Flaky gold nanoparticle aggregate as well as preparation method and application thereof

Abstract

The invention provides a flaky gold nanoparticle aggregate as well as a preparation method and application thereof, the preparation method comprises the following steps: step 10, adding a hydrogen peroxide solution and a sodium borohydride solution into a sodium citrate solution, centrifuging to remove supernate after reaction, and redissolving with deionized water to obtain a seed solution; adding the seed solution into the growth solution, reacting for a period of time, then adding a silver nitrate solution, reacting, and centrifuging to remove supernate; re-dissolving with deionized water to obtain a silver nanosheet solution; and step 20, adding the silver nanosheet solution and the monovalent gold solution into the stabilizer solution, and centrifuging to remove supernate after reaction to obtain the flaky gold nanoparticle aggregate. According to the flaky gold nanoparticle aggregate and the preparation method and application thereof, the sensitivity of a lateral flow immunoassay test strip can be enhanced, the preparation process is simple, and no additional operation is added when the lateral flow immunoassay test strip is used for testing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of lateral flow immunoassay, and particularly relates to a sheet-shaped gold nanoparticle aggregate, a preparation method and application thereof. BACKGROUND

[0002] A lateral flow immunoassay (LFIA) test strip is a solid-phase immunochromatographic assay device based on paper-based chromatographic analysis and antigen-antibody immune response, which has the advantages of low cost, simple operation, convenient use, on-site response, rapidness, and naked-eye detection result, and is widely used in the field of point-of-care testing (POCT).

[0003] Gold nanoparticles (AuNPs) are easy to prepare, chemically stable, have good biocompatibility, the surface is easy to modify biomolecules, and have bright color in the visible light region, which makes gold nanoparticles the most widely used colorimetric probe in commercial LFIA test strips. The detection signal of the LFIA test strip based on gold nanoparticles is the color signal of gold nanoparticles when the gold nanoparticles are aggregated on the detection line or the quality control line. When the size of the gold nanoparticles increases, the extinction cross section also increases, so the color signal after aggregation is more intense under the same number of particles. However, the growth process of preparing large-size gold nanoparticles is difficult to balance, resulting in poor monodispersity of the product. Moreover, large-size gold nanoparticles are easily affected by steric hindrance. Therefore, the LFIA test strip produced in large scale by the current commercialization usually uses 40 nm gold nanoparticles as the immunoprobes, and the optical properties limit the detection ability of the LFIA test strip for low-concentration targets in the sample.

[0004] In the field of developing high-sensitivity LFIA test strips, researchers have carried out a lot of exploration and proposed technical solutions such as aggregation chromogenic probe method, noble metal enhancement method and enzyme-mediated colorimetric enhancement method. However, these methods generally have the problems of complex design, the need for complex operation skills, and high preparation cost, which hinder the promotion of large-scale industrialization and are difficult to meet the batch application demand. Although fluorescent LFIA test strips, surface-enhanced Raman scattering LFIA test strips, photothermal signal LFIA test strips and chemiluminescence LFIA test strips can further improve the sensitivity, they are severely dependent on specific equipment, which greatly limits the detection scene and cannot be applied to scenes with high convenience requirements such as on-site rapid detection and primary medical screening.

[0005] In the current high-sensitivity LFIA test strip research and development field, the existing technical solutions generally have two significant limitations: one is that the complex operation process and high preparation cost limit the promotion of large-scale industrialization; the second is limited by the dependence on specific equipment, which cannot meet the flexible use requirements of on-site rapid detection and grassroots scene application. Therefore, without adding additional operation steps, developing a higher sensitivity LFIA test strip can not only avoid the problems of complex process and cost, but also get rid of the dependence on specific equipment, realize the balance of “high sensitivity” and “convenience and industrialization”, and become the key direction of the current LFIA test strip research. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a sheet-shaped gold nanoparticle aggregate and a preparation method and application thereof, which can enhance the sensitivity of a lateral flow immunoassay test strip, has a simple preparation process, and does not increase additional operations when using the LFIA test strip for testing.

