Ultra-small high-entropy alloy for lateral flow immunochromatography as well as preparation method and application of ultra-small high-entropy alloy

Abstract

The invention relates to the technical field of detection of pathogenic bacteria and disease markers, in particular to an ultra-small high-entropy alloy for lateral flow immunochromatography and a preparation method and application of the ultra-small high-entropy alloy. The preparation method of the high-entropy alloy comprises the following steps: preparing a precursor solution; and the high-entropy alloy is prepared through a low-temperature reduction-diffusion strategy. The high-entropy alloy prepared through the method has excellent catalytic performance and ultrahigh peroxidase simulation activity, staphylococcus aureus can be rapidly and accurately detected when the high-entropy alloy is applied to lateral flow immunochromatography, and the high-entropy alloy has the advantages of being high in sensitivity and specificity. Meanwhile, the application of the high-entropy alloy is successfully popularized to the detection of the renal injury marker cystatin C by modifying a recognition element. The high-entropy alloy for lateral flow immunochromatography provided by the invention has a huge market popularization prospect in the field of detection of pathogenic bacteria and disease markers.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen and disease biomarker detection, and particularly relates to an ultra-small 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 widely present in natural environments such as air, water and soil. After contaminating food, it will produce enterotoxin under appropriate conditions. Consumption of the contaminated food can cause food poisoning, and in severe cases, diseases such as pericarditis, pneumonia, suppurative infection, toxic shock syndrome, acute kidney injury, glomerulonephritis and septicemia, posing a serious threat to human health. There is an urgent need for highly sensitive and specific analytical methods to achieve rapid and accurate detection of this foodborne pathogen, S. aureus.

[0003] Lateral flow immunochromatography assay (LFIA) is a classic point-of-care testing (POCT) technology based on antigen-antibody immune reactions. Due to its advantages of simple operation, rapidity, low cost and direct visual reading of results, it provides a means for rapid, sensitive and effective detection of pathogens and is widely used in fields such as clinical diagnosis, food safety, drug detection and environmental monitoring. Traditional lateral flow immunochromatography test strips mainly rely on colloidal gold nanoparticles (Au NPs) as colorimetric signal reporters, which allow device-free detection and have been successfully commercialized. However, the lateral flow immunochromatography system based on Au NPs is usually limited by poor sensitivity and low accuracy, making it insufficient to meet the strict requirements for screening foodborne pathogens.

[0004] High-entropy alloys (HEAs) are composed of five or more component elements with near-equiatomic ratios. Due to their characteristic effects such as high-entropy effect, lattice distortion effect, sluggish diffusion effect, and "cocktail effect", they have received extensive attention in multiple scientific research fields in recent years. These properties of HEAs endow them with excellent radiation resistance, stability, high catalytic activity, and effective photothermal conversion properties, making them considered strong candidates for applications in fields such as energy systems and the environment. Compared with single binary catalysts or medium-entropy alloys, multi-component HEAs can provide different active sites, making them a new type of catalytic material in multiple research fields. Currently, most research on HEAs focuses on their use as metal structural materials, while there is relatively little research on lateral flow immunoassay based on HEAs for detecting foodborne pathogens and other biomarkers. In addition, HEAs are usually synthesized by mechanical alloying or rapid moving bed pyrolysis, involving high-temperature conditions and long processing times, which to some extent limits their practicality.

[0005] Based on this, this application synthesizes an ultra-small Au-Pt-Ir-Ru-Rh HEA by adopting a low-temperature reduction-diffusion strategy and applies it to lateral flow immunoassay. By creating a nano-tag with high catalytic activity and preparing the corresponding test strip, rapid, efficient, and accurate detection of Staphylococcus aureus can be achieved. At the same time, by changing the recognition element, it is expected to expand the application of this high-entropy alloy to the detection of disease markers. Summary of the Invention

[0006] The purpose of the present invention is to provide an ultra-small high-entropy alloy for lateral flow immunoassay and its preparation method.

[0007] Another purpose of the present invention is to provide the application of the above ultra-small high-entropy alloy in the preparation of nano-tags for detecting pathogenic bacteria or disease markers and their lateral flow immunoassay test strips.

