A method for quantitative detection of vanillin molecules

Abstract

The present invention discloses a quantitative detection method for vanillin molecules with sensitive detection, simple operation method and high accuracy. The quantitative detection method is realized by any one or two of the following methods: 1) After mixing the test solution containing vanillin molecules with Tollens reagent, then adding 3,3',5,5'-tetramethylbenzidine solution to obtain a mixed solution, measuring the absorbance value with a UV-visible spectrometer, and obtaining the content of vanillin in the test solution according to the linear relationship equation between vanillin solutions with known different concentrations and the absorbance value, wherein the vanillin concentration is 0.5-50 μM; 2) After mixing the test solution containing vanillin molecules with Tollens reagent and dropping it on a silicon wafer, drying it and then measuring the Raman signal with a Raman spectrometer, and obtaining the content of vanillin in the test solution according to the linear relationship equation between vanillin solutions with known different concentrations and the Raman signal intensity, wherein the vanillin concentration is 10-4-10-10 M.

Description

Technical Field

[0001] The present invention relates to a method for quantitatively detecting an organic compound, and particularly to a method for quantitatively detecting vanillin molecules, belonging to the field of biotechnology. Background Art

[0002] Vanillin, also known as 3-methoxy-4-hydroxybenzaldehyde, is an aromatic organic compound extracted from vanilla beans, with the aroma of vanilla beans and a strong milk fragrance, and is widely used in industries such as food, beverages, and cosmetics. However, excessive intake of vanillin can cause dizziness, nausea, and difficulty in breathing. Therefore, the development of rapid and ultrasensitive vanillin detection technology is of great significance for food safety and human health.

[0003] In the past few years, various analytical techniques have been used to determine vanillin, such as gas chromatography, high-performance liquid chromatography, capillary electrophoresis, electrochemical sensors, and chemiluminescence methods. Although these analytical techniques have good selectivity and sensitivity, limitations such as high-cost analytical equipment, complex pretreatment, and time-consuming detection processes have restricted their wide application. In addition, single-mode analysis for quantitative determination may lack reliability and precision. In contrast, dual-mode detection methods have higher accuracy and can flexibly cope with different detection conditions.

[0004] Colorimetry is a rapid, simple, and sensitive technique, usually achieved by monitoring the absorbance change caused by the change in analyte concentration at a specific wavelength. In addition, colorimetry can be "visually" detected through color changes and is suitable for rapid detection. Surface-enhanced Raman scattering (SERS) spectroscopy technology is considered a potential bioanalysis and detection platform due to its high specificity and sensitivity. However, the process of preparing SERS-active substrates in most SERS methods is rather cumbersome. Therefore, it is of great significance to develop a rapid, simple, and effective strategy for preparing SERS-active substrates. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a method for quantitatively detecting vanillin molecules with high sensitivity, simple operation method, and high accuracy.

[0006] To solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a method for quantitatively detecting vanillin molecules, and the quantitative detection method is implemented by any one or two of the following methods:

[0007] 1) Mix the test solution containing vanillin molecules with Tollens' reagent, then add 3,3',5,5'-tetramethylbenzidine solution to obtain a mixed solution. Measure the absorbance value using an ultraviolet-visible spectrometer, and obtain the content of vanillin in the test solution according to the linear relationship equation between vanillin solutions with known different concentrations and the absorbance value. The concentration of vanillin is 0.5 - 50 μM;

[0008] 2) Mix the test solution containing vanillin molecules with Tollens' reagent and drop it on a silicon wafer. After drying, measure the Raman signal using a Raman spectrometer, and obtain the content of vanillin in the test solution according to the linear relationship equation between vanillin solutions with known different concentrations and the Raman signal intensity. The concentration of vanillin is 10 -4 -10 -10 M.

[0009] Among them, the vanillin molecules include vanillin and other derivatives with similar structures to vanillin.

[0010] Among them, the derivatives with similar structures to vanillin include ethyl vanillin, methyl vanillin, isovanillin, benzaldehyde, and phenylacetaldehyde.

[0011] Among them, in method 1), the molar ratio of 3,3',5,5'-tetramethylbenzidine solution to Tollens' reagent is 0.2 - 0.35.

