An ultra-small high-entropy alloy for lateral flow immunochromatography and its preparation method and application

Abstract

The present invention relates to the technical field of pathogen and disease marker detection, and specifically to an ultra-small high-entropy alloy for lateral flow immunochromatography, and its preparation method and application. The preparation method of the high-entropy alloy comprises the following steps: preparing a precursor solution; preparing the high-entropy alloy by a low-temperature reduction-diffusion strategy. The high-entropy alloy prepared by the method of the present invention has excellent catalytic performance and ultra-high peroxidase simulation activity. When applied to lateral flow immunochromatography, it can quickly and accurately detect Staphylococcus aureus, and has the characteristics of high sensitivity and strong specificity. At the same time, the present invention successfully extends the application of the high-entropy alloy to the detection of kidney injury marker cystatin C by modifying the recognition element. The high-entropy alloy for lateral flow immunochromatography provided by the present invention has huge market promotion prospects in the field of pathogen and disease marker detection.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen and disease marker detection, and in particular to an ultrasmall high-entropy alloy for lateral flow immunochromatography, a preparation method thereof, and applications thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) is a common foodborne pathogen that is widely present in natural environments such as air, water and soil. After contaminating food, it will produce enterotoxins under appropriate conditions, which can cause food poisoning after consumption. In severe cases, it can cause pericarditis, pneumonia, suppurative infection, toxic shock syndrome, acute kidney injury, glomerulonephritis and sepsis, posing a serious threat to human health. Analytical methods with high sensitivity and strong specificity are urgently needed to achieve rapid and accurate detection of S. aureus, a foodborne pathogen.

[0003] Lateral flow immunochromatography assay (LFIA) is a classic point-of-care (POCT) technology based on antigen-antibody immune reactions. Due to its ease of use, rapidity, low cost, and visually readable results, it provides a means for rapid, sensitive, and effective detection of pathogens and is widely used in clinical diagnosis, food safety, drug testing, and environmental monitoring. Conventional LFIA test strips primarily rely on colloidal gold nanoparticles (Au NPs) as colorimetric signal reporters, allowing for equipment-free detection and successful commercialization. However, Au NPs-based LFIA systems are often limited by poor sensitivity and accuracy, making them insufficient for the stringent requirements of foodborne pathogen screening.

[0004] High-entropy alloys (HEAs) are composed of five or more constituent elements in near-equiatomic ratios. Due to their characteristic properties, including high entropy, lattice distortion, hysteretic diffusion, and the "cocktail effect," they have attracted widespread attention in recent years across multiple research fields. These properties contribute to their excellent radiation resistance, stability, high catalytic activity, and efficient photothermal conversion, making them promising candidates for applications in energy systems and the environment. Compared to single, binary catalysts or intermediate-entropy alloys, multicomponent HEAs offer diverse active sites, making them a novel class of catalytic materials across multiple research areas. Currently, research on HEAs has largely focused on their use as metallic structural materials, while limited research has focused on HEAs-based lateral flow immunochromatography for the detection of foodborne pathogens and other biomarkers. Furthermore, HEAs are typically synthesized via mechanical alloying or rapid moving bed pyrolysis, which involves high temperatures and long processing times, limiting their practical application.

[0005] Based on this, this application synthesizes an ultrasmall Au-Pt-Ir-Ru-RhHEAs using a low-temperature reduction-diffusion strategy and applies it to lateral flow immunochromatography. By creating a nanotag with high catalytic activity and preparing corresponding test paper, it achieves rapid, efficient, and accurate detection of Staphylococcus aureus. At the same time, by modifying the recognition element, the application of this high-entropy alloy is expected to be expanded to the detection of disease markers. Summary of the Invention

[0006] The present invention aims to provide an ultrasmall high entropy alloy for lateral flow immunochromatography and a preparation method thereof.

[0007] Another object of the present invention is to provide an application of the ultrasmall high entropy alloy in the preparation of nanolabels and lateral flow immunochromatographic test strips for the detection of pathogens or disease markers.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The method for preparing the ultrasmall high entropy alloy for lateral flow immunochromatography of the present invention comprises the following steps:

[0010] S1 Preparation of precursor solution:

[0011] Adding chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to water to prepare a precursor solution;

[0012] S2 prepares high entropy alloys through low-temperature reduction-diffusion strategy:

[0013] The precursor solution prepared in the above steps is placed in an ice water bath for ultrasonic treatment, and then sodium borohydride is added. After mechanical stirring, a high entropy alloy is formed through a combined process of reduction and atomic diffusion, and the high entropy alloy is stored under low temperature conditions.

[0014] Preferably, in the method for preparing an ultrasmall high-entropy alloy for lateral flow immunochromatography according to the present invention, step S1 of preparing the precursor solution is specifically as follows: 0.5%-1.5% of chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride are added to 30-50 mL of water respectively to prepare a precursor solution.