[0008] To achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0009] The preparation method of the ultra-small high-entropy alloy for lateral flow immunoassay described in the present invention includes the following steps:

[0010] S1 Prepare the precursor solution:

[0011] Add chloroauric acid, sodium hexachloroplatinate, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to water to prepare the precursor solution;

[0012] S2 Prepare the high-entropy alloy by the low-temperature reduction-diffusion strategy:

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

[0014] Preferably, in the method for preparing the ultra-small high-entropy alloy for lateral flow immunoassay of the present invention, step S1 of preparing the precursor solution is specifically: adding 0.5%-1.5% of chloroauric acid, sodium hexachloroplatinate, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 30-50 mL of water respectively to obtain the precursor solution.

[0015] More preferably, in the method for preparing the ultra-small high-entropy alloy for lateral flow immunoassay of the present invention, step S1 of preparing the precursor solution is specifically: adding 1% of chloroauric acid, sodium hexachloroplatinate, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 40 mL of water respectively to obtain the precursor solution.

[0016] Preferably, in the method for preparing the ultra-small high-entropy alloy for lateral flow immunoassay of the present invention, step S2 of preparing the high-entropy alloy by the low-temperature reduction-diffusion strategy is specifically: placing the precursor solution prepared in step S1 in an ice-water bath and ultrasonically treating it for 2-4 min, then adding 2 mL of sodium borohydride, mechanically stirring at 6000-8000 rpm / min for 4-6 min, forming a high-entropy alloy through a combined process of reduction and atomic diffusion, and storing it under the condition of 2-8 °C.

[0017] More preferably, in the method for preparing the ultra-small high-entropy alloy for lateral flow immunoassay of the present invention, step S2 of preparing the high-entropy alloy by the low-temperature reduction-diffusion strategy is specifically: placing the precursor solution prepared in step S1 in an ice-water bath and ultrasonically treating it for 2 min, then adding 2 mL of sodium borohydride, mechanically stirring at 7000 rpm / min for 5 min, forming a high-entropy alloy through a combined process of reduction and atomic diffusion, and storing it under the condition of 4 °C.

[0018] Application of the ultra-small high-entropy alloy prepared by the above preparation method in the preparation of nano-tags for detecting pathogenic bacteria or disease markers and their lateral flow immunoassay test strips.

[0019] Preferably, the pathogenic bacteria in the present invention are Staphylococcus aureus.

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

[0021] Preferably, in the application of the ultra-small high-entropy alloy of the invention in the preparation of nano-tags for detecting Staphylococcus aureus and their lateral flow immunoassay test strips:

[0022] (1) The method for preparing the nano-tag for detecting Staphylococcus aureus is:

[0023] Add bovine serum albumin to the prepared high-entropy alloy solution and shake at room temperature; subsequently, add K2CO3 and anti-Staphylococcus aureus antibody and shake continuously to obtain a mixture; finally, centrifuge the mixture and resuspend it in a PB solution containing sucrose, bovine serum albumin, and Tween-20 to obtain the product.

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

[0025] Immerse the sample pad in a Tris-HCl buffer solution containing NaCl and Tween-20, and then dry it overnight; distribute anti-Staphylococcus aureus IgG and goat anti-rabbit IgG onto the NC membrane respectively to generate a test line T line and a control line C line; assemble the absorbent pad and the sample pad onto the PVC backplane in sequence; cut the assembled strip to complete the construction of the lateral flow immunochromatographic test strip.

[0026] Further preferably, in the application of the ultra-small high-entropy alloy of the invention in the preparation of nano-tags and their lateral flow immunochromatographic test strips for detecting Staphylococcus aureus:

[0027] (1) The preparation method of the nano-tag for detecting Staphylococcus aureus is as follows:

[0028] Add 40 μL of 1% bovine serum albumin to 1 mL of the prepared high-entropy alloy solution and shake at room temperature for 30 min; subsequently, add 20 μL of 0.1 mol / L K2CO3 and 5 μL of 1 mg / mL anti-Staphylococcus aureus antibody and shake continuously for 60 min to obtain a mixture; finally, centrifuge the mixture at 8000 rpm / min for 15 min and resuspend it in a 10 mM, pH 7.35 PB solution containing 2% sucrose, 1% bovine serum albumin, and 0.05% Tween-20 to obtain the product.

