Visible / ultraviolet dual-light synergistically enhanced substrate, preparation method thereof and application of visible / ultraviolet dual-light synergistically enhanced substrate in rapid detection of veterinary drugs
By using a composite substrate of Bi-doped ZnO nanorod arrays and sea urchin-shaped silver nanoparticles, the problems of fluorescence interference and signal uniformity in the detection of aquatic products by SERS and PIERS technologies were solved, achieving high sensitivity and high accuracy in the detection of veterinary drugs. The substrate is also highly reusable.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING UNIV OF SCI & TECH
- Filing Date
- 2026-01-15
- Publication Date
- 2026-05-05
AI Technical Summary
Existing SERS and PIERS technologies have problems in aquatic product detection, such as fluorescence background interference, poor signal uniformity, poor reusability, and difficulty in distinguishing structural analogs, especially when detecting malachite green and crystal violet.
A composite substrate consisting of Bi-doped ZnO nanorod arrays and urchin-shaped silver nanoparticles was constructed. A uniform and robust load was formed through gas-liquid interface self-assembly and photowelding technology. Visible light response was achieved and fluorescence interference was avoided by utilizing the defect energy levels and dual photoenhanced Raman effect introduced by Bi doping.
It achieves ultrasensitive detection and qualitative and quantitative analysis of multiple veterinary drugs in complex matrices, significantly improves the signal-to-noise ratio, lowers the detection limit to the 10-11M level, has good repeatability, and the substrate can be recycled.
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Figure CN121978074A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of rapid food safety testing technology, specifically relating to a visible / ultraviolet dual-light synergistic enhancement substrate, its preparation method, and its application in rapid veterinary drug testing. Background Technology
[0002] Malachite green, crystal violet, and nitrofurans (such as furacilin) have been widely used in aquaculture to prevent saprolegniasis and bacterial infections in fish. Because these drugs have extremely low residual concentrations in aquatic product matrices and complex compositions, it is essential to develop a rapid, sensitive, and interference-resistant quantitative detection method to ensure food safety.
[0003] Surface-enhanced Raman scattering (SERS) has become a powerful tool for trace substance detection due to its fingerprinting ability and single-molecule-level detection sensitivity. Traditional SERS mainly relies on the electromagnetic field enhancement (EM) of noble metal nanostructures (such as gold and silver) (Xu, H., Aizpurua, J., Käll, M., Apell, P., 2000. Electromagneticcontributions to single-molecule sensitivity in surface-enhanced Ramanscattering. Phys. Rev. E 62, 4318-4324. https: / / doi.org / 10.1103 / PhysRevE.62.4318). To further improve sensitivity, photo-enhanced Raman spectroscopy (PIERS) has been proposed. This technology introduces semiconductor materials that generate charge transfer under illumination, thereby further enhancing the signal through chemical mechanisms (CM) (Panariello, L., Chuen To, K., Khan, Z., et al, 2021. Kinetics-based design of a flow platform for highly reproducible on demand synthesis of gold nanoparticles with controlled size between 50 and 150 nm and their application in SERS and PIERS sensing. Chemical Engineering Journal 423, 129069. https: / / doi.org / 10.1016 / j.cej.2021.129069).
[0004] However, existing SERS and PIERS technologies face significant challenges and limitations in actual aquatic product testing: (1) Fluorescence background interference: Commonly used wide-bandgap semiconductors (such as pure ZnO) usually only respond to ultraviolet light, while ultraviolet light excitation causes strong fluorescence background in biological samples (such as fish meat extract), which seriously reduces the signal-to-noise ratio and affects the accuracy of detection. (2) Signal uniformity and stability: Traditional SERS substrates are often prepared by drop coating, which has a "coffee ring effect" and results in poor signal uniformity; and physically adsorbed noble metal particles are easy to fall off, which limits the reusability of the substrate. (3) Difficulty in distinguishing structural analogs: Malachite green and crystal violet are triphenylmethane substances with highly similar structures, which are difficult to distinguish accurately by the naked eye or conventional spectral analysis.
