Porous gold-silver alloy nanoparticles, and methods of making and using the same

Abstract

The application discloses a kind of porous gold silver alloy nanoparticles and its preparation method and application, belong to instant detection in vitro diagnostic lateral flow immunoassay technical field.Porous gold silver alloy nanoparticles are porous structure, with silver as core, outer layer is wrapped gold silver alloy;Silver core diameter is 15nm, and the diameter of porous gold silver alloy nanoparticles is 20-40nm.Preparation method includes the following steps: (1) preparation Ag seed solution;(2) preparation gold silver alloy nanoparticle colloidal solution;(3) with tetrachloroauric acid solution to gold silver alloy nanoparticles is delidded, namely obtained.Porous gold silver alloy nanoparticles can be applied to lateral flow immunoassay heart-type fatty acid binding protein (H-FABP) in instant detection, and show good linear correlation, excellent specificity, reproducibility and stability and lower economic cost, can greatly improve the sensitivity of detection without sacrificing convenience and reduce cost.

Description

Technical Field

[0001] This invention belongs to the field of point-of-care testing, in vitro diagnostic, and lateral flow immunoassay technology, specifically relating to a porous gold-silver alloy nanoparticle, its preparation method, and its application. Background Technology

[0002] Point-of-care testing (POCT) refers to a testing method that utilizes portable analytical instruments and accompanying reagents to rapidly obtain test results at the sampling site. It is an important area of ​​development in laboratory medicine, offering advantages such as ease of operation, on-site sampling, and rapid testing. The POCT industry has been one of the fastest-growing sub-sectors of the in vitro diagnostics (IVD) industry in recent years. In infectious disease screening and emergency care, POCT is faster and more efficient than traditional pathogen detection methods. With the continuous advancement of biotechnology and industrial technology, a significant development trend has emerged in the medical device industry: moving towards "simplicity, convenience, and personal health management." POCT products characterized by miniaturization, ease of operation, and timely results have emerged and rapidly developed against this backdrop.

[0003] Lateral flow immunochromatographic assay (LFIA) is a strip-based platform that uses colorimetric analysis to display the detection results of the target substance, requiring only 5-30 minutes. It boasts numerous advantages such as ease of operation, speed, and visualization, making it the most widely used technology in point-of-care testing (POCT). LFIA technology primarily uses a large-pore microporous membrane (nitrocellulose membrane, NC membrane) as a carrier. When the sample is added to the sample pad at one end of the test strip, it flows laterally through capillary action, specifically binding to the markers on the surface of colloidal gold or latex microspheres on the conjugation pad. The sample then moves to the NC membrane, where it is captured by the coating material immobilized on the NC membrane surface, accumulating on the detection band. The visual colorimetric result is obtained by observing the aggregated bands of colloidal gold or latex microspheres on the NC membrane surface. Unbound substances cross the detection band and flow into the absorbent pad, achieving automatic separation. LFIA products require no special equipment or trained personnel and are suitable for various common sample types such as blood, serum, saliva, and urine.

[0004] In particular, gold nanoparticle-based lateral flow immunoassay (AuNP LFIA) has become a powerful and practical tool for disease diagnosis, home healthcare, and the detection of pathogens / viruses / drugs / pesticide residues. For example, during the COVID-19 pandemic, AuNP LFIA-based home self-testing provided an effective primary diagnosis, with signals that can be read without the naked eye, aiding in epidemic prevention efforts. LFIA offers advantages such as portability, user-friendliness, and time-saving (test time ≤30 min), but it exhibits relatively low sensitivity. Improving sensitivity without sacrificing convenience and cost / time efficiency remains a challenge. Researching a controllable synthesis strategy for colloidal plasma nanoparticles with nanostructure design, surface cleanliness, and aqueous dispersibility will contribute to their widespread application in the biomedical field. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a porous gold-silver alloy nanoparticle, its preparation method and application, which solves the problem that the existing technology of lateral flow immunoassay based on gold nanoparticles cannot simultaneously achieve convenience, low cost and high sensitivity.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a porous gold-silver alloy nanoparticle is provided. The porous gold-silver alloy nanoparticle has a porous structure with silver as the core and gold-silver alloy as the outer layer. The diameter of the silver core is 15 nm, and the diameter of the porous gold-silver alloy nanoparticle is 20-40 nm.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, the preparation method of the above-mentioned porous gold-silver alloy nanoparticles includes the following steps:

[0009] (1) After boiling the aqueous solution containing the reducing agent, silver nitrate solution is added to obtain Ag seed crystal solution;

[0010] (2) Add trisodium citrate solution, tetrachloroauric acid solution and silver nitrate solution to Ag seed solution in steps, and react at 90-110℃ for 1 h to obtain gold-silver alloy nanoparticles.