[0007] To solve the above technical problems, the present application adopts the following technical solutions: In a first aspect, the present application provides a preparation method of a sheet-shaped gold nanoparticle aggregate, comprising the following steps: Step 10: hydrogen peroxide solution and sodium borohydride solution are added to sodium citrate solution, and after reaction, the supernatant is removed by centrifugation, and then the supernatant is redissolved with deionized water to obtain a seed solution; the seed solution is added to a growth solution, and after reaction for a period of time, silver nitrate solution is added, and after reaction, the supernatant is removed by centrifugation; then the supernatant is redissolved with deionized water to obtain a silver nanoplate solution; wherein the growth solution comprises acetonitrile solution, ascorbic acid solution and sodium citrate solution; Step 20: silver nanoplate solution and monovalent gold solution are added to a stabilizer solution, and after reaction, the supernatant is removed by centrifugation to obtain a sheet-shaped gold nanoparticle aggregate; the stabilizer solution comprises sodium citrate solution and polyvinylpyrrolidone solution.

[0008] As a further improvement of the present application, in step 10, the molar ratio of the seed solution to the silver nitrate solution is 0.83-50:100.

[0009] As a further improvement of the present application, in step 20, the monovalent gold solution is a water-soluble solution of monovalent gold complex ions AuBr2-.

[0010] In a second aspect, the present application further provides a sheet-shaped gold nanoparticle aggregate prepared by the above preparation method.

[0011] In a third aspect, the present application further provides a sheet-shaped gold nanoparticle aggregate immunoprobe, which comprises the above sheet-shaped gold nanoparticle aggregate and a recognition antibody, and the recognition antibody is coupled to the surface of the sheet-shaped gold nanoparticle aggregate.

[0012] As a further improvement of the present invention, the recognition antibody is a cTnI recognition antibody.

[0013] Fourthly, the present invention also provides a method for preparing the above-mentioned sheet-like gold nanoparticle aggregate immune probe, comprising the following steps: Step 10: Redissolve the sheet-like gold nanoparticle aggregates in deionized water to obtain a solution of sheet-like gold nanoparticle aggregates. Step 20: Add potassium carbonate solution to adjust the pH of the sheet gold nanoparticle aggregate solution to 5.8-6.0, add recognition antibody, and after reaction, obtain an immune probe based on sheet gold nanoparticle aggregates.

[0014] Fifthly, the present invention also provides a lateral flow immunoassay test strip based on sheet-like gold nanoparticle aggregates, wherein the gold-labeled conjugate pad of the lateral flow immunoassay test strip is coated with the aforementioned sheet-like gold nanoparticle aggregate immunoprobe.

[0015] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention provides a sheet-like gold nanoparticle aggregate, its preparation method, and its application. Using silver nanosheets as a sacrificial template, a gold precursor is reduced and deposited in situ through a displacement reaction, maintaining the sheet-like morphology of the silver nanosheets, thus preparing sheet-like gold nanoparticle aggregates at the colloidal scale (1~1000 nm). The sheet-like gold nanoparticle aggregates have a large specific surface area, significantly increasing the amount of binding recognition antibodies. When used on LFIA test strips, the recognition antibody, after binding to the antigen, flows to the detection line and has a high probability of being captured by the trapping antibody on the detection line. This sheet-like aggregate is formed by the tight aggregation of gold nanoparticles, exhibiting strong plasmon coupling, further enhancing colorimetric performance. Furthermore, because each sheet-like aggregate contains a large number of gold nanoparticles, compared to the traditional "single particle-single signal" detection mode, the number of gold nanoparticles bound to the detection line far exceeds the number of gold nanoparticles on a traditional LFIA test strip, thereby enhancing the colorimetric signal intensity, i.e., enhancing the detection sensitivity of the LFIA test strip. The preparation process of the sheet-like gold nanoparticle aggregates is simple. After conjugation with the recognition antibody, it is sprayed onto the gold-labeled binding pad of the LFIA test strip. The preparation process of the LFIA test strip is simple and the same as that of existing LFIA test strips. Highly sensitive detection can be achieved without additional operations or equipment. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the preparation process of sheet-like gold nanoparticle aggregates provided in an embodiment of the present invention. Figure 2This is a structural diagram of the lateral flow immunoassay strip based on sheet-like gold nanoparticle aggregates provided in an embodiment of the present invention; Figure 2 a is a schematic diagram of the structure before testing. Figure 2 b is a schematic diagram of the structure during detection; Figure 3 The images show the spectral and scanning electron microscope (SEM) images of the sheet-like gold nanoparticle aggregates prepared in Example 3; among them, Figure 3 a represents the UV-Vis absorption spectra of silver nanosheets and sheet-like gold nanoparticle aggregates prepared from the silver nanosheets. Figure 3 b is a scanning electron microscope image of silver nanosheets. Figure 3 c is a scanning electron microscope image of the sheet-like gold nanoparticle aggregates prepared from the silver nanosheets; Figure 4 The images show the spectral and scanning electron microscope (SEM) images of the sheet-like gold nanoparticle aggregates prepared in Example 4; among them, Figure 4 a represents the UV-Vis absorption spectra of silver nanosheets and sheet-like gold nanoparticle aggregates prepared from the silver nanosheets. Figure 4 b is a scanning electron microscope image of silver nanosheets. Figure 4 c is a scanning electron microscope image of the sheet-like gold nanoparticle aggregates prepared from the silver nanosheets; Figure 5 The results of testing the test solution containing 20 ng / mL cTnI antigen using the lateral flow immunoassay test strips prepared in Example 1, Example 2, Example 3, Example 4 and Comparative Example 1 are shown in the figure. Figure 6 The results of testing with lateral flow immunoassay strips prepared in Examples 1, 2, 3, 4 and Comparative Example 1 on test solutions containing 10 ng / mL cTnI antigen are shown in the figure. Figure 7 The results of testing the test solution containing 5 ng / mL cTnI antigen using the lateral flow immunoassay test strips prepared in Example 1, Example 2, Example 3, Example 4 and Comparative Example 1 are shown in the figure. Figure 8 The results are shown in the graph comparing the detection results of test solutions containing 0.1 ng / mL cTnI antigen using the lateral flow immunoassay test strips prepared in Example 3, Example 4 and Comparative Example 1 respectively. Figure 9 The graph shows a comparison of the results of testing test solutions containing 0.005 ng / mL cTnI antigen using the lateral flow immunoassay test strips prepared in Examples 3, 4 and Comparative Example 1, respectively. Detailed Implementation

