A ventilator-associated pneumonia pathogen rapid detection surface-enhanced raman spectrum chip and a preparation method and application thereof

Abstract

This invention belongs to the field of materials synthesis and biosensing technology, specifically relating to a surface-enhanced Raman spectroscopy chip, its preparation method, and its applications. The surface-enhanced Raman spectroscopy chip provided by this invention includes a substrate and several detection sites disposed on the surface of the substrate. Each detection site comprises a reinforcing substrate material layer and an aptamer-Raman signal probe composite material layer stacked sequentially. The reinforcing substrate material layer is in contact with the substrate. The reinforcing substrate material layer is Ga-doped ZnO nanocrystals with a superlattice structure. The aptamer-Raman signal probe composite material layer includes at least one aptamer-probe complex, which is a complex of a DNA aptamer and a Raman signal probe. The surface-enhanced Raman spectroscopy chip provided by this invention enables rapid, culture-free, and highly specific detection of common pathogenic bacteria of vegetative-associated pneumonia (VAP), and possesses good portability and clinical applicability.

Description

Technical Field

[0001] This invention belongs to the field of materials synthesis and biosensing technology, specifically relating to a surface-enhanced Raman spectroscopy chip for rapid detection of ventilator-associated pneumonia pathogens, its preparation method, and its application. Background Technology

[0002] Ventilator-associated pneumonia (VAP) is the most common type of hospital-acquired pneumonia, typically occurring in critically ill patients on artificial airways and mechanical ventilation for more than 48 hours. It is a significant contributing factor to the increased mortality rate in ICU patients. The main pathogens of VAP include Gram-positive cocci and Gram-negative bacilli such as Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Clinically, early symptoms of VAP are difficult to distinguish from other respiratory infections, and the diverse and easily drug-resistant pathogens necessitate rapid and accurate pathogen detection to guide anti-infective treatment; otherwise, treatment failure or the spread of drug-resistant strains is highly likely.

[0003] Current routine detection methods for VAP pathogens mainly rely on bacterial culture and drug sensitivity testing, which typically take 24–72 hours. These methods suffer from significant drawbacks, including long processing times, low sensitivity, susceptibility to contamination, and inability to achieve point-of-care testing. While some nucleic acid amplification methods can provide results within hours, they often require high-temperature amplification, enzyme systems, complex instruments, or purification processes, hindering rapid on-site application. Furthermore, pathogen concentrations in body fluids are often extremely low, necessitating the development of a detection technology platform that is highly sensitive, selective, rapid-response, structurally stable, and suitable for large-scale application.

[0004] Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive analytical technique based on molecular vibrational signals, capable of amplifying the Raman scattering signal of the analyte molecule up to 10⁻⁶. 6 With a yield exceeding 100 times higher, and possessing advantages such as non-contact, rapid, label-free, and highly selective properties, it represents an important direction for the current development of culture-free microbial detection technologies. However, traditional precious metal-based SERS substrates (such as Au and Ag) suffer from problems such as high cost, large batch-to-batch variability, and strong biological background interference, making them unsuitable for stable clinical detection. Summary of the Invention

[0005] The purpose of this invention is to provide a surface-enhanced Raman spectroscopy chip for rapid detection of ventilator-associated pneumonia (VAP) pathogens, its preparation method, and its application. The surface-enhanced Raman spectroscopy chip provided by this invention can achieve rapid, culture-free, and highly specific detection of common VAP pathogens, and has good portability and clinical applicability.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a surface-enhanced Raman spectroscopy (SERS) chip, comprising a substrate and a plurality of detection sites disposed on the surface of the substrate. Each detection site comprises a reinforcing substrate material layer and an aptamer-Raman signal probe composite material layer stacked sequentially, wherein the reinforcing substrate material layer is in contact with the substrate; the reinforcing substrate material layer is Ga-doped ZnO nanocrystals (GZO SL) with a superlattice structure; the aptamer-Raman signal probe composite material layer comprises at least one aptamer-probe complex, wherein the aptamer-probe complex is a complex of a DNA aptamer and a Raman signal probe.