[0015] Further preferably, in the method for preparing an ultrasmall high-entropy alloy for lateral flow immunochromatography according to the present invention, step S1 of preparing the precursor solution is specifically as follows: 1% of chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride are added to 40 mL of water to prepare a precursor solution.

[0016] Preferably, in the method for preparing an ultrasmall high-entropy alloy for lateral flow immunochromatography described in the present invention, step S2 prepares the high-entropy alloy by a low-temperature reduction-diffusion strategy, specifically: the precursor solution prepared in step S1 is placed in an ice water bath and ultrasonically treated for 2-4 minutes, then 2 mL of sodium borohydride is added, and mechanically stirred at 6000-8000 rpm / min for 4-6 minutes to form a high-entropy alloy through a combined process of reduction and atomic diffusion, and the alloy is stored at 2-8°C.

[0017] Further preferably, in the method for preparing an ultrasmall high-entropy alloy for lateral flow immunochromatography described in the present invention, step S2 prepares the high-entropy alloy by a low-temperature reduction-diffusion strategy, specifically: the precursor solution prepared in step S1 is placed in an ice water bath and ultrasonically treated for 2 minutes, and then 2 mL of sodium borohydride is added, and mechanically stirred at 7000 rpm / min for 5 minutes to form a high-entropy alloy through a combined process of reduction and atomic diffusion, and the alloy is stored at 4°C.

[0018] The ultrasmall high entropy alloy prepared by the above preparation method is used in the preparation of nano-labels for the detection of pathogens or disease markers and their lateral flow immunochromatographic test paper.

[0019] Preferably, the pathogenic bacteria of the present invention is Staphylococcus aureus.

[0020] Preferably, the disease marker of the present invention is cystatin C, a kidney injury marker.

[0021] Preferably, the application of the ultrasmall high entropy alloy in the preparation of nanolabels for Staphylococcus aureus detection and lateral flow immunochromatographic test paper thereof is as follows:

[0022] (1) The preparation method of the nanotag for Staphylococcus aureus detection is as follows:

[0023] Bovine serum albumin is added to the prepared high entropy alloy solution and shaken at room temperature; subsequently, K2CO3 and anti-Staphylococcus aureus antibody are added and shaken continuously to obtain a mixture; finally, the mixture is centrifuged and resuspended in a PB solution containing sucrose, bovine serum albumin and Tween-20 to obtain the product.

[0024] (2) The preparation method of lateral flow immunochromatographic test paper for Staphylococcus aureus detection is as follows:

[0025] The sample pad was immersed in Tris-HCl buffer containing NaCl and Tween-20 and then dried overnight; anti-Staphylococcus aureus IgG and goat anti-rabbit IgG were respectively distributed onto the NC membrane to generate the test line T line and the control line C line; the absorption pad and sample pad were assembled onto the PVC backing in sequence; the assembled strips were cut to complete the construction of the lateral flow immunochromatographic test paper.

[0026] Further preferably, the application of the ultrasmall high entropy alloy in the preparation of nanotags for Staphylococcus aureus detection and lateral flow immunochromatographic test paper thereof is as follows:

[0027] (1) The preparation method of the nanotag for Staphylococcus aureus detection is as follows:

[0028] 40 μL of 1% bovine serum albumin was added to 1 mL of the prepared high-entropy alloy solution and shaken at room temperature for 30 minutes; then, 20 μL of 0.1 mol / L K2CO3 and 5 μL of 1 mg / mL anti-Staphylococcus aureus antibody were added and shaken continuously for 60 minutes to obtain a mixture; finally, the mixture was centrifuged at 8000 rpm / min for 15 minutes and resuspended in a 10 mM PB solution containing 2% sucrose, 1% bovine serum albumin and 0.05% Tween-20 at a pH of 7.35.

[0029] (2) The preparation method of lateral flow immunochromatographic test paper for Staphylococcus aureus detection is as follows:

[0030] The sample pad was immersed in 0.05 M Tris-HCl buffer containing 0.15 M NaCl and 0.25% Tween-20, pH 8.0, and then dried overnight at 37°C. Anti-Staphylococcus aureus IgG at a concentration of 2.5 mg / mL and goat anti-rabbit IgG at a concentration of 1 mg / mL were dispensed onto the NC membrane to generate the test line T and the control line C, respectively. The absorption pad and sample pad were assembled sequentially onto the PVC backing plate, with a 2 mm gap between them and the NC membrane. The assembled strip was cut into 2.5 mm widths to complete the construction of the lateral flow immunochromatographic test strip.

[0031] Beneficial effects of the present invention:

[0032] 1. The present invention successfully synthesized an ultrasmall high-entropy alloy Au-Pt-Ir-Ru-RhHEAs through a low-temperature reduction-diffusion strategy. The high-entropy alloy has excellent catalytic performance and ultra-high peroxidase mimetic activity, which exceeds that of AuNPs, Au-PtNPs, Au-Pt-IrNPs, and Au-Pt-Ir-RuNPs by 15 times, 4 times, 2 times, and 2 times, respectively.