[0017] The technical solution of the present invention will be described in detail below.

[0018] This invention provides a method for preparing sheet-like gold nanoparticle aggregates, such as... Figure 1 As shown, it includes the following steps: Step 10: Add hydrogen peroxide solution and sodium borohydride solution to sodium citrate solution. After reaction, centrifuge to remove the supernatant, then reconstitute with deionized water to obtain the seed solution. Add the seed solution to the growth solution, react for a period of time, then add silver nitrate solution. After reaction, centrifuge to remove the supernatant. Then reconstitute with deionized water to obtain the silver nanosheet solution. The growth solution includes acetonitrile solution, ascorbic acid solution, and sodium citrate solution.

[0019] Step 20: The silver nanosheet solution and the monovalent gold complex ion AuBr2-aqueous phase solution are added to the stabilizer solution. After the reaction, the supernatant is removed by centrifugation to obtain sheet-like gold nanoparticle aggregates. The stabilizer solution includes sodium citrate solution and polyvinylpyrrolidone solution. Specifically, the monovalent gold complex ion AuBr2-aqueous phase solution is sodium dibromoaurate (AuBr2Na) solution or potassium dibromoaurate (AuBr2K) solution.

[0020] Preferably, in step 10, the molar ratio of the seed solution to the silver nitrate solution is 8.3~500:1000. The smaller the molar ratio of the seed solution to the silver nitrate solution, the larger the size of the generated silver nanosheets. The larger the size of the sheet-like gold nanoparticle aggregates prepared using the silver nanosheets as templates, on the one hand, the larger the specific surface area, the significantly increased amount of recombinant recognition antibodies. When used on lateral flow immunoassay strips for detection, the probability of the recognition antibody being captured by the capture antibody on the detection line after binding to the antigen and flowing to the detection line is higher. On the other hand, the higher the content of gold nanoparticles in each sheet-like aggregate, the stronger the colorimetric signal intensity.

[0021] Preferably, in step 20, the monovalent gold solution is an aqueous solution of monovalent gold complex ions (AuBr2). The molar ratio of the silver nanosheet solution to the aqueous solution of monovalent gold complex ions (AuBr2) is 4~25:1.

[0022] This invention also provides a sheet-like gold nanoparticle aggregate, such as... Figure 1 As shown, it was prepared using the preparation method of the above embodiment.

[0023] The sheet-like gold nanoparticle aggregates of this embodiment, This invention also provides an immune probe consisting of sheet-like gold nanoparticle aggregates, comprising the sheet-like gold nanoparticle aggregates described above and a recognition antibody, wherein the recognition antibody is coupled to the surface of the sheet-like gold nanoparticle aggregates.

