Preparation method and application of molecularly imprinted polymer and aptamer double-recognition gold nanoparticle-laser induced graphene composite material modified electrode

Abstract

The invention discloses a preparation method and application of a molecularly imprinted polymer and aptamer double-recognition gold nanoparticle-laser induced graphene composite material modified electrode, and belongs to the technical field of flexible electrochemical sensors and kanamycin detection. According to the invention, a gold nanoparticle-laser-induced graphene composite material modified electrode is used as a base material, a kanamycin aptamer is fixed through a gold-sulfur bond by using gold nanoparticles on the surface of the base material, and a molecularly imprinted polymer layer is further modified; therefore, an electrochemical sensing electrode with a molecularly imprinted polymer and aptamer dual recognition interface is constructed, and the electrochemical sensing electrode is used as a high-performance sensitive element of a sensor and shows good analysis performance in actual fish meat sample kanamycin detection. The prepared modified electrode can be used for sensitively detecting KANA in a concentration range of 10.0 pmol / L-1. 0mmol / L, the detection limit is as low as 3.3 pmol / L, and the modified electrode is good in selectivity and stability.

Description

Technical Field

[0001] This invention belongs to the field of flexible electrochemical sensors and kanamycin detection technology, and particularly relates to the preparation method and application of gold nanoparticle-laser-induced graphene composite modified electrodes with dual recognition of molecularly imprinted polymers and aptamers. Background Technology

[0002] Kanamycin (KANA), a leading representative of aminoglycoside antibiotics, is widely used to treat various infectious diseases caused by Gram-positive and Gram-negative bacteria due to its broad-spectrum antibacterial activity. This class of antibiotics exerts its bactericidal effect by irreversibly binding to the 30S subunit of bacterial ribosomes and inhibiting protein synthesis. However, in livestock farming and aquaculture, the overuse or abuse of KANA can lead to its residues and accumulation in animal tissues, dairy products, and poultry eggs. Long-term ingestion of animal-derived foods containing KANA residues can lead to drug accumulation in the body, potentially causing ototoxicity (such as hearing loss and vestibular dysfunction) and nephrotoxicity (such as renal tubular damage and decreased renal function), posing a potential threat to public health. Therefore, establishing rapid, sensitive, and reliable methods for detecting KANA residues is of great significance for ensuring food safety and human health.

[0003] Currently, commonly used methods for KANA residue analysis include enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), and surface plasmon resonance (SPR). While ELISA offers good specificity, it is susceptible to matrix interference and cross-reactivity. LC-MS and HPLC, although highly sensitive and accurate, rely on expensive instruments, involve complex pretreatment, are time-consuming, and require highly skilled operators. SPR technology is limited by equipment cost and signal stability issues. Overall, these traditional methods generally suffer from limitations such as high reagent consumption, cumbersome procedures, high detection costs, and difficulty in achieving rapid on-site detection.

[0004] In recent years, electrochemical sensing technology has attracted widespread attention in the field of antibiotic residue detection due to its significant advantages, including simple equipment, rapid response, low cost, ease of miniaturization, and real-time detection capabilities. This method leverages the changes in signals such as current, potential, or impedance caused by the electrochemical behavior of the target analyte on the electrode surface to achieve qualitative or quantitative analysis. By designing highly selective recognition interfaces, the recognition ability and detection sensitivity of electrochemical sensors for KANA can be further improved. Therefore, developing new KANA detection methods based on electrochemical sensing not only helps to compensate for the shortcomings of traditional analytical techniques but also provides a promising technical path for on-site and rapid screening of antibiotic residues in food. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode with dual recognition of molecularly imprinted polymers and aptamers, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a gold nanoparticle (AuNPs)-laser-induced graphene (LIG) composite modified electrode with dual recognition of molecularly imprinted polymers (MIPs) and aptamers (Apt), comprising the following steps: (1) Preparation of gold nanoparticle-laser-induced graphene composite modified electrode; (2) The aptamer solution and the kanamycin solution were mixed and incubated, and then applied to the surface of the gold nanoparticle-laser induced graphene composite material modified electrode to obtain the kanamycin aptamer-gold nanoparticle-laser induced graphene electrode. (3) The kanamycin aptamer-gold nanoparticle-laser induced graphene electrode was placed in an aniline solution for electropolymerization and then eluted to obtain the gold nanoparticle-laser induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymer and aptamer.

[0007] Further, in step (1), the preparation of the gold nanoparticle-laser-induced graphene composite material modified electrode includes: mixing chloroauric acid solution and liquid polyimide and coating them onto the substrate surface, then curing them to obtain a flexible precursor film; performing laser-induced treatment on the flexible precursor film according to the pre-designed electrode geometry; coating the reference electrode region of the electrode with silver paste and performing thermal curing treatment to obtain the gold nanoparticle-laser-induced graphene composite material modified electrode; The concentration of the chloroauric acid solution is 10~90 mmol / L; the ratio of the chloroauric acid solution to polyimide is 200 μL: 4 g; The curing temperature is 90~110℃, and the curing time is 20~40min; The laser wavelength for the laser-induced treatment is 450 nm, the engraving depth for the laser-induced treatment is 20-40%, and the laser power for the laser-induced treatment is 1.65-2.48 W. The temperature of the thermosetting treatment is 120~140℃, and the time of the thermosetting treatment is 20~40min.

[0008] Further, in step (2), the concentration of the aptamer solution is 10~20 μmol / L, the concentration of the kanamycin solution is 10~20 μmol / L, and the volume ratio of the aptamer solution to the kanamycin solution is 4:1, 3:2, 1:1, 2:3 or 1:4.

[0009] Further, in step (2), the incubation temperature is 37°C, the incubation liquid volume is 50~150μL, and the incubation time is 10~30min.

[0010] Further, in step (3), the concentration of the aniline solution is 1~5 mmol / L; the aniline solution is a phosphate buffer solution containing aniline, and the pH value of the phosphate buffer solution is 6.0~8.0.

[0011] Further, in step (3), the electropolymerization method is cyclic voltammetry; the number of electropolymerization cycles is 5 to 30, the scan rate of electropolymerization is 0.05 to 0.25 V / s, and the voltage window of electropolymerization is -0.2 to 0.6 V.

[0012] Further, in step (3), the eluent is a mixed solution of methanol and acetic acid, and the volume ratio of methanol to acetic acid is 9:1; the elution time is 10~30 min.

[0013] This invention provides a gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymers and aptamers, prepared according to the preparation method described above.

[0014] This invention provides an electrochemical sensor, including a gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymers and aptamers as described in the above technical solution.

