A nanochannel membrane for small molecule antibiotic detection and a preparation method thereof

Abstract

The application discloses a nanopore membrane for small molecule antibiotic detection and a preparation method thereof, and the nanopore membrane is a gold nanorod / oxidized aluminum composite nanopore membrane, and the surface of the gold nanorod is decorated with nucleic acid aptamer. The gold nanorod array is electrochemically deposited in the regular anodic aluminum oxide (AAO) template channel, and combined with wet chemical etching, the gold nanorod / oxidized aluminum composite nanopore membrane with a sub-10 nm annular gap is successfully constructed, and the inherent limitation that the small pore diameter and high regularity are difficult to be obtained due to the restriction of the thermodynamic barrier of the traditional AAO material is overcome. The method realizes the controllable adjustment of the annular pore diameter by accurately adjusting the key process parameters such as the template size, the electrochemical deposition time and the pore expansion etching time, and the prepared sub-10 nm regular channel array can realize the high sensitivity and high specificity detection of small molecule antibiotics.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a nanoporous membrane for the detection of small molecule antibiotics and its preparation method. Background Technology

[0002] The detection of small-molecule antibiotic residues is a crucial technological need in food safety monitoring and disease prevention. Aminoglycoside antibiotics, such as kanamycin, are widely used in livestock farming and clinical treatment; however, trace residues in food, the environment, or biological samples can induce health risks such as drug resistance, nephrotoxicity, and ototoxicity. Therefore, establishing rapid, sensitive, and specific methods for detecting small-molecule antibiotics is of great significance. In recent years, electrochemical sensing technology based on solid-state nanoporous membranes has gradually become a research hotspot in the field of biochemical detection due to its advantages such as simple detection process, fast response speed, low sample consumption, ease of integration, and high theoretical specificity. This type of technology typically utilizes the sensitive response of ion transport behavior within nanopores to target molecule recognition events to achieve quantitative analysis of target analytes.

[0003] However, existing solid-state nanoporous membranes still face significant limitations in the detection of small-molecule antibiotics. Small-molecule targets are typically smaller than 10 nm, while the pore size of traditional solid-state nanoporous materials is mostly above 70 nm. This large gap between the pore size and the target size makes it difficult to effectively modulate ion transport signals within the pores through target binding or conformational changes. Simultaneously, in practical detection systems, the Debye shielding effect further weakens the influence of small-molecule recognition events on the electrochemical signal, resulting in lower detection sensitivity and specificity than theoretically expected. Anodized alumina templates (AAO) have long been used as solid-state nanoporous detection platforms due to their mature preparation process, regular pore array, good physicochemical stability, and low cost. However, due to thermodynamic barriers and the limitations of the anodizing process, AAO cannot simultaneously achieve sub-10 nm pore sizes and highly regular pore structures, restricting its application in the highly sensitive detection of small-molecule antibiotics such as kanamycin. Therefore, developing nanoporous membranes with sub-10 nm regular pore structures, stable mass transfer performance, and excellent signal amplification capabilities is a key direction for improving the detection performance of small-molecule antibiotics. Summary of the Invention

[0004] The main objective of this invention is to propose a nanoporous membrane for the detection of small molecule antibiotics and its preparation method, aiming to solve the problems of mismatch between pore size and target size, low detection sensitivity and specificity of existing solid nanoporous membranes in the detection of small molecule antibiotics.

[0005] To achieve the above objectives, the present invention proposes a nanoporous membrane for the detection of small molecule antibiotics, wherein the nanoporous membrane is a gold nanorod / anodic aluminum oxide composite nanoporous membrane, and the surface of the gold nanorod is modified with nucleic acid aptamers.

[0006] Preferably, the nucleic acid aptamer includes a kanamycin aptamer.