[0024] Preferably, the recognition antibody is a cTnI recognition antibody. cTnI is one of the three subunits constituting the troponin complex and has high myocardial specificity, which can be used to diagnose acute myocardial injury. The lateral flow immunoassay strip prepared by conjugating sheet-like gold nanoparticle aggregates with a cTnI recognition antibody in this embodiment of the invention can detect trace amounts of cTnI in the blood, meeting the high sensitivity detection requirements for cTnI.

[0025] This invention also provides a method for preparing a sheet-like gold nanoparticle aggregate immune probe, comprising the following steps: Step 10: Redissolve the sheet-like gold nanoparticle aggregates in deionized water to obtain a solution of sheet-like gold nanoparticle aggregates.

[0026] Step 20: Add potassium carbonate solution to adjust the pH of the sheet gold nanoparticle aggregate solution to 5.8-6.0, add recognition antibody, and after reaction, obtain an immune probe based on sheet gold nanoparticle aggregates.

[0027] Preferably, the concentration of the recognition antibody in the reaction solution after adding the recognition antibody is greater than or equal to 10 mg / mL. This ensures that the surface of the sheet-like gold nanoparticle aggregates is adequately covered and coupled by the recognition antibody.

[0028] This invention also provides a lateral flow immunoassay strip based on sheet-like gold nanoparticle aggregates, such as... Figure 2 As shown in diagram a, the device includes a PVC base plate and, sequentially laid along its length, a sample pad, a gold-labeled conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad. One side of the gold-labeled conjugate pad overlaps with the sample pad, and the other side overlaps with one side of the nitrocellulose membrane. The other side of the nitrocellulose membrane overlaps with the absorbent pad. The nitrocellulose membrane has spaced detection lines (T lines) and control lines (C lines), with the detection lines near the gold-labeled conjugate pad and the control lines near the absorbent pad. The gold-labeled conjugate pad is coated with the sheet-like gold nanoparticle aggregate immunoprobe described in the above embodiment. The detection lines are coated with capture antibodies corresponding to the recognition antibodies in the sheet-like gold nanoparticle aggregate immunoprobe described in the above embodiment, and the control lines are coated with goat anti-mouse monoclonal antibody or rabbit anti-mouse monoclonal antibody.

[0029] like Figure 2As shown in b, during testing, the test solution flows from the sample pad towards the absorbent pad. When it passes the gold-labeled binding pad, if the test solution contains antigen, the antigen binds to the recognition antibody of the gold nanoparticle aggregate immunoprobe. Both antigen-bound and antigen-free gold nanoparticle aggregate immunoprobes move to the nitrocellulose membrane under the influence of the test solution. When they reach the detection line, antigen-bound gold nanoparticle aggregate immunoprobes are captured by the capture antibody on the detection line and accumulate there. Antigen-free gold nanoparticle aggregate immunoprobes continue to move towards the absorbent pad and are captured by the goat anti-mouse monoclonal antibody on the control line. Therefore, if the test solution contains the corresponding antigen, both the detection line and the control line will show color; if the test solution does not contain the corresponding antigen, only the control line will show color.

[0030] When using the lateral flow immunoassay test strip of the above embodiment for detection, there is no need to increase the colorimetric signal intensity by secondary aggregation of nanoparticles, and no additional operation is required. Below are four examples and one comparative example.

[0031] Example 1 (The molar ratio of seed solution to silver nitrate solution is 500:1000) Preparation of seed solution: In a 250 mL three-necked flask, 200 mL of deionized water and 9 mL of 0.1 M sodium citrate solution were added and stirred until homogeneous to form a stabilizer solution. Then, 0.2 mL of 0.1 M silver nitrate solution, 0.48 mL of 30% (w / v) hydrogen peroxide solution, and 1.2 mL of 0.1 M sodium borohydride solution were added sequentially. The mixture was reacted with stirring for 45 min, and the supernatant was removed by centrifugation. The solution was then reconstituted with 40 mL of deionized water to obtain the seed solution.

[0032] Preparation of silver nanosheet solution: In a 50 mL Erlenmeyer flask, add 10 mL of deionized water, 5 mL of acetonitrile solution, 0.15 mL of 0.1 M ascorbic acid solution, and 0.1 mL of 0.075 M sodium citrate solution. Stir until homogeneous to form a growth solution, and place the growth solution in an ice-water mixture. While stirring vigorously, add 12 mL of seed solution. React for 5 min while maintaining vigorous stirring, then add 120 μL of 0.1 M silver nitrate solution. React for one hour while stirring, then centrifuge to remove the supernatant. Redissolve the silver nanosheets in 20 mL of deionized water to obtain the silver nanosheet solution.