[0011] (3) The gold-silver alloy nanoparticles are dealloyed using a 0.2-0.4 mM tetrachloroauric acid solution to obtain the desired product.

[0012] Furthermore, the reducing agent is a mixture of trisodium citrate and tannic acid.

[0013] Furthermore, in step (1), the concentration of silver nitrate solution is 20-30 mM, and the ratio of trisodium citrate, tannic acid and silver nitrate solution is 0.25-1 mmol:0.5 mmol:1 mL.

[0014] Furthermore, in step (2), the Ag seed solution is first heated to 90°C, and then trisodium citrate solution, tetrachloroauric acid solution and silver nitrate solution are added sequentially within 10-30 minutes.

[0015] Furthermore, the concentrations of the trisodium citrate solution, tetrachloroauric acid solution, and silver nitrate solution are 170-200 mM, 3-5 mM, and 25-30 mM, respectively; the volume ratio of the trisodium citrate solution, tetrachloroauric acid solution, and silver nitrate solution is 2:1:1.

[0016] Furthermore, in step (3), the concentration of the tetrachloroauric acid solution is 0.3 mM, and the rate of addition of the tetrachloroauric acid solution is 20-40 mL·h. -1 .

[0017] Furthermore, the application of the aforementioned porous gold-silver alloy nanoparticles in lateral flow immunoassay.

[0018] Furthermore, the target of lateral flow immunoassay is heart-shaped fatty acid-binding protein.

[0019] Furthermore, lateral flow immunoassay was performed using a lateral flow immunoassay test card, in which the gold concentration of the porous gold-silver alloy nanoparticles was 90-110 mg·L⁻¹. -1 .

[0020] The beneficial effects of this invention are as follows: Under mild conditions, using Ag as the growth seed, with only an outer layer of gold-silver alloy, and employing a simple and easily scalable redox strategy, this invention successfully synthesized porous gold-silver alloy nanoparticles (porous Ag@AuAg, PGNPs) with uniform dispersion and good stability. This material was then used for the first time in the LFIA detection of heart-shaped fatty acid-binding protein (H-FABP). The synthesis process of this novel colloidal gold is highly compatible with existing commercial colloidal gold production processes, requiring no modification, upgrading, or replacement of existing production equipment. It can be directly used in existing commercial colloidal gold production lines for large-scale production, demonstrating significant industrialization advantages. Thanks to the porous structure and high stability of PGNPs, more antibodies can be bound, significantly amplifying the colorimetric signal of PGNPs-LFIA. Compared to commercial colloidal gold CGNPs, while reducing the concentration by 1.8 times, it still exhibits superior colorimetric ability and a lower detection limit by an order of magnitude. It also demonstrates good linear correlation (R²). 2 =0.99264), exhibiting excellent specificity, reproducibility, and stability, as well as low economic cost. The PGNPs100-LFIA prepared in this invention possesses sensitive, reliable, rapid, and economical H-FABP detection capabilities, and is expected to be widely applied in the field of point-of-care LFIA detection. Attached Figure Description

[0021] Figure 1 These are TEM images of porous gold-silver alloy nanoparticles (PGNPs); where a is a TEM image and b is a HAADF-STEM image.

[0022] Figure 2 These are TEM images of commercial colloidal gold (CGNPs); where a is a TEM image and b is a HAADF-STEM image.

