Fe-ZnO (at) Au-DTNB nano-label with regulated electronic structure and preparation method and application of Fe-ZnO (at) Au-DTNB nano-label

Abstract

The invention provides a Fe-ZnO (at) Au-DTNB nano-label with an electronic structure regulated and controlled as well as a preparation method and application of the Fe-ZnO (at) Au-DTNB nano-label, and relates to the technical field of lateral flow immunoassay. The nano-label comprises iron-doped zinc oxide, and the iron-doped zinc oxide is loaded with gold nano-particles. The nano-label further comprises a Raman reporter molecule DNTB, one end of the Raman reporter molecule DNTB is connected with the gold nano-particles through an Au-S bond, and the other end of the Raman reporter molecule DNTB is coupled with the A-influenza-resistant labeled antibody through modified carboxyl. According to the preparation method, the electronic structure of ZnO is adjusted through iron doping, so that surface charges of Fe-ZnO (at) Au-DTNB are enriched; the surface hot spots and the SERS activity are enhanced, so that the excellent performance is given to the LFIA detection system based on the Fe-ZnO (at) Au-DTNB.

Description

Technical Field

[0001] This invention relates to the field of lateral flow immunoassay technology, and in particular to an electronically structure-regulated Fe-ZnO@Au-DTNB nanotag, its preparation method, and its application. Background Technology

[0002] Respiratory infectious diseases are mainly caused by a variety of pathogenic microorganisms. These diseases are characterized by rapid transmission. They not only pose a serious threat to public health but can also lead to huge economic losses and have a significant impact on health, economic, and social stability. Effective strategies for dealing with infectious diseases caused by pathogens include controlling the source of infection, interrupting transmission routes, and protecting susceptible populations. Among these, controlling the source of infection is particularly crucial. This requires accurate identification of infected and non-infected individuals in the population to achieve early detection, timely isolation, and treatment.

[0003] Currently, the main diagnostic methods for infectious diseases include enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). However, these methods typically require specialized personnel in strictly controlled laboratories using specialized equipment, resulting in drawbacks such as long processing times, high costs, and complex procedures, limiting their application as rapid diagnostic tools for controlling the source of infection during epidemics. Point-of-care testing (POCT) refers to rapid clinical testing performed near the patient and the person being tested. It effectively solves a series of problems existing in traditional clinical laboratory procedures, such as cumbersome steps, long processing times, and the inability to perform rapid on-site testing. POCT meets the higher speed and efficiency requirements of modern society's rapid development in medical diagnosis. Immunochromatographic analysis (ICA, also known as LFIA) is a POCT method, widely used due to its simple operation, visualized results, rapid testing (usually within 30 minutes), and the absence of specialized equipment.

[0004] In recent years, researchers have combined traditional colloidal gold-based LFIA technology with novel nanomaterial labels to overcome its inherent limitations, such as the inability to quantify and low sensitivity. However, the detection sensitivity of SERS-LFIA mainly relies on the surface-enhanced Raman scattering (SERS) effect, which plays a crucial role in improving the sensitivity of traditional colloidal gold LFIA. SERS enhancement is primarily contributed by two mechanisms: electromagnetic (EM) enhancement and chemical (CM) enhancement. The complex coupling between these two mechanisms poses a challenge to further improving the analytical performance of SERS-LFIA.

[0005] Currently, most SERS substrates used for detection are still composed of a single noble metal. Although noble metals possess strong electromagnetic enhancement capabilities, they still have significant drawbacks: they are expensive, exhibit high activity only on rough surfaces, and their enhancement potential is difficult to fully exploit. Novel SERS substrates formed by composites of noble metals and emerging semiconductors are attracting widespread attention because they combine both EM and CM enhancement mechanisms, significantly improving SERS sensitivity. In particular, semiconductor-noble metal heterostructures (such as MnO3, TiO2, ZnO, Cu2O, and perovskite composites with Au or Ag) can promote charge transfer (CT) by modulating the electronic structure of the substrate through band alignment. This structure effectively couples the localized surface plasmon resonance (LSPR) effect of noble metals with the charge transfer resonance mechanism of semiconductors. This synergistic EM-CM interaction can significantly enhance the sensing performance of traditional SERS substrates and greatly improve the sensitivity of SERS-LFIA systems.

[0006] However, some studies have attempted to enhance LSPR by loading noble metal nanoparticles with high density through organic adhesive layers. However, due to the insulating properties of the organic layer, it almost completely blocks the interfacial interaction between the substrate and the noble metal, thus hindering the possibility of modulating noble metal LSPR through substrate properties. Furthermore, many intrinsic substrates, due to the limited number of surface active sites, further limit the loading capacity of Au nanoparticles and their ability to regulate electronic structure. For example, ZnO, due to its excellent biocompatibility, is a highly promising SERS substrate material; however, intrinsic ZnO, with its few surface active sites and wide band gap, exhibits limited performance in both Au NP loading capacity and electronic structure regulation.

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

[0008] The purpose of this invention is to provide an electronically structure-modulated Fe-ZnO@Au-DTNB nanotag, its preparation method, and its applications. The nanotag of this invention utilizes the abundant active sites and narrow bandgap provided by Fe-doped ZnO, enabling it to densely load LSPR-active gold nanoparticles (Au NPs) and generate a strong ZnO-Au-DTNB charge transfer effect, thereby exhibiting significantly enhanced surface-enhanced Raman scattering (SERS) performance. Based on this, this invention constructs an LFIA detection system with both colorimetric and SERS modes, which exhibits high specificity and excellent repeatability, while also achieving ultra-high sensitivity.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides an electronically structure-controlled Fe-ZnO@Au-DTNB nanotag, wherein the nanotag comprises iron-doped zinc oxide and gold nanoparticles are loaded onto the iron-doped zinc oxide. Furthermore, the iron enters the zinc oxide lattice by replacing zinc, and the iron in the iron-doped zinc oxide includes Fe. 2+ and Fe 3+ Two valence states; Furthermore, the nanotag also includes a Raman reporter molecule DNTB, one end of which is connected to the gold nanoparticle via an Au-S bond, and the other end is coupled to an anti-influenza A labeling antibody via a modified carboxyl group.

[0010] Furthermore, the iron-doped zinc oxide has a rough, porous nanoflower structure.

[0011] Furthermore, the pore size of the iron-doped zinc oxide is 15~19 nm.

[0012] Furthermore, the specific surface area of ​​the iron-doped zinc oxide is 60~75 m². 2 / g.

[0013] Furthermore, the amount of iron doping in the iron-doped zinc oxide is 2.5~3.0 wt%.

[0014] Furthermore, the loading of gold nanoparticles in the nanotag is 10~20 wt%.

[0015] In a second aspect, the present invention provides a method for preparing Fe-ZnO@Au-DTNB nanotags with electronically regulated structure as described in the first aspect, the method comprising: (1) Zinc chloride, cuprous oxide, polyvinylpyrrolidone and ethanol aqueous solution are mixed to obtain a mixture; ferric chloride solution is added to the mixture, followed by sodium thiosulfate solution, and the reaction is carried out. After dehydration heat treatment, Fe-ZnO is obtained. (2) Disperse the Fe-ZnO in an aqueous ethanol solution to obtain a dispersion; add chloroauric acid solution to the dispersion and perform a first ultrasonic treatment to obtain Fe-ZnO@Au; mix the Fe-ZnO@Au and DTNB in ​​an ethanol solution and perform a second ultrasonic treatment to obtain Fe-ZnO@Au-DTNB; (3) The Fe-ZnO@Au-DTNB, EDC and NHS are mixed and activated to obtain Fe-ZnO@Au-DTNB activated by carboxyl groups; then mixed with anti-AH influenza labeling antibody and coupled; finally mixed with bovine serum albumin and non-specific blocking reaction to obtain the electronically structured Fe-ZnO@Au-DTNB nanotag.

