Electrochemical sensor for detecting tetracycline based on SiO2 insulation identification coupled immune competition

Abstract

The invention relates to the technical field of inspection and detection of antibiotic residues, in particular to an electrochemical sensor for detecting tetracycline based on SiO2 insulation recognition coupled immune competition. According to the invention, urchin-shaped Au (at) Pt NPs with excellent conductivity and large specific surface area is used as an electrode modification material for high-density loading of an antigen conjugate; insulating SiO2 is used as a signal amplification material and is coupled with a tetracycline antibody through chemical bonding to form a clear insulating recognition interface (SiO2-Ab); when the target object does not exist, the electron transfer inhibition effect of SiO2-Ab on the surface of the electrode reaches the maximum; when a target object exists, SiO2-Ab specifically recognizes the target object, so that the electron transfer inhibition effect of the SiO2-Ab on the surface of the electrode is weakened, current enhancement response is presented, and positive correlation between the TC concentration and the signal intensity is realized. According to the invention, signal response is regulated and controlled by an Au (at) Pt / SiO2-Ab mixed interface, a signal-enabled detection mode is realized, background signal interference can be effectively reduced, the detection sensitivity is enhanced, and reliable data is provided for detection of low-concentration target objects.

Description

Technical Field

[0001] This invention relates to the field of antibiotic residue testing and detection technology, specifically to an electrochemical sensor based on SiO2 insulating recognition coupling for competitive detection of tetracycline. Background Technology

[0002] Antibiotics, as drugs that inhibit and kill bacteria, have been used to address health issues worldwide. In the livestock and poultry farming industry, due to their antibacterial properties, low toxicity, and cost, antibiotics have been widely used as animal feed additives to prevent animal diseases and promote productivity. However, the overuse of antibiotics further leads to the accumulation of drug residues in animal-derived foods such as milk, meat, and honey.

[0003] Tetracycline (TC), a low-cost antibiotic, possesses significant broad-spectrum antibacterial properties against pathogenic microorganisms, including Gram-negative and Gram-positive bacteria. It is used as a veterinary drug additive to prevent animal diseases and promote animal growth. TC residues in food accumulate in animal-derived foods through the food chain, posing serious risks and threats to human liver and bones and leading to increased antibiotic resistance. Therefore, accurate assessment of the dietary antibiotic exposure risk to humans through scientific and highly sensitive detection of tetracycline residues in animal-derived foods is crucial to ensuring the safe entry of animal-derived foods into the market and safeguarding human health.

[0004] Currently, various analytical methods are used to determine antibiotic residues in animal-derived foods, including traditional microbiological detection methods, modern instrumental analysis methods, and biochemical immunoassays. Modern instrumental analysis methods are the gold standard, primarily relying on modern instruments such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). While these methods offer more accurate results, they suffer from drawbacks such as complex and time-consuming sample pretreatment, the need for trained technicians, and the high cost of equipment. Rapid food safety testing technologies, with their advantages of simplicity, speed, and portability, complement modern instrumental analysis methods and are crucial for ensuring food safety. Electrochemical sensors, as the most promising rapid detection technology, are increasingly prominent in antibiotic residue monitoring due to their portability, high sensitivity, ease of miniaturization, and high sample throughput.

[0005] However, due to the limitations of small molecule antigenic epitopes, it is impossible to construct a sandwich-structured recognition mode. Therefore, this invention uses Au@Pt nanoparticles as electrode surface modification materials and green, environmentally friendly, and inexpensive insulating SiO2 as signal amplification materials. Combined with immune competitive recognition, a turn-on competitive electrochemical immunosensor is constructed to detect tetracycline, providing a reference method for ensuring the safety of animal-derived food. Summary of the Invention

[0006] The purpose of this invention is to provide an electrochemical sensor for the detection of tetracycline based on SiO2 insulating recognition coupling and immune competitive detection, in order to solve the problems of low detection sensitivity caused by the limitation of small molecule antigen epitopes in biological small molecules, which prevents the construction of a sandwich structure recognition mode.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: (1) Preparation of Au@Pt nanoparticles (Au@Pt NPs); (2) Preparation of carboxyl-functionalized SiO2 (SiO2-COOH); (3) Preparation of SiO2-conjugated tetracycline antibody (SiO2-Ab); (4) Construct a competitive electrochemical immunosensor to detect tetracycline.

