Zno, zno@au-dtnb nanotags containing oxygen vacancies, and preparation method and application thereof

Abstract

The application provides a kind of oxygen vacancy-containing ZnO, ZnO@Au-DTNB nano label and its preparation method and application, it is related to lateral flow immunoassay (LFIA) detection technology.The preparation method of the oxygen vacancy-containing ZnO includes: in alkaline reducing agent system, Zn salt and cubic Cu2O are reacted to obtain cage-like Zn (OH) 2;The cage-like Zn (OH) 2 is annealed to obtain three-dimensional ZnO;The three-dimensional ZnO is photochemically etched to obtain the oxygen vacancy-containing ZnO.The application realizes the defect engineering regulation of zinc oxide by photochemical etching, and the prepared band structure can be adjusted, and the ZnO@Au loaded with gold nanoparticles exhibits excellent surface-enhanced Raman scattering performance and catalytic activity.At the same time, a multi-mode LFIA detection system is constructed for pathogen instant detection, which has good repeatability, rapid detection, high specificity and high detection sensitivity.

Description

Technical Field

[0001] This invention relates to lateral flow immunoassay (LFIA) detection technology, and more particularly to a ZnO or ZnO@Au-DTNB nanotag containing oxygen vacancies, its preparation method, and its application. Background Technology

[0002] Rapid and efficient bacterial monitoring is of great significance in disease diagnosis, medical prevention, food safety, public health, and environmental protection. Currently, commonly used bacterial detection methods include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and various biosensor technologies. While PCR and ELISA technologies offer high sensitivity and specificity, their cumbersome procedures make them unsuitable for point-of-care testing (POCT), and the reliance on high-precision manufacturing for some biosensors significantly increases usage costs. Therefore, LFIA technology, which combines low cost, high sensitivity, integration, and portability, has become a research hotspot in the POCT field.

[0003] The design and optimization of nanotags are crucial for improving detection performance. In recent years, LFIA has gradually developed novel detection modes, such as catalytic nanotags, fluorescent nanotags, and surface-enhanced Raman scattering (SERS) nanotags, by combining them with nanomaterials with different functions. These novel nanotags have enabled superior detection performance. This demonstrates that multifunctional nanotags can achieve multi-mode detection and multi-functional applications. For example, loading gold (Au) and platinum (Pt) nanoparticles (NPs) onto conventional nanotags can simultaneously endow them with SERS activity and catalytic function; however, these two properties inherently have an inverse relationship.

[0004] Furthermore, although theoretically Au can be reduced and adsorbed through atomic-level surface active sites... 3+ Precursor methods can achieve higher density Au NP loading compared to pre-synthesized Au seed deposition. However, most intrinsic materials lack sufficient natural active sites, and the formation of these sites often requires extreme processing conditions. Even with high-density active sites, their catalytic performance is not necessarily excellent. Although reducing agents can promote Au... 3+ It can grow, but may simultaneously passivate active sites, ultimately affecting the loading density of Au NPs.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One objective of this invention is to provide ZnO nanotags containing oxygen vacancies, ZnO@Au-DTNB nanotags, their preparation methods, and applications. This invention achieves defect engineering control of zinc oxide (ZnO) through photochemical etching technology. The prepared ZnO@Au composite material with tunable band structure and loaded gold nanoparticles exhibits excellent surface-enhanced Raman scattering (SERS) performance and catalytic activity. Furthermore, this invention constructs a system with both bacterial detection and photocatalytic sterilization functions using the ZnO@Au-DTNB nanotags for the immediate detection of pathogens. This system combines good repeatability, rapid detection, high specificity, and high detection sensitivity; it also integrates in-situ sterilization functionality with three detection modes: colorimetric visual recognition mode, catalytic colorimetric enhanced visual recognition mode, and SERS detection mode.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a method for preparing oxygen-vacancy-containing ZnO, the method comprising:

[0010] (a) In an alkaline reducing agent system, Zn salt reacts with cubic Cu2O to obtain cage-like Zn(OH)2;

[0011] (b) Annealing the cage-like Zn(OH)2 to obtain three-dimensional ZnO;

[0012] (c) The three-dimensional ZnO is subjected to photochemical etching to obtain ZnO containing oxygen vacancies.

[0013] In this invention, cubic Cu₂O is first reacted with Zn salt in an alkaline reducing agent system to form cage-like Zn(OH)₂, which is then converted to ZnO by annealing. That is, cubic Cu₂O particles are used as a substrate, and Zn… 2+ A substitution reaction and annealing process were used to obtain three-dimensional ZnO with a nanostructure. The morphology of the three-dimensional ZnO consisted of a mixture of nanoparticles, nanorods, and nanosheets, forming a multi-level structure. Subsequently, a one-step photochemical etching method was used to further increase the reaction sites and regulate the electronic structure of ZnO.

[0014] As an optional implementation, in step (a), the cage-like Zn(OH)2 is specifically prepared by the following steps:

[0015] ZnCl2, cubic Cu2O and PVP were dispersed in an aqueous ethanol solution to obtain a mixture; Na2S2O3 solution was added dropwise to the mixture to carry out the reaction, and then washed, filtered and dried to obtain cage-like Zn(OH)2.

[0016] As an optional implementation, the mass ratio of ZnCl2, cubic Cu2O, PVP, and Na2S2O3 is (2-5):(10-20):(0.333-1):(632-1264); wherein, "2-5" can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.; "10-20" can be, for example, 10, 12, 14, 16, 18, 20, etc.; "0.333-1" can be, for example, 0.333, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.; "632-1264" can be, for example, 632, 700, 800, 900, 1000, 1100, 1200, 1264, etc.

[0017] As an optional implementation, the volume ratio of ethanol to water in the ethanol-water solution is (0.5-2):1, for example, it can be 0.5:1, 0.6:1, 0.8:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, etc.

[0018] As an optional implementation, the reaction temperature is 20-30°C, for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the reaction time is 1-3 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, etc.

[0019] As an optional implementation, in step (a), the cubic Cu2O is specifically prepared by the following steps:

[0020] Under heating conditions, an aqueous solution of NaOH is added to an aqueous solution of CuCl2 to carry out a precipitation reaction and obtain a mixed solution; ascorbic acid solution is added dropwise to the mixed solution to carry out a reduction reaction, and then the solution is washed, filtered and dried to obtain the cubic Cu2O.

[0021] As an optional implementation, the mass ratio of CuCl2, NaOH, and ascorbic acid is (0.1-0.3):(0.1-1):(1-2); wherein, "0.1-0.3" can be, for example, 0.1, 0.15, 0.2, 0.25, 0.3, etc.; "0.1-1" can be, for example, 0.1, 0.2, 0.4, 0.6, 0.8, 1, etc.; and "1-2" can be, for example, 1, 1.2, 1.4, 1.5, 2, etc.

[0022] As an optional implementation, the temperature of the precipitation reaction is 50-60°C, for example, 50°C, 52°C, 54°C, 55°C, 56°C, 58°C, 60°C, etc., and the time of the precipitation reaction is 20-40 min, for example, 20 min, 22 min, 24 min, 25 min, 26 min, 28 min, 30 min, 32 min, 34 min, 35 min, 36 min, 38 min, 40 min, etc.

[0023] As an optional implementation, the temperature of the reduction reaction is 50-60°C, for example, 50°C, 52°C, 54°C, 55°C, 56°C, 58°C, 60°C, etc., and the time of the reduction reaction is 2-4 hours, for example, 2 hours, 2.5 hours, 2.8 hours, 3 hours, 3.2 hours, 3.5 hours, 4 hours, etc.

[0024] As an optional implementation, in step (b), the annealing process is performed in an air environment.

[0025] As an optional implementation, the heating rate of the annealing treatment is 1 to 10°C / min, for example, 1°C / min, 2°C / min, 4°C / min, 5°C / min, 6°C / min, 8°C / min, 10°C / min, etc.; the annealing temperature is 200 to 300°C, for example, 200°C, 220°C, 240°C, 250°C, 260°C, 280°C, 300°C, etc.; and the annealing time is 20 to 40 min, for example, 20 min, 22 min, 25 min, 30 min, 32 min, 35 min, 40 min, etc.

[0026] As an optional implementation, step (c) specifically includes the photochemical etching process:

[0027] Three-dimensional ZnO was dispersed in a photochemical etching solution to obtain a dispersion; the dispersion was then subjected to photo-irradiation to obtain ZnO containing oxygen vacancies.

[0028] As an optional implementation, the concentration of three-dimensional ZnO in the dispersion is 0.5 to 5 mg / mL, for example, it can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, etc.

[0029] As an optional implementation, the photochemical etching solution comprises, by mass percentage: 20-40% K2B4O7, 1-5% Na2SO3, and the balance being water.

[0030] In this invention, a mixed aqueous solution of potassium tetraborate (K₂B₄O₇) and sodium sulfite (Na₂SO₃) is used as the etching environment. On one hand, the weakly alkaline buffer solution composed of K₂B₄O₇ can stabilize the reaction system, and on the other hand, it facilitates the etching process through the presence of borate ions (B₄O₇). 2- The complexation of Zn maintains 2+ On the one hand, sulfite can promote the removal of lattice oxygen induced by photogenerated holes in ZnO, and act as a reducing agent to prevent the occurrence of reverse oxidation reaction.

[0031] As an optional implementation, based on the total mass of the photochemical etching solution as 100%, the content of K2B4O7 is 20% to 40%, for example, it can be 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32%, 34%, 35%, 36%, 38%, 40%, etc.

[0032] As an optional implementation, the content of Na2SO3 is 1-5% based on the total mass of the photochemical etching solution as 100%, for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.

[0033] As an optional implementation, the photochemical etching process uses an AM1.5 light source to simulate sunlight.

[0034] As an optional implementation, the center optical power of the illumination treatment is 310-330mW, for example, it can be 310mW, 312mW, 314mW, 316mW, 318mW, 320mW, 322mW, 324mW, 326mW, 328mW, 330mW, etc.

[0035] As an optional implementation, the light treatment time is 1 to 60 minutes, for example, it can be 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, etc.

