Cascade amplification method based on Zn < 2 + > doped WOX nano enzyme and application of cascade amplification method in clostridium difficile toxin B detection

Abstract

The invention provides a cascade amplification method based on Zn < 2 + > doped WOX nano-enzyme and application of the cascade amplification method in clostridium difficile toxin B detection, and relates to the technical field of lateral flow immunoassay. The cascade amplification method comprises the following steps that firstly, Zn / WOX at Au with reductase-like activity is subjected to immunochromatography to generate an initial colorimetric signal; and Zn / WOX (at) Au (at) Pt with peroxidase-like activity is formed through in-situ light-induced deposition of Pt to amplify a colorimetric signal, and then enzymatic color development is performed, so that the technical problem that clostridium difficile toxin B is difficult to quickly and highly sensitively detect in the prior art is solved, the colorimetric signal is remarkably enhanced while the simplicity and convenience of LFIA operation are maintained, and the detection sensitivity of clostridium difficile toxin B is improved. The technical effect of high-sensitivity colorimetric LFIA detection is realized.

Description

Technical Field

[0001] This invention relates to the technical field of lateral flow immunoassay, and particularly to a method based on Zn. 2+ Doped WO X A cascade amplification method for nanozymes and its application in the detection of Clostridium difficile toxin B. Background Technology

[0002] Clostridium difficile toxin B damages intestinal lining cells, leading to inflammation, cell death, and a range of clinical symptoms. It poses a serious threat to individuals on long-term antibiotic use or with compromised immune systems. Therefore, developing point-of-care testing (POCT) technologies targeting Clostridium difficile toxin B (Tcd B) is crucial for public health and intensive care management.

[0003] Lateral flow immunoassay (LFIA) provides a convenient, rapid, and cost-effective platform, making it ideal for point-of-care testing (POCT). However, the limited sensitivity of LFIA often leads to false negative results in low-concentration samples. To improve LFIA sensitivity, research has explored using nanozymes with peroxidase-like activity as nanotags to detect antigens, combined with chromogenic substrates to enhance the colorimetric signal and improve the detection limit. This method is termed catalytic enhancement colorimetric mode.

[0004] The sensitivity of colorimetric signals is closely dependent on the activity of nanozyme nanotags. Noble metals (such as Au and Pt) and metal oxides (such as Fe3O4 and Co3O4) have been extensively studied as core materials for mimicking POD enzymes. Studies using noble metal nanozymes alone in immunochromatography are limited because highly active nanomaterials tend to aggregate, reducing active sites. Even with initial stable dispersion, they are susceptible to interference from biomolecules. Recent research focuses on using well-dispersible nanomaterials as substrates to load noble metal nanoparticles, thereby exposing highly active surfaces and promoting substrate adsorption and reactions, such as loading Au / Pt nanoparticles on MXene, Pt on Fe3O4, or Au on MOF. Using metal compounds with intrinsic nanozyme activity as supports for noble metal nanocatalysts is an ideal strategy for improving performance. However, connecting noble metal nanoparticles through an interlayer may block substrate active sites, preventing direct contact between the substrate and the metal, and weakening the synergistic catalytic effect mediated by interfacial charge transfer. Therefore, employing in-situ growth methods to allow noble metal nanoparticles to replace some of the substrate's active sites, forming more efficient catalytic centers, is more beneficial for improving catalytic performance.

[0005] Further enhancing catalytic performance requires structural design of the substrate material. For metal oxides, oxygen vacancies are key tunable active sites, promoting the adsorption and dissociation of H₂O₂ or O₂ and facilitating the reduction deposition of noble metal nanoparticles. Effective methods for designing more oxygen vacancies in the substrate include heat treatment under an oxygen-deficient atmosphere, chemical reduction using reducing agents, generating oxygen vacancies and inducing electronic structure changes through charge compensation by doping with low-valence cations or anions, forming physical or chemiphysical vacancies through plasma treatment, photo-induced methods, and electrochemical methods. Simultaneously, the interface effect between the substrate and the metal must be considered, as a matched energy level structure can lower the charge transfer barrier, and the formation of oxygen vacancies is precisely an effective means of regulating the electronic structure of the substrate.

[0006] In summary, by optimizing the performance of nanozymes by modulating the active sites and electronic structure of the substrate material, thereby enhancing the catalytic colorimetric detection performance, a rapid and highly sensitive detection system for Clostridium difficile toxin B based on the LFIA platform can be established.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] One of the objectives of this invention is to provide a highly efficient POD-mimicking nanozyme, which is a POD-mimicking enzyme with high specificity and high catalytic efficiency.

[0009] The second objective of this invention is to provide a highly efficient method for preparing POD-mimicking nanozymes.

[0010] The third objective of this invention is to provide a cascade amplification method, which is a two-step cascade enhanced colorimetric strategy based on LFIA, applicable to the detection of Clostridium difficile toxin B, and beneficial for ultrasensitive lateral flow immunoassay.

[0011] The fourth objective of this invention is to provide an application of a cascade amplification method that, compared to the initial colorimetric mode, can improve the visual detection limit of Clostridium difficile toxin B by 500 times and the fitted detection limit can reach 0.01 ng / mL.

[0012] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: Firstly, a highly efficient POD-mimicking nanozyme has the following structure: Zn / WO X @Au@Pt; The nanozyme is Zn / WO3 X @Au in situ deposited Pt constitutes the composition; The Zn / WO X @Au represents Zn loaded with Au nanoparticles. 2+ Doped WO X .

[0013] Secondly, a method for preparing highly efficient POD-mimicking nanozymes includes the following steps: Zn 2+ Doping into oxygen-vacancy-rich nonstoichiometric WO3 synthesized by solvothermal reduction X To obtain Zn 2+ Doped WO X , for Zn / WO X ; Au nanoparticles loaded onto Zn / WO X Above, we obtain Zn / WO X @Au; Pt nanoclusters were deposited in situ on Zn / WO3 X Highly efficient POD-mimicking nanozymes were obtained on Au seeds, which are Zn / WO3. X @Au@Pt.

[0014] Furthermore, the Au nanoparticles are loaded onto Zn / WO3 X The methods mentioned above include ultrasound-assisted reduction.

[0015] Furthermore, the Pt nanoclusters are deposited in situ on Zn / WO3. X Methods for applying Au seeds to @Au include photoinduced reduction.

[0016] Thirdly, a cascade amplification method includes the following steps: The nanozymes prepared based on the nanozymes described above or any of the preparation methods described above are used to construct a detection system through the LFIA platform to enhance the catalytic colorimetric detection performance.