[0008] The specific steps for preparing Au@Pt NPs in step (1) are as follows: (a) Dissolve 30-50 mg of Pluronic F127 in a mixed solution of 2 mL of 10-30 mM HAuCl4 and 2 mL of 10-30 mM H2PtCl6, and then add 2 mL of 100-300 mM ascorbic acid (AA) solution; (b) Sonicate the mixture from step (a) for 15 min and let it stand for 24 h; (c) After natural cooling, the reaction solution from step (b) was centrifuged to obtain the reactants, which were washed several times with ultrapure water and then freeze-dried to obtain sea urchin-shaped Au@Pt NPs.

[0009] The specific steps for preparing carboxyl-functionalized SiO2 (SiO2-COOH) in step (2) are as follows: (a) Disperse 0.5 mg of silica microspheres in 50 mL of ethanol, and add 0.4 mL of APTES to the solution while stirring vigorously; (b) After reacting the mixture from step (a) at 80°C for 3 hours, centrifuge to collect the precipitate and redisperse it in DMF; (c) Add 0.5~1.5g of succinic anhydride to step (b), stir for 12h, wash three times with ethanol and deionized water respectively, and finally redisperse in 1~2mL of ethanol.

[0010] The specific steps for preparing SiO2-conjugated tetracycline antibody (SiO2-Ab) in step (3) are as follows: (a) Add 200 μL of SiO2-COOH to 1 mL of MES buffer containing EDC / NHS (25 mg / mL of EDC and 25 mg / mL of NHS) and shake for 30 min; (b) Centrifuge the mixture from step (a) to collect the precipitate, wash it three times with PBS buffer, and redisperse it in 500 μL of PBS buffer; (c) Add 150 μL of 50~150 μg / mL tetracycline antibody to the solution obtained in step (b), and incubate overnight at 4°C with shaking; (d) Centrifuge the mixture from step (c) to collect the precipitate, and add 200 μL of 50~150 μg / mL BSA solution. React at 4°C for 1 h. (e) Centrifuge the mixture from step (d) to collect the precipitate, wash the precipitate three times with PBS buffer (pH 7.2-7.4), and finally redisperse it in 500 μL of 5-15 mM PBS.

[0011] The specific steps for constructing a competitive electrochemical immunosensor to detect tetracycline in step (3) are as follows: (a) 8 µL of Au@Pt nanoparticle dispersion was drop-coated onto the electrode (WE) and dried at room temperature to obtain Au@PtNPs / WE; (b) Add 0.5-2% glutaraldehyde solution to step (a), activate for 3 hours, wash with ultrapure water, and dry with N2; (c) Add 8 µL of BSA-TC solution with a concentration of 50~200 μg / mL to step (b), incubate for 3 h to form BSA-TC / Au@Pt NPs / WE; (d) Add 8 µL of BSA solution with a concentration of 40~60 μg / mL to step (c), incubate for 30 min, and wash with ultrapure water to obtain BSA / BSA-TC / Au@Pt NPs / WE; (e) Mix the prepared SiO2-Ab with the test solution and incubate for 30 min; (f) Add 8 μL of the mixture from step (e) to the sensor prepared in step (d), incubate for 30 min, wash with PBS buffer and ultrapure water once each, and record the changes in electrical signal using differential pulse voltammetry (DPV).

[0012] The beneficial effects of this invention are: This invention is the first to utilize the insulating material SiO2 as a signal amplification tag, combined with an immune competition strategy to construct an electrochemical sensor for the detection of tetracycline. The detection range is 20 fg / mL to 20 ng / mL. The detection limit of the GCE-based sensor is as low as 4.55 fg / mL, and the linear correlation coefficient of the prediction model reaches 0.9913. The detection limit of the SPE-based sensor is as low as 11.58 fg / mL, and the linear correlation coefficient of the prediction model reaches 0.9961. The SPE-based sensor provides a simple and reliable reference method for portable electrochemical immunosensing devices for TC detection in food safety. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the principle of the competitive electrochemical immunosensor for detecting tetracycline in this invention; Figure 2 This is a standard operating curve of the competitive electrochemical immunosensor for tetracycline detection based on GCE of this invention; Figure 3 This is a standard operating curve of the competitive electrochemical immunosensor based on SPE for detecting tetracycline in this invention. Detailed Implementation

[0014] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention.

