Application of bifunctional platinum-modified nickel-iron layered double hydroxide in quantitative and qualitative detection of polyphenols

Abstract

The present invention discloses an application of a bifunctional platinum-modified nickel-iron layered double metal hydroxide in the quantitative and qualitative detection of polyphenols. The present invention prepares Pt / NiFe‑LDH, which can be used as a nanozyme material for colorimetric analysis and a matrix for LDI MS. Among them, the abundant hydroxyl groups on LDH can selectively capture hydroxyl-containing molecules such as polyphenols, and Pt / NiFe‑LDH exhibits high peroxidase activity, which can catalyze the decomposition of hydrogen peroxide to oxidize 3,3',5,5'‑tetramethylbenzidine to blue, and the antioxidant properties of polyphenols can affect the color depth of the system, and the linear relationship between the absorbance under the detection signal and the polyphenol content in the system can be used to achieve polyphenol quantification. On the other hand, Pt / NiFe‑LDH-assisted LDI MS has good signal intensity feedback in the monomer detection of polyphenols, and can monitor the oxidation process of polyphenols by virtue of a short detection time interval (a few seconds).

Description

Technical Field

[0001] The invention relates to application of a bifunctional platinum-modified nickel-iron layered double metal hydroxide in quantitative and qualitative detection of polyphenols, belonging to the technical field of metabolite detection. Background Art

[0002] Polyphenols are endogenous metabolites of plants and are essential antioxidants for the human body. In particular, tea polyphenols, due to their phenolic hydroxyl structure, can scavenge free radicals and regulate various oxidases in the body. They have antioxidant, anti-inflammatory, anti-cancer and lipid metabolism regulating properties and have been widely used in the prevention and treatment of diseases. Monitoring the intake and bioavailability of polyphenols is very important. On the one hand, the level of total polyphenols can be used as an important indicator of physiological processes, such as estimating the average daily intake of total polyphenols for nutritional assessment. On the other hand, during the drug treatment of diseases, simple monitoring of the transport and metabolism of polyphenol monomers is crucial for evaluating the efficacy. Therefore, the quantification of total polyphenols and the qualitative fingerprints of different polyphenols are of great significance to the bioavailability of polyphenols.

[0003] However, the instability of polyphenols (for example, tea polyphenols) during storage and administration poses challenges to their quantitative and qualitative analysis. In terms of quantification, colorimetric analysis can quantify total polyphenols, but it faces obstacles in the specific identification and accurate monitoring of individual catechin monomers. The current international standard method is the Folin phenol colorimetric analysis, but its experimental operation is complicated and the detection time is long. Many nanozymes with good performance have been developed to improve the efficiency of colorimetric quantification of polyphenols. For qualitative analysis, high-performance liquid chromatography or liquid chromatography-mass spectrometry is usually used to determine the monomer composition of total polyphenols, but its sample consumption is large, pretreatment is complex, and the procedure is time-consuming, which is limited in application. In contrast, laser desorption ionization mass spectrometry (LDI MS) has the ability to perform rapid analysis within a few seconds and minimal sample consumption, making it an indispensable analytical technique in the study of large and small molecules. The rational design of advanced nanomaterials as a matrix in the process will determine the interaction between metabolites and substrate materials, affecting the analysis of metabolites.

[0004] Advanced nanomaterials can be used as effective tools for building integrated detection platforms due to their adjustable physicochemical properties. Currently, noble metal nanoparticles with excellent local surface plasmon resonance (LSPR) effect and photothermal properties are widely used in the field of catalysis and enhanced LDI MS effect. In particular, platinum nanoparticles can promote the movement of interfacial electrons by reducing the kinetic energy barrier, thereby improving catalytic activity and ionization efficiency. At the same time, platinum nanoparticles have high plasma damping, which promotes the formation of local thermal fields and the desorption of analytes in LDI MS. However, metal nanoparticles are prone to self-aggregation and have a limited surface-to-volume ratio. Recently, inorganic layered materials such as layered double hydroxides (LDHs) with unique optical and electrical properties have been widely used as carrier substrates for metal nanoparticles.

