Satellite structure PDAN-Au composite material and preparation method and application thereof

Abstract

The application belongs to the field of food safety detection and medical examination, and particularly relates to a satellite structure PDAN-Au composite material and a preparation method and application thereof.The preparation method of the composite material comprises the following steps: PDAN and sodium chloroaurate are mixed in a solution system, and the sodium chloroaurate is reduced to AuNP by the phenolic hydroxyl group on the surface of the PDAN under the condition of 50 DEG C-70 DEG C, and finally the satellite structure PDAN-Au composite material is prepared.The satellite structure PDAN-Au with a specific morphology structure is used as a label in an immunochromatography method for the first time, compared with the traditional label 20-40 nm AuNP, the satellite structure PDAN-Au has stronger absorption, good biocompatibility, strong stability and high detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the fields of food safety testing and medical testing, specifically relating to a satellite-structured PDAN-Au composite material, its preparation method, and its application. Background Technology

[0002] Immunochromatography is a rapid detection method combining specific immune reactions and chromatographic techniques. Due to its advantages of simplicity, speed, and low operating cost, it is widely used in food safety testing and clinical diagnosis. The key to establishing a sensitive immunochromatographic method is selecting a suitable label. Currently, the commonly used labels for colorimetric immunochromatographic methods are mostly colored nanomaterials, such as metallic nanomaterials (colloidal gold, colloidal silver, selenium nanoparticles, and magnetic nanoparticles, etc.) and non-metallic nanomaterials (such as carbon black nanoparticles and carbon nanotubes, etc.). Among them, 20-40 nm colloidal gold nanoparticles (AuNP) are the most widely used due to their unique plasma absorption, exhibiting a striking ruby ​​red color. No additional equipment is required; the test results can be directly interpreted with the naked eye, or qualitative detection can be performed using a test strip reader.

[0003] However, due to the limited signal output of AuNPs, low sensitivity is a major drawback in the application of colorimetric immunochromatographic methods. Therefore, using nanomaterials with stronger signal output capabilities as markers is an effective strategy to improve the sensitivity of immunochromatographic methods. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a satellite-structured PDAN-Au composite material, its preparation method, and its application. Specifically, the following technical solution is adopted:

[0005] A method for preparing a satellite-structured PDAN-Au composite material includes the following steps: mixing polydopamine microspheres (PDAN) and sodium chloroaurate in a solution system and reacting them at 50 ℃-70 ℃. Sodium chloroaurate is reduced by phenolic hydroxyl groups on the surface of PDAN to generate AuNP, and finally the satellite-structured PDAN-Au composite material is obtained.

[0006] To address the shortcomings of existing technologies, this invention synthesizes a satellite-structured PDAN-Au composite material with a specific morphology using a surface growth method with PDAN and sodium chloroaurate. This allows a single PDAN to be loaded with tens or even hundreds of AuNPs. Due to the unique surface plasmon resonance effect of AuNPs (with a characteristic UV absorption peak around 525 nm), numerous AuNP arrays on the PDAN surface generate a plasmonic coupling enhancement effect. Therefore, the UV absorption capacity (i.e., colorimetric ability) of the satellite-structured PDAN-Au composite material of this invention is significantly enhanced compared to single AuNPs or polydopamine-coated gold chrysanthemum (AuNC-PDA) nanoparticles, significantly improving signal output capability. Applying this composite material to immunochromatographic methods can improve the detection sensitivity of colorimetric immunochromatographic methods. Currently, there are no reports on establishing immunochromatographic methods using satellite-structured PDAN-Au composite materials with specific morphologies as markers.

[0007] In some preferred embodiments, PDAN and sodium chloroaurate are mixed in solution form, with the PDAN solution concentration being 0.5 mg / mL and the sodium chloroaurate solution mass concentration being 1%, and the volume ratio of sodium chloroaurate solution to PDAN solution being (0.5-1) mL : (50-80) mL; more preferably, it is 0.5 mL : 50 mL. If the sodium chloroaurate solution is too small, the number of AuNPs grown on the PDAN will be small, the plasma coupling enhancement effect will be insufficient, and the ultraviolet absorption capacity (i.e., color development capacity) cannot be effectively improved; the phenolic hydroxyl groups on the PDAN surface are limited, and too much sodium chloroaurate solution will easily lead to waste.