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

[0030] Immerse the sample pad in a 0.05 M, pH 8.0 Tris-HCl buffer solution containing 0.15 M NaCl and 0.25% Tween-20, and then dry it overnight at 37°C; distribute 2.5 mg / mL anti-Staphylococcus aureus IgG and 1 mg / mL goat anti-rabbit IgG onto the NC membrane respectively to generate a test line T line and a control line C line; assemble the absorbent pad and the sample pad onto the PVC backplane in sequence, with a 2 mm gap from the NC membrane; cut the assembled strip into a width of 2.5 mm to complete the construction of the lateral flow immunochromatographic test strip.

[0031] Advantages of the present invention:

[0032] 1. By means of a low-temperature reduction-diffusion strategy, the present invention successfully synthesizes a super-small high-entropy alloy Au-Pt-Ir-Ru-Rh HEAs, which has excellent catalytic performance and ultra-high peroxidase-mimicking activity, exceeding Au NPs, Au-Pt NPs, Au-Pt-Ir NPs, and Au-Pt-Ir-Ru NPs by 15 times, 4 times, 2 times, and 2 times respectively.

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

[0034] 3. Through specific experiments, it is found that Staphylococcus aureus produces signal responses in both HEAs-LFIA and DAB-enhanced HEAs-LFIA, while several other potential interfering microorganisms such as Staphylococcus epidermidis and Escherichia coli can hardly be detected, indicating that the lateral flow immunoassay based on the high-entropy alloy provided by the present invention has strong specificity for the detection of Staphylococcus aureus.

[0035] 4. The LFIA test strip based on HEAs provided by the present invention can quickly and accurately detect Staphylococcus aureus in food samples, and has a low coefficient of variation and an anti-matrix interference effect, showing great application value in the field of screening for pathogenic bacteria in food.

[0036] 5. By modifying the recognition element, the present invention successfully extends the application of the prepared super-small high-entropy alloy to the detection of cystatin C, a kidney injury biomarker, indicating that the super-small high-entropy alloy provided in this application has potential application prospects in the field of disease biomarker detection. Description of the Drawings

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

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

[0039] Figure 3 To compare the catalytic activities of each nanoparticle using TMB and DAB as substrates respectively (in the figure: A is the comparison of catalytic activities using TMB as the substrate; B is the comparison of catalytic activities using DAB as the substrate);

[0040] Figure 4 Feasibility results of LFIA for detecting Staphylococcus aureus based on HEAs-pAb (in the figure: A is the catalytic signal and visual observation results using DAB as the substrate; B is the catalytic signal and visual observation results using TMB as the substrate);

[0041] Figure 5 Sensitivity evaluation results of LFIA based on Au NPs and HEAs (in the figure: A is the test results of Au NPs-LFIA under different concentrations of Staphylococcus aureus; B is the test results of HEAs-LFIA under different concentrations of Staphylococcus aureus; C is the test results of HEAs-LFIA using DAB as the 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 DAB-enhanced LFIA based on HEAs);

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

[0043] Figure 7 Detection results of different samples by LFIA based on HEAs (in the figure: A is the detection results of Staphylococcus aureus in milk samples; B is the detection results of Staphylococcus aureus in orange juice samples; C is the detection results of different concentrations of cystatin C). Specific embodiments

[0044] The technical solutions of the present invention will be described in detail below in combination with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation to the technical solutions of the present invention.

[0045] Example 1

[0046] The ultra-small high-entropy alloy for lateral flow immunoassay is prepared by the following steps:

[0047] S1 Prepare the precursor solution:

[0048] Add 1% chloroauric acid, sodium hexachloroplatinate, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 40 mL of water respectively to prepare the precursor solution;

[0049] S2 Prepare the high-entropy alloy by a low-temperature reduction-diffusion strategy:

[0050] Place the precursor solution prepared in the above step in an ice-water bath and ultrasonically treat it for 2 min, then add 2 mL of sodium borohydride, mechanically stir it at 7000 rpm / min for 5 min, form a high-entropy alloy through a combined process of reduction and atomic diffusion, and store it at 4°C.

