Au (at) M (at) AgNRs nano material and preparation method and application thereof

Abstract

The invention discloses an Au (at) M (at) Ag NRs nano material as well as a preparation method and application thereof. The Au (at) M (at) Ag NRs nano material is composed of an Au (at) Ag NRs core-shell structure and 4-mercaptobenzoic acid coated inside the Au (at) Ag NRs core-shell structure. According to the method, Au (at) M (at) Ag NRs is used as a sensing material for dual-mode detection, an ultraviolet absorption peak is measured by adopting an ultraviolet-visible spectrograph, and the content of kaempferol in a solution to be detected is obtained according to a known linear relation equation between kaempferol solutions with different concentrations and displacement values of the ultraviolet absorption peak. Meanwhile, measuring a Raman signal by adopting a Raman spectrometer, and obtaining the content of kaempferol in the solution to be detected according to a known linear relation equation between kaempferol solutions with different concentrations and the Raman signal intensity. According to the invention, through linear discriminant analysis (LDA), five polyphenols with different antioxidant capacities, namely chlorogenic acid, epicatechin, epigallocatechin gallate, kaempferol and tannic acid, are successfully distinguished.

Description

Technical Field

[0001] The invention relates to an Au@M@Ag NRs nano material and a preparation method and application thereof, belonging to the field of nano material preparation and application. Background Art

[0002] Astringency is a composite sensation of the oral epithelium contracting, stretching or wrinkling after contact with unripe fruits and berries, nuts, red wine, tea and similar substances. Recent studies have shown that taste plays a key role in food selection, often outweighing considerations related to health or nutritional value. Therefore, astringency can significantly affect the palatability of foods and, when increased in intensity, may deter people from consuming certain nutritious foods.

[0003] In fact, astringency is closely associated with beverages and foods that are high in polyphenols. Dietary polyphenols are produced by the secondary metabolism of plants and can serve as a chemical defense to prevent predators. Polyphenols provide the overall taste and color of beverages and foods, but can also cause astringency, which limits food consumption and promotion despite their health benefits. Therefore, the presence of polyphenols poses a challenge to the food industry and has prompted researchers to develop precise methods to identify these compounds. These advances aim to effectively modulate taste properties and ultimately promote the production of healthy foods.

[0004] In the past few years, various analytical techniques have been used to determine polyphenols, such as gas chromatography, high-performance liquid chromatography, capillary electrophoresis, electrochemical sensors, and chemiluminescence. Although these analytical techniques have good selectivity and sensitivity, limitations such as high cost of analytical equipment, complex pretreatment, and time-consuming detection process restrict their widespread application. In addition, single-mode analysis may lack reliability and precision for quantitative determination. In contrast, dual-mode detection methods have higher accuracy and can flexibly cope with different detection conditions.

[0005] Colorimetry is a rapid, simple, and sensitive technique that is usually implemented by monitoring the absorbance changes caused by changes in analyte concentration at a specific wavelength. In addition, colorimetry can be detected "with the naked eye" by color changes, which is suitable for rapid detection. Surface enhanced Raman scattering (SERS) spectroscopy is considered as a potential bioanalysis and detection platform due to its high specificity and sensitivity. At the same time, polyphenols with different antioxidant capacities can be successfully distinguished by linear discriminant analysis (LDA). Therefore, this dual-mode sensor can effectively reduce false positive results and provide broad application prospects for determining and identifying polyphenols in complex food samples. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a preparation method of Au@M@Ag NRs nanomaterials with simple principle, rapidity and simple pre-treatment and its application.

[0007] Technical solution: The present invention provides an Au@M@Ag NRs nanomaterial, wherein the Au@M@Ag NRs nanomaterial comprises an Au@Ag NRs core-shell structure and mercaptobenzoic acid therein.

[0008] Among them, the Au@M@Ag NRs nanomaterial is a core-molecule-shell nanorod structure, the average length of the Au@M@Ag NRs nanomaterial is 36.5nm, the width is 11.1nm, and the core-molecule-shell nanorod structure has a very high surface plasmon resonance, which greatly improves the detection sensitivity of kaempferol.

[0009] The concentration of 4-mercaptobenzoic acid is 0.5-2.0 μM, and 4-MBA is introduced into the core-shell structure as a Raman reporter molecule, so that the quantitative detection of kaempferol is more accurate.

