Preparation of high-performance SERS (Surface Enhanced Raman Scattering) substrate based on colloidal crystal template etching and application of high-performance SERS substrate in detecting benzylpenicillin potassium in medical wastewater

Abstract

The invention provides a preparation method of a high-performance SERS (Surface Enhanced Raman Scattering) substrate based on colloidal crystal template etching and application of the high-performance SERS substrate in detection of benzylpenicillin potassium in medical wastewater. The specific preparation process comprises the following steps: S1, obtaining large-area polystyrene colloid nanospheres which are uniformly, tightly and hexagonally arranged on the water surface by using an inclined plate-assisted gas-liquid interface self-assembly method, and transferring a crystal mask plate to the surface of a clean silicon substrate at a constant speed by using a Czochralski method; s2, putting the silicon wafer integrated with the large-area nanospheres into reactive ion etching (Oxford 100), uniformly etching the nanospheres by adopting oxygen plasma, shortening the distance between the nanospheres, and enhancing local electromagnetic coupling; s3, silver of 60 nm is deposited on the surfaces of the nanospheres in a thermal evaporation mode, and due to the electromagnetic resonance effect of the metal nanoparticles, Raman signals can be further enhanced. And S4, putting the structured SERS substrate and the untreated substrate into a penicillin potassium solution with the concentration of 0.1 mol / L, soaking for 20 minutes, naturally airing, and measuring a Raman signal by using a micro Raman spectrometer.

Description

Technical Field

[0001] The present invention relates to the field of surface enhanced Raman spectroscopy detection, in particular to the preparation of a high-performance SERS substrate based on colloidal crystal template etching and its application in the detection of potassium penicillin in medical wastewater. Background Art

[0002] Raman enhancement technology is based on the phenomenon of Raman scattering, a spectroscopic technique. In the 1920s, Indian physicist C.V. Raman discovered that when light passes through a substance, a small portion of the light scatters and changes frequency. This phenomenon, known as Raman scattering, provides information about the structure and chemical composition of the substance. However, Raman scattering signals are often very weak, making them difficult to detect and analyze. To address this issue, researchers have developed a series of techniques to enhance Raman signals, making them easier to detect and analyze.

[0003] Surface-enhanced Raman scattering (SERS) is one of the most widely known and commonly used Raman enhancement techniques. Scientists Spraman and Livingston independently discovered in the 1970s that the intensity of the Raman scattering signal is significantly enhanced when molecules are adsorbed on a metal surface. This phenomenon sparked the interest of researchers, who began exploring the mechanisms and potential applications of this Raman signal enhancement phenomenon. It utilizes the local electric field effect generated on the surface of metal nanoparticles (usually silver or gold) to enhance the Raman signal. The electric field generated by these nanoparticles can significantly increase the intensity of the scattered light, making the Raman signal easier to detect. Surface-enhanced Raman scattering (SERS) is a Raman enhancement technique that significantly enhances the intensity of the Raman scattering signal through the local electric field effect and surface plasmon resonance effect generated on the surface of metal nanostructures, making weak Raman signals easier to detect and analyze. SERS technology has a wide range of applications in many fields, including molecular detection, biomedical applications, food and beverage analysis, and environmental monitoring. In general, surface-enhanced Raman scattering is a powerful spectroscopic technique that provides high-sensitivity analysis of the structure, composition, and chemical properties of materials through the local electric field effect and surface plasmon resonance effect of metal nanostructures, and has broad application potential.

[0004] Penicillin potassium is an antibiotic widely used in the medical field and belongs to the β-lactam antibiotic class. It has antibacterial effects and can effectively inhibit bacterial cell wall synthesis, leading to bacterial death. Penicillin potassium is used to treat a variety of bacterial infections and is an important antibiotic. However, when penicillin potassium antibiotics enter medical wastewater, if they are not properly treated, they can cause bacteria in the wastewater to develop drug resistance, thereby allowing antibiotic resistance genes to spread to other bacteria in the environment, including pathogens, making antibiotic treatment more difficult. Moreover, penicillin potassium and other medical waste in wastewater can enter natural water bodies such as rivers and lakes, negatively impacting aquatic ecosystems and damaging water quality. SERS technology, with its high sensitivity and non-contact, non-destructive detection characteristics, can be used to track antibiotic residues in medical wastewater, helping to monitor and control the spread and contamination of antibiotics in the environment.

