CN114354581B - Analysis method for detecting vanillin molecules based on host-guest interaction chemiluminescence method and application - Google Patents
Analysis method for detecting vanillin molecules based on host-guest interaction chemiluminescence method and application Download PDFInfo
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses an analysis method for detecting vanillin molecules based on a host-guest interaction chemiluminescence method and application thereof, wherein beta-cyclodextrin solution and luminol solution are mixed, vanillin solution is added to obtain mixed solution, and then H is mixed in situ 2 O 2 The solution is added into the mixed solution to generate luminol chemiluminescence reaction, the electroluminescent analyzer is adopted to measure the change of CL signal along with time at room temperature, and the vanillin content is obtained according to the chemiluminescence intensity. The invention is based on luminol-beta-cyclodextrin-H 2 O 2 The method for realizing sensitive detection of vanillin by chemiluminescent intensity variation and application thereof is characterized in that no catalyst such as metal cobalt ion is added to catalyze H 2 O 2 Instead, the target molecule vanillin directly acts like a catalyst to catalyze the chemiluminescence of luminol. The detection method has the advantages of convenient and simple operation, high sensitivity and high detection speed.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an analysis method for detecting vanillin molecules based on a host-guest interaction chemiluminescence method and application thereof.
Background
At present, vanillin (4-hydroxy-3-methoxybenzaldehyde) has been widely applied to cosmetics, tobacco, milk powder and baked foods due to the unique fragrance, and naturally exists in asparagus, coffee and vanilla, has the sweet bean taste of powdery air, can keep fragrance for a long time, has the effect of increasing and stabilizing fragrance, and is one of the most widely applied fragrances in the world. Vanillin has been reported to have good health benefits for sickle cell anemia patients, such as reduced heart disease mortality and anti-adhesion. However, excessive intake of vanillin can cause headache, nausea and vomiting, and can also affect liver and kidney function. In recent years, the problem of exceeding standard of vanillin in milk powder is attracting attention, and based on the consideration of human health, it is important to explore a rapid and simple method for detecting vanillin.
The detection of vanillin becomes very important due to its influence on human health. There are currently thin layer chromatography, gas chromatography, electrochemical detection, solid phase microextraction-gas chromatography-mass spectrometry, and the like for detecting vanillin, which have many disadvantages although they achieve satisfactory results. If the instrument is complex to operate, the time consumption is long, the cost is high, and the detection is insensitive.
Disclosure of Invention
The invention aims to: the invention aims to provide an analysis method for detecting vanillin molecules based on a host-guest interaction chemiluminescence method.
The invention also solves the technical problem of providing the application of the analysis method in food field detection.
The technical scheme is as follows: in order to solve the technical problems, the invention provides an analysis method for detecting vanillin molecules based on a host-guest interaction chemiluminescence method, which comprises the following steps: mixing beta-cyclodextrin solution and luminol solution, adding vanillin solution to obtain mixed solution, and mixing H in situ 2 O 2 The solution is added into the mixed solution to generate luminol chemiluminescence reaction, the electroluminescent analyzer is adopted to measure the change of CL signal along with time at room temperature, and the vanillin content is obtained according to the chemiluminescence intensity.
Wherein the final concentration of the vanillin solution is 5-110 mu M.
Wherein the final concentration of the luminol solution is 0.1-4 mu M.
Wherein the final concentration of the beta-cyclodextrin solution is 5-70 mu M.
Wherein the H is 2 O 2 The final concentration of the solution is 0-5 mM.
Wherein the in-situ mixing mode is to fix the mixed solution in situ and then adopt a syringe to fix H 2 O 2 The solution was added.
The invention also discloses application of the analysis method for detecting vanillin molecules based on the host-guest interaction chemiluminescence method in the field of food detection.
Wherein the food is milk powder or biscuit.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the invention is based on luminol-beta-cyclodextrin-H 2 O 2 The method for realizing sensitive detection of vanillin by chemiluminescent intensity variation and application thereof is characterized in that no catalyst such as metal cobalt ion is added to catalyze H 2 O 2 Instead, the target molecule vanillin directly acts like a catalyst to catalyze the chemiluminescence of luminol. The detection method has the advantages of convenient and simple operation, high sensitivity and high detection speed.