[0016] Further, in step (1), the mass ratio of zinc chloride, cuprous oxide, polyvinylpyrrolidone, ferric chloride, and sodium thiosulfate is (2~4):(9~12):(0.1~0.5):(1~3):(610~650).

[0017] Furthermore, in step (1), the reaction temperature is 20~30℃ and the reaction time is 0.5~2 h.

[0018] Further, in step (1), the temperature of the dehydration heat treatment is 200~300℃, the heating rate of the dehydration heat treatment is 1~10℃ / min, and the time of the dehydration heat treatment is 1~2 h.

[0019] Further, in step (2), the mass ratio of Fe-ZnO to chloroauric acid is (0.6~1):(9~18).

[0020] Further, in step (2), the molar ratio of Fe-ZnO@Au to DTNB is (1~1.5):(2~5).

[0021] Furthermore, in step (2), the temperature of the first ultrasonic treatment and the second ultrasonic treatment are each 20~30℃, the power is each 100~200 W, and the time is each 0.5~2 h.

[0022] Further, in step (3), the molar ratio of Fe-ZnO@Au-DTNB, EDC and NHS is (2~3):(1~3):(2~3).

[0023] Further, in step (3), the mass ratio of the carboxyl-activated Fe-ZnO@Au-DTNB, the anti-H1N1 labeled antibody, and bovine serum albumin is (0.1~0.5):(0.01~0.05):(10~40).

[0024] Further, in step (3), the activation reaction is carried out under ultrasonic treatment, the temperature of the activation reaction is 20~30℃, the power is 100~200 W, and the time is 10~20 min.

[0025] Furthermore, in step (3), the coupling reaction is carried out under shaking on a shaker at a temperature of 20-30°C, a shaking rate of 700-900 rpm, and a time of 2.5-3 h.

[0026] Furthermore, in step (3), the non-specific blocking reaction is carried out under shaking conditions, with the temperature of the non-specific blocking reaction being 20~30℃, the shaking rate being 700~900 rpm, and the time being 1~2 h.

[0027] Thirdly, the present invention provides the application of the electronically structure-modulated Fe-ZnO@Au-DTNB nanotag as described in the first aspect in the preparation of products for detecting H1N1.

[0028] Furthermore, the product includes SERS lateral flow immunochromatographic test strips.

[0029] Fourthly, the present invention provides a detection kit comprising an electronically structure-regulated Fe-ZnO@Au-DTNB nanotag as described in the first aspect.

[0030] Furthermore, the electronically structure-regulated Fe-ZnO@Au-DTNB nanotag and immunochromatographic test paper; The immunochromatographic test strip includes a sample pad, an absorbent pad, and a nitrocellulose membrane; a detection line (T line) and a control line (C line) are sequentially arranged on the nitrocellulose membrane; the detection line (T line) is coated with an antibody, and the control line (C line) is coated with goat anti-mouse IgG antibody.

[0031] Furthermore, the concentration of goat anti-mouse IgG antibody on the quality control line C is 0.2~1.0 mg / mL, preferably 0.8 mg / mL.

[0032] Furthermore, the concentration of H1N1 antibody on the detection line T is 0.2~1.0 mg / mL, preferably 0.8 mg / mL.

[0033] Compared with the prior art, the present invention has the following beneficial effects: (1) The zinc oxide (ZnO) in the nanotag of the present invention has a large porosity structure and a high specific surface area. Doping it with iron (Fe) yields iron-doped zinc oxide (Fe-ZnO, FZ), thereby introducing more surface active sites and increasing the Fermi level (E). f and reduce the band gap (E) g ); (2) In the nanotag of the present invention, gold nanoparticles (Au NPs) loaded on iron-doped zinc oxide are directly anchored on iron-doped zinc oxide (Fe-ZnO, FZ). The large number of active sites provided by the substrate promotes the efficient in-situ nucleation and growth of Au NPs, while the good band matching enables the charge transfer dynamics in the system to be directionally regulated. Moreover, this structure not only promotes the migration of free charges from FZ to the Au interface, but also increases the defect state density in ZnO, thereby increasing the amount of charge transfer in the system. (3) The nanotags described in this invention are constructed using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a Raman reporter molecule with highly matched energy structure between the band structure of ZnO and FZ, to construct SERS nanotags. DTNB molecules are connected to Au NPs through Au-S bonds, and Au NPs are grown in situ on the FZ substrate by reduction, thereby ensuring that the assembled nanotags have good consistency at the FZ-Au-DTNB interface. Finally, the Schottky junction formed at the FZ-Au and DTNB-Au interface leads to charge accumulation on Au, which further enhances the LSPR effect and produces a significant synergistic enhancement effect. Attached Figure Description

[0034] 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.

[0035] Figure 1 This diagram illustrates the detection process and SERS enhancement mechanism of the SERS-based LFIA sensor of this invention.

[0036] Figure 2A The XRD patterns of FZ with different doping concentrations are shown in Test Example 1 of this invention.

[0037] Figure 2B This is a graph showing the contents of Zn and Fe in FZ with different doping concentrations, as measured by ICP in Test Example 1 of this invention.

[0038] Figure 2C This is a DSC and TG curve of FZ80 in Test Example 1 of the present invention.

[0039] Figure 2D This is an EPR analysis chart of oxygen vacancy content for different doping concentrations of FZ in Test Example 1 of this invention.

[0040] Figure 2EThis is a graph showing the zeta potential changes of different doping concentrations of FZ in Test Example 1 of the present invention.

[0041] Figure 2F This is a BET specific surface area curve of FZ with different doping concentrations in Test Example 1 of the present invention.

[0042] Figure 3 The above are X-ray photoelectron spectroscopy (XPS) characterization images of Fe 2p in Test Example 2 of this invention; where (A~C) are the XPS spectra of Fe 2p in FZ40, FZ80 and FZ120, respectively; (D) are the XPS spectra of Fe in FZ with different doping concentrations. 3+ / Fe 2+ XPS peak area ratio.

[0043] Figure 4 The image shows the SERS performance characterization of FZ@Au-DTNB in ​​Test Example 3 of this invention; where (A) is the TEM image of FZ0@Au; (B) is the TEM image of FZ40@Au; (C) is the TEM image of FZ80@Au; (D) is the TEM image of FZ120@Au; (E) is the HRTEM image of FZ80@Au; (F~H) are the SAED spectra of Au(1 1 1), ZnO(1 0 1) and ZnO(0 0 2), respectively; (I) is the SEM image of FZ0@Au; (J) is the SEM image of FZ40@Au; (K) is the SEM image of FZ80@Au; and (L) is the SEM image of FZ120@Au.

[0044] Figure 5A The SERS enhancement factor of FZ80@Au-DTNB in ​​Test Example 4 of this invention is shown (DTNB concentration is 0.025 mmol / L, and the DTNB concentration in FZ80@Au-DTNB is compared with that of pure DTNB, which is 0.6 mol / L).

[0045] Figure 5B The graph shows the SERS intensity and peak position variations of FZ0@Au, FZ40@Au, FZ80@Au, and FZ120@Au in Test Example 4 of this invention.

[0046] Figure 5C This is a graph showing the elemental content of FZ@Au at different doping concentrations in Test Example 4 of this invention.

[0047] Figure 5D This is a graph showing the SERS peak position changes of FZ0@Au, FZ40@Au, FZ80@Au and FZ120@Au in Test Example 4 of the present invention.

[0048] Figure 5EThis is a diagram showing the band structure and Fermi level positions of FZ0, FZ40, FZ80, FZ120, Au, and DTNB at different doping concentrations in Test Example 4 of this invention.

[0049] Figure 5F This is a schematic diagram of the SERS enhancement mechanism of FZ@Au-DTNB at the microscale in Test Example 4 of the present invention.