[0015] The principle of this invention is as follows: This invention constructs an electrochemical sensor based on SiO2 insulating recognition coupling for the competitive detection of tetracycline.

[0016] like Figure 1 As shown, insulating SiO2 is used as the signal amplification material, and the SiO2 surface is functionalized with carboxyl groups by (3-aminopropyl)triethoxysilane (APTES) and succinic anhydride. Then, tetracycline antibody is anchored on the silicon dioxide layer by covalent bonds, thereby forming a clear insulating recognition interface (SiO2-Ab).

[0017] Sea urchin-like Au@Pt NPs were used as electrode modifiers to increase electron transfer rate and electrode specific surface area, promoting dense and stable anchoring of bovine serum albumin-tetracycline (BSA-TC) conjugates on the electrode surface; BSA was used to seal non-specific adsorption sites on the electrode surface to reduce background interference.

[0018] SiO2-Ab was mixed with the test solution and incubated to allow the antibody to achieve sufficient specific recognition of the target. Then, the mixture was dropped onto the electrode surface and incubated for reaction. The changes in the electrical signal were recorded using DPV.

[0019] When the test solution does not contain the target analyte, a large amount of SiO2-Ab specifically binds to BSA-TC on the electrode surface, resulting in a significant decrease in the electrical signal, which is used as the baseline. When the test solution contains the target analyte, SiO2-Ab first specifically binds to the target analyte, thereby reducing the amount of SiO2-Ab specifically binding to BSA-TC on the electrode surface, making the electrical signal higher than the baseline signal. This enables the detection of the target analyte, and the concentration is positively correlated with the signal.

[0020] In the competitive electrochemical immunosensor of this invention, urchin-like Au@Pt nanoparticles are used to modify the electrode surface to enhance conductivity and increase specific surface area, promoting the dense and stable anchoring of bovine serum albumin-tetracycline (BSA-TC) conjugate on the electrode surface, thereby greatly improving the detection sensitivity of the sensor. The insulating recognition interface formed by the insulating material and antibody and the specific binding of the target on the electrode will change the electron transfer resistance, thereby realizing signal modulation on the electrode surface and realizing the detection mode with signal activation. This can effectively reduce background signal interference, improve sensitivity, and the target concentration is proportional to the output signal, enabling accurate detection of the target at low concentrations.

[0021] Example 1 Preparation of sea urchin-shaped Au@Pt nanoparticles: 40 mg of Pluronic F127 was dissolved in a mixed solution of 2 mL of 20 mM HAuCl4 and 2 mL of 20 mM H2PtCl6, followed by the addition of 2 mL of 200 mM ascorbic acid (AA) solution to synthesize urchin-shaped gold-platinum nanoparticles (Au@Pt NPs). The mixture was sonicated for 15 min and allowed to stand for 24 h. The resulting product was purified by centrifugation at 12500 rpm for 10 min and washed 2-3 times with ultrapure water. The sample was then pre-frozen at -20 °C for 24 h and freeze-dried to obtain a powdered product, which is the urchin-shaped Au@Pt nanoparticle, and stored under vacuum at 4 °C.

[0022] Example 2 Preparation of SiO2-COOH: 0.5 mg of silica microspheres were dispersed in 50 mL of ethanol, and then 0.4 mL of LAPTES was added to the solution under vigorous stirring. After reacting at 80 °C for 3 h, the product was separated by centrifugation and redispersed in DMF (dimethylformamide). Subsequently, 1 g of SA (succinic anhydride) was mixed with the above solution and stirred for 12 h to achieve the optimal carboxyl loading. The obtained SiO2-COOH was purified with ethanol and deionized water and finally dispersed in 1.5 mL of ethanol to obtain a SiO2-COOH dispersion.