[0010] The present invention also provides a method for preparing Au@M@Ag NRs nanomaterial, comprising the following steps:

[0011] 1) CTAB, HAuCl 4 AgNO 3 , hydroquinone and NaBH 4 The solutions are mixed, reacted in a constant temperature water bath, and the reaction products are centrifuged and washed to obtain Au NRs;

[0012] 2) Adding Raman reporter molecules to the prepared Au NRs solution, incubating the solution at a constant temperature on a shaker, and centrifuging and washing the reaction product to obtain Au@M NRs;

[0013] 3) Adding AgCl, Au@M NRs and ascorbic acid solution to the CTAC solution respectively, reacting in a constant temperature water bath, and centrifuging and washing the reaction product to obtain Au@M@Ag NRs nanoparticles.

[0014] Wherein, in step 1), the final concentrations of CTAB, chloroauric acid, silver nitrate, hydroquinone and sodium borohydride solution in the reaction solution are 0.1M, 0.4mM, 0.05-0.15mM, 3-6mM and 0.01-0.05mM respectively, the reaction temperature is 25-35°C, and the reaction time is 10-12h.

[0015] Wherein, the Raman reporter molecule solution in step 2) is a mercaptobenzoic acid solution, and its final concentration is 0.5-2.0 μM, the reaction shaker incubation time is 60-90 min, and the incubation temperature is 25-40°C.

[0016] Wherein, in step 3), the final concentrations of CTAC, silver chloride and ascorbic acid solution in the reaction solution are 60-80 mM, 0-0.5 mM and 50-100 mM respectively, the volume of Au@M NRs used is 1 mL, the water bath temperature is 30-35° C., and the reaction time is 3-4 h.

[0017] The Au@M@Ag NRs nanomaterial can be successfully prepared under the above conditions and can be successfully used to identify, detect and / or differentiate polyphenols, recover polyphenols, and quantitatively detect the concentration of kaempferol in a sample.

[0018] The present invention also provides the use of the Au@M@Ag NRs nanoparticles in identifying, detecting and / or distinguishing polyphenols; the polyphenols include one or more of kaempferol, chlorogenic acid, epicatechin, epigallocatechin gallate or tannic acid solution.

[0019] The present invention also provides a polyphenol detection kit, which contains the Au@M@Ag NRs nanoparticles; the polyphenols include one or more of kaempferol, chlorogenic acid, epicatechin, epigallocatechin gallate or tannic acid solution.

[0020] The present invention also provides the use of the Au@M@Ag NRs nanoparticles or the polyphenol detection kit in recovering polyphenols.

[0021] The present invention also provides a method for quantitatively detecting the concentration of kaempferol based on colorimetry and surface enhanced Raman scattering technology, characterized in that the quantitative detection method is selected from any one or two of the following methods:

[0022] A1) The Au@M@Ag NRs and H 2 O 2 After the solutions are mixed, a kaempferol solution is added to obtain a mixed solution, and the absorbance value is measured by a UV-visible spectrometer. The kaempferol content in the test solution is obtained according to a linear relationship equation between kaempferol solutions of different known concentrations and UV peak shift values, wherein the kaempferol concentration is 0.1-2 μM;

[0023] A2) The Au@M@Ag NRs and H 2 O 2 After the solutions are mixed, kaempferol solution is added to obtain a mixed solution which is then dropped onto a silicon wafer. After drying, a Raman spectrometer is used to measure the Raman signal. The kaempferol content in the solution to be tested is obtained based on a linear relationship equation between known polyphenol solutions of different concentrations and the Raman signal intensity. The kaempferol concentration is 10 -9 -10 - 5 M.

[0024] Wherein, in the method A1), H 2 O 2 The concentration is 100-200mM.

[0025] Wherein, in the method A1), kaempferol solution and Au@M@Ag NRs / H 2 O 2 The reaction time of the mixed solution is 30-50 min.

[0026] The linear relationship equation between the different concentrations of kaempferol solution and the ultraviolet peak shift value in the method A1) is Δλ=34.73+41.08C KAE (R 2 =0.975), Δλ is the ultraviolet absorption peak shift value, and C is the kaempferol concentration.

[0027] Wherein, the volume of the silicon wafer sample drop in the method A2) is 10-20 μL.

[0028] The linear relationship equation between the different concentrations of kaempferol solutions and the Raman signal intensity in the method A2) is ΔI=33093.43+3618.95lgC KAE (R 2 =0.981), where ΔI is 1590 cm -1 is the Raman signal intensity at , and C is the kaempferol concentration.

[0029] The present invention also includes a method for identifying polyphenols and an application thereof, wherein the polyphenols include chlorogenic acid, epicatechin, epigallocatechin gallate and tannic acid solution, and the method includes the following steps: a method for quantitatively detecting the concentration of kaempferol based on colorimetry and surface enhanced Raman scattering technology, replacing the kaempferol solution with polyphenols, and identifying the polyphenols using linear discriminant analysis. Preferably, the application includes the identification of polyphenol compounds in fruit juice and cigarettes.