[0007] Preferably, the method for preparing the Ga-doped ZnO nanocrystals with the superlattice structure includes the following steps: A Zn source, a Ga source, and a solvent are first mixed to obtain a precursor solution; the molar ratio of Zn to Ga in the precursor solution is 1 to 9:1; the solvent includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol. An alkali metal hydroxide solution is added dropwise to the precursor solution to obtain a nanocrystalline nucleus mixture; the ratio of the total molar amount of Zn and Ga elements in the precursor solution to the molar amount of alkali metal hydroxide in the alkali metal hydroxide solution is 1:0.5~2; the solvent in the alkali metal hydroxide solution includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol; The nanocrystal nucleus mixture was mixed with polyvinyl alcohol in the second stage to construct a crystal lattice, thereby obtaining a GaZn lattice layered structure material. The GaZn lattice layered structure material was annealed in an air atmosphere to obtain Ga-doped ZnO nanocrystals with a superlattice structure.

[0008] Preferably, the DNA aptamer is a DNA aptamer with sequence recognition specificity for ventilator-associated pneumonia (VAP) pathogens, including one or more of Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa; the Raman signal probe includes one or more of 4-mercaptobenzoic acid (4-MBA), methylene blue (MB), copper phthalocyanine (CuPc), ferric ferrocyanide (Prussian blue, PB), and tetracyanoquinoline (TCNQ).

[0009] Preferably, the aptamer-probe complex includes one or more of a first aptamer-probe complex, a second aptamer-probe complex, a third aptamer-probe complex, a fourth aptamer-probe complex, and a fifth aptamer-probe complex; the first aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Streptococcus pneumoniae and 4-mercaptobenzoic acid (4-MBA); the second aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Escherichia coli and methylene blue (MB); the third aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Staphylococcus aureus and copper phthalocyanine (CuPc); the fourth aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Acinetobacter baumannii and ferric ferrocyanide (PB); and the fifth aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Pseudomonas aeruginosa and tetracyanoquinoline (TCNQ).

[0010] Preferably, the diameter of the detection site is 1~2mm; when there are multiple detection sites, the spacing between adjacent detection sites is 2~5mm; the surface-enhanced Raman spectroscopy chip further includes a confining capping layer, the confining capping layer has a plurality of detection channels, the detection channels correspond one-to-one with the detection sites, and each detection site is placed in a detection channel.

[0011] This invention provides a method for preparing the surface-enhanced Raman spectroscopy chip described above, comprising the following steps: A dispersion of Ga-doped ZnO nanocrystals with a superlattice structure was coated onto the surface of a substrate, and then subjected to plasma treatment, silane coupling agent activation treatment and heat treatment in sequence to obtain an activated semi-finished product. The aptamer-Raman signal probe complex solution is coated onto the surface of the activated semi-finished product and covalently coupled to obtain the surface-enhanced Raman spectroscopy chip; the aptamer-Raman signal probe complex solution contains at least one aptamer-probe complex, which is a complex of a DNA aptamer and a Raman signal probe.

[0012] This invention provides the application of the surface-enhanced Raman spectroscopy chip described in the above technical solution or the surface-enhanced Raman spectroscopy chip prepared by the preparation method described in the above technical solution in the detection of bacteria.

[0013] The present invention provides a portable real-time detection platform, comprising a surface-enhanced Raman spectroscopy chip as described in the above technical solution or a surface-enhanced Raman spectroscopy chip prepared by the preparation method described in the above technical solution, and a readout module, wherein the readout module includes a Raman spectrometer.

[0014] This invention provides a method for rapid detection of bacteria, comprising the following steps: The sample to be tested is applied to the detection site of the surface-enhanced Raman spectroscopy chip described in the above technical solution or the surface-enhanced Raman spectroscopy chip prepared by the preparation method described in the above technical solution, and incubated. After the incubation is completed, Raman signals are acquired to obtain the probe peak intensity of the Raman signal probe. The ratio of the probe peak intensity of the Raman signal probe to the internal standard peak intensity of the Ga-doped ZnO nanocrystal with a superlattice structure is calculated. The ratio is substituted into the standard curve or standard equation to obtain the detection result of bacteria.

