Fluorescence immunochromatography detection method based on monatomic nano-enzyme enhancement

Abstract

The invention is applicable to the technical field of biological detection, provides a fluorescence immunochromatography detection method based on monatomic nano-enzyme enhancement, and particularly relates to two application forms: in the first form, a detection signal enhanced fluorescence immunochromatography detection card and a sample diluent containing a monatomic nano-enzyme are involved; in the second form, the invention relates to a fluorescence immunochromatography detection card with enhanced detection signals, and a sample pad of the detection card is obtained by soaking a sample pad treatment solution containing a monatomic nano-enzyme solution. The two application forms both relate to a monatomic nano-enzyme solution, and the solution is composed of monatomic nano-enzyme, bio-enzyme composite microspheres, a protective agent, a surfactant, a thickening agent and a preservative. The monatomic nano enzyme is composed of an atomic-scale dispersed metal active center and a carrier material. When the method is used for detecting myocardial and inflammatory markers, the limit of quantitation can reach the pg / mL level, the detection linear range can span three orders of magnitude, and the clinical examination use requirements can be met. The kit can be applied to rapid quantitative detection of myocardial and inflammatory markers, has the advantages of high sensitivity, rapidness, convenience and high efficiency, and provides an auxiliary diagnosis basis for early screening of myocardial infarction, heart failure and infection related diseases.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a method for detecting myocardial and inflammatory markers based on single-atom nanozyme-enhanced fluorescence immunochromatography. Background Technology

[0002] Fluorescence immunochromatographic assay (FICA) is an upgrade of traditional colloidal gold immunochromatography. It involves immobilizing capture antibodies on a nitrocellulose membrane and preloading fluorescently labeled detection antibodies into a fluorescent binding pad. Capillary action is used to achieve chromatographic separation and enrichment of the "sample-antibody-fluorescence" sequence, offering advantages such as high specificity and quantification. It can be coupled with miniaturized fluorescence analyzers for digital readings and has been widely used in point-of-care testing (POCT) scenarios such as myocardial biomarker and inflammatory marker detection. However, current technologies suffer from low signal amplification efficiency and insufficient sensitivity. Existing technologies largely rely on enzyme-catalyzed substrate reactions to generate fluorescence signals, but the signal amplification capability of enzyme systems is limited, making it difficult to detect ultra-low concentrations of biomarkers. The insufficient sensitivity fails to meet the detection requirements of pg / mL levels for myocardial biomarkers or early inflammatory markers. When detecting complex samples such as whole blood and serum, matrix effects may lead to non-specific binding, and existing signal amplification strategies struggle to effectively distinguish between background noise and true signals, resulting in limited linearity. Furthermore, the fluorescence signal of traditional fluorescent dyes decays over time, and under certain conditions, the fluorescent label may quench, affecting the reliability of the results. Summary of the Invention

[0003] The purpose of this application is to provide a fluorescence immunochromatographic detection method based on single-atom nanozymes, which aims to solve the problems of insufficient sensitivity and limited linear range of existing fluorescence immunochromatographic methods for detecting myocardial and inflammatory markers.

[0004] The embodiments of this application are implemented as follows: a fluorescence immunochromatographic detection method based on single-atom nanozymes is provided, including two application forms: the first application form includes a fluorescence immunochromatographic detection card with enhanced detection signal and a sample diluent; the second application form includes only a fluorescence immunochromatographic detection card with enhanced detection signal.

[0005] The test card consists of a sample pad, a fluorescent conjugate pad, an NC membrane, absorbent paper, and a PVC board. The sample pad, fluorescent conjugate pad, NC membrane, and absorbent paper are sequentially stacked on the PVC board. The fluorescent conjugate pad is coated with fluorescent antibody, which is composed of fluorescent nanospheres (Eu). 3+ @PVP is conjugated with labeled antibody; the detection line T on the NC membrane is coated with the antibody; the control line C is coated with secondary antibody.

[0006] The fluorescent antibody mentioned above is composed of fluorescent nanospheres Eu. 3+ @PVP was obtained by conjugation with labeled antibodies. The conjugation method was a chemical cross-linking method, using water-soluble EDC and NHS to cross-link the fluorescent nanospheres Eu. 3+ @PVP crosslinks with labeled antibodies.

[0007] The first application form includes a sample diluent containing a single-atom nanozyme solution, a buffer solution, inorganic salts, a surfactant, and a preservative.

[0008] The second application includes a sample pad, which is obtained by soaking in a sample pad treatment solution containing a single-atom nanozyme solution, a buffer solution, inorganic salts, a surfactant, and a preservative.

[0009] The single-atom nanozyme solution comprises single-atom nanozymes, bio-enzyme composite microspheres, a protective agent, a surfactant, a thickener, and a preservative. The bio-enzyme composite microspheres are synthesized using a coaxial three-layer microfluidic technology. The inner aqueous phase of the bio-enzyme composite microspheres contains glucose oxidase and uric acid oxidase; the middle oil phase of the bio-enzyme composite microspheres contains methylthiophenecarboxylic acid trifluoroacetone (MT-TTFA); and the outer aqueous phase of the bio-enzyme composite microspheres contains CaCl2 and SDS. 2+ It can be cross-linked and cured with the inner layer of sodium alginate.

[0010] Another objective of this application is to provide a card for detecting myocardial and inflammatory markers, the card comprising a sample pad, a fluorescent conjugate pad, an NC membrane, absorbent paper, and a PVC board. The fluorescent conjugate pad is obtained by spraying the aforementioned fluorescent antibody onto the fluorescent conjugate pad. The sample pad is composed of a solution containing the aforementioned single-atom nanozyme.

[0011] The sample is dropped onto the sample pad. The fluorescent antibody in the fluorescent binding pad binds to the target antigen in the sample. The complex migrates forward to the T-line due to capillary action and binds to the coating antibody on the T-line. Glucose and uric acid in the sample are oxidized by glucose oxidase and uric acid oxidase in the sample diluent or the sample pad, respectively, to generate H2O2. Under the catalysis of single-atom nanozymes, the generated H2O2 is further decomposed into highly reactive hydroxyl radicals (·OH). These hydroxyl radicals (·OH) can oxidize Eu. 3+ The chelating agent ligand methylthiophenecarboxylic acid trifluoroacetone (MT-TTFA) relieves Eu 3+ Fluorescence quenching and enhanced energy transfer enhance the detection signal and improve detection sensitivity. Characteristic fluorescence signals at approximately 550–700 nm along the T and C lines are detected using a fluorescence immunoassay analyzer. The content of the target antigen in the sample is quantitatively analyzed based on the signal intensity. (See the principle below.)Figure 2 .

[0012] This application embodiment uses a single-atom nanozyme-enhanced fluorescence immunochromatographic detection method to detect myocardial and inflammatory markers, leveraging the catalytic effect of single-atom nanozymes to amplify Eu... 3+ The fluorescence detection signal significantly improves the sensitivity of the fluorescence immunochromatography method, enabling rapid quantitative detection of myocardial and inflammatory markers. Attached Figure Description

[0013] Figure 1 A schematic diagram of the test card is provided for this application.

[0014] Figure 2 A schematic diagram of the single-atom nanozyme enhancement principle is provided for this application.

[0015] Figure 3 A schematic diagram of the Ce-SA / Al2O3-enhanced single-atom nanozyme is provided for the embodiments of this application.

[0016] Figure 4 A process flow diagram for preparing Ce-SA / Al2O3 solution of single-atom nanozyme is provided for the embodiments of this application.

[0017] Figure 5 This is a process flow diagram of the first application form of this application embodiment.