[0050] Figure 6 The figures shown are DFT and electromagnetic field simulation (full-wave simulation) diagrams for Test Example 5 of this invention; where (A) is the DOS simulation of ZnO; (B) is the DOS simulation of Fe-ZnO; (C) is the atomic-level interface contact model and Bader charge transfer analysis; (D) is the full-wave electromagnetic field simulation of closely packed Au NPs; (E) is the full-wave electromagnetic field simulation of Au NPs with larger spacing; and (F) is the full-wave electromagnetic field simulation of ZnO@Au.

[0051] Figure 7 The image shows the optimization results of the SERS-based LFIA sensor used for H1N1 detection in Test Example 6 of this invention. (A) represents the sample concentration; (B) represents the membrane coating concentration of the T-detection test line; (C) represents the amount of label used per test. LFIA performance analysis: (D) represents specificity (* P<0.1, ** P<0.01, *** P<0.001); (E) represents repeatability; (F) represents antigen detection sensitivity; (G) represents inactivated virus detection sensitivity; (H~I) represent clinical sample detection and the corresponding ROC curves. Detailed Implementation

[0052] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0053] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0054] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] In a first aspect, the present invention provides an electronically structure-controlled Fe-ZnO@Au-DTNB nanotag, wherein the nanotag comprises iron-doped zinc oxide and gold nanoparticles are loaded onto the iron-doped zinc oxide. Furthermore, the iron enters the zinc oxide lattice by replacing zinc, and the iron in the iron-doped zinc oxide includes Fe. 2+ and Fe 3+ Two valence states; Furthermore, the nanotag also includes a Raman reporter molecule DNTB, one end of which is connected to the gold nanoparticle via an Au-S bond, and the other end is coupled to an anti-influenza A labeling antibody via a modified carboxyl group.

[0056] In this invention, the zinc oxide (ZnO) in the nanotag possesses a large porosity structure and a high specific surface area; furthermore, by controlling the doping of Fe and its doping content (doping concentration), iron-doped zinc oxide (Fe-ZnO, FZ) is synthesized, thereby introducing more surface active sites and increasing the Fermi level (E). f and reduce the band gap (E) g Furthermore, this invention employs an in-situ reduction-growth strategy to assist in the loading of gold nanoparticles (Au NPs), enabling the Au NPs to be directly anchored on the FZ. The abundant active sites provided by the substrate promote the efficient in-situ nucleation and growth of the Au NPs, while the excellent bandgap matching allows for the directional regulation of charge transfer dynamics within the system. This structure not only promotes the migration of free charges from the FZ to the Au interface but also increases the defect state density in ZnO, thereby increasing the amount of charge transfer in the system.

[0057] Furthermore, this invention is based on the band structure of ZnO (conduction band E). c =-2.39 eV with E g =3.02 eV) and the band structure of FZ (E c Approximately -2.6 eV, E g (Approximately 2.9 eV), this invention selects the energy-structure highly matched Raman reporter molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, LUMO = -2.262 eV, E...). g =2.843 eV) to construct SERS nanotags. DTNB molecules are connected to Au NPs via Au-S bonds, and the Au NPs are grown in situ on the FZ substrate through reduction, thus ensuring good consistency of the assembled nanotags at the FZ-Au-DTNB interface. Finally, the Schottky junction formed at the FZ-Au and DTNB-Au interfaces leads to charge accumulation on Au, further enhancing the LSPR effect and producing a significant synergistic enhancement. Specifically, the SERS enhancement capability of Fe-ZnO@Au-DTNB (FZ@Au-DTNB) nanotags mainly originates from the following mechanisms: (i) Electromagnetic enhancement (EM) caused by Au LSPR: The highly active Fe and Zn sites in FZ have strong adsorption and reduction capabilities for Au precursors, enabling Au nanoparticles to be loaded in large quantities. (ii) FZ-Au-DTNB heterostructure effect: Under appropriate Fe doping conditions, the conduction band energy level of ZnO gradually shifts downward, reducing the energy barrier of ZnO-Au. At the same time, due to the narrowing of the band gap and the thermal excitation effect, the exciton energy in the valence band is converted into free electrons, which leads to further enhancement of surface charge accumulation of Au, thereby strengthening the LSPR effect. (iii) Charge transfer (CT) contribution of DTNB: The interaction between DTNB and the ZnO-Au interface causes a change in polarizability and enhances the Raman signal through charge transfer. In addition, the formation of the FZ-Au-DTNB heterojunction can reduce the nonradiative carrier recombination rate and prolong the carrier lifetime, both of which further contribute to the enhancement of the SERS effect.

[0058] As an optional implementation, the iron-doped zinc oxide has a rough porous nanoflower structure.

[0059] As an optional implementation, the pore size of the iron-doped zinc oxide is 15~19 nm, for example, it can be 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, etc.

[0060] As an optional implementation, the specific surface area of ​​the iron-doped zinc oxide is 60~75 m². 2 / g, for example, could be 60 m 2 / g、62 m 2 / g、64 m 2 / g、65 m 2 / g、66 m 2 / g、68 m 2 / g、70 m 2 / g、72 m 2 / g、74 m 2 / g、75 m 2 / g etc.

[0061] As an optional implementation, the amount of iron doping in the iron-doped zinc oxide is 2.5~3.0 wt%, for example, it can be 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, etc.

[0062] As an optional implementation, this invention successfully designed and prepared Fe-doped ZnO nanoparticles. By adding different volumes of FeCl3 (0, 40, 80, 120 μL), the corresponding Fe doping amounts in FZ were (0 wt%, 1.2 wt%, 2.6 wt%, 3.6 wt%), and the obtained Fe-doped ZnO nanoparticles were named FZ0, FZ40, FZ80, and FZ120, respectively.

[0063] It should be noted that after in-situ reduction of the Au NPs loaded with FZ0, FZ40, FZ80, and FZ120, there were significant differences in the enhancement effect of the SERS marker DTNB. Among them, FZ80@Au exhibited the strongest SERS signal under the same test conditions. When FZ80 was applied to a biosensor for H1N1 detection, the FZ80@Au-DTNB tag showed an extremely strong SERS signal on the LFIA test strip (Raman enhancement factor EF as high as 4×10⁻⁶). 8 After optimizing the composition of the flow buffer, the antibody concentration on the test line (T line), and the amount of SERS tag used in the LFIA system, high-sensitivity detection of H1N1 was achieved. The limit of detection for antigen visualization reached 10 pg / mL. -1 The fitted LOD was as low as 0.047 pg / mL. -1 The visual detection limit for inactivated viruses reached 10. 5 copies mL -1 The fitted LOD was 196.3 copies / mL. -1 Furthermore, the accuracy rate for testing 20 clinical samples reached 100%.

[0064] As an optional implementation, the loading of gold nanoparticles in the nanotag is 10~20 wt%, for example, it can be 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, etc.

[0065] In a second aspect, the present invention provides a method for preparing Fe-ZnO@Au-DTNB nanotags with electronically regulated structure as described in the first aspect, the method comprising: (1) Zinc chloride, cuprous oxide, polyvinylpyrrolidone and ethanol aqueous solution are mixed to obtain a mixture; ferric chloride solution is added to the mixture, followed by sodium thiosulfate solution, and the reaction is carried out. After dehydration heat treatment, Fe-ZnO is obtained. (2) Disperse the Fe-ZnO in an aqueous ethanol solution to obtain a dispersion; add chloroauric acid solution to the dispersion and perform a first ultrasonic treatment to obtain Fe-ZnO@Au; mix the Fe-ZnO@Au and DTNB in ​​an ethanol solution and perform a second ultrasonic treatment to obtain Fe-ZnO@Au-DTNB; (3) The Fe-ZnO@Au-DTNB, EDC and NHS are mixed and activated to obtain Fe-ZnO@Au-DTNB activated by carboxyl groups; then mixed with anti-AH influenza labeling antibody and coupled; finally mixed with bovine serum albumin and non-specific blocking reaction to obtain the electronically structured Fe-ZnO@Au-DTNB nanotag.