Drawings
FIG. 1 shows a flow chart of a chemiluminescent assay for detecting the target molecule vanillin based on host-guest interactions and a schematic diagram showing chemiluminescence based on host-guest interactions;
FIG. 2 shows a process diagram for detecting target molecule vanillin based on host-guest interaction chemiluminescence;
FIG. 3A shows the feasibility of detecting vanillin molecules by a chemiluminescent method based on interaction of host and guest, experimental purposes are achieved according to chemiluminescent intensity changes, and FIG. 3B shows chemiluminescent intensity signal changes under pairwise combination of different reactants;
FIG. 4 shows a species verification and content comparison of ROS generated within an analytical method system for detecting vanillin based on a host-guest interaction chemiluminescence method;
FIG. 5 shows a conventional metal cobalt ion catalyzed luminol-H 2 O 2 Comparison of CL intensities of chemiluminescent system and detection of Vanillin system based on host-guest interaction chemiluminescent method, FIG. 5A shows that luminol-beta-cyclodextrin-H at a concentration of 1. Mu.M and target molecule Vanillin of 1. Mu.M-110. Mu.M 2 O 2 The chemiluminescent intensity of the system is shown in FIG. 5B, which shows luminol at 1. Mu.M and metal cobalt ion catalyzed luminol-H 2 O 2 The chemiluminescent intensity of the chemiluminescent system is shown in FIG. 5C, which shows luminol at 0.75. Mu.M and metal cobalt ion catalyzed luminol-H 2 O 2 The chemiluminescent intensity of the chemiluminescent system is shown in FIG. 5DLuminol catalyzed by luminol-H at 0.5. Mu.M and metallic cobalt ion 2 The chemiluminescent intensity of the O2 chemiluminescent system;
FIG. 6 shows a conventional metal cobalt ion catalyzed luminol-H 2 O 2 The chemiluminescent system is compared with the detection of the species of ROS generated in the vanillin system based on a host-guest interaction chemiluminescent method;
FIG. 7 shows a comparison of species that generate ROS in a system in the presence or absence of the target molecule vanillin based on a host-guest interaction chemiluminescence method;
FIG. 8 shows a comparison of chemical shift changes in the nuclear magnetic pattern of luminol in a luminol system (a), a luminol-beta-cyclodextrin system (b), a luminol-beta-cyclodextrin-target molecule vanillin system (c) in a heavy water solvent;
FIG. 9 shows a comparison of chemical shift changes in the nuclear magnetic profile of vanillin in a heavy water solvent vanillin system (d), vanillin-beta-cyclodextrin system (e), luminol-beta-cyclodextrin-target molecule vanillin system (f);
FIG. 10 shows the system CL intensity variation under different conditions, FIG. 10A shows the system CL intensity variation with beta-cyclodextrin concentration of 5. Mu.M to 70. Mu.M, FIG. 10B shows the system CL intensity variation with luminol concentration of 0.1. Mu.M to 4. Mu.M, and FIG. 10C shows the system CL intensity variation with target molecule vanillin addition with reaction time of 5 minutes to 120 minutes;
FIG. 11 shows detection of changes in chemical intensity signal of target molecules vanilla 1. Mu.M-110. Mu.M based on host-guest interaction chemiluminescence method;
FIG. 12 shows the selectivity of detection of targets based on the host-guest interaction chemiluminescence method.
Detailed Description
The luminol solution in all the examples of the invention is dissolved by 0.1M NaOH solution and is used after being placed for one week; h 2 O 2 Is a stock solution obtained by diluting with 50mM NaOH; 9, 10-anthracenediyl-bis (methylene) malonic acid (ADA) in DMSO; the rest solution is obtained by dissolving in distilled water.