[0030] Mechanism of the present invention: Figure 1 As shown in Figure 2, 4-MBA, as a Raman reporter molecule, was modified between Au NRs (core) and Ag (shell) to form Au@M@Ag NRs, which exhibited unique UV absorption peaks and Raman activity. 2 O 2 In the presence of , the silver shell of Au@M@AgNRs was oxidized and etched, resulting in changes in its nanostructure. As the silver shell was etched, the color of the solution changed from blue to light pink, and the SERS signal was significantly reduced. However, due to the ability of polyphenols to scavenge H 2 O 2, so the addition of polyphenols inhibits the etching process. The increase in polyphenol concentration can restore the shell-core ratio of Au@M@Ag NRs, resulting in a significant blue shift in UV absorption. Therefore, the light pink color turns into green. At the same time, the SERS signal of 4-MBA gradually increases with the thickening of the silver shell. Therefore, the designed strategy can obtain dual readouts of colorimetry and SERS signals, thereby sensitively detecting polyphenols. By combining the differences in UV absorption and Raman signals, the linear discriminant analysis (LDA) can be applied to identify the five polyphenols with high accuracy, showing great potential in the detection of actual food samples.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0032] 1. The Au@M@Ag NRs nanomaterial prepared by the present invention is based on controllable chemical reactions and exhibits excellent multi-chromatic performance. 2 O 2 The silver shell of the gold nanorods will be etched, causing a significant change in color and a red shift in the ultraviolet absorption peak. At the same time, the Au@Ag NRs core-shell structure can combine the high SERS effect of silver nanoparticles with the uniform stability of gold nanoparticles to maximize the SERS effect, greatly improving the detection sensitivity of polyphenols.

[0033] 2. The present invention also has the advantages of simple principle, rapidity, simple pre-treatment and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Mechanistic diagram of the polyphenol detection method based on colorimetry and surface-enhanced Raman scattering techniques;

[0035] Figure 2 A is the UV-visible spectra of Au@M NRs and Au@M@Ag NRs (the inset is the corresponding solution photo). Figure 2 B is the TEM image of Au@M NRs. Figure 2 C is the TEM image of Au@M@Ag NRs. Figure 2 D is the HAADF-STEM-EDS image of Au@M@Ag NRs;

[0036] Figure 3 A is the zeta potential diagram of Au NRs, Au@M NRs and Au@M@Ag NRs. Figure 3 B is the XRD pattern of the material Au@M@AgNRs;

[0037] Figure 4 Optimization of etching time conditions: Figure 4 A is the absorbance at different etching times, Figure 4B is the change of the ultraviolet absorption peak shift value of Au@M@Ag NRs at different etching times;

[0038] Figure 5 A is a proof of principle diagram for the detection of kaempferol based on colorimetry. Figure 5 B is Au@M@Ag NRs and H 2 O 2 TEM image after reaction with KAE, Figure 5 C is the UV-visible spectra of sample solutions with different concentrations of kaempferol added (the inset is the corresponding solution photo), Figure 5 D is the linear relationship between the UV absorption peak shift value and kaempferol concentration;

[0039] Figure 6 To optimize the conditions of excitation wavelength and 4-MBA concentration, Figure 6 A is the Raman enhancement effect of Au@M@Ag NRs at different excitation wavelengths. Figure 6 B is the SERS signal of Au@M@Ag NRs at different 4-MBA concentrations;

[0040] Figure 7 A is a proof-of-principle diagram for detecting kaempferol based on surface enhanced Raman scattering technology. Figure 7 B is the Raman spectrum of the sample solution with different concentrations of kaempferol added. Figure 7 C is 4-MBA at 1590cm -1 The linear relationship between the Raman signal change value and the logarithm of kaempferol concentration;

[0041] Figure 8 A is the Raman spectra of five polyphenols, kaempferol, tannic acid, chlorogenic acid, epicatechin and epigallocatechin gallate, on Au@M@Ag NRs nanomaterials. Figure 8 B is the three-dimensional standard score map of the dual-mode method for identifying five polyphenols. Figure 8 C is the hierarchical clustering diagram, Figure 8 D is a heat map.