[0066] As an optional implementation, in step (1), the mass ratio of zinc chloride, cuprous oxide, polyvinylpyrrolidone, ferric chloride, and sodium thiosulfate is (2~4):(9~12):(0.1~0.5):(1~3):(610~650).

[0067] As an optional implementation, in step (1), the reaction temperature is 20~30℃, for example, it can be 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc., and the reaction time is 0.5~2 h, for example, it can be 0.5 h, 0.6 h, 0.8 h, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.

[0068] As an optional implementation, in step (1), the temperature of the dehydration heat treatment is 200~300℃, for example, it can be 200℃, 210℃, 220℃, 230℃, 240℃, 250℃, 260℃, 270℃, 280℃, 290℃, 300℃, etc., the heating rate of the dehydration heat treatment is 1~10℃ / min, for example, it can be 1℃ / min, 2℃ / min, 3℃ / min, 4℃ / min, 5℃ / min, 6℃ / min, 7℃ / min, 8℃ / min, 9℃ / min, 10℃ / min, etc., and the time of the dehydration heat treatment is 1~2 h, for example, it can be 1 h, 1.2 h, 1.4 h, 1.5 h, 1.6 h, 1.8 h, 2 h, etc.

[0069] As an optional implementation, in step (2), the mass ratio of Fe-ZnO to chloroauric acid is (0.6~2):(9~18).

[0070] As an optional implementation, in step (2), the molar ratio of Fe-ZnO@Au to DTNB is (1~1.5):(2~5).

[0071] As an optional implementation, in step (2), the temperature of the first ultrasonic treatment and the second ultrasonic treatment are each independently 20~30℃, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc., and the power is each independently 100~200 W, for example, 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 160 W, 170 W, 180 W, 190 W, 200 W, etc., and the time is each independently 0.5~2 h, for example, 0.5 h, 0.6 h, 0.8 h, 1 h, 1.2 h, 1.4 h, 1.5 h, 1.6 h, 1.8 h, 2 h, etc.

[0072] As an optional implementation, in step (3), the molar ratio of Fe-ZnO@Au-DTNB, EDC and NHS is (2~3):(1~3):(2~3).

[0073] As an optional implementation, in step (3), the mass ratio of the carboxyl-activated Fe-ZnO@Au-DTNB, the anti-influenza A labeling antibody, and bovine serum albumin is (0.1~0.5):(0.01~0.05):(10~40).

[0074] As an optional implementation, in step (3), the activation reaction is carried out under ultrasonic treatment. The temperature of the activation reaction is 20~30℃, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc., and the power is 100~200 W, for example, 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 160 W, 170 W, 180 W, 190 W, 200 W, etc., and the time is 10~20 min.

[0075] As an optional implementation, in step (3), the coupling reaction is carried out under shaking on a shaker. The temperature of the coupling reaction is 20~30℃, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc. The shaking rate is 700~900 rpm, for example, 700 rpm, 750 rpm, 800 rpm, 850 rpm, 900 rpm, etc., and the time is 2.5~3h, for example, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, etc.

[0076] As an optional implementation, in step (3), the non-specific blocking reaction is carried out under shaking. The temperature of the non-specific blocking reaction is 20~30℃, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc., the shaking rate is 700~900 rpm, for example, 700 rpm, 750 rpm, 800 rpm, 850 rpm, 900 rpm, etc., and the time is 1~2 h, for example, 1 h, 1.2 h, 1.4 h, 1.5 h, 1.6 h, 1.8 h, 2 h, etc.

[0077] Thirdly, the present invention provides the application of the electronically structure-modulated Fe-ZnO@Au-DTNB nanotag as described in the first aspect in the preparation of products for detecting H1N1.

[0078] As an optional implementation, the product includes SERS lateral flow immunochromatographic test strips.

[0079] Fourthly, the present invention provides a detection kit comprising an electronically structure-regulated Fe-ZnO@Au-DTNB nanotag as described in the first aspect.

[0080] As an optional implementation, the electronically structure-regulated Fe-ZnO@Au-DTNB nanotag and immunochromatographic test paper are used. The immunochromatographic test strip includes a sample pad, an absorbent pad, and a nitrocellulose membrane; a detection line (T line) and a control line (C line) are sequentially arranged on the nitrocellulose membrane; the detection line (T line) is coated with an antibody, and the control line (C line) is coated with goat anti-mouse IgG antibody.

[0081] In this invention, a high-performance LFIA detection platform was constructed through scientific material selection, structural design, and bandgap engineering, which can be used for point-of-care testing (POCT) diagnosis of pathogens. It also provides new ideas for the design of LFIA nanotags (such as...). Figure 1 (As shown).

[0082] This invention designs a novel Fe-ZnO@Au-DTNB nanotag. The abundant active sites and narrow bandgap provided by Fe-doped ZnO enable high-density loading of LSPR-active gold nanoparticles (Au NPs), resulting in a strong ZnO-Au-DTNB charge transfer effect and thus exhibiting significantly enhanced surface-enhanced Raman scattering (SERS) performance. Based on this, we constructed an LFIA detection system with both colorimetric and SERS modes, exhibiting high specificity and excellent repeatability while achieving ultra-high sensitivity. In the detection of H1N1 antigen and inactivated virus, the limits of detection (LOD) in the colorimetric visualization mode reached 10 pg / mL and 10 pg / mL, respectively. 5 The LOD was reduced to 0.047 pg / mL and 196.3 copies / mL in SERS mode; furthermore, the LOD was reduced to 196.3 copies / mL in SERS mode. In addition, the detection of 20 clinical samples in SERS mode showed 100% accuracy. This invention provides a new strategy for further improving the detection sensitivity of LFIA by doping and modulating the electronic structure of novel nanomaterials.

[0083] As an optional implementation, the concentration of goat anti-mouse IgG antibody on the quality control line C is 0.2~1.0 mg / mL, for example, it can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, etc., preferably 0.8 mg / mL.

[0084] As an optional implementation, the concentration of H1N1 antibody on the detection line T is 0.2~1.0 mg / mL, for example, it can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, etc., preferably 0.8 mg / mL.

[0085] 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.

[0086] Example 1 This embodiment provides an electronically structure-modulated Fe-ZnO@Au-DTNB nanotag, which is prepared by the following steps: (1) Preparation of Cu2O: 0.17 g of copper chloride dihydrate (CuCl2·2H2O) was dissolved in 100 mL of deionized water, forming a clear blue solution at 55°C. Then, the solution was magnetically stirred at 55°C, and 10 mL of 2 M sodium hydroxide (NaOH) aqueous solution was added. The mixture was stirred for 30 min, resulting in a darkened solution. Next, 10 mL of 0.6 mol / L ascorbic acid solution was added dropwise to the mixed solution, forming a brick-red precipitate. The mixture was stirred at 55°C for 3 h, then cooled. Finally, the precipitate was separated by filtration, dried overnight at 60°C, and stored in a sealed, dry, ventilated, and cool place.

[0087] (2) Preparation of Fe-doped ZnO In a 250 mL round-bottom flask, 50 mg of Cu₂O and 10 mg of zinc chloride (ZnCl₂) were added and mixed with 50 mL of ethanol / water (1:1) solution (0.1 mg PVP). The mixture was sonicated and stirred for 10 min to ensure uniform dispersion. Then, different volumes (40 / 80 / 120 μL) of 0.1 mol / L ferric chloride (FeCl₃) solution were added. Next, 20 mL of 1 M sodium thiosulfate (Na₂S₂O₃) solution was slowly added through an isobaric funnel for 1 h until the suspension changed from red to pale white. The suspension was then washed three times with water and ethanol, and the samples were separated and collected. The collected products were air-dried and transferred to a porcelain crucible. The obtained products were subjected to dehydration heat treatment in a muffle furnace, with the temperature increased to 250 °C (50 min) at a rate of 5 °C / min and held for 30 min to obtain the target products FZ40, FZ80, and FZ120, respectively.