[0015] The present invention also provides an application of the above-described molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser-induced graphene composite material modified electrode or the electrochemical sensor in the detection of kanamycin.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention uses a gold nanoparticle-laser-induced graphene composite modified electrode as the matrix material. The gold nanoparticles on the surface are used to immobilize the kanamycin aptamer via gold-sulfur bonds. Further modification with a molecularly imprinted polymer layer creates an electrochemical sensing electrode with a dual recognition interface of molecularly imprinted polymer and aptamer. This electrode serves as a high-performance sensitive element in a sensor, demonstrating excellent analytical performance in the detection of kanamycin in actual fish samples. The preparation method of this invention's gold nanoparticle-laser-induced graphene composite modified electrode with dual recognition of molecularly imprinted polymer and aptamer is simple. The resulting modified electrode is portable, inexpensive, and can sensitively detect KANA in the concentration range of 10.0 pmol / L to 1.0 mmol / L, with a detection limit as low as 3.3 pmol / L, exhibiting good selectivity and stability. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 SEM images of AuNPs-LIG prepared in Example 1 and LIG prepared in Comparative Example 1, where A and B are Comparative Example 1, C and D are Example 1, A and C are 1 μm, and B and D are 500 nm. Figure 2 The images shown are TEM images, HAADF images, and elemental mapping images of AuNPs-LIG prepared in Example 1. A and B are TEM images, where A is 50 nm and B is 10 nm. C is a HAADF image and D is an elemental mapping image of C, N, O, and Au. Figure 3 X-ray diffraction patterns and Raman patterns of AuNPs-LIG prepared in Example 1 and LIG prepared in Comparative Example 1 are shown, where A is the X-ray diffraction pattern and B is the Raman pattern. Figure 4 XPS spectra of AuNPs-LIG prepared in Example 1 and LIG prepared in Comparative Example 1, where A is the C 1s spectrum of AuNPs-LIG and LIG, B is the N 1s spectrum of AuNPs-LIG and LIG, C is the O 1s spectrum of AuNPs-LIG and LIG, and D is the Au 4f spectrum of AuNPs-LIG. Figure 5In Figures A-B, the CV curves of AuNPs-LIG prepared in Examples 1 and 3-4 are shown. Figure C shows the CV curves of AuNPs-LIG prepared in Example 1, LIG ​​prepared in Comparative Example 1, Apt / AuNPs-LIG prepared in Comparative Example 2, MIPs / Apt@KANA / AuNPs-LIG and MIPs / Apt / AuNPs-LIG prepared in Example 2, and D shows the CV curves of AuNPs-LIG prepared in Example 1, LIG ​​prepared in Comparative Example 1, and the comparative... Impedance curves of Apt / AuNPs-LIG prepared in Example 2, E~F are the CV curves of AuNPs-LIG prepared in Example 1 and LIG prepared in Comparative Example 1 at different scan rates, G~H are the linear curves of peak current versus the square root of scan rate of AuNPs-LIG prepared in Example 1 and LIG prepared in Comparative Example 1, where A is Example 1 and Example 3, B is Example 1 and Example 4, E is Comparative Example 1, F is Example 1, G is Comparative Example 1, and H is Example 1; Figure 6 The current change when the MIPs / Apt / AuNPs-LIG prepared in Examples 2 and 5-9 detects KANA is shown. In the examples, A is Example 2 and Example 5, B is Example 2 and Example 6, C is Example 2 and Example 7, D is Example 2 and Example 8, and E is Example 2 and Example 9. Figure 7 The DPV curves (A) and the linear relationship between ΔI and KANA concentration (B) of MIPs / Apt / AuNPs-LIG prepared in Example 2 after incubation in KANA solutions of different concentrations, as well as the storage stability (C) and selectivity (D) of MIPs / Apt / AuNPs-LIG prepared in Example 2. Figure 8 DPV curves of MIPs / Apt / AuNPs-LIG prepared in Example 2 in fish samples diluted 50 times with different concentrations of KANA; Figure 9 A schematic diagram illustrating the preparation process of the gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymers and aptamers provided by the present invention. Figure 10 DPV curves (A) and linear relationship between ΔI and KANA concentration (B) of NIPs / Apt / AuNPs-LIG prepared for Comparative Example 3 after incubation in KANA solutions of different concentrations. Figure 11 The CV curves are for the modified electrodes prepared in Example 1 and Comparative Examples 4-5. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] This invention provides a method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode with dual recognition of molecularly imprinted polymers and aptamers, comprising the following steps: (1) Preparation of gold nanoparticle-laser induced graphene composite modified electrode (AuNPs-LIG). (2) The aptamer solution and the kanamycin solution were mixed and incubated, and then applied to the surface of the gold nanoparticle-laser induced graphene composite material modified electrode to obtain the kanamycin aptamer-gold nanoparticle-laser induced graphene electrode. (3) The kanamycin aptamer-gold nanoparticle-laser induced graphene electrode was placed in an aniline solution for electropolymerization and then eluted to obtain the gold nanoparticle-laser induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymer and aptamer.

[0021] Aptamers are single-stranded oligonucleotides capable of highly specific binding to target molecules. Due to their excellent specificity, easily modifiable structure, lack of immunogenicity, and strong affinity, they have been widely used as effective alternatives to antibodies in the field of biosensing. However, as a biorecognition element, aptamers are easily inactivated under harsh detection conditions, resulting in the loss of their ability to specifically recognize target molecules. Molecularly imprinted polymers are polymers prepared using molecular imprinting technology that can specifically recognize target molecules. They achieve high selectivity for template molecules by creating imprinted cavities in the polymer matrix that perfectly match the spatial configuration and functional group combination of the template molecule. During polymerization, functional monomers containing specific functional groups bind to the template molecule covalently or non-covalently. As an artificially synthesized recognition element, molecularly imprinted polymers have advantages such as low preparation cost, wide applicability, and high stability in harsh detection environments. However, molecularly imprinted polymers still have some inherent limitations, such as uneven cavity distribution and template molecule residual leakage due to insufficient elution efficiency. Given the significant advantages of both molecularly imprinted polymers and aptamers, a dual recognition element combining molecularly imprinted polymers and aptamers was developed. This system not only possesses the high specificity of aptamers and the high stability of molecularly imprinted polymers in harsh detection environments, but also combines high sensitivity and high affinity.