[0012] Among them, in method 1), the reaction time of the test solution containing vanillin molecules and Tollens' reagent is 15 - 60 s.

[0013] Among them, in method 1), the reaction time of 3,3',5,5'-tetramethylbenzidine solution and Tollens' reagent is 30 - 120 s.

[0014] Among them, in method 1), the linear relationship equation between vanillin solutions with known different concentrations and the absorbance value includes one or both of the linear relationship equations between vanillin solutions at 350 nm and 490 nm and the absorbance value.

[0015] Among them, the linear relationship equation between vanillin solution and absorbance value at 350 nm is A = 0.0239C + 0.183 (R 2 = 0.9996), A is the absorbance value at 350 nm, and C is the vanillin concentration.

[0016] Among them, the linear relationship equation between vanillin solution and absorbance value at 490 nm is A = -0.0178C + 1.0559 (R 2 = 0.994), A is the absorbance value at 490 nm, and C is the vanillin concentration.

[0017] Among them, the linear relationship equation between the known vanillin solutions with different concentrations and the Raman signal intensity in the method 2) is I = 3171.5 log C + 35133 (R 2 = 0.992), where I is the Raman signal intensity at 1040 cm -1 and C is the vanillin concentration.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. Sensitive detection of vanillin; 2. The detection method of the present invention is convenient to operate, has higher accuracy in detecting vanillin by the colorimetric method, can achieve rapid detection within three minutes, and the obvious color change can be qualitatively analyzed with the naked eye; 3. In-situ rapid preparation of SERS active substrate by SERS method, wide linear range for detecting vanillin, and lower detection limit, reaching 10 -11 M; 4. The two detection methods can cope with different sample detections. Description of the Drawings

[0019] Figure 1 A is the SEM image of the reaction product of vanillin and Tollens reagent, Figure 1 B is the SEM image of the reaction product of TMB and Tollens reagent, Figure 1 C is the TEM image of the reaction product of vanillin and Tollens reagent, Figure 1 D is the TEM image of the reaction product of TMB and Tollens reagent, Figure 1 E is the DLS image of the reaction product of vanillin and Tollens reagent, Figure 1 F is the DLS image of the reaction product of TMB and Tollens reagent, Figure 1 G is the Zeta potential diagram of the reaction product of vanillin and Tollens reagent and the reaction product of TMB and Tollens reagent;

[0020] Figure 2 A is the mechanism diagram of the reaction of vanillin and TMB with Tollens reagent respectively; Figure 2 B is the mechanism diagram of the detection of vanillin by Tollens reagent and TMB;

[0021] Figure 3 A is the image of the sample solution with different concentrations of vanillin added, Figure 3 B is the absorbance spectrum of the sample solution with different concentrations of vanillin added; Figure 3 C is the linear relationship between the sample solution and the vanillin concentration at 350 nm, Figure 3 D is the linear relationship between the sample solution and the vanillin concentration at 490 nm;

[0022] Figure 4 A is the change in absorbance values of different reactants, Figure 4 B is the TEM image of the Tollens reagent-vanillin-TMB system;

[0023] Figure 5 Figure A shows the absorbance value changes of TMB and Tollens reagent solutions with different molar ratios. Figure 5 Figure B shows the absorbance value changes of Tollens reagent and vanillin solutions at different reaction times. Figure 5 Figure C shows the absorbance value changes of Tollens reagent and TMB solutions at different reaction times.

[0024] Figure 6 represents the absorbance at 350 nm of the system after the reaction of different analytes with Tollens reagent;

[0025] Figure 7 Figure A shows the Raman signal spectra of different reactants. Figure 7 Figure B shows the changes in the Raman scattering spectra of the reaction products of vanillin and Tollens reagent at different concentrations. Figure 7 Figure C shows the linear relationship between the Raman signal intensity at 1040 cm -1 and the concentration of vanillin.

[0026] Figure 8 Figure A shows the Raman scattering spectra of the reaction products of different analytes and Tollens reagent. Figure 8 Figure B shows the absorbance at 1040 cm -1 of the reaction products of different analytes and Tollens reagent.