[0036] As an optional implementation, the light treatment time is 20 to 40 minutes, for example, it can be 20 minutes, 22 minutes, 24 minutes, 25 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 35 minutes, 36 minutes, 38 minutes, 40 minutes, etc.

[0037] In this invention, by controlling the photochemical etching time, the Fermi level (EF) of ZnO is reduced while maximizing the oxygen vacancy concentration. This not only lays the foundation for achieving a strong LSPR effect with high-density Au NPs loading, but also further enhances the possibility of charge transfer at the DTNB interface. Furthermore, the differences in electronic structure and oxygen vacancy quantity of ZnO with different etching times have a significant impact on the loading of Au NPs and the SERS effect.

[0038] In a second aspect, the present invention provides an oxygen-vacancy-containing ZnO, which is prepared by the preparation method described in the first aspect.

[0039] Thirdly, the present invention provides a ZnO@Au-DTNB nanotag, wherein the ZnO@Au-DTNB nanotag comprises ZnO with oxygen-containing vacancies loaded with Au nanoparticles; wherein the ZnO with oxygen-containing vacancies comprises ZnO with oxygen-containing vacancies as described in the second aspect; and the ZnO@Au-DTNB nanotag further comprises DNTB, one end of the DTNB being connected to the Au nanoparticles via an Au-S bond, and the other end being connected to a type I antibody via a modified carboxyl group.

[0040] In this invention, based on the synthesized ZnO band structure (conduction band EC = -2.889 eV, band gap Eg = 2.991 eV), a compatible Raman reporter molecule, 5,5'-disulfide bis(2-nitrobenzoic acid) (DTNB, lowest unoccupied molecular orbital LUMO = -2.262 eV, Eg = 2.843 eV), was selected to ensure that both the ZnO-DTNB and ZnO-Au-DTNB interfaces formed staggered energy level arrangements to promote directional charge transfer of DTNB. Ultimately, the Schottky contact induced by the ZnO-Au and DTNB-Au interfaces led to charge accumulation on Au, further amplifying the LSPR effect and producing synergistic enhancement. Furthermore, the formation of the two heterostructures, ZnO-DTNB (staggered) and ZnO-Au-DTNB (Z-type), significantly promoted photogenerated charge separation, optimized the redox potential, and substantially improved the photocatalytic activity of the nanotags.

[0041] Fourthly, the present invention provides a ZnO@Au-DTNB nanotag as described in the second aspect, wherein the preparation method of the ZnO@Au-DTNB nanotag includes:

[0042] (1) Disperse ZnO containing oxygen vacancies in an aqueous ethanol solution to obtain a dispersion; add HAuCl4 aqueous solution to the dispersion, sonicate, centrifuge and wash to obtain ZnO@Au;

[0043] (2) The ethanol solution of ZnO@Au and the ethanol solution of DTNB are mixed, ultrasonically treated, and then centrifuged and washed to obtain ZnO@Au-DTNB;

[0044] (3) The ZnO@Au-DTNB, EDC and NHS are mixed and reacted to activate the carboxyl group of DTNB; then mixed with type I antibody and coupled to obtain the ZnO@Au-DTNB nanotag.

[0045] like Figure 1 The diagram illustrates the preparation process of the ZnO@Au-DTNB SERS tag and its integration with LFIA technology. First, ZnO containing oxygen vacancies is dispersed in an ethanol-water solution, followed by the addition of chloroauric acid (HAuCl4) solution, and then ultrasonic treatment to obtain the ZnO@Au. Subsequently, the obtained ZnO@Au is bound to the Raman reporter molecule DTNB via an Au-S bond under ultrasonic assistance to obtain the ZnO@Au-DTNB nanotag. Next, a type I antibody (MA1-10708) is linked to DTNB via an activation coupling reaction using EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysulfosuccinimide sodium salt) to obtain the ZnO@Au-DTNB nanotag.

[0046] In this invention, to enhance the SERS signal of the LFIA sensor, a photochemically etched ZnO substrate is used as the substrate to load Au nanoparticles, thereby achieving an electromagnetic field enhancement effect. Ultrasonic-assisted in-situ deposition of Au nanoparticles is a convenient method for preparing ZnO-loaded Au (ZnO@Au). Under ultrasonic assistance, ZnO, with its piezoelectric catalytic properties, can generate a large number of electron-hole pairs, thereby accelerating the deposition of Au nanoparticles. 3+ Reduction deposition.

[0047] As an optional implementation, in step (1), the mass ratio of the oxygen-containing vacancy Zn to HAuCl4 is 1:(1 to 1.5), for example, it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.

[0048] As an optional implementation, in step (1), the temperature of the ultrasonic treatment is 20-25°C, for example, 20°C, 22°C, 24°C, 25°C, etc., the power of the ultrasonic treatment is 100-120W, for example, 100W, 105W, 110W, 115W, 120W, etc., and the time of the ultrasonic treatment is 0.5-2h, for example, 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.5h, 1.6h, 1.8h, 2h, etc.

[0049] As an optional implementation, step (1) further includes a resuspension step after washing: resuspending the ZnO@Au in an ethanol solution to obtain an ethanol solution of ZnO@Au-DTNB.

[0050] As an optional implementation, in step (2), the molar ratio of ZnO@Au to DTNB is (5~20):(0.001~0.004); where “5~20” can be, for example, 5, 10, 15, 20, etc.; and “0.001~0.004” can be, for example, 0.001, 0.002, 0.003, 0.004, etc.

[0051] As an optional implementation, in step (2), the temperature of the ultrasonic treatment is 20-25°C, for example, 20°C, 22°C, 24°C, 25°C, etc., the power of the ultrasonic treatment is 100-120W, for example, 100W, 105W, 110W, 115W, 120W, etc., and the time of the ultrasonic treatment is 0.5-2h, for example, 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.5h, 1.6h, 1.8h, 2h, etc.

[0052] As an optional implementation, step (2) further includes a resuspension step after washing: resuspending the ZnO@Au-DTNB in ​​an ethanol solution to obtain an ethanol solution of ZnO@Au-DTNB;

[0053] As an optional implementation, step (3) specifically includes the following steps:

[0054] Carboxyl-activated ZnO@Au-DTNB, PBST buffer solution, and type I antibody are mixed and reacted for 2.5–3 h (e.g., 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h, 3 h, etc.); then BSA is added and reacted for 1–2 h (e.g., 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.); after washing, the ZnO@Au-DTNB nanotag is obtained.

[0055] As an optional implementation, in step (3), the mass ratio of the carboxyl-activated ZnO@Au-DTNB, type I antibody, and BSA is (6-9):(0.006-0.012):(8-12); where “6-9” can be, for example, 6, 6.5, 7, 7.5, 8, 8.5, 9, etc.; where “0.006-0.012” can be, for example, 0.006, 0.008, 0.010, 0.012, etc.

[0056] Fifthly, the present invention provides the application of the oxygen-vacant ZnO or the ZnO@Au-DTNB nanotag described above in the preparation of products for detecting bacteria and / or for in-situ sterilization.

[0057] In a sixth aspect, the present invention provides a system that combines bacterial detection and photocatalytic sterilization functions, the system comprising: the ZnO@Au-DTNB nanotag, immunochromatographic test strip, and TMB colorimetric enhancement system;

[0058] The immunochromatographic test strip includes: a plastic backing plate, on which a glass fiber sample pad, a nitrocellulose membrane, and an absorbent pad are sequentially disposed; a detection line (T line) and a control line (C line) are sequentially disposed on the nitrocellulose membrane; the detection line (T line) is coated with type II antibody, and the control line (C line) is coated with goat anti-mouse IgG antibody;

[0059] The TMB colorimetric enhancement system includes: a device for simulating a sunlight source and a colorimetric solution; the colorimetric solution includes TMB acetone solution, H2O2 and NaAc-HAc buffer.

[0060] In this invention, the ZnO@Au-DTNB nanotag exhibits an extremely strong SERS signal (enhancement factor EF as high as 10⁹) on the LFIA test strip. Furthermore, by optimizing the running buffer composition, the antibody concentration at the detection line (T), and the amount of SERS tag used in the LFIA system, highly sensitive detection of Staphylococcus aureus (S. aureus, SA) is achieved, with a limit of detection (LOD) as low as single digits CFU / mL. In addition, thanks to the photocatalytic activity of the ZnO@Au-DTNB nanotag, the LOD of the catalytic colorimetric enhanced visual recognition mode is improved by 100 times compared to the traditional colorimetric visual recognition mode (from 10⁹ CFU / mL). 4 CFU / mL decreased to 10 2 (CFU / mL). Under solar spectral excitation, the ZnO@Au-DTNB tag on the LFIA test strip can simultaneously achieve in-situ photocatalytic sterilization, providing a clean and simple solution for post-detection contamination control. This study, through rational material selection and electronic structure design, developed a highly efficient and multifunctional LFIA detection platform, providing a high-performance solution for POCT pathogen diagnosis and offering new insights into the design of LFIA nanotags.

[0061] like Figure 1As shown, when using a system that combines bacterial detection and photocatalytic sterilization, the ZnO@Au-DTNB nanotag (carrying type I antibody) is mixed with the antigen and added to the immunochromatographic test strip for detection. The assembled test strip has type II antibody (10-S30B) and goat anti-mouse IgG pre-modified on the test line (T line) and control line (C line). When the SERS tag carrying the antigen flows through the test line, it is captured in an immune sandwich manner, and a valid signal can be detected on the test line by visual observation or Raman spectroscopy. Conversely, if no valid signal is detected on the test line, it is considered negative. Unlike high-sensitivity SERS detection modes that focus on quantitative analysis of pathogens, traditional colorimetric detection modes typically provide only limited positive / negative discrimination sensitivity. However, this situation is significantly improved with the assistance of the ZnO@Au-DTNB nanotag.