[0042] Fig. 9 A is a three-dimensional standard score diagram of five polyphenols in juice samples at a concentration of 1 μM; Fig. 9 B is a three-dimensional standard score diagram of five polyphenols in cigarette samples at a concentration of 1 μM. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0044] Reagents and instruments used in this experiment:

[0045] Chloroauric acid trihydrate (HAuCl 4 ·3H2 O) and sodium borohydride, cetyltrimethylammonium bromide (CTAB) were purchased from J&K Scientific Co. (Beijing, China). Hydroquinone, cetyltrimethylammonium chloride (CTAC), chlorogenic acid (CA), epicatechin (EC), epigallocatechin gallate (EGCG), kaempferol (KAE), tannic acid (TA), and 4-mercaptobenzoic acid (4-MBA) were purchased from MacLean Biochemical Company (Shanghai, China). Silver nitrate and ascorbic acid (AA) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Cigarettes were from China Zhengzhou Tobacco Research Institute (Zhengzhou, China), and fruit juices were purchased from Suguo Supermarket. All aqueous solutions were washed with ultrapure water (18.2 MΩ cm -1 ) preparation.

[0046] TalosF200X high-resolution transmission electron microscope (Thermo Fisher), NanoBrookOmni multi-angle particle size analyzer (Brookhaven), Cary60UV-Vis ultraviolet-visible spectrophotometer (Agilent), SmartLab X-ray diffractometer (Rigaku Co., Ltd.), Renishaw InVia Raman Microscopy confocal micro-Raman spectrometer (Renishaw).

[0047] Example 1 Synthesis and determination of Au@M NRs and Au@M@Ag NRs nanomaterials

[0048] 1. Synthesis of Au@M NRs: The Au NRs in the embodiment of the present invention were prepared according to a typical one-pot method. First, a solution of chloroauric acid (3.04 mL, 0.01 M) was mixed with a solution of hexadecyltrimethylammonium bromide (0.112 M, 68 mL), and then stirred evenly at 32 ° C. Silver chloride solution (0.091 mM, 345.8 μL) and hydroquinone solution (5.26 mM, 2 mL) were added to the above solution in sequence. Five minutes later, sodium borohydride solution (0.017 mM, 2.594 mL) was quickly added to the mixture and allowed to stand overnight at 32 ° C. During this period, the mixture gradually turned brown. Finally, Au NRs were obtained by centrifugal washing and redispersed in 15 mL of ultrapure water. Secondly, take 990 μL of the prepared Au NRs solution into a centrifuge tube, then add 4-mercaptobenzoic acid (4-MBA) solution (100 μM, 10 μL), and put it in a 37°C shaker for incubation for 1 hour; after the reaction is completed, the reaction solution is centrifuged and washed at a speed of 13000 rpm, and the centrifugal product is redispersed to 1 mL and stored in 80 mM CTAC solution for later use to obtain a brown Au@M NRs solution.

[0049] 2. Synthesis of Au@M@Ag NRs: 17 mg of silver nitrate was mixed with hexadecyltrimethylammonium chloride solution (10 mL, 80 mM) to prepare a 10 mM silver chloride solution. Subsequently, silver chloride solution (10 mM, 150 μL), Au@M NRs solution (1 mL) and ascorbic acid solution (250 μL, 0.1 M) were added to hexadecyltrimethylammonium chloride solution (5 mL, 80 mM) solution under oscillation. The mixed solution was allowed to stand in a water bath at 30 ° C for 3 hours. Finally, the reaction product was purified by centrifugation and redispersed in 2 mL of ultrapure water to obtain Au@M@Ag NRs for further use.

[0050] The synthesis of Au@M@Ag NRs was demonstrated using UV-visible spectrophotometry. Figure 2 As shown in Figure A, the Au@M NRs solution is light brown, showing transverse surface plasmon resonance at a wavelength of 510 nm and longitudinal surface plasmon resonance at a wavelength of 805 nm. After the surface is covered with a silver shell, the longitudinal absorption peak of Au@M NRs shifts to 603 nm and the color of the solution turns blue; the morphology and size of Au@M@Ag NRs and intermediates are characterized by high-resolution transmission electron microscopy (HRTEM). Figure 2 As shown in BC, Au@M NRs and Au@M@Ag NRs have uniformly distributed rod-like structures, which is conducive to the generation of gaps and "hot spots" between nanorods, resulting in a strong SERS enhancement effect. The average size of Au@M@Ag NRs is 36.5nm×11.1nm, slightly larger than that of Au@M NRs (28.3nm×7.5nm); then, the elemental atlas image is used to characterize the core-molecule-shell structure of Au@M@Ag NRs. Figure 2 As shown in D, Au, Ag, and S elements are evenly distributed, indicating that the synthesis of Au@M@Ag NRs is very successful; Figure 3 A shows that the zeta potentials of Au NRs, Au@M NRs, and Au@M@Ag NRs are +17.31 mV, +11.53 mV, and +19.28 mV, respectively, which is related to the different modifications on the surface of Au NRs; Figure 3 B shows the XRD spectrum of Au@M@Ag NRs with four peaks at 38.1°, 44.3°, 64.4°, and 77.5°, which are related to the crystal structure of gold and silver. These results confirm that Au@M@AgNRs were successfully prepared.

