Electrochemical aptamer sensor as well as preparation method and application thereof

Abstract

The invention discloses an electrochemical aptamer sensor as well as a preparation method and application thereof. The electrochemical aptamer sensor comprises a substrate, a conductive enhancement layer and an anti-fouling biological recognition layer which are sequentially stacked, the conductive enhancement layer comprises a gold nanostar material; the anti-fouling biological recognition layer comprises a zwitterionic polymer and a sulfhydrylated oxytetracycline aptamer; the zwitterionic polymer is composed of a structural unit as shown in a formula 1, wherein the formula 1 is shown in the description. In the electrochemical aptamer sensor, a sensing interface is constructed by synergistically modifying the gold nanostars and the zwitterionic polymer. Wherein the gold nanostars are stably combined with the zwitterionic polymer and the OTC aptamer through Au-S bonds on the surface while enhancing a current signal by virtue of the high specific surface area and excellent conductivity of the gold nanostars; the zwitterionic polymer endows the interface with remarkable anti-fouling performance, and non-specific adsorption can be effectively inhibited, so that the pretreatment process is greatly simplified in complex sample detection, and the detection reliability is improved.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor detection technology, and particularly relates to an electrochemical aptamer sensor, its preparation method and application. Background Technology

[0002] Oxytetracycline (OTC) is widely used as a broad-spectrum antibiotic in livestock, poultry, and aquaculture, and is often added to feed to promote animal growth. However, overuse of OTC can easily lead to bacterial resistance and may cause direct toxicity to animals. Its oxidation byproducts are even more toxic, damaging the animal's oxidative system, causing liver, pancreas, and tissue damage, and overdose can even lead to death. For human health, long-term consumption of animal-derived foods with excessive OTC residues may cause intestinal flora imbalance, induce secondary infections, and lead to health risks such as hepatotoxicity, nephrotoxicity, allergic reactions, and digestive system diseases. Therefore, developing a sensitive and rapid detection technology for OTC residues in different food samples is of significant practical importance.

[0003] While traditional methods for detecting oxytetracycline (such as high-performance liquid chromatography) offer high accuracy, they rely on large equipment, are cumbersome to operate, and are time-consuming, making them unsuitable for rapid on-site detection. Electrochemical sensor methods, on the other hand, offer advantages such as small equipment, rapid analysis, ease of operation, and low cost, making them suitable for efficient screening and real-time monitoring in food supervision and other applications. However, when applying electrochemical sensors to food media detection, the complex composition of the actual samples makes the sensor surface susceptible to biocontamination. Biocontamination refers to the reversible or irreversible adsorption of biomolecules such as proteins and carbohydrates, or microorganisms, onto the material surface through non-bonded interactions. Contaminant adhesion interferes with electron transfer, significantly reducing the sensor's signal-to-noise ratio, affecting detection accuracy and stability, and in severe cases, even leading to false positives, thus limiting its reliable application in actual sample analysis. Therefore, introducing antifouling materials into the sensor surface construction to improve the electrochemical sensor's antifouling performance is crucial for achieving biocontamination resistance in electrochemical sensors.

[0004] Common antifouling materials include polyethylene glycol (PEG) and its derivatives, polysaccharides, zwitterionic polymers, and peptides. Among these, zwitterionic polymers are a class of high-performance antifouling materials, whose advantages mainly stem from their superior hydration capacity and balanced charge characteristics. Compared to traditional hydrophilic materials (such as PEG), they can form a more robust hydration layer, effectively weakening protein and non-specific adsorption, thus exhibiting superior antifouling performance. However, since zwitterionic polymers are difficult to anchor on sensor surfaces, how to solve the problem of post-functionalization of zwitterionic polymers remains an unsolved problem in the construction of antifouling electrochemical sensors. Summary of the Invention

[0005] In order to overcome at least one of the problems existing in the prior art, one of the objectives of the present invention is to provide an electrochemical aptamer sensor that has both high conductivity and antifouling properties, can achieve stable and highly sensitive detection in complex biological samples, has rapid electron transfer capability, and can be easily and directly used for highly sensitive and rapid detection of oxytetracycline (OTC) in food or biological media.

[0006] A second objective of this invention is to provide a method for preparing the above-mentioned electrochemical aptamer sensor.

[0007] The third objective of this invention is to provide a method for detecting oxytetracycline.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an electrochemical aptamer sensor, comprising a substrate, a conductive reinforcement layer, and an antifouling biorecognition layer stacked sequentially; the conductive reinforcement layer comprises gold nanostar material; the antifouling biorecognition layer comprises a zwitterionic polymer and a mercapto-oxytetracycline aptamer; the zwitterionic polymer is composed of structural units shown in Formula 1: Equation 1; In Equation 1, x, y and z are each independent positive integers from 1 to 100.

[0009] The gold nanostars (AuNS) used in this invention differ from traditional spherical gold nanoparticles. They exhibit a star-shaped structure with unique anisotropic morphology, excellent conductivity, and signal enhancement capabilities. This material can stably bind to the zwitterionic polymer and mercapto-oxytetracycline aptamer in the antifouling biorecognition layer via Au-S bonds, effectively improving sensor performance. The zwitterionic polymer, in turn, imparts significant antifouling properties to the interface, effectively suppressing non-specific adsorption. This greatly simplifies the pretreatment process and improves detection reliability in complex sample detection. The electrochemical aptamer sensor of this invention possesses excellent anti-interference capabilities, high selectivity, high sensitivity, and accuracy, making it suitable for the reliable detection of trace OTC in complex samples.

[0010] In some embodiments of the present invention, the relative molecular weight of the zwitterionic polymer ranges from 9.6 to 23.2 kDa.

