Alloy nano-enzyme with popcorn structure as well as preparation method and application of alloy nano-enzyme

Abstract

The invention discloses a popcorn structure alloy nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of immunochromatography detection. According to the invention, the AuPtCo nano-enzyme is successfully synthesized by adopting a one-step reduction method. The prepared AuPtCo nano-enzyme is in a popcorn shape and serves as a signal probe and an antibody carrier, the larger specific surface area of the AuPtCo nano-enzyme can provide more binding sites for an antibody, and meanwhile, on the basis of the characteristic of catalyzing TMB color development by the activity of AuPtCo nano-enzyme peroxidase, color development signal amplification of a detection line can be achieved, and the detection sensitivity is improved. A'colorimetric / catalytic colorimetric 'immunochromatography detection test strip is constructed by taking AuPtCo as a multi-element signal probe, two signal modes can be flexibly converted and verified, good accuracy and stability are achieved, and the'generality' problem of poor sensitivity and accuracy of a single detection signal mode during immunochromatography detection in the prior art is solved.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic detection technology, and in particular to a popcorn-structured alloy nanozyme, its preparation method, and its application. Background Technology

[0002] Diazepam (DAP), also known as Valium, is a benzodiazepine sedative-hypnotic drug with sedative and anti-anxiety effects. DAP can inhibit the metabolism of fresh live fish and other aquatic animals, reducing their ability to perceive external stimuli and keeping them fresh during transportation. However, DAP residues in aquatic products can be transmitted to humans through the food chain, easily causing symptoms such as fatigue, drowsiness, mental confusion, and arrhythmia. my country's "Maximum Residue Limits for Veterinary Drugs in Animal-Derived Foods" stipulates that DAP must not be detected in animal-derived foods. Therefore, developing a sensitive and accurate DAP detection method is essential for ensuring food safety. Immunochromatographic detection is widely used in rapid food safety screening by food companies and regulatory authorities due to its speed, simplicity, efficiency, and flexibility. Traditional immunochromatographic test strips mainly use colored nanomaterials such as colloidal gold as signal probes, and the intensity of the color development by observing the accumulation of the probe on the test line with the naked eye is used for visual judgment. However, traditional colored probes need to accumulate to a certain amount on the test line to achieve a visible effect, resulting in low detection sensitivity. Although some studies have attempted to improve detection sensitivity by introducing highly sensitive signal probes, single detection signal patterns are susceptible to complex food matrix effects, which in turn affect the accuracy of the test strips in detecting different real-world samples. Therefore, traditional immunochromatographic test strips generally suffer from poor sensitivity and accuracy in practical applications across various scenarios, including food production, distribution, and sales. Summary of the Invention

[0003] The purpose of this invention is to provide a popcorn-shaped alloy nanozyme, its preparation method, and its application, to solve the problems existing in the prior art. The gold-platinum-cobalt (AuPtCo) nanozyme prepared by this invention exhibits a popcorn shape and serves as a signal probe and antibody carrier. Its large specific surface area provides numerous binding sites for antibodies. Furthermore, based on the peroxidase-like catalytic TMB colorimetric properties of the AuPtCo nanozyme provided by this invention, the colorimetric signal of the "detection line" can be amplified, improving detection sensitivity.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a method for preparing popcorn-structured alloy nanoenzymes, comprising the following steps:

[0006] HAuCl4, H2PtCl6 and CoCl2 were added to a 7.5 mg / mL F-127 aqueous solution to obtain mother liquor A; the molar ratio of HAuCl4, H2PtCl6 and CoCl2 was 1:2:2.

[0007] A 3 mg / mL NaOH solution and a 50 mg / mL glucose solution were mixed at a volume ratio of 5:2 to obtain mother liquor B. An equal volume of mother liquor A and the mother liquor were passed through a microfluidic chip to obtain the popcorn-structured alloy nanoenzyme.

[0008] Furthermore, the microfluidic chip has a channel width of 400 μm and a depth of 90 μm.