Example 1 detection of Vanillin, a target, based on a host-guest interaction chemiluminescent method
Firstly, 25 mu L of 1mM beta-cyclodextrin solution and 5 mu L of 0.1mM luminol solution are taken in a centrifuge tube, and the solutions are mixed and oscillated for 30 minutes at room temperature by a constant temperature oscillator; next, 25. Mu.L of 1mM vanillin solution was added to the above system solution, 445. Mu.L of distilled water was added thereto, the total reaction volume was 500. Mu.L, and the mixing and shaking with a constant temperature shaker was continued at room temperature for 30 minutes.
On the basis of the preparation of the above solution, 100. Mu.L of the reaction solution was taken in a centrifuge tube, and H was then immobilized by an in-situ mixing using a self-made immobilization device (as shown in FIG. 2, immobilization of the centrifuge tube by a hollow cylindrical object) 2 O 2 (1 mM, 100. Mu.L) was injected into the above centrifuge tube by a syringe to induce luminol chemiluminescence, and the change of CL signal with time was measured at room temperature by an MPI-E type electroluminescent analyzer (Sesamite analysis apparatus Co., ltd.) to obtain the vanillin content based on the chemiluminescence intensity. FIGS. 1 and 2 show the flow of detection of target molecule vanillin in a homemade in situ mixed chemiluminescent measurement mode; FIG. 11 shows a quantitative analysis of the change in luminous intensity of vanillin as the detection target, whose linear equation is I CL ＝146.7C VAN +2565.47, where I CL Representing the luminous intensity, C VAN Represents the concentration of vanillin, R 2 ＝0.994。
Example 2: CL strength verification under different reactant mixing conditions
And detecting the chemiluminescent intensities of luminol, beta-cyclodextrin and molecular vanillin when the luminol, the beta-cyclodextrin and the molecular vanillin exist alone or are mixed. Based on example 1, the following solutions were prepared separately, and distilled water was added separately to a total reaction volume of 500 μl each: (a) taking 5 μl of 0.1mM luminol; (b) taking 25 μl of 1mM beta-cyclodextrin; (c) taking 25 μl of 1mM vanillin; (d) Taking 5 mu L of 0.1mM luminol, and adding 25 mu L of 1mM beta-cyclodextrin for reaction; (e) Taking 5 mu L of 0.1mM luminol, and then adding 25 mu L of 1mM vanillin for reaction; (f) Taking 25 mu L of 1mM beta-cyclodextrin, and adding 25 mu L of 1mM vanillin for reaction; (g) Taking 5. Mu.L of 0.1mM luminol, then adding 25. Mu.L of 1mM beta-cyclodextrin, and then adding 25. Mu.L of 1mM vanillin for reaction; the above solutions were subjected to CL strength measurement based on example 1. FIG. 3A shows the feasibility of the reaction, and FIG. 3B shows a graph comparing the chemiluminescent intensities of the reactants in combination. The detection results show that the CL strength is enhanced in the presence of the beta-cyclodextrin, which indicates that the cavity of the beta-cyclodextrin provides a stable environment for the excited state of luminol, so that the luminous intensity is enhanced; and demonstrated that the luminescence intensity was maximized only in the presence of beta-cyclodextrin and vanillin at the same time.
Example 3: traditional metal cobalt ion catalyzed luminol-H 2 O 2 CL intensity contrast of chemiluminescent system and detection of vanillin system based on host-guest interaction chemiluminescent method
Based on the results of example 2, a comparison of the CL intensities of the two systems is next carried out, as shown in FIG. 5A. Based on example 1, a series of reaction solutions were prepared at final concentrations of vanillin (final concentration 1. Mu.M) of 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 70. Mu.M, 90. Mu.M, 110. Mu.M, and then CL intensities were measured; FIG. 5B shows the formulation of a series of Co at the concentration of luminol (final concentration 1. Mu.M) 2+ The final concentration of the reaction solution was (0.1. Mu.M, 1. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M, 10. Mu.M), and the CL intensity of the reaction system was measured; FIG. 5C shows the formulation of a series of Co at the concentration of luminol (final concentration 0.75. Mu.M) 2+ The final concentration of the reaction solution was (0.1. Mu.M, 1. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M, 10. Mu.M), and the CL intensity of the reaction system was measured; FIG. 5D shows the formulation of a series of Co at the concentration of luminol (final concentration 0.5. Mu.M) 2+ The final concentration of the reaction solution was (0.1. Mu.M, 1. Mu.M, 3. Mu.M, 5. Mu.M, 7. Mu.M, 10. Mu.M), and the CL intensity of the reaction system was measured; the comparison shows that the intensity of CL produced in the presence of vanillin molecules is similar to that produced when catalyzed by cobalt ions.