[0062] like Figure 2 As shown, the SERS effect of the tag is a key factor in this detection system. The SERS enhancement mechanism mainly includes electromagnetic field enhancement generated by the noble metal localized surface plasmon resonance (LSPR) effect and chemical enhancement effect generated by the charge transfer of the reporter molecule. Furthermore, it should be noted that the essence of SERS lies in its influence on the polarizability change of the reporter molecule. For electromagnetic field enhancement, under a unit electric field, the electric field strength induced by LSPR can reach tens of thousands of times, which leads to significant changes in the polarizability and induced dipole moment of the reporter molecule, resulting in extremely strong Raman signal enhancement. For charge transfer enhancement, when the Raman reporter molecule interacts with other molecules, it triggers a change in polarizability, thereby achieving Raman signal enhancement. Based on this, to obtain a strong SERS signal, the SERS tag must simultaneously possess a high density of LSPR hotspots and a strong charge transfer capability of the reporter molecule.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) The method for preparing oxygen-vacancy ZnO described in this invention can precisely control the oxygen vacancy concentration in ZnO, thereby generating high-density active sites for adsorbing Au. 3+ The precursor was used to achieve Au NPs nucleation through ultrasonic-assisted in-situ reduction by utilizing the piezoelectric catalytic properties of ZnO. Notably, oxygen vacancy engineering not only generates surface active sites but also profoundly alters the electronic band structure of ZnO. Furthermore, the prepared oxygen-vacancy ZnO exhibits a large specific surface area and intrinsic photocatalytic properties.

[0065] (2) This invention utilizes ZnO semiconductor material with large specific surface area and intrinsic photocatalytic properties to construct nanotags with both SERS and catalytic capabilities by loading AuNPs; after AuNPs are deposited, their localized surface plasmon resonance (LSPR) effect can significantly enhance SERS performance (enhancement factor EF up to 10). 9 Meanwhile, the ZnO@Au system exhibits superior photocatalytic activity compared to the original ZnO, which stems from three synergistic mechanisms: (i) SPR-induced light absorption and hot carrier generation, (ii) charge separation promoted by Schottky junction, and (iii) metal-semiconductor catalytic synergy. High-density loading of Au NPs on ZnO can simultaneously enhance the dual functions of the nanotag.

[0066] (3) The present invention further utilizes the Raman reporter molecule 5,5'-disulfide bis(2-nitrobenzoic acid) (DTNB) to further optimize the SERS performance of the nanotag. The photocatalytic ability of the ZnO@Au system can be further enhanced after being combined with the Raman reporter molecule (the SERS effect mainly comes from the LSPR effect of noble metals and the charge transfer between the substrate and the reporter molecule, the latter of which fundamentally affects the change in the polarizability of the reporter molecule), thereby obtaining higher detection sensitivity.

[0067] (4) This invention constructs a multi-mode LFIA detection system for point-of-care testing (POCT) of pathogens. This system combines good repeatability, rapid detection (<15 min), high specificity, and high detection sensitivity. It also integrates in-situ sterilization function (antibacterial rate >99%) and triple detection modes: colorimetric visual recognition mode (detection limit LOD = 10). 4 CFU / mL), catalytic colorimetric enhanced visual recognition mode (LOD=10) 2 The detection modes include CFU / mL and SERS (fitted LOD < 10 CFU / mL). This innovative strategy based on electronic structure design of nanomaterials provides new ideas for improving the detection performance and expanding the functions of LFIA. Attached Figure Description

[0068] 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.

[0069] Figure 1 This is a schematic diagram illustrating the preparation and bacterial detection process of the ZnO@Au-DTNB nanotag described in this invention.

[0070] Figure 2 This is a schematic diagram of the SERS mechanism of the ZnO@Au-DTNB nanotag described in this invention.

[0071] Figure 3A TEM images of cubic Cu2O, cage-like Zn(OH)2, and three-dimensional ZnO provided in Example 1.

[0072] Figure 3B Scanning electron microscope (SEM) images of cubic Cu2O, cage-like Zn(OH)2, and three-dimensional ZnO provided in Example 1.

[0073] Figure 3C X-ray diffraction (XRD) patterns of cubic Cu2O, cage-like Zn(OH)2, and three-dimensional ZnO provided in Example 1.

[0074] Figure 3D The UV-Vis absorption spectra of cubic Cu2O, cage-like Zn(OH)2, and three-dimensional ZnO provided in Example 1 are shown.

[0075] Figure 3E Raman spectra of cubic Cu2O, cage-like Zn(OH)2, and three-dimensional ZnO provided in Example 1.

[0076] Figure 3F Thermogravimetric-differential scanning calorimetry curve of cage-like Zn(OH)2 provided in Example 1.

[0077] Figure 4A Ultraviolet photoelectron energy (UPS) spectra (0V and -5V) of ZnO containing oxygen vacancies obtained by etching three-dimensional ZnO for 0 min as provided in Example 1.

[0078] Figure 4B Ultraviolet photoelectron energy (UPS) spectra (0V and -5V) of ZnO containing oxygen vacancies obtained by etching three-dimensional ZnO for 30 min as provided in Example 1.

[0079] Figure 4C Ultraviolet photoelectron energy (UPS) spectra (0V and -5V) of ZnO containing oxygen vacancies obtained by etching three-dimensional ZnO for 60 min as provided in Example 1.

[0080] Figure 4D Ultraviolet photoelectron energy (UPS) spectra (0V and -5V) of ZnO containing oxygen vacancies obtained by etching three-dimensional ZnO for 120 min as provided in Example 1.

[0081] Figure 4E The ultraviolet photoelectron energy (UPS) spectrum (0V and -5V) of the DTNB provided in Example 1.

[0082] Figure 4FThe UV-Vis absorption spectra of oxygen-vacancy ZnO obtained by etching the three-dimensional ZnO provided in Example 1 for 0 min, 30 min, 60 min and 120 min are shown.

[0083] Figure 4G The image shows the UV-Vis absorption spectrum of DTNB provided in Example 1.

[0084] Figure 5A The diagram shows the band structure of ZnO containing oxygen vacancies obtained by etching three-dimensional ZnO for 0 min, 30 min, 60 min and 120 min as provided in Example 1, and the electronic density of states obtained by DFT simulation.

[0085] Figure 5B Electron paramagnetic resonance (EPR) spectra of oxygen-vacancy ZnO obtained by etching three-dimensional ZnO for 0 min, 30 min, 60 min and 120 min as provided in Example 1.

[0086] Figure 5C Zeta potential (sample size n=3) of ZnO containing oxygen vacancies obtained by etching three-dimensional ZnO for 0 min, 30 min, 60 min and 120 min as provided in Example 1.

[0087] Figure 5D X-ray diffraction patterns of oxygen-vacancy ZnO obtained by etching three-dimensional ZnO provided in Example 1 for 0 min, 30 min, 60 min and 120 min.

[0088] Figure 5E Scanning electron microscope (SEM) images of oxygen-vacancy ZnO obtained by etching three-dimensional ZnO provided in Example 1 for 0 min, 30 min, 60 min and 120 min.

[0089] Figure 6A Fine X-ray photoelectron spectra of the O1s orbitals of ZnO containing oxygen vacancies obtained by etching three-dimensional ZnO with oxygen vacancies for 0 min, 10 min, 20 min and 30 min as provided in Example 1.

[0090] Figure 6B The fine X-ray photoelectron spectra of Zn 2p orbitals of ZnO obtained by etching three-dimensional ZnO with oxygen vacancies at 0 min, 10 min, 20 min and 30 min in the preparation method provided in Example 1 are shown.

[0091] Figure 7 Raman spectra of oxygen-vacancy ZnO obtained by etching three-dimensional ZnO provided in Example 1 for 0 min, 30 min, 60 min and 120 min.

[0092] Figure 8ATEM images of ZnO containing oxygen vacancies, obtained by etching gold nanoparticles provided in Example 1 after 0 min, 30 min, 60 min and 120 min.

[0093] Figure 8B This is a schematic diagram illustrating the consistency between the oxygen vacancy density and the gold nanoparticle density of ZnO@Au provided in Example 1.

[0094] Figure 8C XRD spectra of gold nanoparticles loaded on oxygen-vacant ZnO at different etching times.

[0095] Figure 8D Raman spectra of ZnO@Au-DTNB at different etching times are shown. The annotations in the figure are redshift wavenumbers relative to the -NO2 peak of intrinsic DTNB.

[0096] Figure 8E This is a schematic diagram of the band structure of the contact modes of ZnO@Au-DTNB under different etching times.

[0097] Figure 8F This is a schematic diagram of the band structure of the contact modes of ZnO@Au-DTNB under different etching times.

[0098] Figure 9A High-angle annular dark-field imaging of ZnO@Au at different etching times, and corresponding energy dispersive spectral analysis of Au, O, and Zn elements.

[0099] Figure 9B The energy dispersive spectroscopy (EDS) intensity integral spectra of ZnO@Au composites at different etching times are shown.

[0100] Figure 10A The photoelectric field distribution of a 20 nm thick ZnO thin film array loaded with gold nanoparticles with spacings of 10 nm, 30 nm, 50 nm and 100 nm was shown under the condition of perpendicular incidence of 785 nm wavelength light.

[0101] Figure 10B The photoelectric field distribution of a 20 nm thick gold nanoparticle array with spacings of 10 nm, 30 nm, 50 nm and 100 nm was shown under the condition of perpendicular incidence of 785 nm wavelength light.

[0102] Figure 11A High-resolution transmission electron microscopy (TEM) images and selected area electron diffraction (SED) patterns of ZnO@Au-DTNB at different etching times.

[0103] Figure 11B High-resolution transmission electron microscopy (TEM) images and selected area electron diffraction (SED) patterns of ZnO containing oxygen vacancies at different etching times.

[0104] Figure 12 Fourier transform infrared and Raman scattering spectra of ZnO, ZnO-DTNB, ZnO@Au, ZnO@Au-DTNB, and DTNB containing oxygen vacancies obtained by etching for 30 min.

[0105] Figure 13 Raman spectra of ZnO-DTNB composite materials at different etching times.

[0106] Figure 14 This is a plot of data from a DFT simulation. Note: In the Bader charge analysis plot, the yellow area represents the charge accumulation region, and the blue area represents the charge depletion region.

[0107] Figure 15 Raman spectra of ZnO@Au-DTNB prepared with different amounts of HAuCl4.