[0015] Preferably, the bacteria include pathogens causing ventilator-associated pneumonia, other bacterial lower respiratory tract infections, urinary tract infections, wound infections, blood infections, bacteria on the surface of medical equipment, bacteria from the medical facility environment, bacteria from the food production environment, or bacteria from the pharmaceutical production environment; the other bacterial lower respiratory tract infections include pathogens causing non-ventilator-associated community-acquired pneumonia, hospital-acquired pneumonia, or bacterial infections during acute exacerbations of chronic obstructive pulmonary disease; the urinary tract infections include pathogens causing cystitis or pyelonephritis; the wound infections include pathogens causing burn wound infections; and the blood infections include pathogens causing bacterial sepsis.

[0016] This invention provides a surface-enhanced Raman spectroscopy (SERS) chip, comprising a substrate and a plurality of detection sites disposed on the surface of the substrate. Each detection site comprises a reinforcing substrate material layer and an aptamer-Raman signal probe composite material layer stacked sequentially, the reinforcing substrate material layer being in contact with the substrate. The reinforcing substrate material layer is a Ga-doped ZnO nanocrystal with a superlattice structure (GZO superlattice, GZO SL). The aptamer-Raman signal probe composite material layer comprises at least one aptamer-probe complex, which is a complex of a DNA aptamer and a Raman signal probe. This invention combines the photogenerated charge regulation and aptamer recognition of Ga-doped ZnO nanocrystals with a superlattice structure to achieve rapid and quantitative detection of bacteria (e.g., five types of pathogenic bacteria causing ventilator-associated pneumonia (VAP), such as Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa). The SERS chip provided by this invention utilizes the built-in electric field and quantum confinement effect formed by the GZO superlattice to enhance the Raman signal. Through a triple recognition mechanism (selective charge transfer between GZO and probe molecules, Raman characteristic peak recognition, and DNA aptamer binding), pathogen detection is achieved without culture, with a detection limit as low as 1.27 CFU / mL. The entire process is completed within 30 minutes, with a detection cost of approximately $0.15. The chip is stable at room temperature for over 6 months, making it suitable for rapid bedside screening and guidance on anti-infective medication in ICUs. Compared with existing technologies, the beneficial effects of this invention include: This invention is the first to construct a surface-enhanced Raman spectroscopy (SERS) chip based on a Ga-doped ZnO (GZO) superlattice structure (GZO SL). This superlattice structure significantly enhances the interfacial electric field intensity under non-plasmon conditions by controlling the space charge distribution and electronic state density, thereby significantly improving the Raman scattering signal. This chip has advantages such as high structural uniformity, stable enhancement factor, and good batch reproducibility, solving the problem of unstable morphology-dependent plasmon "hot spots" in traditional noble metal SERS substrates. Building upon this foundation, this invention utilizes covalently immobilized DNA aptamer probes targeting various ventilator-associated pneumonia (VAP) pathogens to simultaneously identify and detect a variety of common clinical pathogens, including Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. The probe design of this invention incorporates Raman signal probe molecules with high Raman scattering cross-sections, coupled with an internal reference analysis strategy, enabling quantitative detection of pathogens down to 1 CFU / mL under enzyme-free and nucleic acid amplification-free conditions. This results in high detection sensitivity, strong specificity, and excellent anti-interference capabilities.

[0017] This invention further integrates standardized multi-array chip design with a portable Raman spectrometer to achieve integrated on-site sample processing, signal acquisition, and result analysis, meeting the needs of point-of-care testing (POCT). The entire detection platform can be stably stored at room temperature for over 30 days, with excellent signal consistency between chip batches, demonstrating promising practical applications and industrialization prospects. This invention not only improves the sensitivity and stability of pathogen SERS detection but also provides a new pathway for the controllable design and device integration of non-precious metal substrates, possessing significant promotional value in the fields of infectious diseases, environmental pathogen monitoring, and biosafety.