[0007] This invention also proposes a method for preparing a nanoporous membrane for the detection of small molecule antibiotics as described above, comprising the following steps: S1. Sputter a layer of silver onto the back of the anodized aluminum template to obtain a silver-backed anodized aluminum template; S2. Using the silver-backed anodized aluminum template as the working electrode, gold nanorod arrays are grown inside the channels of the silver-backed anodized aluminum template by electrochemical deposition to obtain a gold nanorod / silver-backed anodized aluminum composite material. S3. Remove the silver layer of the gold nanorod / silver-backed anodic aluminum oxide composite material to obtain the gold nanorod / anodic aluminum oxide composite material. S4. Pore expansion was performed using an acid solution to obtain the expanded gold nanorod / anodic aluminum oxide composite material. S5. Modify the gold nanorods of the expanded gold nanorod / anodic aluminum oxide composite material with nucleic acid aptamers to obtain a gold nanorod / anodic aluminum oxide membrane, which is a nanoporous membrane for the detection of small molecule antibiotics.

[0008] Preferably, in step S1, the thickness of the silver layer is 395~405 nm; The specific operation of step S2 is as follows: S21. Prepare the precursor solution for growing gold nanorods; S22. Using the back silver anodic aluminum oxide template as the working electrode, the graphite sheet as the cathode, the Hg / HgCl electrode as the reference electrode, and the precursor solution for growing gold nanorods as the electrolyte, an array of gold nanorods is grown inside the pores of the anodic aluminum oxide by electrochemical deposition. The electrochemical deposition time is 18~22 min. S23. After electrochemical deposition, clean and dry.

[0009] Preferably, in step S21, the precursor solution comprises the following components: 10~14 g / L HAuCl4, 28~32 g / L K2HPO4, 0.4~0.6 g / L CoSO4, 4~6 g / L EDTA, and 150~170 g / L NaSO3.

[0010] Preferably, the specific operation of step S3 is as follows: The gold nanorod / silver-backed anodic aluminum oxide composite material was placed with the silver-plated side facing up in a glass petri dish, HNO3 solution was added dropwise, and after washing and drying, the gold nanorod / anodic aluminum oxide composite material was obtained.

[0011] Preferably, the specific operation of step S4 is as follows: Heat a 0.08~0.12 M H3PO4 solution to 38~42℃, then place the gold nanorod / anodic aluminum oxide composite material with the back side facing up in the H3PO4 solution for 4~6 minutes to make it float on the surface of the solution. Keep the temperature constant and remove air bubbles periodically during the process. After the pore expansion is completed, clean and dry.

[0012] Preferably, the specific operation of step S5 is as follows: S51. Reduce the nucleic acid aptamer with TCEP until it is colorless, and then dilute the mixture of reduced nucleic acid aptamer and TCEP. S52. The expanded gold nanorod / anodic aluminum oxide composite material is immersed in a diluted mixture of aptamer and TCEP and incubated at 3~5℃ for 1.5~2.5 h to obtain grafted aptamer gold nanorod / anodic aluminum oxide. S53. Prepare 18-22 mM MCH using phosphate buffer solution, immerse the grafted aptamer gold nanorods / anodic aluminum oxide in the MCH solution, and incubate at 3-5℃ for 7.5-8.5 h to obtain a nanoporous membrane for the detection of small molecule antibiotics.

[0013] The present invention also proposes an application of the nanoporous membrane described above in the detection of small molecule antibiotics.

[0014] Preferably, the application includes the following steps: immersing the nanoporous membrane in a test solution containing a small molecule antibiotic, and using a picoammeter to test the transmembrane current of the nanoporous membrane.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for preparing a nanoporous membrane for the detection of small molecule antibiotics provided by the present invention uses anodic aluminum oxide with a regular structure as a template. Gold nanorod arrays are prepared in the original pores of anodic aluminum oxide by electrochemical deposition. Sub-10 nm annular gaps are etched between the gold nanorods and the inner wall of anodic aluminum oxide by wet chemical etching to construct a gold nanorod / anodic aluminum oxide composite nanopore. This method overcomes the inherent limitation of traditional AAO materials, which are constrained by thermodynamic barriers, making it difficult to achieve both small pore size and high regularity. By controlling key process parameters such as electrochemical deposition time and pore enlargement etching time, the method achieves precise control of pore size. The prepared sub-10 nm regular pore array is highly matched with the size of small molecule antibiotic targets, which can effectively perform physical sieving of molecules entering the pores. This significantly reduces the interference of size mismatch effect and Debye shielding effect, and greatly improves the detection sensitivity of nanoporous sensors for small molecule targets such as kanamycin.