[0027] Figure 9 Figure A shows the recovery rates of the target vanillin at different concentrations in milk powder samples. Figure 9 Figure B shows the recovery rates of the target vanillin at different concentrations in coffee samples. Figure 9 Figure C shows the Raman scattering spectra of the reaction products of vanillin and Tollens reagent in different matrices. Detailed implementation methods

[0028] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0029] Reagents and instruments used in this experiment:

[0030] Silver nitrate and potassium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ammonia water, 3,3',5,5'-tetramethylbenzidine, dimethyl sulfoxide, vanillin, ethyl vanillin, methyl vanillin, and isovanillin were purchased from Aladdin Industrial Corporation (Shanghai, China). Benzaldehyde was purchased from Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China), and phenylacetaldehyde was purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China).

[0031] Navo Nano SEM450 Nova field emission scanning electron microscope (USA), Talos F200X high-resolution transmission electron microscope (Thermo Fisher), NanoBrook Omni multi-angle particle size and zeta potential analyzer (Brookhaven), Cary 60 UV-Vis ultraviolet-visible spectrophotometer (Agilent), Renishaw InVia Raman Microscopy confocal micro-Raman spectrometer (Renishaw).

[0032] In all embodiments of the present invention, Tollens reagent was obtained by successively mixing 500 μL of 10 mM silver nitrate solution, 65 μL of 80 mM potassium hydroxide solution, 35 μL of 1.3% ammonia water, and 400 μL of ultrapure water, and the concentration of Tollens reagent was 5 mM; 3,3',5,5'-tetramethylbenzidine (TMB) solution was prepared in dimethyl sulfoxide (DMSO), and the concentration of TMB was 1 mM; the remaining solutions were all dissolved in distilled water.

[0033] Example 1 Synthesis and determination of two kinds of silver nanoparticles (AgNPs)

[0034] Take 12 μL of 5 mM Tollens reagent in a centrifuge tube, add 200 μL of 100 μM vanillin, react at room temperature for 1 minute, and then dilute to 400 μL with ultrapure water to obtain the first group of colorless AgNPs solution; take another 12 μL of 5 mM Tollens reagent in a centrifuge tube, add 18 μL of 1 mM TMB, react at room temperature for 2 minutes, and then dilute to 400 μL with ultrapure water to obtain the second group of pink AgNPs solution. Finally, SEM and TEM image measurements were performed on the two groups of AgNPs solutions respectively. As Figure 1 shown in A and 1C, vanillin reduces Tollens reagent to obtain silver nanoparticles (Ag 45 NPs) with a size of about 45 nm. The Ag 45 NPs are roughly spherical, with a small size and a smooth surface; as Figure 1 shown in B and 1D, TMB reduces Tollens reagent to obtain silver nanoparticles (Ag 100 NPs) with a size of about 100 nm. The Ag 100 NPs are irregularly spherical, with a large size and a wrinkled surface. Then, dynamic light scattering (DLS) experiments and zeta potential value measurements were performed on the Ag 45 NPs solution and the Ag 100 NPs solution respectively. As Figure 1 shown in E and 1F, the average diameter of Ag 45 NPs is 45 nm, and the average diameter of Ag 100 NPs is 100 nm; as Figure 1 shown in G, Ag 45The Zeta potential value of NPs was -48.4 mV, and that of Ag 100 NPs was -22.1 mV. The above results indicate that vanillin and TMB respectively reduced Tollens' reagent to generate two kinds of AgNPs with different morphologies and sizes, that is, vanillin reduced Tollens' reagent to generate Ag 45 NPs, and the solution was colorless and observable with the naked eye; TMB reduced Tollens' reagent to generate Ag 100 NPs, and the solution was pink and observable with the naked eye.

