ZnO and ZnO-coated Au-DTNB nano-label containing oxygen vacancies and preparation method and application of ZnO and ZnO-coated Au-DTNB nano-label

Abstract

The invention provides a ZnO and ZnO coated Au-DTNB nano-label containing oxygen vacancies as well as a preparation method and application of the ZnO and ZnO coated Au-DTNB nano-label, and relates to a lateral flow immunoassay (LFIA) detection technology. The preparation method of the oxygen vacancy-containing ZnO comprises the following steps: in an alkaline reducing agent system, Zn salt and cubic Cu2O react to obtain cage-shaped Zn (OH) 2; the cage-shaped Zn (OH) 2 is subjected to annealing treatment, and three-dimensional ZnO is obtained; and carrying out photochemical etching treatment on the three-dimensional ZnO to obtain ZnO containing oxygen vacancies. According to the invention, defect engineering regulation and control of zinc oxide are realized through photochemical etching, and the prepared ZnO (at) Au with an adjustable energy band structure and loaded with gold nanoparticles shows excellent surface enhanced Raman scattering performance and catalytic activity. Meanwhile, a multi-mode LFIA detection system is constructed, is used for real-time detection of pathogens, and has good repeatability, rapid detection, high specificity and high detection sensitivity.

Description

TECHNICAL FIELD

[0001] The application relates to a lateral flow immunoassay (LFIA) detection technology, in particular to a ZnO containing oxygen vacancy, a ZnO@Au-DTNB nanolabel and a preparation method and application thereof. BACKGROUND

[0002] Rapid and efficient monitoring of bacteria is of great significance in the fields of disease diagnosis, medical prevention, food safety, public health and ecological environment protection. At present, common bacterial detection methods include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA) and diversified biosensor technology. Although the PCR and ELISA technologies have high sensitivity and specificity, their cumbersome operation process is difficult to meet the needs of point-of-care testing (POCT), and the dependence of some biosensors on high-precision manufacturing greatly increases the use cost. Therefore, the LFIA technology, which has the advantages of low cost, high sensitivity, integration and portability, has become a research hotspot in the field of POCT.

[0003] The design and optimization of nanolabels are the key to improving the detection performance. In recent years, LFIA has gradually developed new detection modes such as catalytic nanolabels, fluorescent nanolabels and surface-enhanced Raman scattering (SERS) nanolabels by combining with nanomaterials with different functions, and has achieved excellent detection performance with the help of new nanolabels. Therefore, nanolabels with multifunctional characteristics can realize multi-mode detection and multifunctional application. For example, loading gold (Au) and platinum (Pt) nanoparticles (NPs) on conventional nanolabels can simultaneously endow SERS activity and catalytic function, but these two performances have an inherent trade-off relationship.

[0004] In addition, although Au NPs can be reduced and adsorbed on the surface of the substrate through atomic-level active sites in theory, 3+ The precursor can realize a higher density of Au NP loading compared with the pre-synthesized Au seed deposition method. However, most intrinsic materials lack sufficient natural active sites, and the generation of these sites usually requires extreme processing conditions. Even if a high density of active sites is obtained, its catalytic performance may not be excellent. Although the reducing agent can promote the growth of Au NPs, it may also passivate the active sites, ultimately affecting the loading density of Au NPs. 3+

[0005] Therefore, the application is proposed. SUMMARY

[0006] ​One of the purposes of the present application is to provide an oxygen vacancy containing ZnO, a ZnO@Au-DTNB nanolabel and a preparation method and application thereof. The present application realizes the defect engineering regulation of zinc oxide (ZnO) through a photochemical etching technology, and the prepared ZnO@Au composite material with a tunable energy band structure and loaded gold nanoparticles exhibits excellent surface enhanced Raman scattering performance and catalytic activity. And the present application constructs a system with bacterial detection and photocatalytic sterilization functions by using the ZnO@Au-DTNB nanolabel, which is used for pathogen real-time detection, and the system has good repeatability, rapid detection, high specificity and high detection sensitivity; at the same time, the system integrates in-situ sterilization function and triple detection mode: colorimetric visual identification mode, catalytic colorimetric enhanced visual identification mode and SERS detection mode.

[0007] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0008] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0009] In the first aspect, the present application provides a preparation method of an oxygen vacancy containing ZnO, which comprises:

[0010] (a) reacting Zn salt and cubic Cu2O in an alkaline reducing agent system to obtain cage-like Zn(OH)2;

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

[0012] (c) photochemically etching the three-dimensional ZnO to obtain the oxygen vacancy containing ZnO.

[0013] In the present application, first, cubic Cu2O reacts with Zn salt in an alkaline reducing agent system to form cage-like cage-like Zn(OH)2, which is then converted into ZnO by annealing. That is, cubic Cu2O particles are used as a substrate, and through a displacement reaction of Zn 2+ and annealing treatment, three-dimensional ZnO with a nanostructure is obtained; the morphology of the three-dimensional ZnO is composed of nanoparticles, nanorods and nanosheets, forming a multi-level structure. Then, through a one-step photochemical etching method of the three-dimensional ZnO, the increase of reaction sites and the regulation of ZnO electronic structure are further realized.

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

[0015] ZnCl2, cubic Cu2O and PVP are dispersed in an ethanol aqueous solution to obtain a mixed solution; a Na2S2O3 solution is added dropwise into the mixed solution to perform a reaction, and then the product is washed, filtered and dried to obtain the cage-like Zn(OH)2.

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

[0017] As an optional embodiment, the volume ratio of ethanol and water in the ethanol aqueous solution is (0.5-2):1, for example, 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 embodiment, the temperature of the reaction is 20-30℃, for example, 20℃, 22℃, 24℃, 25℃, 26℃, 28℃, 30℃, etc., and the time of the reaction is 1-3h, for example, 1h, 1.5h, 2h, 2.5h, 3h, etc.

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

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

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

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

[0023] As an optional embodiment, the temperature of the reduction reaction is 50-60℃, for example, it can be 50℃, 52℃, 54℃, 55℃, 56℃, 58℃, 60℃, etc., and the time of the reduction reaction is 2-4h, for example, it can be 2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h, 4h, etc.

[0024] As an optional embodiment, in step (b), the annealing treatment is carried out in an air environment.