[0005] There is no report on a platinum nanoparticle-modified nickel-iron layered double hydroxide and its use as a nanozyme material for colorimetric analysis and a matrix for LDI MS. Summary of the invention

[0006] The purpose of the present invention is to address the shortcomings of existing quantitative and / or qualitative methods for polyphenol metabolites, and to provide a bifunctional platinum-modified nickel-iron layered double metal hydroxide for use in quantitative and qualitative detection of polyphenols.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a bifunctional platinum-modified nickel-iron layered double hydroxide is provided as a nanozyme material for colorimetric analysis and / or a matrix for LDI MS, wherein the chemical expression of the bifunctional platinum-modified nickel-iron layered double hydroxide is Pt / NiFe-LDH.

[0009] Furthermore, the application includes application in detecting polyphenols;

[0010] And / or, the Pt / NiFe-LDH is a NiFe-LDH material modified with platinum metal nanoparticles obtained by etching the NiFe-LDH material with chloroplatinic acid and reducing the chloroplatinic acid with sodium borohydride.

[0011] Furthermore, the polyphenols include tea polyphenols.

[0012] Furthermore, the preparation method of Pt / NiFe-LDH comprises: dispersing NiFe-LDH in water, adding chloroplatinic acid solution and sodium borohydride solution in sequence for ultrasonic treatment, collecting precipitates, washing and drying to obtain Pt / NiFe-LDH.

[0013] Furthermore, in the preparation method, the raw material ratio of NiFe-LDH to chloroplatinic acid is 50 mg: 0.1-1 mmol.

[0014] Furthermore, in the preparation method, the raw material ratio of NiFe-LDH to chloroplatinic acid is 50 mg: 0.25-0.75 mmol.

[0015] Optimally, the raw material ratio of NiFe-LDH to chloroplatinic acid is 50 mg:0.5 mmol.

[0016] Furthermore, the concentration of the NiFe-LDH dispersed in water is 0.4-0.6 mg / mL, the concentration of the chloroplatinic acid solution is 5-20 mM, and the concentration of the sodium borohydride solution is 2-5 mg / mL.

[0017] Furthermore, the detection includes quantitative detection of the content of polyphenols, qualitative detection of the composition of polyphenols in the sample, and monitoring of the oxidation process of polyphenols.

[0018] Preferably, the quantitative detection of the content of polyphenols is carried out by colorimetry, and the detection principle is: the catalase activity of Pt / NiFe-LDH is used to catalyze the decomposition of hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to turn blue, and the antioxidant properties of polyphenols will change the color depth of the system, and the polyphenol quantification is achieved by relying on the linear relationship between the absorbance under the detection signal and the content of polyphenols in the system.

[0019] Preferably, the qualitative detection of the composition of polyphenols in the sample and the monitoring of the oxidation process of polyphenols are performed by matrix-assisted laser desorption ionization mass spectrometry using Pt / NiFe-LDH as a matrix.

[0020] The second aspect of the present invention provides a method for quantitatively detecting polyphenols, comprising the following steps:

[0021] Step 1: prepare a solution of Pt / NiFe-LDH material with deionized water; the Pt / NiFe-LDH material is a NiFe-LDH material modified with platinum metal nanoparticles, which is prepared by etching NiFe-LDH material with chloroplatinic acid and reducing chloroplatinic acid with sodium borohydride;

[0022] Step 2: Add the Pt / NiFe-LDH solution prepared in step 1 and TMB to the HAc-NaAc buffer;

[0023] Step 3: adding the polyphenol sample solution to be tested into the peroxidase-like system;

[0024] Step 4: Add hydrogen peroxide solution and record the absorbance under the detection signal;

[0025] Step 5: Substitute the absorbance into the standard curve obtained by testing the polyphenols with a standard concentration gradient using the same method to calculate the concentration of polyphenols in the sample.

[0026] A third aspect of the present invention provides a method for monitoring the self-oxidation process of polyphenols, comprising the following steps:

[0027] Step 1: Prepare a solution of the polyphenol sample to be tested with deionized water, add glucose as an internal standard, and spot the sample on a target plate;

[0028] Step 2: Prepare a solution of Pt / NiFe-LDH material with deionized water, and spot the solution on a target plate at different monitoring time periods; the Pt / NiFe-LDH material is a NiFe-LDH material modified with platinum metal nanoparticles, which is prepared by etching NiFe-LDH material with chloroplatinic acid and reducing chloroplatinic acid with sodium borohydride;

[0029] Step 3: Use matrix-assisted laser desorption ionization mass spectrometry for detection, take the ratio of the intensity of the polyphenol sample to be tested to the intensity of the internal standard as the relative intensity, and compare the relative intensity changes of the target within the monitoring time period.