[0035] Example 2 Colorimetric Detection of Vanillin

[0036] According to Figure 2 the steps and mechanism shown, first take 12 μL of 5 mM Tollens' reagent in a centrifuge tube, and add 200 μL of vanillin with concentrations of 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM respectively, and react at room temperature for 1 minute; then, add 18 μL of 1 mM TMB solution to the mixture, react at room temperature for 2 minutes, and finally dilute to 400 μL with ultrapure water to obtain a sample solution. The absorbance of the sample solution was measured in the wavelength range of 200 - 800 nm, and the absorbance of the solution at wavelengths of 350 nm and 490 nm was measured with a UV-visible spectrophotometer. As Figure 2 shown, vanillin reduced Tollens' reagent to obtain Ag 45 NPs, and the solution was colorless and had an ultraviolet absorption signal at 350 nm; TMB reduced Tollens' reagent to obtain Ag 100 NPs, and the solution was pink and had an ultraviolet absorption signal at 490 nm. Therefore, first add the target vanillin to a certain amount of Tollens' reagent to obtain Ag 45 NPs, and then add TMB to the mixed solution to reduce the remaining Tollens' reagent to obtain Ag 100 NPs. As the concentration of the target vanillin increased, the more Ag 45 NPs were obtained, and the fewer Ag 100 NPs were obtained. As Figure 3 shown in A, as the final concentration of the target vanillin increased from 0.5 μM to 50 μM, the color of the sample solution changed from pink to colorless and was observable with the naked eye; as Figure 3 shown in B, as the concentration of vanillin increased, the absorbance value of the sample solution at 350 nm gradually increased, while the absorbance value at 490 nm gradually decreased; as Figure 3 shown in C, the linear relationship between the absorbance value of the sample solution at 350 nm and the concentration of vanillin, and its linear equation is A = 0.0239C + 0.183 (R 2= 0.9996), where A is the absorbance at 350 nm, C is the concentration of vanillin, ranging from 0.5 μM to 50 μM, and the detection limit is 0.411 μM; as Figure 3 shown in D, the linear relationship between the absorbance of the sample solution and the concentration of vanillin at 490 nm, and its linear equation is A = -0.0178C + 1.0559 (R 2 = 0.994). As the concentration of vanillin increases, and at this time the concentration range of vanillin is 0.5 μM to 50 μM, and the detection limit is 0.482 μM.

[0037] Example 3 Verification of Absorbance Intensity under Different Reactant Mixing Conditions

[0038] The absorbance of Tollens reagent, TMB, and the detection molecule vanillin alone or in combination of two or three of them was detected respectively. Based on Example 2, the following solutions were prepared separately and distilled water was added to make the total reaction volume 400 μL: (a) Take 12 μL of 5 mM Tollens reagent; (b) Take 100 μL of 100 μM vanillin; (c) Take 18 μL of 1 mM TMB; (d) Take 100 μL of 100 μM vanillin and then add 18 μL of 1 mM TMB for reaction; (e) Take 12 μL of 5 mM Tollens reagent and then add 100 μL of 100 μM vanillin; (f) Take 12 μL of 5 mM Tollens reagent and then add 18 μL of 1 mM TMB for reaction; (g) Take 12 μL of 5 mM Tollens reagent, then add 100 μL of 100 μM vanillin for reaction, and then add 18 μL of 1 mM TMB for reaction; The above solutions were measured by ultraviolet-visible absorption spectroscopy based on Example 2.

[0039] As Figure 4 shown in A, the reaction product of vanillin and Tollens reagent has an ultraviolet absorption signal at 350 nm; after TMB reacts with Tollens reagent, there is an ultraviolet absorption signal at 490 nm; after Tollens reagent reacts with vanillin and TMB successively, the absorbance at 350 nm remains unchanged, while the absorbance at 490 nm decreases. As Figure 4 shown in B, after Tollens reagent reacts with vanillin and TMB successively, two sizes of AgNPs coexist in the system. From the above detection results, it can be seen that vanillin and TMB can reduce Tollens reagent in the same system to synthesize two kinds of AgNPs with different maximum absorption wavelengths. In the Tollens reagent-vanillin-TMB system, since vanillin first consumes part of Tollens reagent to synthesize Ag 45 NPs, the amount of Tollens reagent that can react with TMB decreases, and then the amount of synthesized Ag 100 NPs decreases, so the absorbance at 490 nm also decreases.