[0023] Example 3 Preparation of SiO2-conjugated tetracycline antibody (SiO2-Ab): 200 μL of the SiO2-COOH dispersion from Example 2 was added to 1 mL of MES buffer containing 25 mg / mL EDC [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] and 25 mg / mL NHS (N-hydroxysuccinimide), and the mixture was shaken for 30 min. After the reaction, the mixture was centrifuged, washed three times with PBS buffer, and resuspended in 500 μL of PBS. Then, 150 μL of tetracycline antibody (Ab-TC, 100 μg / mL, commercially available) was added to the suspension, and the mixture was incubated on a shaker at 4 °C for 12 h. After centrifugation and removal of the supernatant, 200 μL of BSA (100 μg / mL) was added and the mixture was blocked at 4 °C for 1 h. After the reaction, the precipitate was washed three times with PBS and finally resuspended in 500 μL of 10 mM PBS to obtain the SiO2-Ab conjugate, which was stored at 4 °C for later use.

[0024] Example 4 Construction of a competitive electrochemical immunosensor for tetracycline detection: GCE (glassy carbon electrode) was polished sequentially with Al2O3 slurries with particle sizes of 0.5, 0.05, and 0.03 µm, then ultrasonically cleaned in ethanol and ultrapure water, and dried with nitrogen. 8 µL of a 2 mg / mL Au@Pt NPs dispersion (dispersion medium was a 2 mg / mL chitosan solution) was drop-coated onto the GCE and dried at room temperature to obtain Au@Pt NPs / GCE. The electrode was activated in 1% glutaraldehyde solution for 3 h and then washed with ultrapure water. 8 µL of a 100 μg / mL BSA-TC solution (commercially available) was added dropwise to the electrode surface, and incubated for 3 h to form BSA-TC / Au@Pt NPs / GCE. Finally, non-specific binding sites were blocked with 8 µL of a 50 μg / mL BSA solution, and after reacting for 30 min, the electrode was washed with PBS buffer to obtain BSA / BSA-TC / Au@Pt NPs / GCE, which was stored at 4 °C.

[0025] Example 5 Plotting the working curve for the electrochemical immunosensor detection of tetracycline: When detecting tetracycline using a glassy carbon electrode (GCE) as the working electrode: 20 μL of tetracycline at different concentrations (0, 20 fg / mL, 200 fg / mL, 2 pg / mL, 20 pg / mL, 200 pg / mL, 2 ng / mL, 20 ng / mL) was mixed with 20 μL of SiO2-Ab prepared in Example 3 and incubated for 30 min. Then, 8 μL of the mixture was dropped onto the surface of the sensor prepared in Example 4, and incubated for another 30 min. After washing once each with PBS buffer and ultrapure water, the changes in electrical signal were recorded using the DPV signal mode of an electrochemical workstation. Figure 2 A); Plot a standard curve for the detection of tetracycline using this method, with tetracycline concentration on the x-axis and peak oxidation current on the y-axis. Figure 2 B), the regression equation obtained through linear fitting is y=1.962log[TC]+31.038(R). 2 =0.9913), and the detection limit of this method was calculated to be 4.55 fg / mL.

[0026] Similarly, when using a screen-printed electrode (SPE) as the working electrode to detect tetracycline (the sensor construction is the same as in Example 4, except that GCE is replaced with SPE): 20 μL of tetracycline at different concentrations (0, 20 fg / mL, 200 fg / mL, 2 pg / mL, 20 pg / mL, 200 pg / mL, 2 ng / mL) was mixed with 20 μL of SiO2-Ab prepared in Example 3 and incubated for 30 min. Then, 8 μL of the mixture was dropped onto the surface of the prepared sensor, and incubation continued for another 30 min. The sensor was then washed once each with PBS buffer and ultrapure water, and the changes in electrical signal were recorded using the DPV signal mode of an electrochemical workstation. Figure 3 A); Plot a standard curve for the detection of tetracycline using this method, with tetracycline concentration on the x-axis and peak oxidation current on the y-axis. Figure 3 B), the regression equation obtained through linear fitting is y=2.314log[TC]+51.22(R). 2 =0.9961), and the detection limit of this method was calculated to be 11.58 fg / mL.