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

[0026] As an optional embodiment, in step (c), the photochemical etching treatment specifically comprises:

[0027] Disperse the three-dimensional ZnO in a photochemical etching solution to obtain a dispersion liquid; and perform light irradiation treatment on the dispersion liquid to obtain the ZnO containing oxygen vacancies.

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

[0029] As an optional embodiment, the photochemical etching solution comprises, in terms of mass percentage, 20-40% of K2B4O7, 1-5% of Na2SO3, and the balance is water.

[0030] In the present application, 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 maintain the chemical state of Zn 2+ through the complexation of borate ions (B4O7 2- ); on the other hand, the sulfite can promote the lattice oxygen removal induced by the photo-generated holes of ZnO and prevent the occurrence of reverse oxidation reaction as a reducing agent.

[0031] As an optional embodiment, the content of K2B4O7 is 20-40%, for example, 20%, 22%, 24%, 25%, 26%, 28%, 30%, 32%, 34%, 35%, 36%, 38%, 40%, etc., based on the total mass of the photochemical etching liquid being 100%.

[0032] As an optional embodiment, the content of Na2SO3 is 1-5%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc., based on the total mass of the photochemical etching liquid being 100%.

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

[0034] As an optional embodiment, the central light power of the light treatment is 310-330 mW, for example, 310 mW, 312 mW, 314 mW, 316 mW, 318 mW, 320 mW, 322 mW, 324 mW, 326 mW, 328 mW, 330 mW, etc.

[0035] As an optional embodiment, the time of the light treatment is 1-60 min, for example, 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc.

[0036] As an optional embodiment, the time of the light treatment 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.

[0037] In the present application, by controlling the photochemical etching time, the Fermi level (EF) of the ZnO oxygen vacancy concentration is reduced while maximizing it, which not only lays the foundation for high-density Au NPs loading to achieve strong LSPR effect, but also further enhances the possibility of DTNB interface charge transfer. And the difference in electronic structure and oxygen vacancy number of ZnO with different etching time will have an important influence on the loading of Au NPs and SERS effect.

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

[0039] In a third aspect, the present application provides a ZnO@Au-DTNB nanolabel, which comprises an oxygen vacancy-containing ZnO loaded with Au nanoparticles; wherein the oxygen vacancy-containing ZnO comprises the oxygen vacancy-containing ZnO according to the second aspect; and the ZnO@Au-DTNB nanolabel further comprises a DNTB, one end of which is connected to the Au nanoparticles through an Au-S bond, and the other end is connected to a type I antibody through a modified carboxyl group.

[0040] In the present application, based on the synthesized ZnO band structure (conduction band EC=-2.889eV, band gap Eg=2.991eV), a compatible Raman reporter molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, lowest unoccupied molecular orbital LUMO=-2.262eV, Eg=2.843eV) is selected, which ensures that the ZnO-DTNB and ZnO-Au-DTNB interfaces form staggered energy level arrangement to promote the directional charge transfer of DTNB. Finally, the Schottky contact formed between ZnO-Au and DTNB-Au induces the accumulation of charges on Au, further amplifying the LSPR effect and producing synergistic enhancement. In addition, the formation of ZnO-DTNB (staggered type) and ZnO-Au-DTNB (Z type) two heterostructures significantly promotes the photo-induced charge separation, optimizes the redox potential, and greatly improves the photocatalytic activity of the nanolabel.

[0041] In a fourth aspect, the present application provides a ZnO@Au-DTNB nanolabel according to the second aspect, and a preparation method thereof comprises:

[0042] (1) dispersing the oxygen vacancy-containing ZnO in an ethanol aqueous solution to obtain a dispersion liquid; adding an HAuCl4 aqueous solution to the dispersion liquid, ultrasonic treatment, and then centrifuging and washing 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, in step (1), the washing further comprises a resuspension step: resuspending the ZnO@Au in an ethanol solution to obtain a ZnO@Au-DTNB ethanol solution.

[0050] As an optional implementation, in step (2), the molar ratio of ZnO@Au to DTNB is (5-20):(0.001-0.004); wherein, for example, “5-20” can be 5, 10, 15, 20, etc.; and “0.001-0.004” can be 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, in step (2), the washing further comprises a resuspension step: resuspending the ZnO@Au-DTNB in an ethanol solution to obtain a ZnO@Au-DTNB ethanol solution;

[0053] As an optional implementation, in step (3), the coupling reaction specifically comprises the following steps:

[0054] The carboxyl-activated ZnO@Au-DTNB, PBST buffer solution, and type I antibody are mixed, and after reacting for 2.5-3h (for example, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, etc.), BSA is added and reacted for 1-2h (for example, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.), and then washed to obtain the ZnO@Au-DTNB nanolabel;

[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); wherein, for example, “6-9” can be 6, 6.5, 7, 7.5, 8, 8.5, 9, etc.; and “0.006-0.012” can be 0.006, 0.008, 0.010, 0.012, etc.

[0056] In a fifth aspect, the application provides an application of the oxygen vacancy-containing ZnO or the ZnO@Au-DTNB nanolabel in preparation of a product for detecting bacteria and / or a product for in-situ sterilization.

[0057] In a sixth aspect, the application provides a system with both bacterial detection and photocatalytic sterilization functions, which comprises the ZnO@Au-DTNB nanolabel, an immunochromatography test paper and a TMB colorimetric enhancement system.

[0058] The immunochromatography test paper comprises a plastic back plate, on which a glass fiber sample pad, a nitrocellulose membrane and an absorption pad are sequentially arranged; the nitrocellulose membrane is sequentially provided with a test line T and a quality control line C; the test line T is coated with a type II antibody, and the quality control line C is coated with a goat anti-mouse IgG antibody.

