Vanillin SERS (Surface Enhanced Raman Scattering) detection method based on 4-ATP (Adenosine Triphosphate) functionalized Ag nanocube detection chip and application

Abstract

The invention belongs to the technical field of surface enhanced Raman spectroscopy detection, and particularly discloses a vanillin SERS detection method based on a 4-ATP functionalized Ag nanocube detection chip and application, the detection method comprises the following steps: soaking the 4-ATP functionalized Ag nanocube detection chip in a vanillin-containing solution, and carrying out Raman test by using SERS, the preparation method of the 4-ATP functionalized Ag nanocube detection chip comprises the following steps: carrying out centrifugal purification on Ag nanocube sol to prepare Ag nanocube sol which has a surface plasmon resonance effect and is uniform in morphology; adding a ligand molecule 4-ATP into the prepared Ag nanocube sol with uniform morphology, and adjusting the number of times of adding the ligand molecule to obtain the Ag nanocube sol Ag NCs (at) 4-ATP with adjustable surface functionalization degree; and dripping the prepared 4-ATP functionalized Ag nanocube sol Ag NCs (at) 4-ATP onto a gold-plated substrate, and performing constant-temperature drying to prepare the uniform detection chip. According to the SERS detection method disclosed by the invention, the vanillin can be effectively identified.

Description

Technical Field

[0001] This invention belongs to the field of surface-enhanced Raman spectroscopy detection technology, specifically relating to a vanillin SERS detection method and its application based on a 4-ATP-functionalized Ag nanocube detection chip. Background Technology

[0002] Vanillin (4-hydroxy-3-methoxybenzaldehyde), one of the world's most consumed food additives, is naturally found in various natural products such as vanilla pods, orchids, and red pine. It can be extracted through chemical or biochemical methods and possesses a unique aroma and flavor. In the food industry, vanillin is widely used as an additive in products such as tea, biscuits, milk, and ice cream. However, excessive intake of vanillin can have serious negative effects on human health, such as nausea, headaches, allergies, and vomiting, and may also lead to liver and kidney damage. While existing detection technologies for vanillin have good sensitivity, their application in rapid on-site characterization and monitoring is greatly limited by problems such as complex and time-consuming pretreatment steps and expensive equipment.

[0003] Surface-enhanced Raman scattering (SERS) possesses characteristics such as fingerprint recognition, trace non-destructive analysis, rapid response, and high sensitivity, and has been applied in fields such as food safety, environmental pollution, and biomedical analysis, providing safeguards for public safety. Enhancement of the Raman signal typically requires the target molecule to adsorb onto the surface of plasma-enhanced nanomaterials (such as gold and silver nanostructures) or reside in the "hot spot" region of the plasma. Different molecules possess unique SERS fingerprint spectra, which can be used for substance identification and reaction monitoring. However, molecules lacking anchoring groups (such as thiols and amino groups) exhibit poor adsorption on the surface of plasma-enhanced materials, resulting in weak SERS signal responses. For example, vanillin, with its aromatic ring substituted with phenolic hydroxyl, aldehyde, and methoxy groups, hardly adsorbs on the surface of SERS-active materials. Therefore, there is an urgent need to develop new methods to enable non-adsorbing molecules like vanillin to effectively interact with plasma hot spots, thereby achieving molecular sensing.

[0004] The Schiff base reaction, proposed by Hugo Schiff in 1864, is a nucleophilic addition reaction between a carbonyl-containing aldehyde / ketone and a primary amine. This reaction is widely used in catalysis, pharmaceuticals, analytical testing, and functional materials. It involves the formation of a conjugated C=N double bond between the amino and carbonyl groups, linking the aldehyde / ketone to the primary amine to generate a stable imine product. Therefore, the Schiff base reaction is suitable for capturing molecules such as vanillin and can improve detection sensitivity. However, when using the Schiff base reaction for vanillin capture and SERS detection, the core technical bottlenecks are concentrated in the following: the imine bond is easily hydrolyzed under aqueous / acid-base conditions; the presence of aldehyde / ketone impurities in the matrix leads to insufficient selectivity; and the reaction kinetics are slow at low concentrations. When adapted to the SERS system, it faces the challenge of balancing the substrate's "hot spot" activity with its own reactivity, and the ease with which reagents or products can cause Raman signal interference. Furthermore, it is affected by the non-specific adsorption of macromolecules in food matrices, and the reaction conditions conflict with the need for rapid on-site detection, affecting detection stability, reproducibility, and practical applicability.