[0088] (3) Preparation of FZ@Au-DTNB SERS probe: Fe-doped ZnO nanoparticles (FZ40, FZ80, and FZ120) were each added to 10 mL of solvent (ethanol and water in a 1:1 ratio) to prepare 1 mM solutions. After dispersion by ultrasonication for 5 min, the mixture was centrifuged and washed. 800 μL of 1% chloroauric acid (HAuCl4) solution was added to the nanoparticle solution, and ultrasonication was continued for 1 h until the solution turned purplish-black. Subsequently, the nanoparticles were washed twice with ethanol and dispersed in an appropriate amount of ethanol. 2.5 μL of 10 mmol / L DTNB ethanol solution was added to the solution, and ultrasonication was continued for another 1 h. After washing twice with ethanol, the solution was dispersed in an appropriate amount of ethanol to obtain the corresponding probes (FZ40@Au-DTNB SERS probe, FZ80@Au-DTNB SERS probe, and FZ120@Au-DTNB SERS probe).

[0089] (4) Preparation of the immuno-FZ@Au-DTNB SERS probe: Take 1 mL of the above ZnO SERS probe at a concentration of 1 mg / mL, wash with 0.05% PBST (PBS buffer to Tween volume ratio of 0.05%) and centrifuge. Then, add 500 μL of 10 mmol MES (MES solution at pH 5.5 containing a certain amount of Tween), followed by 50 μL of 10 mmol EDC (1.9 mg dissolved in 1 mL of water, molecular weight 190) and 100 μL of 10 mmol NHS (2.1 mg dissolved in 1 mL of solvent, molecular weight 210) for carboxyl activation, and sonicate for 15 min. After centrifugation, 400 μL of 0.05% PBST and 3 μL (4.5 mg / mL) of 37m modified antibody (anti-Anaerobic influenza antibody) were added, followed by sonication and shaking on a shaker (25℃, 800 rpm) for 2.5–3 h. Subsequently, 80–100 μL of 10% BSA was added for non-specific blocking, and shaking was continued (25℃, 800 rpm) for another 1.5 h. After washing with PBST, the sample was centrifuged and resuspended in 200 μL of 0.05% PBST and stored at 4℃.

[0090] Comparative Example 1 This comparative example provides a ZnO@Au-DTNB nanotag, which differs from Example 1 only in that, in step (2), the volume of the ferric chloride (FeCl3) solution is 0 mL, that is, no more Fe is added. The other steps are the same as in Example 1.

[0091] Application Example 1 This application example provides a detection kit, which includes: the electronically structure-regulated Fe-ZnO@Au-DTNB nanotag and an immunochromatographic test strip; the specific assembly method is shown below: (1) The immunochromatographic test strip (SERS LFA test strip) consists of a sample pad, an absorbent pad, and a nitrocellulose (NC) membrane, and has one control line (C) and three detection lines (T) for detecting H1N1 respiratory virus; the antibodies sprayed on the NC membrane include goat anti-mouse IgG (0.8 mg / mL) on the control line (C) and H1N1 antibody (0.8 mg / mL) on the detection line (T); subsequently, the antibody-modified NC membrane is dried in a constant temperature drying oven at 37°C for at least 2 h. The assembled PVC base plate, sample pad, NC membrane, and absorbent pad are cut into strips 3.0 mm wide and stored in a drying oven.

[0092] (2) Incubate 6.6 μL of H1N1 antigen, inactivated virus, or clinical sample with 0.4 μL of FZ80@Au-DTNB SERS probe (2 mg / mL) prepared in Example 1 for 1 min. Then, 7 μL of immune complex is suspended in 63 μL of running buffer (1% PBS-T (PBS:Tween-20 = 99:1):BSA:FBS = 7:2:1) and dropped onto the sample pad of the prepared LFA test strip; use a portable Raman system to capture and measure the SERS signal on the three detection lines with an excitation power of 10 mW, an excitation time of 5 s, and an excitation wavelength of 785 nm.

[0093] Test Example 1 Structural characterization of Fe-doped ZnO Test samples: FZ40, FZ80 and FZ120 provided in Example 1; FZ0 provided in Comparative Example 1.

[0094] Test methods: X-ray diffraction (XRD), energy dispersive spectroscopy (EDS), inductively coupled plasma (ICP), thermogravimetric-differential scanning calorimetry (TG-DSC), electron paramagnetic resonance (EPR), X-ray photoelectron spectroscopy (XPS), and specific surface area measurement (BET).

[0095] Test results: ZnO with a nanoflower-like morphology was synthesized by precipitation method. With increasing Fe doping concentration, the overall morphology of ZnO did not change significantly, but its structure gradually became more fragmented.

[0096] like Figure 2AAs shown, the X-ray diffraction (XRD) patterns of FZ0, FZ40, FZ80, and FZ120 are consistent with the standard card ZnO-PDF#00-003-0888, all exhibiting hexagonal crystal characteristics. Obvious diffraction peaks were observed at 2θ = 37.1°, 40.2°, 42.3°, 55.8°, 67.0°, 74.4°, 78.7°, 80.8°, and 82.2°, corresponding to the (100), (002), (101), (102), (110), (103), (200), (112), and (201) crystal planes, respectively, confirming the typical hexagonal wurtzite structure and indicating the successful preparation of ZnO.

[0097] Energy dispersive spectroscopy (EDS) showed that Fe was uniformly distributed on the surface of the doped sample. EDS further confirmed that Zn, O, and Fe were uniformly distributed on the material surface.

[0098] like Figure 2B As shown, the ratio of Fe to Zn in the sample is as follows: with increasing doping concentration, the Fe content increases while the percentage of Zn decreases. This indicates that Fe enters the lattice in ZnO in the form of substitutional doping, that is, Fe replaces part of Zn, which is consistent with the EDS results, indicating that Fe was successfully incorporated into ZnO.

[0099] After Fe doping, the crystal plane positions of ZnO in the XRD pattern did not shift, but the intensity of the diffraction peaks gradually decreased. Simultaneously, new diffraction peaks appeared at 2θ = 21.3° and 33.3°, corresponding to the (100) and (310) crystal planes of FeO(OH), but their intensity was very weak. This indicates that Fe doping did not generate a large number of new phases, and the phase composition of the system was consistent with expectations. As the Fe doping amount further increased, the crystal structure of ZnO was gradually destroyed, its diffraction peaks decreased and gradually disappeared, macroscopically manifesting as the decomposition of the overall structure. This is mainly due to Fe entering the ZnO lattice and replacing Zn, causing ZnO to gradually transform into an amorphous structure.

[0100] like Figure 2C As shown, thermogravimetric-differential scanning calorimetry (TG-DSC) tests were performed on FZ80 without dehydration heat treatment; the endothermic peaks below 100℃ mainly originated from the volatilization of residual ethanol solvent. An exothermic peak for the crystallization of Zn(OH)₂ particles appeared at around 129.8℃, followed by the removal of water of crystallization at approximately 143.5℃, initiating the transformation to ZnO. A broad exothermic crystallization process occurred in the 300–400℃ range, due to the presence of a large amount of non-uniform amorphous ZnO in the sample. Therefore, samples obtained after hydrothermal treatment at approximately 250℃ still contain incompletely crystallized ZnO.

[0101] like Figure 2DAs shown, oxygen vacancies gradually decrease with increasing Fe doping concentration, which is consistent with the decrease in the proportion of oxygen vacancy peak area in X-ray photoelectron spectroscopy (XPS).

[0102] like Figure 2E As shown, the zeta potential decreases with increasing Fe doping concentration, which may also be due to a reduction in oxygen vacancies.

[0103] like Figure 2F As shown, with the increase of Fe doping amount, the specific surface area of ​​Fe-doped ZnO in the material gradually increases during this process.