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

[0060] In the application, the ZnO@Au-DTNB nanolabel exhibits extremely strong SERS signals (with an enhancement factor EF up to 109) on the LFIA test strip. By optimizing the composition of the running buffer, the concentration of the antibody on the test line (T) and the amount of the SERS label in the LFIA system, high-sensitivity detection of S. aureus (SA) is achieved, with a fitted detection limit (LOD) as low as single-digit CFU / mL. In addition, thanks to the photocatalytic activity of the ZnO@Au-DTNB nanolabel, the catalytic colorimetric enhancement visual recognition mode has an LOD 100 times higher than the traditional colorimetric visual recognition mode (from 10 4 CFU / mL to 10 2 CFU / mL). Under the excitation of sunlight spectrum, the ZnO@Au-DTNB label on the LFIA test strip can also realize in-situ photocatalytic sterilization, providing a clean and simple solution for pollution control after detection. Through reasonable material selection and electronic structure design, the application develops a high-efficiency multifunctional LFIA detection platform, which provides an excellent solution for POCT pathogen diagnosis and also provides a new idea for the design of LFIA nanolabels.

[0061] For example, 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) The application utilizes ZnO semiconductor material with large specific surface area and intrinsic photocatalytic properties, and constructs a nano-tag with SERS and catalytic capabilities by loading Au NPs; after the deposition of Au NPs, the local surface plasmon resonance (LSPR) effect can significantly enhance the SERS performance (the enhancement factor EF is as high as 10 9 ); at the same time, the ZnO@Au system exhibits more excellent photocatalytic activity than the original ZnO, which is due to three synergistic mechanisms: (i) SPR-induced light absorption and hot carrier generation, (ii) Schottky junction-promoted charge separation, and (iii) metal-semiconductor catalytic synergy, and high-density loading of Au NPs on ZnO can simultaneously enhance the dual function of the nano-tag.

[0066] (3) The application further utilizes the Raman reporter molecule 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to further optimize the SERS performance of the nano-tag, and the photocatalytic ability of the ZnO@Au system can be further enhanced in theory after being combined with the Raman reporter molecule (the SERS effect is mainly derived from the LSPR effect of the noble metal and the charge transfer between the substrate and the reporter molecule, which fundamentally affects the change of the polarizability of the reporter molecule), thereby obtaining higher detection sensitivity.

[0067] (4) The application constructs a multi-mode LFIA detection system for pathogen point-of-care testing (POCT), which has good repeatability, rapid detection (<15 min), high specificity and high detection sensitivity, and integrates in-situ sterilization function (antibacterial rate >99%) and triple detection modes: colorimetric visual recognition mode (limit of detection LOD = 10 4 CFU / mL), catalytic colorimetric enhanced visual recognition mode (LOD = 10 2 CFU / mL) and SERS detection mode (the fitting LOD is less than 10 CFU / mL). This innovative strategy of nano-materials based on electronic structure design provides a new idea for the performance improvement and function expansion of LFIA detection. BRIEF DESCRIPTION OF DRAWINGS

[0068] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the following specific embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings without creative labor on the basis of these drawings.

[0069] Figure 1 The preparation of the ZnO@Au-DTNB nano-tag described in the application and the bacterial detection process are shown in the following schematic diagram.

[0070] Figure 2 Schematic diagram of SERS mechanism of ZnO@Au-DTNB nanolabels described in the present application.

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

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

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

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

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

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

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

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

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

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

[0081] Figure 4E Ultraviolet photoelectron energy (UPS) spectra (0 V and -5 V) of DTNB provided for Example 1.

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

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

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

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

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

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

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

[0089] Figure 6A The O 1s orbital X-ray photoelectron spectroscopy fine spectrum of the oxygen vacancy containing ZnO obtained by etching the three-dimensional ZnO provided in Example 1 for 0 min, 10 min, 20 min, and 30 min.

[0090] Figure 6B The Zn 2p orbital X-ray photoelectron spectroscopy fine spectrum of the oxygen vacancy containing ZnO obtained by etching the three-dimensional ZnO provided in Example 1 for 0 min, 10 min, 20 min, and 30 min in the preparation method.

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

[0092] Figure 8ATEM images of ZnO with oxygen vacancies etched for 0 min, 30 min, 60 min and 120 min, on which gold nanoparticles were loaded for Example 1.

[0093] Figure 8B Schematic diagram of the consistency of the oxygen vacancy density of ZnO@Au with the gold nanoparticle density for Example 1.

[0094] Figure 8C XRD spectra of gold nanoparticles loaded on ZnO with oxygen vacancies for different etching times.

[0095] Figure 8D Raman spectra of ZnO@Au-DTNB for different etching times, with the redshift wave number relative to the -NO2 peak of intrinsic DTNB noted in the figure.

[0096] Figure 8E Schematic diagram of the energy band structure of the contact mode of ZnO@Au-DTNB for different etching times.

[0097] Figure 8F Schematic diagram of the energy band structure of the contact mode of ZnO@Au-DTNB for different etching times.

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

[0099] Figure 9B Energy spectrum (EDS) intensity integral spectrum of ZnO@Au composite material for different etching times.

[0100] Figure 10A Under the condition of perpendicular incidence of light at a wavelength of 785 nm, the photoelectric field distribution of a 20 nm thick gold nanoparticle loaded ZnO film array with a spacing of 10 nm, 30 nm, 50 nm and 100 nm is shown.

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

[0102] Figure 11A High-resolution transmission electron microscopy and selected area electron diffraction of ZnO@Au-DTNB for different etching times.

[0103] Figure 11B High-resolution transmission electron microscopy and selected area electron diffraction of ZnO with oxygen vacancies for different etching times.

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

[0105] Figure 13 Raman spectrograms of ZnO-DTNB composites for different etching times.

[0106] Figure 14 DFT simulation diagram. Note: In the Bader charge analysis diagram, the yellow area represents the charge accumulation area, and the blue area represents the charge depletion area.

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

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

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

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

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

[0112] Figure 17C Raman spectrogram of 1M DTNB.

[0113] Figure 18A Signal-to-noise ratio and Raman spectrograms under different buffer ratios.

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

[0115] Figure 18C Signal-to-noise ratio and Raman spectrograms under different antibody concentrations on the detection line.

[0116] Figure 19A Visualized signal diagrams of different SA concentrations in the detection of Staphylococcus aureus (SA) by SERS-based LFIA sensors.

[0117] Figure 19BSchematic diagram of Raman signals for different SA concentrations.

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

[0119] Figure 19D Fitting of the Raman signal at 1331 cm-1by a logistic function using the Levenberg-Marquardt iterative algorithm. -1 Relationship between the intensity of the characteristic peak and the logarithm of the bacterial concentration. The dotted line indicates the limit of detection (LOD), calculated as the average of the blank signal plus three times the standard deviation (Blank + 3SD).