[0051] Example 2 Selection of the optimal reaction conditions for detecting kaempferol based on colorimetry

[0052] In order to obtain a good colorimetric reaction for polyphenol analysis, the etching time was optimized in this example: Based on the step conditions in Example 1, an Au@M@Ag NRs solution was prepared. 100 μL of the prepared Au@M@Ag NRs solution was mixed with 50 μL of H 2 O 2 The solution (150 mM) was mixed evenly, and then 50 μL of kaempferol (1 μM) solution was added to the above solution. After incubation at room temperature for different times (40 min, 50 min), the color of the solution was observed with the naked eye, and the absorbance spectrum was recorded with a UV-visible spectrophotometer.

[0053] Figure 4 As shown in Figures 4A and 4B, as the etching time increases, the UV absorption peak shift value (Δλ) gradually increases and reaches a maximum value at 40 minutes. Therefore, 40 minutes was selected as the ideal etching time for further experiments.

[0054] Example 3 Principle verification of kaempferol detection based on colorimetry

[0055] 100 μL of the Au@M@Ag NRs solution prepared in Example 1 was mixed with 50 μL of H 2 O 2 The (150 mM) solution was mixed evenly, and then different concentrations of kaempferol (0 μM to 10 μM) were added to the above solution and mixed. After incubation at room temperature for 40 minutes, the color of the solution was observed with the naked eye, and the absorbance spectrum was recorded with a UV-visible spectrophotometer.

[0056] Figure 5 The feasibility of using Au@M@Ag NRs as a colorimetric substrate for kaempferol detection was verified. Figure 5 As shown in Figure A, the change in UV absorption (line b) in the presence of kaempferol (1 μM) alone was negligible compared to that in the presence of Au@M@Ag NRs alone (line a), indicating that kaempferol did not affect the Au@M@Ag NRs. 2 O 2 (150mM) can completely oxidize the silver shell of Au@M@AgNRs, making it appear light pink and showing a maximum absorption peak at 805nm (line c). 2 O 2 When kaempferol (1 μM) was introduced into the solution and etched for 40 minutes, the solution changed from light pink to green, and the UV absorption peak had a significant blue shift (line d), which confirmed the effective inhibitory effect of kaempferol in the etching process. These results verify that Au@M@Ag NRs is an effective colorimetric probe for detecting kaempferol. At the same time, the morphology of Au@M@Ag NRs was observed by TEM images ( Figure 5 B) At the optimal etching time, add H2 O 2 After / KAE, the average size of Au@M@Ag NRs increased to 31.34nm×8.72nm, which was due to the recovery of the colorimetric signal. Meanwhile, the sensitivity of Au@M@Ag NRs for the detection of kaempferol was studied by colorimetric reaction under the optimal conditions. Figure 5 C shows that when the final concentrations of kaempferol are 0 μM, 0.01 μM, 0.1 μM, 0.5 μM, 1 μM and 2 μM, the color of the solution gradually changes from light pink to green, and the color change can be easily observed by the naked eye. At the same time, as the concentration of kaempferol increases, the Ag layer gradually becomes thicker, causing an obvious blue shift, and the ultraviolet absorption peak changes from 805 nm to 637 nm. Figure 5 As shown in Figure 4, the linear relationship between the ultraviolet absorption peak shift value and the kaempferol concentration is Δλ=34.73+41.08C KAE (R 2 =0.975), where Δλ represents the ultraviolet absorption peak shift value, C KAE represents the concentration of kaempferol. The detection limit (LOD) of the colorimetric method was calculated to be 43.2 nM. Therefore, the colorimetric method based on Au@M@Ag NRs has good sensitivity for kaempferol detection.

[0057] Example 4 Selection of the optimal reaction conditions for detecting kaempferol based on surface enhanced Raman scattering technology

[0058] In order to obtain a good response to kaempferol concentration, the two experimental parameters of excitation wave and 4-mercaptobenzoic acid were optimized.