[0108] Figure 16A Transmission electron microscopy (TEM) images and energy dispersive spectroscopy (EDS) plots of ZnO@Au-DTNB prepared with different amounts of HAuCl4.

[0109] Figure 16B Energy dispersive spectroscopy (EDS) intensity integral spectra of ZnO@Au-DTNB prepared with different amounts of HAuCl4.

[0110] Figure 17A Raman spectra of ZnO@Au-DTNB nanotags prepared with different concentrations of DTNB.

[0111] Figure 17B Enhancement factor diagram of ZnO@Au-DTNB nanotags prepared with different concentrations of DTNB.

[0112] Figure 17C The image shows the Raman spectrum of 1M DTNB.

[0113] Figure 18A The signal-to-noise ratio and Raman spectra are shown for different buffer ratios.

[0114] Figure 18B Signal-to-noise ratio and Raman spectra for different marker volumes.

[0115] Figure 18C To detect the signal-to-noise ratio and Raman spectra at different antibody concentrations online.

[0116] Figure 19A This is a visualization of different SA concentrations in the detection of Staphylococcus aureus (SA) using a SERS-based LFIA sensor.

[0117] Figure 19BThis is a schematic diagram of Raman signals at different SA concentrations.

[0118] Figure 19C These are Raman spectra for different SA concentrations. Each spectrum is the average of three or more repeated acquisitions.

[0119] Figure 19D To fit the 1331 cm⁻¹ Raman spectrum using the Levenberg-Marquardt iterative algorithm with a logistic function -1 The relationship between characteristic peak intensity and the logarithm of bacterial concentration. The dashed line represents the limit of detection (LOD), calculated by adding three standard deviations (Blank + 3SD) to the mean of the blank signal.

[0120] Figure 19E To evaluate sensor repeatability by analyzing the measurement consistency of four independent sensor groups (test sample size n=4) at a constant SA concentration, the result plot was determined by calculating the relative standard deviation of the average response signal.

[0121] Figure 19F The figure shows the results of evaluating the sensor's specificity for SA by testing five comparable concentrations (all 10⁶ CFU / mL) of bacterial species.

[0122] Figure 20A Three sets of optical images were set up for the detection experiment.

[0123] Figure 20B The images show the Raman spectra of the pure Staphylococcus aureus group and the blank control, as well as the results of three repeated tests.

[0124] Figure 20C The images show the Raman spectra of the mixed bacterial group and the blank control, as well as the results of three repeated tests.

[0125] Figure 21A Schematic diagram of colorimetric enhancement mechanism: When the test strip is added to the TMB colorimetric solution and exposed to light, the ZnO@Au-DTNB tag captured by the positive T line promotes the oxidation and color change of TMB, while the negative T line remains colorless.

[0126] Figure 21B Images of the sensor detecting different concentrations of SA, and images after adding TMB colorimetric solution.

[0127] Figure 21C The EPR spectra of ZnO@Au-DTNB under sunlight and dark field conditions correspond to DMPO-·OH (top) and DMPO-·O2- (bottom), respectively; the enzyme saturation kinetic curve of the ZnO@Au-DTNB tag (sample size n=3).

[0128] Figure 21DWith the H2O2 concentration fixed at 500 mM, the TMB concentration increases geometrically with a first term of 0.5 mM and a common ratio of 2.

[0129] Figure 21E The TMB concentration was fixed at 10 mM, and the H2O2 concentration increased in an arithmetic series with a first term of 0.6 mM and a tolerance of 0.2.

[0130] Figure 21F Thermodynamic path of ORR reaction in ZnO@Au-DTNB calculated by DFT.

[0131] Figure 21G This is a bar chart showing the thermodynamic path of the ORR reaction in ZnO@Au-DTNB.

[0132] Figure 22 This is a schematic diagram illustrating the mechanism by which reactive oxygen species (ROS) generated by ZnO@Au-DTNB oxidize and sterilize TMB under sunlight.

[0133] Figure 23A The UV-Vis absorption spectra of the photocatalytic colorimetric solution composed of H2O2 + TMB + label + NaAc-HAc buffer and the control group are shown.

[0134] Figure 23B UV-Vis absorption spectra of colorimetric solutions prepared for NaAc-HAc buffer solutions at different pH values.

[0135] Figure 23C UV-Vis absorption spectra of colorimetric solutions prepared with different label concentrations.

[0136] Figure 24 The diagram shows the sterilization experiments of three groups of Staphylococcus aureus bacterial solutions at different concentrations. Detailed Implementation

[0137] Unless otherwise defined herein, the scientific and technological terms used in connection with this invention should have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear; however, in any case of potential ambiguity, the definitions provided herein take precedence 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. It should be noted that specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many ways other than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0138] 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.

[0139] 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.

[0140] The sources of some materials and equipment in the following embodiments and test examples are shown below:

[0141] Materials: Copper chloride dihydrate (CuCl2·2H2O, Aladdin, CAS: 10125-13-0), 2M sodium hydroxide solution (CAS: 1310-73-2, Aladdin), ascorbic acid (Sigma-Aldrich), zinc chloride (ZnCl2, Aladdin, CAS: 7646-85-7), polyvinylpyrrolidone (PVP, 40 kDa), anhydrous ethanol (analytical grade), deionized water, 1M sodium thiosulfate Solutions (Aladdin, CAS: 1310-73-2), potassium tetraborate (K2B4O7·4H2O, Aladdin, CAS: 12045-78-2), sodium sulfite (Na2SO3, Aladdin, CAS: 7757-83-7), Tween-20 (Sigma-Aldrich), DuPont phosphate buffer (PBS, Sigma-Aldrich), and acetate-sodium acetate buffer (NaAc-HAc buffer, pH 3.6). Macroporous nitrocellulose membranes (NC membranes) CN95 were purchased from Sartorius (Spain). Glass fiber sample pads (CB08), sample application pads, absorbent pads, and plastic backings were provided by Liangxin Technology and Shanghai Jieyi Biotechnology (China). Goat anti-mouse IgG (catalog number: D111024) was provided by Sangon Biotech (China). Staphylococcus aureus monoclonal antibody (catalog number: MA1-10708, type I antibody) was from Invitrogen. Staphylococcus aureus antibody (catalog number: 10-S30B, type II antibody) was obtained from Fitzgerald. Chloroauric acid tetrahydrate (HAuCl4·4H2O), acetone, and 30% hydrogen peroxide aqueous solution (H2O2) were purchased from Sinopharm Chemical Reagent. 2-(N-morpholino)ethanesulfonic acid (MES), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, ≥99%), 3,3',5,5'-tetramethylbenzidine (TMB, ≥98%), and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, ≥98%) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific. Sodium N-hydroxythiosuccinimide (NHS, 97%) was purchased from Alfaesa. Bovine serum albumin (BSA, pH 7.0) was purchased from GPC Biotechnology.

[0142] Instrumentation: UV-Vis spectrophotometer (UV-Vis, Shimadzu 2600), nanoparticle size and zeta potential analyzer (Malvin Nano-ZS90 Zetasizer), X-ray photoelectron spectrometer (XPS, Thermo Fisher escalab 250Xi), X-ray diffractometer (XRD, D8 ADVANCE X), transmission electron microscope (TEM, Talos-F200s) equipped with energy dispersive spectrometer (EDS, Oxford Super X), scanning electron microscope (SEM, SU8020) equipped with energy dispersive spectrometer (HORIBA EMAX mics2), electron paramagnetic resonance spectrometer (EPR, Bruker A300-10 / 12), multi-functional microplate reader (SPARK, TECAN, Austria) for detecting absorbance changes of oxTMB at 655 nm, Fourier transform infrared spectrometer (FTIR, Bruker ALPHA II), Raman spectrometer (B&WTek i-Raman Plus). BWS465-785H), simultaneous thermal analyzer (TG-DSC, NETZSCH STA 449F3 / F5), ultraviolet photoelectron spectrometer (UPS, Thermo Fisher ESCALAB 250Xi).

[0143] Example 1

[0144] This embodiment provides a ZnO containing oxygen vacancies and a ZnO@Au-DTNB nanotag containing the oxygen vacancies. The preparation method specifically includes the following steps:

[0145] Preparation of S1 and Cu2O cubic crystals:

[0146] Dissolve 0.17 g CuCl2·2H2O in 100 mL of deionized water to form a clear blue solution; heat to 55 °C and add 10 mL of 2 M NaOH solution, stir magnetically for 30 min to obtain a dark black solution; maintain 55 °C and slowly add 10 mL of 0.6 mol ascorbic acid to the mixed solution to react and form a brick-red precipitate; continue stirring for 3 h until the solution is completely brick-red; finally, wash the precipitate four times alternately with deionized water and ethanol, filter and collect; dry the obtained Cu2O cubic crystal product at 60 °C overnight, seal, dry, ventilated and cool storage.

[0147] Preparation of S2 and cage-like Zn(OH)2:

[0148] 10 mg of Cu2O cubic crystals, 2 mg of ZnCl2 and 0.333 mg of PVP were added to 10 mL of a mixture of ethanol and water (volume ratio = 1:1) and dispersed by sonication for 10 min. Then, 4 mL of 1 M Na2S2O3 solution was added dropwise to the mixture and the mixture was magnetically stirred at 25 °C for 2 h until the suspension changed from red to light white. The sample was washed 4 times alternately with deionized water and ethanol, and separated by filtration to obtain cage-like Zn(OH)2. The sample should be air-dried and sealed for storage.

[0149] S3, Preparation of three-dimensional ZnO:

[0150] Zn(OH)2 powder was placed in a crucible and placed in a muffle furnace. The temperature was increased to 250℃ at 5℃ / min and held for 30min in air to finally obtain ZnO nanomaterials.

[0151] S4. Preparation of ZnO containing oxygen vacancies:

[0152] 100 mL of photochemical etching solution was prepared with 30.65 g K₂B₄O₇·4H₂O, 2.52 g Na₂SO₃, and 92.8 mL H₂O. The prepared ZnO powder was mixed with the etching solution at a concentration of 2 mg / mL to form a dispersion, which was then placed in a 96-well plate. The distance between the light source and the plate was maintained at 3 cm (at which point the central light power was 320 mW), and the illumination time (0–120 min) was set according to experimental requirements. After etching, the liquid was collected using a pipette and rinsed three times with deionized water for later use.