[0120] Figure 19E Evaluation of sensor reproducibility by analyzing the consistency of the measurements at a constant SA concentration by four independent sensor groups (n = 4 for the test sample size). The results were determined by calculating the relative standard deviation of the average of the response signals

[0121] Figure 19F Evaluation of the specificity of the sensor for SA by testing five comparable concentrations (all 106CFU / mL) of bacterial species. Results plot

[0122] Figure 20A Optical images of the three groups of detection experiments.

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

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

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

[0126] Figure 21B Photos of the sensor for different concentrations of SA detection and after adding TMB color developing solution.

[0127] Figure 21C EPR spectra of ZnO@Au-DTNB under sunlight and dark field, corresponding to DMPO-·OH (top) and DMPO-·O2- (bottom), respectively; Enzyme saturation kinetic curves of ZnO@Au-DTNB labels (n = 3 for the sample size).

[0128] Figure 21DThe concentration of H2O2 was fixed at 500 mM, and the concentration of TMB was increased in geometric progression with the first term of 0.5 mM and the common ratio of 2.

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

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

[0131] Figure 21G The column chart of the ORR reaction thermodynamic path in ZnO@Au-DTNB.

[0132] Figure 22 The mechanism diagram of the oxidation of TMB and sterilization by reactive oxygen species (ROS) generated by ZnO@Au-DTNB under sunlight irradiation.

[0133] Figure 23A The ultraviolet-visible absorption spectrum of the photocatalytic color developing solution composed of H2O2+TMB+marker+NaAc-HAc buffer and the control group.

[0134] Figure 23B The ultraviolet-visible absorption spectrum of the color developing solution prepared by different pH values of NaAc-HAc buffer.

[0135] Figure 23C The ultraviolet-visible absorption spectrum of the color developing solution prepared by different concentrations of markers.

[0136] Figure 24 The sterilization experiment diagram of three groups of S. aureus bacterial solution under different concentrations. DETAILED DESCRIPTION

[0137] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. In this application, the use of "or" means "and / or" unless specifically stated otherwise, e.g., "comprising A or B" means "comprising A or B or both". Also, the use of "comprising" or other forms of "including", such as "including", "includes", "containing", "contains", or "having", are not intended to be limiting. It is to be further noted that the description herein is made by way of example and not of limitation. The present application is limited only by the claims.

[0138] The technical solutions of the present application will be described clearly and completely below in connection with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0139] The present application will be further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to the existing methods, or directly purchased from the market.

[0140] The sources of the materials and equipment in the following examples and test examples are shown as follows:

[0141] Materials: Copper chloride dihydrate (CuCl2 2H2O, Aldrich, cas: 10125-13-0), 2 M sodium hydroxide solution (cas: 1310-73-2, Aldrich), ascorbic acid (Sigma Aldrich), zinc chloride (ZnCl2, Aldrich, cas: 7646-85-7), polyvinylpyrrolidone (PVP, 40 kDa), absolute ethanol (analytical grade), deionized water, 1 M sodium thiosulfate solution (Aldrich, cas: 1310-73-2), potassium tetraborate (K2B4O7 4H2O, Aldrich, cas: 12045-78-2), sodium sulfite (Na2SO3, Aldrich, cas: 7757-83-7), Tween-20 (Sigma Aldrich), Dulbecco’s phosphate buffered saline (PBS, Sigma), Acetic acid-sodium acetate buffer solution (NaAc-HAc buffer, pH = 3.6). Millipore nitrocellulose membranes (NC membranes) CN95 were purchased from Millipore (Spain). Glass fiber sample pads (CB08), sample application pads, absorbent pads and plastic backings were provided by Life Science (China) and Shanghai Jetway Biotech (China). Goat anti-mouse IgG (Cat. No. D111024) was provided by RiboBio (China). Staphylococcus aureus monoclonal antibody (Cat. No. MA1-10708, Type I antibody) was from Thermo Fisher Scientific. Staphylococcus aureus antibody (Cat. No. 10-S30B, Type II antibody) was from Fitzgerald. Chloroauric acid tetrahydrate (HAuCl4 4H2O), acetone, 30% hydrogen peroxide aqueous solution (H2O2) were purchased from National Medicine 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. N-hydroxysuccinimide sodium salt (NHS, 97%) was purchased from Alfa Aesar. Bovine serum albumin (BSA, pH 7.0) was purchased from GPC Bio.

[0142] Instrumentation: UV-Vis spectrophotometer (UV-Vis, Shimadzu 2600), Nanoparticle size and Zeta potential analyzer (Malvern Nano-ZS90 Zetasizer), X-ray photoelectron spectrometer (XPS, Thermo 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), Multifunctional enzyme label (SPARK, TECAN, Austria) for detecting the absorbance change of oxTMB at 655 nm, Fourier transform infrared spectrometer (FTIR, Bruker ALPHA II), Raman spectrometer (B&W Tek, i-Raman Plus BWS465-785H), Simultaneous thermal analyzer (TG-DSC, NETZSCH STA 449F3 / F5), Ultraviolet photoelectron spectrometer (UPS, Thermo ESCALAB 250Xi).

[0143] Example 1

[0144] The embodiment provides a ZnO containing oxygen vacancy and a ZnO@Au-DTNB nanolabel comprising the ZnO, and a preparation method of the ZnO@Au-DTNB nanolabel specifically includes the following steps.

[0145] S1, preparation of Cu2O cubic crystal:

[0146] 0.17 g of CuCl2·2H2O was dissolved in 100 mL of deionized water to form a blue clear solution; after heating to 55°C, 10 mL of 2M NaOH solution was added, and magnetic stirring was performed for 30 min to obtain a dark black solution; the mixed solution was kept at 55°C, and 10 mL of 0.6 mol ascorbic acid was slowly added dropwise to generate a brick red precipitate; the stirring was continued for 3 h until the solution was completely brick red; finally, the precipitate was washed with deionized water and ethanol alternately for 4 times, and was collected by suction filtration; the obtained Cu2O cubic crystal product was dried at 60°C overnight, and was stored in a sealed dry and cool place.