[0018] Figure 6 This is a process flow diagram of the second application form of this application embodiment. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0020] This application provides a fluorescence immunochromatographic detection method based on single-atom nanozymes, specifically involving two application forms: the first application form involves a fluorescence immunochromatographic detection card with enhanced detection signal and a sample diluent; the second application form involves only a fluorescence immunochromatographic detection card with enhanced detection signal. The detection card consists of a sample pad, a fluorescent conjugate pad, an NC membrane, absorbent paper, and a PVC board. The fluorescent conjugate pad is coated with a fluorescent antibody, which is composed of fluorescent nanospheres (Eu). 3+@PVP is coupled with labeled antibody; the detection line T on the NC membrane is coated with the antibody; the control line C is coated with secondary antibody. In the first application, the sample diluent contains a single-atom nanozyme Ce-SA / Al2O3 solution. In the second application, the sample pad is obtained by soaking in a sample pad treatment solution containing a single-atom nanozyme Ce-SA / Al2O3 solution.

[0021] In this embodiment, the detection method is applied to the rapid quantitative detection of myocardial and inflammatory markers, using a fluorescence immunoassay analyzer to detect the content of myocardial and inflammatory markers in human samples, but is not limited to these biomarker antigens.

[0022] In this embodiment of the application, the sample to be tested is one of human serum, plasma, or whole blood.

[0023] This application provides a Ce-SA / Al2O3 single-atom nanozyme, the preparation of which includes Al2O3 defect construction, Ce(acac)3 (cerium acetylacetonate) precursor solution preparation, photochemical-assisted impregnation, purification and drying, thermal activation, and Ce-SA / Al2O3 dispersion preparation. A flow chart of the Ce-SA / Al2O3 single-atom nanozyme preparation process is shown below. Figure 4 .

[0024] Construction of the Al2O3 defect carrier: 0.088 g ascorbic acid and 0.147 g sodium citrate were pre-dissolved in 5 mL of deionized water, and then a mixed solution containing 25 mL of ethanol and 20 mL of deionized water was added (the final concentration of the mixed solvent was ethanol:water = 1:1 v / v). Finally, 1.0 g of 5-10 nm pseudoboehmite (AlOOH) was added and stirred until homogeneous. The homogeneous solution was added to a three-necked flask and stirred at 60-70 °C for 6-8 hours under a N2 atmosphere. The reaction solution was centrifuged at 8000 × g for 10 minutes, and the supernatant was discarded. The resulting precipitate was washed three times alternately with deionized water and anhydrous ethanol at 8000 × g for 10 minutes each time. The precipitate was then vacuum dried at 60 °C for 12 hours to obtain white Al2O3-Ov powder.

[0025] Preparation of the Ce(acac)3 precursor solution: Weigh 1.8g Ce(acac)3 and dissolve it in 200mL of acetylacetone / ethanol mixed solution (1:9, v / v), the final concentration of Ce(acac)3 is 0.9% (w / v), and stir for 20min in the dark.

[0026] The photochemical-assisted impregnation was carried out by adding 1.0 g of Al2O3-Ov to the above precursor solution, irradiating it with 266 nm ultraviolet light for 2 hours (power 2W, distance from the sample 5 cm), stirring at 300 rpm and introducing N2 for protection.

[0027] Purification and drying: The above reaction solution was centrifuged at 10,000×g for 8 minutes, the supernatant was discarded, and the resulting precipitate was washed three times with anhydrous ethanol at 10,000×g for 8 minutes. It was then vacuum dried at 60°C for 2 hours.

[0028] The thermal activation involved vacuum drying at 120°C for 3 hours to enhance the formation of Ce-O-Al bonds, resulting in a light-white Ce-SA / Al2O3 powder.

[0029] Preparation of the Ce-SA / Al2O3 dispersion: 10.0 mg Ce-SA / Al2O3 powder was added to 100 mL of an aqueous solution containing 0.01–0.1% Tetronic 1307 (w / v), 1–3% glycine (w / v), 1–3% arginine (w / v), 1–3% sucrose (w / v), 5–15% glycerol (w / v), 3–10% anhydrous ethanol (v / v), and 0.1%–0.5% Proclin 300 (w / v). The mixture was stirred at 500 rpm for 10 min and then sonicated at 150 W for 5 min to obtain a 0.1 mg / mL Ce-SA / Al2O3 colloidal dispersion, which was stored at 2–8 °C protected from light.

[0030] This application provides a bio-enzyme composite microsphere as described above. The bio-enzyme composite microsphere is synthesized using microfluidic technology. The bio-enzyme composite microsphere comprises an inner aqueous phase, an intermediate oil phase, and an outer aqueous phase. The inner aqueous phase of the bio-enzyme composite microsphere contains glucose oxidase and uric acid oxidase. The intermediate oil phase of the bio-enzyme composite microsphere contains methylthiophenecarboxylic acid trifluoroacetone (MT-TTFA). The outer aqueous phase of the bio-enzyme composite microsphere contains CaCl2 and SDS.

[0031] In this embodiment, the preparation of bio-enzyme composite microspheres includes the design of a fluid formulation for the bio-enzyme composite microspheres, the preparation of the bio-enzyme composite microspheres using microfluidic technology, and the hydrophilic treatment of the surface of the bio-enzyme composite microspheres. A flow chart of the preparation process of the bio-enzyme composite microspheres is shown below. Figure 4 .

[0032] The bio-enzyme composite microsphere fluid formulation design includes: an inner aqueous phase, an intermediate oil phase, and an outer aqueous phase.

[0033] The inner aqueous phase consists of an aqueous solution containing 9 KU / L glucose oxidase, 100 U / L urate oxidase, 1.5% sodium alginate, and 5% prepolymerized PEGDA575 microgel.

[0034] The intermediate layer oil phase composition is: silicone oil (DC245) containing 1 mM MT-TTFA and 5% Pluronic F127 (w / w);

[0035] The outer aqueous phase consists of an aqueous solution containing 2% CaCl2 and 0.5% SDS.

[0036] The microfluidic technology used to prepare bioenzyme composite microspheres includes: microfluidic chip design, shear flow control, and bioenzyme composite microsphere solidification.

[0037] The microfluidic chip design includes an inner phase consisting of a quartz capillary (50 μm inner diameter, 60 μm outer diameter), an oil phase channel consisting of an FEP tube (150 μm inner diameter), and an outer aqueous phase consisting of a PDMS channel (250 μm inner diameter).

[0038] The shear flow control sets the flow rate ratio of the internal water phase, oil phase, and external water phase to 1:2:10 (μL / min).

[0039] The solidification of the bio-enzyme composite microspheres manifests as Ca in the outer aqueous phase. 2+ The penetration of the oil phase initiates instantaneous cross-linking and curing of sodium alginate in the internal aqueous phase.

[0040] The surface hydrophilization treatment of the aforementioned bio-enzyme composite microspheres is specifically carried out as follows:

[0041] Collect the cross-linked and solidified bio-enzyme composite microsphere solution, centrifuge at 1000×g for 5 min at 2–8℃, and discard the supernatant. Reconstitute with 1% (w / v) carboxylated cellulose nanocrystals (CNC) in 0.01M PBS solution (pH 7.4), and mix continuously on a horizontal shaker at an amplitude of less than 5 cm for 15 min at 2–8℃. Centrifuge at 1000×g for 5 min at 2–8℃, discard the supernatant, reconstitute with pre-cooled PBS, centrifuge at 1000×g for 5 min at 2–8℃, and discard the supernatant. Reconstitute with a preservation solution containing 0.1% CNC and 0.02% CaCl2 to obtain the bio-enzyme composite microsphere solution.

[0042] The preservation solution is an aqueous solution containing 1–3% glycine (w / v), 1–3% arginine (w / v), 1–3% sucrose (w / v), 5–15% glycerol (w / v), 3–10% anhydrous ethanol (v / v), 0.01–0.1% Tetronic 1307 (w / v), and 0.1%–0.5% Proclin 300 (w / v).