[0040] Example 4 Selection of Optimal Reaction Conditions for Colorimetric Detection of Target Molecules

[0041] To make the detection method feasible, it is necessary to ensure that TMB can react completely with Tollens' reagent in the absence of vanillin. Therefore, the concentration ratio of TMB to Tollens' reagent needs to be optimized. Take 12 μL of 5 mM Tollens' reagent, and then add 12 μL, 15 μL, 18 μL, and 21 μL of 1 mM TMB solution respectively. React at room temperature for 2 minutes, and then dilute to 400 μL with ultrapure water. Measure the absorbance values of the system at 490 nm. As Figure 5 shown in A, when the molar ratio of TMB to Tollens' reagent is 0.30, the system reacts completely. Further, optimize the reaction time of Tollens'-vanillin reaction. Take 12 μL of 5 mM Tollens' reagent, and then add 200 μL of 40 μM vanillin. Select a series of reaction times (15 s, 30 s, 45 s, 60 s), and then dilute to 400 μL with ultrapure water. Measure the absorbance values of the system at 350 nm. As Figure 5 shown in B, the reaction between Tollens' reagent and vanillin reaches equilibrium at 60 s. Optimize the reaction time of Tollens' reagent and TMB reaction. Take 12 μL of 5 mM Tollens' reagent, and then add 12 μL, 15 μL, 18 μL, and 21 μL of 1 mM TMB solution respectively. Select a series of reaction times (30 s, 60 s, 90 s, 120 s), and then dilute to 400 μL with ultrapure water. Measure the absorbance values of the system at 490 nm. As Figure 5 shown in C, the reaction between Tollens' reagent and TMB reaches equilibrium at 120 s. Therefore, when the molar ratio of TMB to Tollens' reagent is 0.30, the reaction between Tollens' reagent and vanillin is 60 s, and the reaction between Tollens' reagent and TMB is 120 s, the system reacts completely.

[0042] Example 5 Selectivity of Colorimetric Detection of Target Molecules

[0043] Take 12 μL of 5 mM Tollens' reagent, and then add 200 μL of 40 μM vanillin, 40 μM ethyl vanillin, 40 μM methyl vanillin, 40 μM isovanillin, 40 μM benzaldehyde, and 40 μM phenylacetaldehyde respectively. Then dilute to 400 μL with ultrapure water. React at room temperature for 1 minute, and measure the absorbance values of different systems at 350 nm. As Figure 6As shown, among the aldehyde derivatives with a structure similar to vanillin, the system containing vanillin and ethyl vanillin has a strong ultraviolet absorption peak intensity at 350 nm, while the ultraviolet absorption peak intensity at 350 nm in other systems is very low. Since different aldehydes require different reaction times and temperatures to reduce Tollens' reagent, vanillin and ethyl vanillin can rapidly reduce Tollens' reagent at room temperature within one minute to obtain a large number of silver nanoparticles, while other aldehyde derivatives with similar structures cannot synthesize a large number of silver nanoparticles under the same conditions. Therefore, this quantitative detection method can specifically detect vanillin.

[0044] Example 6 Detection of the target substance vanillin by surface-enhanced Raman scattering (SERS) spectroscopy

[0045] Take 200 μL of 1 mM Tollens' reagent in a centrifuge tube, and add 40 μL of vanillin with concentrations of 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M respectively, and react for 1 minute at room temperature; finally, dilute to 400 μL with ultrapure water. Take 20 μL of the sample and drop it on a silicon wafer. After drying at room temperature, measure the Raman signal using a Raman spectrometer. The excitation wavelength is 785 nm, the laser power is 1%, and the exposure time is 10 s. Figure 7 A shows the Raman spectra of Tollens' reagent, vanillin, and the Tollens'-vanillin system. It can be seen from the figure that the Raman signals of Tollens' reagent and vanillin itself are very weak, while the Raman signal of the Tollens'-vanillin system is very strong, indicating that the silver nanoparticles Ag 45 NPs obtained by reducing Tollens' reagent with vanillin can be used as a SERS substrate to enhance the Raman signal of the system. Figure 7 B shows that as the concentration of vanillin increases, the Raman signal intensity of the system also increases. Therefore, the peak at 1040 cm -1 with the largest Raman signal intensity is selected for the quantitative detection of vanillin. Figure 7 C shows the linear relationship between the Raman signal intensity at 1040 cm -1 and the concentration of vanillin. Its linear equation is I = 3171.5 log C + 35133 (R 2 = 0.992), where I is the Raman signal intensity at 1040 cm -1 , C is the concentration of vanillin, the range is 10 -10 M to 10 -4 M, and the detection limit is 2.58×10 -11M. As the concentration of vanillin increases, the Raman signal intensity of the sample solution gradually increases. This quantitative detection method also provides a new idea for the in-situ rapid preparation of SERS substrates.

