Gold nanoparticles and immunoassay strips
By reacting weakly ionized organic small molecules with chloroauric acid, weakly ionized gold nanoparticles of uniform size are prepared, which solves the problem of insufficient sensitivity in the existing technology and achieves a highly sensitive immunoassay effect.
Patent Information
- Application Number
- CN202111671608.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-12-31
- Publication Date
- 2025-09-16
- Estimated Expiration
- 2041-12-31
AI Technical Summary
Existing gold nanoparticle immunoassay test strips have low sensitivity, existing signal amplification methods increase process complexity and cost, and strongly ionized molecules such as sodium citrate lead to insufficient antibody protein adsorption.
Weakly ionized organic small molecules such as ascorbic acid, gluconic acid, and hydroxybenzoic acid are reacted with chloroauric acid, and the temperature is controlled at 70~100℃ to prepare weakly ionized gold nanoparticles of uniform size, thereby increasing the adsorption capacity of antibody proteins.
The detection sensitivity is significantly improved, the process flow is simplified, the cost is reduced, and high-sensitivity immunoassay is achieved.
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Figure CN114354920B_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of immunoassay technology, and in particular to gold nanoparticles and immunoassay test strips. Background Art
[0002] Most existing immunoassay strips use gold nanoparticles (or colloidal gold) as immunolabeling agents to label proteins, forming gold-labeled antibodies. During detection, color development is observed based on the gold-labeled antibodies on the conjugate pad. Gold nanoparticles are typically reacted with chloroauric acid and reducing agents such as sodium citrate, but the resulting gold-labeled antibodies are difficult to achieve high-sensitivity immunoassays. To enhance the sensitivity of gold nanoparticle immunoassay strips, current improvements primarily involve cascade enzymatic reactions, where the gold nanoparticles undergo color development and are then incubated with a chromogenic or luminescent substrate for secondary signal amplification. These techniques, for example, involve co-modification with horseradish peroxidase or the use of gold nanoparticle composites with peroxidase activity. However, while these techniques achieve signal amplification, they also increase process complexity, labor, and material costs, requiring additional technical effort and reaction time. Therefore, there is a need for a simple method for preparing gold nanoparticles that can significantly improve detection sensitivity when used as markers. Summary of the Invention
[0003] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a simple method for preparing gold nanoparticles. The gold nanoparticles obtained using this method can significantly improve detection sensitivity when used as a marker. This application also proposes an immunoassay test strip based on these gold nanoparticles.
[0004] In a first aspect of the present application, a method for preparing gold nanoparticles is provided, which comprises the following steps: mixing chloroauric acid with a weakly ionized organic small molecule, and reacting the mixture at 70-100° C. to obtain gold nanoparticles.
[0005] The preparation method according to the embodiment of the present application has at least the following beneficial effects:
[0006] During the experiment, the applicant discovered that when sodium citrate and other reducing agents are used, their strong ionization properties cause the gold nanoparticles finally prepared to be in a strongly ionized state, which is not conducive to their adsorption with antibody proteins, resulting in a low total amount of antibodies bound to the surface of the gold nanoparticles and low sensitivity. In contrast, the present solution has made a breakthrough by using weakly ionized reducing organic small molecules as reducing agents for chloroauric acid. The gold nanoparticles obtained by the reaction have weak ionization properties, which can provide a larger total amount of surface antibody protein adsorption for the gold nanoparticles. On the other hand, during the experiment, it was found that the use of weakly ionized small molecule reducing agents to reduce chloroauric acid at room temperature resulted in poor morphology and uneven size of the gold nanoparticles, which affected protein adsorption and subsequent detection sensitivity. However, by adjusting the reaction temperature to 70-100°C, the final weakly ionized gold nanoparticles obtained were more uniform in size and had a better morphology, thereby effectively improving the detection sensitivity from these two aspects.