[0011] Preferably, the zwitterionic polymer is prepared by a method comprising the following steps: subjecting 2-methacryloyloxyethyl phosphocholine, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and methacrylic acid to a free radical polymerization reaction to obtain a polymer precursor; and then subjecting the polymer precursor to a functionalization reaction to form a thiol group to obtain the zwitterionic polymer.

[0012] In the synthesis method of zwitterionic polymer of the present invention, zwitterionic polymer is formed by simple free radical polymerization reaction, and then the carboxyl group in the methacrylic acid unit of the polymer is functionalized to form thiol group. The above simple chemical reaction solves the problem of difficult functionalization of zwitterionic polymer, and realizes the formation of stable antifouling layer of antifouling material through chemical bond connection.

[0013] Preferably, the molar ratio of 2-methacryloyloxyethyl phosphocholine to 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate is 1:(0.05~1); more preferably 1:(0.08~0.5); and even more preferably 1:(0.1~0.2).

[0014] Preferably, the molar ratio of 2-methacryloyloxyethyl phosphocholine to methacrylic acid is 1:(0.05~1); more preferably 1:(0.08~0.5); and even more preferably 1:(0.1~0.2).

[0015] Preferably, the free radical polymerization reaction is carried out in a protective gas atmosphere; specifically, the protective gas includes at least one of nitrogen, argon or helium; in some embodiments of the present invention, the protective gas is selected from nitrogen.

[0016] Preferably, the free radical polymerization reaction is carried out under initiator conditions; in some embodiments of the present invention, the initiator is selected from 4,4'-azobis(4-cyanopentanoic acid).

[0017] Preferably, the free radical polymerization reaction is carried out under solvent conditions; in some embodiments of the present invention, the solvent includes N,N-dimethylformamide and dimethyl sulfoxide.

[0018] Preferably, the temperature of the free radical polymerization reaction is 60~80℃; more preferably 65~75℃.

[0019] Preferably, the free radical polymerization reaction takes 18-30 hours; more preferably 22-26 hours.

[0020] Preferably, the functionalization reaction of the polymer precursor specifically includes the following steps: subjecting the polymer precursor to an amidation reaction with an amine compound containing a disulfide bond, thereby breaking the disulfide bond to form a thiol group.

[0021] A disulfide bond is formed at the carboxyl group position through an amide reaction, and then the disulfide bond is broken to form a free thiol group.

[0022] Preferably, the disulfide-containing amine compound includes cystamine or its salt; specifically, the salt of cystamine may be cystamine dihydrochloride; in some embodiments of the present invention, the disulfide-containing amine compound is selected from cystamine dihydrochloride.

[0023] In some embodiments of the present invention, the compound used to break the disulfide bond is selected from dithiothreitol.

[0024] Preferably, the average particle size of the gold nanostar material is 10-50 nm; more preferably, it is 20-40 nm.

[0025] Preferably, the gold nanostar material has a three-dimensional star-shaped structure.

[0026] Preferably, the gold nanostar material is prepared by a method comprising the following steps: mixing a gold source with a C1-C5 alcohol and performing a photochemical reduction reaction under ultraviolet light to form the gold nanostar material.

[0027] This invention utilizes RO generated by the ionization of C1-C5 alcohols (ROH) As a morphology-directing agent, the anisotropic growth of gold nanoparticles is precisely controlled, ultimately forming a star-shaped structure. Compared with existing photochemical methods that rely on surfactants, the synthesis method of the gold nanostar material in this invention does not use surfactants or other additional stabilizers, but is based on ultraviolet photochemistry, which has significant advantages in simplifying the process, improving product purity, and being environmentally friendly.

[0028] Preferably, the gold source includes at least one of tetrachloroauric acid, tetrachloroauric acid trihydrate, or tetrachloroauric acid tetrahydrate; in some embodiments of the present invention, the gold source is selected from tetrachloroauric acid (HAuCl4).

[0029] Preferably, the C1-C5 alcohols include at least one of methanol, ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, or neopentanol; more preferably, the C1-C5 alcohols are selected from methanol.

[0030] Using methanol as an electron donor, the photochemical reduction process takes only 20 minutes and requires no additional stabilizers, achieving a green and efficient synthesis.

[0031] Preferably, the gold source participates in the reaction in the form of a gold source solution; more preferably, the concentration of the gold source solution is 0.1~0.3 g·L. -1 .

[0032] Preferably, the C1-C5 alcohols participate in the reaction in the form of alcohol solutions; more preferably, the volume concentration of the alcohol solution is 35-45%.

[0033] Preferably, the volume ratio of the gold source solution to the alcohol solution is 1:(0.8~1.2).

[0034] Preferably, the wavelength of the ultraviolet light is 300~400nm.

[0035] Preferably, the irradiation power of the ultraviolet light is 80~100W.

[0036] Preferably, the distance between the ultraviolet light source and the surface of the reaction system liquid is 2-4 cm.

[0037] Preferably, the photochemical reduction reaction takes 10 to 30 minutes.

[0038] Preferably, the photochemical reduction reaction is carried out under alkaline conditions; more preferably, the photochemical reduction reaction is carried out under conditions of pH 11-13; specifically, sodium hydroxide can be used to adjust the pH of the reaction system.

[0039] Preferably, the photochemical reduction reaction is carried out under stirring conditions; more preferably, the stirring speed is 400~600 rpm.

[0040] Preferably, the photochemical reduction reaction further includes centrifugation and washing steps; specifically, the centrifugation speed is 5500~5700 rpm and the time is 8~12 min; the washing reagent is water.

[0041] Preferably, the nucleotide sequence of the thiolated oxytetracycline aptamer is: 5'-SH-(CH2)6-GGA ATT CGCTAG CAC GTT GAC GCT GGT GCC CGG TTG TGG TGC GAG TGT TGT GTG GAT CCG AGC TCCACG TG-3' (SEQ ID NO:1).