[0009] The present invention also provides a popcorn-structured alloy nanoenzyme obtained by the preparation method described above.

[0010] This invention synthesizes a gray-purple AuPtCo nanozyme using a one-step reduction method. The nanomaterial has obvious inherent color characteristics and excellent peroxidase-like activity. Its nanomorphology, dispersibility and composition are suitable for use as an antibody carrier and signal probe to construct immunochromatographic test strips.

[0011] The present invention also provides an immunochromatographic detection kit, comprising an immunoprobe containing the aforementioned popcorn-structured alloy nanozyme and an immunochromatographic test strip; the immunoprobe is composed of the popcorn-structured alloy nanozyme adsorbed and linked to a target antibody.

[0012] Furthermore, it also includes probe working buffer.

[0013] The present invention also provides the application of the popcorn-structured alloy nanozyme or the immunochromatographic detection kit described herein in detecting whether a sample contains diazepam or the amount of diazepam residue.

[0014] The AuPtCo nanozyme provided by this invention can be used as a probe to label antibodies. Based on the change in the number of antibodies that compete with the target to bind to the antigen, and relying on its concentration-dependent peroxidase-like catalytic TMB activity, a quantitative or qualitative immunoassay method can be realized.

[0015] The present invention also provides a method for detecting whether a sample contains diazepam, comprising linking the popcorn-structured alloy nanozyme to a diazepam antibody to prepare an immune probe;

[0016] The sample solution to be tested, the immunoprobe and the probe working buffer are mixed and loaded onto the immunochromatographic test strip for detecting diazepam. After color development, the sample is analyzed by colorimetry or catalytic colorimetry to determine whether it contains diazepam.

[0017] Furthermore, during the catalytic colorimetric analysis, after sample loading, the test strip should be immersed in the TMB-H2O2 colorimetric working solution to observe the color change of the detection line.

[0018] The present invention also provides a method for detecting the residual content of diazepam in a sample, comprising linking the popcorn-structured alloy nanozyme with a diazepam antibody to prepare an immune probe;

[0019] The sample solution to be tested, the immunoprobe, and the probe working buffer are mixed and loaded onto the immunochromatographic test strip for detecting diazepam. After color development, the residual diazepam content in the sample is analyzed by colorimetry or catalytic colorimetry based on the color intensity of the detection line.

[0020] Based on the inherent grayish-purple color characteristic of AuPtCo nanozymes, the colorimetric mode of the test strip has a detection range of 0.97 ng / mL-4000 ng / mL and a detection limit of 0.26 ng / mL.

[0021] Furthermore, during the catalytic colorimetric analysis, after sample loading, the test strip should be immersed in the TMB-H2O2 colorimetric working solution to observe the color change of the detection line.

[0022] AuPtCo can catalyze the production of a blue oxidation product from 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide, exhibiting a characteristic absorption peak at 650 nm. Based on the colorimetric properties of AuPtCo nanozyme-like peroxidase catalyzing TMB, the colorimetric signal of the "detection line" can be amplified. The detection limit of this catalytic colorimetric mode is 13.59 pg / mL.

[0023] The present invention discloses the following technical effects:

[0024] This invention successfully synthesized a stable alloy nanozyme with a popcorn structure—AuPtCo (AuPtCo) nanozyme—using a one-step reduction method. The AuPtCo nanozyme has a distinct inherent color and exhibits excellent peroxidase-like activity.