[0007] In some embodiments of the present application, the weakly ionized organic small molecule is selected from at least one of the following acids and salts thereof: ascorbic acid, gluconic acid, hydroxybenzoic acid, dihydroxybenzoic acid, and trihydroxybenzoic acid. Hydroxybenzoic acids include but are not limited to 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, and 4-hydroxybenzoic acid; dihydroxybenzoic acids include but are not limited to 2,6-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and 3,5-dihydroxybenzoic acid; and trihydroxybenzoic acids include but are not limited to 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, and 3,4,5-trihydroxybenzoic acid.
[0008] In some embodiments of the present application, the molar ratio of the weakly ionized organic small molecule to chloroauric acid is (2-10):1.
[0009] In some embodiments of the present application, the reaction is carried out at 70-100° C. for 0.1-1 h.
[0010] In some embodiments of the present application, the reaction is carried out at 70-90°C, and further at 75-85°C.
[0011] In some embodiments of the present application, the particle size of the gold nanoparticles is 10-100 nm, further 10-50 nm, and further preferably 20-40 nm. The particle size of the gold nanoparticles can be changed by adjusting the preparation conditions to increase protein adsorption and detection sensitivity.
[0012] In a second aspect of the present application, a gold nanoparticle is provided, wherein the gold nanoparticle is prepared using the aforementioned preparation method.
[0013] In a third aspect of the present application, a protein complex is provided, which includes a protein to be labeled and the aforementioned gold nanoparticles bound to the protein to be labeled.
[0014] In a fourth aspect of the present application, a protein labeling method is provided, comprising the following steps: mixing the aforementioned gold nanoparticles with the protein to be labeled and incubating for 1 to 5 hours to obtain a protein complex.
[0015] In some embodiments of the present application, after the gold nanoparticles and the protein to be labeled are mixed and incubated, a blocking solution is added and incubated for a further period of time, and the supernatant is removed by centrifugation to obtain a protein complex. The incubation time after adding the blocking solution can be selected from 1 to 3 hours, preferably 2 hours.
[0016] In some embodiments of the present application, the blocking agent may be a phosphate buffer containing bovine serum albumin (BSA), polyethylene glycol, gelatin or Tween, preferably bovine serum albumin. The specific content of BSA, polyethylene glycol, gelatin or Tween can be adjusted appropriately according to the actual requirements of the preparation.
[0017] In some embodiments of the present application, the protein complex is resuspended in a reconstitution solution to form a dispersion. The reconstitution solution can be a phosphate buffered saline solution containing bovine serum albumin (BSA), polyethylene glycol, gelatin, or Tween, preferably bovine serum albumin. The specific amounts of BSA, polyethylene glycol, gelatin, or Tween can be adjusted according to actual preparation requirements.
[0018] In a fifth aspect of the present application, an immunoassay test strip is provided, which includes a primary antibody, the primary antibody includes the aforementioned protein complex, and the primary antibody is used to specifically bind to the analyte.
[0019] In some embodiments of the present application, a secondary antibody and an anti-antibody are also included. The secondary antibody is used to specifically bind to the analyte, and the anti-antibody (primary anti-protein antibody) is used to specifically bind to the primary antibody. It is understood that in order to form a secondary antibody-analyte-primary antibody complex, the binding sites of the secondary antibody and the primary antibody are different.
[0020] In some embodiments of the present application, an immunoassay test strip is defined as a sample area, a binding area, a detection area, and an adsorption area, which are sequentially connected. The sample area is used for adding the analyte, the binding area is coated with a primary antibody, the detection area is provided with a test line (T line) and a quality control line (C line), the secondary antibody is coated on the test line, and the anti-antibody is coated on the quality control line.
[0021] In some embodiments of the present application, the immunoassay test strip includes a bottom plate, which includes a sample pad, a labeling pad, a chromatography membrane, and a water-absorbing pad connected in sequence.
[0022] In some embodiments of the present application, the base plate is a polymer film, such as a polyvinyl chloride membrane. The sample pad is glass fiber treated with a treatment solution, which may be a phosphate buffered saline solution containing Triton X-100 and a preservative. The labeling pad is glass fiber. The chromatography membrane is a nitrocellulose membrane (NC membrane) with a pore size of 3 to 15 μm, preferably an 8 μm pore size. The absorbent pad is filter paper.