[0005] In existing technologies, Bi-doped zinc oxide (Bi-ZnO) is mostly used in photocatalytic degradation (such as dyes and antibiotics), VOCs gas sensing, and antibacterial applications. Although studies have confirmed that Bi doping can narrow the energy band and increase surface oxygen vacancies, thereby improving photoresponse and charge transfer efficiency, these properties currently mainly serve to improve catalytic and sensing performance (Kazmi, J., Ooi, PC, Goh, BT, wt al, 2020. Bi-doping improves the magnetic properties of zinc oxide nanowires. RSC Adv. 10, 23297-23311. https: / / doi.org / 10.1039 / D0RA03816D). Currently, there are no reports in the literature on using it to construct PIERS substrates, especially for its application in the rapid screening of banned veterinary drugs in aquatic products. Summary of the Invention
[0006] The purpose of this invention is to provide a visible / ultraviolet dual-light synergistic enhancement substrate, its preparation method, and its application in rapid detection of veterinary drugs. This invention constructs a composite substrate (Bi:ZnO-SUSNs) combining a Bi-doped ZnO nanorod array with urchin-shaped silver nanoparticles. Visible light response is achieved by utilizing the defect energy levels introduced by Bi doping, avoiding fluorescence interference. Uniform and robust loading is achieved through gas-liquid interface self-assembly and photowelding. The dual photo-enhanced Raman effect of the doped semiconductor and noble metal composite substrate enables ultrasensitive detection and qualitative and quantitative analysis of various veterinary drugs in complex matrices.
[0007] The technical solution for achieving the objective of this invention is as follows:
[0008] The method for preparing a visible / ultraviolet dual-light synergistic enhancement substrate includes the following steps:
[0009] (1) A three-electrode system was adopted, with a conductive substrate as the working electrode and a mixed aqueous solution of zinc nitrate and bismuth nitrate as the electrolyte. A two-step electrochemical deposition method was used. First, a seed layer was prepared by constant potential deposition in a high-temperature water bath at 65℃~85℃. Then, hexamethylenetetramine was added to the electrolyte, and a nanorod array was prepared by constant current deposition in a high-temperature water bath at 65℃~85℃. After the reaction was completed, the nanorod array was washed with water and dried to obtain Bi-doped ZnO nanorod array (Bi:ZnONRAs).
[0010] (2) The ethanol dispersion of sea urchin-shaped silver nanoparticles (SUSNs) was added dropwise to the surface of silver nitrate solution. The SUSNs self-assembled film was formed by gas-liquid interface self-assembly technology. The SUSNs self-assembled film was then transferred to the surface of Bi:ZnONRAs by dip-coating method. In-situ photowelding was then performed under ultraviolet irradiation. After removal, washing and drying, Bi:ZnO-SUSNs composite Raman substrate was obtained.
[0011] Furthermore, in step (1), the conductive substrate is a substrate with conductive properties commonly used in the field of electrochemical deposition, including but not limited to ITO glass.
[0012] Furthermore, in step (1), the constant potential deposition time is 50 s to 120 s, and the constant potential is -0.5 to -2 V.
[0013] Furthermore, in step (1), the constant current deposition time is 3600 s to 7200 s, and the constant current is -1 mA to -1.5 mA.
[0014] Furthermore, in step (2), the concentration of the ethanol dispersion of the urchin-shaped silver nanoparticles is 0.1~0.5 mg / ml.
[0015] Furthermore, in step (2), the concentration of the silver nitrate solution is 8~15 mM.
[0016] Furthermore, in step (2), the ultraviolet irradiation time is 20~40 min.
[0017] The present invention provides a visible / ultraviolet dual-light synergistic enhancement substrate prepared by the above preparation method.
[0018] The present invention also provides the application of the above-mentioned visible / ultraviolet dual-light synergistic enhancement substrate in SERS or PIERS detection of veterinary drugs.
[0019] Furthermore, veterinary drugs are common medications used to prevent, treat, or diagnose animal diseases or to purposefully regulate animal physiological functions, including but not limited to malachite green, crystal violet, and furacilin.
[0020] Furthermore, the specific application method is as follows:
[0021] (1) The test droplet is added to the Bi:ZnO-SUSNs composite Raman substrate and the Raman spectrum is collected directly, or the substrate is pre-irradiated under visible or ultraviolet light to activate the charge transfer of defect states and then the Raman spectrum is collected.
