Detection method for selectively capturing antibiotics in dairy products based on surface-enhanced Raman nanoimprint chip

Abstract

The invention discloses a surface-enhanced Raman-based detection method for selectively capturing antibiotics in dairy products through a nanoimprint chip. By constructing a periodic nano array and combining precious metal deposition and functional molecule modification, sensitive recognition of various antibiotics is realized. The nanoimprint chip has the characteristics of high repeatability, low detection limit and quick response, can realize quick screening and quantitative analysis of various antibiotics, has the advantages of short detection time, low detection limit, good repeatability and the like, and can be widely applied to the field of detection of antibiotics, and the detection process does not depend on complex instruments. The device is suitable for being used as a core detection part in dairy product processing enterprises, quality inspection mechanisms and on-site rapid detection equipment.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-manufacturing and food safety detection, in particular to a detection method for selectively capturing antibiotics in dairy products using a nano-imprint chip based on surface enhanced Raman. Background Art

[0002] Due to their excellent antibacterial properties, antibiotics are widely used in the livestock and poultry industry to prevent and treat infectious diseases. However, their metabolism in animals is not complete, and some antibiotics and their metabolites may remain in animal-derived foods, especially dairy products such as milk.

[0003] Currently, common methods for detecting antibiotic residues include high-performance liquid chromatography, liquid chromatography-mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Although these methods offer good sensitivity and accuracy, they suffer from limitations such as complex operational procedures, long testing cycles, expensive equipment, and high operator dependence, limiting their widespread application in on-site rapid testing.

[0004] Surface-enhanced Raman scattering (SERS) technology has been widely used in food safety, environmental monitoring, and biological testing due to its advantages, including labeling-free, rapid detection, and ability to provide molecular fingerprint information. SERS combined with precious metal nanostructures can significantly enhance the Raman signal, enabling extremely high sensitivity for detecting trace molecules. However, conventional SERS substrates suffer from structural heterogeneity, complex preparation, unstable signals, and poor reproducibility, hindering their large-scale application in food safety.

[0005] Nanoimprint lithography, a nanofabrication technique with strong controllability, high replication accuracy, and suitability for large-area fabrication, has become a key development direction in the preparation of surface-enhanced Raman substrates. This technique, by constructing periodic nanoarray structures and depositing precious metals on their surfaces, effectively creates high-density electromagnetic hotspots, significantly enhancing the intensity and uniformity of Raman signals.

[0006] At the same time, in order to enhance the target recognition ability of the surface-enhanced Raman platform, a surface functional modification layer needs to be introduced to improve the platform's anti-interference and selective detection capabilities in complex matrices.

[0007] In summary, developing a nanoimprint array surface-enhanced Raman platform with controllable structure, stable performance, multiple recognition functions and suitable for batch preparation is the key to achieving rapid and sensitive detection of antibiotics in dairy products. Summary of the Invention

[0008] The present invention aims to provide a surface-enhanced Raman nanoimprint chip with uniform structure, stable performance and easy batch preparation, and to solve the problems of unstable signal enhancement effect and limited recognition ability when using surface-enhanced Raman technology to detect antibiotic drug molecules in dairy products.

[0009] The present invention provides a method for selectively capturing antibiotics in dairy products using a surface-enhanced Raman nanoimprint chip, which is carried out in the following steps:

[0010] Step (1): The surface-enhanced Raman nanoimprint chip uses a flexible substrate material with good thermal stability and mechanical properties, and constructs a periodic nanostructure of nanopillars and nanopyramids through a thermal nanoimprint process. Then, a thin layer of precious metals such as gold and silver is deposited on its surface to form a "hotspot" active area of ​​surface-enhanced Raman. Step (2): Molecular probes or molecular imprinting polymers are introduced on the surface of the surface-enhanced Raman nanoimprint chip for surface modification, thereby achieving selective capture of multiple antibiotic molecules. Step (3): Add trace amounts of antibiotics 10 to the milk to be tested. -5 -10 -12 mol / L, vortexed, filtered with a 0.45 μm water filter membrane to remove insoluble impurities, and obtained a dairy sample to be tested. Step (4): drop the dairy sample to be tested onto the surface of the surface-enhanced Raman nanoimprint chip, let it stand for 1-5 minutes, and then naturally dry the substrate to obtain the chip to be tested. Step (5): Use a Raman spectrometer to quickly detect the surface-enhanced Raman nanoimprint chip, find characteristic Raman peaks, and achieve qualitative and quantitative detection of multiple antibiotics.

