A gold-silver co-reduced spike-type nanomaterial and its preparation method and application

Abstract

The present invention belongs to the fields of material synthesis, food safety detection and medical inspection, and discloses a gold-silver co-reduced spike-type nanomaterial and its preparation method and application. The preparation method comprises the following steps: (1) adding ascorbic acid to a mixed solution of chloroauric acid solution and silver nitrate solution, and mixing to obtain a reaction solution; (2) adding chloroauric acid solution to the reaction solution in step (1), and obtaining a gold-silver co-reduced spike-type nanomaterial after an electric replacement reaction. The synthesis strategy of the present invention is simple and efficient, and the obtained spike-shaped gold-silver nanomaterial has the advantages of strong extinction ability, high specific surface area and high surface energy. When it is directly used for labeling biological macromolecules such as antibodies, it can significantly save antibody (biological macromolecule) materials, reduce detection costs, and improve the sensitivity of the detection method.

Description

Technical Field

[0001] The invention belongs to the fields of material synthesis, food safety detection and medical inspection, and specifically relates to a gold-silver co-reduced spike-type nanomaterial and a preparation method and application thereof. Background Art

[0002] Immunochromatography technology is a detection method based on antigen-antibody specific reaction. It has the advantages of fast detection speed, good specificity, simple operation and low cost. Compared with other methods, it can better meet the needs of large-scale on-site testing. Therefore, it has developed rapidly in recent years and has been widely used in food safety, medical testing and environmental pollutant monitoring.

[0003] Colloidal gold is a common immunolabeling material and is widely used in immunochromatography technology. However, due to its limited sensitivity, it is very meaningful to develop a more sensitive immunolabeling material. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a gold-silver co-reduced spike-type nanomaterial and a preparation method and application thereof, specifically adopting the following technical solutions:

[0005] A method for preparing a gold-silver co-reduced spike-type nanomaterial comprises the following steps:

[0006] (1) adding ascorbic acid to a mixed solution of chloroauric acid solution and silver nitrate solution, and mixing to obtain a reaction solution;

[0007] (2) Adding chloroauric acid solution to the reaction solution in step (1) to obtain the gold-silver co-reduced spike-type nanomaterial after an electrical replacement reaction.

[0008] The present invention first synthesizes gold-silver hollow nanomaterials, and grows gold on the gold-silver hollow nanomaterials through a replacement reaction in which metal ions with higher electrochemical potential are reduced on metals with lower electrochemical potentials. The obtained gold-silver co-reduced spike-type nanomaterial has a plurality of raised rough structures on the surface, which enhances the plasma exciton resonance near the surface, making it have a stronger extinction ability than spherical colloidal gold. In addition, the complex spike morphology and high surface energy on the surface give the material a higher antibody coupling efficiency, and it can be directly coupled with proteins (antibodies) through electrostatic adsorption and other forces.

[0009] Based on the high extinction ability and high antibody coupling efficiency of the gold-silver co-reduced spike-type nanomaterial of the present invention, it can be used in immunochromatography technology, and the effect is better than the traditional colloidal gold quantitative detection of the concentration of the analyte in the sample. At present, there is no relevant report on the gold-silver co-reduced spike-type nanomaterial on the immunochromatography test strip.

[0010] Preferably, in step (1) of the above preparation method, the volume ratio of the chloroauric acid solution to the silver nitrate solution is (1-10): (1-10).

[0011] Preferably, in the above preparation method, the volume ratio of the chloroauric acid solution in step (1) to the chloroauric acid solution in step (2) is 5 μL:1 mL; and the mass concentration of the chloroauric acid solution in step (2) is 0.1-10%.

[0012] The size of the gold-silver co-reduced spike-type nanomaterial prepared by the above preparation method is 10nm-500nm.

[0013] The present invention also provides an immunochromatographic test strip, comprising a glass fiber pad, on which a gold-silver co-reduced spiking nanomaterial probe is sprayed. The preparation process of the gold-silver co-reduced spiking nanomaterial probe is as follows: the gold-silver co-reduced spiking nanomaterial is mixed with an antibody to be labeled, incubated for the first time, a blocking agent is added, and then a second incubation is performed, and the precipitate is redissolved after centrifugation to obtain the gold-silver co-reduced spiking nanomaterial probe.

[0014] Preferably, in the above preparation process, the first incubation time is 1 hour, and the second incubation time is 0.5 hour. The blocking agent is BSA with a mass concentration of 10%. The antibody to be labeled is a monoclonal antibody, a polyclonal antibody, a nanobody or a phage-expressed antibody.

[0015] The structure of the above-mentioned immunochromatographic test strip specifically includes a bottom plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and a water-absorbing paper which are overlapped and pasted on the bottom plate in sequence.