[0016] (2) The nanoporous membrane for small molecule antibiotic detection provided by this invention is a gold nanorod / anodic aluminum oxide membrane. The surface of the gold nanorods can be stably modified with gold-sulfur bonds to stabilize nucleic acid aptamers, so that aptamers with target-specific recognition capabilities are locally distributed inside the sub-10 nm annular pore array. When small molecule antibiotics such as kanamycin enter the pores, they are subjected to physical sieving by the sub-10 nm annular pores on the one hand, and can be specifically captured by the nucleic acid aptamers inside the pores on the other hand. After the target and aptamer bind, a steric hindrance effect is generated in the confined nanospace, causing changes in ion transport within the pores and forming a detectable electrochemical signal. Therefore, this invention combines pore size sieving with aptamer-specific recognition, which significantly improves the sensitivity, specificity and anti-interference ability of small molecule antibiotic detection, and is suitable for food safety monitoring and related biochemical detection fields. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the preparation process of gold nanorods / anodic aluminum oxide film in an embodiment of the present invention; Figure 2 This is a schematic diagram of the nucleic acid aptamer modification process in an embodiment of the present invention; Figure 3 This is a SEM image of the gold nanorod / anodic aluminum oxide film obtained in the embodiments of the present invention; Figure 4 AFM images of the surface of a blank anodic aluminum oxide film (a) and a gold nanorod / anodic aluminum oxide film (b) prepared according to the embodiments of the present invention; Figure 5 XPS results for the blank anodic aluminum oxide film (ac) and the gold nanorod / anodic aluminum oxide film (df) prepared in the embodiments of the present invention; Figure 6 The graphs shown are: a) Electrochemical signal change curves before and after target binding in the embodiments of the present invention; b) Current-voltage curves for different concentrations of kanamycin; c) Standard curves of kanamycin concentration versus current change rate.

[0019] Figure 7 This is a specific detection diagram of the nanoporous membrane in an embodiment of the present invention.

[0020] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by the present invention. 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.

