Au-DTNB-Ag nanosphere as well as preparation method and application thereof in preparation of SERS (Surface Enhanced Raman Scattering) enhanced immunochromatography test paper

Abstract

The invention provides an Au-DTNB-Ag nanosphere, a preparation method and application of the Au-DTNB-Ag nanosphere in preparation of SERS (Surface Enhanced Raman Scattering) enhanced immunochromatography test paper. Wherein the Au-DTNB-Ag nanosphere comprises a nanogold core, a nanosilver shell and a self-assembly DTNB monomolecular layer located in the middle, the particle size of the nanogold core ranges from 16 nm to 18 nm, and the thickness of the nanosilver shell ranges from 17 nm to 18 nm. The invention also provides an SERS (Surface Enhanced Raman Scattering) immune probe using the nanosphere, DTNB in the Au-DTNB-Ag nanosphere is coupled with the rabbit-derived polyclonal antibody, and the invention also provides an application of the nanosphere or the immune probe in preparation of SERS enhanced immunochromatography test paper. The SERS technology is combined with the immunochromatography test paper, rapid and high-sensitivity quantitative detection and analysis of soybean allergen glycinin are achieved, actual sample detection of soybean products in daily life is achieved, a more convenient, rapid and efficient detection tool is provided for detection of allergens in food of allergic consumers, and the method is suitable for large-scale popularization and application. And reference opinions are provided for selection of consumers.

Description

Technical Field

[0001] The invention relates to the fields of food safety, Raman spectroscopy analysis and immunological detection, and in particular to an Au-DTNB-Ag nanosphere and a preparation method thereof and application in preparing a SERS enhanced immunochromatographic test paper. Background Art

[0002] As a high-quality protein source, soybeans provide animals with abundant plant protein and essential amino acids, becoming an indispensable key ingredient in animal diets. In addition, soybeans are rich in fat, including vitamin E, vitamin D, and linoleic acid required by the human body, which is extremely beneficial to human health. However, it is worth noting that there are also some allergens in soybeans, such as glycinin and β-conglycinin. When the human body ingests these ingredients, some people may have allergic reactions. In mild cases, diarrhea and vomiting may occur, and in severe cases, anaphylactic shock may occur, threatening life and health. Therefore, when people eat soybeans and soy products, they should pay special attention to whether they are allergic to soy products.

[0003] At present, there are many options for detecting soybean allergens, including real-time fluorescence quantitative (rt-qPCR), ELISA, immunochromatographic test strips, protein immunoblotting (WB) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-Q-TOF-MS). Among them, real-time fluorescence quantitative (RT-qPCR) is a commonly used detection method, but its equipment is expensive and needs to rely on a standard curve, which is costly and will introduce large errors. As a commonly used detection method, ELISA has a long detection time and large errors, so it is not convenient for rapid detection. Protein immunoblotting is a common molecular biology technique that is usually used to detect and quantify protein expression levels, but its operation process is complicated and can only achieve relative quantification of the analyte, and cannot accurately measure the absolute concentration. As for ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-Q-TOF-MS), although it has high detection sensitivity and can detect multiple allergens at the same time, its experimental equipment is expensive and requires professional personnel to operate. Although traditional colloidal gold immunochromatographic test strips are fast and easy to operate, the strips have problems such as low detection sensitivity, serious false positives, and inability to accurately quantify. Summary of the invention

[0004] In view of this, the present invention provides Au-DTNB-Ag (gold core silver shell) nanospheres and a preparation method thereof and application in the preparation of SERS enhanced immunochromatographic test paper, combining SERS technology with immunochromatographic test paper to achieve rapid and highly sensitive quantitative detection and analysis of soybean allergen glycinin.

[0005] The present invention is achieved through the following technical solutions: An Au-DTNB-Ag nanosphere is a core-shell structure, including a gold nanoparticle core (AuNPs), a silver nanoparticle shell, and a self-assembled monolayer (SAM) of DTNB located in the middle, wherein one end of the DTNB in ​​the self-assembled DTNB monolayer forms an S-Au bond with the surface of the gold nanoparticle core through a thiol group and is adsorbed on the surface of the gold nanoparticle core, and the other end interacts with the silver nanoparticle shell through a carboxyl group, wherein the particle size of the gold nanoparticle core is 16 to 18 nm, the thickness of the silver nanoparticle shell is 17 to 18 nm, and the coverage of the SAM layer on the gold nanoparticle core is 99% to 100%. This can further enhance the intensity of the SERS (surface enhanced Raman scattering) signal. The full name of DTNB in ​​Chinese is 5,5'-dithiobis-(2-nitrobenzoic acid), and the full name in English is 5,5'-Dithiobis-(2-nitrobenzoic acid).

[0006] Preferably, the particle size of the Au-DTNB-Ag nanospheres is 33-36 nm, and the mass ratio of the nano-gold core to the nano-silver shell is 40.09:53.27-43.04:56.96.

[0007] A preparation method of the above-mentioned Au-DTNB-Ag nanospheres comprises the steps of: firstly stirring a colloidal gold solution and a DTNB ethanol solution at room temperature to react, so as to obtain an Au-DTNP solution; then centrifuging the Au-DTNP solution, then adding hydroquinone and stirring to react, and finally adding an AgNO3 solution and stirring at room temperature until the color of the solution no longer changes, so as to obtain a brown-yellow Au-DTNB-Ag colloidal solution; and centrifuging and drying to obtain the Au-DTNB-Ag nanospheres.