[0022] (2) By comparing the Raman spectra of the veterinary drug standard with the Raman spectra of the test solution, the type of veterinary drug in the test solution is determined;
[0023] (3) Substitute the Raman spectral signal of the test solution into the linear relationship between the veterinary drug concentration and the Raman spectral signal to quantify the veterinary drug concentration in the test solution.
[0024] Furthermore, the pre-irradiation time under visible light is 10-15 min, and the pre-irradiation time under ultraviolet light is 25-30 min.
[0025] Compared with the prior art, the present invention has the following advantages:
[0026] (1) Visible light driven PIERS detection was realized: By utilizing the defect energy level introduced by Bi doping, the substrate can generate significant chemical enhancement under 532nm visible light, effectively avoiding strong fluorescence interference generated by ultraviolet light excitation of biological matrix, and greatly improving the signal-to-noise ratio of actual sample detection.
[0027] (2) Dual photo-enhanced mechanism: The substrate has both ultraviolet response (intrinsic band gap of ZnO) and visible light response capabilities, and the excitation mode can be switched according to actual needs, pushing the detection limit down to 10. -11 M level.
[0028] (3) Optimization of preparation process and signal stability: The high-temperature annealing step that may passivate defect sites was eliminated, and the original high activity of the material was preserved; the gas-liquid interface self-assembly and in-situ photowelding technology were adopted to ensure the uniform distribution and firm binding of silver nanoparticles in the three-dimensional array (resistant to ultrasonic cleaning), which greatly improved the repeatability of detection and the cycle life of the substrate.
[0029] (4) Minimal pretreatment: No expensive and time-consuming solid phase extraction column purification is required; detection can be performed by simple centrifugation or wiping with a cotton swab. Attached Figure Description
[0030] Figure 1 SEM images of Bi:ZnO nanorod array (a), urchin-shaped silver nanoparticles (b), top view of Bi:ZnO-SUSNs composite Raman substrate (c, d), and cross-sectional views of Bi:ZnO-SUSNs composite Raman substrate (e, f).
[0031] Figure 2TEM image of SUSNs (a), cross-sectional view of the photo-welded composite substrate (b), and EDSMapping images of the photo-welded composite substrate (c~f).
[0032] Figure 3 XRD patterns of Bi:ZnO nanorod arrays with different Bi doping concentrations (a), and XPS patterns of Bi:ZnO-SUSNs composite substrates (b~f).
[0033] Figure 4 The Raman spectra of the probe molecule R6 G on the composite substrate under conditions of no light, 365 nm UV pre-irradiation, and 532 nm visible light pre-irradiation are shown in (a), with corresponding characteristic peak concentration gradient waterfall plot (b), signal enhancement formation kinetic curve and relaxation process diagram (c).
[0034] Figure 5 PIERS-enhanced spectra and standard quantitative curves of different veterinary drugs at low concentrations are shown, where a and d represent malachite green, b and e represent crystal violet, and c and f represent furacilin.
[0035] Figure 6 The comparison shows the stability of the photowelding signal (a), the signal comparison of silver nanoparticles with different morphologies at low concentrations (b), and the fluorescence spectrum (c), photocurrent (d), electrochemical impedance (e), and Mott-Schottky curve (f) of zinc oxide nanorod arrays with different doping amounts.
[0036] Figure 7 The substrate stability test includes a, b, and c, which represent the photocatalytic self-cleaning and recyclability performance; d, which represents the signal stability after 30 days of storage; and e, which represents the signal retention rate after ultrasonic treatment. Detailed Implementation
[0037] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0038] The urchin-like silver nanoparticles in this invention were prepared according to the reference (Liu, T., Li, D., Yang, D., Jiang, M., 2011. Fabrication of Flower-Like Silver Structures through Anisotropic Growth. Langmuir 27, 6211–6217. https: / / doi.org / 10.1021 / la200512m). Specifically, under low-temperature ice-water bath conditions, a pre-cooled mixture of formaldehyde and ammonia was rapidly injected into a mixed solution of silver nitrate and PVP. Nucleation was achieved through a burst of instantaneous high supersaturation. After 15 seconds of reaction, saturated sodium chloride was rapidly added to quench the reaction, resulting in small-sized, multi-needle SUSNs, the morphology of which is as follows. Figure 1 b and Figure 2 a.