[0042] Preferably, the thiolated oxytetracycline aptamer is linked to the gold nanostar material via an Au-S bond.

[0043] Preferably, the substrate is a glassy carbon electrode (GCE).

[0044] A second aspect of the present invention provides a method for preparing an electrochemical aptamer sensor as described in the first aspect of the present invention, comprising the following steps: applying gold nanostar material to a substrate surface to form a conductive reinforcement layer; mixing an amphoteric polymer, a mercapto-oxytetracycline aptamer, and a solvent to form a composite modification solution; incubating the composite modification solution with the conductive reinforcement layer to form the antifouling biorecognition layer, thereby obtaining the electrochemical aptamer sensor.

[0045] The electrochemical aptamer sensor of the present invention has a simple fabrication process, is easy to miniaturize, and has excellent anti-interference ability, high selectivity, high sensitivity and accuracy, making it suitable for reliable detection of trace OTC in complex samples.

[0046] Preferably, the substrate is pretreated before the gold nanostar material is applied to the substrate surface; specifically, the pretreatment involves polishing the substrate sequentially with 0.3μm and 0.05μm alumina suspensions, followed by ultrasonic cleaning with anhydrous ethanol and ultrapure water.

[0047] Preferably, the gold nanostar material is applied by drop coating.

[0048] Preferably, the gold nanostar material is applied in the form of a gold nanostar dispersion.

[0049] Preferably, a gold nanostar dispersion with a concentration of 35 to 45 times is used, and the coating amount is 5 to 15 μL.

[0050] Preferably, after the gold nanostar material is applied to the substrate surface, it is dried and rinsed; specifically, the drying method is nitrogen blowing; the rinsing reagent is water; the gold nanostar material that is not physically adsorbed is removed by rinsing.

[0051] Preferably, the solvent in the composite modification solution is a PBS buffer solution; more preferably, the concentration of the PBS buffer solution is 5~15 mmol / L; and the pH value of the PBS buffer solution is 7.2~7.6.

[0052] Preferably, the concentration of the thioglycolic acid aptamer in the composite modification solution is 0.5~5 μmol·L. -1 Further preferred values ​​are 1~3 μmol·L⁻¹ -1 .

[0053] Preferably, the concentration of the zwitterionic polymer in the composite modification solution is 1~7 mg·mL. -1 Further preferred is 3~5 μmol·L -1 .

[0054] Preferably, the temperature at which the composite modification liquid is incubated with the conductive reinforcement layer is 1~10℃; more preferably 2~6℃.

[0055] Preferably, the composite modification liquid is incubated with the conductive reinforcement layer for 8 to 20 hours; more preferably, it is 10 to 14 hours.

[0056] A third aspect of the present invention provides a method for detecting oxytetracycline, comprising the following steps: using the electrochemical aptamer sensor described in the first aspect of the present invention to specifically bind with an analyte containing oxytetracycline to obtain a working electrode to be tested; performing a differential pulse voltammetric scan on the working electrode to be tested to obtain the current difference before and after specific binding; and obtaining the concentration of oxytetracycline in the analyte according to a standard curve of the current difference versus the logarithm of the oxytetracycline concentration.

[0057] Preferably, the electrochemical aptamer sensor binds specifically to the analyte containing oxytetracycline for 50-70 minutes.

[0058] Preferably, the differential pulse voltammetry scan is performed in a three-electrode system placed in an electrochemical probe solution.

[0059] Preferably, the three-electrode system includes the working electrode to be tested, a platinum counter electrode, and a saturated calomel reference electrode.

[0060] Preferably, the electrochemical probe solution contains 0.1~0.3 mol·L⁻¹ 1 KCl and 4~6 mmol·L 1 K3[Fe(CN)6] / K4[Fe(CN)6].

[0061] Preferably, the differential pulse voltammetric scan has a potential range of -0.2V to 0.6V, an amplitude of 40 to 60mV, and a scan rate of 5 to 15mV·s. 1 .

[0062] The current response values ​​before and after incubation, where the signal before incubation is denoted as I0 and the signal after incubation is denoted as I.

[0063] Preferably, the standard curve is a linear relationship curve between the change in current and the logarithm of the oxytetracycline concentration.

[0064] Preferably, the standard curve is obtained by a method comprising the following steps: specifically binding the electrochemical aptamer sensor with oxytetracycline standard solutions of different concentrations to obtain a working electrode to be tested; performing differential pulse voltammetry scanning on the working electrode to be tested to obtain the current difference before and after specific binding; and establishing the standard curve by combining the logarithm of the concentration of the oxytetracycline standard solution.

[0065] Preferably, the oxytetracycline standard solution is prepared by mixing oxytetracycline standard with PBS buffer solution; specifically, the concentration of the PBS buffer solution is 5~15 mmol / L.

[0066] Preferably, the concentration of the oxytetracycline standard solution is 0.05~500 ng·mL.-1 .

[0067] Preferably, the electrochemical aptamer sensor binds specifically to the oxytetracycline standard solution for 50-70 minutes.

[0068] In some embodiments of the present invention, the differential pulse voltammetric scanning method in the standard curve detection process is the same as the differential pulse voltammetric scanning method in the analyte detection process.

[0069] Preferably, the test substance includes food or biological media.

[0070] In some embodiments of the present invention, the food includes at least one of pork, milk, or eggs.

[0071] Preferably, the analyte is directly detected after simple processing and dilution, without the need for complex pretreatment.

[0072] Preferably, the analyte contains 0.05~500 ng·mL of oxytetracycline. -1 .

[0073] Using the detection method of this invention, the target analyte OTC is 0.05~500 ng·mL. -1 It exhibits good linear response within the range.

[0074] Preferably, the detection limit of the detection method is 0.5~0.8 pg·mL. -1 In some embodiments of the present invention, the detection limit of the detection method is 0.61 pg·mL. -1 .