[0022] Laser-induced graphene (LAQ) is a three-dimensional porous graphene material prepared by laser-induced precursor polymerization. It exhibits high design flexibility, mechanical flexibility, and biocompatibility, enabling various surface functionalizations and making it suitable for developing highly sensitive electrochemical sensors. Effective functionalization of LQ through nanomaterial compositing and atomic doping has become an important strategy for improving its sensing performance. After functionalization, the specific surface area, surface hydrophilicity, conductivity, and interfacial diversity of LQ are all improved, effectively enhancing its sensitivity, detection limit, and selectivity in electrochemical sensing applications. Among numerous electrode modification materials, metal nanoparticles have attracted much attention due to their abundant active sites, large specific surface area, and excellent electrocatalytic activity, making them ideal materials for constructing electrochemical sensors. As a representative of noble metal nanoparticles, gold nanoparticles play a crucial role in sensor applications due to their high stability, excellent biocompatibility, and strong adsorption capacity. Modifying the surface of LQ with gold nanoparticles can significantly enhance the electrochemical performance and response sensitivity of the electrode. In addition, gold nanoparticles can be immobilized by connecting aptamers via Au-S bonds.

[0023] Therefore, this invention first synthesizes a gold nanoparticle-laser-induced graphene composite material modified electrode in one step through laser induction, then uses the gold nanoparticles on its surface to fix the kanamycin aptamer through gold-sulfur bonds, and then further modifies the molecularly imprinted polymer layer to construct an electrochemical sensing electrode with a dual recognition interface. This electrode is used as a high-performance sensitive element of the sensor and exhibits good analytical performance in the detection of kanamycin in actual fish meat samples.

[0024] In a preferred embodiment, step (1) of preparing the gold nanoparticle-laser-induced graphene composite modified electrode includes: mixing chloroauric acid solution and liquid polyimide and coating them onto the substrate surface, then curing them to obtain a flexible precursor film; performing laser-induced treatment on the flexible precursor film according to the pre-designed electrode geometry; coating the reference electrode region of the electrode with silver paste and performing thermal curing treatment to obtain the gold nanoparticle-laser-induced graphene composite modified electrode.

[0025] In a preferred embodiment, the concentration of the chloroauric acid solution is 10-90 mmol / L; for example, the concentration of the chloroauric acid solution is 10 mmol / L, 30 mmol / L, 50 mmol / L, 70 mmol / L or 90 mmol / L, more preferably 50 mg / mL; the solvent of the chloroauric acid solution is deionized water.

[0026] In a preferred embodiment, the polyimide is liquid polyimide; the liquid polyimide was purchased from Nantong Suyuan Plastics Co., Ltd.

[0027] In a preferred embodiment, the ratio of chloroauric acid solution to polyimide is 200 μL: 4 g.

[0028] In a preferred embodiment, the substrate is selected from a glass substrate.

[0029] In a preferred embodiment, the curing temperature is 90~110℃, for example, the curing temperature is 90℃, 100℃ or 110℃; the curing time is 20~40min, for example, the curing time is 20min, 30min or 40min.

[0030] In a preferred embodiment, the thickness of the flexible precursor film is 20~22μm.

[0031] In a preferred embodiment, the laser wavelength of the laser-induced treatment is 450 nm; the engraving depth of the laser-induced treatment is 20-40%, for example, the engraving depth of the laser-induced treatment is 20%, 30%, or 40%, more preferably 30%; the laser power of the laser-induced treatment is 1.65-2.48 W, for example, the laser power of the laser-induced treatment is 1.65 W, 2.2 W, or 2.48 W, more preferably 2.2 W.

[0032] In a preferred embodiment, the silver paste is purchased from Shenzhen Saiya Electronic Paste Co., Ltd., and the model number is 01L-2210.

[0033] In a preferred embodiment, the temperature of the thermosetting treatment is 120~140℃, for example, the temperature of the thermosetting treatment is 120℃, 130℃ or 140℃; the time of the thermosetting treatment is 20~40min, for example, the time of the thermosetting treatment is 20min, 30min or 40min.

[0034] In a preferred embodiment, in step (2), the concentration of the aptamer solution is 10~20 μmol / L. For example, the concentration of the aptamer solution is 10 μmol / L, 15 μmol / L, or 20 μmol / L, and more preferably 20 μmol / L; the concentration of the kanamycin solution is 10~20 μmol / L. For example, the concentration of the kanamycin solution is 10 μmol / L, 15 μmol / L, or 20 μmol / L, and more preferably 20 μmol / L; the volume ratio of the aptamer solution to the kanamycin solution is 4:1, 3:2, 1:1, 2:3, or 1:4, and more preferably 1:1; the solvent for both the aptamer solution and the kanamycin solution is TE buffer solution, and the pH value of the TE buffer solution is 7.4. The aptamer concentration determines the density and distribution of recognition sites on the electrode surface, directly affecting the intensity, specificity, and stability of the response signal. If the concentration is too low (<10 μmol / L), the thiol binding sites on the gold nanoparticle surface are unsaturated, resulting in insufficient recognition sites, low target molecule binding capacity, weak response signal, and decreased sensitivity. If the concentration is appropriate (10~20 μmol / L), the aptamer uniformly covers the electrode surface as a monolayer, with strong gold-sulfur bonds and a moderate density of recognition sites, ensuring specific binding while avoiding intermolecular entanglement that obscures active sites, resulting in a stable response signal with minimal interference. If the concentration is too high (>20 μmol / L), the aptamer accumulates in multiple layers on the electrode surface, and molecular chain interactions cause some recognition sites to fail. Simultaneously, the probability of non-specific adsorption increases, significantly increasing the proportion of interference signals. Kanamycin, as a template molecule for molecular imprinting polymerization, requires a concentration that matches the aptamer, as this affects the quality of the imprinted holes and the accuracy of detection. If the concentration is too low (<10 μmol / L), insufficient template molecules bind to the aptamer, resulting in fewer specific imprinted holes during polymerization and fewer target molecule binding sites in subsequent detection. Low-concentration kanamycin can also reduce the response current by more than 40%. At a suitable concentration (10~20 μmol / L), the template molecules bind to the aptamer in a 1:1 ratio, guiding the molecular imprint to form uniformly distributed specific imprinted holes, maximizing the synergistic efficiency of dual recognition. However, if the concentration is too high (>20 μmol / L), excessive kanamycin fails to bind to the aptamer, leading to increased non-specific holes due to free template participation in polymerization, increased elution difficulty, and false positive signals caused by residual template.

[0035] In a preferred embodiment, in step (2), the aptamer is a kanamycin aptamer (5'-SH-TGGGGGTTGAGGCTAAGCCGA-3'); the kanamycin aptamer was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0036] In a preferred embodiment, in step (2), the incubation temperature is 37°C; the volume of the incubation liquid is 50~150μL, for example, the volume of the incubation liquid is 50μL, 100μL or 150μL, more preferably 100μL; the incubation time is 10~30min, for example, the incubation time is 10min, 15min, 20min, 25min or 30min, more preferably 25min.