[0039] Example 1
[0040] 1. A method for preparing a visible / ultraviolet dual-light synergistic enhancement substrate Bi:ZnO-SUSNs, comprising the following steps:
[0041] (1) Fabrication of Bi-doped ZnO nanorod arrays:
[0042] Bi:ZnO nanorod arrays were prepared on ITO glass using a two-step electrochemical deposition method. First, a mixed electrolyte containing zinc nitrate and bismuth nitrate was prepared, with the Bi doping ratio controlled at 5%. Using the cleaned ITO glass as the working electrode and a platinum sheet as the counter electrode, deposition was performed at 70°C with a constant potential of -1.0 V for 60 s. After deposition, the film was rinsed with deionized water and allowed to air dry, yielding a bismuth-doped ZnO seed layer on the ITO. Then, 0.14 g of hexamethylenetetramine was added to the mixed electrolyte and mixed thoroughly using a magnetic stirrer. The dried bismuth-doped ZnO seed layer was then deposited at a constant current of -1.25 mA for 5400 s. After deposition, the prepared film was rinsed with ultrapure water and then dried in an oven to obtain the Bi-doped ZnO nanorod array. No high-temperature annealing was performed throughout the process to preserve oxygen vacancies in the ZnO lattice and the defect energy levels introduced by Bi doping.
[0043] like Figure 1 a and Figure 3 As shown in Figure a, the prepared nanorod array exhibits a regular radial pattern, and the XRD pattern shows a slight shift in the ZnO peak position, confirming that Bi successfully entered the lattice and provided an energy level basis for visible light absorption.
[0044] (2) Loading and photowelding of urchin-shaped silver nanoparticles:
[0045] An ethanol dispersion of urchin-shaped silver nanoparticles was dropwise added to the surface of a silver nitrate solution. Utilizing the miscibility of ethanol and water and the difference in surface tension, SUSNs self-assembled films were formed at the gas-liquid interface. Then, a conductive substrate with deposited Bi:ZnONRAs was immersed in the liquid surface using an dip-pull method. The SUSNs self-assembled film was then horizontally pulled upwards from below the liquid surface to transfer it to the Bi:ZnONRAs surface. Subsequently, it was irradiated under a UV lamp for 20 min. The electrons excited by the UV light reduced the residual silver ions, achieving "in-situ welding" of the contact sites between the silver particles and ZnO nanorods. After removal, washing, and drying, a Bi:ZnO-SUSNs composite Raman substrate was obtained.
[0046] Figure 2 EDM approximation showed that Ag elements were distributed in fixed locations in the gaps and on the surface of ZnO, which corroborated the "welding" effect produced by photoreduction. Figure 7 c confirmed that the substrate signal remained strong after ultrasonic vibration treatment, solving the problem of easy detachment caused by traditional physical adsorption.
[0047] 2. Verification of the dual-mode photo-enhanced mechanism:
[0048] The properties of Bi:ZnO-SUSNs composite Raman substrates were tested using Rhodamine 6G as a probe molecule under no light, ultraviolet light, and visible light conditions. Figure 4 As shown, the signal is significantly improved after illumination compared to the pure SERS signal without illumination.
[0049] Ultraviolet mode (365 nm): Excites the intrinsic band gap of ZnO, generating electron-hole pairs. Electrons are transferred to the Ag Fermi level, enhancing the polarizability of chemisorption molecules.
[0050] Visible light mode (532 nm): Excite the defect level introduced by Bi doping to achieve visible light driven charge transfer (CT).
[0051] Figure 4 The kinetic curves show that after illumination is turned on, the SERS signal gradually increases over time and reaches a plateau after about 25 min (365 nm) and 10 min (532 nm). After illumination is turned off, the signal slowly decays, proving that the enhancement comes from the chemical mechanism of photogenerated carrier transfer (PIERS) rather than a transient thermal effect.