Example 4: reactive Oxygen Species (ROS) contrast generated within the system
According to the results of example 3, luminol-beta-cyclodextrin-vanillin-H was next carried out 2 O 2 System and traditional luminol-H 2 O 2 -Co 2+ A system; luminol-beta-cyclodextrin-vanillin-H 2 O2 system and luminol-beta-cyclodextrinessence-H 2 O 2 Qualitative analysis of ROS produced by the system.
1) First, a solution of β -cyclodextrin (1 mM, 25. Mu.L) and luminol (0.1 mM, 5. Mu.L) was mixed and shaken with a constant temperature shaker at room temperature for 30 minutes; next, a vanillin solution (50. Mu.M) was added to the above system solution, the total reaction volume was 500. Mu.L, and mixed and shaken with a constant temperature shaker at room temperature for 30 minutes; taking 100. Mu.L of the reaction solution in a centrifuge tube, adding the prepared ROS scavenger (wherein Ascorbic Acid (AA) (1 mM, 10. Mu.L) is the total ROS scavenger, thiourea (Thiourea) (1 mM, 10. Mu.L) is the hydroxyl radical scavenger, 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ADA) (1 mM, 10. Mu.L) is the singlet oxygen scavenger, and SOD enzyme (2 mg/ml, 10. Mu.L) is the superoxide anion scavenger) to the reaction solution, mixing uniformly, and then using the fixing device made in example 1, mixing H in situ 2 O 2 (1 mM, 100. Mu.L) is injected into a centrifuge tube by a syringe to induce luminol chemiluminescence, and the change in CL signal with time is measured at room temperature by an MPI-E type electroluminescent analyzer (Sian Rumex analytical instruments Co., ltd.). As can be seen from the results shown in FIG. 4, there is ROS production during the reaction, which is dominated by hydroxyl radicals and superoxide anions, with small amounts of singlet oxygen.
2) Firstly, taking 25 mu L of 1mM beta-cyclodextrin solution and 5 mu L of 0.1mM luminol solution in a centrifuge tube, mixing and oscillating the solutions at room temperature by a constant temperature oscillator for 30 minutes, and adding distilled water until the total reaction volume is 500 mu L; 100. Mu.L of the reaction solution was placed in a centrifuge tube, and then the prepared ROS scavenger was added to the reaction solution as described in 1), respectively, followed by H using the home-made in situ mixing reaction scheme of FIG. 2 2 O 2 (1 mM, 100. Mu.L) was injected from a syringe into a centrifuge tube containing the reaction system to induce luminol chemiluminescence, and the change in CL signal with time was measured at room temperature by an MPI-E type electroluminescent analyzer (Sesamite analysis instruments Co., ltd.).
3) First, 25. Mu.L of a 1mM beta-cyclodextrin solution and 5. Mu.L of a 0.1mM luminol solution were placed in a centrifuge tube, and then 1mM Co was added 2+ 5. Mu.L of distilled water was added to a total reaction volume of 500. Mu.L; the solution was mixed and shaken at room temperature with a constant temperature shaker30 minutes, 100. Mu.L of the reaction solution was taken in a centrifuge tube, and then the prepared ROS scavenger was added to the reaction solution as described in 1), respectively, and H was then added by in situ mixing using a self-made fixture (FIG. 2) 2 O 2 (1 mM, 100. Mu.L) is injected into a centrifuge tube by a syringe to induce luminol chemiluminescence, and the change in CL signal with time is measured at room temperature by an MPI-E type electroluminescent analyzer (Sian Rumex analytical instruments Co., ltd.).