[0147] S2, preparation of cage-like Zn(OH)2:

[0148] 10 mg Cu2O cubic crystal, 2 mg ZnCl2 and 0.333 mg PVP were added into 10 mL mixed solution of ethanol and water (volume ratio = 1:1) and dispersed by ultrasonic for 10 min; then 4 mL 1M Na2S2O3 solution was added dropwise into the above mixed solution, and the suspension was stirred magnetically at 25°C for 2 h until it changed from red to light white; the sample was washed with deionized water and ethanol alternately for 4 times, and separated by suction filtration to obtain the cage-like Zn(OH)2; the sample was naturally dried and stored in a sealed container.

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

[0150] The Zn(OH)2 powder was placed in a crucible and put into a muffle furnace, and then heated to 250°C at a rate of 5°C / min in air and kept for 30 min to finally obtain ZnO nanomaterials.

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

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

[0153] S5, Preparation of ZnO@Au:

[0154] 8 mg etched ZnO was added into a centrifuge tube containing 8 mL ethanol and 8 mL water, and ultrasonicated for 15 min; 1 mL HAuCl4 (10 g / L) aqueous solution was added, 25 at a power of 100W ultrasonic for 1 h until the solution changed from light yellow to deep purple; centrifuged at 8000 rpm, and the precipitate was washed with ethanol and water alternately for 6 times; finally, the ZnO@Au precipitate was resuspended in 400 μL ethanol for standby use.

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

[0156] A 10 mmol / L DTNB ethanol solution was prepared, and 2 μL of the solution was added into the above 400 μL ZnO@Au ethanol solution, and ultrasonicated for 1 h to promote the formation of Au-S bond between DTNB and Au; after centrifugation, the precipitate was washed with ethanol for 2 times, and finally resuspended in 1 mL ethanol for standby use.

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

[0158] Take 1 mL ZnO@Au-DTNB ethanol dispersion liquid centrifugation, wash with 0.05% PBST (PBS: Tween-20 = 0.05:99.95) 1 time after discarding the supernatant; add 500 μL MES (10 mmol, pH = 5.5), 50 μL EDC (10 mmol) and 100 μL NHS (10 mmol), ultrasonic 15 min to activate the DTNB carboxyl; after discarding the supernatant, add 300 μL 0.05% PBST and 3 μL I type antibody (MA1-10708), ultrasonic dispersion and then place in a shaker (25°C, 800 rpm) for 2.5-3 h; add 100 μL 10% BSA (BSA:H2O = 10:90) and vortex mix, continue to block for 1.5 h; finally, wash with 0.05% PBST once, resuspend in 200 μL 0.05% PBST and store at 4°C for standby.

[0159] Example 2

[0160] The embodiment provides a system with bacterial detection and photocatalytic sterilization functions; the system comprises the ZnO@Au-DTNB nanolabel, an immunochromatography test paper and a TMB colorimetric enhancement system.

[0161] The immunochromatography test paper is assembled from a macroporous nitrocellulose membrane (NC membrane), a glass fiber sample pad, an absorbent pad and a plastic back plate; the plastic back plate provides support, the sample pad is used for adding a liquid sample, the NC membrane provides capillary force to push the solution system to flow on the membrane, and the absorbent pad can absorb excess liquid to prevent backflow; the specific process is as follows:

[0162] (1) draw 10-S30B and IgG antibody lines on the NC membrane as a test line (T line) and a quality control line (C line) respectively. The 10-S30B antibody only captures Staphylococcus aureus, and the IgG antibody can capture almost all antigens. The signal change of the T line reflects the detection of the target antigen, and the C line can judge whether the sensor is invalid;

[0163] (2) dry the drawn NC membrane at 37°C, and complete the assembly of each component of the sensor after drying;

[0164] (3) mix an appropriate amount of label, different concentrations of antigens and running buffer, take 70 μL and add to the sample pad. Under the capillary action, the label carrying the antigen flows through the T line and the C line and is captured, and the whole process takes about 15 min. The antigen concentration determines the amount of label retained on the T line, realizing the quantitative detection of SA;

[0165] (4) obtain the detection results by naked eye 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-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-The protection of sulfite reducing protectant is limited to ZnO containing excess oxygen vacancies.

[0178] As shown in Figure 5C , the positive potential of the system is enhanced as the oxygen vacancies increase; but after 60 min, the potential gradually decreases and even forms a negative potential; this is due to over-oxidation and the formation of Zn vacancies; the observed abnormal decrease in Fermi level at the initial stage of photochemical etching may be partly related to the simultaneous generation of Zn vacancies, and more likely the strong interaction between metal Zn-Zn offsets the doping effect of donor Zn.

[0179] As shown in Figure 5D , the products after etching for different lengths of time still belong to the hexagonal crystal system, but long-time etching will cause atomic stacking disorder and crystal face distortion, causing diffraction peak splitting and shift. The relatively weak characteristic peaks in the XRD spectrum indicate that the ZnO has low crystallinity, which corresponds to the weak Zn-O vibration mode in the Raman spectrum. As the etching time is prolonged, the Zn-O vibration mode is further weakened, and the ratio of the ZnO defect state mode peak to the Zn-O vibration mode peak increases significantly, indicating that long-time photochemical etching will cause significant damage to the ZnO crystal structure.

[0180] As shown in Figure 5E , the scanning electron microscope (SEM) images of ZnO after etching for 0 min, 30 min, 60 min and 120 min show that the original three-dimensional hierarchical structure gradually collapses as the etching time is prolonged, which is also due to the destruction of the crystal structure. It shows that photochemical etching also has a significant impact on the crystal structure and morphology of ZnO.

[0181] As shown in Figure 6A , the O1s fine spectrum peaks of the four samples obtained by sampling every 10 min within 30 min also show a consistent trend in the change of oxygen vacancy content with EPR results; the proportion of oxygen vacancy peak area gradually increases over time. As shown in Figure 6B , in the Zn 2p fine spectrum, the proportion of Zn 0 peak area in the 30 min sample increases sharply, and the 4d state transition peak appears at 1025 eV, the Zn LOSS peak appears at 1030 eV, and the plasmon effect peak appears at 1034 eV. These results show that the metal Zn exposed due to the increase in oxygen vacancies is difficult to be shielded by the solution system, and the electronic transition and interaction between Zn-Zn gradually increase. Once the sulfite protectant cannot meet the anti-oxidation needs of Zn, Zn will be re-oxidized to fill the oxygen vacancies, which is also reflected in the Zeta potential characterization.