[0033] 2. The high entropy alloy (HEAs) prepared by the present invention was applied to lateral flow immunoassay (LFIA) for Staphylococcus aureus detection. The detection limits of HEAs-LFIA and DAB-enhanced HEAs-LFIA were 1.5×10 3 CFU / mL and 15 CFU / mL, while the detection limit of Au NPs-LFIA was 1.5×10 6 CFU / mL, indicating that the sensitivity of the lateral flow immunochromatography based on the high entropy alloy provided by the present invention is significantly higher than that of the traditional lateral flow immunochromatography based on Au NPs.

[0034] 3. Through specificity experiments, it was found that Staphylococcus aureus produced signal responses in both HEAs-LFIA and DAB-enhanced HEAs-LFIA, while several other potential interfering microorganisms such as Staphylococcus epidermidis and Escherichia coli were almost undetectable, indicating that the lateral flow immunochromatography based on the high entropy alloy provided by the present invention has strong specificity for the detection of Staphylococcus aureus.

[0035] 4. The HEAs-based LFIA test strip provided by the present invention can quickly and accurately detect Staphylococcus aureus in food samples, and has a low coefficient of variation and resistance to matrix interference effects, and has great promotion and application value in the field of pathogen screening in food.

[0036] 5. By modifying the recognition element, the present invention successfully extends the application of the prepared ultrasmall high-entropy alloy to the detection of the kidney injury marker cystatin C, indicating that the ultrasmall high-entropy alloy for lateral flow immunochromatography provided in this application has potential application prospects in the field of disease marker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the principle of high-entropy alloy-pAb-mediated lateral flow immunochromatographic detection of Staphylococcus aureus (Figure: A is a schematic diagram of HEAs solution preparation; B is a schematic diagram of HEAs-pAb probe preparation; C is a schematic diagram of lateral flow immunochromatographic detection and signal amplification);

[0038] Figure 2Performance characterization of Au NPs and HEAs (Figure: A is the Au NPs solution prepared by citric acid reduction method; B is the SEM image of Au NPs; C is the HEAs solution synthesized by low-temperature reduction-diffusion strategy; D is the HRTEM image of HEAs; E is the element distribution map of HEAs obtained by EDS; F is the interplanar spacing of single HEAs; G is the stability results of HEAs at different preparation temperatures; H is the catalytic activity comparison of HEAs prepared at different preparation temperatures);

[0039] Figure 3 To compare the catalytic activities of the nanoparticles using TMB and DAB as substrates (Figure A: Comparison of catalytic activity using TMB as substrate; B: Comparison of catalytic activity using DAB as substrate);

[0040] Figure 4 The feasibility results of HEAs-pAb-based LFIA for detecting Staphylococcus aureus (Figure: A shows the catalytic signal and visual observation results using DAB as a substrate; B shows the catalytic signal and visual observation results using TMB as a substrate);

[0041] Figure 5 Sensitivity evaluation results of LFIA based on Au NPs and HEAs (Figure: A is the test result of Au NPs-LFIA at different concentrations of Staphylococcus aureus; B is the test result of HEAs-LFIA at different concentrations of Staphylococcus aureus; C is the test result of HEAs-LFIA with DAB as substrate at different concentrations of Staphylococcus aureus; D is the linear regression equation of LFIA based on Au NPs; E is the linear regression equation of LFIA based on HEAs; F is the linear regression equation of LFIA based on HEAs enhanced by DAB);

[0042] Figure 6 The specificity evaluation results of HEAs-LFIA against various interfering strains (in the figure: a is blank; b is Staphylococcus aureus ATCC 29213; c is Staphylococcus epidermidis; d is Pseudomonas aeruginosa; d is Escherichia coli; f is Enterococcus faecium);

[0043] Figure 7 The following are the detection results of different samples using LFIA based on HEAs (in the figure: A is the detection result of Staphylococcus aureus in milk samples; B is the detection result of Staphylococcus aureus in orange juice samples; C is the detection result of different concentrations of cystatin C). DETAILED DESCRIPTION

[0044] The following is a detailed description of the technical solution of the present invention in conjunction with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation of the technical solution of the present invention.

[0045] Example 1

[0046] Ultra-small high entropy alloys for lateral flow immunochromatography are prepared according to the following steps:

[0047] S1 Preparation of precursor solution:

[0048] To 40 mL of water, 1% chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride were added respectively to prepare a precursor solution;

[0049] S2 prepares high entropy alloys through low-temperature reduction-diffusion strategy:

[0050] The precursor solution prepared in the above steps was placed in an ice water bath and ultrasonically treated for 2 minutes. Then, 2 mL of sodium borohydride was added and mechanically stirred at 7000 rpm / min for 5 minutes to form a high entropy alloy through a combined process of reduction and atomic diffusion. The alloy was then stored at 4°C.