[0008] Preferably, to 1 mL of the colloidal gold solution, a DTNB ethanol solution having a concentration of 1 mM and a volume of 10 to 50 μL and a 0.1 mol / L AgNO 3 solution having a volume of 10 to 50 μL are added.

[0009] The colloidal gold is prepared by an existing method, specifically, an AuNPs solution is prepared by a trisodium citrate reduction method.

[0010] A SERS immune probe comprises the Au-DTNB-Ag nanosphere and a rabbit-derived polyclonal antibody, wherein the DTNB Raman signal molecule in the Au-DTNB-Ag nanosphere is coupled with the rabbit-derived polyclonal antibody.

[0011] Preferably, further, the pH value of the SERS immunoprobe is 8.5-9.0.

[0012] A method for preparing the above-mentioned SERS immune probe includes first mixing Au-DTNB-Ag colloidal solution and K2CO3 solution, then adding rabbit polyclonal antibody for one incubation, then adding BSA solution for a second incubation, then centrifuging, discarding the supernatant, obtaining immune probe precipitation, drying, and preparing the SERS immune probe.

[0013] Preferably, 0.2M, 0.5-2.0 μL of the K2CO3 solution is added to 1 mL of Au-DTNB-Ag colloidal solution, and the amount of rabbit polyclonal antibody added is 7-28 μg. The rabbit polyclonal antibody is a soybean globulin rabbit polyclonal antibody.

[0014] An application of the Au-DTNB-Ag nanosphere or SERS immunoprobe in preparing a SERS enhanced immunochromatographic test paper, wherein the SERS enhanced immunochromatographic test paper comprises a conjugate pad on which the SERS immunoprobe is dripped.

[0015] A SERS-enhanced immunochromatography test paper comprises a sample pad, a conjugate pad, an NC membrane and a water absorbent pad, wherein a detection line (T line) and a quality control line (C line) are arranged on the NC membrane, a mouse monoclonal antibody is dripped on the T line, a goat anti-rabbit secondary antibody is dripped on the C line, and the above-mentioned SERS immune probe is dripped on the conjugate pad.

[0016] Preferably, the amount of the mouse monoclonal antibody added is 0.3 μL, 1-4 mg / mL.

[0017] Furthermore, the above-mentioned immunochromatographic test paper also includes a support layer as a bottom layer.

[0018] The present invention also provides an application of the above-mentioned SERS enhanced immunochromatographic test paper in detecting at least one of the following substances: 1) Soybean allergen glycinin; 2) Soy products.

[0019] The present invention also provides a method for preparing the test paper, comprising: Providing Au-DTNB-Ag colloidal solution; Providing a SERS immunoprobe solution: re-dissolving the above immunoprobe with TB buffer to obtain a SERS immunoprobe solution, wherein the TB buffer comprises 0.02 M Na2B4O7, 1% BSA and 0.03% NaN3; Assemble the test paper: stick the sample pad, conjugate pad, NC membrane, and absorbent pad on the support pad, with each layer overlapping by about 1 to 3 mm, drip the SERS immune probe solution on the conjugate pad, drip the mouse monoclonal antibody solution on the T line, and drip the goat anti-rabbit secondary antibody solution on the C line.

[0020] Therefore, the immunochromatographic test paper provided by the present invention is based on the principle of double antibody sandwich, with rabbit polyclonal antibody as capture antibody and mouse monoclonal antibody as detection antibody. Mouse monoclonal antibody has strong specificity and high sensitivity, and can achieve specific and effective binding of antigen and antibody, so that the immunochromatographic test paper has strong specificity and high sensitivity, and will not cross-react with other proteins and has strong stability. Experiments show that the visual detection limit of the immunochromatographic test paper is 1 ng / mL.

[0021] The above-mentioned immunochromatographic test paper provided by the present invention is based on the combination of SERS technology and immunochromatographic technology, showing excellent labeling ability and ultra-sensitivity as well as the advantage of being able to achieve quantitative detection, which can effectively solve the bottleneck problems of low sensitivity, inaccurate quantification, poor anti-base interference ability, and difficulty in meeting the increasingly stringent detection requirements for food safety in the traditional colloidal gold immunochromatographic test paper. The present invention uses SERS as a substrate and introduces the Raman molecule DTNB on the surface of the Au-DTNB-Ag nanomaterial, which can significantly enhance its Raman scattering signal, making the originally weak signal more obvious. Through the auxiliary detection of the Raman spectrometer, compared with the traditional test paper, the sensitivity has been greatly improved, and the analyte at extremely low concentration can be detected without additional complex steps.