[0027] Example 6 Detection of tetracycline in actual samples: The milk samples used in this study were purchased from the local market. In short, 4 mL of milk was thoroughly mixed with 1 mL of chloroform and 2 mL of 1% trichloroacetic acid, and then ultrasonically extracted for 30 min. The mixture was centrifuged at 12000 rpm for 15 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the test solution. The test solution was divided into three equal portions, and tetracycline was added at concentrations of 1 pg / mL, 10 pg / mL, and 100 pg / mL, respectively. The test sample was used instead of the tetracycline standard solution in Example 5, and the reaction was carried out according to the steps in Example 5. Finally, the electrical signal of the detection system was tested using an electrochemical workstation. The obtained current peak value was substituted into the linear regression equation of the standard working curve to calculate the concentration of tetracycline in the test sample, and the spiked recovery rate was calculated (Table 1).

[0028] Table 1. Electrochemical immunosensor detection of tetracycline in actual samples

[0029] As shown in Table 1, the GCE-based sensor achieved a recovery rate of 86.66%–104.63% for tetracycline detection in actual samples, with a relative standard deviation (RSD) of 3.01–4.12%; while the SPE-based sensor achieved a recovery rate of 91.25%–104.69% for tetracycline detection in actual samples, with a relative standard deviation (RSD) of 1.21–4.50%. These results indicate that the present invention has good accuracy in detecting tetracycline in milk samples.

[0030] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. A competitive electrochemical immunosensor, characterized in that: Antigen conjugates were loaded onto electrodes modified with sea urchin-shaped Au@Pt NPs, and insulating SiO2 was chemically bonded to corresponding antibodies to obtain SiO2 antibody conjugates. The antigens in the sample specifically competed with the antigen conjugates for the SiO2 antibody conjugates and regulated the electrical signal of the electrode. The electrical signal of the electrode was positively correlated with the concentration of the antigen in the sample.

2. The competitive electrochemical immunosensor according to claim 1, characterized in that: The electrodes include glassy carbon electrodes and screen-printed electrodes.

3. The competitive electrochemical immunosensor according to claim 1, characterized in that: The antigens include tetracycline, and the antibodies corresponding to tetracycline are tetracycline antibodies.

4. The competitive electrochemical immunosensor according to claim 3, characterized in that: The antigen conjugate is bovine serum albumin-tetracycline.

5. A competitive electrochemical immunosensor according to claim 1, characterized in that: The insulating SiO2, after being functionalized with carboxyl groups, is chemically coupled to the corresponding antibody.

6. The method for preparing the competitive electrochemical immunosensor according to any one of claims 1-5, characterized in that, Includes the following steps: Preparation of S1, sea urchin-like Au@Pt NPs; S2. Preparation of electrodes loaded with antigen conjugates; The electrode was modified using S1 sea urchin-shaped Au@Pt NPs, and an antigen conjugate was loaded onto the modified electrode. Preparation of S3 and SiO2 antibody conjugates; Insulating SiO2 is functionalized with carboxyl groups and then chemically coupled with an antibody to obtain a SiO2 antibody conjugate.

7. The method for preparing the competitive electrochemical immunosensor according to claim 6, characterized in that: In step S1, Pluronic F127 is dissolved in a mixed solution of HAuCl4 and H2PtCl6, and then ascorbic acid solution is added to synthesize urchin-like Au@Pt NPs.

8. The method for preparing the competitive electrochemical immunosensor according to claim 6, characterized in that: In S2, after the SiO2 microspheres react with APTES, they further react with succinic anhydride to achieve carboxyl functionalization of insulating SiO2.

9. The application of the competitive electrochemical immunosensor described or obtained according to any one of claims 1-8 in the detection of tetracycline.

10. The application according to claim 9, characterized in that: The detection limit for tetracycline can be as low as 4.55 fg / mL.

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