[0051] Example 2

[0052] The ultra-small high entropy alloy prepared in Example 1 was used to prepare a nanotag for Staphylococcus aureus detection and a lateral flow immunochromatographic test paper thereof:

[0053] Preparation method of nanotag for Staphylococcus aureus detection:

[0054] 40 μL of 1% bovine serum albumin was added to 1 mL of the prepared high-entropy alloy solution and shaken at room temperature for 30 minutes; then, 20 μL of 0.1 mol / L K2CO3 and 5 μL of 1 mg / mL anti-Staphylococcus aureus antibody were added and shaken continuously for 60 minutes to obtain a mixture; finally, the mixture was centrifuged at 8000 rpm / min for 15 minutes and resuspended in a 10 mM PB solution containing 2% sucrose, 1% bovine serum albumin and 0.05% Tween-20 at a pH of 7.35.

[0055] Preparation method of lateral flow immunochromatographic test paper for Staphylococcus aureus detection:

[0056] The sample pad was immersed in 0.05 M Tris-HCl buffer containing 0.15 M NaCl and 0.25% Tween-20, pH 8.0, and then dried overnight at 37°C. Anti-Staphylococcus aureus IgG at a concentration of 2.5 mg / mL and goat anti-rabbit IgG at a concentration of 1 mg / mL were dispensed onto the NC membrane to generate the test line T and the control line C, respectively. The absorption pad and sample pad were assembled sequentially onto the PVC backing plate, with a 2 mm gap between them and the NC membrane. The assembled strip was cut into 2.5 mm widths to complete the construction of the lateral flow immunochromatographic test strip.

[0057] Example 3

[0058] By functionalizing the cystatin C tag, the ultrasmall high entropy alloy prepared in Example 1 was used to prepare a nanotag for cystatin C detection and a lateral flow immunochromatographic test paper thereof.

[0059] In order to further verify the reliability of the present invention and screen out the best solution, the inventors conducted a series of experiments, as follows:

[0060] 1. Preparation of gold nanoparticles and high entropy alloys

[0061] The research team prepared gold nanoparticles (Au NPs) by using a citric acid reduction method, and high-entropy alloys (Au-Pt-Ir-Ru-Rh HEAs, hereinafter referred to as HEAs), gold-platinum nanoparticles (Au-PtNPs), gold-platinum-iridium nanoparticles (Au-Pt-IrNPs) and gold-platinum-iridium-ruthenium nanoparticles (Au-Pt-Ir-RuNPs) by using a low-temperature reduction-diffusion method. The prepared gold nanoparticles were tested using a scanning electron microscope (SEM), and the structure and properties of the high-entropy alloys were studied using a high-resolution transmission electron microscope (HRTEM) and an energy dispersive spectrometer (EDS).

[0062] 1.1 Preparation of gold nanoparticles

[0063] Gold nanoparticles (Au NPs) were prepared using the sodium citrate reduction method. First, 16 mL of a 0.25% HAuCl4 stock solution was added to a flask. DNAse- and RNAse-free water was then added to bring the total volume to 200 mL. The solution was heated to boiling under mechanical stirring. Subsequently, 12 mL of a 1% (weight-to-volume) sodium citrate aqueous solution was quickly added. The solution was boiled for 15 minutes until the color of the solution changed from transparent to deep purple-red. Heating was stopped and stirring continued for 15 minutes. Finally, the volume of the colloidal solution was brought to 200 mL by adding DNAse- and RNAse-free water. The solution was stored at 4°C until ready for use.

[0064] 1.2 Preparation of high entropy alloys

[0065] High-entropy alloys (HEAs) were prepared using a low-temperature reduction-diffusion strategy. A precursor solution was prepared by adding 1% chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 40 mL of water. The resulting mixture was sonicated in an ice-water bath for 2 minutes, followed by the addition of 2 mL of sodium borohydride (NaBH4) and mechanical stirring at 7000 rpm / min for 5 minutes. The HEAs formed through a combined reduction and atomic diffusion process and were stored at 4°C until ready for use.

[0066] In addition, the research team also synthesized gold-platinum nanoparticles (Au-PtNPs), gold-platinum-iridium nanoparticles (Au-Pt-IrNPs), and gold-platinum-iridium-ruthenium nanoparticles (Au-Pt-Ir-RuNPs) using the same method.

[0067] 2. Experimental methods

[0068] 2.1 Catalytic activity test

[0069] In this study, control experiments were conducted to elucidate the catalytic activity of high-entropy alloys (HEAs). TMB (3,3',5,5'-tetramethylbenzidine) and DAB (diaminobenzidine) were added to various nanoparticle solutions and HEAs, including AuNPs, Au-PtNPs, Au-Pt-IrNPs, Au-Pt-Ir-RuNPs, and HEAs. The catalytic activity of each solution was determined by measuring the absorbance of these solutions in the 300-800 nm wavelength range using a UV-visible spectrophotometer and monitoring the color changes of TMB and DAB. For the colorimetric detection of HEAs, 100 μL of TMB or DAB solution was mixed with 100 μL of HEAs and incubated at 25°C for 5 minutes. The resulting oxidized TMB or DAB precipitate was then analyzed for color.