[0005] In summary, there is an urgent need to develop new methods to obtain more refined and comprehensive spectral information, thereby breaking through the current technological bottlenecks. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a vanillin SERS detection method and application based on a 4-ATP-functionalized Ag nanocube detection chip.

[0007] To achieve the above objectives, one of the technical solutions of the present invention is: a vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip, comprising the following steps: immersing the 4-ATP-functionalized Ag nanocube detection chip in a solution containing vanillin and performing Raman spectroscopy using SERS, wherein the 4-ATP-functionalized Ag nanocube detection chip is prepared by the following method:

[0008] (1) Preparation of Ag nanocube sol: Ag nanocube sol was purified by centrifugation to prepare Ag nanocube sol with uniform morphology and surface plasmon resonance effect.

[0009] (2) Preparation of 4-ATP-functionalized Ag nanocubes: The ligand molecule 4-aminothiophenol (4-ATP) was added to the uniform Ag nanocube sol prepared in step (1). By adjusting the number of times the ligand molecule was added, Ag nanocube sol Ag NCs@4-ATP with adjustable surface functionalization was obtained.

[0010] (3) Preparation of 4-ATP-functionalized Ag nanocube detection chip: The 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP obtained in step (2) is dropped onto a gold-plated substrate and dried at a constant temperature to prepare a uniform (high signal stability) detection chip.

[0011] In a preferred embodiment of the present invention, the laser used for SERS detection can effectively excite the hot spots on the surface of nanoparticles and between particles, as well as the photoinduced charge transfer resonance of the system. The Raman test conditions are a laser wavelength of 500-700 nm, a power of 20-30 mW, and an exposure time of 3-7 s.

[0012] In a preferred embodiment of the present invention, the Ag nanocube sol in step (1) is synthesized by adjusting the polyol reduction method.

[0013] In a preferred embodiment of the present invention, the particle size of the Ag nanocube sol in step (1) is about 70-90 nm.

[0014] In a preferred embodiment of the present invention, the mass ratio of ligand molecule 4-ATP to Ag nanocube sol in step (2) is 1:(180-220).

[0015] In a preferred embodiment of the present invention, in step (2), the ligand molecule 4-ATP can directly capture the vanillin molecule to be tested, and 4-ATP can be anchored on the surface of Ag NCs by Ag-S bonds; the 4-ATP has a uniform coverage on the surface of Ag nanocubes and the ligand molecule is added 1-2 times.

[0016] In a preferred embodiment of the present invention, in step (3), the 4-ATP-functionalized Ag nanocube sol AgNCs@4-ATP is added dropwise according to the target coverage and thickness of the functionalized Ag nanocubes on the substrate surface.

[0017] In a preferred embodiment of the present invention, the constant temperature drying temperature in step (3) is 20-30 ℃.

[0018] The ligand molecule 4-ATP was modified onto the surface of Ag nanocubes exhibiting surface plasmon resonance (SPR). The uniform coverage of 4-ATP on the Ag nanocube surface can be modulated by the number of ligand replacements. 4-ATP possesses amino groups and a large Raman scattering cross-section, allowing vanillin molecules to be captured and anchored to the sensor chip surface via Schiff base reactions. Detection is achieved through significant changes in the SERS spectrum of the ligand molecules. When appropriate 4-ATP coverage is achieved, it provides abundant capture sites and maximizes the amplification of spectral changes caused by the adsorption of small analytes. This structure is applicable to the detection of vanillin molecules and vanillin derivatives in various teas, and effectively identifies vanillin derivatives based on specific Raman peaks, confirming the value of this strategy in vanillin detection.

[0019] To achieve the above objectives, the second technical solution of the present invention is: the application of a vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip in the detection of flavorings and fragrances in tea.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. In this invention, 4-ATP, as a surface ligand, can directly capture vanillin molecules to be tested. Furthermore, 4-ATP can be anchored to the surface of Ag NCs via Ag-S bonds. At the same time, the SERS effect of the Ag nanocube core and the large scattering cross-section of the 4-ATP molecule effectively enhance the signal changes brought about by the analyte molecule. After capturing vanillin molecules through the Schiff base reaction, 4-ATP will exhibit unique Raman characteristic peak changes. This surface-functionalized Ag nanocube particle combined with Raman technology can effectively identify vanillin.

[0022] 2. The raw materials for synthesizing the 4-ATP functionalized Ag nanocubes of this invention are simple, the coverage of the ligand molecule 4-ATP on the surface of the Ag nanocubes is controllable and adjustable, and the edges and particles of the Ag nanocubes can generate a strong SERS coupling effect; 4-ATP, as a surface ligand, can directly capture the vanillin molecule to be tested, and 4-ATP can be anchored on the surface of Ag NCs through Ag-S bonds.