[0017] Furthermore, the cascade amplification method includes the following steps: First, Zn / WO3 with reductase-like activity X @Au generates an initial colorimetric signal through immunochromatography, and then deposits Pt in situ via photoinduced deposition to form Zn / WO3 with peroxidase-like activity. X @Au@Pt is used to amplify the colorimetric signal, followed by enzymatic color development.

[0018] Furthermore, enzymatic color development is achieved through substrate reactions catalyzed by peroxidase.

[0019] Fourthly, the application of any of the above-described cascade amplification methods in the detection of Clostridium difficile toxin B.

[0020] Furthermore, the visual detection limit of the Clostridium difficile toxin B is increased by at least 500 times.

[0021] Furthermore, based on gray-scale analysis, the fitted detection limit of Clostridium difficile toxin B reaches 0.01 ng / mL.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: The highly efficient POD-mimicking nanozyme provided by the present invention is a Zn / WO X @Au@Pt structure, which is a POD-mimicking enzyme with high specificity and efficient catalytic function. Under optimized electronic structure conditions, Zn / WO X -Au-Pt heterostructure is conducive to the directional transfer of thermal and non-equilibrium carriers from Zn / WO X to Au and then to Pt; at the same time, the defect states near the conduction band edge and the narrow bandgap of Zn / WO X also promote the generation of thermally generated carriers under environmental conditions, which is beneficial to the accumulation of more charge carriers on Pt; the charge accumulation on Pt optimizes its d-band center, further enhancing the catalytic activity. Experimental characterization confirms that Zn / WO X @Au@Pt of the present invention exhibits stronger POD-like catalytic efficiency than other materials, and its POD-like catalytic efficiency is stronger than that of Zn / WO X @Au and Zn / WO X ; Simulations based on density functional theory further prove that Zn / WO X @Au@Pt has a lower energy barrier for the rate-determining step of H2O2 homolysis.

[0023] For the preparation method of the highly efficient POD-mimicking nanozyme provided by the present invention, the WO X synthesized by solvothermal reduction itself has a large number of oxygen vacancies, and the doping of Zn 2+ further increases the oxygen vacancies to maintain charge balance, meeting the requirement of rich active sites and narrowing the bandgap of WO X ; this doping also localizes the defect state electron density formed by low-valent W 5+ ions, reduces the energy gap between the defect state and the energy band edge, and enhances the valence-interband charge transfer; compared with directly loading Pt on Zn / WO X , the pre-grown Au seeds in the present invention can optimize the lattice mismatch between Pt and Zn / WO X , stabilize Pt on the Au surface, and act as a charge transfer mediator to alleviate the large work function difference between Pt and Zn / WO X , thereby improving the charge transfer efficiency; in addition, the local surface plasmon resonance effect of Au and the narrow bandgap of Zn / WO X can promote the efficient deposition of Pt under visible light; at the same time, modifying Au with Pt can significantly improve the substrate affinity and catalytic efficiency of the POD-mimicking enzyme.

[0024] The cascade amplification method provided by this invention is based on a two-step cascade enhancement colorimetric strategy using LFIA, which integrates high-performance Zn / WO3... X The integration of @Au@Pt nanozymes (nanotags) with LFIA technology employs a two-stage cascaded colorimetric enhancement strategy, which is beneficial for the ultrasensitive lateral flow immunoassay of Clostridium difficile toxin B.

[0025] The application of the cascade amplification method provided by this invention can improve the visual detection limit of Clostridium difficile toxin B by 500 times compared with the initial colorimetric mode, and the fitted detection limit can reach 0.01 ng / mL. Attached Figure Description

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

[0027] Figure 1 WO provided for one embodiment of the present invention X A diagram illustrating the modulation of the electronic band structure; Figure 2 Zn / WO provided for one embodiment of the present invention X The color change of (Zn / W=0.05) after annealing at 150°C for 30 minutes and then irradiating with an AM 1.5 light source for 10 minutes; Figure 3 Different Zn provided for one embodiment of the present invention 2+ Zn / WO doping concentration X XRD pattern and magnified view of (010) crystal plane; Figure 4 Zn / WO provided for one embodiment of the present invention X TEM image and EDS surface distribution of the sample (Zn / W=0.05); Figure 5 Different Zn provided for one embodiment of the present invention 2+ Doping concentration Zn / WO X Zn 2p XPS fine spectrum; Figure 6 Different Zn provided for one embodiment of the present invention 2+ Doping concentration Zn / WO X O 1s XPS fine spectrum and different Zn 2+ Doping concentration Zn / WO X W 4f XPS fine spectrum; Figure 7 For different Zn 2+ doping concentrations of Zn / WO X FTIR spectra; Figure 8 For different Zn 2+ doping concentrations of Zn / WO X Raman spectra; Figure 9 For different Zn 2+ doping concentrations of Zn / WO X UV-Vis absorption spectra, direct band gap Tauc plots, and indirect band gap Tauc plots; Figure 10 For different Zn 2+ doping concentrations of Zn / WO X XPS valence band spectra; Figure 11 For different Zn 2+ doping concentrations of Zn / WO X Photoluminescence emission spectra and excitation spectra; Figure 12 Schematic diagram of the two-step cascade enhanced colorimetric strategy provided by one embodiment of the present invention; Figure 13 TEM images, SAED patterns, and lattice fringe images of Zn / WO X provided by one embodiment of the present invention; [[ID=4�]] Figure 14 TEM images of Zn / WO X @Au@Pt and enlarged images of Pt nanoclusters provided by one embodiment of the present invention; Figure 15 For different Zn 2+ doping concentrations of Zn / WO X @Au@Pt catalytic performance and mechanism analysis diagrams; Figure 16 Catalytic performance and mechanism analysis diagrams of Zn / WO X , Zn / WO X @Au, and Zn / WO X @Au@Pt provided by one embodiment of the present invention; Figure 17 Schematic diagram and result diagram for the detection of Clostridium difficile toxin B (Tcd B) provided by one embodiment of the present invention. Detailed implementation manners

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Clostridioides difficile is one of the main pathogens of antibiotic-related diseases in hospitals. The toxins it produces can damage the intestinal wall cells, posing a serious potential risk. Early detection and prevention are effective strategies to curb the spread of the disease, which usually requires highly sensitive point-of-care testing methods. Although lateral flow immunoassay has attracted much attention in POCT for its convenience, rapidity, and cost-effectiveness, its relatively low sensitivity limits its application in early screening. Therefore, the technical solution of the present invention is specifically proposed.

[0030] According to the first aspect of the present invention, there is provided a highly efficient POD-mimicking nanozyme with the following structure: Zn / WO X @Au@Pt; This nanozyme is composed of Zn / WO X @Au with Pt deposited in-situ; Among them, Zn / WO X @Au is Zn 2+ doped with WO X .