[0037] In a preferred embodiment, in step (3), the concentration of the aniline solution is 1~5 mmol / L, for example, the concentration of the aniline solution is 1.0 mmol / L, 2.0 mmol / L, 3.0 mmol / L, 4.0 mmol / L or 5.0 mmol / L, more preferably 2.0 mmol / L; the aniline solution is an aniline-containing phosphate buffer solution; the concentration of the phosphate buffer solution is 0.01~0.20 mol / L, for example, the concentration of the phosphate buffer solution is 0.01 mol / L, 0.10 mol / L or 0.20 mol / L, more preferably 0.20 mol / L; the pH value of the phosphate buffer solution is 6.0~8.0, for example, the pH value of the phosphate buffer solution is 6.0, 6.5, 7.0, 7.5 or 8.0, more preferably 6.5. This invention uses aniline to prepare molecularly imprinted polymers. Aniline and kanamycin aptamers have a dual synergistic effect of recognition complementarity and structural support: (1) Recognition complementarity: The targeted binding of the aptamer to kanamycin can accurately anchor the target molecule to the electrode surface, providing guidance for the uniform distribution of template molecules during aniline polymerization, so that the binding sites of imprinted holes and aptamers are in a point-to-point correspondence, thereby improving the synergistic efficiency of dual recognition; (2) Structural support: The MIP film formed by aniline electropolymerization can partially encapsulate the aptamer molecules, fix their spatial conformation through physical action, avoid the decrease in binding activity of the aptamer due to molecular chain swing, and isolate the non-specific binding of external interfering substances to the aptamer, thereby improving the recognition stability of the aptamer.

[0038] In a preferred embodiment, in step (3), the electropolymerization method is cyclic voltammetry; the number of electropolymerization cycles is 5 to 30, for example, 5, 10, 15, 20, 25, or 30, more preferably 15; the scan rate of the electropolymerization is 0.05 to 0.25 V / s, for example, 0.05 V / s, 0.10 V / s, 0.15 V / s, 0.20 V / s, or 0.25 V / s, more preferably 0.15 V / s; the voltage window of the electropolymerization is -0.2 to 0.6 V.

[0039] In a preferred embodiment, in step (3), the eluent is a mixed solution of methanol and acetic acid, and the volume ratio of methanol to acetic acid is 9:1; the elution time is 10 to 30 minutes, for example, the elution time is 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes, and more preferably 20 minutes; the elution method is soaking.

[0040] This invention provides a gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymers and aptamers, prepared according to the preparation method described above.

[0041] This invention provides an electrochemical sensor, including a gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymers and aptamers as described in the above technical solution.

[0042] The present invention also provides an application of the above-described molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser-induced graphene composite material modified electrode or the electrochemical sensor in the detection of kanamycin.

[0043] In this embodiment of the invention, room temperature refers to "25±2℃".

[0044] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0045] Example 1 A method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode (AuNPs-LIG) includes the following steps: (1) Preparation of flexible precursor membrane: Chloroauric acid was dissolved in deionized water to prepare a chloroauric acid solution with a concentration of 50 mmol / L. 200 μL of the above chloroauric acid solution was mixed with 4 g of liquid polyimide. The resulting mixture was then coated on a glass plate and cured in a vacuum oven at 100 °C for 30 min to obtain a uniform and transparent flexible precursor membrane with a thickness of about 20.5 μm. (2) Preparation of AuNPs-LIG: Using laser-induced technology, the flexible precursor film prepared in step (1) was laser-induced on the surface of the film according to the pre-designed electrode geometry. The etching depth was 30% and the laser power was 2.2W. Then, the reference electrode area of ​​the electrode was coated with silver paste and placed in an oven at 140℃ for 20 min to obtain the gold nanoparticle-laser-induced graphene composite modified electrode (AuNPs-LIG).

[0046] Comparative Example 1 A method for fabricating a laser-induced graphene-modified electrode (LIG): (1) Preparation of flexible precursor film: Liquid polyimide was coated on a glass plate and cured in a vacuum oven at 100°C for 30 min to obtain a uniform and transparent flexible precursor film with a thickness of about 20.5 μm. Step (2) is the same as in Example 1.

[0047] Figure 1 SEM images of AuNPs-LIG prepared in Example 1 and LIG prepared in Comparative Example 1 are shown, where A and B are from Comparative Example 1, and C and D are from Example 1. A and C are 1 μm images, and B and D are 500 nm images. Figure 1 As can be seen from parts A to B, the LIG prepared in Comparative Example 1 exhibits a porous, layered, three-dimensional structure. The pores are formed due to the high temperature and rapid release of gaseous products during laser combustion. Figure 1 As can be seen from parts C to D, when chloroauric acid is doped into the flexible precursor film, the laser-induced AuNPs-LIG is a three-dimensional network structure of surface-loaded particles.

[0048] Figure 2 TEM images, HAADF images, and elemental mapping images of the AuNPs-LIG prepared in Example 1 are shown. A and B are TEM images (A = 50 nm, B = 10 nm), C is the HAADF image, and D is the elemental mapping image of C, N, O, and Au. Figure 1 As can be seen from part A, the gold nanoparticles are encapsulated in a graphene layer. Figure 1 The lattice spacing of 0.334 nm and 0.249 nm shown in Part B may correspond to the (002) crystal plane of graphene (PDF#41-1487) and the (220) crystal plane of Au (PDF#04-0784). Figure 1 The EDS elemental distribution diagram shown in section D shows that carbon (C), nitrogen (N), oxygen (O), and gold (Au) are uniformly distributed throughout the nanocomposite material, confirming the successful preparation of AuNPs-LIG.

[0049] Figure 3 X-ray diffraction (XRD) and Raman spectroscopy (RMS) patterns of the AuNPs-LIG prepared in Example 1 and the LIG prepared in Comparative Example 1 are shown, where A is the XRD pattern and B is the Raman spectroscopy pattern. Figure 3 As can be seen in section A, both materials exhibit characteristic diffraction peaks at 25.9°, corresponding to the (002) crystal plane of hexagonal graphitic carbon, indicating a high degree of graphitization (PDF#41-1487). Figure 3 As shown in section B, the characteristic D band (1343 cm⁻¹) was observed in both LIG and AuNPs-LIG. -1 ) and G-band (1581cm)-1 These spectral features confirm the successful formation of laser-induced graphitization. Typically, the D and G peaks correspond to the sp... of the carbon phase, respectively. 2 Coordination defect states and sp 2 E of the bonded carbon atom 2g Pattern-related. Furthermore, the intensity ratio (ID) between the D and G peaks... D / I G Ig is a key parameter for the disorder of the graphite lattice. Comparative analysis revealed that Ig in AuNPs-LIG... D / I G The ratio is 0.97, LIG's I D / I G The ratio of 0.86 indicates that the introduction of AuNPs enhances lattice defects, which is beneficial for higher electron transfer and electrochemical activity.