[0059] 1. Optimize the excitation wavelength: Based on the conditions in Example 1, prepare Au@M@Ag NRs solution. Mix 100 μL Au@M@Ag NRs solution with 50 μL H 2 O 2 The solution (150mM) was mixed evenly, and then 50μL of kaempferol (4μM) solution was injected into the above solution to mix. After incubation at room temperature for 40 minutes, 20μL of the mixed sample was dropped on a silicon wafer and dried for Raman detection. Raman detection of the sample was performed using three different excitation wavelengths: 785nm, 633nm, and 532nm.

[0060] 2. Optimize the concentration of 4-mercaptobenzoic acid: Based on the step conditions in Example 1, the added Raman signal molecule 4-MBA was changed to 10 μL with a concentration of 10 -2 μM, 10 -1The Au@M@Ag NRs solution with different Raman reporter molecule concentrations was prepared by preparing solutions of 1 μM, 1 μM, 10 μM, and 100 μM. 100 μL of the prepared Au@M@Ag NRs solution was evenly mixed with 100 μL of ultrapure water, and 20 μL of the mixed sample was dropped on a silicon wafer after incubation at room temperature for 40 minutes. After drying at room temperature, it was used for SERS detection. When the Raman spectrometer was used for detection, the excitation wavelength was 785 nm.

[0061] like Figure 6 As shown in Figure A, only laser excitation at 785 nm can achieve the best Raman enhancement effect for Au@M@Ag NRs (100 μM, 10 μL of 4-MBA). Therefore, the optimal laser wavelength for Raman sensors is 785 nm. Figure 6 As shown in B, with the increase of 4-MBA concentration, the -1 When the final concentration of 4-MBA reached 1 μM, the SERS signal was obvious and reached a stable state.

[0062] Example 5 Principle verification of kaempferol detection based on surface enhanced Raman scattering technology

[0063] Based on the conditions in Example 1, Au@M@Ag NRs solution was prepared. 100 μL of Au@M@AgNRs solution was mixed with H 2 O 2 The solution (50 μL, 150 mM) was mixed evenly, and then 50 μL of different concentrations (10 -3 μM, 10 -2 μM, 10 -1 μM, 1μM, and 10μM kaempferol were added to the sample and incubated at room temperature for 40 minutes. 20μL of the mixed sample was dropped on a silicon wafer and dried for SERS detection. When using a Raman spectrometer for detection, the excitation wavelength was 785nm, the laser power was 0.5%, the exposure time was 10s, and the spectral range was 300cm -1 -1800cm -1 .

[0066] like Figure 7 As shown in A, the Raman reporter molecule 4-MBA is at 1080 cm -1 and 1590cm -1There are two characteristic peaks at the edges of the nanomaterials. In the case of Au@M NRs alone, the Raman signal was found to be poor (line a), while on Au@M@Ag NRs, a clear 4-MBA Raman characteristic peak was shown (line b). The Raman signal enhancement is attributed to the synergistic effect between the bimetallic components, which can provide a large number of SERS active "hot spots" at the edge of the nanomaterials, thus bringing strong Raman signals to the analytes. 2 O 2 After that, the silver shell on the surface of Au@M@Ag NRs was oxidized and etched, resulting in a weakening of the Raman signal of 4-MBA (line d). 2 O 2 Compared with Au@M@Ag NRs / H 2 O 2 +KAE (1μM) SERS signal increased significantly (line e), indicating that kaempferol can inhibit the above etching process and restore the two Raman characteristic peak signals of 4-MBA. At the same time, the introduction of kaempferol (1μM) into Au@M@Ag NRs has little effect on the Raman intensity of 4-MBA (line c). These results confirm that it is feasible to detect kaempferol by observing the changes in the 4-MBA SERS signal. At the same time, under the optimal conditions, Figure 7 B shows the interaction of different concentrations of kaempferol with Au@M@AgNRs / H 2 O 2 The Raman spectrum of the mixed samples showed that the Raman signal intensity of 4-MBA increased with the increase of kaempferol concentration. Figure 7 C shows 1590cm -1 There is a linear relationship between the Raman signal intensity at 100 μg / cm2 and the kaempferol concentration, and its linear equation is ΔI = 33093.43 + 3618.95lgC KAE (R 2 =0.981). Here, ΔI represents the relative humidity of 4-MBA at 1590 cm -1 The SERS signal is shown in Figure 2, and C represents the concentration of kaempferol. The detection limit of kaempferol using the SERS method is about 0.786 nM. Therefore, the proposed dual-mode detection method can provide more help for the actual identification of kaempferol.