[0153] Preparation of S5 and ZnO@Au:

[0154] Add 8 mg of etched ZnO to a centrifuge tube containing 8 mL of ethanol and 8 mL of water, and sonicate for 15 min; then add 1 mL of HAuCl4 (10 g / L) aqueous solution. 25 At ℃, with 100W The solution was sonicated for 1 hour until it changed from light yellow to dark purple; centrifuged at 8000 rpm, and the precipitate was washed 6 times with alternating ethanol and water; finally, the ZnO@Au precipitate was resuspended in 400 μL of ethanol for later use.

[0155] Preparation of S6 and ZnO@Au-DTNB:

[0156] Prepare a 10 mmol / L DTNB ethanol solution, add 2 μL to the above 400 μL ZnO@Au ethanol solution, and sonicate for 1 h to promote the formation of Au-S bonds between DTNB and Au; after centrifugation, wash twice with ethanol, and finally resuspend the ZnO@Au-DTNB precipitate in 1 mL of ethanol for later use.

[0157] Preparation of S7, ZnO@Au-DTNB nanotags:

[0158] Centrifuge 1 mL of ZnO@Au-DTNB ethanol dispersion, wash once with 0.05% PBST (PBS: Tween-20 = 0.05: 99.95) and discard the supernatant; add 500 μL MES (10 mmol, pH = 5.5), 50 μL EDC (10 mmol) and 100 μL NHS (10 mmol), and sonicate for 15 min to activate the DTNB carboxyl group; discard the supernatant and add 300 μL 0.05% PBST and 3 μL L type antibody (MA1-10708), sonicate and react in a shaker (25℃, 800 rpm) for 2.5–3 h; add 100 μL 10% BSA (BSA: H2O = 10: 90) and vortex to mix, continue blocking for 1.5 h; finally, wash once with 0.05% PBST, resuspend in 200 μL 0.05% PBST, and store at 4℃ for later use.

[0159] Example 2

[0160] This embodiment provides a system that combines bacterial detection and photocatalytic sterilization functions; the system includes: the ZnO@Au-DTNB nanotag, immunochromatographic test strip, and TMB colorimetric enhancement system.

[0161] The immunochromatographic test strip is assembled from components such as a macroporous nitrocellulose membrane (NC membrane), a glass fiber sample pad, an absorbent pad, and a plastic backing plate. The plastic backing plate provides support, the sample pad is used to add liquid samples, the NC membrane provides capillary force to propel the solution system across the membrane, and the absorbent pad absorbs excess liquid to prevent backflow. Specific procedure:

[0162] (1) 10-S30B and IgG antibodies were streaked onto the NC membrane as the detection line (T line) and control line (C line), respectively. The 10-S30B antibody only captures Staphylococcus aureus, while the IgG antibody can capture almost all antigens. Changes in the T line signal reflect the detection status of the target antigen, and the C line can determine whether the sensor is malfunctioning;

[0163] (2) Dry the scribing NC film at 37℃, and then assemble the sensor components.

[0164] (3) Mix an appropriate amount of label, antigens of different concentrations, and running buffer, and add 70 μL to the sample pad. Under capillary action, the label carrying the antigen flows through the T and C lines and is captured, with the entire process taking approximately 15 minutes. The antigen concentration determines the amount of label retained in the T line, thus achieving quantitative detection of SA;

[0165] (4) Detection results are obtained through visual observation and Raman signal acquisition.

[0166] Note: The concentration of T-line antibody, running buffer, and tag addition amount need to be optimized in advance to obtain the best signal-to-noise ratio; in this detection system, the concentration of T-line antibody is 1.4 mg / mL, the tag addition amount is 1 μL, and the running buffer is a mixture of 1% PBST (PBS: Tween-20 = 99:1), 10% BSA (BSA: H2O = 1:9), and FBS at a volume ratio of 8:1:1.

[0167] Configuration process of the TMB colorimetric enhancement system:

[0168] After the sensor ran for 15 minutes, it was illuminated with simulated sunlight using AM 1.5 (light source 3 cm from the test strip, central light power 320 mW). Immediately, 1 μL of the colorimetric solution was added to the T line, and the reaction was allowed to proceed for 1 minute before capturing the colorimetric signal. The colorimetric solution consisted of 10 mM TMB acetone solution, 30% H2O2, and 0.2 M NaAc-HAc buffer (pH = 3.6). In the catalytic kinetics experiment, the total solution volume in the 96-well plate was 200 μL, containing 1 μL H2O2, 10 μL TMB acetone solution, and 189 μL NaAc-HAc buffer. Different concentrations of H2O2 and TMB solutions needed to be prepared in advance (stored at 4℃).

[0169] Test Example 1

[0170] Test methods: ultraviolet photoelectron spectroscopy, ultraviolet-visible absorption spectroscopy, electron paramagnetic resonance, zeta potential, X-ray diffraction, and scanning electron microscopy.

[0171] Test results:

[0172] like Figures 3A-3E As shown, this invention uses cubic Cu2O particles as a substrate. The cubic Cu2O reacts with ZnCl2 in an alkaline reducing agent system, through Zn... 2+ The substitution reaction forms cage-like Zn(OH)2, which is then annealed to obtain three-dimensional ZnO mainly composed of plate-like structures. The morphology of this three-dimensional ZnO is composed of a mixture of nanoparticles, nanorods and nanosheets, forming a multi-level structure.

[0173] To further verify the influence of oxygen vacancy content on the electronic structure of ZnO, we constructed ZnO models with low, medium, and high oxygen vacancy densities and calculated their respective densities of states (DOS) using density functional theory (DFT). With increasing oxygen vacancy numbers, the ordered arrangement of surface atoms gradually becomes disordered, with a large number of Zn atoms significantly delocalized. The total density of states (TDOS) indicates that the atomic densities of states of Zn and O in the system are non-degenerate superpositions. In intrinsic ZnO, all bonds are Zn-O, and O has a stronger electronegativity; therefore, the valence band apex is mainly dominated by the electron density of the O 2p orbitals. With increasing oxygen vacancy numbers, the hybridization effect between the O 2p orbitals and Zn 4s orbitals weakens, thus decreasing the local density of states (DOS) of O at the valence band apex, while Zn exhibits a relatively high DOS due to the localization of 4s orbital electrons. However, Zn's DOS is mainly concentrated in the 3d orbitals, so the TDOS still decreases as the O-dominated DOS decreases. The decrease in DOS due to the increase in oxygen vacancies means that the localized states of the top electrons in the valence band weaken, the electronic energy states will broaden, and the band gap will decrease. The calculated results are in perfect agreement with the experimental results.

[0174] like Figure 3F As shown, Zn(OH)₂ dehydrates at 170℃ and crystallizes at 375℃. Therefore, the above-mentioned suitable annealing temperature was selected to preserve the multi-level structure.

[0175] Furthermore, based on ultraviolet photoelectron spectroscopy (UPS), Figures 4A-4E ) and ultraviolet-visible absorption spectroscopy (UV-Vis, Figures 4F to 4G Based on the results, the band gap (Eg), Fermi level (EF), and valence band top (EV) of ZnO at different etching times were calculated.

[0176] like Figure 5A As shown, with the extension of etching time, the band gap of ZnO gradually decreases, and the Fermi level gradually decreases; the band gap of ZnO reaches its minimum at 30 min, at which point the Fermi level is also at its lowest; further extending the etching time leads to an increase in the band gap; when it reaches 120 min, the band structure almost returns to the state of unetched ZnO. The above changes indicate that the electronic structure modulation of ZnO is successful.

[0177] like Figure 5B As shown, the electron paramagnetic resonance (EPR) spectrum of the oxygen-vacancy-containing ZnO confirms the consistency between the bandgap change and the oxygen vacancy change. This indicates that the oxygen vacancy content in ZnO increases with etching time, reaching a maximum at 30 min; further extending the etching time leads to a decrease in oxygen vacancy content, and by 120 min, the oxygen vacancy count has almost recovered to the same level as unetched ZnO. The main reason for the decrease in oxygen vacancy content after 30 min is likely B4O7. 2-Sulfite reducing protectants have limited protective effects on ZnO containing excess oxygen vacancies.

[0178] like Figure 5C As shown, the positive potential of the system increases with the increase of oxygen vacancies; however, after 60 min, the potential gradually decreases and even forms a negative potential. This is attributed to excessive oxidation and the formation of Zn vacancies. The abnormal drop in Fermi level observed in the early stage of photochemical etching may be partly related to the synchronous generation of Zn vacancies, but more likely it is due to the strong interaction between metallic Zn and Zn offsetting the doping effect of donor Zn.

[0179] like Figure 5D As shown, the products etched for different durations still belong to the hexagonal crystal system, but long-term etching leads to disordered atomic stacking and crystal plane distortion, causing diffraction peak splitting and shifting. The relatively weak characteristic peaks in the XRD spectrum indicate that ZnO has low crystallinity, which corresponds to the weak Zn-O vibrational mode in the Raman spectrum. With the extension of etching time, the Zn-O vibrational mode further weakens, while the ratio of ZnO defect state mode peaks to Zn-O vibrational mode peaks increases significantly, indicating that long-term photochemical etching causes significant damage to the ZnO crystal structure.

[0180] like Figure 5E As shown in the scanning electron microscope (SEM) images of ZnO after etching for 0 min, 30 min, 60 min, and 120 min, it can be observed that the original three-dimensional hierarchical structure gradually collapses with increasing etching time, which is due to the destruction of the crystal structure. This indicates that photochemical etching also has a significant impact on the crystal structure and morphology of ZnO.