[0050] Figure 4 XPS spectra of AuNPs-LIG prepared in Example 1 and LIG prepared in Comparative Example 1 are shown, where A is the C 1s spectrum of AuNPs-LIG and LIG, B is the N 1s spectrum of AuNPs-LIG and LIG, C is the O 1s spectrum of AuNPs-LIG and LIG, and D is the Au 4f spectrum of AuNPs-LIG. Figure 4 It can be seen that the XPS spectra of both materials show characteristic peaks corresponding to C 1s, N 1s and O 1s, and peaks related to Au 4f were also observed in AuNPs-LIG. Figure 4 The C 1s spectrum in part A shows four distinct peaks at 284.7 eV, 285.4 eV, 288.4 eV, and 291.1 eV, corresponding to C, COC, O=CN, and CO=C bonds, respectively, confirming the successful graphitization of the precursor. Figure 4 Part B shows the N 1s spectrum, with three peaks at 398.9 eV, 400.3 eV, and 402.1 eV, corresponding to pyridine N, pyrrole N, and graphite N, respectively. Figure 4 The O1s spectrum in the C portion shows two peaks at 533.9 eV and 532.4 eV, which are attributed to CO and C=O, respectively. Figure 4 Part D shows the Au4f spectrum, with characteristic peaks at 85.1 eV and 89.1 eV, corresponding to 4f, respectively. 5 / 2 and 4f 7 / 2 This indicates that gold nanoparticles have been successfully incorporated into the nanocomposite material.

[0051] Example 2 A method for preparing a molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser-induced graphene composite modified electrode (MIPs / Apt / AuNPs-LIG), the specific steps of which are as follows: Steps (1)-(2) are the same as in Example 1; (3) The aptamer and kanamycin were dissolved in TE buffer solution with pH 6.5 to obtain aptamer solution and kanamycin solution with a concentration of 20 μmol / L respectively; 50 μL of aptamer solution and 50 μL of kanamycin solution were mixed and incubated at 37℃ for 25 min to obtain Apt@KANA complex; then 10 μL of the above Apt@KANA complex was dropped onto the surface of AuNPs-LIG obtained in step (2), the 5'-SH of the aptamer formed Au-S bond with AuNPs and was fixed on the surface of gold nanoparticles to obtain kanamycin aptamer-gold nanoparticle-laser induced graphene composite modified electrode (Apt@KANA / AuNPs-LIG); (4) Apt@KANA / AuNPs-LIG was electropolymerized for 15 cycles at a scan rate of 0.15 V / s in a phosphate buffer solution containing 2 mM aniline (the concentration of the phosphate buffer solution was 0.01 mol / L and the pH value was 7.5) to obtain MIPs / Apt@KANA / AuNPs-LIG; (5) The obtained MIPs / Apt@KANA / AuNPs-LIG was immersed in an eluent with a volume ratio of methanol to acetic acid of 9:1 for 20 min, and then the electrode was gently rinsed with ultrapure water to obtain a gold nanoparticle-laser induced graphene composite modified electrode (MIPs / Apt / AuNPs-LIG) with dual recognition of molecularly imprinted polymer and aptamer.

[0052] Comparative Example 2 The preparation method of an aptamer-recognized gold nanoparticle-laser-induced graphene composite modified electrode (Apt / AuNPs-LIG) differs from Example 2 only in that step (4) is omitted, while the rest is the same as Example 2.

[0053] Example 3 A method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode (AuNPs-LIG) differs from Example 1 in that, in step (2), the engraving depth is 30%, and the laser power is 1.65W and 2.48W respectively, while the rest is the same as in Example 1.

[0054] Example 4 A method for preparing a gold nanoparticle-laser induced graphene composite modified electrode (AuNPs-LIG) differs from Example 1 in that, in step (2), the engraving depth is 20% and 40%, the laser power is 2.2W, and the rest is the same as in Example 1.

[0055] The CV method was used to analyze the 5.0 mmol / L [Fe(CN)6] solution containing 0.1 mol / L KCl. 3- / 4- AuNPs-LIG prepared in Examples 1 and 3-4 were tested in redox probe solution at a scan rate of 0.1 V / s. The results are shown in [Figure number missing]. Figure 5 Parts A and B in the text; CV method was used in a 5.0 mmol / L [Fe(CN)6] solution containing 0.1 mol / L KCl. 3- / 4- The AuNPs-LIG prepared in Example 1, the LIG prepared in Comparative Example 1, the Apt / AuNPs-LIG prepared in Comparative Example 2, and the MIPs / Apt@KANA / AuNPs-LIG and MIPs / Apt / AuNPs-LIG prepared in Example 2 were tested in redox probe solutions at a scan rate of 0.1 V / s. The results are shown in [Figure 1]. Figure 5 Part C of the report; the AuNPs-LIG prepared in Example 1, the LIG prepared in Comparative Example 1, and the Apt / AuNPs-LIG prepared in Comparative Example 2 were tested at a scan rate of 0.1 V / s. The results are shown in [the table below]. Figure 5 Part D of the diagram; the AuNPs-LIG prepared in Example 1 and the LIG prepared in Comparative Example 1 were tested using the CV method at scan rates of 0.08 V / s, 0.1 V / s, 0.15 V / s, 0.2 V / s, 0.25 V / s, 0.3 V / s, 0.35 V / s, 0.4 V / s, 0.45 V / s, and 0.5 V / s. The results are shown in [Figure number missing]. Figure 5 Parts E and F in the text; according to Figure 5 Plot the peak current (I) in the E and F sections. p ) and the square root of the scan rate (υ) 1 / 2 The linear curve of ) is shown in the figure. Figure 5 The G and H parts in it.