[0031] The nanozyme of the present invention has a Zn / WO X @Au@Pt structure and is a POD-mimicking enzyme with high specificity and efficient catalytic function. Under optimized electronic structure conditions, the Zn / WO X -Au-Pt heterostructure is conducive to the directional transfer of thermal equilibrium and non-equilibrium carriers from Zn / WO X to Au and then to Pt; at the same time, the defect states near the conduction band edge and the narrow bandgap of Zn / WO X also promote the generation of thermally generated carriers under environmental conditions, which is conducive to the accumulation of more charge carriers on Pt; the charge accumulation on Pt optimizes its d-band center, further enhancing the catalytic activity.

[0032] Experimental characterization confirms that the Zn / WO X @Au@Pt of the present invention exhibits stronger POD-like catalytic efficiency than other materials, and its POD-like catalytic efficiency is stronger than that of Zn / WO X @Au and Zn / WO X ; Simulations based on density functional theory further prove that Zn / WO X @Au@Pt has a lower energy barrier in the rate-determining step of H2O2 homolysis.

[0033] According to a second aspect of the present invention, a method for preparing the above-described highly efficient POD-mimicking nanozyme is provided, comprising the following steps: Zn 2+ Doping into oxygen-vacancy-rich nonstoichiometric WO3 synthesized by solvothermal reduction X To obtain Zn 2+ Doped WO X , for Zn / WO X ; Au nanoparticles loaded onto Zn / WO X Above, we obtain Zn / WO X @Au; Pt nanoclusters were deposited in situ on Zn / WO3 X Highly efficient POD-mimicking nanozymes were obtained on Au seeds, which are Zn / WO3. X @Au@Pt.

[0034] In a preferred embodiment, first through Zn 2+ Non-stoichiometric WO3 rich in oxygen vacancies was synthesized by a doped solvothermal reduction method. X To obtain Zn 2+ Doped WO X , for Zn / WO X Au nanoparticles were then loaded onto Zn / WO3 using an ultrasonic-assisted reduction method. X The above process yields Zn / WO3 with reductase-like activity. X @Au, then ultrafine Pt nanoclusters were deposited in situ on Au seeds using a photoinduced reduction method to form Zn / WO. X @Au@Pt, thus obtaining a highly efficient POD nanozyme.

[0035] WO3 synthesized by solvothermal reduction method X Zn itself has a large number of oxygen vacancies 2+ Doping further increases oxygen vacancies to maintain charge balance, satisfying the need for abundant active sites and reducing WO3 size. X The band gap, this doping also localizes the low-valence W 5+ The defect state electron density formed by ions reduces the band gap between the defect state and the band edge, thus enhancing the transfer of charge between valence states.