[0075] The beneficial effects of this invention are as follows: In the electrochemical aptamer sensor of this invention, the sensing interface is constructed by synergistic modification of gold nanostars and zwitterionic polymers. Gold nanostars, with their high specific surface area and excellent conductivity, enhance the current signal while achieving stable binding with zwitterionic polymers and OTC aptamers through surface Au-S bonds. The zwitterionic polymers impart significant anti-fouling properties to the interface, effectively suppressing non-specific adsorption, thereby greatly simplifying the pretreatment process and improving detection reliability in complex sample detection. Attached Figure Description

[0076] Figure 1 This is a schematic diagram of the preparation and detection process in the examples.

[0077] Figure 2 The images show the 1H NMR spectra of the products prepared in Examples 1 and 2.

[0078] Figure 3 TEM and EDS images of the gold nanostars prepared in Example 1.

[0079] Figure 4 SEM images of different electrode materials prepared in Example 1.

[0080] Figure 5 EDS diagrams of different electrode materials prepared in Example 1.

[0081] Figure 6 The graph shows the signal suppression rate of the electrodes prepared in Examples 1-2 in different matrices.

[0082] Figure 7 The image shows the DPV signal curves of the electrodes prepared in Examples 1 and 3.

[0083] Figure 8 Figures showing the antifouling performance of the GCE electrode and the MCM-SH / AuNS / GCE electrode in different food samples.

[0084] Figure 9 The graphs show the DPV curves of the electrode in Example 3 incubated in OTC standard solutions of different concentrations and the relationship between OTC concentration and signal change. Detailed Implementation

[0085] The following specific embodiments further illustrate the content of the present invention in detail. It should also be understood that the following embodiments are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the principles described herein are all within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make selections within a suitable range based on the description herein, and are not intended to be limited to the specific data in the examples below. Unless otherwise specified, the raw materials, reagents, or apparatus used in the following embodiments and comparative examples can be obtained from conventional commercial sources or by existing known methods.

[0086] It should be noted that the information on some of the raw materials and instruments involved in the embodiments of the present invention is as follows: (1) Raw materials: 2-methacryloyloxyethyl phosphocholine (MPC), 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), methacrylic acid (MAA), 4,4'-azobis(4-cyanopentanoic acid), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), cystamine dihydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, methanol (CH3OH), and oxytetracycline (OTC) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; glycogen (Gn) and dithiothreitol (DTT) were purchased from Ron Reagent; tetrachloroauric acid trihydrate (HAuCl4) .3H2O was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; bovine serum albumin (BSA) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; lysozyme (Lys) and bovine hemoglobin (Hb) were purchased from Boster Biologics; oxytetracycline aptamer was purchased from Shanghai Sangon Biotech Co., Ltd.; D (+) Glucose (Glu), D Fructose (Fru), α Lactose (Lac) was purchased from Shanghai Lanji Technology Co., Ltd.; ultrapure water (18.2 MΩ·cm) 1 The ingredients were prepared in the laboratory; milk, eggs, and pork were purchased from local supermarkets.

[0087] (2) Detection instruments: The DPV value and CV signal of the reaction were recorded by using a CHI440c electrochemical workstation, and the EIS signal of the reaction was recorded by using a VersaSTAT3 electrochemical workstation.

[0088] (3) The detection principle is as follows: In this embodiment of the invention, the detection principle of the electrochemical aptamer sensor is based on the change in interfacial charge transfer current caused by target recognition. When OTC specifically binds to the aptamer fixed on the electrode surface, the conformational change of the aptamer leads to the densification of the interfacial layer structure, generating a steric hindrance effect, which significantly inhibits charge transfer during the sensing interface detection process. This causes the current signal detected by the differential pulse voltammetry to exhibit a concentration-dependent attenuation, and the signal change value is related to the logarithm of the OTC concentration in the range of 0.05~500 ng·mL. 1 A good linear negative correlation is observed within the range. The sensor interface is constructed using gold nanostars and zwitterionic polymers. Gold nanostars enhance the current response due to their high specific surface area, and the zwitterionic polymer and aptamer are simultaneously immobilized via Au-S bonds. The zwitterionic polymer forms a hydrated antifouling layer, effectively inhibiting the non-specific adsorption of biomolecules such as proteins. This allows the sensor to maintain an excellent signal-to-noise ratio even in complex sample matrices, while significantly simplifying the sample pretreatment process. By measuring the signal change and substituting it into a pre-established standard curve, accurate quantitative analysis of OTC in complex samples can be achieved.

[0089] (4) Preparation and reaction: All electrochemical tests were performed in a specific probe solution system consisting of 5 mmol / L K3[Fe(CN)6 / K4[Fe(CN)6] and 0.2 mol / L KCl.

[0090] The preparation process of PBS buffer solution (0.2 mol / L, pH=7.4) is as follows: Weigh 35.8 g Na2HPO4·12H2O and 15.6 g NaH2PO4·2H2O, dissolve them separately in ultrapure water, and then transfer them to a 500 mL volumetric flask and make up to volume to obtain 0.2 mol / L Na2HPO4 and NaH2PO4 stock solutions. Measure 324 mL of Na2HPO4 solution and 76 mL of NaH2PO4 solution, mix them, add 3.6 g NaCl, and stir until completely dissolved to obtain 400 mL of PBS working solution, which is stored at room temperature for later use.

[0091] The electrochemical probe solution was prepared as follows: Accurately weigh 164.62 mg K3[Fe(CN)6], 184.17 mg K4[Fe(CN)6], and 1491.02 mg KCl. Using 0.01 mol / L PBS (diluted from 0.2 mol / L PBS) as the solvent, dilute to volume in a 100 mL volumetric flask to obtain a 5 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.2 mol / L KCl. The prepared probe solution is bright yellow and should be wrapped in aluminum foil to protect it from light and stored at 4°C until use.