[0033] Preparation of sheet-like gold nanoparticle aggregates: In a 100 mL three-necked flask, add 10 mL of deionized water, 10 mL of 0.06 M sodium citrate solution, and 20 mL of 1% (w / v) polyvinylpyrrolidone (Mw = 58000) solution. Stir until homogeneous to form a stabilizer solution. Under vigorous stirring, add 10 mL of 1 mM AuBrNa solution and 8 mL of silver nanosheet solution sequentially. After reacting for two hours with stirring, centrifuge, remove the supernatant, and reconstitute with 20 mL of deionized water to obtain a solution of sheet-like gold nanoparticle aggregates.

[0034] Preparation of sheet-like gold nanoparticle aggregate immune probes: Take 1 mL of the sheet-like gold nanoparticle aggregate solution, adjust the pH to 5.8–6.0 with 0.2 M potassium carbonate solution, mix thoroughly, and then add 10 μg of cTnI recognition antibody for conjugation. Add 100 μL of blocking buffer containing 10% (w / v) bovine serum albumin in tris(hydroxymethyl)aminomethane buffer (0.2 M pH = 8.2). Store the sheet-like gold nanoparticle aggregate immunoprobe in a tris(hydroxymethyl)aminomethane buffer containing 10% (w / v) sucrose, 10% (w / v) trehalose, 1% (w / v) bovine serum albumin, and 0.1% (w / v) Tween (10 mM pH = 8.2).

[0035] Preparation of lateral flow immunoassay strips based on sheet-like gold nanoparticle aggregates: Dissolve 0.9% (w / v) sodium chloride, 0.25% (w / v) Triton X-100, 1% (w / v) bovine serum albumin, 1% (w / v) sucrose, 0.1% (w / v) polyvinylpyrrolidone (Mw = 10000), and 0.05% (w / v) Tween in 25 mM tris(hydroxymethyl)aminomethane buffer solution at pH 7.4.

[0036] Dissolve 0.05% (w / v) sodium chloride and 0.5% (w / v) Triton X-100 in 25 mM tris(hydroxymethyl)aminomethane buffer solution at pH = 7.4.

[0037] The sample pad was cut into strips of 2.4 × 30 cm, and the gold-labeled conjugate pad was cut into strips of 1.2 × 30 cm. Then, they were soaked in the corresponding treatment buffer solution and stored at room temperature for 2 h. After that, they were dried in an oven at 28 °C for 24 h.

[0038] The pretreated sample pad, gold-labeled conjugate pad, nitrocellulose membrane, and absorbent paper were attached to a PVC support plate. Specifically, the nitrocellulose membrane was first attached to the corresponding position on the PVC base plate; then, the pretreated gold-labeled conjugate pad was attached to the PVC base plate, overlapping the left side of the nitrocellulose membrane by 2 mm; next, the pretreated sample pad was attached to the PVC plate, overlapping the gold-labeled conjugate pad by 2 mm; finally, absorbent paper (3×30 cm) was attached to the far right side of the PVC base plate, overlapping the right side of the nitrocellulose membrane by 2 mm.

[0039] The cTnI capture antibody was uniformly sprayed onto the nitrocellulose membrane detection line (T line) at a speed of 0.8 μL / cm using an XYZ 3D stretching and gold spraying apparatus. Goat anti-mouse monoclonal antibody was also uniformly sprayed onto the nitrocellulose membrane control line (C line) at the same speed. The C line was 6 mm away from the T line. Additionally, sheet-like gold nanoparticle aggregates of immunoprobes were sprayed onto the gold-labeled binding pad. Finally, the sprayed test strips were cut into 4 mm wide strips using a CNC high-speed cutter and stored in a dry environment at room temperature for later use. Example 2 (molar ratio of seed solution to silver nitrate solution is 200:1000) The difference from Example 1 is that 4 mL of seed solution was added when preparing the silver nanosheet solution; the rest of the steps are the same as in Example 1. Example 3 (The molar ratio of seed solution to silver nitrate solution is 50:1000) The difference from Example 1 is that 1 mL of seed solution is added when preparing the silver nanosheet solution; the rest of the steps are the same as in Example 1. Example 4 (The molar ratio of seed solution to silver nitrate solution is 8.3:1000) The difference from Example 1 is that 0.2 mL of seed solution was added when preparing the silver nanosheet solution; the rest of the steps are the same as in Example 1. Comparative Example 1 Preparation of gold nanoparticle immunoprobes: Take 1 mL of a solution of gold nanoparticles with a particle size of 40 nm, and follow the same steps as in Example 1.