[0043] This application provides a single-atom nanozyme solution, which comprises single-atom nanozyme Ce-SA / Al2O3, bio-enzyme composite microspheres, a protective agent, a surfactant, a thickener, and a preservative. It is obtained by mixing a Ce-SA / Al2O3 dispersion and a bio-enzyme composite microsphere solution at a ratio of 1:9 (v / v). A flow chart of the single-atom nanozyme Ce-SA / Al2O3 solution preparation process is shown below. Figure 4 .

[0044] The sample is dropped onto the sample pad. The fluorescent antibody in the fluorescent binding pad binds to the target antigen in the sample. The complex migrates forward to the T-line due to capillary action and binds to the coating antibody on the T-line. Glucose and uric acid in the sample are oxidized by glucose oxidase and uric acid oxidase in the sample diluent or sample pad, respectively, to produce H2O2. The single-atom nanozyme Ce-SA / Al2O3 from the sample diluent or sample pad is oxidized by the carrier effect of Al2O3 and Ce... 3+ -Ce 4+ The reversible valence state cycle exerts POD-like enzyme activity, catalyzing the decomposition of H2O2 into highly active hydroxyl radicals (·OH), which can oxidize Eu. 3+ The chelating agent ligand MT-TTFA, relieves Eu 3+ Fluorescence quenching and enhanced energy transfer enhance the detection signal and improve detection sensitivity. Characteristic fluorescence signals at approximately 550–700 nm along the T and C lines are detected using a fluorescence immunoassay analyzer. The content of the target antigen in the sample is quantitatively analyzed based on the signal intensity. The principle is as follows: Figure 3 As shown.

[0045] This application provides a first application of the above-mentioned fluorescence enhancement detection method, comprising a detection card and a sample diluent. The preparation process includes: solution preparation, detection card preparation, and sample diluent dispensing. A process flow diagram is shown below. Figure 5 .

[0046] The solution preparation specifically includes the preparation of sample diluent, fluorescent conjugate pad treatment solution, coating solution, fluorescent conjugate diluent, fluorescent conjugate activation solution, fluorescent conjugate labeling solution, and fluorescent conjugate blocking solution. The specific preparation process is as follows:

[0047] (1) Preparation of sample diluent: Taking the preparation of 1000mL sample diluent as an example, add 800mL of purified water, 3.58g Na2HPO4·12H2O, 1.56g NaH2PO4·2H2O, 9.0g NaCl, 1.0g TWEEN-20, 20mL single-atom nanozyme solution, and 1.0g Proclin 300 to a beaker. Stir until completely dissolved, adjust the pH of the solution to 7.30±0.1, add purified water to make up to 1000mL, mix well, and filter with a 0.45μm filter membrane.

[0048] (2) Preparation of fluorescent conjugate pad treatment solution: Taking the preparation of 1000mL fluorescent conjugate pad treatment solution as an example, add 800mL of purified water, 3.58g Na2HPO4·12H2O, 1.56g NaH2PO4·2H2O, 50.0g sucrose, 1.0g TWEEN-20 and 1.0g Proclin300 to a beaker, stir until completely dissolved, adjust the pH of the solution to 7.30±0.1, add purified water to make up to 1000mL, mix well and filter with a 0.45μm filter membrane.

[0049] (3) Preparation of coating solution: Taking the preparation of 1000mL coating solution as an example, add 800mL of purified water, 3.58g Na2HPO4·12H2O, 1.56g NaH2PO4·2H2O, 10.0g D-trehalose and 1.0g Proclin 300 to a beaker, stir until completely dissolved, adjust the pH of the solution to 7.30±0.1, add purified water to make up to 1000mL, mix well and filter with a 0.45μm filter membrane.

[0050] (4) Preparation of fluorescent conjugate dilution solution

[0051] Taking the preparation of 1000mL of fluorescent conjugate dilution solution as an example, add 800mL of purified water to a beaker, add 3.58-6.79g of Na2HPO4·12H2O, 0.17-1.56g of NaH2PO4·2H2O, 1.0-5.0g of BSA, 5.0-10.0g of fluorescent microspheres, 1.0-5.0g of TWEEN-20 and 1.0g of Proclin 300, stir until completely dissolved, adjust the pH of the solution to 7.10-8.1, add purified water to make up to 1000mL, mix well and filter through a 0.45μm filter membrane.

[0052] (5) Preparation of fluorescent conjugate activation solution

[0053] Taking the preparation of 1000mL of fluorescent conjugate activation solution as an example, add 800mL of purified water to a beaker, add 10.66g of MES, stir until completely dissolved, adjust the pH of the solution to 6.00±0.1, add purified water to make up to 1000mL, mix well, and filter with a 0.45μm filter membrane.

[0054] (6) Preparation of fluorescent conjugate labeling solution

[0055] Taking the preparation of 1000mL of fluorescent conjugate labeled solution as an example, add 800mL of purified water to a beaker, add 17.9g Na2HPO4·12H2O, 7.8g NaH2PO4·2H2O and 1.0g Proclin 300, stir until completely dissolved, adjust the pH of the solution to 7.30±0.1, add purified water to make up to 1000mL, mix well and filter with a 0.45μm filter membrane.

[0056] (7) Fluorescent conjugate blocking solution

[0057] Taking the preparation of 100mL of fluorescent conjugate blocking solution as an example, add 80mL of purified water, 10g of BSA and 0.1g of Proclin300 to a beaker, stir until completely dissolved, adjust the pH of the solution to 7.00±0.1, add purified water to make up to 1000mL, mix well and filter with a 0.45μm filter membrane.

[0058] The preparation of the test card includes the treatment of the fluorescent binding pad, antibody streaking on the NC membrane, preparation of the fluorescent antibody, preparation of the fluorescent binding pad, pasting the test strip plate, and assembly of the test card. The specific preparation process is as follows:

[0059] (1) Treatment of fluorescent conjugate pads:

[0060] The fluorescent conjugate pad was completely immersed in the fluorescent conjugate pad treatment solution for 10±1 min, during which the sample pad was turned over once every 5±1 min. After immersion, it was dried for 8±0.5 hours at a temperature of 37℃±2℃ and a humidity of ≤30%.

[0061] (2) Antibody streaking on NC membrane:

[0062] Dilute the coating antibody and secondary antibody to the required concentrations using coating buffer. For the detection line (T line), use a coating antibody concentration of 1.0–1.6 mg / mL; for the control line (C line), use a secondary antibody concentration of 0.15–0.8 mg / mL. After appropriately cutting the NC membrane, peel off the 2.5 cm section of backing paper from the PVC board to expose the adhesive. Attach the NC membrane to the adhesive area on the PVC board. Use a continuous streaking apparatus to streak the membrane at a flow rate of 1 μL / cm. After streaking, place the coated plate in an environment at 37±2℃ and ≤30% humidity for 24±1 hours to dry.

[0063] (3) Preparation of fluorescent antibodies

[0064] Take the preparation of 10 mL of fluorescent antibody as an example.

[0065] ① Take 10 mL of fluorescent conjugate activation solution, add 300 μL of fluorescent nanospheres, and mix by sonication at 480 W for 30 s.

[0066] ② Add 30 μL of 10 mg / mL EDC and 30 μL of 10 mg / mL NHS solution, sonicate at 480 W for 30 seconds to mix, and activate in the dark for 15 minutes.

[0067] ③ Centrifuge at 20000×g for 20 min at 2~8℃, and discard the supernatant.

[0068] ④ Add 10 mL of fluorescent conjugate labeling solution to the precipitate and sonicate until the fluorescent solution is mixed.