[0070] 2.2 Modification of AuNPs and high entropy alloys

[0071] The AuNPs were thoroughly mixed with 20 μL of 0.1 mol / L K2CO3 solution, and 5 μL of anti-Staphylococcus aureus IgG was added to reach a final concentration of 5 μg / mL. The mixture was shaken on a mixer for 60 min, and then bovine serum albumin (BSA) was added as a blocking agent at a final concentration of 0.1% (w / v), and then shaken at room temperature for 30 min. The mixture was then centrifuged at 10,000 rpm / min for 12 min, and the red precipitate was collected. The anti-Staphylococcus aureus IgG-modified AuNPs precipitate was resuspended in a PB solution (10 mM, pH 7.35) containing 2% sucrose, 1% BSA, and 0.05% Tween-20 to obtain the AuNPs-pAb probe for later use.

[0072] Anti-Staphylococcus aureus IgG (rabbit IgG) was coupled to the HEAs solution via electrostatic adsorption. The detailed procedure is as follows: 40 μL of BSA (1%, w / v) was added to 1 mL of the HEAs solution, and the mixture was shaken at room temperature for 30 minutes. Subsequently, 20 μL of 0.1 mol / L K2CO3 and 5 μL of anti-Staphylococcus aureus IgG (1 mg / mL) were added to the solution and shaken continuously for 60 minutes. The mixture was then centrifuged at 8000 rpm / min for 15 minutes and resuspended in a 10 mM PB solution containing 2% sucrose, 1% BSA, and 0.05% Tween-20 at a pH of 7.35 to obtain the HEAs-pAb probe, which was then used for further preparation.

[0073] 2.3 Preparation of lateral flow immunochromatographic test strips

[0074] The sample pad was immersed in 0.05 M Tris-HCl (pH 8.0) buffer containing 0.15 M NaCl and 0.25% Tween-20 (v / v), and then dried at 37°C overnight. Anti-Staphylococcus aureus IgG (2.5 mg / mL) and goat anti-rabbit IgG (1 mg / mL) were dispensed onto the NC membrane to generate a test line (T test line) and a control line (C line), respectively. The absorption pad and sample pad were assembled onto the PVC backing in sequence, with a gap of approximately 2 mm from the NC membrane. The assembled strips were cut into a width of 2.5 mm to complete the construction of the lateral flow immunochromatography.

[0075] 2.4 Lateral flow immunochromatographic detection based on high entropy alloy

[0076] 5 μL of the HEAs-pAb probe was added to a sample solution containing the target bacteria and reacted in a phosphate buffer (PB) solution for 5 minutes. The reaction solution was then introduced onto the sample pad of a lateral flow immunochromatography instrument. After running under capillary force for 10 minutes, the colorimetric results were recorded. After the addition of the DAB mixed solution, the catalytic signal on the T test line was amplified, and an image was captured using a smartphone. Finally, the grayscale value on the T test line was quantitatively analyzed using a GIC-H1 portable colloidal gold immunoassay analyzer.

[0077] 2.5 Strain culture

[0078] Standard strains, including Staphylococcus aureus (ATCC 29213 and ATCC 25923) and additional clinical strains (Pseudomonas aeruginosa, Staphylococcus epidermidis, Escherichia coli, and Enterococcus faecium), were obtained from the Microbiology Laboratory of Guizhou Provincial People's Hospital (Guizhou, China). The use of clinical strains was approved by the Ethics Committee of Guizhou Provincial People's Hospital (No.

[2024] 171). These strains were cultured on Columbia blood agar and incubated at 35°C with 5% carbon dioxide for 24 h.

[0079] 2.6 Determination of Staphylococcus aureus in food samples

[0080] In order to explore the applicability of the established HEAs-based LFIA sensor in real sample analysis, Staphylococcus aureus was introduced into milk and orange juice samples, with 9×10 8 CFU of Staphylococcus aureus and mix thoroughly, then add 5 μL of buffer containing HEAs-pAb probe to 50 μL of milk or orange juice mixture solution and react for 5 minutes, then transfer to the sample pad of the lateral flow immunochromatographic immunosensor and record its signal response on the T detection line.

[0081] 3. Results and Discussion

[0082] 3.1 Principle of High Entropy Alloy-pAb Mediated Lateral Flow Immunochromatographic Detection of Staphylococcus aureus