[0040] Preparation of lateral flow immunoassay strips based on gold nanoparticles: The preparation process is the same as in Example 1, except that the gold nanoparticle immune probe is sprayed onto the gold-labeled binding pad. Figure 3 b shows a scanning electron microscope image of the silver nanosheets prepared in Example 3. Figure 3 c shows a scanning electron microscope image of the sheet-like gold nanoparticle aggregates prepared from the silver nanosheets. Figure 3a shows the UV-Vis absorption spectra of the silver nanosheets prepared in Example 1 and the sheet-like gold nanoparticle aggregates prepared from the silver nanosheets.

[0041] Figure 4 b shows a scanning electron microscope image of the silver nanosheets prepared in Example 4. Figure 4 c shows a scanning electron microscope image of the sheet-like gold nanoparticle aggregates prepared from the silver nanosheets. Figure 4 a shows the UV-Vis absorption spectra of the silver nanosheets prepared in Example 2 and the sheet-like gold nanoparticle aggregates prepared from the silver nanosheets.

[0042] from Figure 3 As can be seen from a, when the number of silver nanosheets and plate-like gold nanoparticle aggregates is the same, the absorption peak intensity of the plate-like gold nanoparticle aggregates increases significantly due to the strong plasmon coupling between particles within the aggregates, reaching twice that of the silver nanosheets. From Figure 4 As can be seen, when the number of silver nanosheets and plate-like gold nanoparticle aggregates is the same, the absorption peak intensity of the plate-like gold nanoparticle aggregates increases to 5 times that of the silver nanosheets due to the strong plasmon coupling between particles inside the plate-like gold nanoparticle aggregates.

[0043] from Figure 3 b and Figure 3 c and Figure 4 b and Figure 4 As can be seen from c, the sheet-like gold nanoparticle aggregates generated using silver nanosheets as templates maintain the shape and structure of the silver nanosheets. The less seed solution added during the preparation of silver nanosheets, the larger the size of the silver nanosheets, the larger the size of the prepared sheet-like gold nanoparticle aggregates, the higher the peak intensity of the absorption peak of the sheet-like gold nanoparticle aggregates, and the better the color development effect. The following tests were conducted using lateral flow immunoassay strips prepared in Examples 1, 2, 3, 4, and Comparative Example 1 to detect test solutions containing different concentrations of cTnI antigen. cTnI is currently the most specific and sensitive biomarker internationally recognized for diagnosing acute coronary syndrome (ACS).

[0044] Using the lateral flow immunoassay test strips prepared in Examples 1, 2, 3, 4, and Comparative Example 1, 50 μL of test solution containing 20 ng / mL cTnI antigen was added, and the results were obtained after 15 min. Figure 5 The test results are shown. It can be seen that the T-line color of the lateral flow immunoassay test strips prepared in Examples 1, 2, 3 and 4 is deeper and easier to distinguish than that in Comparative Example 1, and the results of Examples 1, 2, 3 and 4 are better in that order.

[0045] Using the lateral flow immunoassay test strips prepared in Examples 1, 2, 3, 4, and Comparative Example 1, 50 μL of test solution containing 10 ng / mL cTnI antigen was added, and the results were obtained after 15 min. Figure 6 The test results are shown. It can be seen that the T-line color of the lateral flow immunoassay test strips in Examples 1, 2, 3, and 4 is deeper and easier to distinguish than that in Comparative Example 1, and the results of Examples 1, 2, 3, and 4 are better in that order.

[0046] Using the lateral flow immunoassay test strips prepared in Examples 1, 2, 3, 4, and Comparative Example 1, 50 μL of test solution containing 5 ng / mL cTnI antigen was added, and the results were obtained after waiting for 15 min. Figure 7 The test results are shown. It can be seen that the T-line color of the lateral flow immunoassay test strips prepared in Examples 1, 2, 3 and 4 is deeper and easier to distinguish than that in Comparative Example 1, and the results of Examples 1, 2, 3 and 4 are better in that order.