[0008] PDAN is formed by the oxidative polymerization of dopamine hydrochloride under alkaline conditions. The specific process is as follows: dopamine hydrochloride, ammonia, and an aqueous ethanol solution are mixed and reacted. More preferably, the ratio of dopamine hydrochloride, ammonia, and the aqueous ethanol solution is 25 mg: 50 μL: 15 mL; the mass concentration of the aqueous ethanol solution is 25%. Alkaline conditions (pH=8.5) are the optimal conditions for the oxidative polymerization of dopamine hydrochloride to form PDAN. The role of ammonia is to adjust the pH of the reaction, and the role of ethanol is to slow down the reaction rate, resulting in more uniform particle size of the synthesized PDAN.

[0009] Based on the above-mentioned satellite-structured PDAN-Au composite material, the present invention also provides an immunochromatographic test strip, including a base plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper sequentially overlapped and pasted on the base plate; the glass fiber pad is coated with a probe of the analyte, and the probe of the analyte is made from the above-mentioned satellite-structured PDAN-Au composite material and the antibody of the analyte.

[0010] The preparation process of the analyte probe is as follows: The analyte antibody is added to the solution of the satellite-structured PDAN-Au composite material, mixed, and reacted at room temperature for 1.5 h. Then, a blocking agent is added, and the reaction is continued at room temperature for 1.5 h. The precipitate is collected by centrifugation and reconstituted to obtain the analyte probe (specifically, the centrifugation speed is 4000-9000 r / min, and the time is 5-20 min; the precipitate obtained after centrifugation is reconstituted with 0.01 M, pH=7.4 phosphate buffer). After adding the analyte antibody, the final concentration of the analyte antibody is 10 μg / mL-100 μg / mL; after adding the blocking agent, the final mass concentration of the blocking agent is 1%-20%. The blocking agent is any one of bovine serum albumin, casein, or ovalbumin.

[0011] Before adding the antibody to be tested to the satellite-structured PDAN-Au composite material solution, the satellite-structured PDAN-Au is pretreated. The pretreatment process is as follows: the satellite-structured PDAN-Au is sonicated for 1-3 min, and then the concentration of the satellite-structured PDAN-Au is adjusted to 0.01-0.05 mg / mL with 0.02-0.2 M borate buffer at pH 6-8.

[0012] In the above-mentioned immunochromatographic test strip, the nitrocellulose membrane is coated with an artificially conjugated antigen or antibody as a detection line, and an anti-mouse antibody or anti-rabbit antibody as a control line. The preparation method of the nitrocellulose membrane is as follows:

[0013] (1) Use 0.01 M, pH=7.4 phosphate buffer solution to adjust the concentration of the artificial conjugated antigen and the secondary antibody (anti-mouse antibody or anti-rabbit antibody) to 0.01-1.0 mg / mL respectively;

[0014] (2) The artificially conjugated antigen of the analyte after adjusting the concentration is sprayed on the upper part of the nitrocellulose membrane as the detection line, and the anti-mouse antibody or anti-rabbit antibody is sprayed on the lower part of the nitrocellulose membrane as the quality control line; wherein, the detection line and the quality control line are separated by a certain distance, and the spraying amount of both is 0.4-0.8 μL / cm;

[0015] (3) After drying the nitrocellulose membrane coated with the detection line and quality control line at 37 °C for 6 h, store it in a room temperature drying oven for later use.

[0016] Detection principle: The sample solution is dropped onto the sample pad of the immunochromatographic test strip. The sample solution is chromatographically separated by the absorbent paper. When the sample solution does not contain the analyte, the analyte probe will be captured by the artificial conjugate antigen on the detection line, the detection line will show color, and the test result will be negative. When the sample solution contains the analyte, because the analyte probe specifically binds to the analyte, it cannot be captured by the artificial conjugate antigen on the detection line, the detection line will not show color, and the test result will be positive.

[0017] The beneficial effects of this invention are as follows: This invention is the first to use a satellite-structured PDAN-Au with a specific morphology as a marker in an immunochromatographic assay. Compared with the traditional marker AuNP of 20-40 nm, the satellite-structured PDAN-Au has stronger absorption, better biocompatibility, stronger stability and higher detection sensitivity. Attached Figure Description

[0018] Figure 1 The diagram shows a schematic of an immunochromatographic test strip based on a satellite-structured PDAN-Au composite material.

[0019] Figure 2 The image shown is a transmission electron microscope (TEM) characterization of the satellite-type PDAN-Au structure.

[0020] Figure 3 The image shows the ultraviolet absorption spectra of satellite-structured PDAN-Au, AuNC-PDA, and AuNP.

[0021] Figure 4 The figure shows the standard curves for AFM1 detection based on satellite-structured PDAN-Au (A), AuNC-PDA (B), and AuNP (C);

[0022] Figure 5 The images shown are actual images of AFM1 detected by satellite-based PDAN-Au (A), AuNC-PDA (B), and AuNP (C); 1-10 represent AFM1 concentrations of 0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.5 ng / mL, respectively.