FIGS. 6-7 show conventional luminol-H, respectively 2 O 2 -Co 2+ Systems based on host-guest interaction chemiluminescent luminol-beta-cyclodextrin-H 2 O2 system for detecting vanillin and luminol-beta-cyclodextrin-H alone 2 O 2 ROS species comparison in the absence of target molecule from the system. The results showed that chemiluminescent luminol-beta-cyclodextrin-H was based on host guest interaction 2 The increase of ROS in O2 system in the presence of target molecule vanillin than in the absence of vanillin is compared with that of traditional luminol-H 2 O 2 -Co 2+ The system relative ratio generates ROS which mainly comprises hydroxyl radicals and superoxide anions, and the generation of singlet oxygen is slightly increased.
Example 5: influence of changes in the reaction Environment on chemical shifts in 1H NMR spectra of luminol and vanillin
5mg of luminol and vanillin were dissolved with 500. Mu.L of heavy water, respectively, pH > 11 was adjusted with deuterium loaded sodium hydroxide, and then the separate luminol solution (5 mg, 500. Mu.L) and vanillin solution (5 mg, 500. Mu.L) were added to the nuclear magnetic resonance tube, respectively, and nuclear magnetic analysis was performed using an AVANED AV-600 nuclear magnetic resonance spectrometer. 5mg of luminol, 5mg of beta-cyclodextrin were dissolved in 500. Mu.L of heavy water, the pH was adjusted to > 11 with deuterium-loaded sodium hydroxide, the solution was mixed and shaken for 30 minutes at room temperature with a constant temperature shaker, and then 500. Mu.L of the solution was added to a nuclear magnetic tube and nuclear magnetic analysis was performed with an AVANED AV-600 nuclear magnetic resonance spectrometer. 5mg of vanillin and 5mg of beta-cyclodextrin are dissolved in 500 mu L of heavy water, the PH is adjusted to be more than 11 by deuterium-loaded sodium hydroxide, the solutions are mixed and oscillated for 30 minutes at room temperature by a constant temperature oscillator, then 500 mu L of the solution is added into a nuclear magnetic tube, and nuclear magnetic analysis is carried out by using an AVANED AV-600 nuclear magnetic resonance spectrometer. 5mg of luminol, 5mg of beta-cyclodextrin was dissolved in 500. Mu.L of heavy water, the pH was adjusted to > 11 with deuterium-loaded sodium hydroxide, the solution was mixed and shaken with a constant temperature shaker at room temperature for 30 minutes, then 5mg of vanillin was added and then mixed and shaken with a constant temperature shaker at room temperature for 30 minutes, 500. Mu.L of the solution was added to a nuclear magnetic tube, and nuclear magnetic analysis was performed with an AVANED AV-600 nuclear magnetic resonance spectrometer.
The results are shown in FIG. 8, which shows luminol (a), luminol-beta-cyclodextrin (b); the chemical shift of three H of benzene ring on luminol in luminol-beta-cyclodextrin-vanillin (c) is compared, the chemical shift of H is changed, which indicates that the chemical environment of luminol is changed to a certain extent, the added beta-cyclodextrin and luminol have certain interaction, and luminol molecules enter into the cavity of beta-cyclodextrin. Whereas vanillin (d) shown in fig. 9; vanillin-beta-cyclodextrin (e); the three H chemical shifts on the benzene ring on the vanillin in the vanillin-beta-cyclodextrin-luminol (f) are compared, the chemical shift of the H is changed, and the chemical environment where the vanillin is positioned is also changed, so that the beta-cyclodextrin and vanillin molecules have certain interaction, and the vanillin molecules can enter the cavity of the beta-cyclodextrin.