[0069] ⑤ Add 450 μg of labeled antibody and sonicate at 480 W for 30 seconds to mix. Incubate in the dark for 60 minutes, sonicating at 480 W for 15 seconds every 20 minutes.

[0070] ⑥ Add 2.5 mL of fluorescent conjugate blocking solution and let stand in the dark for 60 min.

[0071] ⑦ Centrifuge at 16000×g for 15 min at 2-8℃ and discard the supernatant.

[0072] ⑧ Add 10 ml of fluorescent conjugate diluent to the precipitate and sonicate until the fluorescent solution is mixed.

[0073] (4) Preparation of fluorescent binding pads:

[0074] Using a gold spraying apparatus, fluorescent antibodies were sprayed onto pretreated fluorescent binding pads. The spraying length was set to 300 mm / strip, the interval to 10, and the speed to 5 μL / cm. After spraying, the fluorescent binding pads were placed in an environment with a temperature of 37±2℃ and a humidity of ≤30% for drying for 24±1 hours.

[0075] (5) Attach the test strip sheet:

[0076] Cut the dried fluorescent bonding pad into strips according to the spraying position, with a width of 8mm; cut the absorbent paper into strips, with a width of 25mm; cut the treated sample pad into strips, with a width of 25mm.

[0077] A PVC board has four backing papers, with the NC film already glued to backing paper #2. Remove backing paper #1 from the top of the PVC board, press the NC film 1-2mm down, and attach absorbent paper. Remove backing paper #3 from the PVC board, press the NC film 1-2mm down, and attach the fluorescent binding pad. Remove backing paper #4 from the PVC board, press the fluorescent binding pad 1-2mm down, and attach the sample pad (humidity ≤30%). The assembled test strip looks like this. Figure 1 .

[0078] (6) Assembly of the test card:

[0079] Cut the test strip into pieces and insert the cut test strips into the test card housing.

[0080] The sample diluent dispensing process is as follows: the sample diluent is dispensed into centrifuge tubes.

[0081] This application provides a second application of the above-described fluorescence enhancement detection method, which includes a detection card. The preparation process includes: solution preparation and detection card preparation. A process flow diagram is shown below. Figure 6 .

[0082] The second type of solution preparation process includes sample pad treatment solution, fluorescent conjugate pad treatment solution, coating solution, fluorescent conjugate diluent, fluorescent conjugate activation solution, fluorescent conjugate labeling solution, and fluorescent conjugate blocking solution. Except for the sample pad treatment solution, the preparation process for the other solutions is the same as that in the first type. The specific preparation process for the sample pad treatment solution is as follows:

[0083] Taking the preparation of 1000mL sample pad treatment solution as an example, add 800mL of purified water, 3.58g Na2HPO4·12H2O, 1.56g NaH2PO4·2H2O, 9.0g NaCl, 1.0~5.0g TWEEN-20, 20mL single-atom nanozyme solution, and 1.0g Proclin 300 to a beaker. Stir until completely dissolved, adjust the pH of the solution to 7.30±0.1, add purified water to make up to 1000mL, mix well, and filter through a 0.45μm filter membrane.

[0084] The preparation of the second type of test card includes sample pad treatment, fluorescent binding pad treatment, antibody streaking on the NC membrane, preparation of the fluorescent binding pad, and attaching the test strip plate. Except for the sample pad treatment, the remaining processes are the same as those in the first type. The specific process for sample pad treatment is as follows:

[0085] Completely immerse the sample pad in the sample pad treatment solution for 10±1 min, turning the sample pad over once every 5±1 min during the immersion. After immersion, dry for 8±0.5 hours at a temperature of 37℃±2℃ and a humidity of ≤30%.

[0086] The following specific examples demonstrate the detection of cardiac troponin I (cTnI), N-terminal pro-brain natriuretic peptide (NT-ProBNP), procalcitonin (PCT), and interleukin-6 (IL-6) in human serum samples, but are not intended to limit the scope of this application. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0087] Furthermore, when the aforementioned fluorescence immunochromatographic detection method based on single-atom nanozymes is used to determine myocardial and inflammatory markers in human samples, the sample diluent and detection card in the first application form or the detection card in the second application form are used, along with the matching calibrator ID card, for detection on a fluorescence immunochromatographic analyzer.

[0088] Example 1: A method for detecting cardiac troponin I using fluorescence immunochromatography enhanced by single-atom nanozymes.

[0089] This method is used for the in vitro quantitative determination of cardiac troponin I (cTnI) levels in human serum or plasma samples. Clinically, it is mainly used as an adjunct to the diagnosis of myocardial infarction.

[0090] Troponin (Tn) is a group of contractile proteins found in skeletal muscle and cardiomyocytes. Cardiac troponin (cTn) is a group of proteins in the troponin complex that are involved in myocardial contractile function. It is a protein complex composed of three subunits: cardiac troponin T (cTnT), cardiac troponin I (cTnI), and cardiac troponin C (TnC). cTnI is found only on the thin filaments of cardiac contractile proteins and plays an important regulatory role in muscle contraction and expansion. In the early stages of cardiomyocyte injury, cTnI levels rise rapidly in the blood. It has high specificity and sensitivity, thus becoming the gold standard for diagnosing acute myocardial infarction (AMI).

[0091] The reference range for cTnI in general fluorescence immunochromatographic methods is <0.3 ng / mL.

[0092] 1) Using the first application method, prepare the test card and sample diluent.

[0093] ① Solution preparation: Preparation of sample diluent, fluorescent conjugate pad treatment solution, coating solution, fluorescent conjugate diluent, fluorescent conjugate activation solution, fluorescent conjugate labeling solution, and fluorescent conjugate blocking solution.

[0094] ②Preparation of test cards: treatment of fluorescent conjugate pads, antibody streaking on NC membranes, preparation of fluorescent antibodies, preparation of fluorescent conjugate pads, pasting of test strip plates, and assembly of test cards.

[0095] ③ Sample diluent aliquoting: Dispense the sample diluent into EP tubes.

[0096] 2) Validation of the limit of quantitation of cardiac troponin I reagent card

[0097] Experimental Methods: Five samples with a limit of quantitation (LOQ) concentration were tested using a single-atom nanozyme-enhanced fluorescent immunochromatographic assay card. Each sample was tested three times, with testing conducted over three days. The allowable error range for the detection value was defined as 0.06 ± 25% (ng / mL) of the LOQ. The number of samples whose results fell within this allowable error range was calculated, thus determining the percentage of all samples meeting the acceptable target standard of a LOQ of 0.06 ng / mL. This percentage was compared with the lower limit of observation in Table 1. If the percentage was greater than or equal to the corresponding result in Table 1, the validation was considered successful, and the LOQ was determined to be 0.06 ng / mL. Simultaneously, the same five samples were tested using a conventional fluorescent immunochromatographic assay card according to the same experimental method. The number of samples whose results fell within the allowable error range was also calculated, thus determining the percentage of all samples meeting the acceptable target standard of a LOQ of 0.06 ng / mL. The validation results of the LOQ of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card and the conventional fluorescent immunochromatographic assay card were compared.

[0098] Table 1. Correspondence between the total number of measurements and the lower limit of the proportion of observations in the limit of quantitation verification.

[0099]

[0100]

[0101] The experimental data and results are shown in Table 2-3.

[0102] Table 2. Analysis of Quantitative Limit Validation Data for Single-Atom Nanozyme-Enhanced Fluorescent Immunochromatographic Detection Card

[0103]

[0104] The experimental results show that the quantitation limit of cardiac troponin I detected by the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card meets the accuracy target of 95.56%, which is greater than the minimum percentage (88%) required for a sample size of 45 in the consensus. The quantitation limit is 0.06 ng / mL, which is a successful verification.