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0023] Example 1

[0024] A nanoporous membrane for the detection of small molecule antibiotics, wherein the nanoporous membrane is a gold nanorod / anodic aluminum oxide composite nanoporous membrane, and the surface of the gold nanorod is modified with nucleic acid aptamers; The method for preparing the nanoporous membrane for small molecule antibiotic detection includes the following steps: (1) Place the AAO template in a vacuum coating apparatus at a vacuum level of 1×10⁻⁶. -4 A 400 nm thick Ag layer was magnetron sputtered on the back of the AAO template at a speed of 0.1 nm / s in an mbar environment to obtain a silver-backed AAO template. (2) Prepare 10 mL of precursor solution for growing gold nanorods. The precursor solution for growing gold nanorods includes: HAuCl4 at a concentration of 12 g / L, K2HPO4 at a concentration of 30 g / L, CoSO4 at a concentration of 0.5 g / L, EDTA at a concentration of 5 g / L, and NaSO3 at a concentration of 160 g / L. (4) Using the Ag-sprayed surface of the back-silver AAO template as the working electrode, the graphite sheet as the cathode, the Hg / HgCl electrode as the reference electrode, and the precursor solution for growing gold nanorods as the electrolyte, an array of gold nanorods is grown inside the pores of the back-silver AAO template by electrochemical deposition. The electrochemical deposition of gold nanorods was performed using a chronoamperometry method on an electrochemical workstation at a rate of 5 μA / cm. 2 Electrochemical deposition was performed at a constant current density for 20 minutes. (5) After electrochemical deposition, a gold nanorod / silver-backed anodic aluminum oxide composite material with a silver layer on the surface is obtained. After washing the composite material with deionized water, it is sent to an oven and dried at 60°C. (6) Place the gold nanorod / silver-backed anodic aluminum oxide composite material with the silver-plated side facing up in a glass petri dish, add 1 M HNO3 to remove the Ag layer, wash it again with deionized water, and then dry it in a 60°C oven to obtain the gold nanorod / anodic aluminum oxide composite material. (7) Place 10 mL of 0.1 M H3PO4 on a heating table and heat it to 40°C. Then gently place the gold nanorod / anodic aluminum oxide composite material with the back side facing up in the solution for 5 minutes to make it float on the surface of the solution. Keep the temperature constant and use tweezers to periodically remove air bubbles. After the pore expansion is completed, wash with deionized water and dry at 60°C to obtain the expanded gold nanorod / anodic aluminum oxide composite material. (8) Take 2 μL each of 100 μM kanamycin aptamer and 20 mM TCEP (Tris (2-carboxyethyl) phosphine hydrochloride) after centrifugation, mix them evenly in a volume ratio of 1:1, reduce them at room temperature for 2 h until colorless, use TCEP to reduce the nucleic acid aptamer, and cleave the disulfide bond at the 5-terminus of the DNA oligonucleotide through TCEP. (9) Dilute the mixture of aptamer and TCEP 100 times with phosphate buffer solution and mix well; (10) Cut the expanded gold nanorod / anodic aluminum oxide composite material into squares with a side length of about 2 mm with a blade, immerse them in a mixture of diluted aptamer and TCEP, and incubate the aptamer for 2 h in a refrigerator at 4°C, so that the Au nanorods and the disulfide bonds after reduction and cleavage at the 5-end form gold-sulfur bonds, thereby leaving the aptamer on the Au nanorods. (11) Take 2.8 μL of MCH (6-mercapto-1-hexanol), dissolve it in 1 mM phosphate buffer solution and mix well to prepare MCH with a concentration of 20 mM. Soak the Au nanorods / anodic aluminum oxide grafted with aptamers in MCH with a concentration of 20 mM and incubate at 4℃ for 8 h to obtain gold nanorods / anodic aluminum oxide membrane, which is a nanoporous membrane for the detection of small molecule antibiotics.

[0025] Figure 1 This is a schematic diagram of the preparation process of gold nanorods / anodic aluminum oxide film in this embodiment. Figure 2 This is a flowchart illustrating the nucleic acid aptamer modification process in steps (9) to (11) of this embodiment.