[0056] Figure 5In Figures A-B, the CV curves of AuNPs-LIG prepared in Examples 1 and 3-4 are shown. Figure C shows the CV curves of AuNPs-LIG prepared in Example 1, LIG ​​prepared in Comparative Example 1, Apt / AuNPs-LIG prepared in Comparative Example 2, MIPs / Apt@KANA / AuNPs-LIG and MIPs / Apt / AuNPs-LIG prepared in Example 2, and D shows the CV curves of AuNPs-LIG prepared in Example 1, LIG ​​prepared in Comparative Example 1, and the comparative... Impedance curves of the Apt / AuNPs-LIG prepared in Example 2 are shown. E~F are the CV curves of the AuNPs-LIG prepared in Example 1 and the LIG prepared in Comparative Example 1 at different scan rates. G~H are the linear curves of the peak current versus the square root of the scan rate for the AuNPs-LIG prepared in Example 1 and the LIG prepared in Comparative Example 1. Wherein, A represents Examples 1 and 3, B represents Examples 1 and 4, E represents Comparative Example 1, F represents Example 1, G represents Comparative Example 1, and H represents Example 1. Figure 5 As can be seen from parts A and B, AuNPs-LIG achieves the maximum peak current when the laser power is 2.2W and the engraving depth is 30%, which represents the optimal laser parameters for AuNPs-LIG fabrication. Figure 5 As can be seen from part C, LIG ​​exhibits the lowest current response, indicating its limited conductivity. However, the current response of the AuNPs-LIG modified electrode, prepared after adding AuNPs, increases significantly, indicating that AuNPs increase the electron transfer rate and improve conductivity. Figure 5 As can be seen from part D in the Nyquist plot, the fabricated electrode exhibits semicircles of different diameters in the high-frequency region, indicating that the charge transfer resistance (R) is... ct Among the various electrode compositions, the AuNPs-LIG electrode exhibited the smallest semicircle, demonstrating its superior electrochemical activity, likely due to the excellent conductivity and charge transfer capabilities of AuNPs. Further modification of the AuNPs-LIG electrode surface with the Apt@KANA complex hindered probe solution penetration and electron transfer, resulting in an increased Rct value. Figure 5 The E and F sections show that the redox peak currents of LIG and AuNPs-LIG gradually increase with increasing scan rate. From... Figure 5 As can be seen from the G and H portions, both LIG and AuNPs-LIG exhibit a pair of reversible redox peaks, and I... p With the square root of the scan rate (υ) 1 / 2 The relationship exhibits a significant linearity, indicating a diffusion-controlled process. This relationship can be described by the Randle-Sevcik equation. pa =2.69×10 5 n 3 / 2 ACD 1 / 2 V1 / 2 , where I pa [Fe(CN)6] 3- / 4- (A) is the anodic peak current value, where n is the number of transferred electrons and A is the effective surface area (cm²). 2 C is [Fe(CN)6] 3- / 4- Concentration (mol / cm) 3 ), D is the diffusion coefficient (7.6 × 10⁻⁶). -6 cm 2 / s), where V is the scan rate (V / s), and the calculated EASA (electrochemically active area) of AuNPs-LIG and LIG are 0.296 cm². 2 and 0.245cm 2 This indicates that there are more electrochemical reaction active sites on the electrode, and the modification of AuNPs can improve the conductivity, electron transport capability and electrochemical active area of ​​LIG.

[0057] Example 5 A method for preparing a molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser induced graphene composite modified electrode (MIPs / Apt / AuNPs-LIG) differs from Example 2 only in that, in step (4), the concentration of the phosphate buffer solution is 0.01 mol / L, and the pH values ​​are 6.0, 7.0, 7.5, and 8.0, respectively. The other steps are the same as in Example 2.

[0058] Example 6 A method for preparing a molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser-induced graphene composite modified electrode (MIPs / Apt / AuNPs-LIG) differs from Example 2 only in that, in step (4), electropolymerization is performed for 15 cycles by cyclic voltammetry at scan rates of 0.05 V / s, 0.10 V / s, 0.20 V / s, and 0.25 V / s, respectively. The rest is the same as in Example 2.

[0059] Example 7 A method for preparing a molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser-induced graphene composite modified electrode (MIPs / Apt / AuNPs-LIG) differs from Example 2 only in that, in step (4), electropolymerization is performed by cyclic voltammetry at a scan rate of 0.15V / s for 5, 10, 20, 25, and 30 cycles respectively, while the rest is the same as in Example 2.

[0060] Example 8 A method for preparing a molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser induced graphene composite modified electrode (MIPs / Apt / AuNPs-LIG) differs from Example 2 only in that, in step (5), the obtained MIPs / Apt@KANA / AuNPs-LIG is immersed in an elution solution with a volume ratio of methanol to acetic acid of 9:1 and eluted for 10 min, 15 min, 25 min, and 30 min, respectively, while the rest is the same as in Example 2.

[0061] Example 9 A method for preparing a molecularly imprinted polymer and aptamer dual-recognition gold nanoparticle-laser induced graphene composite modified electrode (MIPs / Apt / AuNPs-LIG) differs from Example 2 only in that, in step (3), 50 μL of aptamer solution and 50 μL of kanamycin solution are mixed and incubated at 37°C for 10 min, 15 min, 20 min and 30 min respectively, and the rest is the same as in Example 2.

[0062] The MIPs / Apt / AuNPs-LIGs prepared in Examples 2 and 5-9 were used as the sensitive elements of the electrochemical sensor, respectively. These were connected to an electrochemical workstation to fabricate the electrochemical sensor, which was then used to detect KANA. The test was conducted in a 5.0 mmol / L [Fe(CN)6] solution containing 0.1 mol / L KCl. 3- / 4- The reaction was carried out in solution, and the signal was recorded using differential pulse voltammetry (DPV). The peak response current after elution was defined as I0, and the peak response current after incubation with the target analyte was defined as I. The change in current ΔI (ΔI = I0 - I) was used as the evaluation index of sensor performance. The results are shown in [Figure number missing]. Figure 6 .

[0063] Figure 6 The current changes when detecting KANA using MIPs / Apt / AuNPs-LIG prepared in Examples 2 and 5-9 are shown. Where A represents Examples 2 and 5, B represents Examples 2 and 6, C represents Examples 2 and 7, D represents Examples 2 and 8, and E represents Examples 2 and 9. Figure 6 As shown in Part A, the ΔI current gradually increases with increasing pH of the phosphate buffer solution, reaching its maximum at pH 6.5. However, when the pH exceeds 6.5, the ΔI current drops sharply. This is because slightly acidic solutions promote the formation of dense and uniform polyaniline structures, while excessively acidic or alkaline conditions hinder its formation. Figure 6 As can be seen from part B, the ΔI current increases significantly with increasing scan rate, reaching its maximum at a scan rate of 0.15V / s. Therefore, 0.15V / s is chosen as the optimal scan rate. Figure 6 As can be seen from section C, the ΔI current increases with the number of polymerization cycles, reaching its maximum at 15 cycles. After 15 cycles, ΔI decreases rapidly, possibly due to incomplete template molecule removal caused by excessive MIP film thickness. Therefore, 15 cycles were determined to be the optimal number of polymerization cycles. Figure 6 As can be seen from part D, the ΔI current increases with increasing elution time, reaching a maximum at 20 min. This is attributed to the formation of an imprinted cavity with a size matching the KANA structure at this time, enhancing the penetration of the probe molecules and facilitating electron transfer. However, after 20 min, the current drops sharply, possibly due to the prolonged elution time damaging or disrupting the three-dimensional structure of the MIP membrane. Therefore, 20 min was chosen as the optimal elution time. Figure 6 As can be seen from part E, the ΔI current increases with the increase of incubation time, reaching a maximum at 25 min. After this time, the current begins to decrease. This is attributed to the structural deformation of the molecularly imprinted polymer induced by swelling, which can cause KANA to partially detach from the imprinted binding site. Therefore, 25 min was selected as the optimal incubation time.