[0022] The above-mentioned immunochromatographic test paper provided by the present invention can also be used to detect actual samples of soybean products in daily life. After simple pretreatment of the purchased food, it can detect whether the relevant food samples contain soybean allergen glycinin, providing a more convenient, quick and efficient detection tool for the detection of allergens in the food of allergic consumers, and providing reference opinions for consumers' choices. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A flow chart of the preparation of the SERS immune probe provided by the present invention and a detection principle diagram of the immune chromatography test paper based on the probe; Figure 2 (A) UV analysis spectrum of AuNPs; (B) UV analysis spectrum of AgNPs; (C) UV analysis spectrum of AuNP-DTNB-Ag; (D) UV analysis spectrum of Au-Ag-DTNB; (E) UV analysis spectrum of different volume DTNB coverage; (F) Relationship between DTNB volume and LSPR peak red shift value of AuNPs; Figure 3(A) Raman spectrum of Au-DTNB-Ag; (B) Raman spectrum of Au-Ag-DTNB; Figure 4 (A) TEM image of Au-DTNB-Ag NPs; (B) particle size image of Au-DTNB-Ag NPs; (C) high-angle annular dark field image of Au-DTNB-AgNPs; (D) ~ (F) energy spectrum X-ray diffraction image of Au-DTNB-Ag NPs; (G) mass ratio of Au to Ag; Figure 5 (A) X-ray diffraction pattern of Au-DTNB-Ag NPs; (B) X-ray diffraction pattern of AuNPs and Au-DTNB-Ag NPs; (C) X-ray photoelectron spectrum of Au-DTNB-Ag NPs; (D) S2p fine spectrum; (E) Au4f fine spectrum; (F) Ad3d fine spectrum; Figure 6 Screening diagram for exploring the optimal pH value (+ for positive, - for negative) for preparing immune probes; Figure 7 Prepare a screening plot for the optimal antibody labeling amount (+ for positive, - for negative) for the probe immunoprobe; Figure 8 This is a screening diagram for optimizing the amount of antibody added to the T line (detection line) on the test strip (+ for positive, - for negative); Fig. 9 (A) UV analysis spectrum of the immunoprobe; (B) Zeta potential of the immunoprobe; Fig.10 Figure 1 shows the test results of soybean globulin standard solution with different concentrations; 1, 10 4 ng / mL; 2, 10 3 ng / mL; 3, 10 2 ng / mL; 4, 10 ng / mL; 5, 1 ng / mL; 6, 0.1 ng / mL; 7, 0 ng / mL; Fig.11 The results of the specificity test of the test strips are shown below: 1. Soybean globulin; 2. β-conglycinin; 3. Peanut protein; 4. Sesame protein; 5. Bovine serum albumin; 6. Wheat protein; Fig.12 This is a graph of the test strip repeatability test results (+ for positive, - for negative); Fig.13 This is the test paper stability test result diagram; Fig.14 (A) Raman signal diagram of the T line of the test paper; (B) Standard curve diagram of soybean globulin detection; Fig.15 This is the actual sample test result diagram. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0025] The terms used in the present invention are all commonly used terms in the relevant field, and the raw materials, equipment, preparation processes, test methods, etc. used, unless otherwise specified, are all prior art in the relevant field. The rabbit-derived polyclonal antibody used in each embodiment is a soybean globulin polyclonal antibody with a concentration of 9.25 mg / mL.

[0026] See also Figure 1 In order to achieve rapid and highly sensitive quantitative detection and analysis of soybean allergen glycinin, the present invention adopts spherical colloidal gold AuNPs particles prepared by trisodium citrate reduction method; AuNPs form S-Au bonds with the thiol group in the Raman molecule DTNB, so that DTNB is stably adsorbed on the surface of AuNPs to form a self-assembled DTNB monolayer SAM, which is wrapped on the surface of AuNPs to obtain AuNPs-DTNB particles; the AuNPs-DTNB particles are uniformly mixed with silver nitrate, and the silver nitrate undergoes an in-situ decomposition reaction on the surface of AuNPs-DTNB to form nanosilver, which is wrapped on the surface of AuNPs-DTNB to form a nanosilver shell, thereby forming nanospheres Au-DTNB-Ag NPs, and the silver shell in the nanosphere interacts with the carboxyl group at the other end of DTNB, which can further enhance the intensity of the SERS signal; Au-DTNB-Ag NPs are coupled with rabbit polyclonal antibodies (PAb) to form Au-DTNB-Ag-PAb NPs, Au-DTNB-Ag-PAb NPs were blocked by BSA solution to obtain SERS immunoprobe.

[0027] The above-mentioned SERS immunoprobe is combined with the existing technology to prepare a SERSERS enhanced immunochromatography test paper, which uses the double antibody sandwich principle to detect samples. First, the test solution is dripped on the sample pad of the test paper. Then, due to the chromatography effect, the test solution will diffuse to the NC membrane together with the SERS immunoprobe in the binding pad, and finally reach the absorbent pad. In this process, the soybean allergenic protein first binds to the SERS immunoprobe, and then binds to the T line on the NC membrane. Since the SERS immunoprobe is labeled with a rabbit polyclonal antibody and the T line is labeled with a mouse monoclonal antibody, the two antibodies will bind to different antigenic determinants on the soybean allergenic protein, so that the soybean allergenic protein forms an antigen detection mode between a monoclonal antibody and a polyclonal antibody, that is, a double antibody sandwich. Next, the C line on the NC membrane is labeled with a goat anti-rabbit secondary antibody, which will bind to the SERS immunoprobe. In the presence of soybean allergenic protein, both the T line and the C line will show color; on the contrary, if the object to be detected does not contain allergenic protein, the T line will not show color, but the C line will show color. If the C line does not show color, the test paper is considered invalid.