[0005] In summary, there have been many studies on the detection of antibiotics in aqueous solutions, especially the detection of antibiotics in medical wastewater based on SERS, which has been proven to have unique advantages such as extremely high sensitivity, excellent specificity, diversified analytical capabilities, good stability, non-destructive and non-destructive detection, and resistance to fluorescence interference. Summary of the Invention

[0006] To meet the needs of the existing technology, the present invention provides a method for preparing a high-performance SERS substrate based on colloidal crystal template etching, which solves the problem that penicillin potassium is difficult to detect in trace amounts due to its small Raman scattering cross section. The accuracy, signal-to-noise ratio, sensitivity, repeatability and stability of the SERS detection system are improved, laying the foundation for on-site monitoring of antibiotics in medical wastewater.

[0007] The technical solution of the present invention includes the following manufacturing steps: S1. Fabrication of a large-area colloidal crystal nanosphere mask plate by self-assembly at the gas-liquid interface: First, the silicon wafer required for the experiment was placed in a piranha solution (99% H2SO4: 30% H2O2 = 7:3) and soaked for 2 hours for hydrophilic treatment. The residual piranha solution was then removed by ultrasonic treatment with acetone, ethanol, and deionized water for 20 minutes in sequence. Second, a polystyrene nanosphere solution with a solid content of 5% was mixed with ultrapure water and anhydrous ethanol in a volume ratio of 1:1:2, and the mixed solution was fully ultrasonically vibrated to disperse the nanospheres. Third, the colloidal template was manufactured in a standard glass culture dish filled with deionized water. The silicon wafer was partially immersed in the water and tilted at 15° relative to the water surface. A pipette was used to slowly drip 100 μL of the nanosphere mixed solution onto the silicon wafer used as the tilted plate. Under the combined action of capillary force, van der Waals force, and friction, the nanospheres self-assembled on the water surface to form a floating hexagonal tightly packed monolayer structure. Then, 20 μL of sodium dodecyl sulfate surfactant was added to one edge, causing a dramatic change in the liquid's surface tension, which in turn pushed the nanospheres closer together. Finally, an electrically controlled translation stage was used to pull the silicon substrate vertically at a constant speed of 0.09 mm / s, resulting in a high-quality, hexagonally densely packed silicon nanosphere array. S2, oxygen plasma dry etching: The silicon-based single-layer polystyrene nanospheres obtained in S1 were placed in a reactive ion etching chamber, and the etching time was set to 220s, the etching power to 50W, and the oxygen flow rate to 50sccm. Finally, a nanosphere array with a spacing of 310nm and a period of 500nm was obtained on the silicon wafer surface. S3, thermal evaporation deposition of silver nanoparticles to obtain nanosphere core-shell structure: thermal evaporation deposition of silver film on the silicon wafer single-layer nanosphere mask obtained in S2, the thermal evaporation rate is controlled at To ensure the uniformity of the silver nanoparticles, the working gas pressure is controlled at 3×10-3Pa to prevent silver oxidation. The thickness of the silver film on the silicon substrate is controlled by adjusting the thermal evaporation time. During the thermal evaporation process, as the polystyrene nanospheres melt the core-shell structure of the silver nanoparticles, tips are formed on the surface of the nanospheres, greatly enhancing the local electromagnetic field intensity. The presence of smaller nanogaps between particles further enhances the electromagnetic field intensity. Since the resonant wavelengths of the plasma resonance bodies generated on the surfaces of silver nanoparticles of different sizes are different, the strongest Raman spectroscopy enhancement effect can only be produced when the resonant wavelength matches the wavelength of the Raman incident laser. The final silver nanoparticle film with a thickness of 60nm has the best Raman spectroscopy enhancement effect on the core-shell structure silicon substrate. In step S4, an appropriate amount of 99% pure potassium penicillin solid was prepared into a 0.1 mol / L potassium penicillin solution. The silicon-based core-shell silver nanostructure obtained in step S3 was then immersed in the potassium penicillin solution for 20 minutes. After air drying, the structured substrate was placed under a Raman microscope for Raman spectroscopy. The Raman spectrometer used an excitation wavelength of 635 nm, a laser power of 40 mW, an integration time of 10 seconds, and two integrations.

[0008] Silicon-based core-shell silver nanostructures such as Figure 1 As shown, a single nanosphere core-shell silver metal nanostructure excites localized surface plasmons under the action of incident laser light, enhancing the interaction between light and penicillin potassium molecules. Furthermore, the electromagnetic field is greatly enhanced by the smaller and sharper Ag nanoprotrusions on the nanosphere's surface. This is further enhanced by the tiny gaps between the nanospheres and the presence of gap resonance modes within the Ag nanoparticles on the nanosphere's surface.