[0025] The AuPtCo nanozyme prepared in this invention exhibits a popcorn-like shape and serves as both a signal probe and antibody carrier. Its large specific surface area provides numerous binding sites for antibodies. Furthermore, based on the peroxidase-catalyzing TMB colorimetric properties of the AuPtCo nanozyme provided by this invention, the colorimetric signal of the "detection line" can be amplified, improving detection sensitivity. A "colorimetric / catalytic colorimetric" immunochromatographic test strip can be constructed using AuPtCo as a multivariate signal probe. The two signal modes can be flexibly switched for verification, demonstrating good accuracy and stability, and can be applied to the detection of DAP residues in animal-derived foods. This solves the common problem in existing technologies using immunochromatographic detection where the sensitivity and accuracy of a single detection signal mode are insufficient. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The images show TEM images and elemental scan results for AuPtCo nanomaterials; where a and b are TEM images at scales of 100 nm and 50 nm, respectively; c is the elemental scan result for the metals in the AuPtCo nanomaterials; d is the elemental scan result for the Au in the AuPtCo nanomaterials; e is the elemental scan result for the Pt in the AuPtCo nanomaterials; and f is the elemental scan result for the Co in the AuPtCo nanomaterials.

[0028] Figure 2 XPS analysis results for AuPtCo nanomaterials;

[0029] Figure 3 XRD analysis results for AuPtCo nanomaterials;

[0030] Figure 4 The UV-Vis absorption spectrum of the TMB-H2O2 reaction system;

[0031] Figure 5 The curves show the changes in AuPtCo enzyme activity over time at different concentrations.

[0032] Figure 6 Steady-state kinetic analysis of AuPtCo and AuPtCo-IgG catalyzing TMB (a) and linear kinetic relationship of AuPtCo and AuPtCo-IgG with TMB as substrate (b).

[0033] Figure 7 This is the standard curve for colorimetric mode;

[0034] Figure 8 This is the standard curve for the catalytic colorimetric mode. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example

[0041] I. Experimental Methods

[0042] 1. Synthesis of AuPtCo

[0043] 30 mg of block polyether F-127 (ST501-10g, Beyotime) was added to 4 mL of water and mixed with 2 mL of HAuCl4 (10 mM), 4 mL of H2PtCl6 (10 mM), and 4 mL of CoCl2 (10 mM) to obtain mother solution A. 500 µL of sodium hydroxide (3 mg / mL) and 200 µL of glucose (50 mg / mL) were mixed to obtain mother solution B. Equal volumes of mother solutions A and B were injected into the two inlets of the microfluidic chip at a flow rate of 500 μL / min using a syringe pump. After passing through the microfluidic chip, a grayish-purple AuPtCo nanoparticle solution was obtained. The microchannel PDMS substrate of this microfluidic chip has a thickness of 0.5 cm and includes two reaction solution injection inlets, one serpentine channel mixing zone, one spiral channel reaction zone, and one reaction solution outlet; the microfluidic chip channel width is 400 μm and the depth is 90 μm.

[0044] 2. Characterization of AuPtCo

[0045] After successfully preparing AuPtCo nanomaterials, their morphology was characterized using transmission electron microscopy (TEM) to visually demonstrate the particle shape, size, and dispersion state of the AuPtCo nanomaterials. To investigate the crystal structure characteristics and phase composition of the material samples, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were used to characterize the crystal structure and elemental composition of the AuPtCo nanomaterials. To examine the surface plasmon resonance effect of the AuPtCo nanomaterials, the ultraviolet-visible spectrophotometer (UV-Vis) was used to scan and characterize the ultraviolet absorption spectrum of the material's aqueous solution.

[0046] 3. Study on the activity of AuPtCo peroxidases

[0047] Peroxidases catalyze a redox reaction between H₂O₂ and TMB, generating a blue oxidation product (oxTMB). oxTMB exhibits a characteristic UV-Vis absorption peak at 650 nm. This invention uses TMB as a chromogenic substrate. A solution of prepared AuPtCo nanoparticles is mixed with a TMB chromogenic solution containing H₂O₂. The absorbance changes of the system are measured using a UV-Vis spectrophotometer within the wavelength range of 400-800 nm, and the change in absorbance of AuPtCo nanoparticles at 650 nm over time is observed. Simultaneously, horseradish peroxidase (HRP) is mixed with a TMB chromogenic solution containing H₂O₂ for a control experiment. The absorbance changes of the system are measured using a UV-Vis spectrophotometer within the wavelength range of 400-800 nm to evaluate the activity of AuPtCo peroxidases.