[0046] Example 7 Selectivity of Detecting Target Molecules by Surface-Enhanced Raman Scattering Method

[0047] Based on the step conditions in Example 6, replace the target molecule vanillin (concentration 10 -3 M) with ethyl vanillin at a concentration of 10 -3 M, methyl vanillin at a concentration of 10 -3 M, isovanillin at a concentration of 10 -3 M, benzaldehyde at a concentration of 10 -3 M, and phenylacetaldehyde at a concentration of 10 -3 M, measure the Raman scattering spectra of different systems and the Raman signal intensity at 1040 cm -1 . As can be seen from Figure 8 A, for the aldehyde derivatives similar in structure to vanillin, after reacting with Tollens' reagent, the positions of the characteristic peaks of the Raman scattering in the system are similar to those of vanillin, but the signal intensities of the characteristic peaks are different. Figure 8 B shows that the Raman signal intensity of the reaction products of the aldehyde derivatives similar in structure to vanillin and Tollens' reagent at 1040 cm -1 is much lower than that of vanillin. This is because the reaction conditions required for different aldehydes to reduce Tollens' reagent are different, and the amount of silver nanoparticles synthesized under the same conditions is different, which leads to different SERS enhancement effects, and the different molecular structures of vanillin and its aldehyde derivatives similar in structure will cause differences in their Raman spectra. Therefore, this quantitative detection method can specifically detect vanillin.

[0048] Example 8 Detection of Spiked Recovery of Vanillin in Milk Powder and Coffee

[0049] To apply this method to actual samples, instant whole milk powder and coffee (both containing vanillin, < 0.5 mg / L in the ingredients) were purchased from a supermarket. The actual samples (10 mg / ml) were dissolved and shaken with water, and the supernatant was taken for use. The spiking experiment was carried out within the linear range, and the actual samples were added at 10% of the total volume.

[0050] The experimental steps for the spiked recovery of vanillin by colorimetry are as follows: Take 12 μL of 5 mM Tollens reagent in a centrifuge tube, add 40 μL of the actual sample solution containing vanillin at concentrations of 50 μM, 250 μM, and 500 μM respectively, and react at room temperature for 1 minute; then, add 18 μL of 1 mM TMB to the mixture and react at room temperature for 2 minutes. Finally, dilute it to 400 μL with ultrapure water to obtain vanillin solutions with final concentrations of 5 μM, 25 μM, and 50 μM. The absorbance of the sample is measured in the wavelength range of 200 - 800 nm. Use a UV-visible spectrometer to measure the absorbance values of the solution at wavelengths of 350 nm and 490 nm, and calculate the recovered concentration (Found) and recovery rate (Recovery) of vanillin in the samples with different vanillin concentrations (Added). The experimental results are shown in Table 1, Figure 9 A Figure 9 and

[0051] Table 1

[0052]

[0053] The experimental steps for the spiked recovery of vanillin by SERS are as follows: Take 200 μL of 1 mM Tollens reagent in a centrifuge tube, add 40 μL of the actual sample solution containing vanillin at concentrations of 10 -9 mM, 10 -6 mM, 10 -3 mM respectively, and react at room temperature for 1 minute. Finally, dilute it to 400 μL with ultrapure water. Take 20 μL of the sample and drop it on a silicon wafer. After drying at room temperature, use a Raman spectrometer to measure the Raman signal. Figure 9 Figure C shows the Raman scattering spectra of the reaction products of vanillin solution and vanillin solution added to milk powder and coffee matrix with Tollens reagent. It can be seen from the figure that the milk powder and coffee matrix do not affect the peak at 1040 cm -1 of the product, indicating that the milk powder and coffee matrix do not interfere with this detection system.