[0023] The components include: 1. Sample pad; 2. Glass fiber pad; 3. PDAN-Au antibody probe; 4. Nitrocellulose membrane; 5. Artificial conjugated antigen of the analyte; 6. Anti-mouse antibody or anti-rabbit antibody; 7. Absorbent paper; 8. PVC base plate; 9. Detection line; and 10. Quality control line. Detailed Implementation

[0024] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution and effects of the present invention.

[0025] Example 1: Preparation of satellite-structured PDAN-Au composite material

[0026] 1. Synthesis of PDAN

[0027] 50 μL of ammonia and 25 mg of dopamine hydrochloride were added to 15 mL of ethanol aqueous solution (mass concentration of 25%) and mixed and stirred for 24 h. Dopamine hydrochloride was oxidatively polymerized under alkaline conditions to form polydopamine. The mixture was centrifuged and the supernatant was discarded. The precipitate was redissolved in 50 mL of ultrapure water to obtain PDAN.

[0028] 2. Synthesis of satellite-structured PDAN-Au

[0029] 0.5 mL of sodium chloroaurate (1% by mass) was added to 50 mL of PDAN solution (0.5 mg / mL), heated to 60 °C and stirred for 30 min. Sodium chloroaurate was reduced by the phenolic hydroxyl groups on the surface of PDAN to generate a large amount of AuNP. Then the mixture was cooled to room temperature. After the reaction was completed, the mixture was centrifuged (5000 r / min for 20 min), the supernatant was discarded, the precipitate was washed, and the precipitate was redissolved in ultrapure water to obtain the satellite structure PDAN-Au, which was stored at 4 °C.

[0030] Among them, the transmission electron microscopy characterization image of the satellite-type structure PDAN-Au is as follows: Figure 2 As shown; by Figure 2 It can be seen that the satellite-structured PDAN-Au composite material has good uniformity, with the average particle size of PDAN being 160 nm and its surface loaded with a large number of AuNPs of about 30 nm.

[0031] The ultraviolet absorption spectrum of the satellite-structured PDAN-Au is shown below. Figure 3 As shown; by Figure 3 It can be seen that, at the same concentration, due to the enhanced plasma coupling effect, the ultraviolet absorption intensity of satellite-structured PDAN-Au at 525 nm is about 1.5 times and 2.5 times higher than that of AuNC-PDA and AuNP, respectively. The overall absorption intensity (in the 300-800 nm range) is about 3 times and 5 times higher, respectively, indicating that satellite-structured PDAN-Au has a stronger signal output capability than AuNC-PDA and AuNP.

[0032] Example 2: Preparation method of immunochromatographic test strip based on satellite-structured PDAN-Au composite material

[0033] 1. Preparation of nitrocellulose membranes

[0034] The AFM1 antigen and anti-mouse antibody were diluted to 0.3 mg / mL and 0.4 mg / mL with 0.01 M PBS (pH 7.4), respectively. The solutions were then sprayed onto a nitrocellulose membrane as the test line and control line, respectively. The spray volume for both lines was 0.74 μL / cm. The test line was 10 mm away from the top edge of the membrane, and the two lines were 5 mm apart. The membrane was dried at 37 °C for 6 h and then stored in a desiccator for later use.

[0035] 2. Preparation of satellite-structured PDAN-Au antibody probe glass fiber pad

[0036] Take 0.2 mg of PDAN-Au (prepared in Example 1) and sonicate for 3 min. Adjust the concentration of satellite-structured PDAN-Au to 0.02 mg / mL with 0.1 M borate buffer (pH=6.0). After vortexing and mixing, add 4 μg of AFM1 monoclonal antibody to 1 mL of satellite-structured PDAN-Au. Mix thoroughly and stir at 4 °C for 1.5 h. Add 100 μL of 2% casein and block at room temperature for 1.5 h. Centrifuge at 6000 r / min for 10 min. Redissolve the precipitate in 100 μL of PBS (0.01 M pH 7.4) and spray it onto a glass fiber pad at a volume of 3.5 μL / cm. Vacuum dry for 3 h.

[0037] 3. Assemble the test strips

[0038] (1) Sample pad, with dimensions of 0.8 × 30 cm;

[0039] (2) Fiberglass mat, with a specification of 0.7×30 cm;

[0040] (3) A nitrocellulose membrane with test lines and quality control lines sprayed on, with a specification of 2.5×30 cm;

[0041] (4) Absorbent paper, with dimensions of 1.5 × 30 cm;

[0042] (5) PVC base plate, with a specification of 5.5×30 cm.