[0048] 4. Kinetics of AuPtCo enzyme reaction

[0049] To investigate the affinity and catalytic rate of AuPtCo nanomaterials for the substrate TMB, enzyme reaction kinetics were studied. First, under the condition of constant H2O2 concentration in the system, the concentration of TMB was adjusted to explore the specific effects of TMB concentration changes on the enzyme kinetic parameters of AuPtCo nanomaterials.

[0050] 0.4 µL of H₂O₂ was completely mixed with different concentrations of TMB (0.08–1.3 mM) to obtain a colorimetric solution. 50 µL of the colorimetric solution was then mixed with 100 µL of AuPtCo nanoparticle solution and AuPtCo-IgG, respectively. The absorbance at 650 nm was recorded every 40 seconds, and each group was performed in triplicate.

[0051] Calculations were performed based on Beer-Lambert's law, and the Michaelis-Menten kinetic curve was constructed to obtain the maximum reaction rate (V). max ) and Michaelis constant (K m ), where V max K represents the maximum catalytic rate of an enzyme at saturated substrate concentration and is an important indicator for evaluating enzyme catalytic efficiency; while K m K refers to the substrate concentration required for an enzyme-catalyzed reaction to reach half of its maximum rate; it is often used to express the strength of an enzyme's affinity for its substrate. m The magnitude of the K value is closely related to the enzyme's affinity for the substrate. m A higher K value means that the enzyme requires a higher substrate concentration to reach half of its maximum catalytic rate, reflecting a relatively weak affinity of the enzyme for the substrate; conversely, a lower K value indicates a lower affinity. m A smaller value indicates that the enzyme can exhibit significant catalytic activity at a lower substrate concentration, reflecting a strong binding ability between the enzyme and the substrate.

[0052] The rate of increase in absorbance per minute was converted into the rate of change in substrate concentration, expressed in micromoles per minute. This rate was then plotted against the corresponding substrate concentration, yielding the Michaelis-Menten kinetic curve. By mathematically fitting this curve with the reaction rate as the dependent variable and the substrate concentration as the independent variable, the Michaelis-Menten equation can be derived, as shown in Equation 1:

[0053]

[0054] The double reciprocal method (Lineweaver-Burk plot) is a linearization method for the Michaelis-Menu equation. By taking the reciprocals of both sides of the Michaelis-Menu equation, a linear equation is obtained, making it convenient to derive V from experimental data. max and Km The equation for the double reciprocal method is shown in Formula 2:

[0055]

[0056] In Equations 1 and 2, V represents the reaction rate; V max K represents the maximum reaction rate. m represents the Michaelis constant; S represents the substrate concentration.

[0057] 5. Preparation of probe working buffer

[0058] Dissolve 0.25 g PVP and 0.5 g BSA in 100 mL of deionized water until fully dissolved. Then add 500 µL PEG 200 and 1 mL Tween-20 and dissolve until fully dissolved. Store at 4°C for later use.

[0059] 6. Construction of AuPtCo-IgG immune probe

[0060] The pH of the AuPtCo nanoparticle solution was adjusted to 7 using potassium carbonate (0.2 M) solution. 100 µL of the AuPtCo nanoparticle solution was thoroughly mixed with 8 µg of DAP-IgG (1 mg / mL) and incubated at 4°C for 1 hour. To eliminate non-specific antibody binding, 3 µL of 20% BSA was added, and the mixture was thoroughly mixed by pipetting and incubated at 4°C for another 30 min. Finally, the mixture was centrifuged at 6437 g for 10 min to remove unbound protein. The supernatant was discarded, and the AuPtCo-IgG immunoprobe was resuspended in 20 µL of PBS to obtain the probe, which was then stored at 4°C for future use.