[0023] 3. The 4-ATP-functionalized Ag nanocube detection chip in this invention can detect vanillin molecules in complex environments, proving that this technology has great potential in practical applications.

[0024] 4. The present invention has high reproducibility and can effectively avoid the problem of different batches of samples producing different results in Raman testing. Attached Figure Description

[0025] Figure 1The image shows a scanning electron microscope (SEM) image of the 4-ATP-functionalized Ag nanocube detection chip prepared in Example 1.

[0026] Figure 2 The 4-ATP-functionalized Ag nanocube detection chip prepared in Example 1 has 30 random points between 1077 cm⁻¹. -1 The relative standard deviation of the Raman intensity of the characteristic peak;

[0027] Figure 3 The SERS spectra of the detection chip before and after immersion in a vanillin solution environment in the vanillin sensing sensitivity test of Example 3 are shown.

[0028] Figure 4 This is a fitted curve of the target peak intensity ratio as a function of vanillin molecule concentration in Example 3;

[0029] Figure 5 This demonstrates the anti-interference ability of the 4-ATP-functionalized Ag nanocube detection chip in Example 3 in different interfering environments in tea.

[0030] Figure 6 This demonstrates the performance of the method in actual tea sample testing in Example 3. Detailed Implementation

[0031] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0032] A vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip includes the following steps: immersing the 4-ATP-functionalized Ag nanocube detection chip in a solution containing vanillin and performing Raman spectroscopy using SERS, wherein the 4-ATP-functionalized Ag nanocube detection chip is prepared by the following method:

[0033] (1) Preparation of Ag nanocube sol: Ag nanocube sol was purified by centrifugation to prepare Ag nanocube sol with uniform morphology and surface plasmon resonance effect.

[0034] (2) Preparation of 4-ATP-functionalized Ag nanocubes: The ligand molecule 4-aminothiophenol (4-ATP) was added to the uniform Ag nanocube sol prepared in step (1). By adjusting the number of times the ligand molecule was added, Ag nanocube sol Ag NCs@4-ATP with adjustable surface functionalization was obtained.

[0035] (3) Preparation of 4-ATP-functionalized Ag nanocube detection chip: The 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP obtained in step (2) is dropped onto a gold-plated substrate and dried at a constant temperature to prepare a uniform (high signal stability) detection chip.

[0036] The laser used in the SERS detection can effectively excite hot spots on the surface of nanoparticles and between particles, as well as the photoinduced charge transfer resonance of the system. The Raman test conditions are a laser wavelength of 500-700 nm, a power of 20-30 mW, and an exposure time of 3-7 s.

[0037] In step (1), the Ag nanocube sol was synthesized by adjusting the polyol reduction method.

[0038] The particle size of the Ag nanocube sol in step (1) is approximately 70-90 nm.

[0039] In step (2), the mass ratio of ligand molecule 4-ATP to Ag nanocube sol is 1:(180-220).

[0040] In step (2), the ligand molecule 4-ATP can directly capture the vanillin molecule to be tested, and 4-ATP can be anchored on the surface of Ag NCs through Ag-S bonds;

[0041] The 4-ATP has a uniform coverage on the surface of the Ag nanocubes, and the ligand molecules are added 1-2 times.

[0042] In step (3), the 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP is dropped on according to the target coverage and thickness of the functionalized Ag nanocubes on the substrate surface.

[0043] The constant temperature drying temperature in step (3) is 20-30 ℃.

[0044] An application of a vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip in the detection of flavorings and fragrances in tea.

[0045] Example 1

[0046] A 4-ATP-functionalized Ag nanocube detection chip was prepared by the following method:

[0047] (1) Preparation of 80 nm Ag nanocube sol: Ag nanocubes were prepared by adjusting the polyol reduction method. First, the precursor solutions were prepared: solution a was prepared by dissolving 0.8 g AgNO3 and 0.78 mg CuCl2·2H2O in 20 mL 1,5-pentanediol, and solution b was prepared by dissolving 0.2 g polyvinylpyrrolidone (PVP) in 20 mL 1,5-pentanediol. After preparing the precursor solutions, 20 mL of 1,5-pentanediol was heated from room temperature to 195 °C under magnetic stirring for 20 min. Then, the prepared solutions a and b were simultaneously injected into the magnetically stirred 1,5-pentanediol at a rate of 500 μL / min to start the growth of Ag nanocubes. Finally, the reaction was terminated with an ice bath to obtain Ag nanocube sol with a size of about 80 nm. The prepared Ag nanocube sol was centrifuged and purified three times to obtain the nanocubes with a size concentrated at 80 nm. A uniformly morphological Ag nanocube sol with surface plasmon resonance effect at nm.