[0051] Example 2

[0052] Use the ultra-small high-entropy alloy prepared in Example 1 to prepare a nanolabel for Staphylococcus aureus detection and its lateral flow immunoassay strip:

[0053] Preparation method of the nanolabel for Staphylococcus aureus detection:

[0054] Add 40 μL of 1% bovine serum albumin to 1 mL of the prepared high-entropy alloy solution, shake it at room temperature for 30 min; subsequently, add 20 μL of 0.1 mol / L K2CO3 and 5 μL of 1 mg / mL anti-Staphylococcus aureus antibody and continuously shake it for 60 min to obtain a mixture; finally, centrifuge the mixture at 8000 rpm / min for 15 min and resuspend it in a PB solution with a concentration of 10 mM, pH value of 7.35, containing 2% sucrose, 1% bovine serum albumin, and 0.05% Tween-20, and that is it.

[0055] Preparation method of the lateral flow immunoassay strip for Staphylococcus aureus detection:

[0056] Immerse the sample pad in a 0.05 M, pH 8.0 Tris-HCl buffer solution containing 0.15 M NaCl and 0.25% Tween-20, and then dry it overnight at 37°C; dispense 2.5 mg / mL anti-Staphylococcus aureus IgG and 1 mg / mL goat anti-rabbit IgG onto the NC membrane to generate a test line T line and a control line C line respectively; assemble the absorbent pad and the sample pad onto the PVC backplane in sequence, with a 2 mm gap from the NC membrane; cut the assembled strip into a width of 2.5 mm to complete the construction of the lateral flow immunoassay strip.

[0057] Example 3

[0058] Through the functionalization of cystatin C tags, the ultra-small high-entropy alloy prepared in Example 1 was used to prepare nano-tags for cystatin C detection and its lateral flow immunoassay test strips.

[0059] To further verify the reliability of the present invention and screen out the best scheme, the inventors conducted a series of tests, which are as follows:

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

[0061] The research team prepared gold nanoparticles (Au NPs) by using the citrate reduction method, and prepared high-entropy alloys (Au-Pt-Ir-Ru-Rh HEAs, hereinafter referred to as HEAs), gold-platinum nanoparticles (Au-Pt NPs), gold-platinum-iridium nanoparticles (Au-Pt-Ir NPs), and gold-platinum-iridium-ruthenium nanoparticles (Au-Pt-Ir-Ru NPs) by using the low-temperature reduction-diffusion method. The prepared gold nanoparticles were tested by using a scanning electron microscope (SEM), and the structure and properties of the high-entropy alloy were studied by 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 by using the sodium citrate reduction method. First, 16 mL of 0.25% HAuCl4 stock solution was added to a flask, and then DNAse / RNAse-free water was added to make the total volume reach 200 mL, and the solution was heated to boiling under mechanical stirring. Subsequently, 12 mL of 1% (weight / volume) sodium citrate aqueous solution was quickly added, and it was continuously boiled for 15 min until the solution color changed from transparent to dark purple-red, then the heating was stopped and stirring was continued for 15 min. Finally, DNAse / RNAse-free water was added to make up the volume of the colloidal solution to 200 mL, and it was stored at 4 °C for later use.

[0064] 1.2 Preparation of high-entropy alloys

[0065] High-entropy alloys were prepared by using the low-temperature reduction-diffusion strategy. A precursor solution was prepared by adding 1% chloroauric acid, sodium hexachloroplatinate, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 40 mL of water. The obtained mixture was ultrasonicated in an ice-water bath for 2 min, then 2 mL of sodium borohydride (NaBH4) was added, and it was mechanically stirred at 7000 rpm / min for 5 min. HEAs were formed through a combined process of reduction and atomic diffusion, and it was stored at 4 °C for later use.

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

[0067] 2. Experimental Methods

[0068] 2.1 Catalytic Activity Test

[0069] In this study, control experiments were conducted to clarify the catalytic activity of high-entropy alloys. TMB (3,3’,5,5’-tetramethylbenzidine) and DAB (diaminobenzidine) were added to various nanoparticle solutions and high-entropy alloy solutions, including Au NPs, Au-Pt NPs, Au-Pt-Ir NPs, Au-Pt-Ir-Ru NPs, and HEAs. The absorbance of the above solutions in the wavelength range of 300 - 800 nm was detected using an ultraviolet-visible spectrophotometer, and the color changes of TMB and DAB were monitored to judge the catalytic activity of each group of solutions. 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 min. Then, the resulting oxidized TMB or DAB precipitate was used for color analysis.