[0083] The detection principle of high entropy alloy (HEAs)-pAb mediated lateral flow immunoassay (lateral flow immunochromatography) is based on the sandwich lateral flow immunochromatography model. Figure 1 (In the figure: A is a schematic diagram of HEAs solution preparation; B is a schematic diagram of HEAs-pAb probe preparation; C is a schematic diagram of lateral flow immunochromatography detection and signal amplification). HEAs are synthesized by low-temperature reduction-diffusion strategy. The synthesis process is as follows. Figure 1 As shown in A. The precursor solution is ultrasonically treated in an ice-water bath, and then a reducing agent is added to prepare HEAs. The anti-Staphylococcus aureus polyclonal antibody (pAb) binds to the HEAs solution through electrostatic interaction to prepare the HEAs-pAb probe ( Figure 1 B) Schematic diagram of lateral flow immunochromatographic detection and signal amplification Figure 1As shown in Figure C, anti-Staphylococcus aureus IgG (2.5 mg / mL) and goat anti-rabbit IgG (1 mg / mL) were dispensed onto a nitrocellulose (NC) membrane to prepare a test line (T test line) and a control line (C line). In the presence of S. aureus, the pathogen binds to the recognition site on the HEAs-pAb probe, resulting in the accumulation of the nanoprobes at the T test line. The color intensity of the T test line increases with increasing S. aureus concentration. To further amplify the signal and enhance detection sensitivity, a DAB solution was applied to the test area. Due to the strong catalytic activity of the HEAs, the initial gray band transformed into a distinct brown band. In the absence of S. aureus, the HEAs-pAb probe migrated upward in the test tube toward the absorbent pad, where its recognition site became occupied by goat anti-rabbit IgG, resulting in coloration of the control line (C line). The C line remained visible in all cases, serving as a functional indicator of the reliability of the lateral flow immunochromatography assay. The entire lateral flow immunochromatography assay process can be visually evaluated within 10 minutes. Quantitative analysis is performed using the GIC-H1 portable colloidal gold immunoassay analyzer, and results can be interpreted without the need for specialized personnel or advanced equipment.

[0084] 3.2 Characterization of Au NPs and High Entropy Alloys

[0085] The results are as follows Figure 2 (In the figure: A is the Au NPs solution prepared by citric acid reduction method; B is the SEM image of Au NPs; C is the HEAs solution synthesized by low-temperature reduction-diffusion strategy; D is the HRTEM image of HEAs; E is the element distribution map of HEAs obtained by EDS; F is the interplanar spacing of single HEAs; G is the stability results of HEAs at different preparation temperatures; H is the catalytic activity comparison of HEAs prepared at different preparation temperatures). The colloidal gold solution prepared by citrate reduction shows a distinct red color (as shown in Figure 1). Figure 2 A), the SEM image of gold nanoparticles shows that they have a uniformly dispersed spherical structure (as shown in Figure 2 B). The ultra-small high entropy alloy synthesized using the low-temperature reduction diffusion strategy exhibits a gray-black solution without precipitate (as shown in Figure 2 C), the high-resolution TEM (HRTEM) image shows that the obtained Au-Pt-Ir-Ru-Rh HEAs have an obvious elliptical structure and a size distribution of about 5 nm (as shown in Figure 2 D). The elemental map of energy dispersive X-ray spectroscopy (EDS) shows that all elements are uniformly distributed in HEAs (e.g. Figure 2 E). The d-row spacing of a single HEAs nanoparticle is 0.225 nm (as shown in Figure 2 F).

[0086] The research team investigated the stability of HEAs prepared at 0℃, 4℃ and 25℃ respectively (e.g. Figure 2 G). The results show that when the preparation temperature is 0°C, no precipitation is observed in the solution, while precipitation occurs when the preparation temperature is 4°C and 25°C, indicating that HEAs prepared at 0°C have better stability. Subsequently, the research team compared the catalytic activity of the nanoparticles prepared under the above temperature conditions (such as Figure 2 The results show that the nanoparticles prepared at 0 ° C exhibit significantly higher catalytic activity than those prepared at 4 ° C and 25 ° C. Therefore, 0 ° C was determined to be the optimal preparation temperature for HEAs provided in this study.

[0087] 3.3 Catalytic activity of high entropy alloys

[0088] In this study, control experiments were conducted to elucidate the catalytic activity of high entropy alloys. TMB and DAB were added to various nanoparticle solutions and high entropy alloy solutions, including AuNPs (tube a), Au-PtNPs (tube b), Au-Pt-IrNPs (tube c), Au-Pt-Ir-RuNPs (tube d), and HEAs (tube e). The results are shown in Figure 2. Figure 3 As shown (Figure: A is the comparison of catalytic activity using TMB as a substrate; B is the comparison of catalytic activity using DAB as a substrate). The results show that HEAs (tube e) exhibit excellent peroxidase mimetic activity, with catalytic performance exceeding that of AuNPs (tube a), Au-PtNPs (tube b), Au-Pt-IrNPs (tube c), and Au-Pt-Ir-RuNPs (tube d) by 15 times, 4 times, 2 times, and 2 times, respectively. In addition, after the addition of the DAB system, HEAs (tube e) produced a distinct dark brown precipitate with no observable absorption peak, further confirming the superior catalytic activity of HEAs compared to other nanoparticles.

[0089] 3.4 Feasibility of HEAs-pAb-based LFIA for Staphylococcus aureus Detection

[0090] To evaluate the feasibility of the lateral flow test strip based on the HEAs-pAb probe, the research team conducted a comparative analysis using standard strains and clinical isolates (a: blank; b: Staphylococcus aureus ATCC 29213; c: Staphylococcus aureus ATCC25923; d: clinical isolate-1; e: clinical isolate-2). The results are as follows: Figure 4 As shown (in the figure, A is the catalytic signal and visual observation result using DAB as substrate; B is the catalytic signal and visual observation result using TMB as substrate).