[0067] Example 6: Spiked recovery test of kaempferol in juice and cigarettes

[0068] In order to apply this method to real samples, juice and cigarettes were purchased from a supermarket, and cigarettes were obtained by peeling off the cigarette paper. 0.1 g of cigarette was dissolved in 10 ml of ultrapure water, and then the cigarette solution and juice were centrifuged and filtered separately. The spike experiment was performed within the linear range.

[0069] The experimental steps for the spike recovery of kaempferol by colorimetry are as follows: 100 μL of Au@M@Ag NRs solution was mixed with H 2 O 2 The solution (50 μL, 150 mM) was mixed evenly in a centrifuge tube, and 50 μL of the actual sample solution containing 0.8 μM, 4.0 μM, and 7.2 μM kaempferol was added. After incubation at room temperature for 40 minutes, the color of the solution was observed with the naked eye, and the absorbance spectrum was recorded with a UV-visible spectrophotometer. The absorbance of the sample was measured in the wavelength range of 200 to 900 nm, and the kaempferol recovery concentration (Found) and recovery rate (Recovery) of the samples containing different kaempferol concentrations (Added) were calculated. The experimental results are shown in Table 1. The spiked recovery rate of the detection method in juice and cigarette samples was between 90.60% and 109.44%, and the relative standard deviation RSD was less than 4.88%, indicating that the prepared method has good performance and can be used to detect kaempferol in juice and cigarette samples.

[0070] Table 1

[0071]

[0072] The experimental steps for the spike recovery of kaempferol using SERS are as follows: 100 μL Au@M@Ag NRs solution (the final concentration of 4-MBA used in the preparation is 1 μM) is mixed with H 2 O 2 The solution (50 μL, 150 mM) was mixed evenly in a centrifuge tube, and the -3 μM, 4.00×10 -1 The actual sample solution of 50 μL of 10 μM and 4.00×10 μM kaempferol was incubated at room temperature for 40 minutes, and 20 μL of the sample was dropped on a silicon wafer, dried at room temperature and used for SERS detection. The corresponding Raman signal was detected by Raman spectrometer, and the concentration of kaempferol in the solution was obtained by combining the corresponding linear fitting equation. The recovery concentration of kaempferol (Founded) was obtained by subtracting the unspiked result from the spiked result, and then the recovery concentration of kaempferol was compared with the theoretical concentration of kaempferol added to obtain the spiked recovery rate of kaempferol (Recovery). The experimental results are shown in Table 2.

[0073] Table 2

[0074]

[0075] As shown in Table 2, the recovery rate of the detection method in actual samples of juice and cigarettes is between 90.43% and 109.22%, and the relative standard deviation RSD is less than 5.62%. The above results show that the method of the present invention has good performance and can be used for the detection of kaempferol in juice and cigarettes.

[0076] Example 7 Differentiation of polyphenols based on linear discriminant analysis (LDA)

[0077] 1. Based on the experimental steps in Example 3, the kaempferol solution was replaced with 4 μM tannic acid, 4 μM chlorogenic acid, 4 μM epicatechin and 4 μM epigallocatechin gallate solutions, respectively. The color of the solution was observed with the naked eye, and the absorbance spectrum was recorded with a UV-visible spectrophotometer.

[0078] 2. Based on the experimental steps in Example 5, the kaempferol solution was replaced with 4 μM tannic acid, 4 μM chlorogenic acid, 4 μM epicatechin and 4 μM epigallocatechin gallate solutions, and the Raman scattering spectra of different systems and 1080 cm -1 and 1590cm -1 The Raman signal intensity at .

[0079] A dual-mode sensor based on Au@M@Ag NRs was constructed, and the resolution ability of the sensor was investigated by detecting five polyphenols, including chlorogenic acid (CA), epicatechin (EC), epigallocatechin gallate (EGCG), kaempferol (KAE), and tannic acid (TA). Figure 8 A shows that the Raman response behaviors of different polyphenols are different due to their different inhibition effects on silver shell etching. Then, the multidimensional response patterns of the dual-mode sensor were recorded and subjected to deep three-dimensional linear discriminant analysis (LDA) to perform discriminant analysis for each polyphenol. Figure 8 B shows that the five groups of polyphenols can be successfully separated without overlap. Figure 8 The hierarchical clustering diagram (HCA) shown in C also confirmed the effective differentiation of the five polyphenols without any misclassification. Figure 8 D heat map also showed that the dual-mode sensor could effectively identify the five polyphenols.