[0092] Differential pulse voltammetry (DPV) was used to measure the current response values ​​of the modified electrode before and after incubation with the target material. The signal before incubation was denoted as I0, and the signal after incubation was denoted as I. Based on the obtained data, the signal suppression rate (%) was calculated using the formula: Signal suppression rate (%) = [(I0) / I0] The corresponding signal suppression rate is calculated by multiplying I) / I0] by 100%.

[0093] Example 1 An electrochemical aptamer sensor is prepared according to the following steps: (1) Preparation of antifouling materials, the reaction equation is as follows:

[0094] The synthesis of zwitterionic polymers (MCM-SH) is achieved through free radical copolymerization and subsequent thiol functionalization, with the specific steps as follows: The specific steps for S1 and MCM-SH are as follows: First, MPC (349 mg, 1.18 mmol), CBMA (30 mg, 0.13 mmol), and MAA (12.3 mg, 0.14 mmol) are placed in a polymerization tube (MPC:CBMA:MAA = 8:1:1, molar ratio). ACVA (4.20 mg, 0.015 mmol) dissolved in 0.5 mL DMF is added as an initiator (the amount of initiator is approximately 1% of the total molar amount of the three monomers). Then, a mixed solvent of 20 mL deionized water and 0.5 mL DMSO is injected, the reaction system is sealed, and after degassing with nitrogen for 30 min, the reaction is stirred in a 70℃ oil bath for 24 h. After the reaction is complete, polymerization is terminated with liquid nitrogen. The crude product is dialyzed through a 12 kDa molecular weight cutoff cellulose membrane for 3 days to remove unreacted monomers and solvent, yielding the MCM polymer. S2. To introduce thiol groups, the above MCM polymer was mixed with cystamine dihydrochloride (120 mg, 0.53 mmol), the pH was adjusted to 4.75, and EDAC·HCl (126.5 mg, 0.66 mmol) was added for coupling reaction. The reaction was maintained at pH 4.5-5.0 for 6 h with stirring at 350 rpm, and the resulting product was dialyzed against deionized water at pH 4.0 for 2 days. Subsequently, DTT (31 mg, 0.20 mmol) was added, and the reaction was carried out at pH 8.0 and 400 rpm for 6 h to break disulfide bonds and generate free thiol groups. The final reaction solution was adjusted to pH 3.5, dialyzed against 0.1 M NaCl solution (pH 3.5) for 2 days, and then freeze-dried to obtain the MCM-SH polymer, which was stored at 4°C for later use. The relative molecular weight range of the MCM-SH polymer is 9.6~23.2 kDa.

[0095] (2) Preparation of sensitizing materials The gold nanostars (AuNS) material was prepared using a UV-photochemical reduction method. The specific steps were as follows: 5 mL of 0.2 g / L HAuCl4 solution was mixed thoroughly with 5 mL of 40% (v / v) methanol solution (pH adjusted to 12.0 with NaOH), and then transferred to a 25 mL beaker pretreated with aqua regia. The mixture was irradiated under a 365 nm UV lamp at 3.0 cm for 20 min with magnetic stirring at 500 rpm. After the reaction, the product was centrifuged at 5600 r / min for 10 min, washed twice with ultrapure water for purification, and finally concentrated to a 40× stock concentration to obtain an AuNS dispersion, which was stored at 4℃ for later use.

[0096] (3) Construction of anti-fouling sensing interface The method for constructing an anti-fouling sensing interface includes the following steps: S1. Pretreatment of substrate electrode: Using glassy carbon electrode (GCE) as working electrode, the electrode is polished stepwise with 0.3 μm and 0.05 μm alumina suspensions, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water respectively to obtain a clean and activated electrode surface. S2. Preparation of gold nanostar modification layer: Take 10 μL of 40× AuNS dispersion prepared in step (2) and uniformly coat it on the pretreated GCE surface. Dry and solidify it under nitrogen flow to form a uniform nanostructure layer. Then rinse it three times with ultrapure water to remove the nanomaterials that are not physically adsorbed to obtain AuNS / GCE electrode. S3, Antifouling Interface Assembly: The MCM-SH polymer prepared in step (1) is dissolved in 10 mM PBS buffer to form a concentration of 4 mg·mL⁻¹ -1 The AuNS / GCE electrode was immersed in 200 μL of the modification solution and incubated for 12 h. The zwitterionic polymer was stably immobilized on the electrode surface through Au-S bond interaction, and the MCM-SH / AuNS / GCE sensor was finally constructed.

[0097] Example 2 An electrochemical aptamer sensor differs from Example 1 in that the amounts of MPC, CBMA, and MAA are changed, as shown in Table 1 below, while other conditions remain the same as in Example 1.

[0098] Table 1. Material dosage for different monomer polymerization ratios in Examples 1-2

[0099] Example 3 An electrochemical aptamer sensor differs from Example 1 in that step S3 of step (3) is modified as follows: aptamer / antifouling composite interface assembly: a thiolized OTC aptamer (aptamer sequence 5'-SH-(CH2)6-GGA ATT CGCTAG CAC GTT GAC GCT GGT GCC CGG TTG TGG TGC GAG TGT TGT GTG GAT CCG AGC TCCACG TG-3', SEQ ID NO:1) and an MCM-SH zwitterionic polymer are co-dissolved in 10 mM PBS buffer to prepare a composite modification solution, wherein the concentration of the thiolized OTC aptamer is 2 μmol·L⁻¹. -1 The concentration of the MCM-SH zwitterionic polymer was 4 mg·mL. -1The AuNS / GCE electrode was immersed in 200 μL of the solution and incubated at 4°C for 12 h. The aptamer and the antifouling polymer were simultaneously immobilized through Au-S bond interaction to construct the Apt-OTC / MCM-SH / AuNS / GCE sensor. Other conditions were the same as in Example 1.