[0052] 3. Rapid detection procedure for veterinary drug residues:
[0053] (1) Sample pretreatment: Take a fish meat sample, homogenize it, add 5 times the volume of acetonitrile, vortex and centrifuge, and take the supernatant. Alternatively, wipe the surface of the fish body directly with a cotton swab and immerse the cotton swab in acetonitrile to wash it off.
[0054] (2) SERS / PIERS spectral acquisition:
[0055] A small amount of the test solution was added to the Bi:ZnO-SUSNs composite substrate.
[0056] For high-concentration samples, the signal is directly acquired using a Raman spectrometer (785 nm or 532 nm laser).
[0057] For trace samples (<10) -10 (M) First, the substrate is pre-irradiated with a 532 nm visible light source for 10 min or with a 365 nm ultraviolet light source for 25 min to activate defect state charge transfer, and then Raman spectra are collected. The former mode effectively avoids the problem of ultraviolet light exciting biofluorescence.
[0058] like Figure 5 As shown, after enabling PIERS mode, even at 10 -11 Even at extremely low concentrations of M, the characteristic peaks of malachite green, crystal violet, and furazolidone remain clearly visible. The R value of the calibration curve... 2 All are greater than 0.96.
[0059] 4. Recycling of the substrate:
[0060] After the test was completed, the Bi:ZnO-SUSNs composite substrate was immersed in deionized water and irradiated under a UV lamp for 30 minutes. The photocatalytic properties of Bi:ZnO were used to degrade the organic molecules adsorbed on the surface. Figure 7 The results show that after multiple cycles of "adsorption-detection-photocatalytic degradation", the SERS signal intensity of the substrate to the standard molecule still remains above 70% of the initial value, proving its recyclability.
[0061] Comparative Example 1
[0062] Comparative Example 1 provides a traditional physical drop-coating method, distinct from photowelding, for loading noble metal nanoparticles onto the surface of a doped semiconductor nanoarray. The specific preparation method is as follows:
[0063] (1) Preparation of SUSNs ethanol suspension: The pre-synthesized urchin-shaped silver nanoparticles were centrifuged and redispersed, and the solvent was replaced with anhydrous ethanol to prepare a concentration of 0.2 mg / mL. The mixture was then ultrasonically treated with a 40 kHz water bath for 30 minutes to ensure dispersion, thus obtaining the SUSNs ethanol suspension.
[0064] (2) Drop coating: The Bi-doped ZnO nanorod array / ITO substrate (1 cm × 1.5 cm) was preheated on a 70°C constant-temperature heating plate. Then, 5 µL of SUSNs ethanol suspension was added drop by drop using a micropipette. Each drop was allowed to evaporate completely before adding the next drop, repeating until the entire area was covered, with a total volume of approximately 30 µL. Finally, post-treatment: After drop coating, the substrate was kept at 70°C for 15 minutes to remove residual solvent, and then allowed to cool naturally to room temperature.
[0065] Figure 7 e and Figure 6 a compares the signal retention of substrates treated with photowelding and traditional physical drop coating after ultrasonic treatment. The signal of the substrate after welding only declines slightly, but the signal of the traditional physical drop coating method decreases significantly due to the problem of easy adsorption and detachment.
[0066] Comparative Example 2
[0067] To investigate the influence of different morphologies on the detection signal during the growth of urchin-like silver nanoparticles, a time-resolved sampling experiment was conducted. The experiment followed the original protocol: silver nitrate solution was preheated and then formaldehyde was added; ammonia was rapidly injected at t=0 to trigger the reaction. Samples were taken at 5, 15, 30, 90, and 300 seconds, and the samples were immediately injected into PVP solution to lock the particle morphology. After centrifugation and washing, the samples were dispersed in ethanol for characterization. Observations showed that: quasi-spherical crystal nuclei formed at 5 seconds (S5); tiny protrusions appeared at 15 seconds (S4); short, thick, spiky "daisy-like" structures formed at 30 seconds (S3); the protrusions sharpened into a urchin-like shape at 90 seconds (S2); and larger urchin-like particles matured at 300 seconds (S1). Figure 6 b represents silver nanoparticles with different growth morphologies at 10 -9 A comparison of the detection signals of Rhodamine 6 G solution at M-level concentrations shows that sample number two has the best signal at low concentrations.