[0030] The present invention utilizes the advantages and synergistic effects of platinum metal and nickel-iron layered double hydroxides to develop an integrated platform for polyphenol detection - bifunctional platinum-modified nickel-iron layered double hydroxides. Using LDH as a carrier can not only reduce the size of metal nanoparticles, but also prevent their agglomeration, thereby improving the catalytic effect of metal-LDH mixtures. Various metal-LDH hybrids show high catalytic activity, large specific surface area and synergistic effects.

[0031] The platinum nanoparticle-modified nickel-iron layered double hydroxide (hereinafter referred to as Pt / NiFe-LDH) provided by the present invention can be used as a nanozyme material for colorimetric analysis and a matrix for LDI MS. Importantly, the abundant hydroxyl groups on LDH can selectively capture hydroxyl-containing molecules such as polyphenols. On the one hand, Pt / NiFe-LDH exhibits high peroxidase activity and can catalyze the decomposition of hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to blue, and the antioxidant properties of polyphenols can affect the color depth of the system. The linear relationship between the absorbance at 652nm and the polyphenol content in the system can be used to achieve polyphenol quantification. On the other hand, Pt / NiFe-LDH-assisted LDI MS has good signal intensity feedback in the monomer detection of polyphenols, and can monitor the oxidation process of polyphenols by virtue of a short detection time interval (a few seconds).

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention can efficiently quantify polyphenols by Pt / NiFe-LDH catalytic colorimetry: The present invention synthesizes Pt / NiFe-LDH with high catalase activity for rapid colorimetric quantification of the total amount of tea polyphenols; Pt / NiFe-LDH exhibits high peroxidase activity and can catalyze the decomposition of hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to blue, and the antioxidant properties of polyphenols can affect the color depth of the system. The quantification of polyphenols can be achieved by relying on the linear relationship between the absorbance at 652 nm and the polyphenol content in the system;

[0034] (2) The present invention utilizes Pt / NiFe-LDH-assisted laser desorption ionization mass spectrometry to qualitatively determine the polyphenol composition of the sample: the LDH surface has abundant hydroxyl groups, which is conducive to the adsorption of polyphenols; platinum nanoparticles have high plasma damping, which promotes the formation of local thermal fields and the desorption of analytes. At the same time, Pt / NiFe-LDH can promote the movement of interface electrons, thereby improving catalytic activity and ionization efficiency; using Pt / NiFe-LDH-assisted LDI MS, m / z signals of multiple polyphenol monomers in the sample can be obtained;

[0035] (3) The present invention utilizes Pt / NiFe-LDH-assisted laser desorption ionization mass spectrometry to monitor the self-oxidation reaction of polyphenols: Pt / NiFe-LDH-assisted LDI MS has a short detection interval, and after adding an internal standard, the reduction of polyphenol monomers and the increase of oxides can be monitored by the ratio of the target signal intensity to the internal standard signal intensity;

[0036] (4) The present invention synthesized Pt / NiFe-LDH with a catalase activity of 33.472 U / mg, significantly improving the single polyphenol quantitative reaction time to 1 minute, reducing the single nanozyme consumption to 1 microgram, and the quantitative results were not significantly different from those of the standard method;

[0037] (5) The present invention uses Pt / NiFe-LDH as a matrix-assisted LDI MS to achieve a signal effect in the detection of polyphenol monomers that is significantly better than its constituent monomers (Pt NPs, NiFe-LDH) and commonly used matrices (CHCA, DHB, Au NPs). BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a scanning electron microscopic image of Pt / NiFe-LDH material;

[0039] Figure 2 Transmission electron microscopy image of Pt / NiFe-LDH material (the circle represents platinum nanoparticles);

[0040] Figure 3 is the enzyme activity curve of Pt / NiFe-LDH-50;

[0041] Figure 4 The mass spectrometry detection signal intensity of different polyphenols was compared for NiFe-LDH modified with different concentrations of platinum nanoparticles;

[0042] Figure 5 The standard curve for determining the content of tea polyphenols using the Pt / NiFe-LDH colorimetric method;

[0043] Figure 6 To compare the quantitative results of tea polyphenols in actual samples with those of standard methods;

[0044] Figure 7 This is the LDI MS qualitative spectrum of polyphenol monomers;

[0045] Figure 8 It is the monitoring result of polyphenol monomer self-oxidation (the relative intensity change of target mass spectrum detection);