[0181] like Figure 6A As shown, the O1s fine spectrum peaks of the four samples obtained by sampling every 10 minutes within 30 minutes also showed a trend of oxygen vacancy content change consistent with the EPR results; the proportion of oxygen vacancy peak area gradually increased over time. Figure 6B As shown, in the Zn 2p fine spectrum, the peak area ratio of Zn 0 in the 30-minute sample increases sharply, and a 4d state transition peak appears at 1025 eV, a Zn LOSS peak at 1030 eV, and a plasmon effect peak at 1034 eV. These results indicate that overexposed metallic Zn due to increased oxygen vacancies is difficult to shield by the solution system, and the electronic transitions and interactions between Zn and Zn gradually increase. Once the sulfite protectant can no longer meet the antioxidant requirements of Zn, Zn will be re-oxidized to fill the oxygen vacancies, a phenomenon also reflected in the Zeta potential characterization.

[0182] like Figure 7 As shown, the Raman spectrum at 100 cm⁻¹ -1 and 445cm -1It contains two peaks representing the Zn-O vibration, and at 300–350 cm⁻¹ -1 The peaks within the range represent the defect states of ZnO.

[0183] Test Example 2

[0184] Test methods: transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Raman scattering spectroscopy, and Fourier transform infrared spectroscopy.

[0185] Test results:

[0186] ZnO@Au products obtained by ultrasonic-assisted deposition based on ZnO with different etching times were captured and characterized by transmission electron microscopy (TEM).

[0187] like Figure 8A as well as Figure 9A Based on the above, it can be determined that the light-colored layer is a specific view of plate-like ZnO, and the black material on it is Au NPs. The deposition density of Au NPs is significantly affected by different etching times of ZnO.

[0188] like Figure 8B As shown, ZnO with high-density oxygen vacancies is loaded with more dense Au NPs, reflecting the close correlation between Au NP reduction deposition and oxygen vacancy sites. The main reason is that oxygen vacancies serve as excellent reaction sites, acting as carrier trapping centers.

[0189] like Figure 8C As shown, the XRD pattern clearly shows the diffraction peak of the Au(111) crystal plane at 38.3° and the diffraction peak of the ZnO(101) crystal plane at 36.2°.

[0190] like Figure 8D The image shows the Raman spectra of ZnO@Au-DTNB derived from ZnO at different etching times. It can be observed that ZnO@Au-DTNB obtained from ZnO etched for 30 min (i.e., ZnO-30) exhibits the highest Raman spectra at approximately 1331 cm⁻¹. 1 The strongest -NO2 characteristic peak intensity is observed at this location. This is due to two main reasons: firstly, the high-density oxygen vacancies on ZnO are filled by denser gold nanoparticles, resulting in a stronger electromagnetic field enhancement effect.

[0191] Based on transmission electron microscopy images, this invention further constructed a ZnO model loaded with gold nanoparticles of different density distributions, and used the FDTD (finite-difference time domain) algorithm to simulate the electric field distribution of 785nm laser under the condition of perpendicular incidence of 785nm continuous wave plane laser.

[0192] like Figure 10AAs shown, under the condition of perpendicular incidence of a 785nm continuous-wave plane laser, an array of gold nanoparticles with smaller spacing and higher density will generate a stronger electric field. This verifies the conclusion that the high-density distribution of gold nanoparticles leads to a stronger electromagnetic field enhancement.

[0193] Another reason is that ZnO with different electronic structures can also significantly affect the charge transfer of DTNB. For example... Figure 8E The diagram illustrates the band alignment of ZnO after contact with Au and DTNB at different etch times. Without high-energy photon excitation, Au, with its larger work function, forms an N-type semiconductor-Au Schottky contact at the interface with DTNB and ZnO at different photo-etch times. Considering the charge balance of ZnO-Au-DTNB, charge injection by ZnO and DTNB to Au is actually competitive. The side with a work function closer to Au is more likely to inject charge into Au and form a smaller potential barrier. Since ZnO-30 has the smallest work function difference with Au, it competes least with DTNB for charge injection into Au, which favors more charge transfer and a larger potential barrier between DTNB and Au.

[0194] And in Figure 8D Among the four curves, the ZnO@Au-DTNB curve obtained from ZnO-30 shows the largest redshift of the NO2 peak, indicating the strongest hybridization effect of DTNB in ​​this structure and reflecting the enhanced charge transfer of DTNB. Furthermore, the built-in electric field between ZnO-30 and Au is the weakest, making it more likely that hot carriers from Au will be injected into ZnO-30 rather than DTNB, thus reducing the negative impact on DTNB charge transfer. In addition, the charge accumulation on the Au surface caused by charge transfer further enhances its LSPR effect, generating a stronger localized electromagnetic field under laser excitation, thereby increasing the change in DTNB molecular polarizability and forming a typical synergistic enhancement effect.

[0195] like Figure 10B As shown, FDTD simulations of Au nanoparticle arrays with different spacings but without a ZnO substrate are presented. It was found that the presence of ZnO enhances the electric field in the gaps between Au nanoparticles to some extent, which is completely consistent with the synergistic enhancement effect.

[0196] like Figure 8FAs shown, possible ZnO-DTNB contact modes exist: when ZnO and DTNB with different band structures come into contact, significant charge transfer occurs. The work function difference between ZnO-30 and DTNB is the largest, implying the maximum charge transfer between them. This hypothesis can be confirmed by the changes in the vibrational energy levels of ZnO@Au molecules before and after DTNB modification. The changes in the characteristic peaks of DTNB are closely related to its charge distribution.

[0197] like Figure 11A and Figure 11B As shown, selected area electron diffraction (SAED) clearly reveals the diffraction spots of the ZnO(101) and Au(111) crystal planes. Furthermore, a lattice transition from ZnO to Au was observed through inverse Fourier transform reconstruction. All of these clearly confirm the successful loading of Au nanoparticles.

[0198] like Figure 12 As shown, Raman scattering and Fourier transform infrared (FTIR) spectroscopy strongly confirm the successful modification of DTNB, as the originally smooth ZnO@Au spectrum displays characteristic peaks belonging to DTNB. Figure 12 As shown, after modification with DTNB on ZnO@Au, the chromatic alumina content is located in the 3000–3300 cm⁻¹ region. -1 The OH vibration peak is located at 2800–3000 cm⁻¹. -1 The CH stretching vibration peak almost disappears. This may be due to the deprotonation effect of delocalized Zn active sites in ZnO and the SPR shielding effect of the Au surface; located at 1300–1600 cm⁻¹. -1 The absorption peak of the nitro stretching vibration within the range became more pronounced, and a 10 cm⁻¹ peak occurred. -1 The blue shift is likely due to the electrostatic interaction between ZnO@Au and the nitro group; and the CO(1042cm) in ZnO@Au-DTNB... -1 ) and CN (875cm -1 The disappearance of the stretching vibration peak may be due to the coordination of the carboxyl group with the zinc site and the electrostatic attraction between the nitro nitrogen and gold. Figure 12 As shown, 1340cm in ZnO@Au-DTNB -1 The nitro vibration peak at that location was redshifted by 10 cm compared to DTNB. -1 This is also due to the hybridization effect between ZnO@Au and DTNB.

[0199] like Figure 13 As shown, ZnO-DTNB derived from ZnO-30 exhibits the largest redshift of the -NO2 peak, indicating that it has the strongest hybridization with DTNB and the highest charge transfer efficiency among all configurations.

[0200] Test Example 3

[0201] Test method: Charge transfer in ZnO-Au-DTNB was studied by DFT calculation.

[0202] Test results:

[0203] like Figure 14 As shown, schematic diagrams of atomic-level interface contact models for DTNB-Au, Au-ZnO, DTNB-Au-ZnO, and DTNB-ZnO, as well as Bader charge analysis diagrams for each material, are presented.

[0204] The Bader charge analysis at the DTNB-Au interface shows the Bader charge distribution between DTNB and Au, with the yellow area representing charge accumulation and the blue area representing charge depletion. Significant charge transfer from DTNB to the Au surface is visible at the interface. Further Bader charge calculations were used to analyze the charge transfer phenomena at the Au-ZnO, DTNB-Au-ZnO, and DTNB-ZnO interfaces formed by high-density oxygen-vacancy ZnO (ZnO-high) and low-density oxygen-vacancy ZnO (ZnO-low).

[0205] Among them, the charge transfer phenomena at the Au-ZnO, DTNB-Au-ZnO, and DTNB-ZnO interfaces formed by ZnO with high and low oxygen vacancy densities are consistent: 1. For Au-ZnO, charge mainly accumulates on the Au surface; 2. In the DTNB-ZnO contact, charge mainly accumulates at defect sites; 3. For the DTNB-Au-ZnO contact, the charge accumulation on the Au surface is the strongest, indicating that both ZnO and DTNB are injecting charge into Au. Due to the presence of ZnO, the charge transfer between DTNB and Au is less than at the pure DTNB-Au interface, indicating that there is indeed competition between ZnO and DTNB in ​​injecting charge into Au; 4. In the DTNB-ZnO contact, the charge polarization of DTNB is very obvious, and more charge accumulates at ZnO defect sites.

[0206] Further comparison of charge transfer differences at the two types of ZnO formation interfaces revealed the following: 1. In the DTNB-Au-ZnO contact, when ZnO-high is involved, the charge depletion region of DTNB is more pronounced, indicating weaker competition for charge injection from DTNB to Au; 2. In the DTNB-ZnO contact, when ZnO-high is involved, charge is more easily transferred to deeper parts of the ZnO crystal; while when ZnO-low is involved, the charge is restricted by surface oxygen dangling bonds, hindering charge transfer between them. The DFT calculation results are largely consistent with the experimental characterization analysis results, providing a sound theoretical basis. In summary, due to the high density of oxygen vacancies and suitable band structure, ZnO etched for 30 min should exhibit the strongest SERS performance. Furthermore, sufficient Au is required. 3+ The source fully covers the abundant active sites on ZnO and supports the growth of Au seeds to form hotspots. The improvement of the SERS effect is partly related to Au. 3+ The content of the source is significantly correlated.