[0028] The present invention combines SERS technology with immunochromatographic test paper, which can not only realize rapid and highly sensitive quantitative detection and analysis of soybean allergen glycinin, but also realize actual sample detection of soybean products in daily life, providing a more convenient, quick and efficient detection tool for the detection of allergens in food of allergic consumers, and providing reference opinions for consumers' choices.

[0029] Example 1 Preparation of Au-DTNB-Ag spherical nanomaterials 1.1 Preparation of AuNPs Take 1 mL of 1% w / v chloroauric acid solution and add it to a flask with 99 mL of deionized water. Then, when it is heated to boiling, quickly add 1.5 mL of 1% w / v sodium citrate solution. Stop heating after 15 minutes. At this time, the colloidal gold solution is wine red. Store it in a refrigerator at 4°C. Observe with the naked eye that the prepared colloidal gold solution is wine red and remains transparent without suspended precipitated particles. In addition, Figure 2 The ultraviolet spectroscopic analysis shown in (A) shows an obvious absorption peak at 525 nm. By analyzing the width of the absorption peak, it can be preliminarily determined that the prepared colloidal gold particles have a uniform size of about 20 nm. The colloidal gold solution prepared in this example was stored in a refrigerator at 4°C for use in the following examples.

[0030] 1.2 Preparation of Au-DTNB-Ag NPs The present invention uses DTNB as a Raman molecule, and each end of DTNB has a thiol group and a carboxyl group. The thiol group interacts with the S-Au bond formed on the surface of colloidal gold, so that DTNB can be stably adsorbed on the surface of colloidal gold to form a SAM, which can wrap the colloidal gold. At the same time, the carboxyl group at the other end of DTNB can interact with the silver shell, further enhancing the intensity of the SERS signal. The specific preparation method is as follows: Take 1 mL of colloidal gold solution and add 30 μL of 1 mM DTNB ethanol solution, then stir at room temperature for 4 hours. After stirring, place the mixture in a 4°C refrigerator overnight to ensure the formation of DTNB SAM on the surface of colloidal gold. Centrifuge 1 mL of Au-DTNP solution at 9500 r / min for 15 min to remove excess DTNB molecules. Then add 30 μL of 10 mM hydroquinone to 1 mL of Au-DTNP solution and stir for 5 min. After that, quickly add 40 μL of 100 mM AgNO3 solution and stir at room temperature for 1 h until the color of the solution no longer changes. At this time, the colloidal solution is brown-yellow, indicating that the gold-silver core-shell nanosphere particles Au-DTNB-Ag NPs were successfully prepared to obtain the Au-DTNB-Ag colloidal solution. By Figure 2 (B) It can be analyzed that Ag has an obvious characteristic peak between 390 and 410 nm, and as the volume of AgNO3 increases, the characteristic peak of Ag gradually appears, while the characteristic peak of Au begins to shift to the blue and gradually disappears, while the characteristic peak of Ag begins to show a red shift phenomenon, indicating that the hydroquinone reducing agent used successfully reduces AgNO3 to Ag 0 , and a silver shell was formed on the surface of AuNPs-DTNP.

[0031] 1.3 Preparation of Au-Ag NPs-DTNB Take 1 mL of colloidal gold solution and add 80 μL of 100 mM ascorbic acid solution and stir for 5 min. Then, slowly add 30 μL of 100 mM AgNO3 solution and stir at room temperature for 1 h until the color is stable and no longer changes. Then, centrifuge the prepared Au-Ag NPs at a centrifugal force of 3420 r / min for 8 min to remove excess ascorbic acid and AgNO3 solution, and re-dissolve with deionized water to the initial volume. The subsequent DTNB addition steps are the same as the aforementioned "1.2 Preparation of Au-DTNB-AgNPs". Figure 2 (D) It can be seen that Ag has an obvious characteristic peak between 390 and 410 nm, and as the volume of AgNO3 increases, the characteristic peak of Ag gradually appears, while the characteristic peak of Au begins to shift to the blue and gradually disappears, while the characteristic peak of Ag begins to show a red shift phenomenon, which indicates that the ascorbic acid reducing agent used successfully reduces AgNO3 to Ag 0 , and a silver shell was formed on the surface of AuNPs.

[0032] 1.4 Exploring the coverage of the Raman molecule DTNB In the Raman molecule step of modifying the surface of AuNPs, colloidal solutions with different DTNB molecule coverage can be obtained by adding different volumes of DTNB molecule solution to the colloidal gold solution. The specific operation is as follows: add 10, 20, 30, 40, and 50 μL of 1 mM DTNB ethanol solution to 1 mL of colloidal gold solution, and supplement with pure ethanol solution so that the total amount of ethanol added to each sample is 50 μL. Subsequently, stir at room temperature for 4 h, and then store in a 4°C refrigerator overnight. The next day, centrifuge at 9500 r / min for 15 min to remove excess DTNB, and then perform UV-visible light detection. After the DTNB molecules are modified on the surface of gold nanoparticles, the localized plasmon resonance (LSPR) peak on the surface of AuNPs will produce a red shift due to the change in the refractive index of the surrounding environment. Therefore, the adsorption of DTNB on colloidal gold can be monitored by real-time measurement of the UV-visible spectrum of the AuNPs-DTNB mixture. According to Figure 2 (E) The results show that when the concentration of added DTNB increases to 30 μL, the UV-visible spectrum of colloidal gold continues to red-shift. However, once the concentration exceeds 30 μL, the degree of red-shift hardly changes. Figure 2 (F) shows that even if the amount of DTNB is increased, the amount of DTNB adsorption cannot be increased. This is mainly because the Raman molecule DTNB has reached a monolayer saturated adsorption on the surface of AuNPs. Therefore, 30 μL of DTNB was finally selected to label 1 mL of colloidal gold solution.