[0011] Preferably, the substrate material in step (1) is made of polycarbonate, polydimethylsiloxane or other flexible materials with good thermal stability and mechanical properties as the supporting basis of the array structure.

[0012] Preferably, the periodic nanoarray structure in step (1) is formed by transferring the pre-designed nanopattern to the substrate surface using hot pressing nanoimprinting technology to form a uniformly arranged pyramidal and columnar structure, with the array spacing controlled at 50-300 nm to construct a local electromagnetic hotspot.

[0013] Preferably, the precious metal deposition layer in step (1) is deposited on the surface of the nanostructure by a physical vapor deposition method, such as magnetron sputtering or electron beam evaporation, with a thickness of 5-25 nm to form an active layer with surface enhanced Raman enhancement capability.

[0014] Preferably, in step (2), the surface functionalized recognition layer is formed by introducing molecular probes or molecular imprinting polymers onto the metal surface to enhance the target molecule recognition selectivity.

[0015] Preferably, the dairy products in step (3) include but are not limited to fresh cow milk, cow milk powder, fresh goat milk, and goat milk powder.

[0016] The invention discloses a detection method for selectively capturing antibiotics in dairy products using a nanoimprint chip based on surface enhanced Raman spectroscopy, and is applied to the rapid screening and quantitative detection of trace antibiotics such as tetracycline, chloramphenicol or ciprofloxacin.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The technical approach of the present invention integrates a highly controllable nanostructure construction method, a high-efficiency precious metal deposition technology, and a molecular-level recognition mechanism, which not only achieves a high enhancement effect of the surface-enhanced Raman signal, but also ensures the batch consistency, repeatability, and specificity of the detection platform.

[0019] (2) The platform can realize the rapid screening and quantitative analysis of multiple types of antibiotics (such as tetracycline, chloramphenicol, and ciprofloxacin). The detection process does not rely on complex instruments and has the advantages of short detection time, low detection limit, and good repeatability. It is suitable for use as a core detection component in dairy processing companies, quality inspection agencies, and on-site rapid testing equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Schematic diagram of the nanoimprint chip structure based on surface-enhanced Raman

[0021] Figure 2 :Scanning electron microscope side view of nanoimprinted chip based on surface-enhanced Raman

[0022] Figure 3 :Tetracycline in milk (10 -6 mol / L) Raman spectrum

[0023] Figure 4 :Standard curve (log[C] vs. Raman intensity)

[0024] Figure 5 :Detection repeatability of different batches of nanoimprint chips DETAILED DESCRIPTION

[0025] Example 1: Fabrication of a Nanoimprint Chip for Surface-Enhanced Raman Spectroscopy. A 20 mm × 20 mm × 1 mm polycarbonate sheet was selected as the substrate. After cleaning and drying, it was placed on a non-stick silicone mold with a periodic nanostructure. The heat press was set at 160°C and a pressure of 0.6 MPa. After 5 minutes of imprinting, the chip was cooled and released from the mold, resulting in a uniform array of columnar nanostructures with a spacing between 80 and 300 nm.

[0026] Example 2: Metal deposition and construction of surface-enhanced Raman active layer. The array substrate obtained in Example 1 was placed in a vacuum coating system and metallic silver was deposited using magnetron sputtering. Specific conditions were: working pressure 0.5 Pa, power 100 W, time 8 min, and film thickness controlled at 80 nm. The metallic silver layer formed a uniform covering structure on the array surface. Scanning electron microscope images showed that the array structure was uniform, without collapse, and had good replication accuracy ( Figure 2 ).

[0027] Example 3: Surface Recognition Modification. A tetracycline nucleic acid recognition probe, 4-aminobutane-1-thiol (10 μM concentration), was dissolved in methanol and evenly added to the metal surface in 20 μL drops. The solution was incubated at room temperature for 10 minutes to stabilize the metal-sulfur bond. After modification, the surface was rinsed three times with deionized water, dried, and set aside.