[0104] Test Example 2 Elemental composition and chemical state of materials Test samples: FZ40, FZ80 and FZ120 provided in Example 1; FZ0 provided in Comparative Example 1.

[0105] Test method: X-ray photoelectron spectroscopy (XPS) characterization.

[0106] Test results: like Figure 3 The figure shows high-resolution XPS spectra in the Zn 2p region (approximately 1015–1055 eV), O 1s region (approximately 527–537 eV), and Fe 2p region (approximately 705–740 eV). In the Zn 2p spectrum, two characteristic peaks appear at 1021.85 eV and 1044.62 eV, corresponding to Zn 2p³ / 2 and Zn 2p¹ / 2, respectively. This is due to spin-orbit splitting, indicating that Zn is in the +2 valence state. The O 1s spectrum can be decomposed into three peaks at 530.6, 531.8, and 534.0 eV, corresponding to oxygen, oxygen vacancies, and adsorbed oxygen species or hydroxyl groups in the ZnO lattice. In the Fe-doped sample, a significant peak appears in the O 1s region at approximately 529.9 eV, attributed to the Fe-O bond, further indicating that Fe atoms have successfully entered the ZnO lattice by substituting Zn. The high-resolution Fe 2p spectrum shows peaks at 724.1 eV and 711.4 eV, corresponding to Fe24.1 eV and 711.4 eV, respectively. 3+ The peaks at 723.1 eV and 709.7 eV correspond to Fe1 / 2 and 2p3 / 2, respectively. 2+ The presence of 2p1 / 2 and 2p3 / 2 indicates the simultaneous presence of Fe in the material. 2+ and Fe 3+ Two valence states.

[0107] Meanwhile, combined XRD and XPS analysis results show that with increasing Fe doping concentration, the XRD diffraction peaks of ZnO shift to the left at low doping levels (e.g., FZ40); while with further increases in doping concentration, the diffraction peaks shift to the right. In the XPS spectrum of Fe, Fe...3+ / Fe 2+ The peak area ratio gradually increases, indicating that a certain proportion of Fe exists in the sample in the early stage of doping. 2+ This suggests that Fe is at low concentrations of doping. 2+ This is the primary doping form. According to Bragg's law, the leftward shift of the diffraction peak corresponds to lattice expansion in ZnO, which can be attributed to Fe. 2+ (0.077 nm) Ionic radius is greater than that of Zn 2+ (0.074 nm). This result indicates that Fe 2+ Fe successfully entered the ZnO lattice and accompanied by lattice expansion. With increasing doping concentration, Fe... 3+ It gradually became the main doping form. Because Fe 3+ (0.064 nm) Ionic radius is smaller than Zn 2+ The shrinkage of the ZnO unit cell causes an overall rightward shift of the diffraction peaks. This change in unit cell volume further validates the hypothesis that Fe is doped in ZnO via substitution.

[0108] Furthermore, using absorption spectroscopy (UV-Vis) and ultraviolet photoelectron spectroscopy (UPS), the band gap (E) of ZnO under different Fe doping concentrations was calculated. g ), Fermi level (E f The doping concentration of ZnO was analyzed, and its work function (W) and maximum valence band level (Ev) were also analyzed. As the doping concentration increased, the band gap of ZnO gradually decreased, while the Fermi level shifted upwards relative to the vacuum level, indicating a significant change in its electronic structure. These results demonstrate that the electronic structure of ZnO can be effectively controlled through doping, providing a theoretical basis for its application in the design of band structures for electronic devices.

[0109] Test Example 3 SERS performance of FZ@Au-DTNB Test samples: FZ40@Au-DTNB SERS probe, FZ80@Au-DTNB SERS probe and FZ120@Au-DTNB SERS probe provided in Example 1; FZ0@Au-DTNB SERS probe provided in Comparative Example 1.

[0110] Test methods: EDS test, transmission electron microscopy (TEM), scanning electron microscopy (SEM), high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) pattern.

[0111] Test results: To enhance the SERS signal of the LFIA sensor, this invention uses Fe-doped ZnO as a substrate to support the in-situ growth of Au nanoparticles (Au NPs). Au NPs were deposited on the ZnO surface using an ultrasound-assisted in-situ reduction method. EDS testing confirmed successful Au anchorage on the ZnO surface. Quantitative analysis showed that the Au content was highest in the FZ80 sample.

[0112] like Figure 4 A~ Figure 4 As shown in Figure L, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) characterized Au-loaded FZ0, FZ40, FZ80, and FZ120, revealing that the distribution density of Au NPs varied with Fe doping concentration. FZ80 exhibited the most uniform and dense distribution of Au nanoparticles. This may be attributed to the additional active sites provided by Fe ions, promoting the adsorption of more Au NPs. Furthermore, high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) patterns showed the presence of diffraction points corresponding to the (101) and (002) crystal planes of ZnO and the (111) crystal plane of Au in FZ80, further confirming the successful loading of Au NPs. Simultaneously, ZnO lattice distortion was also observed.

[0113] Test Example 4 Raman spectroscopy analysis of FZ@Au-DTNB Test samples: FZ40@Au-DTNB SERS probe, FZ80@Au-DTNB SERS probe and FZ120@Au-DTNB SERS probe provided in Example 1; FZ0@Au-DTNB SERS probe provided in Comparative Example 1.

[0114] Test method: Raman spectroscopy analysis.

[0115] Test results: In the nanotag described in this invention, DTNB is bonded to ZnO@Au via Au-S bonds. A ZnO@Au-DTNB SERS tag was prepared by ultrasonic treatment and then subjected to Raman spectroscopy analysis.

[0116] like Figure 5A As shown, at 1331 cm -1 A strong Raman peak appeared at the point, confirming the successful modification by DTNB and indicating a significant SERS enhancement effect.

[0117] like Figure 5B As shown, the SERS intensity gradually increases with increasing doping concentration, reaching a maximum at 80 μL, but gradually weakens at 120 μL.

[0118] first, Figure 5E The band structures of ZnO, Au, and DTNB with different doping levels are shown. Figure 5F The electromagnetic enhancement mechanism was explained. Under laser irradiation, ZnO and DTNB come into contact with Au, forming a Schottky junction due to the Fermi level difference between the n-type ZnO semiconductor and the metallic Au. This energy level difference drives charge transfer from ZnO to Au. Similarly, since the Fermi level of DTNB is higher than that of Au, electrons also transfer from DTNB to Au. These effects work together to increase the charge density on the Au surface, significantly enhancing the local electromagnetic field. Therefore, the polarizability of DTNB molecules adsorbed on the composite substrate increases, thereby improving the LSPR coupling efficiency of Au nanoparticles, amplifying the near-field enhancement effect, and directly enhancing the SERS signal of DTNB.

[0119] Under 785 nm laser irradiation, the photon energy is insufficient to excite intrinsic transitions from the valence band to the conduction band in ZnO. However, due to the subbandgap absorption of the semiconductor, a small number of electrons can still be excited from the valence band to form excitons. These excitons are thermally dissociated into free electrons due to the photothermal effect of ZnO. Based on the band structure and the position of the Fermi level, the material behaves as a substitution-doped n-type semiconductor. With increasing Fe doping concentration, the Fermi level gradually shifts towards the conduction band, indicating an increase in the free carrier density in the material. When the doping concentration reaches 80 μL, the Fermi level approaches the bottom of the conduction band, suggesting that the carrier concentration is close to saturation. Figure 5E These free carriers can also transfer to Au nanoparticles, further enhancing the interfacial electromagnetic field and synergistically strengthening the LSPR effect of Au.

[0120] Furthermore, SEM images revealed that the prepared ZnO possessed a rough, porous nanoflower structure. This rough surface and abundant protrusions contributed to enhanced local electromagnetic fields and inherently supported stronger surface plasmon resonance (SPR) effects. With increasing Fe doping concentration, both the Fe content and the specific surface area of ​​ZnO increased, providing more active sites for Au nanoparticle deposition. Quantitative analysis by EDS confirmed that the surface density of Au NPs was highest at a doping concentration of 80 μL, which significantly contributed to SPR enhancement. Figure 5C ).