[0061] 7. Preparation of Immunochromatographic Test Strips

[0062] The DAP immunochromatographic test strip consists of five parts: an absorbent pad, a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and a PVC base plate. The NC membrane is fixed to the PVC base plate. DAP-BSA and goat anti-mouse IgG are diluted to 0.5 mg / mL and 1 mg / mL, respectively, in PBS buffer (pH=7.4). Using a scrubbing apparatus, 0.5 mg / mL DAP-BSA and 1 mg / mL goat anti-mouse IgG are uniformly sprayed onto the NC membrane at a scrubbing rate of 0.8 µL / cm, serving as the "detection line" and "control line" of the immunochromatographic test strip. The strips are dried in a 37°C incubator for 10 hours. Subsequently, the conjugate pad, sample pad, and absorbent pad are sequentially glued onto the PVC plate, ensuring a slight overlap (1-2 mm) between each part. Finally, the assembly is cut into uniform strips and stored at room temperature in the dark for later use.

[0063] 8. Establishment of standard curve for multimodal immunochromatographic test strips

[0064] To verify the detection performance of the immunochromatographic test strip for DAP, two detection modes were constructed: colorimetric and catalytic colorimetric. Based on the optimized conditions, a standard curve for the colorimetric mode was first established. The specific steps are as follows: DAP standards at nine gradients (4000 ng / mL, 500 ng / mL, 62.5 ng / mL, 7.812 ng / mL, 0.976 ng / mL, 0.122 ng / mL, 0.0152 ng / mL, 0.0019 ng / mL, and 0.000238 ng / mL) were mixed with AuPtCo-IgG probe and probe working buffer and loaded onto the sample. Three parallel groups and a blank control were set up for each gradient. The loading volume was: 40 µL DAP standard / PBS + 6 µL AuPtCo-IgG probe + 14 µL probe working buffer.

[0065] Based on the capillary effect, the solution diffuses on the test strip and is allowed to stand at 25°C for 30 min. A colorimetric model for the test strip is established using the inherent gray-purple color characteristic of AuPtCo nanozymes. The color development of the test strip is observed visually. After color development, the test strip is photographed using a gel electrophoresis imaging system with a CCD camera to reduce interference from the shooting environment. The grayscale value is measured using ImageJ software to quantitatively determine the color intensity of the detection line. A standard curve for the colorimetric model is established with the logarithm of the DAP standard concentration as the x-axis and the corresponding grayscale value as the y-axis.

[0066] A catalytic colorimetric mode was established based on the peroxidase activity of AuPtCo nanomaterials. Using this mode, the test strip was immersed in TMB-H2O2 colorimetric working solution, and the color change of the detection line was observed. After color development, the test strip was photographed using a gel electrophoresis imaging system with a CCD camera to reduce interference from the shooting environment. The color intensity of the detection line was measured using ImageJ software. A standard curve for the catalytic colorimetric mode was established with the logarithm of the DAP standard concentration as the abscissa and the corresponding grayscale value as the ordinate.

[0067] 9. Sample preparation and sample addition and recovery tests

[0068] To ensure the practical application of the test strips in actual sample testing, crucian carp was selected as the sample in this invention. The sample processing method for animal-derived food was as follows: First, 4 g of sample was placed in an extraction solution containing 1 mL hydrochloric acid and 4 mL acetonitrile, and ultrasonically broken up for 5 min. Then, 1 g of calcium chloride was added, and the mixture was ultrasonically broken up for 5 min. The mixture was then centrifuged at 5000 rpm for 10 min. The supernatant was purged with nitrogen at 70°C, and the dried residue was reconstituted in PBS (0.01 M, pH 7.4).

[0069] II. Experimental Results and Analysis

[0070] 1. AuPtCo characterization results

[0071] TEM images from AuPtCo ( Figure 1 As shown in (ab), the AuPtCo particles are popcorn-shaped, with a particle size of approximately 20 nm, and are uniformly dispersed, exhibiting good water solubility. Elemental scanning results show ( Figure 1 The three elements Au, Pt, and Co are uniformly distributed on the surface of the nanoparticles. The results show that the AuPtCo nanozyme exhibits a popcorn shape. As a signal probe and antibody carrier, its large specific surface area can provide more binding sites for antibodies. Furthermore, its particle size and good dispersibility make it suitable as a nano-signal carrier for constructing immunochromatographic test strips.