[0068] Comparative Example 3
[0069] The preparation of this comparative example is roughly the same as that of the Bi-doped ZnO nanorod array in Example 1 (1), except that the Bi doping ratio in the Bi-doped ZnO nanorod array is controlled to be 0%, 2.5% and 10%, respectively.
[0070] Figure 6 c~f shows the fluorescence spectra, photocurrent, electrochemical impedance spectroscopy, and Mott-Schottky curves of ZnO nanorod arrays with different Bi doping concentrations. Figure 6 c indicates that the carrier recombination rate of the Bi-ZnO samples is reduced and the photoelectrochemical performance is improved, with 5% Bi-ZnO showing the lowest fluorescence intensity and the best performance. Figure 6The results show that Bi-ZnO has higher photocurrent density and instantaneous photoresponse capability than pure ZnO, with 5% Bi-ZnO having the highest photocurrent. Figure 6 d also indicates that Bi-ZnO has a lower charge transfer resistance and stronger charge separation and transfer capabilities. Figure 6 f shows that both pure ZnO and Bi-ZnO are N-type semiconductors, with Bi-ZnO having a higher carrier concentration.
Claims
1. A method for preparing a visible / ultraviolet dual-light synergistic enhancement substrate, characterized in that, Includes the following steps: (1) A three-electrode system was adopted, with a conductive substrate as the working electrode and a mixed aqueous solution of zinc nitrate and bismuth nitrate as the electrolyte. A two-step electrochemical deposition method was adopted. First, a seed layer was prepared by constant potential deposition in a high-temperature water bath at 65℃~85℃. Then, hexamethylenetetramine was added to the electrolyte, and a nanorod array was prepared by constant current deposition in a high-temperature water bath at 65℃~85℃. After the reaction was completed, the nanorods were washed with water and dried to obtain Bi:ZnONRAs. (2) The ethanol dispersion of urchin-shaped silver nanoparticles was added dropwise to the surface of silver nitrate solution. SUSNs self-assembled film was formed by gas-liquid interface self-assembly technology. The SUSNs self-assembled film was then transferred to the surface of Bi:ZnONRAs by dip-coating method. In-situ photowelding was then performed under ultraviolet irradiation. After removal, washing and drying were performed to obtain Bi:ZnO-SUSNs composite Raman substrate.
2. The preparation method according to claim 1, characterized in that, In step (1), the conductive substrate is ITO glass.
3. The preparation method according to claim 1, characterized in that, In step (1), the constant potential deposition time is 50 s to 120 s, and the constant potential is -0.5 to -2 V; The constant current deposition time is 3600 s to 7200 s, and the constant current is -1 mA to -1.5 mA.
4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the ethanol dispersion of the sea urchin-shaped silver nanoparticles is 0.1~0.5 mg / ml, and the concentration of the silver nitrate solution is 8~15 mM.
5. The preparation method according to claim 1, characterized in that, In step (2), the ultraviolet irradiation time is 20~40 min.
6. The visible / ultraviolet dual-light synergistic enhancement substrate prepared by the preparation method according to any one of claims 1 to 5.
7. The application of the visible / ultraviolet dual-light synergistic enhancement substrate according to claim 6 in SERS or PIERS detection of veterinary drugs.
8. The application according to claim 7, characterized in that, The veterinary drug is selected from one or more of malachite green, crystal violet and furazolidone.
9. The application according to claim 8, characterized in that, The specific application method is as follows: (1) The test droplet is added to the Bi:ZnO-SUSNs composite Raman substrate and the Raman spectrum is collected directly, or the substrate is pre-irradiated under visible or ultraviolet light to activate the charge transfer of defect states and then the Raman spectrum is collected. (2) By comparing the Raman spectra of the veterinary drug standard with the Raman spectra of the test solution, the type of veterinary drug in the test solution is determined; (3) Substitute the Raman spectral signal of the test solution into the linear relationship between the veterinary drug concentration and the Raman spectral signal to quantify the veterinary drug concentration in the test solution.
10. The application according to claim 9, characterized in that, In step (1), the pre-irradiation time under visible light is 10-15 min, and the pre-irradiation time under ultraviolet light is 25-30 min.