[0105] Table 3. Analysis of Quantitative Limit Validation Data for Conventional Fluorescent Immunochromatographic Cards

[0106]

[0107]

[0108] The experimental results show that the percentage of the quantitation limit of cardiac troponin I detection using the conventional fluorescent immunochromatographic card that meets the accuracy target is 86.67%. Compared with the minimum percentage (88%) required for a sample size of 45 in the consensus, this percentage is less than the minimum required percentage. The quantitation limit of 0.06 ng / mL fails the verification. Therefore, the quantitation limit of the conventional fluorescent immunochromatographic card for cardiac troponin I detection is higher than 0.06 ng / mL.

[0109] The results above indicate that the quantitation limit of the single-atom nanozyme-enhanced fluorescence immunochromatographic cardiotropin I detection strip is [not specified].

[0110] The results were lower than those obtained using a conventional fluorescent immunochromatographic cardiac troponin I detection card.

[0111] 3) Validation of the linear interval of the cardiac troponin I reagent card

[0112] Experimental Method: High-value samples close to the upper limit of the linear range were diluted to at least five concentrations, with low-value samples close to the lower limit of the linear range. Following the kit instructions, each concentration was tested three times, and the average value was calculated. A regression analysis was performed between the measured average concentration (y) and the corresponding theoretical concentration or dilution factor (x) to determine the linear regression equation and calculate the linear correlation coefficient (r). The result should be within the range of [0.1, 50] ng / mL, with a linear correlation coefficient (r) greater than 0.99. The relative bias between each measured value and the theoretical concentration value was calculated, and the relative bias should be within ±10%.

[0113] The experimental data and results are shown in Table 4.

[0114] Table 4. Analysis of Linear Interval Validation Data

[0115]

[0116] The experimental results show that, through linear interval verification of the cardiac troponin I detection card prepared by the above method, the linear correlation coefficient (r) is ≥0.99 within the range of [0.1, 50] ng / mL, and the linear relative deviation does not exceed ±10%. This meets the clinical requirements for the linearity of cardiac troponin I.

[0117] 4) Analytical specificity verification

[0118] Experimental Methods: Interference samples were prepared by adding interfering agents to low-value samples (0.3 ng / mL) and high-value samples (5 ng / mL), respectively, while control samples were prepared by adding an equal volume of interfering agent matrix. Each sample was measured six times using both a single-atom nanozyme-enhanced fluorescent immunochromatographic cardiac troponin I detection card and a conventional fluorescent immunochromatographic cardiac troponin I detection card. The relative deviation B between the mean measurements of the added samples and the control samples was calculated according to formula (1).

[0119]

[0120] The calculated result (B) should not interfere with the analyte if the interfering substance is within the pre-set clinically acceptable bias of 10% (at the corresponding concentration levels); otherwise, it will cause interference.

[0121] The experimental data and results are shown in Table 5.

[0122] Table 5. Data Analysis Table for Specificity Analysis

[0123]

[0124]

[0125] The experimental results showed that samples containing high concentrations of rheumatoid factor, immunoglobulin E, immunoglobulin A, immunoglobulin G, immunoglobulin M, β2-microglobulin, α1-microglobulin, neutrophil gelatinase-associated lipotransferase, insulin, metformin hydrochloride, aspirin, digoxin, and valproic acid interfered with the detection of cardiac troponin I using conventional fluorescent immunochromatographic assay cards. However, they did not significantly interfere with the detection of cardiac troponin I using single-atom nanozyme-enhanced fluorescent immunochromatographic assay cards. This verifies that the analytical specificity of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card for cardiac troponin I is superior to that of the conventional fluorescent immunochromatographic assay card for cardiac troponin I.

[0126] Example 2: A method for detecting myoglobin based on single-atom nanozyme-enhanced fluorescence immunochromatography

[0127] This method is used for the in vitro quantitative determination of myoglobin (MYO) levels in human serum or plasma samples. Clinically, it is mainly used as an adjunct to the diagnosis of myocardial infarction.

[0128] Myoglobin (MYO) is a small heme-containing protein mainly found in skeletal muscle, responsible for oxygen storage and distribution in cardiomyocytes. While not a heart-specific biomarker, MYO is released more rapidly than cardiac troponin I (cTnI) in myocardial infarction, allowing for detection of myocardial damage as early as 2 hours after symptom onset. Blood MYO concentration rises rapidly 1–3 hours after the onset of acute myocardial infarction (AMI), peaking at 6–7 hours, and almost all AMI patients show elevated MYO levels within 12 hours, making it a useful biomarker for early AMI diagnosis. The degree and duration of MYO elevation are closely related to the prognosis of myocardial infarction. A low peak value, with recovery to normal levels within 24 hours, indicates a good prognosis; conversely, a significant peak value lasting 72–96 hours indicates a poor prognosis and higher mortality rate. Furthermore, MYO's short half-life, with no increase 6–12 hours after chest pain onset, helps rule out AMI and is a good screening indicator.

[0129] In general fluorescence immunochromatographic methods, the reference range for MYO is ≤58 ng / mL.

[0130] 1) Using the first application method, prepare the test card and sample diluent.

[0131] ① Solution preparation: Preparation of sample diluent, fluorescent conjugate pad treatment solution, coating solution, fluorescent conjugate diluent, fluorescent conjugate activation solution, fluorescent conjugate labeling solution, and fluorescent conjugate blocking solution.

[0132] ②Preparation of test cards: treatment of fluorescent conjugate pads, antibody streaking on NC membranes, preparation of fluorescent antibodies, preparation of fluorescent conjugate pads, pasting of test strip plates, and assembly of test cards.

[0133] ③ Sample diluent aliquoting: Dispense the sample diluent into EP tubes.

[0134] 2) Validation of the limit of quantitation for myoglobin reagent cards

[0135] Experimental Methods: Five samples with a limit of quantitation (LOQ) concentration were tested using a single-atom nanozyme-enhanced fluorescent immunochromatographic assay card. Each sample was tested three times, with testing conducted over three days. The allowable error range for the detection value was set at 6 ± 25% (ng / mL) of the LOQ. The number of samples whose results fell within this allowable error range was calculated, thus determining the percentage of all samples meeting the acceptable LOQ of 6 ng / mL. This percentage was compared with the lower limit of observation in Table 1. If the percentage was greater than or equal to the corresponding result in Table 1, the validation was considered successful, and the LOQ was determined to be 6 ng / mL. Simultaneously, the same five samples were tested using a conventional fluorescent immunochromatographic assay card according to the same method. The number of samples whose results fell within the allowable error range was also calculated, thus determining the percentage of all samples meeting the acceptable LOQ of 6 ng / mL. The validation results of the LOQ of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card and the conventional fluorescent immunochromatographic assay card were compared.

[0136] The experimental data and results are shown in Tables 6-7.

[0137] Table 6. Analysis of Quantitation Limit Validation Data for Single-Atom Nanozyme-Enhanced Fluorescent Immunochromatographic Detection Card

[0138]

[0139]

[0140] The experimental results show that the percentage of the single-atom nanozyme-enhanced fluorescence immunochromatographic detection card that meets the accuracy target is 97.78%, which is greater than the minimum percentage required for a sample size of 45 (88%) in the consensus. The limit of quantitation is 6 ng / mL, which is a successful verification.

[0141] Table 7. Analysis of Quantitative Limit Validation Data for Conventional Fluorescent Immunochromatographic Cards

[0142]

[0143] The experimental results show that the percentage of myoglobin detection limits met by the conventional fluorescent immunochromatographic card was 84.44%, which is less than the minimum percentage (88%) required for a sample size of 45 in the consensus. The limit of quantitation of 6 ng / mL failed the verification. Therefore, the limit of quantitation of the conventional fluorescent immunochromatographic card for myoglobin detection is higher than 6 ng / mL.