[0072] Figure 2 XPS analysis results for AuPtCo nanomaterials, by Figure 2 XPS analysis revealed that the AuPtCo nanomaterials prepared by a one-step reduction method using glucose as a reducing agent at room temperature contain gold, platinum, and cobalt, and that these three elements are present in their elemental form. This demonstrates that glucose can reduce gold, platinum, and cobalt ions to zero valence at room temperature, thus producing AuPtCo nanozymes. The room-temperature one-step preparation method for AuPtCo nanozymes proposed in this invention is simple, rapid, and convenient, requiring no additional heating. It is suitable for large-scale preparation of AuPtCo, effectively ensuring the availability of this nanoprobe for subsequent experiments.

[0073] Figure 3 This is the X-ray diffraction (XRD) spectrum of AuPtCo nanomaterials. The characteristic XRD diffraction peaks of AuPtCo nanomaterials can be indexed as Au (PDF#97-004-4362), Pt (PDF#97-006-4917; PDF#97-006-4921), and Co (PDF#97-004-2684). The diffraction peaks at 2θ = 38.178°, 44.374°, and 64.559° belong to the (111), (200), and (220) crystal planes of Au crystal; the diffraction peaks at 2θ = 39.892° (38.687°), 46.396° (44.974°), and 67.708° (65.489°) belong to the (111), (200), and (220) crystal planes of Pt crystal; the diffraction peaks at 2θ = 38.178°, 44.374°, and 64.559° belong to the (111), (200), and (220) crystal planes of Au crystal, and the 2θ of the material is highly consistent with the crystal plane of Co crystal (PDF#97-004-2684).

[0074] The characterization results above indicate that AuPtCo nanozymes can be successfully and stably prepared using the room temperature one-step reduction method proposed in this invention, and their nanomorphology, dispersibility and composition are suitable for use as antibody carriers and signal probes to construct immunochromatographic test strips.

[0075] 2. AuPtCo peroxidase activity

[0076] To investigate the peroxidase-like activity of AuPtCo nanomaterials, this invention selected the commonly used peroxidase substrate TMB as the test substrate for colorimetric verification of peroxidase activity. This experiment compared the TMB catalytic performance of horseradish peroxidase (HRP), commonly used in biosensing, with that of AuPtCo nanomaterials. In the presence of hydrogen peroxide, HRP catalyzed TMB to produce the blue oxidation product oxTMB, which exhibited a characteristic absorption peak at 650 nm. Figure 4 As shown, the AuPtCo nanoparticle solution exhibits a characteristic absorption peak at 480 nm. When a TMB substrate containing H2O2 is added, the AuPtCo nanozyme catalyzes the production of a blue product from colorless TMB, showing a strong oxTMB absorption peak at 650 nm, which is highly consistent with the peroxidase catalytic activity of HRP. In contrast, the control group (TMB and H2O2 only, or only TMB) shows almost no significant absorption near 650 nm, verifying the peroxidase-like activity of the AuPtCo nanomaterials. Furthermore, the characteristic peak of the AuPtCo nanoparticle solution (480 nm) and the characteristic absorption peak of the catalytic product (650 nm) do not interfere with each other, indicating that the AuPtCo nanomaterials possess excellent catalytic performance for TMB in the presence of H2O2 and can serve as a potential catalytic colorimetric signal amplification probe for constructing immunoassay methods.