[0033] 1.5 Raman spectroscopy analysis According to the different positions of Raman molecule addition, the present invention designs the above-mentioned Au-DTNB-Ag NPs and Au-Ag NPs-DTNB, the two inner and outer nanomaterials, and then explores the Raman signal intensity of the two materials respectively. This test method is basically the same as the aforementioned "1.2 Preparation of Au-DTNB-Ag NPs" and "1.3 Preparation of Au-Ag NPs-DTNB", the main difference is that the added volume of AgNO3 solution is 10, 20, 30, 40, 50 μL concentration, respectively. The results are as follows Figure 3 shown.

[0034] from Figure 3 It can be seen that the Raman molecule DTNB is at 1061cm -1 , 1335cm -1 , 1557cm -1 There are obvious characteristic peaks corresponding to CN bending, CN stretching mode, and symmetric nitro stretching to NO2. In the Raman peak of DTNB, 1335 cm -1 The signal peak at is the strongest, so this peak is selected for quantitative analysis. Figure 3 (A) is the Raman spectrum of Au-DTNB-Ag NPs. From this figure, it can be found that as the volume of AgNO3 increases, the Raman signal intensity first increases and then decreases. When the volume of AgNO3 is 40 μL, its Raman signal reaches the highest value. It is speculated that this may be because more "hot spots" are generated between the gold core and the silver shell as the silver shell gradually increases. "Hot spots" are a very strong signal enhancement phenomenon in Raman spectroscopy detection and can be used to improve the intensity and detection sensitivity of Raman signals. "Hot spots" usually appear at the tip or edge of nanomaterials, where the electric field is the strongest. When Raman molecules exist near the "hot spot" area, the strong electromagnetic field generated by the hot spot can interact with the molecules, increase the vibration scattering cross section of the molecules, and thus increase the Raman signal intensity.

[0035] However, when the thickness of the silver shell is too high, the SERS signal may be shielded. Therefore, the optimal addition amount of AgNO3 is 40 μL in 1 mL of Au-DTNB solution. Figure 3 (B) corresponds to the Raman spectrum of Au-Ag NP-DTNB. Similar rules can be found from this figure. In a 1mL solution system, when the addition amount of AgNO3 is 30 μL, the Raman signal reaches the highest value. Analysis shows that this result may be caused by the following factors: (1) Shielding effect: When the thickness of the silver shell is too thick, the excitation light cannot directly enter the core area, thereby weakening the Raman signal intensity. (2) Surface enhanced Raman scattering effect: Because silver also has the ability of surface plasmon resonance, when the thickness of the silver shell increases, the resonance frequency of the surface plasmon will change. Only when the thickness of the silver shell is moderate, the resonance frequency is close to the frequency of the Raman excitation light, and the optimal surface enhanced Raman scattering effect will be achieved, resulting in an increase in the Raman signal, otherwise the Raman signal will decrease. By comparing the Raman signal values ​​of the two nanomaterials, Au-DTNB-Ag NPs were finally selected.

[0036] 1.6 Characterization and analysis of nanomaterials Through transmission electron microscopy observation, Figure 4 (A) It can be seen that Au-DTNB-Ag NPs present a spherical structure. Figure 4 (B) shows the particle size distribution diagram. When the volume of AgNO3 solution is 40 μL, the particle size of Au-DTNB-Ag NPs is 36 nm, the particle size of the gold core is 18 nm, and the thickness of the silver shell is also 18 nm. In addition, Figure 4 (C) shows the high-angle annular dark field image (HAADF-STEM) of Au-DTNB-Ag NPs. Figure 4(D) to (F) show the energy-dispersive X-ray diffraction (EDX) patterns of Au-DTNB-Ag NPs, which indicate that the silver shell is successfully coated on the outside of the gold core. Figure 4 The results of (G) determined that the mass ratio of gold and silver is 43.04:56.96.

[0037] The prepared gold-silver alloy material was analyzed by X-ray diffraction (XRD). Figure 5 From the XRD pattern of (A), we can observe that there are diffraction peaks at 38°, 44°, 64°, 77° and 81°, which correspond to the (111), (200), (220), (311) and (222) crystal planes of Au and Ag materials with metal face-centered cubic structures. Figure 5 As can be seen in (B), the diffraction peaks of Au-AgNPs are the same as those of AuNPs. This is because Au and Ag have the same face-centered cubic crystal structure, and the lattice constant and atomic radius are also similar. Therefore, it is impossible to accurately distinguish Au and Ag by the XRD method. In addition, the sharpness and intensity of the diffraction peaks in the XRD spectrum are related to the crystallinity and grain size. Figure 5 In the comparison of (C), it can be found that the diffraction peak of the nanomaterial prepared by the present invention is very sharp, which indicates that the two materials have high crystallinity. At the same time, the diffraction peak intensity of Au-AgNPs is significantly higher than that of AuNPs, which indirectly proves that a layer of silver shell is formed on the surface of AuNPs under the action of hydroquinone and AgNO3.