[0023] Figure 3 This is a graph illustrating the sensitivity performance of LFIA; where 'a' represents an Au content of 180 mg·L⁻¹. -1 The commercially available colloidal gold test card (CGNPs180-LFIA) shows H-FABP antigen concentrations of 80, 40, 20, 10, 5, 2.5, and 1 ng / mL from left to right. -1 The test results; b represents an Au content of 100 mg·L⁻¹. -1 The test card (PGNPs100-LFIA) prepared with a novel colloidal gold in purple color, from left to right, shows H-FABP antigen concentrations of 80, 40, 20, 10, 5, 2.5, 1, 0.5, and 0.25 ng·mL. -1 The test results;

[0024] Figure 4 A graph illustrating the linear range of LFIA performance testing for commercial colloidal gold (CGNPs);

[0025] Figure 5 A graph showing the linear range of LFIA performance testing for porous gold-silver alloy nanoparticles (PGNPs);

[0026] Figure 6 The graph shows the specific performance of LFIA and the test results of blank samples; where a represents an Au content of 180 mg·L⁻¹. -1 The commercially available colloidal gold test card (CGNPs180-LFIA) shows, from left to right, the results of serum albumin (ALB), natural human D-dimer (ADD02), N-terminal brain natriuretic peptide monoclonal antibody (ANT03), lipoprotein-associated phospholipase (ALP03), and blank human serum assays; b represents the results of Au content of 100 mg·L⁻¹. -1 The test card (PGNPs100-LFIA) prepared with purple novel colloidal gold, from left to right, shows the detection results of serum albumin (ALB), natural human D-dimer (ADD02), N-terminal brain natriuretic peptide monoclonal antibody (ANT03), lipoprotein-associated phospholipase (ALP03), and blank human serum group. Detailed Implementation

[0027] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0028] Example 1

[0029] A porous gold-silver alloy nanoparticle (PGNPs) comprises a porous silver core coated with a porous gold-silver alloy. The preparation of the porous gold-silver nanoparticles includes the following steps:

[0030] (1) Synthesis of 15nm silver nanoparticles Ag NPs

[0031] Heat 50 mL of an aqueous solution containing trisodium citrate (5 mM) and tannic acid (10 mM) to boiling. Add 1 mL of AgNO3 solution (30 mM) while stirring vigorously. After 30 s, remove the reaction flask and cool to room temperature. Centrifuge at 11000 rpm for 10 min to remove excess reducing agent. Resuspend in ultrapure water to a final volume of 50 mL and store at 4 °C for later use.

[0032] (2) Preparation of 25-36nm Ag@AuAg nanoparticles

[0033] Take 25 mL of the 15 nm silver nanoparticles synthesized in step (1) and add them to 210 mL of ultrapure water. Heat to 90 °C and, under stirring, add 1080 μL of trisodium citrate solution (170 mM), 540 μL of HAuCl4 solution (3 mM), and 540 μL of AgNO3 solution (30 mM) in three steps over 15 min. Then heat to 100 °C and react for 1 h. After the reaction is complete, centrifuge at 11000 rpm for 10 min to remove 80% of the supernatant. Add 10% ultrapure water and disperse evenly. Mix and set aside.

[0034] (3) Preparation of porous Ag@AuAg nanoparticles (PGNPs)

[0035] Using 0.3 mM HAuCl4 solution as the dealloying agent, the HAuCl4 solution was dispensed using a syringe and injected at a rate of 40 mL / h using a syringe pump. -1 The HAuCl4 solution was injected into a rapidly stirred Ag@AuAg solution at a certain rate for dealloying treatment. When the solution turned purple, the addition was stopped and the reaction was continued for 30 minutes to obtain a novel purple colloidal gold solution (porous Ag@AuAg, PGNPs).

[0036] Example 2

[0037] A porous gold-silver alloy nanoparticle (PGNPs) comprises a porous silver core coated with a porous gold-silver alloy. The preparation of the porous gold-silver nanoparticles includes the following steps:

[0038] (1) Synthesis of 15nm silver nanoparticles Ag NPs

[0039] Heat 50 mL of an aqueous solution containing trisodium citrate (20 mM) and tannic acid (10 mM) to boiling. Add 1 mL of AgNO3 solution (20 mM) while stirring vigorously. After 30 s, remove the reaction flask and cool to room temperature. Centrifuge at 11000 rpm for 10 min to remove excess reducing agent. Resuspend in ultrapure water to a final volume of 50 mL and store at 4 °C for later use.

[0040] (2) Preparation of 25-36nm Ag@AuAg nanoparticles

[0041] Take 25 mL of the 15 nm silver nanoparticles synthesized in step (1) and add them to 210 mL of ultrapure water. Heat to 90 °C and, under stirring, add 1080 μL of trisodium citrate solution (200 mM), 540 μL of HAuCl4 solution (5 mM), and 540 μL of AgNO3 solution (25 mM) in three steps over 30 min. Then heat to 110 °C and react for 1 h. After the reaction is complete, centrifuge at 11000 rpm for 10 min to remove 80% of the supernatant. Add 10% ultrapure water and disperse evenly. Mix and set aside.