[0016] Furthermore, the nitrocellulose membrane is coated with an artificially coupled antigen of the object to be detected or an antibody of the object to be detected (i.e., an antibody to be labeled) as a detection line, and is coated with an anti-mouse antibody or an anti-rabbit antibody (secondary antibody) as a quality control line; wherein the preparation method of the nitrocellulose membrane comprises the following steps:

[0017] (1) Using 0.01M–0.5M PBS (pH 6.0–8.0) solution, adjust the concentration of the coated analyte artificially coupled antigen or analyte antibody, anti-mouse antibody or anti-rabbit antibody to 0.01 mg / mL–10.0 mg / mL respectively;

[0018] (2) Spraying the artificially coupled antigen or antibody of the test object after adjusting the concentration onto the upper part of the nitrocellulose membrane as the test line, and spraying the anti-mouse antibody or anti-rabbit antibody onto the lower part of the nitrocellulose membrane as the quality control line; wherein the test line and the quality control line are separated by a certain distance, and the spray volume of both is 0.25 μL / cm-0.74 μL / cm;

[0019] (3) The nitrocellulose membrane sprayed with the test line and the quality control line is dried at 37°C overnight and stored in a dry environment at room temperature for future use.

[0020] The beneficial effects of the present invention are as follows: the synthesis strategy of the present invention is simple and efficient, and the obtained spiky gold and silver nanomaterials have the advantages of strong extinction ability, high specific surface area and high surface energy. When directly used for labeling biological macromolecules such as antibodies, it can significantly save antibody (biological macromolecule) materials, reduce detection costs, and improve the sensitivity of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure shows the preparation principle diagram of gold-silver co-reduction spike-type nanomaterials;

[0022] Figure 2 Shown is the application principle diagram and structure diagram of gold-silver co-reduction spike-type nanomaterials in test strips;

[0023] Figure 3 Shown is the transmission electron microscopy characterization of gold and silver co-reduced spike-type nanomaterials;

[0024] Figure 4 Shown is the UV absorption spectrum of the gold-silver co-reduced spike-type nanomaterial;

[0025] Figure 5 Shown is a physical comparison of the positive test strips of gold-silver co-reduced spike-type nanomaterials and colloidal gold coupled with antibodies of different concentrations;

[0026] Figure 6 Shown is a physical comparison of the test strips of gold-silver co-reduced spike-type nanomaterials and colloidal gold for detecting different concentrations of Escherichia coli O157:H7. DETAILED DESCRIPTION

[0027] 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 drawings, so as to fully understand the purpose, scheme and effects of the present invention.

[0028] Embodiment 1:

[0029] A method for preparing a gold-silver co-reduction spike-type nanomaterial, the principle of which is as follows Figure 1 As shown, the following steps are included:

[0030] (1) Add 5 μL of chloroauric acid solution and 10 μL of silver nitrate solution to the enzyme-labeled well, then add ascorbic acid thereto, and mix well to obtain a reaction solution;

[0031] (2) Add 1 mL of chloroauric acid solution to the reaction solution in step (1), and obtain a gold-silver co-reduced spike-type nanomaterial after an electrical replacement reaction.

[0032] The transmission electron microscopy characterization of the prepared gold-silver co-reduced spike-type nanomaterial is shown in the figure Figure 3 As shown in the figure, the gold and silver nanomaterials with obvious spikes were obtained, and the particle size was about 82nm. The spike-type nanomaterials were scanned by UV spectrophotometer, and the UV absorption spectrum was as shown in the figure Figure 4 As shown, it has high absorption from 500nm-1000nm, showing remarkable optical properties, and its maximum ultraviolet absorption peak wavelength is 835nm.

[0033] Embodiment 2:

[0034] Application of sandwich immunochromatographic test strips using the gold-silver co-reduced spike-type nanomaterial prepared in Example 1 as an immune marker for detecting Escherichia coli O157:H7:

[0035] 1. The structure and principle of immunochromatographic test strips (double antibody sandwich mode) Figure 2 As shown, it specifically includes a base plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are overlapped and pasted on the base plate in sequence.

[0036] (1) Preparation of nitrocellulose membrane: E. coli O157:H7 polyclonal antibody and anti-mouse antibody were coated on nitrocellulose membrane: E. coli O157:H7 polyclonal antibody was diluted to 1 mg / mL with 0.01 M PBS (pH 7.5), and the resulting solution was sprayed on the membrane as a test line; the anti-mouse antibody was diluted to 0.5 mg / mL, and the resulting solution was sprayed on the membrane as a quality control line. The spray volume of both lines was 0.74 μL / cm2, the test line was 10 mm away from the top edge of the membrane, and the interval between the two lines was 5 mm. The membrane was dried at 37°C for 12 h and stored in a drying cabinet for future use.