[0144] The results above show that the limit of quantitation of the single-atom nanozyme-enhanced fluorescence immunochromatographic myoglobin detection card is lower than that of the conventional fluorescence immunochromatographic myoglobin detection card.

[0145] 3) Validation of the linear interval of the myoglobin reagent card

[0146] Experimental Method: High-value samples close to the upper limit of the linear range were diluted to at least five concentrations, with low-value samples close to the lower limit of the linear range. The procedure was followed according to the kit instructions. Each concentration was tested three times, and the average value was calculated. Regression analysis was performed on the average measured concentration (y) and the corresponding theoretical concentration or dilution factor (x) to determine the linear regression equation and calculate the linear correlation coefficient r. The result should be within the range of [10, 400] ng / mL, with a linear correlation coefficient (r) greater than 0.99. The relative bias between each measured value and the theoretical concentration value was calculated, and the relative bias should be within ±10%.

[0147] The experimental data and results are shown in Table 8.

[0148] Table 8. Analysis of Linear Interval Validation Data

[0149]

[0150] The experimental results show that, through linear interval verification of the myoglobin detection card prepared by the above method, the linear correlation coefficient (r) is ≥0.99 within the range of [10, 400] ng / mL, and the relative deviation of linearity does not exceed ±10%. This meets the clinical requirements for the linearity of myoglobin.

[0151] 4) Analytical specificity verification

[0152] Experimental Methods: Interference samples were prepared by adding interfering agents to low-value samples (60 ng / mL) and high-value samples (200 ng / mL), respectively, while control samples were prepared by adding an equal volume of interfering agent matrix. Each sample was measured six times using a single-atom nanozyme-enhanced fluorescence immunochromatographic myoglobin detection card and a conventional fluorescence immunochromatographic myoglobin detection card. The relative deviation B between the mean measurements of the added samples and the control samples was calculated according to formula (1).

[0153] The calculated result (B) should not interfere with the analyte if the interfering substance is within the pre-set clinically acceptable bias of 10% (at the corresponding concentration levels); otherwise, it will cause interference.

[0154] The experimental data and results are shown in Table 9.

[0155] Table 9. Data Analysis Table for Specificity Analysis

[0156]

[0157]

[0158]

[0159] The experimental results showed that samples containing high concentrations of rheumatoid factor, immunoglobulin E, immunoglobulin A, immunoglobulin G, immunoglobulin M, β2-microglobulin, α1-microglobulin, insulin, aspirin, nifedipine sustained-release tablets, vancomycin, and valproic acid interfered with the detection of myoglobin using conventional fluorescent immunochromatographic assay cards. However, they did not significantly interfere with the detection of myoglobin using single-atom nanozyme-enhanced fluorescent immunochromatographic assay cards. This verifies that the analytical specificity of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card for myoglobin is superior to that of the conventional fluorescent immunochromatographic assay card for myoglobin.

[0160] Example 3: A method for detecting procalcitonin based on single-atom nanozyme-enhanced fluorescence immunochromatography

[0161] This method is used for the in vitro quantitative determination of procalcitonin (PCT) levels in human serum or plasma samples. Clinically, it is mainly used as an adjunct to the diagnosis of bacterial infectious diseases.

[0162] Procalcitonin (PCT) is the precursor peptide of the hormone calcitonin (CT). Normal serum PCT levels are extremely low. After microbial infection, the CALC-I gene is induced to express PCT, promoting its secretion, which peaks after 8 hours. Currently, PCT is considered a biomarker for systemic inflammatory response syndrome, guiding treatment, reducing antibiotic use, diagnosing sepsis, and improving long-term outcomes.

[0163] World Health Organization research indicates that only 20 out of every 100 respiratory infections require antibiotic treatment. However, traditional bacterial culture methods for diagnosis often result in delays. Therefore, a biomarker specifically for bacterial infections is most helpful. Studies have found that PCT can serve as an important indicator for differentiating between bacterial and non-infectious inflammatory causes, as well as between bacterial and viral infections. Moreover, dynamic PCT levels have significant prognostic implications.

[0164] In severe infections, most pro-inflammatory cytokines (such as TNF-α, IL-1β, or IL-6) increase only transiently or intermittently. However, in sepsis, PCT levels increase several times to thousands of times, and this increase at admission is often associated with disease severity and subsequent mortality. Various studies and reviews have shown that PCT has higher diagnostic accuracy compared to other parameters for diagnosing sepsis.

[0165] In general fluorescence immunochromatographic methods, the reference range for PCT is ≤0.5 ng / ml.

[0166] 1) Using the first application method, prepare the test card and sample diluent.

[0167] ① Solution preparation: Preparation of sample diluent, fluorescent conjugate pad treatment solution, coating solution, fluorescent conjugate diluent, fluorescent conjugate activation solution, fluorescent conjugate labeling solution, and fluorescent conjugate blocking solution.

[0168] ②Preparation of test cards: treatment of fluorescent conjugate pads, antibody streaking on NC membranes, preparation of fluorescent antibodies, preparation of fluorescent conjugate pads, pasting of test strip plates, and assembly of test cards.

[0169] ③ Sample diluent aliquoting: Dispense the sample diluent into EP tubes.

[0170] The prepared procalcitonin test card, along with the matching calibrator ID card and sample diluent, was used to perform the test on a fluorescence immunoassay analyzer.

[0171] 2) Validation of the limit of quantitation for procalcitonin reagent card

[0172] Experimental Methods: Five samples with a limit of quantitation (LOQ) concentration were tested using a single-atom nanozyme-enhanced fluorescent immunochromatographic assay card. Each sample was tested three times, with testing conducted over three days. The allowable error range for the detection value was defined as 0.07 ± 25% (ng / mL) of the LOQ. The number of samples whose results fell within this allowable error range was calculated, thus determining the percentage of all samples meeting the acceptable target standard of a LOQ of 0.07 ng / mL. This percentage was compared with the lower limit of observation in Table 1. If the percentage was greater than or equal to the corresponding result in Table 1, the validation was considered successful, and the LOQ was determined to be 0.07 ng / mL. Simultaneously, the same five samples were tested using a conventional fluorescent immunochromatographic assay card according to the same experimental method. The number of samples whose results fell within the allowable error range was also calculated, thus determining the percentage of all samples meeting the acceptable target standard of a LOQ of 0.07 ng / mL. The validation results of the LOQ of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card and the conventional fluorescent immunochromatographic assay card were compared.

[0173] The experimental data and results are shown in Table 10-11.

[0174] Table 10 Analysis of Quantitation Limit Validation Data for Single-Atom Nanozyme-Enhanced Fluorescent Immunochromatographic Detection Card

[0175]

[0176] The experimental results show that the percentage of the fluorescence immunochromatographic detection card enhanced with single-atom nanozymes that meets the accuracy target is 95.56%, which is greater than the minimum percentage required for a sample size of 45 (88%) in the consensus. The limit of quantitation is 0.07 ng / mL, which is a successful verification.

[0177] Table 11 Analysis of Quantitation Limit Validation Data for Conventional Fluorescent Immunochromatographic Cards

[0178]

[0179] The experimental results show that the percentage of the quantitation limit of procalcitonin detection that meets the accuracy target using the conventional fluorescent immunochromatographic card is 86.67%. Compared with the minimum percentage (88%) required for a sample size of 45 in the consensus, this percentage is lower than the minimum required percentage. The quantitation limit of 0.07 ng / mL does not pass the verification. Therefore, the quantitation limit of the conventional fluorescent immunochromatographic procalcitonin detection card is higher than 0.07 ng / mL.

[0180] The results above indicate that the limit of quantitation of the single-atom nanozyme-enhanced fluorescent immunochromatographic procalcitonin assay card is lower than that of the conventional fluorescent immunochromatographic procalcitonin assay card.