[0023] In some embodiments of the present application, the size of the immunoassay test strip is (40-100) × (1-10) mm.
[0024] In some embodiments of the present application, the chromatographic membrane is marked with detection lines and quality control lines at intervals of 5 to 10 mm.
[0025] In some embodiments of the present application, the width of the detection line and the quality control line is 0.25-0.5 mm, preferably 0.35 mm.
[0026] In some embodiments of the present application, the immunoassay test strips include the following raw materials in parts by mass when prepared and used: 50-100 parts of gold nanoparticles, 1-3 parts of primary antibody protein, 10-30 parts of blocking solution, 1-5 parts of reconstitution solution, 0.1-0.5 parts of secondary antibody protein, 0.1-0.5 parts of primary anti-protein antibody, and 1-5 parts of working buffer.
[0027] In some embodiments of the present application, the working buffer is used to support chromatographic flow, and a phosphate buffer containing Triton X-100 can be optionally used. The specific content of Triton X-100 can be appropriately adjusted according to the actual needs of the test.
[0028] In some embodiments of the present application, the method for using the immunoassay test strip is: applying the sample to be tested to the sample area of the immunoassay test strip, determining the color signal of the immunoassay test strip, and determining the content of the analyte in the sample to be tested based on the color signal.
[0029] In some embodiments of the present application, the method for using the immunoassay test strip is as follows: applying the sample to be tested to the sample area of the immunoassay test strip, then taking the working buffer and applying it to the sample area, performing chromatography, determining the color signal of the immunoassay test strip after the chromatography is completed, and determining the content of the analyte in the sample to be tested based on the color signal.
[0030] In some embodiments of the present application, the method for determining the content of the analyte in the test sample based on the colorimetric signal is as follows: determining the colorimetric signals of the test line and the quality control line, and determining the content of the analyte in the test sample based on the ratio of the colorimetric signal of the test line to the colorimetric signal of the quality control line.
[0031] In a sixth aspect of the present application, a kit is provided, which includes the aforementioned gold nanoparticles or the aforementioned protein complex.
[0032] In a seventh aspect of the present application, a detection method for non-diagnostic purposes is provided, which uses the aforementioned gold nanoparticles, or the aforementioned protein complex, or the aforementioned immunoassay test strip, or the aforementioned kit for detection.
[0033] The present application also includes the use of the aforementioned gold nanoparticles, or the aforementioned protein complex, or the aforementioned immunoassay test strip, or the aforementioned kit in a detection process for non-diagnostic purposes.
[0034] Traditional immunoassay test strips use sodium citrate to reduce chloroauric acid to prepare gold nanoparticles, but sodium citrate is a strongly ionized molecule, so that the prepared gold nanoparticles are in a strongly ionized state as a whole, so pH adjustment must be performed in the process of modifying antibodies to prevent the aggregation of gold nanoparticles. This not only increases the complexity of the overall process and consumes expensive antibody reagents, but also the strongly ionized state is unfavorable for the adsorption of antibody proteins, resulting in a lower total amount of gold nanoparticle surface modified antibodies, making it difficult to achieve high-sensitivity immunoassays. For this reason, the present application reduces chloroauric acid by weakly ionized organic small molecules, designs and synthesizes weakly ionized gold nanoparticles, optimizes the overall process, can increase the total amount of antibody protein adsorbed on the surface of gold nanoparticles, and effectively improves the final detection sensitivity.
[0035] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is an electron microscope image of the gold nanoparticles prepared in Example 1 of the present application.
[0037] Figure 2 This is a comparison of the test results of the immunoassay strips in Example 5 of the present application. The left side shows the relationship between the color signal ratio and the concentration of the standard, and the right side shows the color development results of different immunoassay strips for different concentrations of the standard. Among them, A represents the weakly ionized organic small molecule-gold nanoparticles in the example, and B represents the sodium citrate-gold nanoparticles of the same size in Comparative Example 1. DETAILED DESCRIPTION
[0038] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.