[0064] The MIPs / Apt / AuNPs-LIG prepared in Example 2 was mixed with different concentrations of KANA (1×10⁻⁶). -11 mol / L, 1×10 - 10 mol / L, 1×10 -9 mol / L, 1×10 -8 mol / L, 1×10 -7 mol / L, 1×10 -6 mol / L, 1×10 -5 mol / L, 1×10 - 4 mol / L, 1×10 -3 The solution (mol / L) was incubated in PBS buffer at 37°C for 25 min, and its response was characterized by changes in DPV peak current. The results are shown in [Figure number missing]. Figure 7 Parts A to B in the text; The stability of the electrochemical sensor was tested using DPV. The MIPs / Apt / AuNPs-LIG prepared in Example 2 was stored in a vacuum drying oven at 25°C for 9 days, and every two days it was dried in 5.0 mM [Fe(CN)6] with 0.1 M KCl. 3- / 4- DPV scanning was performed in the solution, and the results are shown below. Figure 7 Part C in the text; Selectivity testing of the electrochemical sensor was performed using DPV. The MIPs / Apt / AuNPs-LIG prepared in Example 2 was incubated at 37°C for 25 min in a 1.0 mmol / L KANA solution containing four common antibiotic interfering agents: streptomycin (SM), chloramphenicol (CAP), ciprofloxacin (CFX), and tetracycline (TC). The concentration of each interfering agent was 5.0 mmol / L. The results are shown in [Figure number missing]. Figure 7 Part D in the text.

[0065] Figure 7 The DPV curves (A) and the linear relationship between ΔI and KANA concentration (B) of the MIPs / Apt / AuNPs-LIG prepared in Example 2 after incubation in KANA solutions of different concentrations, and the storage stability (C) and selectivity (D) of the MIPs / Apt / AuNPs-LIG prepared in Example 2. Figure 7 As can be seen from Part A, with increasing KANA concentration, more molecularly imprinted cavities are occupied, and the DPV peak current gradually decreases. This is attributed to the fact that more and more KANA molecules enter the imprinted cavity, hindering the penetration of probe molecules and electron transfer, thus causing the peak current to gradually decrease. Figure 7 As shown in Part B, MIPs / Apt / AuNPs-LIG exhibits a good linear response to KANA concentrations in the range of 10 pmol / L to 1 mmol / L, with the corresponding linear regression equation being ΔI(μA) = 10.94 log[C(mol / L)] + 132.05(R). 2 =0.995), the detection limit (LOD) was 3.3 pmol / L (based on a 3x signal-to-noise ratio, 3S / N). From Figure 7 As can be seen from section C, the electrochemical sensor retained 93.3% of its initial current response after being stored under controlled conditions (vacuum drying oven at 25°C, RH < 10%) for 9 days, indicating that the MIPs / Apt / AuNPs-LIG prepared in Example 2 has good stability. Figure 7 As can be seen from part D, after the addition of interfering antibiotics, the changes in peak current caused by the antibiotics were 8.7% (SM), 2.7% (CFX), 12.6% (TC), and 5.8% (CAP), respectively, indicating that MIPs / Apt / AuNPs-LIG has good selectivity and does not interfere with the detection of KANA.

[0066] The MIPs / Apt / AuNPs-LIG prepared in Example 2 was applied to the determination of KANA in actual fish samples using a spiked recovery method. Specifically, fish samples without KANA were purchased from Guilin Yang Market, mixed with a handheld homogenizer, and extracted with a methanol-water mixture at a volume ratio of 80:20 at 1800 rpm for 5 min. The volume ratio of the mixture to the fish sample was 0.6 g / mL. The crude extract was centrifuged at 4000 rpm for 10 min, and the supernatant was retained. The supernatant was diluted 50 times with 0.2 mol / L PBS buffer (pH 6.5). Then, a known concentration (1 × 10⁻⁶) of PBS buffer was added to the resulting sample solution. -8 mol / L, 1×10 -9 mol / L, 1×10 -10 The KANA standard (mol / L) was incubated at room temperature for 25 min, and then measured using the MIPs / Apt / AuNPs-LIG prepared in Example 2. The average value of the peak current was read and substituted into... Figure 7 The linear equation in Part B is used to calculate the KANA content in the spiked sample solution. The results are shown in [Figure 1]. Figure 8 .

[0067] Figure 8 DPV curves of MIPs / Apt / AuNPs-LIG prepared in Example 2 in fish samples diluted 50 times with different concentrations of KANA. Figure 8 The results showed that the concentrations of KANA in fish samples diluted 50 times were 0.10 nM, 1.00 nM, and 10.00 nM, respectively. The recovery rate of the modified electrode was 89.1%–110.0% as determined by the spike recovery experiment, indicating that MIPs / Apt / AuNPs-LIG can be used to detect the KANA content in actual fish samples.

[0068] Figure 9 This is a schematic diagram illustrating the preparation process of the gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymers and aptamers provided by the present invention.

[0069] Comparative Example 3 A method for preparing a non-molecularly imprinted polymer-aptamer-gold nanoparticle-laser-induced graphene composite modified electrode (NIPs / Apt / AuNPs-LIG) differs from Example 2 only in that kanamycin solution is omitted in step (4), while the rest is the same as in Example 2.

[0070] The NIPs / Apt / AuNPs-LIG prepared in Comparative Example 3 was mixed with different concentrations of KANA (1×10⁻⁶). -7 mol / L, 1×10 - 6mol / L, 1×10 -5 mol / L, 1×10 -4 mol / L, 1×10 -3 The solution (mol / L) was incubated in PBS buffer at 37°C for 25 min, and its response was characterized by changes in DPV peak current. The results are shown in [Figure number missing]. Figure 10 .