[0042] (3) Preparation of porous Ag@AuAg nanoparticles (PGNPs)

[0043] Using 0.2 mM HAuCl4 solution as the dealloying agent, the HAuCl4 solution was dispensed using a syringe and injected at 20 mL / h using a syringe pump. -1 The HAuCl4 solution was injected into a rapidly stirred Ag@AuAg solution at a certain rate for dealloying treatment. When the solution turned purple, the addition was stopped and the reaction was continued for 30 minutes to obtain a novel purple colloidal gold solution (porous Ag@AuAg, PGNPs).

[0044] The porous gold-silver nanoparticles (PGNPs) prepared in Examples 1 and 2 have similar properties. The porous gold-silver nanoparticles (PGNPs) prepared in Example 1 are used as an example to carry out relevant detection and subsequent experiments.

[0045] The TEM detection results of the porous gold-silver nanoparticles (PGNPs) prepared in Example 1 are as follows: Figure 1 As shown, the nanoparticles have a unique porous structure, exhibit a monodisperse state, and have a particle size of 30.86±4.42 nm.

[0046] Comparative Example 1

[0047] The preparation of commercially available colloidal gold (CGNPs) includes the following steps:

[0048] (1) Synthesis of 15nm gold nanoparticles Au NPs

[0049] Add 300 μL of tetrachloroauric acid solution (30 mM) to 28 mL of ultrapure water, heat to boiling, then add 200 μL of trisodium citrate solution (170 mM), and continue the reaction at 100 °C for 30 min.

[0050] (2) Preparation of 30-40nm Au nanoparticles

[0051] Take 3 mL of the 15 nm Au NPs obtained in step (1), add ultrapure water to a final volume of 60 mL and heat to 90 °C. Then, in 10 steps over 1 hour, add 500 μL of trisodium citrate solution (170 mM) and 270 μL of tetrachloroauric acid solution (30 mM) respectively. Then, continue the reaction at 90 °C for 1 hour to obtain the final product.

[0052] TEM detection results of the prepared commercial colloidal gold (CGNPs) are as follows: Figure 2 As shown, commercial colloidal gold exhibits a solid nanosphere structure with relatively uniform particle size, an average particle size of 30 nm, and a monodisperse state.

[0053] Example 3

[0054] The application of porous gold and silver nanoparticles (PGNPs) in lateral flow immunoassay includes the following steps:

[0055] (1) Preparation of antibody-labeled conjugates of CGNPs-dAb and PGNPs-dAb

[0056] Take Au content of 180mg L respectively -1 Commercial colloidal gold (CGNPs, prepared in Example 1), with an Au content of 100 mg / L. -1 1.2 mL each of the novel purple colloidal gold solution (PGNPs) (the Au content in CGNPs and PGNPs was determined by inductively coupled plasma spectroscopy (ICP)) was added. The pH was adjusted to approximately 7.2 using a pH adjuster (S005 solution, purchased from Zhongyuan Huiji Co., Ltd.). Then, 3.58 μL of gold-labeled conjugate antibody (dAb, H-FABP 13G11) was added to CGNPs180, and 4 μL of gold-labeled conjugate antibody (dAb, H-FABP 13G11) was added to PGNPs100. The mixture was then rapidly inverted to ensure a final antibody concentration of 20 μg / mL. -1 The reaction was allowed to proceed for 8 minutes. Then, 12 μL of blocking agent (10 wt% BSA solution) was added separately and quickly mixed, and allowed to stand for 8 minutes. The supernatant was removed by centrifugation, and the colloidal gold nanoparticles were reconstituted to 1.2 mL. Using a pipette, the resulting CGNPs-dAb and PGNPs-dAb antibody-labeled conjugate solutions were evenly spread onto glass fiber membranes, vacuum dried for 3 hours, and stored in a vacuum drying room, ensuring the humidity was less than 30%.