[0036] in WO X In the solvothermal synthesis process, a specific amount of ZnCl2 is introduced into the precursor solution to react together, yielding Zn 2+ Doped WO X ,like Figure 1 a (Zn) 2+ Doped WO XThe synthesis process is illustrated in the diagram. The product is named according to the molar ratio of Zn to W used; for example, Zn / W = 0.00 indicates that no Zn was added. 2+ The sample. To reveal the differences in Zn 2+ Doping level on WO X The effects and results were determined using a variety of material characterization techniques and simulation calculations. Figure 1 c (different Zn) 2+ Doping concentration WO X The X-ray diffraction pattern shown in the XRD pattern confirms that Zn / WO3 X The crystal structure of the product mainly corresponds to the γ phase W of the monoclinic crystal system. 18 O 49 The diffraction peaks at 23.20° and 47.90° are attributed to the (010) and (020) crystal planes, respectively. Given that based on W... 18 O 49 The "smart window" material possesses a highly efficient photochromic / electrochromic effect, and the coloring-bleaching cycle induced by light irradiation and thermal annealing can also serve as a visual method to verify its identity, such as... Figure 2 As shown, the observed changes are related to W. 18 O 49 The material exhibits typical bleaching-dyeing cycles. No Zn-related abnormalities were observed in the XRD pattern. 2+ The diffraction peaks of the compound. However, the energy-dispersive X-ray spectroscopy results are shown in [reference needed]. Figure 3 This confirms Zn 2+ The successful incorporation of Zn indicates 2+ The doping did not form a second phase, or any second phase was long-range disorder. For quantitative analysis of Zn 2p X-ray photoelectron spectroscopy data, see [link to relevant documentation]. Figure 4 This further confirms that Zn 2+ As a trace dopant, even at the highest zinc chloride addition, the atomic percentage of Zn is only 0.70%. This is considering factors such as the coordination number of Zn being 4. 2+ The ionic radius is 0.74 Å, and increases with increasing coordination number, reaching 0.82 Å at a coordination number of 5 and 0.88 Å at a coordination number of 6; in W 18 O 49 In the middle, W 6+ / W 5+ It mainly occupies sites with a coordination number of 6, and its ionic radius ranges from 0.65 Å to 0.74 Å; W 18 O 49 The structure of Zn itself is loose, with abundant voids and large interlayer spacing. 2+ To replace W with a coordination number of 6 6+ / W 5+At certain sites, significant lattice distortion occurs, which contradicts the principle of energy minimization. Therefore, Zn 2+ Doping should primarily be carried out through interstitial doping. Figure 1 A magnified image of the (010) diffraction peak of C is shown below. Figure 3 As shown, with Zn 2+ With increasing doping concentration, the (010) peak shifts slightly towards a lower angle, indicating an increase in interplanar spacing, consistent with interstitial doping. The largest peak shift occurs in the sample with Zn / W = 0.05. Beyond this doping level (e.g., Zn / W = 0.10), the shift suddenly decreases, possibly due to exceeding the solid solubility limit and causing Zn... 2+ This is due to precipitation and partial recovery of the (010) peak position. 2+ These particles may migrate to high-energy sites such as grain boundaries, dislocations, or surfaces, forming a nanoscale second phase that is undetectable due to its small size or disordered nature. Furthermore, the increase of such nanoscale second phases at defects enhances lattice distortion, which may explain the reappearance of a low-angle shift in the (010) peak in samples with Zn / W ≥ 0.10. Figure 1 The middle d (EPR spectrum) shows different Zn 2+ Doping level Zn / WO X The electron paramagnetic resonance spectrum of Zn. The signal at g = 2.003 corresponds to an oxygen vacancy defect. Semi-quantitative analysis shows that the oxygen vacancy content increases with Zn content. 2+ The doping concentration increases with increasing concentration, until a sharp decrease is observed at Zn / W = 0.10, which proves that Zn... 2+ Doping of WO X Significant impact of oxygen vacancies. Figure 5 For different Zn 2+ Doping concentration Zn / WO X The fine XPS spectrum of Zn 2p. Figure 6 A similar trend was observed in the high-resolution XPS spectra of O 1s and W 4f shown. The proportion of the sub-peak at 532.51 eV attributed to oxygen vacancies increased with Zn. 2+ The ratio increases with doping, then drops sharply when Zn / W = 0.10. Similarly, this is attributed to low-valence W. 5+ The proportions of sub-peaks at 37.35 eV and 34.45 eV also increased with Zn. 2+ The solubility increases with doping and decreases sharply at Zn / W = 0.10. These observations are consistent with the solid solubility limit conclusions derived from XRD analysis. To maintain charge balance, Zn... 2+ Doping induces oxygen vacancies and low-valence W 5+ The formation of Zn. The observed change up to Zn / W = 0.10 is continuous. 2+ Doping increases oxygen vacancies and W5+ The content results. At Zn / W = 0.10, Zn 2+ When the solubility exceeds the solid solubility limit, Zn precipitates out, leading to the formation of Zn in the bulk phase. 2+ The content decreased sharply. This phenomenon also reduced the availability of oxygen vacancies and W. 5+ The need to compensate for charge leads to a sudden drop in its concentration. The slight change in oxygen vacancy content in samples with Zn / W > 0.10 may be attributed to interfacial defects introduced by the precipitated phase. Figure 1 The zeta potential value (e) indicates the different Zn values. 2+ Doping level Zn / WO X The zeta potential value. Non-stoichiometric WO X The negative charge in water is mainly due to the defect state W. 5+ / W 4+ Induced surface hydroxylation ionization is the cause. This is induced in oxygen-vacancy-rich Zn / WO3. X Contains more low-priced W 5+ / W 4+ This further enhances its surface hydroxylation ionization in aqueous systems, resulting in a more negative potential. Therefore, with the development of Zn... 2+ With increasing doping concentration, the Zeta potential of the Zn / W = 0.05 sample reaches its most negative value. This is because Zn 2+ Doping-induced charge compensation increases the low-valence W 5+ / W 4+ This promotes surface hydroxylation ionization, which is consistent with XPS results. However, with Zn... 2+ With further increases in doping concentration, the potential of the Zn / W = 0.10 sample became more positive. Based on previous analysis, this can be attributed to two reasons: Zn 2+ The precipitation of W decreased with low price 5+ / W 4+ The relevant local electron density weakens surface hydroxylation ionization; the surface-deposited Zn 2+ WO is blocked X Some high-energy sites reduce the degree of surface hydroxylation ionization. The potential of the Zn / W = 0.50 and Zn / W = 1.00 samples shows a further negative shift, which can be achieved by the precipitation of Zn. 2+ This can be explained by the hydroxylation ionization of the relevant phase. O 1s high-resolution XPS spectra ( Figure 6 (a) FTIR spectrum ( Figure 7 ) and Raman spectroscopy ( Figure 8 This further supports the point. Figure 6 In the middle of the af, it belongs to the low-priced W 5+ The proportion of the sub-peak at 531.31 eV of the relevant surface -OH groups also increases with Zn. 2+The doping increases, then decreases sharply at Zn / W = 0.10, before increasing again. This trend is perfectly consistent with the change in the Zeta potential. In the FTIR spectrum ( Figure 7 ), 1261 cm -1 and 1095 cm -1 The two peaks at that location change with Zn 2+ This becomes more pronounced with increasing doping concentration. The former is attributed to the surface hydroxyl vibrational mode, while the latter corresponds to the doped Zn. 2+ The Zn-OW vibrational mode formed between WO⁻ and WO⁻. In WO⁻ with high oxygen vacancy content... X In the bulk phase, it is difficult to retain a large number of W–O⁻ dangling bonds. Therefore, the Zn–O–W vibrational mode should be mainly related to the precipitated Zn. 2+ Related. For W 18 O 49 Its disordered structure contains WOW bonds of varying lengths, resulting in a range of 100–400 cm. -1 and 600–900cm -1 Two broad, featureless Raman peaks appear within the range. However, under ambient oxygen conditions, the laser-induced thermal effect promotes oxidation and gradually transforms the structure into WO3 (monoclinic γ-phase), leading to the appearance of characteristic peaks. During partial oxidation, characteristic peaks appear at 131, 267, 714, and 803 cm⁻¹. -1 Four main peaks appear at 87 and 327 cm⁻¹, while after complete oxidation, peaks appear at 87 and 327 cm⁻¹. -1 A sharp peak appears there. Therefore, Figure 8 The Raman spectrum shown actually corresponds to oxidized WO3. X Zn 2+ Doping affects the vibrational modes associated with WO, causing corresponding peak shifts. With the addition of Zn... 2+ With increasing doping concentration, the peak shift was most significant in the sample with Zn / W = 0.05. At Zn / W = 0.10, the peak shift decreased significantly, and all peaks broadened significantly or almost disappeared. X The broadening and disappearance of the mid-Raman peaks indicate a reduced degree of oxidation, meaning that samples with Zn / W > 0.10 experienced significantly less laser-induced thermal oxidation. The only possible factor compensating for shielding thermal oxidation is likely the precipitation of Zn. 2+ Related second phase. Besides affecting WO X In addition to its crystal structure, oxygen vacancy content, chemical state, and molecular vibrational modes, Zn 2+ Doping further modulates its electronic band structure. Figure 1 f (different Zn) 2+ Doping concentration WO X The optical photograph (top) and schematic performance band structure diagram (bottom) show the structure based on ultraviolet-visible absorption spectroscopy (UV-Vis). Figure 9 ) and XPS valence band spectrum ( Figure 10 The schematic diagram of the bandgap structure constructed is shown. It can be observed that the bandgap increases with Zn. 2+ The doping concentration gradually narrows with increasing concentration, reaching a minimum at Zn / W = 0.05, and then gradually widens again. This trend is consistent with that of Zn. 2+ The consistent changes in oxygen vacancy content induced by doping and precipitation indicate interstitial Zn 2+ Doping reduces WO3 by promoting oxygen vacancy formation. X The band gap, and then Zn 2+ Precipitation weakens this effect. Compared to the parent monoclinic WO3, W 18 O 49 It exhibits a more disordered structure; its amorphous properties and high defect density result in a broad electronic state density near the band edges and introduce defect energy levels, often exhibiting characteristics of direct and indirect bandgap mixing. This is reflected in UV-Vis absorption spectra ( Figure 9 In section a), there is a low-slope absorbing edge with a tail (purple area) and a feature attributed to W. 5+ Defect state and W 6+ A broad near-infrared absorption peak (red region) indicating polaron transitions between sites. With Zn doping, the Zn / WO3 ratio... X The ultraviolet absorption edge becomes smoother and steeper, reflecting the band edge localization induced by Zn doping. This is combined with the changes in the defect state peak positions in the valence band spectrum (…). Figure 10 It is clear that Zn doping also affects WO3. X The defect energy level. This is related to the low valence W induced by charge compensation. 5+ / W 4+ The changes in content correspond precisely. Furthermore, W 18 O 49 The main defect states in it are precisely these low-priced W 5+ / W 4+ It is composed of species. This also explains why Figure 1 Zn / WO in the upper part of the optical photograph X The blue color deepens. Furthermore, the photoluminescence excitation and emission spectra of Zn... 2+ to WO X The emphasis on the controlling role of electronic structure provides additional evidence, such as Figure 11 As shown, the broad peak centered at 440 nm in the emission spectrum indicates band broadening and delocalization, suggesting that the excited electrons underwent diverse and complex relaxation processes before radiative recombination. The broad near-infrared emission near 715 nm likely originates from low-valence W 5+ / W 4+The relaxation-recombination emission of the formed defect energy levels is evident from the broad near-infrared absorption in the UV-Vis spectrum, indicating that these defect states are widely distributed and sufficient to form near-infrared emission of approximately 715 nm with the delocalized band edges. Zn 2+ After doping, the intensity and range of the emission peaks at 440 nm and 715 nm were suppressed and reduced, indicating that Zn 2+ This promotes the localization of electron distribution at the band edge and defect states. In the excitation spectra, the optimal excitation wavelength for interband electron-hole recombination emission of all samples is around 376 nm. The tailing peak below 375 nm corresponds to relaxation-recognition emission of shallower energy levels above the conduction band edge, which may involve a cooperative continuum process of interband and intraband excitation. With the development of Zn... 2+ Doping enables bandgap localization, significantly suppressing the tailing portion of the excitation spectrum. Furthermore, with Zn... 2+ With increasing doping concentration, the optimal excitation wavelength redshifts, and the degree of redshift decreases after Zn / W > 0.10, which is consistent with the bandgap change obtained from UV-Vis absorption spectroscopy.