[0071] Figure 10 The DPV curves (A) and the linear relationship between ΔI and KANA concentration (B) of NIPs / Apt / AuNPs-LIG prepared for Comparative Example 3 after incubation in KANA solutions of different concentrations are shown. Figure 10 It can be seen that the modified electrode prepared in Comparative Example 3 has a detection limit of 3.3 μmol / L for KANA and a linear range of 10 μmol / L to 1 mmol / L.

[0072] Comparative Example 4 A method for preparing a silver nanoparticle-laser-induced graphene composite modified electrode (AgNPs-LIG) differs from Example 1 only in that the chloroauric acid solution in step (1) is replaced with a silver nitrate solution of equal concentration, while the rest is the same as in Example 1.

[0073] Comparative Example 5 A method for preparing a platinum nanoparticle-laser-induced graphene composite modified electrode (PtNPs-LIG) differs from Example 1 only in that the chloroauric acid solution in step (1) is replaced with a chloroplatinic acid solution of equal concentration, while the rest is the same as in Example 1.

[0074] The CV method was used to analyze the 5.0 mmol / L [Fe(CN)6] solution containing 0.1 mol / L KCl. 3- / 4- The modified electrodes prepared in Example 1 and Comparative Examples 4-5 were tested in a redox probe solution at a scan rate of 0.1 V / s. The results are shown in [Figure number missing]. Figure 11 .

[0075] Figure 11 The CV curves are for the modified electrodes prepared in Example 1 and Comparative Examples 4-5. Figure 11 The results showed that the modified electrode prepared in Example 1 had better electrochemical performance than the modified electrodes prepared in Comparative Examples 4 and 5.

[0076] Comparative Example 6 A method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode with single-molecule imprint recognition is different from Example 2 in that step (3) is omitted, and the rest is the same as Example 2.

[0077] The CV method was used to analyze the 5.0 mmol / L [Fe(CN)6] solution containing 0.1 mol / L KCl. 3- / 4- The modified electrode prepared in Comparative Example 6 was tested in a redox probe solution at a scan rate of 0.1 V / s. The response speed of the electrode (time to reach a stable signal) and the response current of low-concentration kanamycin were compared. The results showed that the response time of Comparative Example 6 was twice that of Example 2, and the response current of low-concentration kanamycin was reduced by 50%, indicating the rapid advantage of aptamer targeted binding.

[0078] Comparative Example 7 A method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode with dual recognition of molecularly imprinted polymer and aptamer is different from Example 2 only in that the aniline in step (4) is replaced with an equal concentration of o-phenylenediamine, and the rest is the same as Example 2.

[0079] The modified electrode prepared in Comparative Example 7 was tested using the same test method as in Example 2. The results showed that the response saturation time of Comparative Example 7 was extended by 2 times compared with Example 2, indicating the advantages and stability of using aniline to prepare the polymer film.

[0080] Comparative Example 8 A method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode with dual recognition of molecularly imprinted polymer and aptamer is different from Example 2 only in that the aniline in step (4) is replaced with pyrrole of equal concentration, and the rest is the same as Example 2.

[0081] The modified electrode prepared in Comparative Example 8 was tested using the same test method as in Example 2. The selectivity and conductivity of kanamycin were compared. The results showed that the selectivity ratio of Comparative Example 8 was reduced to 1 / 3 of that of Example 2. Although the conductivity of the film was slightly higher, non-specific adsorption was serious, indicating that the interaction between aniline and kanamycin had a specific advantage.

[0082] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a gold nanoparticle-laser-induced graphene composite modified electrode with dual recognition of molecularly imprinted polymers and aptamers, characterized in that, Includes the following steps: (1) Preparation of gold nanoparticle-laser-induced graphene composite modified electrode; (2) The aptamer solution and the kanamycin solution were mixed and incubated, and then applied to the surface of the gold nanoparticle-laser induced graphene composite material modified electrode to obtain the kanamycin aptamer-gold nanoparticle-laser induced graphene electrode. (3) The kanamycin aptamer-gold nanoparticle-laser induced graphene electrode was placed in an aniline solution for electropolymerization and then eluted to obtain the gold nanoparticle-laser induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymer and aptamer.

2. The preparation method according to claim 1, characterized in that, In step (1), the preparation of the gold nanoparticle-laser-induced graphene composite modified electrode includes: mixing chloroauric acid solution and liquid polyimide and coating them onto the substrate surface, then curing them to obtain a flexible precursor film; performing laser-induced treatment on the flexible precursor film according to the pre-designed electrode geometry; coating the reference electrode region of the electrode with silver paste and performing thermal curing treatment to obtain the gold nanoparticle-laser-induced graphene composite modified electrode; The concentration of the chloroauric acid solution is 10~90 mmol / L; the ratio of the chloroauric acid solution to polyimide is 200 μL: 4 g; The curing temperature is 90~110℃, and the curing time is 20~40min; The laser wavelength for the laser-induced treatment is 450 nm, the engraving depth for the laser-induced treatment is 20-40%, and the laser power for the laser-induced treatment is 1.65-2.48 W. The temperature of the thermosetting treatment is 120~140℃, and the time of the thermosetting treatment is 20~40min.

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the aptamer solution is 10~20 μmol / L, the concentration of the kanamycin solution is 10~20 μmol / L, and the volume ratio of the aptamer solution to the kanamycin solution is 4:1, 3:2, 1:1, 2:3 or 1:

4.

4. The preparation method according to claim 1, characterized in that, In step (2), the incubation temperature is 37°C, the incubation liquid volume is 50~150μL, and the incubation time is 10~30min.

5. The preparation method according to claim 1, characterized in that, In step (3), the concentration of the aniline solution is 1~5 mmol / L; the aniline solution is a phosphate buffer solution containing aniline, and the pH value of the phosphate buffer solution is 6.0~8.

0.

6. The preparation method according to claim 1, characterized in that, In step (3), the electropolymerization method is cyclic voltammetry; the number of electropolymerization cycles is 5 to 30, the scan rate of electropolymerization is 0.05 to 0.25 V / s, and the voltage window of electropolymerization is -0.2 to 0.6 V.

7. The preparation method according to claim 1, characterized in that, In step (3), the eluent is a mixture of methanol and acetic acid, and the volume ratio of methanol to acetic acid is 9:1; the elution time is 10~30 min.

8. A gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymer and aptamer, prepared by the preparation method according to any one of claims 1 to 7.

9. An electrochemical sensor, characterized in that, The electrode is modified with a gold nanoparticle-laser-induced graphene composite material that features dual recognition of molecularly imprinted polymers and aptamers, as described in claim 8.

10. The application of a gold nanoparticle-laser-induced graphene composite material modified electrode with dual recognition of molecularly imprinted polymer and aptamer as described in claim 8, or the electrochemical sensor as described in claim 9, in the detection of kanamycin.

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