[0100] Example 4 A method for detecting oxytetracycline OTC is provided, utilizing the Apt-OTC / MCM-SH / AuNS / GCE sensor prepared in Example 3. A schematic diagram of the entire preparation and detection process is shown below. Figure 1 As shown, the specific steps are as follows: S1. Detection Method and Standard Curve Establishment: The Apt-OTC / MCM-SH / AuNS / GCE sensors were placed in 200 μL of a series of OTC standard solutions (0.05-500 ng·mL⁻¹). -1 Incubate in 10 mM PBS for 60 min, rinse with ultrapure water, and then form a three-electrode system. In a solution containing 0.2 mol·L⁻¹ -1 5 mmol·L⁻¹ KCl -1 Differential pulse voltammetry (DPV) was performed in a K3[Fe(CN)6] / K4[Fe(CN)6] probe solution in the potential range of -0.2V to 0.6V, with parameters of 50mV amplitude and 10mV·s scan rate. -1 A quantitative detection standard curve was established by measuring the correlation between the change in current (ΔI=I0-I) and the logarithm of OTC concentration.

[0101] S2. For OTC detection in real samples: When conducting OTC detection in actual food samples (milk, eggs, pork), the pretreated sample is diluted 100-fold and prepared to contain 20, 100, and 200 ng / mL. -1 OTC-spiked samples were analyzed using an Apt-OTC / MCM-SH / AuNS / GCE sensor. The difference in electrical signal response was substituted into an established standard curve to calculate the actual concentration of OTC in the sample and the spiked recovery rate.

[0102] Meanwhile, parallel analyses of the same spiked samples were performed using high-performance liquid chromatography (HPLC) to verify the accuracy of the sensor's analytical results and ensure the reliability of the detection method. The results are shown in Table 2.

[0103] Table 2. Results of OTC spiked recovery in three food samples

[0104] As shown in Table 2, Example 4 uses the electrochemical aptamer sensor constructed in Example 3 to detect oxytetracycline (OTC), which shows excellent detection sensitivity. Combined with its good anti-interference performance in complex food matrices, it shows that this method has reliable application potential in actual sample detection.

[0105] Performance testing (1) Verification of zwitterionic polymer synthesis: by nuclear magnetic resonance hydrogen spectroscopy (NMR spectroscopy) 1 The structures of MPC monomer, MPC-MAA-SH copolymer (the copolymer synthesized in Example 2 with MPC:CBMA:MAA=9:0:1) and MCM-SH copolymer were characterized by H NMR.

[0106] Figure 2 The images show the 1H NMR spectra of the products prepared in Examples 1 and 2; where a is the MPC monomer; b is the MPC-MAA-SH copolymer; and c is the MCM-SH copolymer. Figure 2 As shown in a, MPC monomers exhibit typical olefin proton signals in the δ range of 5.5-6.5 ppm; while MPC-MAA-SH ( Figure 2 b) and MCM-SH Figure 2 In spectrum c), the signal completely disappears, indicating that the monomer has fully participated in the polymerization reaction, with no unreacted monomer remaining. Further analysis shows that... Figure 2 Both b and c in the sample appeared around δ 2.90 ppm. The CH2SH characteristic peak confirms the successful introduction of terminal thiol groups into both polymers. Additionally, the MCM-SH spectrum shows additional characteristic peaks at δ 3.00 ppm (-CH2COO-), 3.60 ppm (-NCH2-), and 3.90 ppm (-CH2N-). These signals belong to the CBMA structural unit, indicating that CBMA has successfully participated in the copolymerization reaction to form the ternary polymer MCM-SH.

[0107] (2) Validation of gold nanostar (AuNS) synthesis: The morphology, elemental composition and distribution of the synthesized product were characterized by transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS).

[0108] Figure 3 TEM and EDS images of the gold nanostars prepared in Example 1 are shown; where a1, a2, and a3 are TEM images of the gold nanostars at different magnifications, respectively; b1, b2, and b3 are TEM images of the gold nanostars and their corresponding elemental distribution maps, respectively. TEM images ( Figure 3 As shown in a), the gold nanostar material prepared in Example 1 exhibits a distinct star-shaped irregular morphology, with a statistically average particle size of approximately 30 nm. (EDS) Figure 3Analysis b) in the paper shows that gold accounts for more than 90% of the total elements in the gold nanostar material, and its spatial distribution is uniform. The combined morphological and elemental analysis results confirm the successful preparation of the gold nanostar.

[0109] (3) Characterization and analysis of electrode modification interface: The electrode surface modification process was systematically characterized by scanning electron microscopy (SEM) combined with energy dispersive spectroscopy (EDS).

[0110] Figure 4 SEM images of different electrode materials prepared in Example 1 are shown; where a is glassy carbon electrode (GCE); b is AuNS / GCE electrode; and c is MCM-SH / AuNS / GCE electrode. Figure 5 EDS diagrams of different electrode materials prepared in Example 1 are shown; where a is a glassy carbon electrode (GCE); b is an AuNS / GCE electrode; and c is an MCM-SH / AuNS / GCE electrode. Figure 4 As shown in Figure a, the surface of the polished glassy carbon electrode (GCE) exhibits a smooth morphology. Figure 4 As shown in b, after modification with gold nanostars (AuNS), the surface roughness of the AuNS / GCE electrode increased significantly, as indicated by EDS analysis. Figure 5 As shown in b), the Au element distribution area accounts for more than 88%, indicating that AuNS forms a uniform coverage on the electrode surface. Figure 4 As shown in c, after further modification of MCM-SH, fine spherical particles can be observed uniformly distributed on the interface of the MCM-SH / AuNS / GCE electrode surface, while the Au element distribution area ( Figure 5 The concentration of c) decreased to approximately 53%, confirming that MCM-SH was successfully immobilized on the AuNS / GCE interface via Au-S bonds. The aforementioned changes in morphology and elemental distribution validate the successful layer-by-layer construction of the antifouling sensing interface.