[0057] (2) Preparation of T and C line scribing membrane plates

[0058] Add 3 μL of H-FABP antibody (4 mg·mL) -1 4 μL of 20 wt% sucrose solution and 23 μL of coating buffer (S002, NC membrane coating buffer; purchased from Zhongyuan Huiji Co., Ltd.) were mixed evenly to prepare the T-line streaking solution; 3 μL of H-FABP antigen (2 mg / mL) was added. -1 ), 6 μL of goat anti-mouse antibody (2 mg / mL) -1 Mix 4 μL of 20 wt% sucrose solution and 17 μL of coating buffer thoroughly to prepare the C-line streaking solution; streaking is performed using a gold sputtering streaking apparatus, and the film is dried and stored in a drying room for later use.

[0059] (3) Evaluation of LFIA test card assembly

[0060] Using a microcomputer-controlled cutter, the sample pad, conjugate pad, NC membrane, absorbent paper, and other materials are cut into test strips with a width of about 4 mm and then assembled to obtain PGNPs 100-LFIA and CGNPs 180-LFIA kits. After assembly, the reagent cards are stored in a drying room.

[0061] Under optimal testing conditions, a blank test was performed using commercial negative human serum; H-FABP antigen was diluted with negative human serum to different concentrations (0-80 ng / mL). -1 Each reagent card was sampled at a concentration of 70 μL for linearity testing. The samples were incubated for 15 minutes using an external immunoassay analyzer. Color changes and brightness contrast were observed visually, and T and C signal values ​​were recorded using a Q7 immunoassay analyzer. Data were then processed and linearity analysis was performed. Simultaneously, the same method was used to sample and incubate other antigens: natural human D-dimer (ADD02), a coagulation function indicator; lipoprotein-associated phospholipase A2 (ALP03), a cardiovascular function indicator; N-terminal brain natriuretic peptide monoclonal antibody (ANT03), a cardiac function indicator; and serum albumin (ALB), a liver function indicator, to determine the specific adsorption performance of the novel colloidal gold reagent strip (where the concentrations of other antigens were higher than the concentration of the test antigen H-FABP, and within the corresponding linear range of commercial LFIA detection).

[0062] The LFIA sensitivity performance is shown in the following figure. Figure 3As shown, direct visual observation reveals that even with a 1.8-fold reduction in Au concentration compared to commercial colloidal gold CGNPs 180-LFIA, PGNPs100-LFIA still exhibits slightly better color development ability. Furthermore, the T-line color development ability increases with increasing H-FABP antigen concentration. The PGNPs100-LFIA T-line can be developed up to 0.5 ng / mL. -1 At H-FABP, CGNPs180-LFIA T-line was developed to 5 ng / mL. -1 At H-FABP, compared with CGNPs180-LFIA, the detection limit of PGNPs100-LFIA under naked-eye observation is reduced by 10 times, which shows that the detection sensitivity of PGNPs 100-LFIA is significantly improved.

[0063] Linearity graph of LFIA performance test of commercial colloidal gold (CGNPs) is shown below. Figure 4 As shown, a standard curve was plotted using the H-FABP antigen concentration and the T / C value obtained from the Q7 immunoassay analyzer, within the range of 2.5-80 ng / mL. -1 Within the H-FABP antigen concentration range, this method showed an independent linear correlation in the quantitative detection of H-FABP antigen, with the corresponding regression equation being Y = 0.0081X + 0.06892, and the linear regression correlation coefficient (R0) being [missing value]. 2 The value is 0.99078.

[0064] Linear graph of LFIA performance test of porous gold-silver alloy nanoparticles (PGNPs) is shown below. Figure 5 As shown, a standard curve was plotted using the H-FABP antigen concentration and the T / C value obtained from the Q7 immunoassay analyzer, within the range of 0.25-40 ng / mL. -1 Within the H-FABP antigen concentration range, this method showed an independent linear correlation in the quantitative detection of H-FABP antigen, with the corresponding regression equation being Y = 0.01843X + 0.07738, and the linear regression correlation coefficient (R0) being [missing value]. 2 The value was 0.99264. In various cardiovascular disease studies, the clinical levels of H-FABP ranged from 1.6 to 19 ng / mL. -1 The porous gold-silver alloy nanoparticles (PGNPs) provided by this invention can achieve accurate detection of H-FABP antigen levels.