[0047] Using the lateral flow immunoassay test strips prepared in Examples 3, 4, and Comparative Example 1, 50 μL of test solution containing 0.1 ng / mL cTnI antigen was added, and the results were obtained after 15 min. Figure 8 The test results are shown. It can be seen that the T line of the lateral flow immunoassay test strips prepared in Examples 3 and 4 is clearly visible, and the effects of Examples 3 and 4 are comparable, while the lateral flow immunoassay test strip of Comparative Example 1 only shows the C line.

[0048] Using the lateral flow immunoassay test strips prepared in Examples 3, 4, and Comparative Example 1, 50 μL of test solution containing 0.005 ng / mL cTnI antigen was added, and the results were obtained after 15 min. Figure 9 The test results are shown. It can be seen that the T line of the lateral flow immunoassay test strips prepared in Examples 3 and 4 is clearly visible, and the effects of Examples 3 and 4 are comparable. The lateral flow immunoassay test strip of Comparative Example 1 only shows the C line.

[0049] It can be concluded that silver nanosheets are generated when the molar ratio of seed solution to silver nitrate solution is 8.3–500:1000. Using these silver nanosheets as templates to prepare sheet-like gold nanoparticle aggregates, the lateral flow immunoassay strips based on these sheet-like gold nanoparticle aggregates exhibit better detection performance than those based on lateral flow immunoassay strips using 40 nm gold nanoparticles. Furthermore, the detection limit of the lateral flow immunoassay strips based on a silver nanosheet generation method with a seed solution to silver nitrate solution molar ratio of 8.3:1000 is 5 pg / mL.

[0050] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the specific embodiments described above. The specific embodiments and descriptions in the specification are merely for further illustrating the principles of the invention. The basic principles, main features, and advantages of the present invention have been shown and described above without departing from the spirit and scope of the invention. Those skilled in the art should understand that various changes and modifications will be made, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing sheet-like gold nanoparticle aggregates, characterized in that, Includes the following steps: Step 10: Add hydrogen peroxide solution and sodium borohydride solution to sodium citrate solution, centrifuge after reaction to remove supernatant, and then reconstitute with deionized water to obtain seed solution; The seed solution was added to the growth solution, and after reacting for a period of time, silver nitrate solution was added. After the reaction, the supernatant was removed by centrifugation. The solution was then reconstituted with deionized water to obtain a silver nanosheet solution. The growth solution included acetonitrile solution, ascorbic acid solution, and sodium citrate solution. Step 20: Add the silver nanosheet solution and the monovalent gold solution to the stabilizer solution, and centrifuge to remove the supernatant after the reaction to obtain the sheet-like gold nanoparticle aggregates. The stabilizer solution includes sodium citrate solution and polyvinylpyrrolidone solution.

2. The preparation method according to claim 1, characterized in that, In step 10, the molar ratio of seed solution to silver nitrate solution is 0.83~50:

100.

3. The preparation method according to claim 1, characterized in that, In step 20, the monovalent gold solution is an aqueous solution of monovalent gold complex ions AuBr2-.

4. A sheet-like gold nanoparticle aggregate, characterized in that, It was prepared by the method described in claim 1.

5. A sheet-like gold nanoparticle aggregate immune probe, characterized in that, It includes the sheet-like gold nanoparticle aggregates of claim 4 and a recognition antibody, wherein the recognition antibody is coupled to the surface of the sheet-like gold nanoparticle aggregates.

6. The sheet-like gold nanoparticle aggregate immune probe according to claim 5, characterized in that, The recognition antibody is a cTnI recognition antibody.

7. A method for preparing the sheet-like gold nanoparticle aggregate immune probe according to claim 5, characterized in that, Includes the following steps: Step 10: Redissolve the sheet-like gold nanoparticle aggregates in deionized water to obtain a solution of sheet-like gold nanoparticle aggregates. Step 20: Add potassium carbonate solution to adjust the pH of the sheet gold nanoparticle aggregate solution to 5.8-6.0, add recognition antibody, and after reaction, obtain an immune probe based on sheet gold nanoparticle aggregates.

8. A lateral flow immunoassay strip based on sheet-like gold nanoparticle aggregates, characterized in that, The gold-labeled conjugate pad of the lateral flow immunoassay strip is coated with the sheet-like gold nanoparticle aggregate immunoprobe as described in claim 5.

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