[0077] Figure 5The curves showing the change of AuPtCo enzyme activity over time at different concentrations were displayed. The changes in absorbance at 650 nm in the TMB-H₂O₂ reaction system with different concentrations of AuPtCo nanomaterials revealed a positive correlation between the concentration of the AuPtCo nanoparticle solution and its catalytic ability to TMB: as the concentration of the AuPtCo nanoparticle solution increased, the absorbance at 650 nm also increased. This indicates that the catalytic activity of AuPtCo depends on the concentration of the AuPtCo nanoparticles. Furthermore, based on the changes in absorbance at 650 nm at different reaction time points, it was found that the catalytic reaction rate first increased and then decreased with increasing time, verifying that when TMB is used as a substrate, AuPtCo nanomaterials exhibit excellent concentration-dependent catalytic performance. These results suggest that AuPtCo nanomaterials can be used as probes to label antibodies. Based on the changes in the number of antibodies that compete with the target for binding to the antigen, and relying on their concentration-dependent peroxidase-like catalytic activity to TMB, quantitative or qualitative immunoassay methods can be established.

[0078] 3. AuPtCo enzyme reaction kinetics

[0079] To investigate the changes in the enzymatic catalytic performance of AuPtCo before and after antibody modification, this invention explored the peroxidase-like reaction kinetics of AuPtCo nanomaterials and AuPtCo antibody-modified products (AuPtCo-IgG). Figure 6 As shown in (a), both AuPtCo and AuPtCo-IgG catalyze TMB according to typical Michaelis-Menten kinetics. Figure 6 As shown in (b), the Vt of the TMB substrate catalyzed by AuPtCo nanomaterials was calculated using Lineweaver-Burk. max The efficiency was 0.192 µM / s; the AuPtCo-IgG immunoprobe V obtained by physically adsorbing nanomaterials onto IgG was... max The value is 0.105 µM / s. Additionally, it can be observed that the K0 of AuPtCo is... m (0.22 mM) lower than the K of AuPtCo-IgG m (0.24 mM) indicates that both AuPtCo nanomaterials and AuPtCo-IgG exhibit strong peroxidase activity, good TMB affinity, and catalytic performance.

[0080] 4. Establishment of the standard curve for multimodal immunochromatographic test strips

[0081] Immunochromatographic analysis of DAP standard solutions of different concentrations was performed using four signal detection modes of AuPtCo-IgG: colorimetric, photothermal, catalytic colorimetric, and catalytic photothermal. Figure 7As shown, a colorimetric model was established based on the inherent grayish-purple color characteristic of AuPtCo nanomaterials. It can be observed that as the concentration of the DAP standard solution gradually increases, the number of AuPtCo-IgG immunoprobes captured on the detection line decreases, and the color of the detection line on the test strip exhibits a regular change: the color gradually weakens until it eventually disappears completely. This is because when the concentration of the DAP standard solution increases, the number of free "antigen-antibody" complexes generated by binding with the antibodies on the probes increases, leading to a decrease in the number of probes on the detection line.

[0082] As the DAP concentration increased from 0.97 ng / mL to 4000 ng / mL, the signal intensity of the detection line gradually decreased. Quantitative analysis using ImageJ software showed that within the range of 0.97-4000 ng / mL, the correlation regression equation between the detection line gray value (Y) and the DAP concentration (X) was Y = -8.80X + 32.00, exhibiting a good linear relationship (R²). 2 =0.998), and the limit of detection (LOD, calculated by dividing the absolute slope of the standard curve by three times the standard deviation of the blank signal) is 0.26 ng / mL.

[0083] The prepared test strips were first subjected to conventional colorimetric determination, and then immersed in TMB colorimetric working solution. Leveraging the peroxidase-like catalytic activity of AuPtCo nanomaterials, they catalyze colorimetric determination of the immunoprobe in immunochromatographic assays. During this process, the test line on the test strip changed from its original gray-black color to a deep blue-green. After the catalytic effect of the AuPtCo nanomaterials, the naked-eye signal intensity of the test line was significantly enhanced, further improving the sensitivity of visual detection.

[0084] like Figure 8 As shown, compared with the traditional basic colorimetric readout mode, the catalytic colorimetric readout mode significantly improves the grayscale value of the detection line, achieving signal amplification. The linear regression equation in this mode is Y = -10.77X + 43.27 (R² + π / 4). 2 =0.998), with good fit in the range of 0.12-4000 ng / mL, and LOD improved to 13.59 pg / mL, which is 19.1 times higher than that of the traditional colorimetric mode.