[0070] 2.2 Modification of Au NPs and High-Entropy Alloys

[0071] Au NPs were thoroughly mixed with 20 μL of a 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) with a final concentration of 0.1% (w / v) was added as a blocking agent, and the mixture was shaken at room temperature for 30 min. Then, the mixture was centrifuged at 10000 rpm / min for 12 min, and the red precipitate was collected. The precipitate of anti-Staphylococcus aureus IgG-modified Au NPs was resuspended in a PB solution (10 mM, pH 7.35) containing 2% sucrose, 1% BSA, and 0.05% Tween-20 to obtain the Au NPs-pAb probe for standby.

[0072] Staphylococcus aureus - specific IgG (rabbit IgG) was conjugated to the HEAs solution through electrostatic adsorption. The detailed procedure was 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 min. Subsequently, 20 μL of 0.1 mol / L K2CO3 and 5 μL of Staphylococcus aureus - specific IgG (1 mg / mL) were added to the solution and shaken continuously for 60 min. Then the mixture was centrifuged at 8000 rpm / min for 15 min and resuspended in a PB solution with a concentration of 10 mM, pH 7.35, containing 2% sucrose, 1% BSA, and 0.05% Tween - 20 to obtain the HEAs - pAb probe for standby.

[0073] 2.3 Fabrication of lateral - flow immunochromatographic test strips

[0074] The sample pad was immersed in a 0.05 M Tris - HCl (pH 8.0) buffer solution containing 0.15 M NaCl and 0.25% Tween - 20 (v / v), and then dried overnight at 37 °C. Staphylococcus aureus - specific IgG (2.5 mg / mL) and goat anti - rabbit IgG (1 mg / mL) were respectively dispensed onto the NC membrane to generate the test line (T test line) and the control line (C line). The absorbent pad and the sample pad were sequentially assembled onto the PVC backboard with a gap of approximately 2 mm from the NC membrane. The assembled strip was 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 alloys

[0076] 5 μL of the HEAs - pAb probe was added to the sample solution containing the target bacteria and reacted in a phosphate - buffered saline (PB) solution for 5 min. Then the reaction solution was introduced onto the sample pad of the lateral - flow immunochromatography. After running for 10 min under capillary force, the colorimetric results were recorded. After adding the DAB mixed solution, the catalytic signal on the T test line was amplified and the image was captured by a smartphone. Finally, the gray - scale 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 other 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] To explore the applicability of the established HEAs-based LFIA sensor in the analysis of real samples, Staphylococcus aureus was introduced into milk and orange juice samples. 9×10 8 CFU of Staphylococcus aureus was added to each 1 mL of milk or orange juice sample and mixed well. Then, a buffer solution containing 5 μL of HEAs-pAb probe was added to 50 μL of the milk or orange juice mixture solution. After reacting for 5 min, it was transferred to the sample pad of the lateral flow immunoassay immunosensor and the signal response at the T test line was recorded.

[0081] 3. Results and Discussion

[0082] 3.1 Principle of lateral flow immunoassay for Staphylococcus aureus detection mediated by high-entropy alloy-pAb

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

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

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

[0086] The research team investigated the stability of HEAs prepared at temperatures of 0 °C, 4 °C, and 25 °C, respectively (as shown in Figure 2 Figure G). The results showed that when the preparation temperature was 0 °C, no precipitation was observed in the solution, while precipitation occurred at 4 °C and 25 °C, indicating that the HEAs prepared at 0 °C had better stability. Subsequently, the research team compared the catalytic activities of the nanoparticles prepared under the above temperature conditions (as shown in Figure 2 Figure H). The results showed that the nanoparticles prepared at 0 °C exhibited 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 the HEAs provided in this study.

[0087] 3.3 Catalytic activity of high-entropy alloys

[0088] In this study, control experiments were conducted to clarify 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 as shown in Figure 3 (In the figure: A is the comparison of catalytic activities with TMB as the substrate; B is the comparison of catalytic activities with DAB as the substrate). The results showed that HEAs (tube e) exhibited excellent peroxidase-mimicking activity, and its catalytic performance exceeded 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 adding the DAB system, HEAs (tube e) produced obvious dark brown precipitates and no observable absorption peaks, further confirming the superior catalytic activity of HEAs compared with other nanoparticles.