[0037] Constructing undoped WO X and Zn 2+ Doped WO X The models were constructed, and their electronic densities of states (DOS) were calculated based on first-principles calculations, such as... Figure 1 Chinese b (pure WO) X and Zn / WO X As shown in the atomic model and calculated density of states, the label "Zn" represents an interstitial doped Zn. 2+ The DOS diagram shows that Zn 2+ After doping, WO X The original band edge DOS, dominated by O 2p and W 3d orbitals and located at -0.518 eV and 0.716 eV respectively, became more localized towards the band gap, resulting in a narrower band gap. Simultaneously, DOS outside the -0.518 eV to 0.716 eV range shifted deeper into the band gap. These observations primarily indicate that Zn 2+ Doping strongly affects WO X The electronic structure of the outer orbitals is as observed experimentally in Zn. 2+ Doping-induced band edge localization provides solid theoretical support. Although doped Zn 2+ The DOS contribution of its own 4s and 3d orbits is low, but it still triggered WO X The significant reconstruction of electronic states confirms that even at trace levels of interstitial Zn... 2+ Doping can also effectively regulate WO3 X The electronic band structure.

[0038] In this invention, on the one hand, compared with directly loading Pt onto Zn / WO3... X In comparison, pre-grown Au seeds can optimize the Pt / Zn / WO3 ratio. X The lattice mismatch between Pt and Zn / WO3 stabilizes Pt on the Au surface and acts as a charge transfer mediator, mitigating the interaction between Pt and Zn / WO3. X The significant difference in work function between them improves charge transfer efficiency; on the other hand, the localized surface plasmon resonance effect of Au and Zn / WO3... X The narrow band gap can promote the efficient deposition of Pt under visible light; Pt modification of Au can significantly improve the substrate affinity and catalytic efficiency of POD-mimicking enzymes.

[0039] According to a third aspect of the present invention, a cascade amplification method is provided, comprising the following steps: The nanozymes prepared based on the nanozymes described above or any of the preparation methods described above are used to construct a detection system through the LFIA platform to enhance the catalytic colorimetric detection performance.

[0040] A two-step cascaded enhancement colorimetric strategy based on LFIA will enable high-performance Zn / WO3. X The integration of @Au@Pt nanozymes with LFIA technology, employing a two-stage cascaded colorimetric enhancement strategy, facilitates ultrasensitive lateral flow immunoassay of Clostridium difficile toxin B.

[0041] In a preferred embodiment, Zn / WO3 with reductase-like activity is first... X @Au generates an initial colorimetric signal through immunochromatography, and then deposits Pt in situ via photoinduced deposition to form Zn / WO3 with peroxidase-like activity. X @Au@Pt is used to amplify the colorimetric signal, followed by enzymatic color development.

[0042] In this invention, enzymatic color development can be achieved through substrate reactions catalyzed by peroxidase.

[0043] This invention will utilize high-performance Zn / WO X The @Au@Pt nanozyme (nanotag) is integrated with LFIA technology, employing a two-stage cascaded colorimetric enhancement strategy: firstly, it utilizes Zn / WO3 with reductase-like activity... X @Au generates the initial colorimetric signal, and then Pt nanoparticles are deposited in situ through a photoinduced reduction process. The smaller size of the nanotags is beneficial to the flow properties of LFIA, while the deposition of Pt itself can also enhance the colorimetric signal, compared with using Zn / WO4 alone. X Compared to the colorimetric signal generated by @Au (detection limit = 50 ng / mL), the light-induced Zn / WO3 X @Au@Pt can increase the visual detection limit by 5 times, reaching 10 ng / mL; then Zn / WOX The POD-like activity of @Au@Pt catalyzed enzymatic colorimetric reaction further increased the visual detection limit to 0.1 ng / mL, and the visual sensitivity was improved by another 100-fold.