[0207] Test Example 4

[0208] Test method:

[0209] (1) Raman spectroscopy, TEM, and EDS: Effect of different amounts of HAuCl4 on the SERS effect of ZnO@Au-DTNB tags; Sample group: ZnO@Au-DTNB composites prepared with different amounts of HAuCl4; Control group: Au NPs-DTNB and 1M DTNB. The synthesis method of Au NPs-DTNB is the same as that of ZnO@Au-DTNB, but without the addition of ZnO.

[0210] (2) Raman spectroscopy: The effect of different concentrations of DTNB on the SERS effect of ZnO@Au-DTNB tag.

[0211] The enhancement effect of SERS is usually evaluated by the enhancement factor (EF). The specific calculation method is as follows: Except for Raman characterization used in material vibration mode analysis, all Raman signal tests on the test strips were conducted using a 785nm laser, 1% laser intensity (1mW), a 50x objective lens, a 500ms excitation time, and a single acquisition. The ideal formula for calculating the enhancement factor (EF) is EF = (ISERS / NSERS), where ISERS and NSERS represent the Raman characteristic peak intensity and the number of molecules under the SERS effect, respectively, and INormal and NNormal represent the corresponding values ​​under normal conditions. In practice, molar concentration is used instead of the number of molecules, and the corrected formula is: EF = (ISERS / CSERS) * (INormal / CNormal). Under the condition of strictly ensuring consistent acquisition parameters, the EF of lower concentrations of reporter molecules is closer to the true value.

[0212] Test results:

[0213] like Figure 15 As shown, with the increase of HAuCl4 solution, 1331 cm -1 The peak intensity continuously increased. The pH of the 3000 μL HAuCl4 solution was approximately 5.5, close to the lower limit of ZnO solubility, and therefore it was set as the maximum addition amount. Au NPs-DTNB were prepared by adding 3000 μL chloroauric acid in the same manner without ZnO support. Due to the lack of a three-dimensional ZnO substrate to support the Au NPs, the SERS effect of Au NPs-DTNB was far inferior to that of ZnO@Au.

[0214] like Figure 16A and Figure 16B As shown, with the increase of HAuCl4 solution, Au NPs grow rapidly and accumulate densely, almost covering the signals of Zn and O elements.

[0215] like Figures 17A-17C Raman signals of ZnO@Au modified with different concentrations of DTNB are shown. Ideally, DTNB molecules should be located at hot spots of Au NPs and loaded onto ZnO to achieve a synergistic SERS effect. However, excessive DTNB molecules cannot guarantee that they are in the ideal positions. DTNB molecules in non-ideal positions will lose the electromagnetic field enhancement effect and have a lower EF, which is inaccurate for the evaluation of the SERS effect. Therefore, only ZnO@Au modified with relatively low concentrations of DTNB can show the true value of E, which is approximately 10⁹ for ZnO@Au-DTNB.

[0216] Test Example 5

[0217] Test method:

[0218] (1) Under different buffer ratios: PBST / BSA / FBS = 8:1:1; 7:2:1; 7:1:2; 9:0:1; 9:1:0;

[0219] (2) Different label volumes: 1; 2; 3; 4 μL;

[0220] (3) Detection of different antibody concentrations on the online test: 1.0; 1.2; 1.4; 1.6 mg / mL.

[0221] Test results:

[0222] The optimal operating conditions for LFIA were determined using Figure 18. The buffer ratio was set as PBST / BSA / FBS = 8:1:1; the labeled volume was 1 μL; and the antibody concentration on the detection line was 1.4 mg / mL.

[0223] Test Example 6

[0224] Test method:

[0225] (1) The SERS-based LFIA sensor was used for bacterial detection. Data were subjected to Shapiro-Wilk normality test, one-way ANOVA, and then a two-tailed t-test with a blank control. The significantly smaller p-values ​​marked in the figure demonstrate statistically robust selectivity for SA detection.

[0226] (2) The detection experiment was set up with three groups: a blank control group, a pure Staphylococcus aureus group, and a mixed bacterial group (containing Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium), with each strain having a concentration of approximately 10. 6 CFU / mL.

[0227] SA bacteria were cultured using commercial blood agar plates, and the bacterial concentration was assessed using a McFarland turbidimeter. The ZnO@Au-DTNB tag was synthesized according to the aforementioned steps, but ultimately dispersed in 1 mL PBS (pH=7). In the antibacterial experiment, 10 μL of the tag dispersion was mixed with 490 μL of bacterial suspension and irradiated with light for 30 min (320 mW), then plated onto blood agar plates and incubated for 16 h before counting.

[0228] The procedures for each experimental group and the control group were identical. The variables were whether or not light was applied and whether or not labels were added: the Normal group indicated untreated bacterial culture cultured using standard methods; the Sunlight 30mins group indicated bacterial culture treated with light for 30 minutes before inoculation; the Tags addition group indicated bacterial culture added with ZnO@Au-DTNB and immediately inoculated; and the Tags addition and Sunlight 30min group indicated bacterial culture added with ZnO@Au-DTNB and light-treated for 30 minutes before inoculation. The sterilization rate was calculated using the formula R = (N0 - N) / N0 * 100%, where R is the sterilization rate, N0 represents the average viable count in the Normal control group, and N represents the average remaining viable count in the Tags addition and Sunlight 30min sterilization experimental group.

[0229] Test results:

[0230] ZnO@Au-DTNB tags that capture different concentrations of Staphylococcus aureus (SA) are further captured after flowing through the detection line. Tags with higher SA concentrations are captured more, leaving a deeper, more visible mark on the detection line, such as... Figure 19A As shown, 104 CFU / mL SA is the visually perceptible limit of detection (LOD). Labels with strong SERS effects exhibit lower LODs in Raman assays. For example... Figure 19B As shown, even when the SA concentration reaches 10 1 Even at CFU / mL, a distinguishable Raman signal is still present. For example... Figure 19C As shown, the Raman spectra obtained after detecting different concentrations of SA are presented, with each spectral line being the average of three repeated tests. From Figure 19C Extracting 1331cm at different SA concentrations -1 The relationship between Raman peak intensity and SA concentration was investigated, and a logistic model was used for nonlinear fitting. Figure 19D As shown, the final statistically obtained LOD reached single digits CFU / mL, demonstrating extremely high sensitivity. Figure 19E As shown, the repeatability of the SERS-based LFIA sensor is evaluated, with 10 samples respectively. 4 CFU / mL and 10 6 Four assays were performed using SA bacteria at a concentration of CFU / mL. It was found that the relative standard deviation (RSD) of all four sensor groups was very small (RSD < 1%) at both SA bacterial concentrations, indicating no significant signal differences between different batches of sensors and demonstrating excellent repeatability. Next, five concentrations of 10... 6 The specificity of the sensor was validated using CFU / mL of bacteria. For example... Figure 19F As shown, the detection signal of SA bacteria is much greater than that of other bacterial species, and the significantly smaller p-value (marked in the figure) indicates that the sensor has statistically robust selectivity for SA detection, suggesting that the sensor has good specificity for SA detection.

[0231] like Figure 20A Image~ Figure 20C As shown, 10 4 CFU / mL SA represents the visually perceptible limit of detection (LOD). The label exhibiting a strong SERS effect demonstrated specific detection results in Raman spectroscopy, simulating mixed bacterial cultures in real-world applications, and the results also indicated high specificity for SA.

[0232] Test Example 7

[0233] Test method: colorimetric enhancement visual recognition and in-situ sterilization.

[0234] Test results:

[0235] Under standard AM 1.5 illumination, adding a colorimetric solution containing TMB (3,3',5,5'-tetramethylbenzidine) to the test strip can further enhance the visual signal. Figure 21A The visual colorimetric detection limit was increased from 10. 4CFU / mL increased to 10 2 CFU / mL Figure 21B This peroxidase-like (POD) activity primarily originates from the nanotags used.

[0236] Under high-energy photon excitation, the ZnO-Au-DTNB contact mode forms a typical Z-type heterojunction. Au, acting as the charge transport layer, promotes efficient separation of photogenerated carriers between ZnO and DTNB, and enhances light-harvesting efficiency based on the LSPR effect. Photogenerated holes and electrons are enriched in ZnO (with a higher oxidation potential) and DTNB (with a higher reduction potential), respectively, thereby optimizing the redox potential and further promoting potential redox reactions.

[0237] Thanks to this photocatalytic property, the test strip capturing the ZnO@Au-DTNB tag can generate strong redox capabilities under light to oxidize the TMB substrate, thereby enhancing the colorimetric signal. In this system, TMB may react directly with holes or be oxidized by reactive oxygen species (ROS). However, considering that a large number of active sites of ZnO are occupied by deposited Au particles, it is difficult for TMB to form a stable adsorption with ZnO on a large scale. Therefore, a large amount of ROS is the key to TMB oxidation.

[0238] like Figure 22 As shown, photogenerated holes and electrons can promote the conversion of H2O and H2O2 into ·OH radicals, while oxygen molecules can generate superoxide radicals (·O2-) at higher reduction potentials. The corresponding reactive oxygen species were also confirmed by EPR characterization. Figure 21C In an acidic buffer solution, the reduction of H2O2 to ·OH radicals is promoted, thereby greatly accelerating the oxidation of TMB.

[0239] To further evaluate the catalytic performance of the tag for TMB, this invention investigated the reaction kinetics. At low concentrations, according to the Beer-Lambert law, absorbance exhibits a positive linear relationship with substrate concentration. Therefore, based on... Figures 23A-23C The ultraviolet-visible spectrum was used, and the OD (optical density) value at 655 nm was selected to reflect the substrate concentration. Figure 21D and 21E A typical Michaelis-Menten curve was obtained by fitting the curve to calculate the reaction rates of H₂O₂ and TMB at different concentrations. max and K m Dynamic parameters. V max K represents the maximum reaction rate, reflecting the catalytic efficiency of the enzyme; m λ is the Michaelis constant, reflecting the affinity between the enzyme and the substrate. Compared to TMB substrates, the ZnO@Au-DTNB tag exhibits a smaller KB ratio for H2O2. m Value and larger V max The value indicates that the label has a stronger affinity for H2O2 and higher catalytic efficiency. Combined with... Figures 23A-23C The ultraviolet-visible absorption spectrum also shows that the presence of H2O2 significantly promotes the oxidation and discoloration of TMB, which is consistent with the mechanism by which H2O2 generates ·OH to oxidize TMB.