[0038] The surface properties of the gold-silver alloy material were studied using X-ray photoelectron spectroscopy (XPS). The presence of three elements, Au, Ag and S, can be clearly observed from the full spectrum of XPS, where the S element comes from the Raman molecule DTNB. In the full spectrum, the characteristic peaks with binding energies of 368eV and 573eV correspond to the 3d and 3p electronic energy levels of Ag, the characteristic peak with a binding energy of 84eV corresponds to the 4f electronic energy level of Au, and the characteristic peak with a binding energy of 168.5eV corresponds to the 2p electronic energy level of S. Figure 5 (F) shows the detailed spectrum of the 3d electron energy level of Ag. The characteristic peaks at 367.8 eV and 373.8 eV correspond to the 3d 5 / 2 and 3D 3 / 2 Energy level, from this spectrum, we can observe not only Ag 0 The presence of AgO can also be found. Since Ag has strong activity, it can react with O2 in the air. This shows that under the action of hydroquinone, AgNO3 is successfully reduced to elemental Ag and adsorbed on the surface of AuNPs. Figure 5(E) shows the detailed spectrum of the 4f electron energy level of Au, and the characteristic peaks at 84.2 eV and 87.8 eV correspond to the existence of Au-S bonds. Figure 5 (D) shows the detailed spectrum of the 2p electron energy level of S. The characteristic peaks at 168.5 eV and 163 eV correspond to SO4 2- These XPS spectra indicate that the prepared gold-silver alloy nanomaterials have stable chemical properties.

[0039] Example 2: Au-DTNB-Ag immunochromatographic test paper based on SERS and its preparation This embodiment provides a SERS immune probe, which is prepared by coupling the Au-DTNB-Ag nanoparticles provided in Example 1 and rabbit polyclonal antibodies.

[0040] This embodiment also provides a method for preparing the SERS immune probe, including adding 0.2 M K2CO3 to 1 mL of the Au-DTNB-Ag colloidal solution prepared in the aforementioned "Preparation of 1.2 Au-DTNB-Ag NPs". Then, a rabbit-derived polyclonal antibody of soybean globulin is added and stirred for 1 h, followed by adding 100 μL of a 10% BSA solution to block the excess sites on the surface of the nanomaterial. The mixed solution is placed at 4°C overnight. The next day, centrifuge at 9700 g for 20 min, remove the supernatant, obtain the immune probe, and re-dissolve with 50 μL of TB resuspension to obtain a SERS immune probe solution. Among them, the TB resuspension in this embodiment consists of 0.02 M Na2B4O7, 1% BSA and 0.03% NaN3.

[0041] This embodiment also provides an immunochromatographic test paper based on the above-mentioned SERS immune probe, wherein the bottom layer of the test paper is a support layer, and an adsorption layer consisting of a sample pad, a conjugation pad, an NC membrane and a water-absorbing pad is arranged on the support layer. Wherein, a detection line T line and a quality control line C line are arranged on the NC membrane, mouse monoclonal antibody is dripped on the T line, and goat anti-rabbit secondary antibody is dripped on the C line. The above-mentioned SERS immune probe solution is dripped on the conjugation pad.

[0042] The present invention also provides a method for preparing the above-mentioned immunochromatographic test paper, comprising the steps of assembling the test paper: pasting the sample pad, conjugate pad, NC membrane, and absorbent pad on a support pad, with each layer having a stacking thickness of about 1 to 3 mm, dripping 1.5 μL of the above-mentioned SERS immune probe solution on the conjugate pad, dripping 0.3 μL of mouse monoclonal antibody solution on the T line, dripping 0.3 μL of 1 mg / mL goat anti-rabbit secondary antibody solution on the C line, placing in an oven to dry for 5 hours, adding a desiccant and sealing, and storing at room temperature.

[0043] The pH of the Au-DTNB-Ag colloidal solution and the rabbit polyclonal antibody have an important influence on the performance of the SERS immunoprobe. The optimal pH value and optimal antibody labeling amount of the SERS immunoprobe are determined by dropping different volumes of K2CO3 and different masses of rabbit polyclonal antibodies, adding the antigen to the test paper, and observing the color development of the T line.

[0044] 2.1 Optimal pH value for Au-DTNB-Ag labeled rabbit polyclonal antibody Optimized conditions: K2CO3 volumes were 0.5, 1.0, 1.5, and 2.0 μL, respectively, rabbit polyclonal antibody to soybean globulin was 21 μg, and mouse monoclonal antibody concentration was 4.02 mg / mL. Figure 6 As shown in the figure, when the amount of K2CO3 added is 1 μL, the color imprint at the T line of the test paper is the darkest. Therefore, the optimal amount of K2CO3 added to 1 mL of Au-DTNB-Ag colloidal solution is determined to be 1 μL.

[0045] 2.2 Optimal labeling amount of rabbit polyclonal antibody Optimized conditions: K2CO3 volume was 1.0 μL, the amount of soybean globulin rabbit polyclonal antibody added was 7, 14, 21, and 28 μg, respectively, and the concentration of mouse monoclonal antibody was 4.02 mg / mL. The results are as follows Figure 7 As shown in the figure, when the amount of polyclonal antibody is 21 μg, the color imprint at the T line of the test paper is the darkest. Therefore, the optimal labeling amount of rabbit polyclonal antibody is 21 μg.