[0026] Example 2

[0027] A nanoporous membrane for the detection of small molecule antibiotics, wherein the nanoporous membrane is a gold nanorod / anodic aluminum oxide composite nanoporous membrane, and the surface of the gold nanorod is modified with nucleic acid aptamers; The method for preparing the nanoporous membrane for small molecule antibiotic detection includes the following steps: (1) Place the AAO template in a vacuum coating apparatus at a vacuum level of 1×10⁻⁶. -4 A 395 nm thick Ag layer was magnetron sputtered on the back of the AAO template at a speed of 0.1 nm / s in an mbar environment to obtain a back-silvered AAO template. (2) Prepare 10 mL of precursor solution for growing gold nanorods. The precursor solution for growing gold nanorods includes: HAuCl4 at a concentration of 10 g / L, K2HPO4 at a concentration of 28 g / L, CoSO4 at a concentration of 0.4 g / L, EDTA at a concentration of 4 g / L, and NaSO3 at a concentration of 150 g / L. (4) Using the Ag-sprayed surface of the back-silver AAO template as the working electrode, the graphite sheet as the cathode, the Hg / HgCl electrode as the reference electrode, and the precursor solution for growing gold nanorods as the electrolyte, an array of gold nanorods is grown inside the pores of the back-silver AAO template by electrochemical deposition. The electrochemical deposition of gold nanorods was performed using a chronoamperometry method on an electrochemical workstation at a rate of 5 μA / cm. 2 Electrochemical deposition was performed at a constant current density for 18 minutes. (5) After electrochemical deposition, a gold nanorod / silver-backed anodic aluminum oxide composite material with a silver layer on the surface is obtained. After washing the composite material with deionized water, it is sent to an oven and dried at 60°C. (6) Place the gold nanorod / silver-backed anodic aluminum oxide composite material with the silver-plated side facing up in a glass petri dish, add 1 M HNO3 to remove the Ag layer, wash it again with deionized water, and then dry it in a 60°C oven to obtain the gold nanorod / anodic aluminum oxide composite material. (7) Place 10 mL of 0.08 M H3PO4 on a heating table and heat it to 38°C. Then gently place the gold nanorod / anodic aluminum oxide composite material with the back side facing up in the solution for 4 minutes to make it float on the surface of the solution. Keep the temperature constant and use tweezers to periodically remove air bubbles. After the pore expansion is completed, wash with deionized water and dry at 60°C to obtain the expanded gold nanorod / anodic aluminum oxide composite material. (8) Take 2 μL each of 100 μM kanamycin aptamer and 20 mM TCEP (Tris (2-carboxyethyl) phosphine hydrochloride) after centrifugation, mix them evenly in a volume ratio of 1:1, reduce them at room temperature for 2 h until colorless, use TCEP to reduce the nucleic acid aptamer, and cleave the disulfide bond at the 5-terminus of the DNA oligonucleotide through TCEP. (9) Dilute the mixture of aptamer and TCEP 100 times with phosphate buffer solution and mix well; (10) Cut the expanded gold nanorod / anodic aluminum oxide composite material into squares with a side length of about 2 mm with a blade, immerse them in a mixture of diluted aptamer and TCEP, and incubate the aptamer for 2.5 h in a refrigerator at 3°C, so that the Au nanorods and the disulfide bonds after reduction and cleavage at the 5-end form gold-sulfur bonds, thereby leaving the aptamer on the Au nanorods. (11) Take 2.8 μL of MCH (6-mercapto-1-hexanol), dissolve it in 1 mM phosphate buffer solution and mix well to prepare MCH with a concentration of 18 mM. Soak the Au nanorods / anodic aluminum oxide grafted with aptamers in MCH with a concentration of 18 mM and incubate at 3℃ for 8.5 h to obtain gold nanorods / anodic aluminum oxide membrane, which is a nanoporous membrane for the detection of small molecule antibiotics.