[0080] Example 8 Identification of multiple polyphenols in real samples

[0081] Based on the experimental steps in Example 6, the kaempferol solution was replaced with 4 μM tannic acid, 4 μM chlorogenic acid, 4 μM epicatechin and 4 μM epigallocatechin gallate solutions. Fig. 9As shown in Figures A and 9B, the present invention evaluates the ability of the dual-mode sensor to identify five polyphenols (CA, EC, EGCG, KAE, and TA) in complex samples (including juice and cigarettes). The results based on LDA analysis show that the five polyphenols are distributed in five independent clusters without overlap, which confirms the application ability of the dual-mode sensor to identify polyphenols in actual samples.

Claims

1. An Au@M@Ag NRs nanomaterial, characterized in that: The Au@M@Ag NRs nanomaterial consists of an Au@Ag NRs core-shell structure and 4-mercaptobenzoic acid coated inside.

2. A method for preparing the Au@M@Ag NRs nanomaterial according to claim 1, characterized in that: The following steps are involved: 1) CTAB solution, chloroauric acid solution, AgNO3 solution, hydroquinone solution and NaBH4 solution are mixed, reacted in a constant temperature water bath, and the reaction product is centrifuged and washed to obtain Au NRs solution; 2) adding 4-mercaptobenzoic acid solution to the Au NRs solution in step (1), incubating the Au NRs solution under constant temperature shaking on a shaker, and washing the reaction product by centrifugation to obtain an Au@M NRs solution; 3) Adding AgCl solution, Au@M NRs solution and ascorbic acid solution to the CTAC solution respectively, reacting in a constant temperature water bath, and washing the reaction product by centrifugation to obtain Au@M@Ag NRs nanoparticles.

3. The preparation method according to claim 2, characterized in that: The final concentration ratio of the CTAB solution, chloroauric acid solution, AgNO3 solution, hydroquinone solution and NaBH4 solution in step 1) is 0.1:0.4:0.05-0.15:3-6:0.01-0.05, the shaking incubation temperature is 25-35°C, and the shaking incubation time is 10-12h.

4. The preparation method according to claim 2, characterized in that: The final concentration of the 4-mercaptobenzoic acid solution in step 2) is 0.5-2.0 μM.

5. The preparation method according to claim 2, characterized in that: In step 3), the final concentration ratio of the CTAC solution, the AgCl solution, and the ascorbic acid solution is 60-80:0-0.5:50-100, the temperature of the water bath is 30-35° C., and the time is 3-4 h.

6. Use of the Au@M@Ag NRs nanoparticles according to claim 1 in identifying, detecting and / or distinguishing polyphenols; the polyphenols include one or more of kaempferol, chlorogenic acid, epicatechin, epigallocatechin gallate or tannic acid solution.

7. A polyphenol detection kit, characterized in that: It contains the Au@M@Ag NRs nanoparticles according to claim 1; the polyphenols include one or more of kaempferol, chlorogenic acid, epicatechin, epigallocatechin gallate or tannic acid solution.

8. Use of the Au@M@Ag NRs nanoparticles according to claim 1 or the polyphenol detection kit according to claim 7 in recovering polyphenols.

9. A method for quantitatively detecting the concentration of kaempferol, characterized in that: The quantitative detection method is selected from any one or both of the following methods: A1) After mixing the Au@M@Ag NRs nanoparticles described in claim 1 and H2O2 solution, a kaempferol solution is added and reacted for 30-50 min, and the absorbance is measured by a UV-visible spectrometer. The kaempferol content in the solution to be tested is obtained according to a linear relationship equation between kaempferol solutions of different known concentrations and the shift value of the UV absorption peak; the kaempferol concentration is 10 -2 ~100μM; the linear relationship equation between the different concentrations of kaempferol solution and the ultraviolet absorption peak shift value is Δλ=34.73+41.08C KAE (R 2 =0.975), Δλ is the ultraviolet absorption peak shift value, and C is the kaempferol concentration. A2) After mixing the Au@M@Ag NRs nanoparticles described in claim 1 and H2O2 solution, adding kaempferol solution, dripping on a silicon wafer, and measuring the Raman signal with a Raman spectrometer after drying, the content of kaempferol in the test solution is obtained according to the linear relationship equation between the polyphenol solutions of different known concentrations and the Raman signal intensity, wherein the final concentration of kaempferol is 10 -9 -10 -5 M; the linear relationship equation between the known different concentrations of kaempferol solution and the Raman signal intensity is ΔI = 33093.43 + 3618.95lgC KAE (R 2 =0.981), where ΔI is 1590 cm -1 is the Raman signal intensity at , and C is the kaempferol concentration.

10. The method according to claim 9, characterized in that: The concentration of H2O2 in method A1) is 100-200 mM.

Images