Example 7: optimal selection of reaction conditions
The concentration of beta-cyclodextrin was first optimized, the reaction conditions were controlled in the same manner as in example 1, a series of beta-cyclodextrin solutions were selected with final concentrations (5. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 50. Mu.M, 70. Mu.M), and then the CL intensities were measured using a self-made fixture (FIG. 2), and FIG. 10A shows that the CL intensities were highest for beta-cyclodextrin (50. Mu.M); the concentration of luminol was then optimized and the reaction conditions were controlled in the same manner as in example 1, a series of luminol solutions were selected with final concentrations (0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 3. Mu.M, 4. Mu.M) and then the CL intensities were measured, and FIG. 10B shows that the CL intensities were highest under the conditions of luminol (1. Mu.M); fig. 10C shows that CL intensity is strongest at 30 minutes with the optimal reaction time selected.
Example 7: detection of target molecule selectivity by chemiluminescent method
Based on the step conditions in example 1The target molecules vanillin (final concentration 50. Mu.M) were replaced by sucrose at a concentration of 0.1M, 0.1M Na + ，0.1M K + ，0.1M Cl - 0.1M glucose, 0.1M citric acid, 0.1M p-hydroxybenzaldehyde and 0.1M benzaldehyde. FIG. 12 shows the high selectivity of detection of vanillin based on host guest interaction chemiluminescence.
Example 9: labeling recovery detection of vanillin in milk powder and biscuits
In order to apply this method to the actual sample, gold Australian instant whole milk powder and biscuits (containing vanillin in the ingredients, < 0.5 mg/L) were purchased from supermarkets, the biscuits were first crushed into powder with a mortar, then the actual samples (10 mg/ml) were dissolved and shaken with water respectively, the supernatant was taken for use, the addition standard experiment was performed in a linear range, and the actual samples were added at 10% of the total volume. Taking 25 mu L of 1mM beta-cyclodextrin solution and 5 mu L of 0.1mM luminol solution in a centrifuge tube, mixing and oscillating the solutions at room temperature by a constant temperature oscillator for 30 minutes, then adding actual samples into the system solution to dilute the actual samples to the vanillin solutions with the final concentrations of 5 mu M,20 mu M and 50 mu M respectively, and continuously mixing and oscillating the actual samples at room temperature by the constant temperature oscillator for 30 minutes, wherein the total reaction volume is 500 mu L; finally, H is fixed in a self-made fixing mode 2 O 2 (1 mM, 100. Mu.L) by syringe into a centrifuge tube to induce luminol chemiluminescence, and measuring the change in CL signal over time by an electroluminescent analyzer at room temperature; the experimental results are shown in table 1, and these results demonstrate acceptable recovery and relative standard deviation, indicating that the prepared methods have good performance for detecting vanillin in food.
TABLE 1
Claims (7)
1. An analytical method for detecting vanillin molecules based on a host-guest interaction chemiluminescence method is characterized by comprising the following steps: mixing beta-cyclodextrin solution and luminol solution, and adding vanillin solution to obtainMixing the solutions, and then mixing H in situ 2 O 2 Adding the solution into the mixed solution to generate luminol chemiluminescence reaction, measuring the change of CL signals with time by adopting an electroluminescence analyzer at room temperature, and obtaining the content of vanillin according to the chemiluminescence intensity, wherein the final concentration of the vanillin solution is 5-110 mu M.
2. The method for detecting vanillin molecules based on host-guest interaction chemiluminescence according to claim 1, wherein the final concentration of the luminol solution is 0.1-4 μm.
3. The method for detecting vanillin molecules according to claim 1, wherein said β -cyclodextrin solution has a final concentration of 5 to 70 μm.
4. The method for detecting vanillin molecules according to claim 1, wherein said H 2 O 2 The final concentration of the solution is 0-5 mM.
5. The method for detecting vanillin molecules according to claim 1, wherein the in-situ mixing method is to fix the mixed solution in situ and then to use a syringe to fix H 2 O 2 The solution was added.
6. The use of the analytical method for detecting vanillin molecules based on the host-guest interaction chemiluminescence method of any of claims 1-5 in the field of food detection.
7. The use according to claim 6, wherein the food product is a milk powder or a biscuit.
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| A novel electrochemiluminescence immunosensor based onfunctional -cyclodextrin-ferrocene host-guest complex withmultiple signal amplification;Xiyue Xie等;Sensors and Actuators B: Chemical;第258卷;1146–1151 * |
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