[0037] (2) Preparation of gold-silver co-reduced spike-type nanomaterial probe glass fiber mat: 10 μg of Escherichia coli O157:H7 monoclonal antibody was added to the prepared gold-silver co-reduced spike-type nanomaterial, mixed and incubated for 1 h, then 10% BSA blocking agent was added, incubated at room temperature for 0.5 h, and the precipitate was collected by centrifugation. The obtained precipitate was re-dissolved with 0.01 M PBS, pH 7.0, to 1 / 10 of the initial volume to prepare a gold-silver co-reduced spike-type nanomaterial probe, which was sprayed onto the glass fiber mat at a volume of 3 μL / cm and vacuum dried for 2 h.

[0038] 2. Use the above-mentioned immunochromatographic test strips and colloidal gold immunochromatographic test strips to detect Escherichia coli O157:H7.

[0039] Adjust the standard curve: add the spike to the negative matrix, the concentration of E. coli O157:H7 in the standard curve is: 2.5×10 3 , 5×10 3 , 10 4, 2.5×10 4 , 5×10 4 , 10 5 , 2.5×10 5 and 5×10 5 CFU / mL, calculate R 2 It is 0.963, and the linear regression equation is: y=7507.35log(x)-25794.51.

[0040] Figure 5 This is a positive comparison of the test strips of gold-silver co-reduced spike-type nanomaterials and colloidal gold coupled with antibodies of different concentrations. Figure 5 It can be seen that compared with traditional colloidal gold nanomaterials, gold-silver co-reduced spike-type nanomaterials can achieve stronger colorimetric signals on the test line and quality control line on the test strip with less antibody labeling, demonstrating that gold-silver co-reduced spike-type nanomaterials have better advantages in coupling antibodies, reducing the use of expensive antibodies and reducing the preparation cost of test strips.

[0041] Figure 6 This is a comparison of the gold-silver co-reduced spike nanomaterial and colloidal gold test strips for detecting different concentrations of E. coli O157:H7. Figure 6 It can be seen that the detection limit of the gold-silver co-reduced spike-type nanomaterial test strip for detecting Escherichia coli O157:H7 is about 5-10 times lower than that of the colloidal gold test strip, indicating that the application of gold-silver co-reduced spike-type nanomaterials on test strips has higher sensitivity and has good application prospects.

[0042] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. A method for preparing a gold-silver co-reduced spike-type nanomaterial, characterized in that: The following steps are involved: (1) adding ascorbic acid to a mixed solution of chloroauric acid solution and silver nitrate solution, and mixing to obtain a reaction solution; (2) adding chloroauric acid solution to the reaction solution in step (1) to obtain the gold-silver co-reduced spike-type nanomaterial after an electrical replacement reaction; In the step (1), the volume ratio of the chloroauric acid solution to the silver nitrate solution is (1-10): (1-10); The volume ratio of the chloroauric acid solution in step (1) to the chloroauric acid solution in step (2) is 5 μL:1 mL; the mass concentration of the chloroauric acid solution in step (2) is 0.1-10%.

2. A gold-silver co-reduced spike-type nanomaterial, characterized in that: Prepared by the preparation method described in claim 1.

3. The gold-silver co-reduced spike-type nanomaterial according to claim 2, characterized in that: The size of the gold-silver co-reduced spike-type nanomaterial is 10nm-500nm.

4. Use of the gold-silver co-reduced spike-type nanomaterial according to claim 2 or 3 in the preparation of immunochromatographic test strips.

5. An immunochromatographic test strip, characterized in that: The invention comprises a glass fiber mat on which a gold-silver co-reduced spiking nanomaterial probe is sprayed. The preparation process of the gold-silver co-reduced spiking nanomaterial probe is as follows: the gold-silver co-reduced spiking nanomaterial according to claim 2 or 3 is mixed with an antibody to be labeled, incubated for the first time, a blocking agent is added and then incubated for the second time, and the precipitate is redissolved after centrifugation to obtain the gold-silver co-reduced spiking nanomaterial probe.

6. The immunochromatographic test strip according to claim 5, characterized in that: The first incubation time was 1 h, and the second The second incubation time was 0.5 h.

7. The immunochromatographic test strip according to claim 6, characterized in that: The blocking agent is BS with a mass concentration of 10% A。 8. The immunochromatographic test strip according to claim 5, characterized in that: Including a base plate, and on the base plate Overlap taped sample pads, fiberglass pads, nitrocellulose membranes, and absorbent paper.

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