[0121] However, when the doping concentration exceeds 80 μL, the structural integrity of the ZnO nanosheets decreases. The lattice becomes increasingly disordered, and the density of surface-loaded Au nanoparticles decreases. Although higher doping concentrations increase the specific surface area and introduce more Au nanoparticles... 3+While providing active sites for precursor adsorption and reduction, it also disrupts the ZnO crystal structure, weakening its catalytic reduction ability. The LSPR effect reaches its maximum at a doping concentration of 80 μL, after which the SERS intensity begins to decline due to structural degradation and reduced Au loading.

[0122] The chemical enhancement mechanism is supported by the observed changes in the position of the NO2-related Raman peak in DTNB. Figure 5D This indicates a hybridization interaction between DTNB molecules and the ZnO@Au substrate. This suggests charge transfer from DTNB to Au. Simultaneously, the built-in electric field at the ZnO-Au interface promotes partial injection of hot carriers from Au to ZnO, rather than into DTNB. This process helps suppress nonradiative recombination and prolong carrier lifetime, thereby minimizing the negative impact on DTNB charge transfer and enhancing the overall chemical contribution to the SERS effect.

[0123] Test Example 5 The effect of Fe doping on the electronic structure of ZnO Test method: Density functional theory (DFT).

[0124] Test results: This invention constructs undoped and Fe-doped ZnO models and calculates their densities of states (DOS) using density functional theory (DFT). Figure 6 A and Figure 6 (B) It is evident that spin-down Fe d orbital electrons introduce new defect levels near the Fermi level and conduction band edge. Furthermore, the Fe d orbitals reshape the valence band DOS of ZnO. At -5.0 eV, the hybridization contribution from Zn d orbital electrons is strongly suppressed, clearly indicating that Fe doping withdraws hybridization from the Zn-O sp orbitals, forming new Fe-O sp hybrid orbitals. Moreover, the asymmetric redistribution of DOS strongly depends on spin-up Fe d orbital electrons. These changes collectively contribute to the upward shift of the Fermi level to the conduction band minimum, leading to n-type doping and a narrowing of the band gap. The simulation results are consistent with experimental observations. Furthermore, the calculations of this invention also show that the charge transfer between doped ZnO-Au and DTNB-Au is 1.35 eV. - and 2.34e - This result is largely consistent with previous analyses in terms of the direction and amount of charge transfer. Figure 6 C). Furthermore, full-wave simulations conducted by this invention on Au nanoparticles (NPs) with different particle spacings and ZnO-Au surfaces show a significant enhancement of the surface electromagnetic field at the interface between the ZnO substrate and Au NPs. The large amount of charge accumulated on the Au surface through charge transfer further enhances the LSPR effect, thus verifying the proposed SERS enhancement mechanism (C). Figure 6D~ Figure 6 E).

[0125] Based on previous results, this invention selected ZF80@Au–DTNB, which exhibited the strongest SERS signal, as the subject of subsequent experiments. The enhancement performance of SERS is typically evaluated by the enhancement factor (EF), which ideally can be calculated using the following formula: EF = (I sulf / N sulf )×(I norm / N norm ). Among them, I sulf The SERS signal intensity obtained from the enhanced substrate, N sulf The concentration of substances contributing to the SERS signal; while I norm and N norm These represent the Raman signal intensity and substance concentration under unenhanced conditions, respectively. In practice, it is difficult to accurately determine the number of DTNB molecules successfully bound to the Au surface after multiple washing and treatment steps. Therefore, under strictly controlled experimental conditions, the calculated EF value of ZF80@Au is approximately 4 × 10⁻⁶. 8 ( Figure 5B This figure may underestimate the actual level of enhancement.

[0126] Test Example 6 Application of Immuno-FZ80@Au-DTNB in ​​LFIA test strips for respiratory virus detection Test sample: The immune-FZ@Au-DTNB SERS probe provided in Example 1.

[0127] Test results: like Figure 7 As shown, several key parameters in the detection process were optimized to achieve ultrasensitive detection of respiratory viruses using the FZ80@Au-DTNB SERS nanoprobe. The antibody concentration on the detection (T) line must be sufficient to effectively capture the antigen-antibody-nanoparticle immune complex while minimizing non-specific binding. The optimal spray concentration of the H1N1 detection antibody was 0.8 mg / mL, which exhibited the highest signal-to-noise ratio (SNR) and was therefore considered the optimal concentration. The optimal run buffer composition was 1% PBS-T (PBS:Tween-20 = 99:1) mixed with BSA and FBS in a 7:2:1 volume ratio. 0.4 μL (2 mg / mL) of the SERS tag was added for each test. After a 10-minute incubation period, a further 15-minute reaction period was allowed, sufficient for the formation of the virus-nanoparticle immune complex and its specific binding to the detection antibody on the test strip.

[0128] Detection capability evaluation based on optimized conditions: Under the optimized conditions described above, the detection capability of the proposed method was further evaluated by simultaneously testing low concentrations (0.1 ng / mL) and high concentrations (100 ng / mL) of respiratory viruses, including SARS, MERS, FLU B, PIV-I, PIV-II, PIV-III, and ADV, under the same conditions. The results are as follows: Figure 7 As shown in Figure D, the LFIA stripe for H1N1 exhibits a distinct dark T-line band and a corresponding strong SERS signal, while in other samples, neither the visible T-line signal nor the SERS signal was observed due to the lack of specific antigen. These results confirm that the synthetic FZ80@Au–DTNBSERS tag demonstrates excellent specificity in H1N1 detection.

[0129] Repeatability and stability testing: To evaluate the stability and repeatability of the SERS-based LFA strip, different concentrations of H1N1 virus were added to the samples, and five replicate tests were performed. All tests produced a visible signal and a strong SERS response at the T line, with relative standard deviations (RSD) of less than 10%, demonstrating the high repeatability and robustness of the detection method. Figure 7 E).

[0130] Sensitivity and quantification capability evaluation: The sensitivity and quantification capability of this method were evaluated by detecting different concentrations of H1N1 antigen. Figure 7 As shown in Figure F, the visual limit of detection (VDL) for H1N1 antigen using the FZ80@Au–DTNB strip kit is 10 pg / mL. This was determined by measuring the SERS intensity (1331 cm⁻¹) on the T line. -1 Quantitative analysis was completed. The SERS spectra in the figure correspond to the SERS signals of the T line of the reagent strip at different antigen concentrations. By plotting the relationship between SERS intensity and antigen concentration, a standard calibration curve for H1N1 was established.

[0131] Limit of Detection (LOD) Calculation: The LOD is calculated using the following formula according to the standards of the International Union of Pure and Applied Chemistry (IUPAC): .in, The blank control group was at 1331 cm. -1 The average SERS intensity, This is the standard deviation of the blank signal. Based on this calculation, the LOD of H1N1 is 0.047 pg / mL, demonstrating the high sensitivity of this SERS-based LFIA platform.

[0132] Sensitivity assessment of inactivated H1N1 virus detection: Figure 7G demonstrated the sensitivity of the FZ80@Au-DTNB SERS LFIA strip for detecting inactivated H1N1 virus. The visual detection limit for inactivated H1N1 was 10. 5 The LOD of inactivated H1N1 virus was calculated to be 196.3 copies / mL through signal-to-noise ratio analysis and extrapolation from the calibration curve, further demonstrating the high sensitivity of the SERS-based LFIA platform in detecting real biological samples.

[0133] Clinical Sample Testing: To further validate the practical performance of the SERS tag, clinical samples were tested using the proposed method. Throat swab samples were collected from 20 clinical patients and evaluated using the SERS testing platform. Photos of the corresponding test strips are shown below. Figure 7 As shown in Figure I, receiver operating characteristic (ROC) curve analysis revealed that the area under the curve (AUC) for the Raman-based detection method was 1.00, indicating 100% diagnostic accuracy. The FZ80@Au–DTNB SERS LFIA strip successfully achieved 100% accurate identification of clinical samples, demonstrating the significant potential of this method in practical diagnostic applications.