[0048] (2) Preparation of 4-ATP-functionalized Ag nanocubes: 3.125 mg of the uniform Ag nanocube sol obtained in step (1) was added to 10 μL of 10 mM ligand molecule 4-ATP solution and magnetically stirred to remove surfactant PVP and perform surface functionalization on Ag nanocubes; after stirring, centrifugation was performed to obtain 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP;

[0049] (3) Preparation of 4-ATP functionalized Ag nanocube detection chip: Take 4 μL of 4-ATP functionalized Ag nanocube sol Ag NCs@4-ATP (double layer arrangement, 100% coverage) obtained in step (2) and drop it onto a gold-plated substrate cleaned with piranha solution. After drying at a constant temperature of 26 °C, a detection chip with uniform signal stability is obtained. The gold-plated substrate is prepared by depositing gold nanofilms on a silicon wafer using an electron beam evaporation system.

[0050] In this embodiment, the coverage of 4-ATP on the surface of Ag nanocubes is controlled by adjusting the number of times the ligand molecule 4-ATP is added. In this embodiment, the number of additions is once.

[0051] Figure 1 This is an SEM image of a high-uniformity detection chip assembled from Ag nanocubes with 4-ATP functionalization on the surface in this embodiment. As can be seen from the image, the functionalized Ag nanocubes are densely and uniformly arranged on the gold-plated substrate.

[0052] The Raman signal stability of the 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP detection chip obtained in the above synthesis was characterized using a laser wavelength of 633 nm, a power of 25 mW, and an exposure time of 5 s. The results are as follows: Figure 2 SERS intensity was measured at 30 randomly selected points on the AgNCs@4-ATP detection chip, with a relative standard deviation of only 5.07%. This indicates that the SERS platform has high reproducibility and uniformity. This is because fluctuations caused by randomness and disorder are effectively suppressed when the hotspot density exceeds the threshold. Three types of coupling exist in the chip: adjacent Ag nanocubes, interlayer Ag nanocubes, and Ag nanocubes coupled to the substrate Au film. Due to the morphological characteristics and dense arrangement of the Ag nanocubes, the local photoelectric field strength and hotspot density are extremely high. Therefore, this chip exhibits outstanding signal uniformity and high SERS enhancement, providing a basis for amplifying and reproducing the subtle spectral changes caused by the capture of vanillin molecules.

[0053] Example 2

[0054] A 4-ATP-functionalized Ag nanocube detection chip is prepared using the same method as in Example 1, except that a high 4-ATP surface coverage Ag nanocube sol is prepared in step (2):

[0055] 3.125 mg of uniformly shaped Ag nanocube sol was added to 10 μL of 10 mM ligand molecule 4-ATP solution, and the mixture was magnetically stirred to remove the surfactant PVP and perform surface functionalization on the Ag nanocubes. After stirring, the mixture was centrifuged to obtain 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP. Another 10 μL of 10 mM 4-ATP solution was added, and the mixture was magnetically stirred. After stirring, the mixture was centrifuged to obtain highly surface-functionalized Ag nanocube sol.

[0056] Example 3

[0057] A vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip:

[0058] The 4-ATP-functionalized Ag nanocube detection chip prepared in Example 1 was immersed in vanillin standard solutions of different concentrations to observe the vanillin sensing sensitivity of this strategy. Raman spectroscopy was performed using a 633 nm laser with a power of 25 mW and an exposure time of 5 s. Figure 3The figures, from bottom to top, show the Raman spectral results of the detection chip before and after immersion in a 100 ppb vanillin standard solution. As can be seen from the figures, the new peak observed at 1650 cm⁻¹ can be attributed to the C=N bond formed after the Schiff base reaction between the aldehyde and amino groups. This characteristic peak has been identified in previous studies as key spectral evidence for vanillin capture and has been used in subsequent in-depth research. Figure 4 It can be seen that it is 1077 cm -1 Compared to the reference peak intensity, 1650 cm⁻¹ -1 The spectral intensity at a certain point changes steadily with the concentration of vanillin solution, and the fitted R0 value shows this. 2 The lowest distinguishable concentration of vanillin molecules is 10 ppb, with a value of 0.995.