[0091] 3.5 Sensitivity evaluation of LFIA based on Au NPs and HEAs

[0092] The concentration of the standard Staphylococcus aureus suspension in PB solution was 1.5×10 5 to 15×10 8 CFU / mL and 1.5×10 1 to 9×10 8 CFU / mL, and PB solution was used as a negative control to determine the sensitivity of LFIA based on Au NPs and HEAs. Figure 5 (In the figure: A is the test result of Au NPs-LFIA under different concentrations of Staphylococcus aureus; B is the test result of HEAs-LFIA under different concentrations of Staphylococcus aureus; C is the test result of HEAs-LFIA with DAB as substrate under different concentrations of Staphylococcus aureus; D is the linear regression equation of LFIA based on Au NPs; E is the linear regression equation of LFIA based on HEAs; F is the linear regression equation of LFIA based on DAB enhanced HEAs). For LFIA based on Au NPs, no color is observed on the T test line of negative samples, and the signal intensity is close to zero; the color intensity of the T test line of positive samples gradually increases with the increase of Staphylococcus aureus concentration (as shown in Figure 1). Figure 5 The intensity of the red band on the T detection line is proportional to the logarithm of the concentration of Staphylococcus aureus, and the linear equation is y = -788.36 + 176.37 lgx (R 2 =0.977)(e.g. Figure 5 D); Based on visual evaluation, the detection limit of Au NPs-LFIA was 1.5×10 6 For HEAs-based LFIA, as the concentration of Staphylococcus aureus increases, the detection signal of HEAs-LFIA gradually increases (e.g. Figure 5 B), the signal intensity was also linearly correlated with the logarithm of the Staphylococcus aureus concentration, and the linear equations were y = 120.48 + 40.95lgx (standard curve before catalysis, R 2 =0.988) and y = -816.5 + 159.81 gx (standard curve after catalysis, R 2 =0.977)(e.g. Figure 5 E). In addition, the HEAs-LFIA results using DAB as substrate are shown in Figure 5 As shown in C, the detection signal intensity is also linearly correlated with the logarithm of the concentration of Staphylococcus aureus, and the linear equations are y=395.63+68.06lgx (standard curve before catalysis, R 2 =0.953) and y=3.94+107.341 gx (standard curve after catalysis, R 2=0.993). Based on the visual inspection results, the detection limits of HEAs-LFIA and DAB-enhanced HEAs-LFIA were 1.5×10 3 CFU / mL and 15 CFU / mL, indicating that the sensitivity of HEAs-LFIA and DAB-enhanced HEAs-LFIA was significantly higher than that of Au NPs-based LFIA, and the detection method showed the ability to perform on-site detection without additional instruments.

[0093] 3.6 Specificity analysis of HEAs-based LFIA

[0094] The specificity of HEAs-LFIA was evaluated by testing several potential interfering organisms, such as Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecium. Figure 6 (As shown in the figure: a is a blank; b is Staphylococcus aureus ATCC29213; c is Staphylococcus epidermidis; d is Pseudomonas aeruginosa; d is Escherichia coli; f is Enterococcus faecium). The results show that Staphylococcus aureus produces a signal response in both HEAs-LFIA and DAB-enhanced HEAs-LFIA, while other bacteria are barely detectable, demonstrating the excellent specificity of HEAs-based LFIA.

[0095] 3.7 Application in real samples

[0096] In order to evaluate the practical applicability of the HEAs-based LFIA test strip proposed in this study, it was used for rapid bacterial detection in milk and orange juice samples. Since Staphylococcus aureus was not detected in the selected milk and orange juice original samples, in order to verify the effectiveness of the detection method, standard addition experiments were carried out on milk and orange juice in parallel for 5 times. The results are shown in Figure 2. Figure 7 As shown in the figure (Figure: A shows the detection results of Staphylococcus aureus in milk samples; B shows the detection results of Staphylococcus aureus in orange juice samples; C shows the detection results of different concentrations of cystatin C). Known concentrations of S. aureus were added to the above liquid samples and tested on test strips. The recovery concentration was calculated using the standard curve. The results showed that the recovery rates were 102.2% and 118.0%, respectively, and the relative standard deviations were 8.1% and 7.3%, respectively. This demonstrates that the HEAs-based LFIA proposed in this study has a low coefficient of variation and is resistant to matrix interference. The HEAs-LFIA test strips can quickly and accurately detect S. aureus in food samples and have great potential in the field of pathogen screening.