[0182] As shown in Figure 7 , the Raman spectrum is in the range of 100 cm -1 and 445 cm -1two peaks representing Zn-O vibration, and a peak representing ZnO defect state in the range of 300-350 cm -1 .

[0183] Test Example 2

[0184] Test method: Transmission electron microscope (TEM), Energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Raman scattering spectrum, Fourier transform infrared spectrum.

[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 microscope (TEM).

[0187] As shown in Figure 8A and Figure 9A It can be determined that the light-colored layer is a special view of flaky ZnO, and the black substance above is Au NPs. The deposition density of Au NPs is obviously affected by different etching times of ZnO.

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

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

[0190] As shown in Figure 8D Raman spectra of ZnO@Au-DTNB derived from ZnO under different etching times are shown. It can be observed that ZnO@Au-DTNB obtained from ZnO etched for 30 min (i.e. ZnO-30) has the strongest -NO2 characteristic peak intensity at about 1331 cm- 1 . The reasons for this are as follows: first, the high-density oxygen vacancies on ZnO are filled with more dense gold nanoparticles, thereby achieving a stronger electromagnetic field enhancement effect.

[0191] Based on the transmission electron microscope images, the present application further constructs a ZnO model loaded with gold nanoparticles with different density distributions, and uses FDTD (finite difference time domain) algorithm to simulate the electric field distribution of 785 nm laser under the condition of 785 nm continuous wave plane laser vertical incidence.

[0192] As shown in 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] As shown in Figure 14 , respectively, atomic-level interface contact model diagrams of DTNB-Au, Au-ZnO, DTNB-Au-ZnO and DTNB-ZnO, and Bader charge analysis diagrams of each material are shown.

[0204] Among them, in the Bader charge analysis of the DTNB-Au interface: the Bader charge distribution between DTNB and Au is shown, the yellow area is the charge accumulation area, and the blue area is the charge depletion area. It can be seen that there is significant charge transfer from DTNB to the surface of Au at the interface. Further through Bader charge calculation, the present application analyzes the charge transfer phenomenon of 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 Au-ZnO, DTNB-Au-ZnO and DTNB-ZnO interface charge transfer phenomena formed by high and low oxygen vacancy density ZnO are consistent: 1. For Au-ZnO, the charge is mainly accumulated on the Au surface; 2. In the DTNB-ZnO contact, the charge is mainly gathered at the defect site; 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 charges into Au. Due to the presence of ZnO, the charge transfer between DTNB and Au is less than that of pure DTNB-Au interface, which indicates that there is indeed competition between ZnO and DTNB in injecting charges into Au; 4. In the DTNB-ZnO contact, the charge polarization of DTNB is very obvious, and the charge accumulation of the defect site of ZnO is more.

[0206] By further comparing the charge transfer differences of the two types of ZnO interface, it is found that: 1. In the DTNB-Au-ZnO contact, when ZnO-high participates, the charge depletion zone of DTNB is more obvious, indicating that its competition for charge injection to Au is weaker; 2. In the DTNB-ZnO contact, when ZnO-high participates, the charge is more easily transferred to the deeper part of the ZnO crystal; while when ZnO-low participates, the charge is limited by the surface oxygen dangling bond, resulting in the hindering of the charge transfer between them. The DFT calculation results are basically consistent with the experimental characterization analysis results, providing a good theoretical basis. In summary, due to the high density of oxygen vacancies and the appropriate band structure, ZnO etched for 30 min should exhibit the strongest SERS performance. In addition, sufficient Au 3+ source is needed to fully cover the abundant active sites on ZnO and support the growth of Au seeds to form hot spots. The improvement of SERS effect is significantly related to the content of Au 3+ source.

[0207] Test Example 4

[0208] Test Method:

[0209] (1) Raman spectrum, TEM and EDS: Effect of different amounts of HAuCl4 on the SERS effect of ZnO@Au-DTNB label; sample group: ZnO@Au-DTNB composite materials prepared by different amounts of HAuCl4; control group: Au NPs-DTNB and 1M DTNB. Among them, the synthesis method of Au NPs-DTNB is the same as that of ZnO@Au-DTNB, but no ZnO is added.

[0210] (2) Raman spectrum: Effect of different concentrations of DTNB on the SERS effect of ZnO@Au-DTNB label.

[0211] Among them, the enhancement effect of SERS is usually evaluated by the enhancement factor (EF), and the specific calculation method is: In addition to part of the Raman characterization for material vibration mode analysis, all Raman signal tests on test strips are carried out under the conditions of 785nm laser, 1% laser intensity (1mW), 50 times objective, 500ms excitation time and single collection. The ideal calculation formula of enhancement factor (EF) is EF=(ISERS / NSERS), where ISERS and NSERS represent the Raman characteristic peak intensity and the number of molecules under SERS effect, INormal and NNormal represent the corresponding values under normal conditions. In actual operation, molar concentration is used instead of molecule number, and the modified formula is: EF=(ISERS / CSERS)*(INormal / CNormal). Under the condition of strictly ensuring the consistency of collection parameters, the EF of low concentration reporter molecule 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) SERS-based LFIA sensor for bacteria detection. Data were subjected to Shapiro-Wilk normality test, one-way ANOVA, followed by two-tailed t-test against the blank control. The significantly smaller p-value annotated in the figure demonstrated statistically robust selectivity for SA detection.

[0226] (2) Detection experiment setup three groups: blank control group, pure S. aureus group (S. aureus) and mixed bacteria group (containing S. aureus, E. coli, S. pneumoniae, P. aeruginosa and Salmonella Typhimurium), each strain concentration is about 10 6 CFU / mL.

[0227] SA bacteria were inoculated using commercial blood agar plates, and the bacterial liquid concentration was evaluated by McFarland Turbidity Meter. ZnO@Au-DTNB labels were synthesized according to the previous steps, but finally dispersed in 1 mL PBS (pH = 7). In the antibacterial experiment, 10 μL of label dispersion was mixed with 490 μL of bacterial liquid and then irradiated with light for 30 min (light power 320 mW), and then coated on the blood agar plate for 16 h before counting.