[0065] LFIA specificity performance and blank test results are as follows: Figure 6As shown, other proteins (ADD02, ALP03, ANT03, and ALB) were detected, and the results were observed after 15 minutes. These samples showed consistent results with the blank human serum samples; none showed color at the T line. However, the H-FABP antigen exhibited a very strong signal value in the performance test, showing color at both the T and C lines (as shown). Figure 3 (As shown). The results indicate that PGNPs 100-LFIA and CGNPs 180-LFIA can specifically identify H-FABP and have good specific detection capabilities.

[0066] This invention provides porous gold-silver alloy nanoparticles (porous Ag@AuAg, PGNPs) and their preparation method, and for the first time applies them to the detection of heart-shaped fatty acid-binding protein (H-FABP) LFIA. Benefiting from the porous structure and high stability of PGNPs, the porous gold-silver alloy nanoparticles (PGNPs) can bind more antibodies, significantly amplifying the colorimetric signal of PGNPs-LFIA. Compared with commercial colloidal gold CGNPs, while reducing the Au concentration by 1.8 times, they still exhibit superior colorimetric ability and a lower detection limit by an order of magnitude. They also demonstrate good linear correlation (R0). 2 =0.99264), exhibiting excellent specificity, reproducibility, and stability, as well as low economic cost. The PGNPs100-LFIA prepared in this invention possesses sensitive, reliable, rapid, and economical H-FABP detection capabilities, and is expected to be widely applied in the field of point-of-care LFIA detection.

[0067] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A method for preparing porous gold-silver alloy nanoparticles, characterized in that, Includes the following steps: (1) After boiling the aqueous solution containing the reducing agent, silver nitrate solution is added to obtain Ag seed crystal solution; (2) Add the trisodium citrate solution, tetrachloroauric acid solution and silver nitrate solution to the Ag seed solution in steps, and react at 90-110 °C for 1 h to obtain gold-silver alloy nanoparticles. (3) The gold-silver alloy nanoparticles are dealloyed using a 0.2-0.4 mM tetrachloroauric acid solution to obtain the desired product. The porous gold-silver alloy nanoparticles have a porous structure with silver as the core and gold-silver alloy as the outer layer; the diameter of the silver core is 15 nm, and the diameter of the porous gold-silver alloy nanoparticles is 20-40 nm.

2. The method for preparing porous gold-silver alloy nanoparticles according to claim 1, characterized in that: The reducing agent is a mixture of trisodium citrate and tannic acid.

3. The method for preparing porous gold-silver alloy nanoparticles according to claim 2, characterized in that: In step (1), the concentration of silver nitrate solution is 20-30 mM, and the ratio of trisodium citrate, tannic acid and silver nitrate solution is 0.25-1 mmol:0.5 mmol:1 mL.

4. The method for preparing porous gold-silver alloy nanoparticles according to claim 1, characterized in that: In step (2), the Ag seed solution is first heated to 90°C, and then the trisodium citrate solution, tetrachloroauric acid solution and silver nitrate solution are added sequentially within 10-30 min.

5. The method for preparing porous gold-silver alloy nanoparticles according to claim 4, characterized in that: The concentrations of the trisodium citrate solution, tetrachloroauric acid solution, and silver nitrate solution are 170-200 mM, 3-5 mM, and 25-30 mM, respectively; the volume ratio of the trisodium citrate solution, tetrachloroauric acid solution, and silver nitrate solution is 2:1:

1.

6. The method for preparing porous gold-silver alloy nanoparticles according to claim 1, characterized in that: In step (3), the concentration of the tetrachloroauric acid solution is 0.3 mM, and the rate of addition of the tetrachloroauric acid solution is 20-40 mL·h. -1 .

7. The porous gold-silver alloy nanoparticles prepared by the method according to any one of claims 1-6, wherein the porous gold-silver alloy nanoparticles have a porous structure with silver as the core and gold-silver alloy as the outer layer; the diameter of the silver core is 15 nm, and the diameter of the porous gold-silver alloy nanoparticles is 20-40 nm.

8. The application of the porous gold-silver alloy nanoparticles according to claim 7 in lateral flow immunoassay.

9. The application according to claim 8, characterized in that: The target of the lateral flow immunoassay is heart-shaped fatty acid-binding protein.

10. The application according to claim 9, characterized in that: The lateral flow immunoassay was performed using a lateral flow immunoassay test card, wherein the gold concentration of the porous gold-silver alloy nanoparticles in the lateral flow immunoassay test card was 90-110 mg·L⁻¹. -1 .

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