[0043] Please arrange the above materials as follows: Figure 1 As shown in the schematic diagram of the test strip structure, the components are pasted in sequence (1. Sample pad, 2. Glass fiber pad, 3. PDAN-Au antibody probe, 4. Nitrocellulose membrane, 5. Artificial conjugate antigen of the analyte, 6. Anti-mouse antibody or anti-rabbit antibody, 7. Absorbent paper, 8. PVC base plate, 9. Detection line, 10. Control line). After assembly, cut the strips into 4×55 mm strips, put them into a self-sealing bag, add desiccant, seal and store. The shelf life is 6 months at room temperature.

[0044] Example 3: Detection of aflatoxin M1 in milk using an immunochromatographic test strip based on a satellite-structured PDAN-Au composite material.

[0045] The method for detecting aflatoxin M1 (AFM1) in milk samples using the above-mentioned immunochromatographic test strip with satellite-structured PDAN-Au composite material as a marker includes the following steps:

[0046] 1. Dilute the milk sample 10 times with PBS (0.01 M pH 7.4) to obtain the sample solution for testing;

[0047] 2. Add 100 μL of the above sample solution to the sample well of the test strip and react for 15 min;

[0048] 3. Using a smartphone and ImageJ software, take a picture and read the gray value of the T line on the test strip. Based on the established standard curve, the content of AFM1 in the sample can be calculated, thus realizing the quantitative detection of positive samples.

[0049] 4. Establish a standard curve: Add the standard to the negative matrix at AFM1 concentrations of: 0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, and 2.5 ng / mL. Figure 4 and Figure 5 The standard curve established using immunochromatographic test strips with satellite-structured PDAN-Au composite material as the marker has the following linear regression equation: y = -1479ln(x) - 545.68 (R² = 0.9898), with a limit of detection (LOD) of 0.0076 ng / mL and a limit of detection (LOD) of 0.5 ng / mL. The standard curve established using immunochromatographic test strips with AuNC-PDA as the marker has the following linear regression equation: y = -1635ln(x) + 552.47 (R² = 0.9803), with a LOD of 0.019 ng / mL and a LOD of 1.0 ng / mL. The standard curve established using the immunochromatographic test strip with AuNP as a marker had the following linear regression equation: y = -1506ln(x) + 1149.2 (R² = 0.9805), with a limit of quantification of 0.031 ng / mL and a limit of qualitative detection of 2.5 ng / mL.

[0050] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention using the same means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. The application of a satellite-structured PDAN-Au composite material as a marker in immunochromatographic test strips, characterized in that, The preparation method of the satellite-structured PDAN-Au composite material includes the following steps: mixing polydopamine microspheres (PDAN) and sodium chloroaurate in a solution system and reacting them at 50 ℃-70 ℃. Sodium chloroaurate is reduced by phenolic hydroxyl groups on the surface of PDAN to generate AuNP, and finally the satellite-structured PDAN-Au composite material is obtained. PDAN is formed by the oxidative polymerization of dopamine hydrochloride under alkaline conditions; the specific process is as follows: dopamine hydrochloride, ammonia and ethanol aqueous solution are mixed and reacted in a ratio of 25 mg: 50 μL: 15 mL; The volume ratio of sodium chloroaurate solution to PDAN solution is (0.5-1) mL: (50-80) mL.

2. The application according to claim 1, characterized in that, PDAN and sodium chloroaurate were mixed in solution form, with the concentration of PDAN solution being 0.5 mg / mL and the mass concentration of sodium chloroaurate solution being 1%.

3. The application according to claim 1, characterized in that, The volume ratio of sodium chloroaurate solution to PDAN solution is 0.5 mL: 50 mL.

4. The application according to claim 1, characterized in that, The mass concentration of the ethanol aqueous solution is 25%.

5. An immunochromatographic test strip, characterized in that, It includes a base plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper that are sequentially overlapped and pasted on the base plate; the glass fiber pad is coated with a probe for the analyte, and the probe for the analyte is made from the satellite-structured PDAN-Au composite material as described in claim 1 and the antibody for the analyte.

6. The immunochromatographic test strip according to claim 5, characterized in that, The preparation process of the test probe is as follows: Add the test antibody to the solution of the satellite-structured PDAN-Au composite material, mix well, react at room temperature for 1.5 h, add the blocking agent, react at room temperature for 1.5 h, and obtain the test probe.

7. The immunochromatographic test strip according to claim 6, characterized in that, After adding the antibody to be tested, the final concentration of the antibody to be tested is 10 μg / mL-100 μg / mL; after adding the blocking agent, the final mass concentration of the blocking agent is 1%-20%.

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