[0039] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.
[0040] In the description of this application, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.
[0041] In the description of this application, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.
[0042] Example 1
[0043] This embodiment provides a gold nanoparticle, and the preparation method of the gold nanoparticle is as follows: ascorbic acid and chloroauric acid are mixed in a molar ratio of 5:1, reacted in an 80°C water bath for 30 minutes, centrifuged, and redissolved in deionized water to obtain a dispersion of gold nanoparticles. Figure 1 The electron microscopy results showed that the gold nanoparticles of this embodiment had a particle size of 20-40 nm, were relatively uniform, and were generally spherical in shape.
[0044] Example 2
[0045] This embodiment provides gold nanoparticles, which differ from Example 1 in that potassium gluconate is used in place of ascorbic acid in the same molar amount, so that the molar ratio of potassium gluconate to chloroauric acid in the reaction raw materials is 5:1.
[0046] Example 3
[0047] This embodiment provides gold nanoparticles, which differ from Example 1 in that sodium salicylate (sodium 2-hydroxybenzoate) and chloroauric acid are mixed in a molar ratio of 10:1 for reaction.
[0048] Example 4
[0049] This embodiment provides gold nanoparticles, which differ from Example 1 in that gallic acid (3, 4, 5-trihydroxybenzoic acid) and chloroauric acid are mixed in a molar ratio of 10:1 for reaction.
[0050] Example 5
[0051] This embodiment provides gold nanoparticles, which differ from embodiment 1 in that a 70° C. water bath reaction is performed for 40 minutes followed by centrifugation during the preparation process.
[0052] Example 6
[0053] This embodiment provides a gold nanoparticle, which differs from Example 1 in that, during the preparation process, a 100° C. water bath reaction is performed for 15 minutes followed by centrifugation.
[0054] Example 7
[0055] This embodiment provides an immunoassay test strip, which is an alpha-fetoprotein (AFP) test strip. The raw materials for its preparation are shown in Table 1 below in parts by volume:
[0056] Table 1. Raw materials for immunoassay test strip preparation
[0057]
[0058] The gold nanoparticles were those prepared in Example 1, the blocking solution and the reconstitution solution were 0.02 M phosphate buffered saline (PBS, pH 7.0) containing 1 w / v% BSA (bovine serum albumin), and the working buffer was 0.02 M phosphate buffered saline containing 0.1 w / v% Triton X-100.
[0059] The specific preparation process of the immunoassay test strip is as follows:
[0060] (1) The primary antibody protein AFP-Ab1 was directly mixed with gold nanoparticles, incubated on a shaker for 2 hours, and then blocking solution was added and incubated for another 1 hour. After centrifugation at 8000 rpm for 10 minutes, the supernatant was removed and the mixture was resuspended with a resuspending solution to obtain a gold-labeled antibody solution.
[0061] (2) Spray the gold-labeled antibody solution onto the labeling pad and dry it for later use; take the secondary antibody protein AFP-Ab2 and the primary antibody protein Anti-AFP-Ab1 and draw lines on the chromatographic membrane respectively to form a 0.35 mm wide T line and C line, and dry it for later use; after the sample pad is pretreated with 0.02 M PBS treatment solution (2 w / v% Triton X-100, 0.03 wt% NaN3, pH = 7.0), it is assembled with the labeling pad, chromatographic membrane, absorbent pad, and polyvinyl chloride bottom plate into an immunoassay chromatography plate, and then cut into 60 × 3 mm immunoassay test strips and placed in a dry and sealed aluminum foil bag for later use.
[0062] Add 0 ng / mL, 0.5 ng / mL, 5 ng / mL, and 10 ng / mL of alpha-fetoprotein standard solutions to the sample pad, then add the corresponding working buffer to the sample pad. Observe the chromatography until the C line / T line stabilizes. Repeat 3 times for each standard.