[0044] For LFIA, the smooth migration of nanotags is crucial. All else being equal, smaller nanotags are generally more favorable for capillary-driven chromatography processes. However, smaller nanotags also mean a reduction in active components, which may lead to a decrease in colorimetric signal intensity. From a hydrodynamic perspective, WO4 with a one-dimensional nanowire morphology... X It can migrate smoothly on nitrocellulose membranes with a pore size of 20 μm. Furthermore, compared to nanoparticles, its linear structure is more easily captured by specific sites, thus improving the signal-to-noise ratio. However, in WO X Loading a large number of nanoparticles onto the surface can hinder this smooth migration, thus requiring a sacrifice of some signal intensity to maintain flow performance. The two-step cascaded enhanced colorimetric strategy of this invention effectively solves the above problems, such as... Figure 12 As shown in diagram a (a two-step cascaded enhanced colorimetric process), Au seed crystals with a size of approximately 10 nm are first loaded onto Zn / WO3 using an ultrasonic-assisted reduction method. X Zn / WO3 with reductase-mimicking activity was obtained on nanowires. X @Au nanozyme (nanotag) was subsequently functionalized for conjugation with 10-1276 antibody; specifically, 3-mercaptopropionic acid was linked to Zn / WO3 via an Au-S bond. X @Au surface, and then covalently coupled between the carboxyl group of MPA and the amino group of 10-1276 antibody was achieved using the carbodiimide crosslinking method; antibody-coupled Zn / WO X @Au can specifically capture Clostridium difficile toxin B (Tcd B) antigen. Simultaneously, the detection line on the LFIA test strip is also modified with 10-1274 antibody, enabling it to specifically capture Tcd B antigen. The Tcd B antigen is then mixed with Zn / WO4. X After the mixture of @Au-antibodies was added to the test strip, a 10-minute chromatography process was performed to determine the Zn / WO4 antibody composition. X @Au is firmly captured on the T-line, generating a colorimetric signal. For clarity, Zn / WO will be used. X The detection mode of the @Au reductase-mimicking nanozyme nanotag is defined as colorimetric mode (CM); next, 30 µL of 0.0122 mol / L chloroplatinic acid solution was added to the test strip and allowed to migrate, followed by irradiation of the T region with an AM 1.5 light source (light power: 320 mW) for 5 minutes. Using Zn / WO3... X@Au reductase-mimicking activity and photocatalytic effect enable the rapid in-situ growth of Pt nanoclusters on Au seeds, forming Zn / WO3 with POD-mimicking activity. X @Au@Pt nanozymes. Although extending the irradiation time could further increase the Pt loading, 5 minutes of irradiation was chosen to meet POCT requirements and maintain high catalytic activity. The deposition of Pt nanoclusters further enhanced the colorimetric signal generated in the CM, and this new stage was defined as enhanced colorimetric mode ECM; the prepared TMB chromogenic solution was added to the functionalized Zn / WO3 solution. X @Au@Pt POD simulates the T region of nanozymes. Highly efficient POD-like activity rapidly oxidizes colorless TMB into a blue product, further amplifying the colorimetric signal. This final stage is defined as catalytically enhanced colorimetric mode (CECM). This two-step strategy, first deploying small-sized nanotags for migration, and then growing fully functional nanotags in situ at the T line, effectively avoids the flow blockage problem commonly encountered with pre-loaded high-content nanotags. This confirms the successful loading of Au onto Zn / WO3. X Pt was deposited on and subsequently onto Zn / WO3. X On @Au, a series of characterization techniques were performed, including transmission electron microscopy, high-resolution TEM, energy-dispersive X-ray spectroscopy, and selected area electron diffraction. Figure 12 Image b shows the Zn / WO3 ratio after 15 minutes of ultrasound-assisted reduction. X TEM images of @Au confirm the successful loading of Au seeds; SAED (Self-Enhanced Electrochemical Emission) was performed on the dark nanoparticles. Figure 12 (b1), the diffraction spots obtained ( Figure 12 b2) is labeled as the Au crystal plane. Further IFFT analysis revealed the lattice fringes corresponding to the Au crystal plane ( Figure 12 The interplanar spacing (b3) was measured to be 2.356 Å, consistent with the standard value for Au. SAED and corresponding IFFT lattice fringe analyses were also performed on the nanowire region (e.g., [missing information]). Figure 13 As shown), this further confirms the Zn / WO3 ratio. X The structure belongs to W 18 O 49 Crystal phase. Furthermore... Figure 12 The EDS mapping shown in Figure c visually confirms the presence of Au in Zn / WO3. X The presence on the carrier. For Zn / WO X @Au@Pt nanotags (by using Zn / WO) X The synthesis of Pt by photoinduced deposition over 5 minutes (@Au) was fully characterized. Figure 12 The TEM image of the middle d clearly shows nanoparticles anchored on the nanowires; Figure 12The close-up image of d1 clearly reveals the growth of a second phase composed of Pt nanoclusters around the Au seed crystals; SAED and IFFT analyses of the characteristic region identify diffraction spots and lattice fringes corresponding to the Au and Pt crystal planes. Figure 12 (d2 and d3) confirmed the successful deposition of Pt on Au seed crystals. Figure 12 The EDS mapping of Pt in Zn / WO3 also intuitively verifies that Pt in Zn / WO3... X The presence of Pt nanoclusters on an Au substrate. Furthermore, by extending the photoinduced deposition time to 60 minutes, the heterogeneous growth of Pt nanoclusters around the Au seeds can be observed more clearly, such as... Figure 14 As shown.

[0045] Beyond stepwise operation and in-situ growth, the more significant aspect of the two-step cascade enhanced colorimetric strategy lies in its ability to continuously amplify the colorimetric signal. The transformation from CM to ECM and then to CECM fundamentally depends on the catalytic activity of the precursor material. Therefore, selecting Zn / WO3 with the narrowest band gap and the highest oxygen vacancy concentration is crucial. X As the best foundation and playing a key role. First, Zn / WO X The sample's abundant oxygen vacancy sites and narrow bandgap facilitated loading higher-density Au seeds during a short 15-minute ultrasonic treatment, theoretically resulting in a stronger colorimetric signal in CM; secondly, Zn / WO X The high density of Au seed sites on the @Au surface enables the subsequent deposition of more Pt nanoclusters; in addition, Zn / WO X The narrow bandgap enhances Zn / WO X The Au heterostructure's efficient use of light promotes the generation of photogenerated charge carriers, enabling Pt to rapidly nucleate and grow around Au seeds within 5 minutes of irradiation, resulting in a stronger ECM colorimetric signal and higher catalytic activity. For example... Figure 15 As shown in a, b, and c, it can be observed that based on Zn / WO X The sample with (Zn / W=0.05) achieved a higher Au seed loading, thus supporting the deposition of more Pt nanoclusters. Using Zn / WO X High Zn / WO content formed on the substrate after 5 minutes of light irradiation X @Au@Pt ensures sufficient catalyst availability. From a kinetic perspective, in Zn / WO X The heterojunction formed in Au–Pt facilitates a more efficient directional transfer of thermally balanced and unbalanced charge carriers to Pt, thereby optimizing the catalytic efficiency of Pt for substrate molecules. Figure 15 (d). DFT-based Bader charge analysis and charge density difference calculations further validated this charge transfer path. Figure 15 As shown in e, approximately 7.10 e comes from Zn / WO. XThe electrons are transferred to Au, followed by approximately 1.37 e from Au to Pt. This is to experimentally verify the Zn / WO3-based... X Zn / WO X @Au@Pt nanotags exhibit excellent POD-like catalytic activity, and quantitative EPR tests were performed (measuring the homolytic cracking of H2O2). Figure 15 (f) and enzyme kinetics assay using H2O2 and TMB as substrates ( Figure 15 (g and h), comparing Zn / WO3 ratios of 0.00, 0.05, and 0.10. X The prepared sample. For example... Figure 15 As shown in Figure f, equal masses of various Zn / WO3... X @Au@Pt nanotags, after being added to an equal volume of H₂O₂ and reacting for 1 minute, captured and detected ·OH radicals. The results indicate that, based on Zn / WO₂... X Zn / WO X @Au@Pt most effectively catalyzes the homolytic cleavage of H2O2 to generate ·OH, producing the strongest EPR signal corresponding to the DMPO-·OH adduct. Enzyme kinetics experiments using TMB and H2O2 as substrates are shown below. Figure 15 As shown in g and h, at low concentrations, absorbance is linearly related to substrate concentration according to the Beer-Lambert law. Therefore, the OD value at 655 nm obtained from the UV-Vis spectrum of oxidized TMB reflects the change in substrate concentration. Figure 15 The Michaelis-Menten curves in g and h represent the oxidation rates of TMB at different concentrations of H₂O₂ and TMB, respectively. The kinetic parameters Vmax and Km extracted from the Michaelis-Menten curves represent the maximum reaction rate (catalytic efficiency) and the Michaelis constant (substrate affinity), respectively. The results indicate that, based on Zn / WO₂... X (Zn / W=0.05) Zn / WO X @Au@Pt exhibits the smallest Km and the largest Vmax for both H2O2 and TMB substrates, demonstrating its most effective POD-like catalytic activity.