[0134] In summary, this invention developed a large specific surface area FZ80@Au-DTNB nanoprobe based on iron-doped ZnO, and demonstrated its significant SERS enhancement effect. It was successfully applied to the LFIA (lateral flow immunoassay) platform, achieving high-sensitivity detection of H1N1 virus.

[0135] Iron doping allows for precise modulation of the surface active sites and electronic structure of the ZnO substrate. Simultaneously, its large specific surface area and abundant active sites promote high-density loading of gold nanoparticles, generating numerous plasmonic "hot spots" on the surface and significantly enhancing the SERS signal. Furthermore, the heterostructure formed between FZ80, Au, and DTNB optimizes band alignment, promoting charge transfer and accumulation from FZ80 and DTNB to Au, further enhancing the local surface plasmon resonance (LSPR) effect of Au, thereby amplifying the SERS effect.

[0136] Therefore, these SERS nanoprobes achieved up to 4 × 10⁻⁶ 8 The enhancement factor demonstrated its potential in highly sensitive detection. Integrating the FZ80@Au–DTNB nanoprobe into the LFIA platform achieved highly sensitive detection of the H1N1 virus. Its visual limit of detection (VDL) was as low as 10 pg / mL, while the fitted limit of detection (LOD) for SERS detection mode was 0.047 pg / mL, achieving an approximately 200-fold increase in sensitivity. For inactivated virus detection, the VDL was 10... 5The fitted LOD was 196.3 copies / mL.

[0137] Furthermore, clinical validation using 20 clinical pharyngeal swab samples showed that the diagnostic accuracy of the testing platform was 100%. These results fully demonstrate the significant advantages of rationally designed and precisely engineered SERS probes in the LFIA platform, providing a powerful development strategy for next-generation high-sensitivity rapid diagnostic systems suitable for the rapid detection of pathogens such as H1N1.

[0138] 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 Fe-ZnO@Au-DTNB nanotag with electronically regulated structure, characterized in that, The nanotag comprises iron-doped zinc oxide, wherein the iron-doped zinc oxide is loaded with gold nanoparticles. Furthermore, the iron enters the zinc oxide lattice by replacing zinc, and the iron in the iron-doped zinc oxide includes Fe. 2+ and Fe 3+ Two valence states; Furthermore, the nanotag also includes a Raman reporter molecule DNTB, one end of which is connected to the gold nanoparticle via an Au-S bond, and the other end is coupled to an anti-influenza A labeling antibody via a modified carboxyl group.

2. The Fe-ZnO@Au-DTNB nanotag with electronically regulated structure according to claim 1, characterized in that, The iron-doped zinc oxide has a rough porous nanoflower structure. Preferably, the pore size of the iron-doped zinc oxide is 15~19 nm; Preferably, the specific surface area of ​​the iron-doped zinc oxide is 60-75 m². 2 / g; Preferably, the iron doping amount in the iron-doped zinc oxide is 2.5~3.0 wt%; Preferably, the loading of gold nanoparticles in the nanotag is 10~20 wt%.

3. A method for preparing Fe-ZnO@Au-DTNB nanotags with electronically regulated structure according to claim 1 or 2, characterized in that, The preparation method includes: (1) Zinc chloride, cuprous oxide, polyvinylpyrrolidone and ethanol aqueous solution are mixed to obtain a mixture; ferric chloride solution is added to the mixture, followed by sodium thiosulfate solution, and the reaction is carried out. After dehydration heat treatment, Fe-ZnO is obtained. (2) Disperse the Fe-ZnO in an aqueous ethanol solution to obtain a dispersion; add chloroauric acid solution to the dispersion and perform a first ultrasonic treatment to obtain Fe-ZnO@Au; mix the Fe-ZnO@Au and DTNB in ​​an ethanol solution and perform a second ultrasonic treatment to obtain Fe-ZnO@Au-DTNB; (3) The Fe-ZnO@Au-DTNB, EDC and NHS are mixed and activated to obtain Fe-ZnO@Au-DTNB activated by carboxyl groups; then mixed with anti-AH influenza labeling antibody and coupled; finally mixed with bovine serum albumin and non-specific blocking reaction to obtain the electronically structured Fe-ZnO@Au-DTNB nanotag.

4. The method for preparing the electronically structure-controlled Fe-ZnO@Au-DTNB nanotag according to claim 3, characterized in that, In step (1), the mass ratio of zinc chloride, cuprous oxide, polyvinylpyrrolidone, ferric chloride, and sodium thiosulfate is (2~4):(9~12):(0.1~0.5):(1~3):(610~650). Preferably, in step (1), the reaction temperature is 20~30℃ and the reaction time is 0.5~2 h; Preferably, in step (1), the temperature of the dehydration heat treatment is 200~300℃, the heating rate of the dehydration heat treatment is 1~10℃ / min, and the time of the dehydration heat treatment is 1~2 h.

5. The method for preparing the electronically structure-controlled Fe-ZnO@Au-DTNB nanotag according to claim 3, characterized in that, In step (2), the mass ratio of Fe-ZnO to chloroauric acid is (0.6~2):(9~18); Preferably, in step (2), the molar ratio of Fe-ZnO@Au to DTNB is (1~1.5):(2~5); Preferably, in step (2), the temperature of the first ultrasonic treatment and the second ultrasonic treatment are each independently 20~30℃, the power is each independently 100~200 W, and the time is each independently 0.5~2 h.

6. The method for preparing the electronically structure-controlled Fe-ZnO@Au-DTNB nanotag according to claim 3, characterized in that, In step (3), the molar ratio of Fe-ZnO@Au-DTNB, EDC and NHS is (2~3):(1~3):(2~3); Preferably, in step (3), the mass ratio of the carboxyl-activated Fe-ZnO@Au-DTNB, the anti-H1N1 labeled antibody, and bovine serum albumin is (0.1~0.5):(0.01~0.05):(10~40); Preferably, in step (3), the activation reaction is carried out under ultrasonic treatment, the temperature of the activation reaction is 20~30℃, the power is 100~200 W, and the time is 10~20 min; Preferably, in step (3), the coupling reaction is carried out under shaking on a shaker, the temperature of the coupling reaction is 20~30℃, the shaking rate is 700~900 rpm, and the time is 2.5~3 h; Preferably, in step (3), the nonspecific blocking reaction is carried out under shaking, the temperature of the nonspecific blocking reaction is 20~30℃, the shaking rate is 700~900 rpm, and the time is 1~2 h.

7. The application of an electronically structure-modulated Fe-ZnO@Au-DTNB nanotag according to claim 1 or 2 in the preparation of products for detecting H1N1; And / or, the product includes SERS lateral flow immunochromatographic test strips.

8. A test kit, characterized in that, The detection kit includes the electronically structure-modulated Fe-ZnO@Au-DTNB nanotag as described in claim 1 or 2.

9. The detection kit according to claim 8, characterized in that, The detection kit includes: the electronically structure-regulated Fe-ZnO@Au-DTNB nanotag and immunochromatographic test strip; The immunochromatographic test strip includes a sample pad, an absorbent pad, and a nitrocellulose membrane; a detection line (T line) and a control line (C line) are sequentially arranged on the nitrocellulose membrane; the detection line (T line) is coated with an antibody, and the control line (C line) is coated with goat anti-mouse IgG antibody.

10. The detection kit according to claim 9, characterized in that, The concentration of goat anti-mouse IgG antibody on the quality control line C is 0.2~1.0 mg / mL, preferably 0.8 mg / mL; The concentration of H1N1 antibody on the detection line T is 0.2~1.0 mg / mL, preferably 0.8 mg / mL.

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