[0063] Comparative Example 1: The weakly ionized gold nanoparticles in Example 1 were replaced with strongly ionized sodium citrate gold nanoparticles of the same size (commercially available), and an immunoassay test strip was prepared according to the above method, and the same standard was used for detection.
[0064] The experimental results of Example 1 and Comparative Example 1 are as follows Figure 2 As shown, the left side is I T / I C The relationship between the concentration of the standard and the color development of the standard at different concentrations on the immunoassay test strip is shown on the right. A represents the weakly ionized organic small molecule-gold nanoparticles in the example, and B represents the sodium citrate-gold nanoparticles of the same size in Comparative Example 1. As can be seen from the figure, the test strip prepared using the gold nanoparticles provided in the examples of this application shows a relatively clear and discernible T line when testing the 0.5 ng / mL alpha-fetoprotein standard. In contrast, when testing the 5 ng / mL alpha-fetoprotein standard in Comparative Example 1, although the T line is colored, it is difficult to discern with the naked eye.
[0065] It can be seen from the results of the embodiments and comparative examples that the detection limit of the immunoassay test strip provided in the present application can reach 0.5 ng / mL or even lower, which is more than 10 times the sensitivity of the comparative example.
[0066] Example 8
[0067] This example provides a procalcitonin (PCT) immunoassay test strip, which differs from Example 7 in that the primary anti-protein is a PCT monoclonal antibody (mAb-PCT-18D2), the secondary anti-protein is a PCT monoclonal antibody (mAb-PCT-16A1), and the primary anti-protein antibody is a goat anti-mouse IgG polyclonal antibody.
[0068] PCT standard solutions at concentrations of 0 pg / mL, 1 pg / mL, 2 pg / mL, 5 pg / mL, and 10 pg / mL were added dropwise to the sample pad, and then the corresponding working buffer was added dropwise to the sample pad. The chromatography was observed until the C line / T line developed stable color.
[0069] It was observed that the immunoassay strip corresponding to the 2 pg / mL standard had a clear T line visible to the naked eye, while the immunoassay strip corresponding to the 1 pg / mL standard had a T line visible to the naked eye but not obvious. Therefore, the detection limit of this immunoassay strip for PCT can reach 2 pg / mL or even lower.
[0070] Example 9
[0071] This example provides a C-reactive protein (CRP) immunoassay test strip, which differs from Example 7 in that the primary anti-protein is a CRP monoclonal antibody (Anti-CRP-C6), the secondary anti-protein is a CRP monoclonal antibody (Anti-CRP-C2), and the primary anti-protein antibody is a goat anti-mouse IgG polyclonal antibody.
[0072] CRP standard solutions at concentrations of 0 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, and 500 pg / mL were added dropwise to the sample pad, and then the corresponding working buffer was added dropwise to the sample pad. The chromatography was observed until the C line / T line showed stable color.
[0073] It was observed that a clear T line was visible to the naked eye on the immunoassay strip corresponding to the 100 pg / mL standard, while a T line was visible to the naked eye but not obvious on the immunoassay strip corresponding to the 50 pg / mL standard. Therefore, the detection limit of this immunoassay strip for CRP can reach 200 pg / mL (0.2 ng / mL) or even lower.
[0074] Examples 10-14
[0075] In Examples 10 to 14, the gold nanoparticles prepared in Examples 2 to 6 were respectively prepared according to the method in Example 7 to form immunoassay test strips. The sensitivity thereof was close to that of Example 7 after testing.
[0076] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.
Claims
1. A protein labeling method, characterized in that: The steps are as follows: weakly ionized gold nanoparticles are mixed with the protein to be labeled and incubated for 1 to 5 hours to obtain a protein-labeled complex; The preparation method of the weakly ionized gold nanoparticles comprises the following steps: mixing chloroauric acid with a weakly ionized organic small molecule, reacting at 75-85°C for 0.1-1h to obtain weakly ionized gold nanoparticles, wherein the weakly ionized organic small molecule is ascorbic acid, and the molar ratio of the weakly ionized organic small molecule to the chloroauric acid is (5-10):1.