[0046] like Figure 16 As shown in Figure a, the nanotags used in CECM are achieved through a sequential material construction process based on CM and ECM, serving as the most critical element in the entire detection process and making a major contribution to the enhancement of the colorimetric signal; using Zn / WO4 X and Zn / WO X @Au was used as a control group to further verify Zn / WO X @Au@Pt Due to Zn / WO X The heterogeneous interface formed in the –Au–Pt system exhibits stronger POD-like activity. Firstly, a Zn / WO3 interface was theoretically constructed. X ( Figure 16 in b), Zn / WO X @Au( Figure 16 in c) and Zn / WO X @Au@Pt( Figure 16 The atomic structure models in d). Based on first-principles calculations, the thermodynamic feasibility and kinetic processes of the three materials for catalyzing the homolytic cleavage of H2O2 were evaluated, as shown in Figure 16 e to g. All three materials satisfy the thermodynamic conditions for the homolytic cleavage of H2O2, meaning the reaction can proceed spontaneously. However, Zn / WO X @Au@Pt exhibits significantly lower energy barriers in the rate-determining step of H2O2 homolytic cleavage, which is kinetically favorable for more efficient generation of ·OH radicals. In the TMB colorimetric system based on POD activity, the rate of H2O2 homolytic cleavage to generate ·OH directly determines the oxidation and color development rate of TMB. Therefore, theoretically, it is expected that Zn / WO X @Au@Pt can produce the strongest colorimetric signal; subsequently, EPR spectra were used to compare the ·OH generation capabilities of equal masses of Zn / WO X 、Zn / WO X @Au and Zn / WO X @Au@Pt after reacting with the same amount of H2O2 for the same time. As shown in Figure 16 h, all three materials can catalyze H2O2 to produce ·OH, but the sample containing Zn / WO X @Au@Pt shows the strongest ·OH capture signal, indicating the highest catalytic efficiency for ·OH generation; finally, enzyme kinetics assays with TMB and H2O2 as substrates were performed to further evaluate the catalytic efficiencies of the three materials, as shown in Figure 16 i and j. For the H2O2 substrate, Zn / WO X @Au@Pt shows the smallest Km and the largest Vmax, indicating the highest affinity and catalytic efficiency for H2O2, and thus the strongest POD-like activity. For the TMB substrate, Zn / WO X @Au@Pt exhibits the smallest Km, indicating the strongest affinity for TMB. However, its lowest Vmax value indicates that under limited H2O2 conditions, the direct oxidation efficiency of Zn / WO X @Au@Pt for TMB is lower than that of the other two materials. This observation is consistent with the proposed mechanism, that is, Zn / WO X @Au@Pt relies on its efficient catalysis of H2O2 homolytic cleavage to generate ·OH, which then oxidizes TMB. This confirms that Zn / WO X @Au@Pt acts as a POD mimetic enzyme with high specificity and efficient catalytic function.

[0047] According to a fourth aspect of the present invention, an application of the cascade amplification method described in any of the preceding claims in the detection of Clostridium difficile toxin B is provided.

[0048] The application of the cascade amplification method of this invention can improve the visual detection limit of Clostridium difficile toxin B by 500 times compared with the initial colorimetric mode, and the fitted detection limit can reach 0.01 ng / mL. This solves the technical problem of difficulty in rapid and highly sensitive detection of Clostridium difficile toxin B in the prior art, and achieves the technical effect of significantly enhancing the colorimetric signal and realizing highly sensitive colorimetric LFIA detection while maintaining the ease of LFIA operation.

[0049] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0050] Example A method for preparing highly efficient POD-mimicking nanozymes includes the following steps: 0.198 g of WCl6 was dissolved in 60 mL of anhydrous ethanol. A 1 M ZnCl2 solution dispersed in anhydrous ethanol was added to the above solution. The resulting mixture was transferred to a 100 mL high-pressure reactor and reacted overnight at 180°C for 12 hours. The precipitate was collected, washed twice alternately with anhydrous ethanol and deionized water, and dried in the dark in air to obtain ZnCl2. 2+ Doped WO X , for Zn / WO X ; 8 mg of the synthesized Zn / WO3 was added. X Add 30 mL of a mixed solvent (ethanol and water in a 1:1 ratio), sonicate for 15 minutes, add 400 μL of chloroauric acid solution, and sonicate for 15 minutes to load Au seed crystals onto the Zn / WO3 mixture. X The product was washed twice with anhydrous ethanol and deionized water by alternating centrifugation to obtain Zn / WO4. X @Au; The Zn / WO obtained in the previous step X The Au precipitate was redispersed in 30 mL of a mixed solvent (ethanol and water in a 1:1 ratio) and sonicated. 200 μL of chloroplatinic acid solution (1 g / 100 mL) was added and thoroughly mixed. Under AM 1.5 light (320 mW) irradiation, Pt nanoclusters were reduced and loaded onto Au seeds to form Zn / WO3. X @Au@Pt yielded a highly efficient POD-mimicking nanozyme.