[0111] (4) Antifouling performance: To evaluate the antifouling performance of the constructed sensing interface, the non-specific adsorption behavior of non-target substances on the electrode surface was studied using a differential pulse voltammetry (DPV) system. Bare glassy carbon electrode (GCE), AuNS / GCE electrode, and sensor electrodes prepared in Examples 1-2 were placed in single protein solutions of different concentrations (negatively charged BSA, positively charged Lys, and neutrally charged Hb, with concentration gradients of 1.0, 5.0, and 10.0 mg·mL⁻¹). -1 The sensor was immersed in extracts of different carbohydrate solutions (glucose, lactose, and starch, with the same concentration gradient as above) and different volume concentrations (1.00%, 5.00%, and 10.00%) of actual food samples (pork, eggs, and milk) for 30 minutes. The inhibition rate of DPV signal before and after immersion was calculated to quantitatively characterize the resistance of the sensor interface to non-specific adsorption in complex matrices.

[0112] Figure 6 The graph shows the signal suppression rate of the electrodes prepared in Examples 1-2 in different matrices; where a represents casein; b represents whey protein; and c represents glycogen. Figure 6 As shown, the concentrations of casein, whey protein, and glycogen were all 5 mg / mL. Electrodes prepared using different molar ratios of MPC, CBMA, and MAA showed the best antifouling effect with the 8:1:1 sample, followed by the 7:2:1, 9:0:1, and 0:9:1 samples. It can be concluded that the antifouling effect is optimal when the MPC:CBMA:MAA ratio is 8:1:1 (molar ratio), with signal inhibition rates of 2.16%, 1.71%, and 0.44% in the three solutions, respectively.

[0113] (5) Feasibility verification of electrochemical sensor detection of OTC Figure 7 The graphs show the DPV signal curves of the electrodes prepared in Examples 1 and 3. Figure 7 As shown, the differential pulse voltammetry (DPV) current signal of the AuNS / GCE electrode, constructed by modifying the GCE electrode with gold nanostars (AuNS), increased from 322.6 μA to 374.7 μA, indicating that AuNS effectively enhances the electrode's electronic conductivity. Subsequently, the zwitterionic polymer MCM-SH was fixed on the AuNS / GCE surface via Au-S bonds to construct the MCM-SH / AuNS / GCE electrode. Its current signal slightly decreased to 361 μA, indicating that the polymer has some influence on charge transfer, but it only causes a signal attenuation of about 10 μA, far lower than the significant hindering effect of traditional zwitterionic materials on conductivity, demonstrating the material's advantage in maintaining electrode transport performance. Further modification of the electrode surface with MCM-SH and an OTC aptamer to construct the Apt-OTC / MCM-SH / AuNS / GCE electrode resulted in a further decrease in current signal to 346.6 μA, mainly attributed to the insulating properties of the aptamer itself. The Apt-OTC / MCM-SH / AuNS / GCE electrode was heated at 50 ng·mL⁻¹ -1 After incubation in the OTC standard solution for 60 min, the DPV current signal further decreased. This is because the OTC molecule has poor conductivity, and its binding with the aptamer causes a denser interfacial structure, both of which hinder electron transfer on the electrode surface. These signal changes systematically verify the feasibility of this sensor for OTC detection.

[0114] (6) Evaluation of the antifouling performance of the coating in actual food samples: The antifouling performance of the bare GCE electrode and the MCM-SH / AuNS / GCE electrode was systematically evaluated by placing them in diluted samples of pork, eggs, and milk at concentrations ranging from 0.00% to 10.00%. The test results are shown in Table 3 and... Figure 8 As shown. Figure 8 The graphs show the antifouling performance of the GCE electrode and the MCM-SH / AuNS / GCE electrode in different food samples. Specifically, a1, a2, and a3 represent the signal inhibition rate, DPV signal curve of the GCE electrode, and the DPV signal curve of the MCM-SH / AuNS / GCE electrode in the pork sample, respectively; b1, b2, and b3 represent the signal inhibition rate, DPV signal curve of the GCE electrode, and the DPV signal curve of the MCM-SH / AuNS / GCE electrode in the egg sample, respectively; and c1, c2, and c3 represent the signal inhibition rate, DPV signal curve of the GCE electrode, and the DPV signal curve of the MCM-SH / AuNS / GCE electrode in the milk sample, respectively.

[0115] Table 3. Signal inhibition rate data of GCE electrode and MCM-SH / AuNS / GCE electrode in different food samples.

[0116] The results showed that the current signal of the bare GCE decreased significantly after incubation with food samples, with inhibition rates of 51.19%, 65.3%, and 81.2% in 10.00% pork, eggs, and milk, respectively. This indicates that contaminants such as proteins in the food matrix undergo severe non-specific adsorption on the electrode surface. In contrast, the signal inhibition rates of the MCM-SH / AuNS / GCE modified electrode were only 1.28%, 2.47%, and 2.07% under the same conditions, demonstrating its ability to effectively maintain electrode interface stability. These results prove that the constructed antifouling coating possesses excellent antifouling performance, providing a reliable guarantee for the practical application of the sensor in complex food samples.