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

[0090] To evaluate the feasibility of the lateral flow test strip based on the HEAs-pAb probe, the research team used standard strains and clinically isolated strains (a is blank; b is Staphylococcus aureus ATCC 29213; c is Staphylococcus aureus ATCC25923; d is clinically isolated strain -1; e is clinically isolated strain -2) for comparative analysis. The results are as shown in Figure 4 (In the figure, A is the catalytic signal and visual observation results with DAB as the substrate; B is the catalytic signal and visual observation results with TMB as the substrate).

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

[0092] By detecting the concentration of standard Staphylococcus aureus suspension in PB solution at 1.5×10 5 to 15×10 8 CFU / mL and 1.5×10 1 to 9×10 8 CFU / mL, and using PB solution as the negative control, the sensitivity of LFIA based on Au NPs and HEAs was determined. The results are as 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 the 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 DAB-enhanced LFIA based on HEAs) shown. For the Au NPs-based LFIA, no color was observed on the T test line of the negative sample, and the signal intensity was close to zero; the color intensity of the T test line of the positive sample gradually increased with the increase of the Staphylococcus aureus concentration (as Figure 5 shown in A); the intensity of the red band on the T test line was proportional to the logarithm of the Staphylococcus aureus concentration, and the linear equation was y = -788.36 + 176.37lgx (R 2 = 0.977) (as Figure 5 shown in D); based on visual evaluation, the detection limit of Au NPs-LFIA was 1.5×10 6 CFU / mL. For the HEAs-based LFIA, with the increase of the Staphylococcus aureus concentration, the detection signal of HEAs-LFIA gradually increased (as Figure 5 shown in B), and the signal intensity was also linearly correlated with the logarithm of the Staphylococcus aureus concentration. The linear equations were y = 120.48 + 40.95lgx (standard curve before catalysis, R 2 = 0.988) and y = -816.5 + 159.8lgx (standard curve after catalysis, R 2 = 0.977) (as Figure 5 shown in E). In addition, the results of HEAs-LFIA with DAB as the substrate are as Figure 5 shown in C, and its detection signal intensity was also linearly correlated with the logarithm of the Staphylococcus aureus concentration. The linear equations were y = 395.63 + 68.06lgx (standard curve before catalysis, R 2 = 0.953) and y = 3.94 + 107.34lgx (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, respectively, indicating that the sensitivities of HEAs-LFIA and DAB-enhanced HEAs-LFIA were significantly higher than those of Au NPs-based LFIA, and this detection method demonstrated 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 faecalis. The results are as Figure 6 (In the figure: a is blank; b is Staphylococcus aureus ATCC29213; c is Staphylococcus epidermidis; d is Pseudomonas aeruginosa; d is Escherichia coli; f is Enterococcus faecalis). From the results, it can be seen that Staphylococcus aureus produced signal responses in both HEAs-LFIA and DAB-enhanced HEAs-LFIA, while other bacteria were hardly detectable, proving that the HEAs-based LFIA has excellent specificity.

[0095] 3.7 Application in actual samples

[0096] 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 original milk and orange juice samples, to verify the effectiveness of the detection method, standard addition experiments were conducted on milk and orange juice, with 5 parallels each. The results are as Figure 7 shown (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). Known concentrations of Staphylococcus aureus were added to the above liquid samples and tested on the test strip, and the recovery concentrations were 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 indicates that the HEAs-based LFIA proposed in this study has a low coefficient of variation and anti-matrix interference effect. The HEAs-LFIA test strip can quickly and accurately detect Staphylococcus aureus in food samples and has great potential in the field of pathogen screening.

[0097] To further explore the practicality of HEAs-LFIA, the research team extended the HEAs label for lateral flow chromatography detection of cystatin C (the results are as Figure 7As shown in C, the cystatin C antibody-functionalized HEAs tags were respectively applied to the detection of cystatin C at concentrations of 0, 0.5, 1.0, 1.8, and 2.0 mg / L. The results showed that after the catalysis of HEAs nanozymes, the detection signals were significantly enhanced, and the color development became stronger with the increase in the detection concentration. This result indicates that the HEAs nanozymes prepared in this study are expected to be used for the detection of early renal tubular injury and other disease markers by functionalizing different recognition units.

