernatant was collected and dried under nitrogen at 50-60°C. 1 mL of 0.01 mol / L PBS was added. -1 The solution was reconstituted with PBS buffer and centrifuged at 4000 rpm for 5 minutes at room temperature. The aqueous phase was then diluted 1% with PBS buffer. A 0.9% physiological saline solution was prepared using sodium chloride. Different concentrations of sodium chloride standard solution were added to the three diluted food samples to ensure that the spiked solution contained 0.5 ng / mL of sodium chloride. -1 9 ng mL -1 and 90.0 ng mL -1 Chloramphenicol was then detected using Apt-Ag-Cu NDs / PTMA-SBMA / GCE to obtain the spiked recovery rate in the samples. The spiked samples were then analyzed using enzyme-linked immunosorbent assay (ELISA) to validate the results obtained from the constructed sensor and ensure the accuracy of the analysis. Specific results are shown in Table 1 below: Table 1

[0076] As can be seen from Table 1 above, the antifouling electrochemical sensor prepared by this invention can sensitively detect chloramphenicol in the sample to be tested, and this method has broad application prospects in food matrices.

[0077] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A zwitterionic polymer, characterized in that, The structural formula of the zwitterionic polymer is shown in Formula 1 below: Formula 1; The zwitterionic polymer is prepared by a method comprising the following steps: oxidizing N-[(3-(dimethylamino)propyl]methacrylamide to obtain a zwitterionic monomer, and photopolymerizing the zwitterionic monomer with methacrylate sulfobetaine to obtain the zwitterionic polymer. The mass ratio of the zwitterionic monomer to methacrylate sulfobetaine is (1~9):

1.

2. The method for preparing the zwitterionic polymer according to claim 1, characterized in that, The process includes the following steps: oxidizing N-[(3-(dimethylamino)propyl]methacrylamide to obtain an amphoteric monomer, and then photopolymerizing the amphoteric monomer with methacrylate sulfobetaine to obtain the amphoteric polymer.

3. An electrochemical sensor, characterized in that, The device includes an electrode, the surface of which is an antifouling layer formed from the zwitterionic polymer of claim 1, the surface of which is loaded with silver-copper nanoparticles and thiol aptamers.

4. The electrochemical sensor according to claim 3, characterized in that, The thiol aptamer is a thiol chloramphenicol aptamer.

5. The method for preparing the electrochemical sensor according to any one of claims 3-4, characterized in that, The steps include the following: S1. N-[(3-(dimethylamino)propyl]methacrylamide is oxidized to obtain a zwitterionic monomer. The zwitterionic monomer, methacrylate sulfobetaine and photoinitiator are mixed in a solvent to obtain a mixture. The mixture is then coated on the surface of the electrode and photopolymerization is carried out to obtain electrode 1 with an antifouling layer on the surface. S2. Coat the surface of the antifouling layer with a dispersion containing silver-copper nanoparticles, and react to modify the surface of the antifouling layer with silver-copper nanoparticles to obtain electrode 2; place electrode 2 in a mercapto aptamer solution for incubation to obtain the electrochemical sensor.

6. The method for preparing the electrochemical sensor according to claim 5, characterized in that, The silver-copper nanoparticles were synthesized using a seed growth method based on silver nanoparticles.

7. The method for preparing the electrochemical sensor according to claim 5, characterized in that, In step S1, the photopolymerization reaction is carried out under ultraviolet light; and / or, the reaction time of the photopolymerization reaction is 20~40 min.

8. The method for preparing the electrochemical sensor according to claim 5, characterized in that, In step S2, a dispersion containing silver-copper nanoparticles is coated on the surface of the antifouling layer, and after standing for a period of time, a self-assembly reaction is carried out to modify the surface of the antifouling layer with silver-copper nanoparticles, thus obtaining electrode 2. And / or, the incubation time is 8-12 h; the incubation temperature is 0-5℃.

9. The application of the electrochemical sensor according to any one of claims 3-4 in the detection of chloramphenicol.

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