[0117] (7) Linearity verification of electrochemical sensors Figure 9 The graphs shown are (a) and (b) of the relationship between OTC concentration and signal change, respectively, for the electrode of Example 3 incubated in OTC standard solutions of different concentrations. The inset in b represents the corresponding standard curve. Figure 9 As shown, the range is 0.05~500 ng·mL -1 Within the concentration range, the change in current response of the sensor shows a good linear relationship with the logarithm of the OTC concentration, and the resulting standard curve equation is y = 6.94x + 27.54 (R0). 2 =0.994), and the detection limit was calculated to be 0.61 pg·mL based on the signal-to-noise ratio S / N=3. -1 .

[0118] This invention provides an electrochemical aptamer sensor capable of rapid and accurate detection of oxytetracycline (OTC) in complex food samples. A composite interface of Apt-OTC / MCM-SH / AuNS / GCE is fabricated using a sensing interface construction strategy that combines high conductivity and strong antifouling properties. The high specific surface area and excellent conductivity of gold nanostars (AuNS) enhance the signal response, while a highly efficient antifouling layer is formed using zwitterionic polymers (MCM-SH). The fabrication method includes three key steps: electrode pretreatment, gold nanostar modification, and aptamer / polymer co-immobilization. This sensor, combined with the specific recognition capabilities of the aptamer, enables highly sensitive detection of OTC in actual food samples such as pork, milk, and eggs without complex sample pretreatment, achieving a synergistic improvement in conductivity and antifouling performance.

[0119] In summary, the sensing interface of the electrochemical aptamer sensor of this invention is constructed by synergistic modification of gold nanostars and zwitterionic polymers. Gold nanostars, with their high specific surface area and excellent conductivity, enhance the current signal while achieving stable binding with the zwitterionic polymer and OTC aptamers through surface Au-S bonds. The zwitterionic polymer imparts significant anti-fouling properties to the interface, effectively suppressing non-specific adsorption, thereby greatly simplifying the pretreatment process and improving detection reliability in complex sample detection.

Claims

1. An electrochemical aptamer sensor, characterized in that, The system comprises a substrate, a conductive reinforcement layer, and an antifouling biorecognition layer stacked sequentially; the conductive reinforcement layer contains gold nanostar material; the antifouling biorecognition layer contains a zwitterionic polymer and a mercapto-oxytetracycline aptamer; the zwitterionic polymer is composed of structural units shown in Formula 1. Equation 1; In Equation 1, x, y and z are each independent positive integers from 1 to 100.

2. The electrochemical aptamer sensor according to claim 1, characterized in that, The zwitterionic polymer is prepared by a method comprising the following steps: 2-methacryloyloxyethyl phosphocholine, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate and methacrylic acid are subjected to free radical polymerization to obtain a polymer precursor; the polymer precursor is then subjected to a functionalization reaction to form a thiol group to obtain the zwitterionic polymer.

3. The electrochemical aptamer sensor according to claim 2, characterized in that, The molar ratio of 2-methacryloyloxyethyl phosphocholine to 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate is 1:(0.1~1). And / or, the molar ratio of 2-methacryloyloxyethyl phosphocholine to methacrylic acid is 1:(0.1~1). And / or, the functionalization reaction of the polymer precursor specifically includes the following steps: subjecting the polymer precursor to an amidation reaction with an amine compound containing a disulfide bond, thereby breaking the disulfide bond to form a thiol group.

4. The electrochemical aptamer sensor according to claim 1, characterized in that, The average particle size of the gold nanostar material is 10~50 nm; And / or, the gold nanostar material is prepared by a method comprising the following steps: mixing a gold source with a C1-C5 alcohol and performing a photochemical reduction reaction under ultraviolet light to form the gold nanostar material.

5. The electrochemical aptamer sensor according to claim 1, characterized in that, The nucleotide sequence of the thiolated oxytetracycline aptamer is: 5'-SH-(CH2)6-GGA ATT CGC TAG CAC GTT GAC GCT GGT GCC CGG TTGTGG TGC GAG TGT TGT GTG GAT CCG AGC TCC ACG TG-3'.

6. A method for preparing an electrochemical aptamer sensor as described in any one of claims 1 to 5, characterized in that, Includes the following steps: Gold nanostar materials are applied to the substrate surface to form a conductive reinforcement layer; A zwitterionic polymer, a mercapto-oxytetracycline aptamer, and a solvent are mixed to form a composite modification solution. The composite modification solution is then incubated with the conductive reinforcement layer to form the antifouling biorecognition layer, thus obtaining the electrochemical aptamer sensor.

7. The preparation method according to claim 6, characterized in that, The concentration of the thioglycolic acid aptamer in the composite modification solution is 0.5~5 μmol·L. -1 ; And / or, the concentration of the zwitterionic polymer in the composite modification solution is 1~7 mg·mL. -1 ; And / or, the temperature at which the composite modification liquid is incubated in contact with the conductive reinforcement layer is 1~10℃; And / or, the composite modification liquid is incubated with the conductive reinforcement layer for 8 to 20 hours.

8. A method for detecting oxytetracycline, characterized in that, The process includes the following steps: specifically binding the electrochemical aptamer sensor according to any one of claims 1 to 5 with the analyte containing oxytetracycline to obtain the working electrode to be tested; performing differential pulse voltammetry scanning on the working electrode to be tested to obtain the current difference before and after specific binding. The concentration of oxytetracycline in the analyte was obtained by using a standard curve of current difference versus logarithm of oxytetracycline concentration.

9. The detection method according to claim 8, characterized in that, The electrochemical aptamer sensor binds specifically to the analyte containing oxytetracycline for 50-70 minutes. And / or, the differential pulse voltammetric scan has a potential range of -0.2V to 0.6V, an amplitude of 40 to 60mV, and a scan rate of 5 to 15mV·s. 1 .

10. The detection method according to claim 8, characterized in that, The test substance includes food or biological media; And / or, the analyte contains oxytetracycline at a concentration of 0.05~500 ng·mL. -1 ; And / or, the detection limit of the detection method is 0.5~0.8 pg·mL. -1 .

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