[0009] The Raman spectrum of silicon-based nano core-shell structure is shown in the figure Figure 2 As shown, the Raman spectrometer excitation wavelength is 635nm, the laser power is 40mW, the integration time is 10s, and the integration times are 2 times. The Raman characteristic peak of potassium penicillin is located at 1000cm-1, Figure 2 It is clearly evident that when the silver film thickness is 60nm, the strongest Raman spectral enhancement effect is achieved when the resonant wavelength matches the wavelength of the incident Raman laser. Compared to 99% solid penicillin potassium, the 60nm silicon-based nanocore-shell structure exhibits a stronger Raman spectral enhancement factor, which has significant application value in the detection of low-concentration penicillin potassium in medical wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Silicon-based 60nm silver nanocore-shell structure.

[0011] Figure 2 The Raman spectra of 0.1 mol / L penicillin potassium solution and 99% solid penicillin potassium were measured on silicon-based silver nanocore-shell structures with different film thicknesses and unstructured substrates. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0013] Reference Figure 1-2 The preparation of high-performance SERS substrate based on colloidal crystal template etching and its application in the detection of potassium penicillin in medical wastewater includes the following steps: S1. Fabrication of a large-area colloidal crystal nanosphere mask plate by self-assembly at the gas-liquid interface: First, the silicon wafer required for the experiment was placed in a piranha solution (99% H2SO4: 30% H2O2 = 7:3) and soaked for 2 hours for hydrophilic treatment. The residual piranha solution was then removed by ultrasonic treatment with acetone, ethanol, and deionized water for 20 minutes in sequence. Second, a polystyrene nanosphere solution with a solid content of 5% was mixed with ultrapure water and anhydrous ethanol in a volume ratio of 1:1:2, and the mixed solution was fully ultrasonically vibrated to disperse the nanospheres. Third, the colloidal template was manufactured in a standard glass culture dish filled with deionized water. The silicon wafer was partially immersed in the water and tilted at 15° relative to the water surface. A pipette was used to slowly drip 100 μL of the nanosphere mixed solution onto the silicon wafer used as the tilted plate. Under the combined action of capillary force, van der Waals force, and friction, the nanospheres self-assembled on the water surface to form a floating hexagonal tightly packed monolayer structure. Then, 20 μL of sodium dodecyl sulfate surfactant was added to one edge, causing a dramatic change in the liquid's surface tension, which in turn pushed the nanospheres closer together. Finally, an electrically controlled translation stage was used to pull the silicon substrate vertically at a constant speed of 0.09 mm / s, resulting in a high-quality, hexagonally densely packed silicon nanosphere array. S2, oxygen plasma dry etching: The silicon-based single-layer polystyrene nanospheres obtained in S1 were placed in a reactive ion etching chamber, and the etching time was set to 220s, the etching power to 50W, and the oxygen flow rate to 50sccm. Finally, a nanosphere array with a spacing of 310nm and a period of 500nm was obtained on the silicon wafer surface. S3, thermal evaporation deposition of silver nanoparticles to obtain nanosphere core-shell structure: thermal evaporation deposition of silver film on the silicon wafer single-layer nanosphere mask obtained in S2, the thermal evaporation rate is controlled at To ensure the uniformity of the silver nanoparticles, the working gas pressure is controlled at 3×10-3Pa to prevent silver oxidation. The thickness of the silver film on the silicon substrate is controlled by adjusting the thermal evaporation time. During the thermal evaporation process, as the polystyrene nanospheres melt the core-shell structure of the silver nanoparticles, tips are formed on the surface of the nanospheres, greatly enhancing the local electromagnetic field intensity. The presence of smaller nanogaps between particles further enhances the electromagnetic field intensity. Since the resonant wavelengths of the plasma resonance bodies generated on the surfaces of silver nanoparticles of different sizes are different, the strongest Raman spectroscopy enhancement effect can only be produced when the resonant wavelength matches the wavelength of the Raman incident laser. The final silver nanoparticle film with a thickness of 60nm has the best Raman spectroscopy enhancement effect on the core-shell structure silicon substrate. In step S4, an appropriate amount of 99% pure potassium penicillin solid was prepared into a 0.1 mol / L potassium penicillin solution. The silicon-based core-shell silver nanostructure obtained in step S3 was then immersed in the potassium penicillin solution for 20 minutes. After air drying, the structured substrate was placed under a Raman microscope for Raman spectroscopy. The Raman spectrometer used an excitation wavelength of 635 nm, a laser power of 40 mW, an integration time of 10 seconds, and two integrations.