[0097] To further explore the practicality of HEAs-LFIA, the research team expanded the HEAs tag to lateral flow chromatography detection of cystatin C (results as shown in Figure 7C), the cystatin C antibody-functionalized HEAs tag was applied to the detection of cystatin C at concentrations of 0, 0.5, 1.0, 1.8, and 2.0 mg / L, respectively. The results showed that after catalysis by the HEAs nanozyme, the detection signal was significantly enhanced, and the color development became stronger with increasing detection concentration. This result suggests that the HEAs nanozyme prepared in this study has the potential to be used for the detection of early renal tubular damage and other disease markers by functionalizing different recognition units.

[0098] 4. Conclusion

[0099] In summary, we successfully synthesized ultrasmall high-entropy alloys (HEAs) via a low-temperature reduction-diffusion strategy and integrated them into lateral flow immunochromatography for the detection and specific discrimination of Staphylococcus aureus. The results showed that the HEAs provided in this study possessed excellent catalytic performance and ultrahigh peroxidase mimetic activity, exceeding those of AuNPs, Au-PtNPs, Au-Pt-IrNPs, and Au-Pt-Ir-RuNPs by 15 times, 4 times, 2 times, and 2 times, respectively, indicating their potential as effective signal amplification nanolabels. The detection limits of HEAs-LFIA and DAB-enhanced HEAs-LFIA for Staphylococcus aureus were 1.5×10 3 The sensitivity of the HEAs-based LFIA was significantly lower than that of the Au NPs-based LFIA, indicating that the HEAs-based LFIA provided in this study has high sensitivity. In addition, the research team functionalized the high-entropy alloy with a cystatin C tag and extended its application to the detection of cystatin C, which is expected to be used for the early diagnosis of renal injury. This shows that HEAs-based lateral flow immunochromatography has broad application prospects in the on-site detection of pathogens and disease markers.

[0100] Although the present invention has been described in detail above using general descriptions, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing an ultra-small high entropy alloy for lateral flow immunochromatography, characterized in that: The steps include: S1 Preparation of precursor solution: A precursor solution was prepared by adding 1% chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 40 mL of water, respectively; S2 prepares high entropy alloys through low-temperature reduction-diffusion strategy: The precursor solution prepared in step S1 was placed in an ice-water bath and ultrasonically treated for 2 min. Then, 2 mL of sodium borohydride was added and mechanically stirred at 7000 rpm / min for 5 min to form a high-entropy alloy through a combined process of reduction and atomic diffusion. The alloy was then stored at 4°C.

2. Use of the ultrasmall high entropy alloy prepared by the preparation method as claimed in claim 1 in the preparation of nanotags for the detection of pathogens or disease markers and their lateral flow immunochromatographic test paper.

3. The use according to claim 2, characterized in that The pathogen is Staphylococcus aureus.

4. The use according to claim 2, characterized in that The disease marker is the kidney injury marker cystatin C.

5. The use according to claim 3, characterized in that: The preparation method of the nanotag for Staphylococcus aureus detection is as follows: Add bovine serum albumin to the prepared high entropy alloy solution and shake it at room temperature; Subsequently, K2CO3 and anti-Staphylococcus aureus antibody were added and shaken continuously for 60 min to obtain a mixture; finally, the mixture was centrifuged and resuspended in a PB solution containing sucrose, bovine serum albumin, and Tween-20 to obtain; The preparation method of the lateral flow immunochromatographic test paper for Staphylococcus aureus detection is as follows: The sample pad was immersed in Tris-HCl buffer containing NaCl and Tween-20 and then dried overnight; anti-Staphylococcus aureus IgG and goat anti-rabbit IgG were respectively distributed onto the NC membrane to generate the test line T line and the control line C line; the absorption pad and sample pad were assembled onto the PVC backing in sequence; the assembled strips were cut to complete the construction of the lateral flow immunochromatographic test paper.

6. The use according to claim 5, characterized in that: The preparation method of the nanotag for Staphylococcus aureus detection is as follows: 40 μL of 1% bovine serum albumin was added to 1 mL of the prepared high-entropy alloy solution and shaken at room temperature for 30 min. Subsequently, 20 μL of 0.1 mol / L K2CO3 and 5 μL of 1 mg / mL anti-Staphylococcus aureus antibody were added and shaken continuously for 60 min to obtain a mixture. Finally, the mixture was centrifuged at 8000 rpm / min for 15 min and resuspended in 10 mM PB solution containing 2% sucrose, 1% bovine serum albumin, and 0.05% Tween-20 at a pH of 7.35 to obtain the product. The preparation method of the lateral flow immunochromatographic test paper for Staphylococcus aureus detection is as follows: The sample pad was immersed in 0.05 M Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl and 0.25% Tween-20 and then dried overnight at 37°C. Anti-Staphylococcus aureus IgG at a concentration of 2.5 mg / mL and goat anti-rabbit IgG at a concentration of 1 mg / mL were dispensed onto the NC membrane to generate the test line T and the control line C, respectively. The absorption pad and sample pad were sequentially assembled onto a PVC backing plate, with a 2 mm gap between them and the NC membrane. The assembled strip was cut into 2.5 mm widths to complete the construction of the lateral flow immunochromatographic test strip.

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