[0028] Example 3

[0029] A nanoporous membrane for the detection of small molecule antibiotics, wherein the nanoporous membrane is a gold nanorod / anodic aluminum oxide composite nanoporous membrane, and the surface of the gold nanorod is modified with nucleic acid aptamers; The method for preparing the nanoporous membrane for small molecule antibiotic detection includes the following steps: (1) Place the AAO template in a vacuum coating apparatus at a vacuum level of 1×10⁻⁶. -4 A 405 nm thick Ag layer was magnetron sputtered on the back of the AAO template at a speed of 0.1 nm / s in an mbar environment to obtain a back-silvered AAO template. (2) Prepare 10 mL of precursor solution for growing gold nanorods. The precursor solution for growing gold nanorods includes: HAuCl4 at a concentration of 14 g / L, K2HPO4 at a concentration of 32 g / L, CoSO4 at a concentration of 0.6 g / L, EDTA at a concentration of 6 g / L, and NaSO3 at a concentration of 170 g / L. (4) Using the Ag-sprayed surface of the back-silver AAO template as the working electrode, the graphite sheet as the cathode, the Hg / HgCl electrode as the reference electrode, and the precursor solution for growing gold nanorods as the electrolyte, an array of gold nanorods is grown inside the pores of the back-silver AAO template by electrochemical deposition. The electrochemical deposition of gold nanorods was performed using a chronoamperometry method on an electrochemical workstation at a rate of 5 μA / cm. 2 Electrochemical deposition was performed at a constant current density for 22 minutes. (5) After electrochemical deposition, a gold nanorod / silver-backed anodic aluminum oxide composite material with a silver layer on the surface is obtained. After washing the composite material with deionized water, it is sent to an oven and dried at 60°C. (6) Place the gold nanorod / silver-backed anodic aluminum oxide composite material with the silver-plated side facing up in a glass petri dish, add 1 M HNO3 to remove the Ag layer, wash it again with deionized water, and then dry it in a 60°C oven to obtain the gold nanorod / anodic aluminum oxide composite material. (7) Place 10 mL of 0.12 M H3PO4 on a heating table and heat it to 42°C. Then gently place the gold nanorod / anodic aluminum oxide composite material with the back side facing up in the solution for 6 minutes to make it float on the surface of the solution. Keep the temperature constant and use tweezers to periodically remove air bubbles. After the pore expansion is completed, wash with deionized water and dry at 60°C to obtain the expanded gold nanorod / anodic aluminum oxide composite material. (8) Take 2 μL each of 100 μM kanamycin aptamer and 20 mM TCEP (Tris (2-carboxyethyl) phosphine hydrochloride) after centrifugation, mix them evenly in a volume ratio of 1:1, reduce them at room temperature for 2 h until colorless, use TCEP to reduce the nucleic acid aptamer, and cleave the disulfide bond at the 5-terminus of the DNA oligonucleotide through TCEP. (9) Dilute the mixture of aptamer and TCEP 100 times with phosphate buffer solution and mix well; (10) Cut the expanded gold nanorod / anodic aluminum oxide composite material into squares with a side length of about 2 mm with a blade, immerse them in a mixture of diluted aptamer and TCEP, and incubate the aptamer for 1.5 h in a refrigerator at 5°C, so that the Au nanorods and the disulfide bonds after reduction and cleavage at the 5-end form gold-sulfur bonds, thereby leaving the aptamer on the Au nanorods. (11) Take 2.8 μL of MCH (6-mercapto-1-hexanol), dissolve it in 1 mM phosphate buffer solution and mix well to prepare MCH with a concentration of 22 mM. Soak the Au nanorods / anodic aluminum oxide grafted with aptamers in MCH with a concentration of 22 mM and incubate at 5℃ for 8.5 h to obtain gold nanorods / anodic aluminum oxide membrane, which is a nanoporous membrane for the detection of small molecule antibiotics.

[0030] Application test cases 1. The nanoporous membrane for small molecule antibiotic detection prepared in Example 1 was observed using a scanning electron microscope, and the results were obtained. Figure 3 The image shows that the gold nanorods grow uniformly within the pores, with flat and dense tips, exhibiting a highly ordered array arrangement. Tiny nanopores appear between the gold nanorods and the pore walls of the anodic alumina, while the alumina framework maintains its complete honeycomb structure. The pore wall thickness is moderate, providing sufficient mechanical support. Furthermore, the gold nanorods stand firmly upright in the center of the pores without collapsing or detaching. These tiny annular gaps represent the strong confinement space sought in this study, ensuring that analyte molecules can enter while providing physical size sieving capabilities through sub-10 nm dimensions.

[0031] 2. The surfaces of the blank anodic alumina template and the nanoporous membrane for small molecule antibiotic detection prepared in Example 1 of this invention were observed using an atomic force microscope to obtain... Figure 4 As shown in the image, the anodic aluminum oxide template exhibits a typical hexagonal close-packed structure, with the pores appearing as dark, recessed areas, while the alumina framework presents as a bright, raised mesh. The pore size distribution is uniform, and the surface is relatively smooth. This highly ordered pore structure provides an excellent geometric template for the subsequent confined growth of gold nanorods. After electrodeposition and subsequent processing, compared to the blank template, the deep black depressions in the anodic aluminum oxide template disappear, replaced by obvious solid fillers in the pore centers. This indicates that the gold nanorods have successfully filled the pore areas of the anodic aluminum oxide without significant overgrowth, and the surface maintains good smoothness. At the interface between the gold nanorods and the anodic aluminum oxide framework, there is a distinct annular low-lying transition zone, corresponding to the annular gap position formed after sacrificial layer removal or chemical pore enlargement mentioned earlier. AFM characterization results confirm that the gold nanorods achieved high-quality, dense filling within the anodic aluminum oxide pores. The gold nanorods exhibit a small relative height difference between their tips and the pore surface, while maintaining a high degree of array order, which is highly consistent with the cross-sectional observations obtained by SEM.