Claims

1. A high-performance SERS substrate prepared by colloidal crystal template etching and its application in the detection of potassium penicillin in medical wastewater, characterized in that : Based on oxygen plasma dry etching of silicon-based polystyrene colloidal crystals to self-assemble nanosphere core-shell structure, a core-shell structure with a larger specific surface area is obtained. Under the action of Raman signal light, a "hotspot" is generated at the tip of the nanosphere structure, and the surrounding local electromagnetic field is greatly enhanced, which plays the role of an optical antenna, absorbing the far field and converting it into the near field. In addition, there are smaller gaps between particles, which can not only generate "hotspots" again, but also adsorb a large number of signal molecules to further amplify the signal. The method for manufacturing a high-performance SERS substrate is as follows.

2. Fabrication of large-area colloidal crystal nanosphere mask plates by self-assembly at the gas-liquid interface: First, the silicon wafers needed in the experiment were immersed in piranha solution (99% H2SO4: 30% H2O2 = 7:3) for 2 hours for hydrophilic treatment. The residual piranha solution was then removed by ultrasonic treatment with acetone, ethanol, and deionized water for 20 minutes. Secondly, a polystyrene nanosphere solution with a solid content of 5% was mixed with ultrapure water and anhydrous ethanol in a volume ratio of 1:1:2, and the mixed solution was fully ultrasonically vibrated to disperse the nanospheres. Thirdly, the colloidal template was manufactured in a standard glass culture dish filled with deionized water. , the silicon wafer was partially immersed in the water and tilted at 15° relative to the water surface. A pipette was used to slowly drip 100 μL of the nanosphere mixed solution onto the silicon wafer used as a tilted plate. Under the combined action of capillary force, van der Waals force and friction, the nanospheres self-assembled on the water surface to form a floating hexagonal closely packed single-layer arrangement structure; then, 20 μL of sodium dodecyl sulfate surfactant was added to one side of the edge, and the surface tension of the liquid changed dramatically. The surface tension pushed the nanospheres to arrange more closely, and the silicon substrate was pulled out vertically at a uniform speed of 0.09 mm / s using an electric-controlled translation stage to obtain a high-quality silicon-based hexagonal closely packed nanosphere array.

3. Oxygen plasma dry etching: The silicon-based single-layer polystyrene nanospheres obtained in S1 were placed in a reactive ion etching chamber. The etching time was set to 220s, the etching power was 50W, and the oxygen flow rate was 50sccm. Finally, a nanosphere array with a spacing of 310nm and a period of 500nm was obtained on the silicon wafer surface.

4. Thermal evaporation deposition of silver nanoparticles to obtain nanosphere core-shell structure: thermal evaporation deposition of silver film on the silicon wafer single-layer nanosphere mask obtained in S2, the thermal evaporation rate is controlled at To ensure the uniformity of silver nanoparticles, the working pressure was controlled at 3×10 -3 Pa prevents silver from being oxidized, and the thickness of the silver film on the silicon-based surface is controlled by adjusting the thermal evaporation time. During the thermal evaporation process, as the polystyrene nanospheres melt the silver nanoparticle core-shell structure, tips are formed on the surface of the nanospheres, which greatly enhances the local electromagnetic field intensity. The smaller nanogaps between particles further enhance the electromagnetic field intensity. Since the resonant wavelengths of the plasma resonance bodies generated on the surfaces of silver nanoparticles of different sizes are different, the strongest Raman spectrum enhancement effect can only be produced when the resonant wavelength and the wavelength of the Raman incident laser match each other. The final silver nanoparticle film layer thickness is 60nm, and the core-shell structure silicon-based Raman spectrum enhancement effect is the best.

5. Take out an appropriate amount of 99% pure potassium penicillin solid and prepare it into a 0.1 mol / L potassium penicillin solution. Then immerse the silicon-based core-shell silver nanostructure obtained by S3 in the potassium penicillin solution for 20 minutes. After natural drying, place the structured substrate under a micro Raman spectrometer for Raman spectroscopy testing. The Raman spectrometer has an excitation wavelength of 635 nm, a laser power of 40 mW, an integration time of 10 s, and two integration times.

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