[0046] Fig. 9 The figure shows a comparison of the assisted mass spectrometry monitoring effects of Pt / NiFe-LDH-50 and commonly used matrices (CHCA, DHB, Au NPs); CHCA: α-cyano-4-hydroxycinnamic acid; DHB: 2,5-dihydroxybenzoic acid; Au NPs: gold nanoparticles. DETAILED DESCRIPTION

[0047] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] Example 1

[0050] Prepare Pt / NiFe-LDH nanomaterials and optimize the concentration of modified platinum to obtain the best detection effect:

[0051] Step 1: Dispersing NiFe-LDH material (purchased from Jiangsu Pioneer Nanomaterials). 50 mg of LDH was added to 100 mL of ultrapure water and subjected to 30 min of ultrasound (300 W power) for dispersion and exfoliation.

[0052] Step 2: Modify platinum nanoparticles. Slowly add 50 mL of chloroplatinic acid (10 mM, 10 mmol / L) to the above system, ultrasonicate for 10 min, then slowly drop 50 mL of freshly prepared sodium borohydride solution (3.5 mg / mL) in an ice bath, the solution becomes black and turbid, and ultrasonicate for 30 min. Changing the volume of chloroplatinic acid in the reaction to 10, 25, 75, and 100 mL can synthesize Pt / NiFe-LDH with different platinum modification amounts.

[0053] Step 3: Centrifugation, washing and drying of materials. The solution after the above reaction was centrifuged at 10000rpm for 10min, washed with an appropriate amount of ethanol, ultrasonicated for 10min and then centrifuged at 10000rpm for 10min, the supernatant was removed, an appropriate amount of ultrapure water was added for washing, ultrasonicated for 10min and then centrifuged at 10000rpm for 10min, the supernatant was removed, and the precipitate was dried at 60°C for 10 hours. NiFe-LDH sheets modified with platinum nanoparticles of different concentrations were obtained, and the materials were recorded as Pt / NiFe-LDH-50 (10, 25, 75, 100), and the numbers correspond to the volume of chloroplatinic acid used in step 2; the characterization results of the materials are shown in Figure 2. Figure 1-2 The catalase activity of the material was tested, where the enzyme activity of Pt / NiFe-LDH-50 was 33.472U / mg. The enzyme activity test curve was shown in Figure 3 shown.

[0054] The prepared NiFe-LDH modified with different concentrations of platinum nanoparticles was used for mass spectrometry detection of different polyphenols: standard (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG). The comparative statistical results of the mass spectrometry detection signal intensity are shown in FIG. Figure 4 As shown, from Figure 4 It can be seen that Pt / NiFe-LDH-50 can obtain better mass spectrometry detection signals for the detection of polyphenols, followed by Pt / NiFe-LDH-25 and Pt / NiFe-LDH-75.

[0055] Example 2

[0056] Polyphenol quantification based on optimized Pt / NiFe-LDH (Pt / NiFe-LDH-50):

[0057] Step 1: Prepare a 1 mg / mL solution of Pt / NiFe-LDH material with deionized water.

[0058] Step 2: Add 1 μL Pt / NiFe-LDH-50 (1 mg / mL) and 50 μL TMB (1 mM) to 1 mL HAc-NaAc buffer.

[0059] Step 3: Add 50 μL of tea polyphenols sample solution to the peroxidase sample system.

[0060] Step 4: Add 25 μL HO 2 O 2 Mix with the above system and record the absorbance at λmax of 652 nm after 1 minute.

[0061] Step 5: Substitute the absorbance into the standard curve obtained by testing the standard concentration gradient of tea polyphenols using the same method ( Figure 5 ), the tea polyphenols concentration in the sample was calculated and compared with the results of the commercial kit of Folin phenol method ( Figure 6 ).

[0062] Example 3

[0063] Detection and monitoring of polyphenol monomers based on optimized Pt / NiFe-LDH (Pt / NiFe-LDH-50) assisted laser desorption ionization mass spectrometry:

[0064] Step 1: Prepare the standard substances (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) into a 500 μg / mL solution with deionized water, and add 500 μg / mL glucose to the sample as an internal standard in the experiment of monitoring polyphenol monomers.

[0065] Step 2: Prepare the sample on a 384 Polish target plate, spot 1 μL of each tea polyphenol standard sample, and dry naturally at room temperature.