[0228] The operation method of each experimental group and the control group is completely consistent, the variable is whether to irradiate and whether to add labels: Normal group represents the untreated bacterial liquid cultured by the conventional method; Sunlight 30 minutes group represents that the bacterial liquid is treated with 30 minutes of light before inoculation; Tags addition group represents that the bacterial liquid is inoculated immediately after adding ZnO@Au-DTNB; Tags addition and Sunlight 30 min group represents that the bacterial liquid is inoculated after adding ZnO@Au-DTNB and irradiating for 30 minutes. The sterilization rate calculation formula is R = (N0-N) / N0*100%, where R is the sterilization rate, N0 represents the average number of viable bacteria in the Normal control group, and N represents the average number of remaining viable bacteria in the Tags addition and Sunlight 30 min sterilization experiment group.

[0229] Test results:

[0230] ZnO@Au-DTNB labels capturing different concentrations of S. aureus (SA) will be further captured after flowing through the detection line. Labels with higher SA concentrations will be captured more, leaving deeper marks on the detection line with the naked eye, such as Figure 19A 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 nonlinear fitting was performed using a logistic model. 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 was increased to 10 2 CFU / mL Figure 21B ). This peroxidase-like (POD) activity mainly comes from the used nanolabels.

[0236] Under high-energy photon excitation, the ZnO-Au-DTNB contact mode forms a typical Z-type heterojunction. Au as a charge transport layer can promote the efficient separation of photo-generated carriers of ZnO and DTNB and enhance the light trapping efficiency based on the LSPR effect. Photo-generated holes and electrons are enriched in ZnO with a higher oxidation potential and DTNB with a higher reduction potential, thereby optimizing the redox potential and further promoting possible redox reactions.

[0237] Benefiting from this photocatalytic property, the test strip capturing ZnO@Au-DTNB labels can produce strong redox ability under light to oxidize the TMB substrate, thereby enhancing the colorimetric signal. In this system, TMB can be directly reacted with holes or oxidized by reactive oxygen species (ROS). But considering that a large number of active sites of ZnO are occupied by deposited Au particles, TMB is difficult to form stable adsorption with ZnO widely, and therefore a large amount of ROS is the key to TMB oxidation.

[0238] As shown in Figure 22 , photo-generated holes and electrons can promote the conversion of H2O and H2O2 into ·OH free radicals, while oxygen molecules can produce superoxide free radicals (·O2-) at a higher reduction potential, and the corresponding active oxygen is also verified by EPR characterization Figure 21C . If in an acidic buffer solution, the reaction of H2O2 reduction to ·OH free radicals will be promoted, thereby greatly accelerating the oxidation of TMB.

[0239] To further evaluate the catalytic performance of the label on TMB, the present application studies the reaction kinetics process. At low concentration, according to the Beer-Lambert law, the absorbance is positively linearly related to the substrate concentration. Therefore, based on the UV-Vis spectrum Figures 23A-23C , the OD (optical density) value at 655 nm is selected to reflect the substrate concentration. Figure 21D and 21E are typical Michaelis curves, and the V max and K m kinetic parameters are obtained by fitting the reaction rates of H2O2 and TMB at different concentrations. V max represents the maximum reaction rate, reflecting the catalytic efficiency of the enzyme; K m is the Michaelis constant, reflecting the affinity of the enzyme to the substrate. Compared with the TMB substrate, the ZnO@Au-DTNB label shows smaller K m value and larger V max value, indicating that the label has stronger affinity and catalytic efficiency for H2O2. Combined with the fact that the ZnO@Au-DTNB label has a larger specific surface area and more active sites, it is concluded that the label has stronger catalytic performance for H2O2.Figures 23A-23C The UV-visible absorption spectrum of the ZnO@Au-DTNB SERS tag also shows that the presence of H2O2 significantly promotes the oxidative discoloration of TMB, which is consistent with the mechanism of H2O2 generating ·OH to oxidize TMB.

[0240] The large amount of ROS generated by the SERS tag is also conducive to sterilization, which endows the device with the function of in-situ sterilization. Figure 22 Under the neutral incubation condition maintained by PBS buffer, both the reversible reactions of oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) can form transition state free radicals. Among them, free radicals such as ·OH and ·O2-which have strong oxidizing properties can inactivate bacteria. The generation of free radicals is reflected in the EPR characterization of Figure 21C : the spectrum lines corresponding to ·OH and ·O2-are weak in the dark field, but the spectrum line intensity increases significantly under illumination. The present application further calculates the reaction thermodynamic path of the ZnO@Au-DTNB tag in the air environment by DFT simulation Figure 21F and Figure 21G . Based on the thermodynamic analysis of Gibbs free energy change, the product with lower free energy is more likely to be spontaneously generated. Therefore, under the catalysis of the SERS tag, the four-electron path of ORR can spontaneously proceed to form intermediate state ROS, and in the rate-determining step, illumination will significantly reduce the energy barrier to ·OH conversion, making the reaction more likely to proceed.

[0241] Test Example 8

[0242] Test method: In order to further verify the antibacterial effect of the SERS tag, the present application carries out an antibacterial experiment by using the viable cell counting method.

[0243] Sterilization experiment of three groups of S. aureus bacterial solution under different concentrations: untreated group (Normal): untreated control bacterial solution, directly incubated; sunlight 30min group: bacterial solution received 30min illumination and then incubated; tag addition group (Tags addition): bacterial solution added with ZnO@Au-DTNB complex and then incubated; tag addition + sunlight 30min group (Tags addition and sunlight 30min): bacterial solution added with ZnO@Au-DTNB and then received 30min illumination and incubated.

[0244] Test results:

[0245] As Figure 24As shown, under normal culture conditions, all three SA concentration groups formed large and dense bacterial colonies; the bacterial colonies were still relatively dense after 30 min of light, indicating that simulated sunlight did not produce a significant sterilization effect in a short time; in the control group with only the label added, the number of viable bacterial colonies decreased slightly, which may be due to the intrinsic catalytic ability of the label under dark field or the effect of indoor weak light on the label during the culture process; and after the label was added and cultured under light conditions for 30 min, the number of viable bacterial colonies decreased significantly. According to the counting results of the culture plates, the sterilization rate of all three SA concentration groups reached more than 99.8%, indicating that the ZnO@Au-DTNB label has good sterilization effect.