[0085] 5. Sample addition and recovery test

[0086] To verify the practicality of the multimodal immunochromatographic assay for DAP detection in complex environments and food matrices, a representative animal-derived food (crucian carp) was selected as the test sample. Simultaneously, natural lake water was selected as the environmental sample, and a spiked recovery experiment was conducted.

[0087] As shown in Tables 1 and 2, the average recoveries of the traditional colorimetric method for detecting DAP in aquatic products and lake water ranged from 100.70% to 101.21%, while the average recoveries of the catalytic colorimetric method ranged from 100.64% to 106.64%. Furthermore, compared to commercial ELISA kits, all these results met acceptable levels for DAP detection, with the added DAP concentration being essentially consistent with the spiked DAP concentration. The results indicate that the DAP multi-reading immunochromatographic strip is effective for the quantitative analysis of DAP residues in food and the environment, and various methods can be cross-validated, improving the accuracy of detection.

[0088] Table 1. Spike Recovery Tests for Immunochromatographic Detection Techniques

[0089]

[0090] Table 2 Validation of Commercial ELISA Kits

[0091]

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a popcorn structure alloy nanoszyme, characterized in that, It comprises the following steps: HAuCl4, H2PtCl6 and CoCl2 are added into 7.5 mg / mL F-127 aqueous solution respectively to obtain mother liquor A; the mass ratio of the HAuCl4, the H2PtCl6 and the CoCl2 is 1:2:2; 3 mg / mL NaOH solution and 50 mg / mL glucose are mixed in a volume ratio of 5:2 to obtain mother liquor B; the mother liquor A and the mother liquor B are mixed in equal volume and passed through a microfluidic chip to obtain the popcorn structure alloy nanoscale enzyme.

2. The production method according to claim 1, characterized by, The channel width of the microfluidic chip is 400 μm, and the depth is 90 μm.

3. The popcorn structure alloy nanoscale enzyme obtained by the preparation method of claim 1 or 2.

4. An immunochromatographic test kit, characterized by, It comprises an immunological probe and an immunochromatographic test strip containing the popcorn structure alloy nanoscale enzyme of claim 3; the immunological probe is composed of the popcorn structure alloy nanoscale enzyme and target antibody adsorbed connection.

5. The immunochromatographic test kit according to claim 4, characterized by It also comprises a probe working buffer.

6. The application of the popcorn structure alloy nanoscale enzyme of claim 3 or the immunochromatographic detection kit of claim 4 or 5 in detecting whether the sample contains diazepam or diazepam residue content.

7. A method of detecting the presence of diazepam in a sample, characterized in that, It comprises connecting the popcorn structure alloy nanoscale enzyme of claim 3 with diazepam antibody to prepare an immunological probe; The sample solution to be tested, the immunological probe and the probe working buffer are mixed and loaded, and then dropped on the immunochromatographic test strip for detecting diazepam; after color development, whether the sample contains diazepam is analyzed by colorimetry or catalytic colorimetry.

8. The method of claim 7, wherein, In the catalytic colorimetry, after loading, the test strip is immersed in TMB-H2O2 color development working solution to observe the color change of the detection line.

9. A method of detecting the residual content of diazepam in a sample, characterized in that, It comprises connecting the popcorn structure alloy nanoscale enzyme of claim 3 with diazepam antibody to prepare an immunological probe; The sample solution to be tested, the immunological probe and the probe working buffer are mixed and loaded, and then dropped on the immunochromatographic test strip for detecting diazepam; after color development, the residual content of diazepam in the sample is analyzed according to the color intensity of the detection line by colorimetry or catalytic colorimetry.

10. The method of claim 9, wherein, In the catalytic colorimetry, after loading, the test strip is immersed in TMB-H2O2 color development working solution to observe the color change of the detection line.

Images