[0098] 4. Conclusions

[0099] In summary, we successfully synthesized ultra-small high-entropy alloys through a low-temperature reduction-diffusion strategy and integrated them into lateral flow immunoassay for the detection and specific discrimination of Staphylococcus aureus. The results showed that the HEAs provided in this study have excellent catalytic performance and ultra-high peroxidase-mimicking activity, exceeding 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 CFU / mL and 15 CFU / mL, respectively, which were significantly lower than those of LFIA based on Au NPs, indicating that the HEAs-based LFIA provided in this study has high sensitivity. In addition, the research team functionalized the high-entropy alloy with cystatin C tags and extended its application to the detection of cystatin C, which is expected to be used for the early diagnosis of kidney injury. This indicates that HEAs-based lateral flow immunoassay has broad application prospects in the on-site detection of pathogenic bacteria and disease markers.

[0100] Although the present invention has been described in detail with general descriptions, specific embodiments, and experiments above, some modifications or improvements can be made on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

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: Adding chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride into water to prepare a precursor solution; S2 Preparation of high entropy alloys by low temperature reduction-diffusion strategy: 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.

2. The method for preparing the ultra-small high entropy alloy for lateral flow immunochromatography according to claim 1, characterized in that: Step S1 of preparing the precursor solution specifically comprises: adding 0.5%-1.5% of chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 30-50 mL of water, respectively, to prepare a precursor solution.

3. The method for preparing the ultra-small high entropy alloy for lateral flow immunochromatography according to claim 2, characterized in that: Step S1 of preparing the precursor solution specifically comprises: adding 1% of chloroauric acid, sodium platinum hexachloride, sodium iridium chloride, potassium rhodium chloride, and potassium ruthenium chloride to 40 mL of water, respectively, to prepare a precursor solution.

4. The method for preparing the ultra-small high entropy alloy for lateral flow immunochromatography according to claim 1, characterized in that: Step S2 prepares a 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 for ultrasonic treatment for 2-4 minutes, then 2 mL of sodium borohydride is added, and mechanical stirring is performed 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.

5. The method for preparing the ultra-small high entropy alloy for lateral flow immunochromatography according to claim 4, characterized in that: Step S2 prepares a 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 for ultrasonic treatment for 2 minutes, and then 2 mL of sodium borohydride is added, and mechanical stirring is performed 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.

6. Use of the ultrasmall high entropy alloy prepared by the preparation method according to any one of claims 1 to 5 in the preparation of nano-tags for the detection of pathogens or disease markers and lateral flow immunochromatographic test paper thereof.

7. The use according to claim 6, characterized in that: The pathogen is Staphylococcus aureus.

8. The use according to claim 6, characterized in that: The disease marker is cystatin C, a kidney injury marker.

9. The use according to claim 7, characterized in that: The preparation method of the nanotag for Staphylococcus aureus detection is as follows: Adding bovine serum albumin to the prepared high entropy alloy solution and shaking it at room temperature; Subsequently, K2CO3 and anti-Staphylococcus aureus antibody were added and shaken continuously for 60 minutes 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 on the NC membrane to generate the test line T line and the control line C line; the absorption pad and the sample pad were assembled on the PVC back plate in sequence; the assembled strips were cut to complete the construction of the lateral flow immunochromatography test paper.

10. The use according to claim 9, 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; 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 min to obtain a mixture; finally, the mixture was centrifuged at 8000 rpm / min for 15 min and resuspended in a PB solution containing 2% sucrose, 1% bovine serum albumin and 0.05% Tween-20 with a concentration of 10 mM and a pH value of 7.35 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 a 0.05 M Tris-HCl buffer containing 0.15 M NaCl and 0.25% Tween-20 at a pH of 8.0, and then dried at 37°C overnight; 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 respectively distributed on the NC membrane to generate a test line T line and a control line C line; the absorption pad and the sample pad were assembled on the PVC back plate in sequence, with a gap of 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 test paper.

Images