[0046] 2.3 Screening of SESR immunoprobes Au-DTNB-Ag and Ab-Au-DTNB-Ag, which were prepared at the optimal pH value and optimal labeling amount of Au-DTNB-Ag labeled rabbit polyclonal antibody, were scanned in the ultraviolet wavelength range of 300 to 700 nm. The results are as follows: Figure 8 As shown, Figure 8 (B) shows that when the rabbit polyclonal antibody successfully combines with the Au-DTNB-Ag nanomaterial to form a complex, its maximum absorption peak wavelength undergoes a red shift. This red shift phenomenon indicates that the coupling reaction between Au-DTNB-Ag and the rabbit polyclonal antibody is successful. At the same time, Figure 8 (A) The observed zeta potential change also further verified the successful coupling of Au-DTNB-Ag with anti-rabbit polyclonal Ab.

[0047] 2.4 Optimization of the labeling amount of the test paper T line (detection line) The amount of monoclonal antibody added at the T line will also affect the performance of the test strip. Optimized conditions: K2CO3 volume is 1.0 μL, the amount of soybean globulin rabbit polyclonal antibody added is 21 μg, and the concentration of mouse monoclonal antibody is 1, 2, 3, and 4 mg / mL. The results are as follows: Fig. 9 As shown in the figure, when the concentration of mouse monoclonal antibody coated on the T line is lower than 2 mg / mL, the T line does not show color. Excessive addition of antibodies will not only cause waste of raw materials, but also increase costs. Therefore, adding 0.3 μL of 2 mg / mL mouse monoclonal antibody at the T line is optimal.

[0048] Example 3 Performance test of Au-DTNB-Ag immunochromatographic test paper based on SERS 3.1 Sensitivity test Set seven gradient soybean globulin solution concentrations of 10 4 ng / mL, 10 3 ng / mL, 10 2 ng / mL, 10ng / mL, 1 ng / mL, 0.1ng / mL and 0 ng / mL, take 100 μL of each sample and drop it on the test paper, and observe the color development results of the T line. Fig.10 As shown, when the concentration of soybean globulin was 1 ng / mL, the T line still showed an obvious color development effect, so 1 ng / mL was determined as the visual detection limit of the test paper, with high sensitivity.

[0049] 3.2 Specificity test Soybean globulin (1), β-conglycinin (2), peanut globulin (3), sesame protein (4), bovine serum albumin (5) and wheat protein (6) were taken and diluted to 10% with PBS solution. 4 ng / mL. Then, take 100 μL of sample and drop it onto the sample pad, and observe the T line color development results as shown in the figure. Fig.11 As shown, it was found that the test paper only reacted with soybean globulin, and the T line showed color, while the other five groups did not show color, indicating that the test paper did not cross-react with the other five proteins, showing that it has good specificity.

[0050] 3.3 Repeatability test Samples were taken from three different batches of test strips, with concentrations of 10 4 ng / mL, 10 2 ng / mL and 0 ng / mL samples were tested. The color development results of the test paper are as follows Fig.12 As shown, it can be confirmed that the test results of the three batches of test strips at the same concentration are consistent, indicating that the test results of different batches of test strips are stable and reliable with good repeatability.

[0051] 3.4 Stability Test In order to evaluate the storage stability of the immunochromatographic test strips, the test strips were placed in a dark and dry environment at room temperature for 7 days, 14 days, and 21 days. The experiment was repeated three times at each time point. The results are shown in Figure 2. Fig.13 As shown, it was found that the test paper could still maintain a good color development effect at different storage times, further proving that the prepared test paper has good stability.

[0052] Example 4 Raman signal analysis The logarithm of soybean globulin concentration was used as the horizontal coordinate, and Fig.14 The natural logarithm of the Raman signal intensity at 1335 cm⁻¹ shown in (A) is used as the vertical coordinate to plot the fitting curve. Fig.14 (B) shows the linear equation ln Y = 0.396 log X + 8.614 (R2 = 0.997), where X represents the concentration of glycinin and Y represents the SERS signal intensity. This linear relationship is applicable to the glycinin concentration range of 10 4 According to the Raman intensity of the negative sample, the LOD of the established SERS-LFIA was calculated to be 0.04 ng / mL using the formula LOD = negative sample average + 3SD.