[0032] 3. To further investigate the elemental composition and chemical valence state of the nanoporous membrane surface prepared in Example 1 for the detection of small molecule antibiotics, and to confirm the successful growth of gold nanorods within the pores of the anodic alumina template, this test example used XPS to compare and characterize the blank anodic alumina template and the gold nanorod / AAO composite membrane. Figure 5As shown, distinct Al 2p, C 1s, and O 1s characteristic peaks were detected on the surfaces of both the blank anodic alumina template and the composite film. The Al and O elements mainly originate from the inorganic framework of the alumina template itself, while the C element is primarily attributed to unavoidable adsorption of carbon in the test environment. After modification and assembly with gold nanorods, a significant new characteristic peak appeared in the full spectrum of the composite film at a binding energy of approximately 84 eV. This peak belongs to the Au 4f orbital, preliminarily demonstrating that gold has been successfully introduced into the anodic alumina nanoporous system.

[0033] 4. The nanoporous membrane prepared in Example 1 for detecting small molecule antibiotics was used to detect kanamycin in the liquid. The nanoporous membrane was immersed in phosphate buffer solutions containing different concentrations (0, 0.1, 1, 10, 100, 1000 nM) of kanamycin for 4 h.

[0034] The transmembrane current of the nanoporous membrane was measured using a picoammeter with an Ag / AgCl electrode; the electrolytic cell was an acrylic mold. Figure 6 . Figure 6 To detect the electrochemical response of kanamycin solutions at different concentrations, the experimental current-voltage (IV) characteristic curves showed that the transmembrane ion current of the pores exhibited a significant concentration-dependent decreasing trend after the introduction of the target molecule. This inhibition effect on the transmembrane current is mainly attributed to the specific trapping of kanamycin in the annular gaps within the complex pores. The formation of the macromolecular complex not only significantly increases the physical steric hindrance within the pores, but the charge characteristics of kanamycin itself also alter the local charge distribution on the pore walls, thereby synergistically hindering the free migration of electrolyte ions through electrostatic interactions and volume exclusion effects. In the embodiments of this invention, the linear response range for the detection of kanamycin was 2.96 × 10⁻⁶. -12 ~1.0×10 -6 M corresponds to the linear regression equation y = 197.49 + 16.90x, R0 2 =0.9959. The detection limit is 2.3 pM.

[0035] 5. To verify the detection specificity of the embodiments for kanamycin (K), five common biomolecules and potential interfering substances were selected as control test groups, namely BSA, PSA, ATP, CRP and miRNA. Figure 7The bar chart shown illustrates the differences in the response of the nanoporous membrane prepared in Example 1 to different target analytes. When the sensor was used to detect the 2 μM target molecule kanamycin, the transmembrane ion current exhibited extremely significant resistance, with the output signal change rate reaching approximately 85%. However, when the examples were exposed to solutions with the same high concentration of interfering substances, their current change rates were suppressed to extremely low levels. The highest response in the ATP group was only about 10%, while the responses in the BSA, CRP, and miRNA groups were all around 5%, and the response in the PSA group was even lower, down to about 1%. These results demonstrate that the examples can effectively resist interference from high concentrations of impurity molecules in common environmental and biological samples, and are fully capable of performing accurate quantitative detection in complex real-world matrices.

[0036] This test case demonstrates that the method of the present invention can construct a regular, high-density sub-10 nm pore array by utilizing pore reconstruction, and promote the localization of nucleic acid aptamers within the sub-10 nm pore array. Through the dual effects of physical sieving and nucleic acid aptamer-specific capture, the specificity of small molecule antibiotic detection is improved, providing a new method for high-sensitivity and high-specificity detection of small molecule antibiotics.

[0037] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A nanoporous membrane for the detection of small molecule antibiotics, characterized in that, The nanoporous membrane is a gold nanorod / anodic aluminum oxide composite nanoporous membrane, and the surface of the gold nanorod is modified with nucleic acid aptamers.