[0054] Table 2

[0055]

[0056] The results of the standard addition recovery experiment are shown in Table 2. The spiked recoveries of this detection method in milk powder and coffee samples are between 94.5% and 110.9%, and the coefficient of variation (CV) is less than 12.3%, indicating that the prepared method has good performance and can be used to detect vanillin in milk powder and coffee samples.

[0057] Comparative example

[0058] Compare with the research results of Wang Yongping et al. (Synthesis of nitrogen-doped carbon dots and their application in the detection of vanillin, 2019) (Serial number 1), Mohamed Amin Elaguech et al. (Label-free sensitive determination of vanillin in food samples based on nanopore-based aptazyme sensors, 2022) (Serial number 2), Jiang Jiamin et al. (Quantitative recoverable SERS detection induced by tunable Raman internal standard embedded in nanosilicon, 2022) (Serial number 3), Zhang Yan et al. (Bimetallic molecularly imprinted nanozyme: a dual-mode detection platform, 2022) (Serial numbers 4 and 5).

[0059] Table 3

[0060]

[0061] As shown in Table 3, among the above five existing detection methods, the detection limit of the electrochemical method of Serial number 2 is the lowest, which is 500 pM, much higher than 25.8 pM in the method of the present invention (Serial number 7), and the accuracy is far less than that of the method of the present invention; the response time (3 min) of the method of the present invention (Serial number 6) is also faster than the above six detection methods, and the operation is simple and fast; in addition, the present invention can also perform qualitative analysis on vanillin in the sample to be tested by colorimetry (Serial number 6), and then perform quantitative analysis by SERS (Serial number 7). The above schemes can all achieve efficient, fast and accurate detection of vanillin, and the detection effect is much higher than that of the prior art.

Claims

1. A quantitative detection method for vanillin molecules, characterized in that, the quantitative detection method comprises the following steps: after mixing a test solution containing vanillin molecules with Tollens' reagent, then adding a 3,3',5,5'-tetramethylbenzidine solution to obtain a mixed solution, measuring the absorbance value using an ultraviolet-visible spectrometer, and obtaining the content of vanillin in the test solution according to the linear relationship equation between vanillin solutions with known different concentrations and the absorbance value, wherein the vanillin concentration is 0.5 - 50 μM.

2. The quantitative detection method for vanillin molecules according to claim 1, characterized in that, the vanillin molecules include vanillin and other aldehyde derivatives with similar structures to vanillin.

3. The quantitative detection method for vanillin molecules according to claim 1, characterized in that, the derivatives with similar structures to vanillin include ethyl vanillin, methyl vanillin, isovanillin, benzaldehyde, and phenylacetaldehyde.

4. The quantitative detection method for vanillin molecules according to claim 1, characterized in that, in method 1), the molar ratio of the 3,3',5,5'-tetramethylbenzidine solution to Tollens' reagent is 0.2 - 0.

35.

5. The quantitative detection method for vanillin molecules according to claim 1, characterized in that, in method 1), the reaction time of the test solution containing vanillin molecules and Tollens' reagent is 15 - 60 s.

6. The quantitative detection method for vanillin molecules according to claim 1, characterized in that, in method 1), the reaction time of the 3,3',5,5'-tetramethylbenzidine solution and Tollens' reagent is 30 - 120 s.

7. The quantitative detection method for vanillin molecules according to claim 1, characterized in that, in method 1), the linear relationship equation between vanillin solutions with known different concentrations and the absorbance value includes one or both of the linear relationship equations between vanillin solutions at 350 nm and 490 nm and the absorbance value.

8. The quantitative detection method for vanillin molecules according to claim 7, characterized in that, the linear relationship equation between the vanillin solution and the absorbance value at 350 nm is A = 0.0239C + 0.183, where A is the absorbance value at 350 nm and C is the vanillin concentration.

9. The quantitative detection method for vanillin molecules according to claim 7, characterized in that, the linear relationship equation between the vanillin solution and the absorbance value at 490 nm is A = -0.0178C + 1.0559, where A is the absorbance value at 490 nm and C is the vanillin concentration.

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