[0246] In summary, when zinc oxide semiconductor is combined with low-cost and simple-to-operate low-field immunoassay technology, it exhibits excellent surface-enhanced Raman scattering (SERS) and catalytic colorimetric enhancement dual-mode detection performance, and also has good antibacterial properties. Through photochemical etching and ultrasonic-assisted deposition technology, a ZnO@Au-DTNB label with excellent SERS effect is successfully prepared. Three-dimensional zinc oxide rich in oxygen vacancies provides a basis for high-density deposition of gold nanoparticles, thereby realizing strong electromagnetic field enhancement; based on the adjustment of the electronic structure of zinc oxide, the charge transfer between ZnO, gold and DTNB is optimized to obtain the maximum polarizability change of the DTNB reporter molecule; with the aid of zinc oxide, the LSPR effect of gold and the charge transfer ability of DTNB are synergistically enhanced. In the SERS detection mode, the detection limit of Staphylococcus aureus can reach a single trillionth unit / milliliter. The Z-type heterojunction formed by ZnO@Au-DTNB has excellent photocatalytic performance. The ·OH generated in the four-electron path greatly promotes the oxidation reaction of TMB, making the detection limit of the naked-eye colorimetric identification mode increase by two orders of magnitude. Harmless treatment after pathogen detection is very important. With the help of ZnO@Au-DTNB, 99% sterilization rate can be achieved in a short time of irradiation. Reasonable selection and fine design of the label will enable the LFIA platform to have excellent performance and multifunctional characteristics, providing a new idea for the development of new 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 application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing ZnO containing oxygen vacancies, characterized in that, The method for preparing the oxygen-vacancy-containing ZnO includes: (a) In an alkaline reducing agent system, Zn salt reacts with cubic Cu2O to obtain cage-like Zn(OH)2; (b) Annealing the cage-like Zn(OH)2 to obtain three-dimensional ZnO; (c) The three-dimensional ZnO is subjected to photochemical etching to obtain ZnO containing oxygen vacancies.

2. The method for preparing oxygen-vacancy-containing ZnO according to claim 1, characterized in that, In step (a), the cage-like Zn(OH)2 is specifically prepared by the following steps: 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; Preferably, the mass ratio of ZnCl2, cubic Cu2O, PVP and Na2S2O3 is (2-5):(10-20):(0.333-1):(632-1264); Preferably, the volume ratio of ethanol to water in the ethanol-water solution is (0.5–2):1; Preferably, the reaction temperature is 20–30°C, and the reaction time is 1–3 hours.

3. The method for preparing oxygen-vacancy-containing ZnO 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, resulting in a mixed solution; ascorbic acid solution is added dropwise to the mixed solution to carry out a reduction reaction, followed by washing, filtration and drying to obtain the cubic Cu2O; Preferably, the mass ratio of CuCl2, NaOH, and ascorbic acid is (0.1–0.3):(0.1–1):(1–2); Preferably, the precipitation reaction is carried out at a temperature of 50–60°C for 20–40 minutes. Preferably, the temperature of the reduction reaction is 50-60°C, and the time of the reduction reaction is 2-4 hours.

4. The method for preparing oxygen-vacancy-containing ZnO according to claim 1, characterized in that, In step (b), the annealing process is performed in an air environment; Preferably, the heating rate of the annealing treatment is 1-10℃ / min, the annealing temperature is 200-300℃, and the annealing time is 20-40min; Preferably, in step (c), the photochemical etching process specifically includes: 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. Preferably, the concentration of three-dimensional ZnO in the dispersion is 0.5–5 mg / mL; Preferably, the photochemical etching solution comprises, by mass percentage: 20-40% K2B4O7, 1-5% Na2SO3, and the balance being water; Preferably, the center optical power of the illumination treatment is 310-330mW; Preferably, the light treatment time is 1 to 60 minutes, more preferably 20 to 40 minutes.

5. A ZnO containing oxygen vacancies, characterized in that, The oxygen-vacant ZnO is prepared by the preparation method according to any one of claims 1 to 4.

6. A ZnO@Au-DTNB nanotag, characterized in that, The ZnO@Au-DTNB nanotag comprises ZnO with oxygen vacancies loaded with Au nanoparticles; wherein the ZnO with oxygen vacancies comprises the ZnO with oxygen vacancies as described in claim 5. 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.

7. A ZnO@Au-DTNB nanotag according to claim 6, characterized in that, 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.

8. The ZnO@Au-DTNB nanotag according to claim 7, characterized in that, In step (1), the mass ratio of the oxygen-vacant Zn to HAuCl4 is 1:(1~1.5); Preferably, in step (1), the temperature of the ultrasonic treatment is 20-25°C, the power of the ultrasonic treatment is 100-120W, and the time of the ultrasonic treatment is 0.5-2h. Preferably, in step (1), the washing process further includes a resuspension step: resuspending the ZnO@Au in an ethanol solution to obtain an ethanol solution of ZnO@Au-DTNB; Preferably, in step (2), the molar ratio of ZnO@Au to DTNB is (5-20):(0.001-0.004); Preferably, in step (2), the temperature of the ultrasonic treatment is 20-25°C, the power of the ultrasonic treatment is 100-120W, and the time of the ultrasonic treatment is 0.5-2h. Preferably, in step (2), the washing process further includes a resuspension step: resuspending the ZnO@Au-DTNB in ​​an ethanol solution to obtain an ethanol solution of ZnO@Au-DTNB; Preferably, 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, followed by washing to obtain the ZnO@Au-DTNB nanotag. Preferably, 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).

9. The use of an oxygen-vacancy-containing ZnO according to claim 5, or a ZnO@Au-DTNB nanotag according to claim 6, in the preparation of products for detecting bacteria and / or for in-situ sterilization.

10. A system that combines bacterial detection and photocatalytic sterilization functions, characterized in that, The system comprises: the ZnO@Au-DTNB nanotag, immunochromatographic test strip, and TMB colorimetric enhancement system as described in claim 6; 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.

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