2. The nanoporous membrane for detecting small molecule antibiotics according to claim 1, characterized in that, The nucleic acid aptamers include kanamycin aptamers.

3. A method for preparing a nanoporous membrane for the detection of small molecule antibiotics as described in any one of claims 1-2, characterized in that, Includes the following steps: S1. Sputter a layer of silver onto the back of the anodized aluminum template to obtain a silver-backed anodized aluminum template; S2. Using the silver-backed anodized aluminum template as the working electrode, gold nanorod arrays are grown inside the channels of the silver-backed anodized aluminum template by electrochemical deposition to obtain a gold nanorod / silver-backed anodized aluminum composite material. S3. Remove the silver layer of the gold nanorod / silver-backed anodic aluminum oxide composite material to obtain the gold nanorod / anodic aluminum oxide composite material. S4. Pore expansion was performed using an acid solution to obtain the expanded gold nanorod / anodic aluminum oxide composite material. S5. Modify the gold nanorods of the expanded gold nanorod / anodic aluminum oxide composite material with nucleic acid aptamers to obtain a gold nanorod / anodic aluminum oxide membrane, which is a nanoporous membrane for the detection of small molecule antibiotics.

4. The preparation method according to claim 3, characterized in that, In step S1, the thickness of the silver layer is 395~405 nm; The specific operation of step S2 is as follows: S21. Prepare the precursor solution for growing gold nanorods; S22. Using the back silver anodic aluminum oxide template as the working electrode, the graphite sheet as the cathode, the Hg / HgCl electrode as the reference electrode, and the precursor solution for growing gold nanorods as the electrolyte, an array of gold nanorods is grown inside the pores of the anodic aluminum oxide by electrochemical deposition. The electrochemical deposition time is 18~22 min. S23. After electrochemical deposition, clean and dry.

5. The preparation method according to claim 4, characterized in that, In step S21, the precursor solution comprises the following components: 10~14 g / L HAuCl4, 28~32 g / L K2HPO4, 0.4~0.6 g / L CoSO4, 4~6 g / L EDTA, and 150~170 g / L NaSO3.

6. The preparation method according to claim 3, characterized in that, The specific operation of step S3 is as follows: The gold nanorod / silver-backed anodic aluminum oxide composite material was placed with the silver-plated side facing up in a glass petri dish, HNO3 solution was added dropwise, and after washing and drying, the gold nanorod / anodic aluminum oxide composite material was obtained.

7. The preparation method according to claim 3, characterized in that, The specific operation of step S4 is as follows: Heat a 0.08~0.12 M H3PO4 solution to 38~42℃, then place the gold nanorod / anodic aluminum oxide composite material with the back side facing up in the H3PO4 solution for 4~6 minutes to make it float on the surface of the solution. Keep the temperature constant and remove air bubbles periodically during the process. After the pore expansion is completed, clean and dry.

8. The preparation method according to claim 3, characterized in that, The specific operation of step S5 is as follows: S51. Reduce the nucleic acid aptamer with TCEP until it is colorless, and then dilute the mixture of reduced nucleic acid aptamer and TCEP. S52. The expanded gold nanorod / anodic aluminum oxide composite material is immersed in a diluted mixture of aptamer and TCEP and incubated at 3~5℃ for 1.5~2.5 h to obtain grafted aptamer gold nanorod / anodic aluminum oxide. S53. Prepare 18-22 mM MCH using phosphate buffer solution, immerse the grafted aptamer gold nanorods / anodic aluminum oxide in the MCH solution, and incubate at 3-5℃ for 7.5-8.5 h to obtain a nanoporous membrane for the detection of small molecule antibiotics.

9. An application of the nanoporous membrane as described in any one of claims 1-2, characterized in that, It is used in the detection of small molecule antibiotics.

10. The application according to claim 9, characterized in that, The application includes the following steps: immersing the nanoporous membrane in a test solution containing small molecule antibiotics, and using a picoammeter to test the transmembrane current of the nanoporous membrane.

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