[0059] By detecting sucrose and magnesium in tea 2+ Fumei Shuang, Ca 2+ The presence of common components such as benzyl alcohol verified the selectivity and anti-interference ability of the aldehyde detection chip in tea in the vanillin detection method. The results are as follows: Figure 5 As shown, the high-uniformity detection chip was placed in a centrifuge tube, and an interfering solution was added. Raman spectroscopy was performed under a 633 nm laser with a power of 25 mW and an exposure time of 5 s. When the detection chip was in a liquid environment where the interfering analytes were all in high concentrations, it only exhibited a low target peak intensity ratio. This demonstrates the strong specificity of this sensing strategy in actual tea samples.

[0060] Example 4

[0061] A vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip was applied to the detection of flavorings and fragrances in tea.

[0062] This embodiment provides a comprehensive and systematic analysis of actual tea samples: a high-uniformity detection chip was placed in a centrifuge tube, and the actual tea sample extract was added for immersion. Raman spectroscopy was performed using a 633 nm laser with a power of 25 mW and an exposure time of 5 s. Using the... Figure 6 Spectroscopic data analysis clearly shows that this strategy can accurately detect vanillin molecules in tea leaves with a high sensitivity of 10 ppb.

[0063] This invention first modifies the surface of Ag nanocubes with the ligand 4-ATP to obtain a surface-functionalized Ag nanocube sol, which exhibits good stability and surface plasmon resonance (SPR). Next, a highly uniform detection chip is fabricated from the nanoparticle sol. After immersion in a vanillin molecule solution, Raman spectroscopy using SERS technology confirms the sensitivity of this invention in the analysis and detection of vanillin molecules. Finally, tests on vanillin in different teas demonstrate the specificity of this method in practical applications. This invention is simple to operate, low in cost, fast in detection, and highly versatile, and can be applied to the preparation of detection chips for flavorings and fragrances in tea.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for vanillin SERS detection based on a 4-ATP-functionalized Ag nanocube detection chip, characterized in that, The process includes the following steps: immersing a 4-ATP-functionalized Ag nanocube detection chip in a solution containing vanillin and performing Raman spectroscopy using SERS. The 4-ATP-functionalized Ag nanocube detection chip is prepared using the following method: (1) Preparation of Ag nanocube sol: Ag nanocube sol was purified by centrifugation to prepare Ag nanocube sol with uniform morphology and surface plasmon resonance effect. (2) Preparation of 4-ATP-functionalized Ag nanocubes: The ligand molecule 4-aminothiophenol (4-ATP) was added to the uniform Ag nanocube sol prepared in step (1). By adjusting the number of times the ligand molecule was added, Ag nanocube sol Ag NCs@4-ATP with adjustable surface functionalization was obtained. (3) Preparation of 4-ATP-functionalized Ag nanocube detection chip: The 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP obtained in step (2) is dropped onto a gold-plated substrate and dried at a constant temperature to prepare a uniform (high signal stability) detection chip.

2. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 1, characterized in that, The laser used in the SERS detection can effectively excite hot spots on the surface of nanoparticles and between particles, as well as the photoinduced charge transfer resonance of the system. The Raman test conditions are a laser wavelength of 500-700 nm, a power of 20-30 mW, and an exposure time of 3-7 s.

3. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 1, characterized in that, In step (1), the Ag nanocube sol was synthesized by adjusting the polyol reduction method.

4. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 1, characterized in that, The particle size of the Ag nanocube sol in step (1) is approximately 70-90 nm.

5. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 1, characterized in that, In step (2), the mass ratio of ligand molecule 4-ATP to Ag nanocube sol is 1:(180-220).

6. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 1, characterized in that, In step (2), the ligand molecule 4-ATP can directly capture the vanillin molecule to be tested, and 4-ATP can be anchored on the surface of Ag NCs through Ag-S bonds.

7. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 5, characterized in that, The 4-ATP has a uniform coverage on the surface of the Ag nanocubes and the ligand molecules are added 1-2 times.

8. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 1, characterized in that, In step (3), the 4-ATP-functionalized Ag nanocube sol Ag NCs@4-ATP is added dropwise according to the target coverage and thickness of the functionalized Ag nanocubes on the substrate surface.

9. The vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in claim 1, characterized in that, The constant temperature drying temperature in step (3) is 20-30 ℃.

10. The application of the vanillin SERS detection method based on a 4-ATP-functionalized Ag nanocube detection chip as described in any one of claims 1-9 in the detection of flavorings and fragrances in tea.

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