[0181] 3) Validation of the linear range of the procalcitonin reagent card

[0182] Experimental Method: High-value samples close to the upper limit of the linear range were diluted to at least five concentrations, with low-value samples close to the lower limit of the linear range. Following the kit instructions, each concentration was tested three times, and the average value was calculated. A regression analysis was performed between the measured average concentration (y) and the corresponding theoretical concentration or dilution factor (x) to determine the linear regression equation and calculate the linear correlation coefficient r. The result should be within the range of [0.1, 100] ng / mL, with a linear correlation coefficient (r) greater than 0.99. The relative bias between each measured value and the theoretical concentration value was calculated, and the relative bias should be within ±10%.

[0183] The experimental data and results are shown in Table 12.

[0184] Table 12 Analysis of Linear Interval Validation Data

[0185]

[0186]

[0187] The experimental results show that, through linear interval verification of the procalcitonin test card prepared by the above method, the linear correlation coefficient (r) is ≥0.99 within the range of [0.1, 100] ng / mL, and the linear relative deviation does not exceed ±10%. This meets the clinical requirements for the linearity of procalcitonin.

[0188] 4) Analytical specificity verification

[0189] Experimental Methods: Interference samples were prepared by adding interfering agents to low-value samples (0.5 ng / mL) and high-value samples (10 ng / mL), respectively, while control samples were prepared by adding an equal volume of interfering agent matrix. Each sample was measured six times using a single-atom nanozyme-enhanced fluorescent immunochromatographic procalcitonin detection card and a conventional fluorescent immunochromatographic procalcitonin detection card. The relative deviation B between the mean measurements of the added samples and the control samples was calculated according to formula (1).

[0190] The calculated result (B) should not interfere with the analyte if the interfering substance is within the pre-set clinically acceptable bias of 10% (at the corresponding concentration levels); otherwise, it will cause interference.

[0191] The experimental data and results are shown in Table 13.

[0192] Table 13 Data Analysis Table for Specificity Analysis

[0193]

[0194]

[0195] The experimental results showed that samples containing high concentrations of rheumatoid factor, retinol-binding protein, β2-microglobulin, α1-microglobulin, neutrophil gelatinase-associated lipotransferase, insulin, aspirin, nifedipine sustained-release tablets, furosemide, and valproic acid interfered with the detection of procalcitonin using conventional fluorescent immunochromatographic assay cards. However, they did not significantly interfere with the detection of procalcitonin using single-atom nanozyme-enhanced fluorescent immunochromatographic assay cards. This verifies that the analytical specificity of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card for procalcitonin is superior to that of the conventional fluorescent immunochromatographic assay card for procalcitonin.

[0196] Example 4: A method for detecting interleukin-6 based on single-atom nanozyme-enhanced fluorescence immunochromatography

[0197] This method is used for the in vitro quantitative determination of interleukin-6 (IL-6) levels in human serum or plasma samples. Clinically, it is mainly used to monitor the body's immune status and inflammatory response.

[0198] Interleukin-6 (IL-6) is a single-chain glycoprotein with a relative molecular mass of approximately 21-30 kDa. IL-6 possesses a variety of biological functions, playing a crucial role in anti-infection, tumorigenesis, immune diseases, and immune regulation. Under abnormal conditions, elevated IL-6 levels may have adverse effects on the body, causing tissue damage and exacerbating disease progression. Studies have shown that IL-6 is highly expressed and transiently upregulated in pathological states such as multiple myeloma, rheumatoid arthritis, acute bacterial infections, and HIV infection. The pathological role of IL-6 in disease development has been validated in various diseases, including multiple myeloma, rheumatoid arthritis, AIDS, mesangial proliferative glomerulonephritis, sepsis, and osteoporosis.

[0199] In general fluorescence immunochromatographic methods, the reference range for IL-6 is ≤10 pg / mL.

[0200] 1) Prepare the detection card using the second application method.

[0201] ① Solution preparation: Prepare sample pad treatment solution, fluorescent conjugate pad treatment solution, coating solution, fluorescent conjugate diluent, fluorescent conjugate activation solution, fluorescent conjugate labeling solution, and fluorescent conjugate blocking solution.

[0202] ②Preparation of test cards: processing of sample pads, processing of fluorescent conjugate pads, antibody streaking on NC membranes, preparation of fluorescent conjugate pads, pasting of test strips, and assembly of test cards.

[0203] 2) Validation of the limit of quantitation for the interleukin-6 detection card

[0204] Experimental Methods: Five samples with a limit of quantitation (LOQ) concentration were tested using a single-atom nanozyme-enhanced fluorescent immunochromatographic assay card. Each sample was tested three times, with testing conducted over three days. The allowable error range for the detection value was defined as 3 ± 25% (pg / mL) of the LOQ. The number of samples whose results fell within this allowable error range was calculated, thus determining the percentage of all samples meeting the acceptable target standard of a LOQ of 3 pg / mL. This percentage was compared with the lower limit of observation in Table 1. If the percentage was greater than or equal to the corresponding result in Table 1, the validation was considered successful, and the LOQ was determined to be 3 pg / mL. Simultaneously, the same five samples were tested using a conventional fluorescent immunochromatographic assay card according to the same experimental method. The number of samples whose results fell within the allowable error range was also calculated, thus determining the percentage of all samples meeting the acceptable target standard of a LOQ of 3 pg / mL. The validation results of the LOQ of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card and the conventional fluorescent immunochromatographic assay card were compared.

[0205] The experimental data and results are shown in Tables 14-15.

[0206] Table 14 Analysis of Quantitation Limit Validation Data for Single-Atom Nanozyme-Enhanced Fluorescent Immunochromatographic Detection Card

[0207]

[0208]

[0209] The experimental results show that the percentage of the single-atom nanozyme-enhanced fluorescence immunochromatographic detection card that meets the accuracy target is 95.56%, which is greater than the minimum percentage required for a sample size of 45 (88%) in the consensus. The limit of quantitation is 3 pg / mL, which is a successful verification.

[0210] Table 15 Analysis of Quantitation Limit Validation Data for Conventional Fluorescent Immunochromatographic Cards

[0211]

[0212] The experimental results show that the percentage of interleukin-6 detection that meets the accuracy target by using the conventional fluorescent immunochromatographic assay card is 84.44%. Compared with the minimum percentage (88%) required for a sample size of 45 in the consensus, this percentage is less than the minimum required percentage. Therefore, the verification of the limit of quantitation (LOQ) of 3 pg / mL fails. Thus, the LOQ of the conventional fluorescent immunochromatographic assay card for interleukin-6 detection is higher than 3 pg / mL.

[0213] The results above show that the limit of quantitation of the single-atom nanozyme-enhanced fluorescence immunochromatographic interleukin-6 detection card is lower than that of the conventional fluorescence immunochromatographic interleukin-6 detection card.

[0214] 3) Validation of the linear interval of the interleukin-6 detection card

[0215] Experimental Method: High-value samples close to the upper limit of the linear range were diluted to at least five concentrations, with low-value samples close to the lower limit of the linear range. Following the kit instructions, each concentration was tested three times, and the average value was calculated. A regression analysis was performed between the measured average concentration (y) and the corresponding theoretical concentration or dilution factor (x) to determine the linear regression equation and calculate the linear correlation coefficient r. The result should be within the range of [4, 4000] pg / mL, with a linear correlation coefficient (r) greater than 0.99. The relative bias between each measured value and the theoretical concentration value was calculated, and the relative bias should be within ±10%.

[0216] The experimental data and results are shown in Table 16.