[0053] Example 5 Detection of soy-containing foods based on SERS combined with Au-DTNB-Ag immunochromatographic test paper The five food samples used in the experiment were purchased from a local supermarket. The purchased food samples were simply pre-treated. First, the solid samples were crushed. Then, the protein was extracted with Tris-HCl buffer solution (10mM, pH 7.9), and finally the extract was filtered through a 0.45μm filter membrane. After that, all sample solutions were diluted to 10 times, 100 times, and 1000 times, respectively, and then each sample was tested using test paper. The test results are shown in the figure. Fig.15 As shown: The food sample tested in Sample 1 is a soy milk product (containing a large amount of soybean globulin), and the test paper shows an obvious color reaction. The soybean content in the ingredient list of the food samples in Samples 2 and 3 is about 2% (that is, the soybean globulin content is low), and the color development effect of the test paper is not as obvious as that of Sample 1, but it still shows a weak positive, indicating that soybean globulin can be detected. The ingredient list of the food samples in Samples 4 and 5 does not contain soybeans, so the test results are negative. This shows that this test paper can realize the rapid detection of soybean foods, providing a more convenient, fast and efficient detection tool for the detection of allergens in food for allergic consumers.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. An Au-DTNB-Ag nanosphere, characterized in that: It is a core-shell structure, including a nanogold core, a nanosilver shell and a self-assembled DTNB monolayer in the middle, wherein one end of the DTNB in ​​the self-assembled DTNB monolayer forms an S-Au bond with the surface of the nanogold core through a thiol group and is adsorbed on the surface of the nanogold core, and the other end interacts with the nanosilver shell through a carboxyl group, wherein the particle size of the nanogold core is 16-18 nm, the thickness of the nanosilver shell is 17-18 nm, and the coverage rate of the SAM layer on the nanogold core is 99%-100%; Preferably, the particle size of the Au-DTNB-Ag nanospheres is 33-36 nm, and the mass ratio of the nano-gold core to the nano-silver shell is 40.09:53.27-43.04:56.

96.

2. A method for preparing Au-DTNB-Ag nanospheres according to claim 1, comprising the steps of: firstly stirring a colloidal gold solution and a DTNB ethanol solution at room temperature to react to obtain an Au-DTNP solution; then centrifuging the Au-DTNP solution, then adding hydroquinone and stirring to react, and finally adding an AgNO3 solution and stirring at room temperature until the color of the solution no longer changes, thereby obtaining a brown-yellow Au-DTNB-Ag colloidal solution; centrifuging and drying to obtain Au-DTNB-Ag nanospheres.

3. The preparation method according to claim 2, characterized in that To 1 mL of the colloidal gold solution, add the DTNB ethanol solution with a concentration of 1 mM and a volume of 10 to 50 μL, and the AgNO3 solution with a volume of 0.1 mol / L and 10 to 50 μL.

4. A SERS immune probe, characterized in that: The method comprises the Au-DTNB-Ag nanospheres and rabbit polyclonal antibodies as claimed in claim 1, wherein the DTNB in ​​the Au-DTNB-Ag nanospheres is coupled to the rabbit polyclonal antibodies; Preferably, the pH value of the SERS immunoprobe is 8.5-9.

0.

5. A method for preparing the SERS immune probe according to claim 4, comprising first mixing an Au-DTNB-Ag colloidal solution and a K2CO3 solution, then adding a rabbit polyclonal antibody for a first incubation, then adding a BSA solution for a second incubation, then centrifuging, discarding the supernatant, obtaining an immune probe precipitate, and drying to obtain a SERS immune probe; Preferably, 0.2M, 0.5-2.0 μL of the K2CO3 solution is added to 1 mL of the Au-DTNB-Ag colloidal solution; Preferably, the amount of rabbit polyclonal antibody added to 1 mL of the Au-DTNB-Ag colloidal solution is 7-28 μg.

6. Use of the Au-DTNB-Ag nanospheres according to claim 1 or the SERS immune probe according to claim 4 in preparing SERS enhanced immunochromatographic test paper, characterized in that: The SERS enhanced immunochromatographic test paper comprises a conjugate pad, on which the SERS immunoprobe according to claim 4 is dropped.

7. A SERS enhanced immunochromatographic test paper, characterized in that: It comprises a sample pad, a conjugate pad, an NC membrane and a water absorbent pad, wherein the SERS immunoprobe according to claim 4 is dripped on the conjugate pad, a detection line and a quality control line are set on the NC membrane, a mouse monoclonal antibody is dripped on the T line, and a goat anti-rabbit secondary antibody is dripped on the C line; Preferably, the amount of the mouse monoclonal antibody added is 0.3 μL, 1-4 mg / mL.

8. The SERS enhanced immunochromatographic test paper according to claim 7, characterized in that: The logarithm of the concentration of soybean globulin was used as the horizontal coordinate, and the natural logarithm of the Raman signal intensity at 1335 cm⁻¹ was used as the vertical coordinate. The linear equation was obtained as follows: ln Y = 0.396 log X + 8.614 (R2 = 0.997), where X represents the concentration of soybean globulin. 4 ~1 ng / mL, Y represents the SERS signal intensity; Preferably, the LOD of the linear equation is 0.04 ng / mL.

9. Use of the SERS enhanced immunochromatographic test paper according to claim 7 or 8 in detecting at least one of the following substances: 1) Soybean allergen glycinin; 2) Soy products.

10. A method for preparing the SERS enhanced immunochromatographic test paper according to claim 7 or 8, comprising: Providing Au-DTNB-Ag colloidal solution; Providing a SERS immunoprobe solution: re-dissolving the above immunoprobe with TB buffer to obtain a SERS immunoprobe solution, wherein the TB buffer comprises 0.02 M Na2B4O7, 1% BSA and 0.03% NaN3; Assemble the test paper: stick the sample pad, conjugate pad, NC membrane, and absorbent pad on the support pad, with each layer overlapping by about 1 to 3 mm, drip the SERS immune probe solution on the conjugate pad, drip the mouse monoclonal antibody solution on the T line, and drip the goat anti-rabbit secondary antibody solution on the C line.

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