[0240] The large amount of ROS generated by the SERS tag is also beneficial for sterilization, thus giving the device an in-situ sterilization function. Figure 22 Under neutral incubation conditions maintained by PBS buffer, both the reversible reactions of the oxygen reduction reaction (ORR) and the oxygen evolution reaction (OER) can generate transition state free radicals. Among these, highly oxidizing free radicals such as ·OH and ·O2- can inactivate bacteria. The generation of free radicals is manifested in… Figure 21C In the EPR characterization: the spectral lines corresponding to ·OH and ·O2- are weaker under dark conditions, while the spectral line intensities increase significantly under illumination. This invention further uses DFT simulation to calculate the reaction thermodynamic path of the ZnO@Au-DTNB tag in an air environment. Figure 21F and Figure 21G Based on thermodynamic analysis of the Gibbs free energy change, products with lower free energy are more likely to spontaneously form. Therefore, under SERS-tagged catalysis, the four-electron pathway of ORR can spontaneously form intermediate ROS, and in the rate-determining step, light irradiation will significantly lower the energy barrier to the conversion to ·OH, making the reaction easier to proceed.

[0241] Test Example 8

[0242] Test method: In order to further verify the antibacterial effect of SERS tag, the present invention used the live cell counting method to conduct antibacterial experiment.

[0243] Sterilization experiments of three groups of Staphylococcus aureus bacterial suspensions at different concentrations: Normal group: untreated control bacterial suspension, directly incubated; Sunlight 30 min group: bacterial suspension incubated after 30 minutes of light exposure; Tags addition group: bacterial suspension incubated after ZnO@Au-DTNB complex was added; Tags addition and Sunlight 30 min group: bacterial suspension incubated after ZnO@Au-DTNB was added and exposed to light for 30 minutes.

[0244] Test results:

[0245] like Figure 24As shown, under normal culture conditions, all three SA concentration groups formed large and dense bacterial colonies. Even after 30 minutes of light exposure, the bacterial colonies remained relatively dense, indicating that simulated sunlight did not produce a significant bactericidal effect in a short period. In the control group with only the label added, the number of viable colonies decreased slightly, possibly due to the label's inherent catalytic ability in dark fields or the influence of weak indoor light on the label during culture. However, after adding the label and culturing under light for 30 minutes, the number of viable colonies decreased significantly. Based on the counting results of the culture plates, the sterilization rate of all three SA concentration groups reached over 99.8%, indicating that the ZnO@Au-DTNB label has a good bactericidal effect.

[0246] In summary, when zinc oxide semiconductors are combined with low-cost and easy-to-operate low-field immunoassay techniques, they exhibit superior dual-mode detection performance in both surface-enhanced Raman scattering (SERS) and catalytic colorimetric enhancement, while also possessing excellent antibacterial properties. A ZnO@Au-DTNB tag with excellent SERS effect was successfully fabricated using photochemical etching and ultrasound-assisted deposition techniques. The oxygen-vacancy-rich three-dimensional zinc oxide provides the basis for high-density deposition of gold nanoparticles, thereby achieving strong electromagnetic field enhancement. Based on the electronic structure modulation of zinc oxide, the charge transfer between ZnO, gold, and DTNB was optimized to obtain the maximum polarizability change of the DTNB reporter molecule. With the assistance of zinc oxide, the LSPR effect of gold and the charge transfer ability of DTNB were synergistically enhanced. In SERS detection mode, the detection limit for Staphylococcus aureus reached one part per billion unit / mL. The Z-shaped heterojunction formed by ZnO@Au-DTNB exhibits excellent photocatalytic performance. The ·OH generated in the four-electron pathway significantly promotes the oxidation reaction of TMB, increasing the detection limit of the visual colorimetric recognition mode by two orders of magnitude. Harmless treatment after pathogen detection is crucial. Using ZnO@Au-DTNB, a 99% sterilization rate can be achieved with short-term irradiation. Appropriate selection and meticulous design of markers will endow the LFIA platform with excellent performance and multifunctionality, providing new ideas for the development of novel detection systems based on LFIA technology.

[0247] 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 ZnO@Au-DTNB nanotag, characterized in that, The ZnO@Au-DTNB nanotag comprises ZnO with oxygen vacancies loaded with Au nanoparticles; Furthermore, the ZnO@Au-DTNB nanotag also includes DNTB, one end of which is connected to the Au nanoparticle via an Au-S bond, and the other end is connected to a type I antibody via a modified carboxyl group; The oxygen-vacancy-containing ZnO is prepared by the following steps: (a) In an alkaline reducing agent system, Zn salt and cubic Cu2O react to obtain cage-like Zn(OH)2; the cage-like Zn(OH)2 is specifically prepared by the following steps: dispersing ZnCl2, cubic Cu2O and PVP in an aqueous ethanol solution to obtain a mixture; adding Na2S2O3 solution dropwise to the mixture to carry out the reaction, and then washing, filtering and drying to obtain cage-like Zn(OH)2; (b) Annealing the cage-like Zn(OH)2 to obtain three-dimensional ZnO; (c) Dispersing three-dimensional ZnO in a photochemical etching solution to obtain a dispersion; subjecting the dispersion to photochemical treatment to obtain ZnO containing oxygen vacancies: the concentration of three-dimensional ZnO in the dispersion is 0.5~5 mg / mL; the photochemical etching solution comprises, by mass percentage: 20~40% K2B4O7, 1~5% Na2SO3, and the balance being water; the central optical power of the photochemical treatment is 310~330 mW; the photochemical treatment time is 20~40 min; The preparation method of the ZnO@Au-DTNB nanotag includes: (1) Disperse ZnO containing oxygen vacancies in an aqueous ethanol solution to obtain a dispersion; add HAuCl4 aqueous solution to the dispersion, sonicate, centrifuge and wash to obtain ZnO@Au; (2) The ethanol solution of ZnO@Au and the ethanol solution of DTNB are mixed, ultrasonically treated, and then centrifuged and washed to obtain ZnO@Au-DTNB; (3) The ZnO@Au-DTNB, EDC and NHS are mixed and reacted to activate the carboxyl group of DTNB; then mixed with type I antibody and coupled to obtain the ZnO@Au-DTNB nanotag.

2. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (a), the mass ratio of ZnCl2, cubic Cu2O, PVP and Na2S2O3 is (2~5):(10~20):(0.333~1):(632~1264).

3. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (a), the volume ratio of ethanol to water in the ethanol-water solution is (0.5~2):

1.

4. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (a), the reaction temperature is 20~30℃ and the reaction time is 1~3 h.

5. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (a), the cubic Cu2O is specifically prepared by the following steps: Under heating conditions, an aqueous solution of NaOH is added to an aqueous solution of CuCl2 to carry out a precipitation reaction and obtain a mixed solution; ascorbic acid solution is added dropwise to the mixed solution to carry out a reduction reaction, and then the solution is washed, filtered and dried to obtain the cubic Cu2O.

6. The ZnO@Au-DTNB nanotag according to claim 5, characterized in that, The mass ratio of CuCl2, NaOH and ascorbic acid is (0.1~0.3):(0.1~1):(1~2).

7. The ZnO@Au-DTNB nanotag according to claim 5, characterized in that, The precipitation reaction is carried out at a temperature of 50-60°C for 20-40 minutes.

8. The ZnO@Au-DTNB nanotag according to claim 5, characterized in that, The reduction reaction is carried out at a temperature of 50-60°C for 2-4 hours.

9. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (b), the annealing process is performed in an air environment.

10. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (b), the heating rate of the annealing treatment is 1~10℃ / min, the temperature of the annealing treatment is 200~300℃, and the time of the annealing treatment is 20~40 min.

11. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (1), the mass ratio of the oxygen-containing vacancy Zn to HAuCl4 is 1:(1~1.5).

12. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (1), the temperature of the ultrasonic treatment is 20~25℃, the power of the ultrasonic treatment is 100~120 W, and the time of the ultrasonic treatment is 0.5~2h.

13. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (2), the molar ratio of ZnO@Au to DTNB is (5~20):(0.001~0.004).

14. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (2), the temperature of the ultrasonic treatment is 20~25℃, the power of the ultrasonic treatment is 100~120 W, and the time of the ultrasonic treatment is 0.5~2h.

15. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (2), the washing process further includes a resuspension step: the ZnO@Au-DTNB is resuspended in an ethanol solution to obtain an ethanol solution of ZnO@Au-DTNB.

16. The ZnO@Au-DTNB nanotag according to claim 1, characterized in that, In step (3), the coupling reaction specifically includes the following steps: The carboxyl-activated ZnO@Au-DTNB, PBST buffer solution, and type I antibody were mixed and reacted for 2.5-3 h. Then BSA was added and reacted for 1-2 h. After washing, the ZnO@Au-DTNB nanotag was obtained.

17. The ZnO@Au-DTNB nanotag according to claim 16, characterized in that, In step (3), the mass ratio of the carboxyl-activated ZnO@Au-DTNB, type I antibody and BSA is (6~9):(0.006~0.012):(8~12).

18. The use of a ZnO@Au-DTNB nanotag according to any one of claims 1 to 17 in the preparation of products for detecting bacteria and / or for in-situ sterilization.

19. A system that combines bacterial detection and photocatalytic sterilization functions, characterized in that, The system comprises: the ZnO@Au-DTNB nanotag, the immunochromatographic test strip, and the TMB colorimetric enhancement system as described in any one of claims 1 to 17; The immunochromatographic test strip includes: a plastic backing plate, on which a glass fiber sample pad, a nitrocellulose membrane, and an absorbent pad are sequentially disposed; a detection line (T line) and a control line (C line) are sequentially disposed on the nitrocellulose membrane; the detection line (T line) is coated with type II antibody, and the control line (C line) is coated with goat anti-mouse IgG antibody; The TMB colorimetric enhancement system includes: a device for simulating a sunlight source and a colorimetric solution; the colorimetric solution includes TMB acetone solution, H2O2 and NaAc-HAc buffer.

Images