[0217] Table 16 Analysis of Linear Interval Validation Data

[0218]

[0219] The experimental results show that, through linear interval verification of the interleukin-6 detection card prepared by the above method, the linear correlation coefficient (r) is ≥0.99 within the range of [4, 4000] pg / mL, and the relative deviation of linearity does not exceed ±10%. This meets the clinical requirements for the linearity of interleukin-6.

[0220] 4) Analytical specificity verification

[0221] Experimental Methods: Interference samples were prepared by adding interfering agents to low-value samples (15 pg / mL) and high-value samples (250 pg / mL), respectively, and control samples were prepared by adding an equal volume of interfering agent matrix. Each sample was measured six times using a single-atom nanozyme-enhanced fluorescence immunochromatographic interleukin-6 detection card and a conventional fluorescence immunochromatographic interleukin-6 detection card. The relative deviation B between the measured mean values ​​of the added samples and the control samples was calculated according to formula (1).

[0222] The calculated result (B) should not interfere with the analyte if the interfering substance is within the pre-set clinically acceptable bias of 10% (at the corresponding concentration levels); otherwise, it will cause interference.

[0223] The experimental data and results are shown in Table 17.

[0224] Table 17 Data Analysis Table for Specificity Analysis

[0225]

[0226]

[0227] The experimental results showed that samples containing high concentrations of retinol-binding protein, neutrophil gelatinase-associated lipotransferase, insulin, nifedipine sustained-release tablets, vancomycin, and furosemide interfered with the detection of interleukin-6 using conventional fluorescent immunochromatographic assay cards. However, they did not significantly interfere with the detection of interleukin-6 using single-atom nanozyme-enhanced fluorescent immunochromatographic assay cards. This verifies that the analytical specificity of the single-atom nanozyme-enhanced fluorescent immunochromatographic assay card for interleukin-6 is superior to that of the conventional fluorescent immunochromatographic assay card for interleukin-6.

Claims

1. A fluorescence immunochromatographic detection method based on single-atom nanozymes, characterized in that, There are two application forms: the first application form involves a fluorescence immunochromatographic detection card with enhanced detection signal and a sample diluent; the second application form involves only a fluorescence immunochromatographic detection card with enhanced detection signal. The detection card consists of a sample pad, a fluorescence binding pad, an NC membrane, absorbent paper, and a PVC board. The characteristic fluorescence signals of the T line and C line are detected by a fluorescence immunoassay analyzer, and the analyte is quantitatively analyzed based on the signal intensity.

2. The first application form according to claim 1, characterized in that, The application includes a sample diluent, which comprises: (a) Single-atom nanoenzyme solution; (b) Inorganic salts; (c) Surfactants; (d) Buffer solution; (e) Preservatives; The concentration of the single-atom nanozyme solution is 10–50 mL / L; The inorganic salt contains at least one of NaCl, KCl, MgCl2, and CaCl2, and the total ionic strength is 50–500 mmol / L. The surfactant is a nonionic surfactant, comprising at least one of Tween-20, Tween-80, Triton X-100, NP-40, Pluronic F-68, Brij-35, and Tetronic 1307, at a concentration of 0.01%–0.5% (w / v); The buffer solution may be one of the following: phosphate buffer, Tris-HCl buffer, HEPES buffer, MOPS buffer, MES buffer, citrate buffer, carbonate buffer, and borate buffer. The preservative contains at least one of Proclin 300, NaN3, KN3, potassium sorbate, and phenoxyethanol, with a final concentration of 0.1%–0.5% (w / v).

3. The second application form according to claim 1, characterized in that, The application includes a sample pad, which is obtained by soaking in a sample pad treatment solution, the sample pad treatment solution comprising: (a) Single-atom nanoenzyme solution; (b) Inorganic salts; (c) Surfactants; (d) Buffer solution; (e) Preservatives; The concentration of the single-atom nanozyme solution is 10–50 mL / L; The inorganic salt contains at least one of NaCl, KCl, MgCl2, and CaCl2, and the total ionic strength is 50–500 mmol / L. The surfactant is a nonionic surfactant, comprising at least one of Tween-20, Tween-80, Triton X-100, NP-40, Pluronic F-68, Brij-35, and Tetronic 1307, with a final concentration of 0.01%–0.5% (w / v). The buffer solution may be one of the following: phosphate buffer, Tris-HCl buffer, HEPES buffer, MOPS buffer, MES buffer, citrate buffer, carbonate buffer, and borate buffer. The preservative contains at least one of Proclin 300, NaN3, KN3, potassium sorbate, and phenoxyethanol, with a final concentration of 0.1%–0.5% (w / v).

4. The single-atom nanoenzyme solution according to claims 2 and 3, characterized in that, The single-atom nanoenzyme solution contains: (a) Single-atom nanozymes; (b) Bioenzyme composite microspheres; (c) Protective agents; (d) Surfactants; (e) Thickeners; (f) Preservatives; The final concentration of the single-atom nanozyme is 5–50 μg / mL; The protective agent comprises at least one of glycine, arginine, L-aspartic acid, and cysteine. The surfactant comprises at least one of Tween-20, Tween-80, Triton X-100, NP-40, Pluronic F-68, Brij-35, and Tetronic 1307, with a final concentration of 0.01%–0.5% (w / v). The thickener comprises at least one of sucrose, glycerin, polyethylene glycol, and ethylene glycol. The preservative contains at least one of Proclin 300, NaN3, KN3, potassium sorbate, and phenoxyethanol, with a final concentration of 0.1%–0.5% (w / v).

5. The single-atom nanozyme according to claim 4 is composed of atomically dispersed metal active centers and a support material. The metal active centers are selected from one or more of Ce, Fe, Co, Ni, Cu, Mn, Pt, Pd, Ru, and Ir as active center atoms. The support material is selected from one of N-SiO2, TiO2, Al2O3, CeO2, sulfur-doped mesoporous carbon, and graphene. Through precise control of the electronic structure and catalytic performance of the active centers via metal-support electronic interactions, the single-atom nanozyme can catalyze the decomposition of H2O2 into highly active hydroxyl radicals (·OH), which can oxidize Eu. 3+ The chelate ligands, relieving Eu 3+ Fluorescence quenching and enhanced energy transfer enhance the detection signal.

6. The bio-enzyme composite microspheres according to claim 4, characterized in that, The bio-enzyme composite microspheres consist of a three-layer structure: an inner aqueous phase, a middle oil phase, and an outer aqueous phase. The inner aqueous phase contains bio-enzymes, the middle oil phase contains methylthiophene trifluoroacetone (MT-TTFA), and the outer aqueous phase contains CaCl2 and SDS.

7. The biological enzyme according to claim 6, characterized in that, The bioenzyme includes glucose oxidase and urate oxidase, with a final concentration of glucose oxidase of 5-10 KU / L and a final concentration of urate oxidase of 50-300 U / L. Glucose oxidase and urate oxidase can catalyze the production of H2O2 from glucose and uric acid in the sample, respectively.

8. The Eu according to claim 5 3+ The chelate ligand, characterized in that, The Eu 3+ The chelate ligand is methylthiophenecarboxylic acid trifluoroacetone (MT-TTFA).

9. The fluorescence immunochromatographic detection method based on single-atom nanozymes enhanced according to claim 1, characterized in that, The aforementioned detection method is applied to the rapid quantitative detection of myocardial and inflammatory markers. The myocardial markers include troponin I (cTnI), troponin T (cTnT), myoglobin (Myo), creatine kinase isoenzyme (CK-MB), B-type natriuretic peptide (BNP), N-terminal pro-brain natriuretic peptide (NT-proBNP), soluble growth-stimulating gene 2 protein (ST2), and heart-type fatty acid-binding protein (H-FABP). The inflammatory markers include C-reactive protein (CRP), high-sensitivity C-reactive protein (hs-CRP), serum amyloid A (SAA), procalcitonin (PCT), and interleukin-6 (IL-6).

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