[0066] Step 3: Prepare the matrix on a 384 Polish target plate, drop 1 μL of the matrix solution on the dried blood sample, and dry it naturally at room temperature. In the experiment of monitoring the self-oxidation of polyphenol monomers, the matrix was prepared after 0, 1, and 2 hours of self-oxidation at a single sample point.

[0067] Step 4: Use matrix-assisted laser desorption ionization mass spectrometer to collect mass spectral fingerprints of polyphenol monomers ( Figure 7 ).

[0068] Step 5: Calculate the ratio of the intensity of the target polyphenol monomer to the internal standard intensity as the relative intensity. Comparing the relative intensity of the target product oxidized for 0-2 hours can monitor its degree of auto-oxidation ( Figure 8 ).

[0069] In addition, in this example, the assisted mass spectrometry monitoring effects of Pt / NiFe-LDH-50 and common commercial matrices were compared. Pt / NiFe-LDH as a matrix-assisted LDI MS obtained a significantly better signal effect in the detection of polyphenol monomers than its constituent monomers (Pt NPs, NiFe-LDH) and common matrices (CHCA, DHB, Au NPs), such as Fig. 9 shown.

[0070] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Application of a bifunctional platinum-modified nickel-iron layered double metal hydroxide as a nanozyme material for colorimetric analysis and a matrix for LDIMS, characterized in that: The expression of the bifunctional platinum-modified nickel-iron layered double hydroxide is Pt / NiFe-LDH; The application is the application in detecting polyphenols; The preparation method of Pt / NiFe-LDH comprises: dispersing NiFe-LDH in water, adding chloroplatinic acid solution and sodium borohydride solution in sequence for ultrasonic treatment, collecting precipitates, washing and drying to obtain the product; In the preparation method, the raw material ratio of NiFe-LDH to chloroplatinic acid is 50 mg: 0.25-0.75 mmol.

2. The use according to claim 1, characterized in that The polyphenols include tea polyphenols.

3. The use according to claim 2, characterized in that The detection includes quantitative detection of the content of polyphenols, qualitative detection of the composition of polyphenols in the sample, and monitoring of the oxidation process of polyphenols.

4. The use according to claim 3, characterized in that The quantitative detection of the content of polyphenols adopts a colorimetric method, and the detection principle is: the catalase activity of Pt / NiFe-LDH is used to catalyze the decomposition of hydrogen peroxide to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to blue, and the antioxidant properties of polyphenols will cause the color depth of the system to change. The linear relationship between the absorbance under the detection signal and the content of polyphenols in the system is relied upon to achieve polyphenol quantification.

5. The use according to claim 3, characterized in that The method for qualitatively detecting the composition of polyphenols in the sample and monitoring the oxidation process of polyphenols is to use Pt / NiFe-LDH as a matrix to perform matrix-assisted laser desorption ionization mass spectrometry detection.

6. The use according to claim 3, characterized in that The method for quantitatively detecting the content of polyphenols comprises the following steps: Step 1: prepare a solution of Pt / NiFe-LDH material with deionized water; the Pt / NiFe-LDH material is a NiFe-LDH material modified with platinum metal nanoparticles, which is prepared by etching NiFe-LDH material with chloroplatinic acid and reducing chloroplatinic acid with sodium borohydride; Step 2: Add the Pt / NiFe-LDH solution prepared in step 1 and TMB to the HAc-NaAc buffer; Step 3: adding the polyphenol sample solution to be tested into the peroxidase-like system; Step 4: Add hydrogen peroxide solution and record the absorbance under the detection signal; Step 5: Substitute the absorbance into the standard curve obtained by testing the polyphenols with a standard concentration gradient using the same method to calculate the concentration of polyphenols in the sample.

7. The use according to claim 3, characterized in that The method for monitoring the self-oxidation process of polyphenols comprises the following steps: Step 1: Prepare a solution of the polyphenol sample to be tested with deionized water, add glucose as an internal standard, and spot the sample on a target plate; Step 2: Prepare a solution of Pt / NiFe-LDH material with deionized water, and spot the solution on a target plate at different monitoring time periods; the Pt / NiFe-LDH material is a NiFe-LDH material modified with platinum metal nanoparticles, which is prepared by etching NiFe-LDH material with chloroplatinic acid and reducing chloroplatinic acid with sodium borohydride; Step 3: Use matrix-assisted laser desorption ionization mass spectrometry for detection, take the ratio of the intensity of the polyphenol sample to be tested to the intensity of the internal standard as the relative intensity, and compare the relative intensity changes of the target within the monitoring time period.

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