[0051] Application examples A Zn-based 2+ Doped WOX The application of nanozyme cascade amplification method in the detection of Clostridium difficile toxin B (Tcd B) includes the following steps: like Figure 17 As shown in Figure a, using Zn / W = 0.05 Zn / WO X Prepared Zn / WO X After the @Au-antibody nanotag binds to the TcdB antigen, it is mixed with the running buffer and then added to the sample pad. After 10 minutes of migration, the mixture passes through the detection line and the control line. The 10-1274 antibody pre-immobilized on the T line captures the nanotag, producing a visible colorimetric signal (in contrast, no colorimetric signal is observed when testing negative samples that do not contain the TcdB antigen). The detection pattern produced by this process is called CM. Subsequently, 30 μL of a 0.0122 mol / L chloroplatinic acid solution dispersed in a 1:1 ethanol-water mixture was added to the sample pad and allowed to migrate for 5 minutes. The T region was then irradiated with an AM 1.5 light source, which promoted the formation of Pt nanoclusters in Zn / WO3. X @Au reductase mimics in-situ reduction and growth on nanozymes to form Zn / WO X @Au@Pt nanozymes (nanotags), this light-induced Pt deposition to enhance the detection mode after colorimetric signal is defined as ECM; Figure 17 Figure a1 shows photographs of test strips obtained by CM and ECM in detecting different concentrations of Tcd B. The visual detection limit of CM is 50 ng / mL, while that of ECM is 10 ng / mL, which indicates that the visual detection sensitivity is improved by 5 times. Figure 17 Figure a2 shows the gray values ​​of the T zone of test strips with different Tcd B concentrations under ECM mode and their corresponding fitting curves. The detection limit based on the gray values ​​reaches 5 ng / mL. After ECM treatment, add 1 µL of colorimetric solution (prepared by mixing 3 µL of 200 mM H2O2 and 10 µL of 200 mM TMB) to the T zone of the test strip. Take a picture after approximately 1 minute of reaction. Figure 17 As shown in Figure b, if the sample is positive, due to Zn / WO X The presence of @Au@PtPOD mimic enzymes causes TMB to be oxidized and change color, resulting in a further enhancement of the colorimetric signal; otherwise, there is no change. This detection mode that enhances the colorimetric signal through TMB oxidation based on POD mimic enzymes is defined as CECM. Figure 17 Image b1 shows a photograph of the test strips detecting different concentrations of Tcd B in CECM mode. The visual detection limit reached 0.1 ng / mL, which means that the sensitivity is 100 times higher than ECM and 500 times higher than CM. Figure 17 b2 shows from Figure 17 The gray values ​​of different Tcd B concentrations in the T region extracted from b1 under CECM mode and their corresponding fitting curves were obtained. The detection limit based on the gray value fitting reached 0.01 ng / mL. Figure 17 Figure c shows the gray values ​​extracted from the T region after five repeated detections of 50 ng / mL Tcd B in CM, ECM and CECM modes. The relative standard deviation of the five gray values ​​in each mode is less than 2%, indicating that all three modes have excellent detection reproducibility and the two-step cascaded enhancement colorimetric strategy has high consistency. Figure 17 The image shows the detection of four enterotoxins (100 ng / mL) in CM, ECM, and CECM modes: TcdB, Clostridium perfringens type A toxin, cholera toxin B, Staphylococcus aureus enterotoxin B, and two enterotoxin-producing bacteria (10 ng / mL). 6 The CFU / mL values ​​were extracted from the T region for Bacillus cereus and enterotoxigenic Escherichia coli, respectively. The results fully demonstrate the high specificity of each detection mode and the consistency of the two-step cascaded enhanced colorimetric strategy.

[0052] In summary, in this invention, Zn / WO X The optimized band structure between Au and Pt promotes the best dynamic properties and charge transfer efficiency, guiding the directional transfer of electrons to Pt, which significantly improves the Zn / WO3 ratio. X The catalytic performance of @Au@Pt increases the visual detection limit of Clostridium difficile toxin B by 500 times, and the fitted detection limit of Clostridium difficile toxin B based on grayscale analysis also reaches 0.01 ng / mL. This solves the technical problem of rapid and highly sensitive detection of Clostridium difficile toxin B in existing technologies, and achieves the technical effect of significantly enhancing the colorimetric signal and realizing highly sensitive colorimetric LFIA detection while maintaining the ease of operation of LFIA.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-efficiency mimic POD nanoszyme, characterized in that, The structure is as follows: Zn / WO X @Au@Pt; The nanoenzyme is Zn / WO X @Au in situ deposition of Pt constitutes; The Zn / WO X @Au is Zn loaded with Au nanoparticles 2+ Doped WO X .

2. The method for preparing high-efficiency mimic POD nanoszyme according to claim 1, characterized in that, The method comprises the following steps: Zn 2+ doped to the non-stoichiometric WO X , obtaining Zn 2+ doped WO X , as Zn / WO X ; Loading of Au nanoparticles to Zn / WO X Zn / WO X @Au; Pt nanoclusters were deposited in-situ on Zn / WO X @Au seeds to obtain high-efficiency POD mimicking nanozyme, which is Zn / WO X @Au@Pt.

3. The preparation method according to claim 2, characterized in that, The Au nanoparticles are loaded onto Zn / WO X The method includes an ultrasonic-assisted reduction method.

4. The production method according to claim 2, characterized by, The Pt nanoclusters are deposited in situ on Zn / WO X The method on Au seeds of Au includes a photo-induced reduction method.

5. A method of cascode amplification, characterized by, The method comprises the following steps: The nano-enzyme prepared by the method of any one of claims 1-4 is used to construct a detection system through an LFIA platform, and the catalytic colorimetric detection performance is enhanced.

6. The method of claim 5, wherein, The cascade amplification method comprises the following steps: Zn / WO X @Au generates initial colorimetric signal by immunochromatography, then forms Zn / WO X @Au@Pt to amplify colorimetric signal, followed by enzymatic color development.

7. The method of claim 6, wherein the step of amplifying the signal comprises the step of: The enzymatic color development is performed through a substrate reaction catalyzed by a peroxidase.

8. Use of the cascade amplification method of any one of claims 5-7 in detection of Clostridium difficile toxin B.

9. Use according to claim 8, characterized in that, The visual detection limit of the Clostridium difficile toxin B is increased by at least 500 times.

10. Use according to claim 8, characterized in that, Based on gray scale analysis, the fitting detection limit of the Clostridium difficile toxin B reaches 0.01 ng / mL.

Images