[0028] Example 4: Detection of tetracycline in milk. After diluting the commercial whole milk sample at a volume ratio of 3:1, add an equal volume of acetonitrile and shake to precipitate the protein. Centrifuge at 10000 rpm for 10 minutes and take the supernatant as the test sample. Add it dropwise to the surface of the modified surface enhanced Raman nanoimprint chip, incubate it for 5 minutes, dry it naturally and use a Raman spectrometer (laser wavelength 785nm) to detect it. The test shows that it has significant surface enhanced Raman capability at 1560cm -1 Tetracycline characteristic peaks can be observed at concentrations up to 10 -6 mol / L when the signal-to-noise ratio is greater than 5( Figure 3 ).

[0029] Example 5: Standard curve establishment. Prepare different concentrations of tetracycline standard solutions (10 -9 to 10 -6 mol / L), and the Raman signals were detected and recorded at 1560 cm -1 The results show that the Raman intensity is well linear with the logarithmic concentration, and the fitting equation is I=8366*log[C]+79998, R 2 >0.9975( Figure 4 ).

[0030] Example 6: Repeatability analysis. Prepare different concentrations of tetracycline standard solutions (10 -9 to 10 -6 mol / L), and Raman spectroscopy was performed on three independent batches of surface-enhanced Raman nanoimprinting chips, and the 1560 cm -1 Peak intensity, assess batch consistency ( Figure 5 ).

Claims

1. A method for selectively capturing antibiotics in dairy products using a surface-enhanced Raman nanoimprint chip, characterized in that: The surface-enhanced Raman nanoimprint chip is prepared by the following steps: Step (1): The surface-enhanced Raman nanoimprint chip uses a flexible substrate material with good thermal stability and mechanical properties, constructs a periodic nanostructure of nanopillars and nanopyramids through a thermal nanoimprint process, and then deposits a thin layer of precious metals such as gold and silver on its surface to form a "hotspot" active area of ​​surface-enhanced Raman; Step (2): Molecular probes or molecular imprinting polymers are introduced into the surface of the surface-enhanced Raman nanoimprint chip for surface modification, thereby achieving selective capture of multiple antibiotic molecules; Step (3): Trace amounts of antibiotics 10-10% are added to the milk to be tested. 6 -10-12mol / L, after vortex oscillation, filter with a 0.45μm water filter membrane to remove insoluble impurities to obtain a dairy sample to be tested; step (4): drop the dairy sample to be tested onto the surface of a surface-enhanced Raman nanoimprint chip, let it stand for 1-5 minutes, and then naturally dry the substrate to obtain a chip to be tested; step (5): use a Raman spectrometer to quickly detect the surface-enhanced Raman nanoimprint chip, find characteristic Raman peaks, and realize qualitative and quantitative detection of multiple antibiotics.

2. The method for selectively capturing antibiotics in dairy products using a surface-enhanced Raman nanoimprint chip according to claim 1, characterized in that: The substrate material in step (1) is made of polycarbonate, polydimethylsiloxane or other flexible materials with good thermal stability and mechanical properties as the supporting basis of the array structure.

3. The method for selectively capturing antibiotics in dairy products using a surface-enhanced Raman nanoimprint chip according to claim 1, wherein: The periodic nanoarray structure in step (1) is formed by transferring the pre-designed nanopattern to the substrate surface using hot pressing nanoimprinting technology to form a uniformly arranged pyramidal and columnar structure with an array spacing controlled at 50–300 nm to construct a local electromagnetic hotspot.

4. The method for detecting antibiotics in dairy products by selectively capturing them using a surface-enhanced Raman nanoimprint chip according to claim 1, wherein: The precious metal deposition layer in step (1) is deposited on the surface of the nanostructure by physical vapor deposition, such as magnetron sputtering or electron beam evaporation, with a thickness of 5-25 nm, to form an active layer with surface-enhanced Raman enhancement capability.

5. The method for detecting antibiotics in dairy products by selectively capturing them using a surface-enhanced Raman nanoimprint chip according to claim 1, wherein: Step (2) surface functionalization recognition layer, introducing molecular probes or molecular imprinting polymers on the metal surface to enhance the target molecule recognition selectivity.

6. The method for detecting antibiotics in dairy products by selectively capturing them using a surface-enhanced Raman nanoimprint chip according to claim 1, wherein: The dairy products in step (3) include but are not limited to fresh cow milk, cow milk powder, fresh goat milk, and goat milk powder.

7. The method for selectively capturing antibiotics in dairy products using a surface-enhanced Raman nanoimprint chip according to claim 6 is applied to the rapid screening and quantitative detection of trace antibiotics such as tetracycline, chloramphenicol or ciprofloxacin in dairy products.

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