[0018] Furthermore, this invention uses Zn and Ga as metal sources, ethylene glycol as a solvent and reducing agent, alkali metal hydroxide as a pH adjuster for precipitation, and polyvinyl alcohol as a stabilizer to construct the crystal structure. The crystal structure is optimized through annealing to obtain Ga-doped ZnO nanocrystals with a superlattice structure (abbreviated as GZO superlattice material, GZO SL). Ethylene glycol, acting as both a solvent and reducing agent, possesses excellent coordination ability, which facilitates the stable coexistence of Zn and Ga metal ions in solution and controls subsequent reactivity, providing a good foundation for subsequent reactions. This invention uses a dropwise addition method to add the alkali metal hydroxide solution to adjust the alkalinity of the reaction system and induce nanocrystal nuclei formation. It also ensures that local pH changes do not trigger large-scale aggregation or abrupt precipitation changes, ensuring the uniformity of the reaction system and controllable particle size. This invention controls the pH value to 9-10, a range that facilitates the gradual transformation of the Zn(OH)2 precursor phase into ZnO crystal nuclei while suppressing the uneven precipitation of Ga(OH)3. This ensures the orderliness and controllability of the crystal formation process, laying the morphological foundation for the subsequent formation of highly regular Ga-doped ZnO nanocrystals. By adding polyvinyl alcohol (PVA) to the system, this invention can control the product morphology and prevent excessive particle size growth. Furthermore, PVA, as a water-soluble polymer, possesses excellent film-forming and complexing abilities, allowing it to coat the particle surface through its molecular chains and inhibit agglomeration between nanocrystals. In addition, the adsorption of PVA molecular chains on the anisotropic growth surfaces of the crystals helps the crystals expand orderly in specific directions, thereby inducing the formation of periodically modulated nanostructures and creating the physical conditions for superlattice arrangement. 2+ and Ga 3+ Under the synergistic effect of ethylene glycol reduction and PVA coordination regulation, precipitation and crystallization gradually occur, forming a nanocrystalline structure dominated by ZnO and stably doped with Ga. Within the aforementioned temperature and time range, the Ga content in the reaction system... 3+ Zn can preferentially enter the ZnO lattice through heterogeneous nucleation mechanism. 2+The site promotes crystal growth and the spontaneous formation of periodically distributed Ga-rich regions at ordered interfaces, thereby inducing the crystal to self-assemble and align along the

[0001] direction, initially establishing a periodically distributed superlattice modulation structure. This invention, through annealing, promotes Ga growth and the spontaneous formation of periodically distributed Ga-rich regions at ordered interfaces, thereby inducing the crystal to self-assemble and align along the

[0001] direction, initially establishing a periodically distributed superlattice modulation structure. 3+ The ZnO lattice is fully doped by substitution and its periodic distribution within the crystal is stabilized. On the other hand, annealing can effectively release the residual stress inside the crystal and improve the integrity and order of the crystal. Based on this, the modulation structure arranged along the

[0001] direction is formally formed, thus completing the construction of the high-order superlattice. By improving the integrity of the crystal, enhancing the doping stability of metal ions, and activating the periodic arrangement between superlattice layers, Ga-doped ZnO nanocrystals with a high-order superlattice structure are finally obtained. The Ga-doped ZnO nanocrystals with a superlattice structure exhibit a cubic nanoparticle morphology with a side length of 10~14nm and good dispersion and lattice regularity.

[0019] This invention introduces Ga into the ZnO lattice for doping, adjusting the carrier concentration and band structure of ZnO. This allows the material to maintain excellent chemical stability while shrinking its band gap to match that of a visible light excitation source, thus enhancing its absorption capacity for excitation laser light. Based on this, a periodic Ga-doped ZnO superlattice structure is constructed through layer-by-layer heterogeneous modulation, forming multiple sets of continuous built-in electric field regions within the crystal. This significantly improves the spatial separation efficiency of excited-state electrons and holes, providing a favorable electron transport environment for subsequent enhancement processes. This invention utilizes the coupling of Ga doping modulation and the superlattice structure to achieve precise control over the band structure, built-in electric field distribution, and excited-state electronic behavior of ZnO. This enables significant enhancement of Raman signals for typical probe molecules without the need for noble metal plasmon structures.

[0020] This invention introduces additional charge carriers and modulates the bandgap to 1.8–2.0 eV by doping ZnO with Ga, thereby achieving excitation wavelength matching. The Ga-doped ZnO nanocrystals prepared by this invention possess a superlattice structure. By introducing a built-in electric field through periodic interface modulation, the spatial separation efficiency of photogenerated electron-hole pairs can be effectively improved, enhancing excited-state lifetime and charge injection behavior. Furthermore, superlattice modulation can induce exciton-phonon coupling to form self-trapped states (STS), providing a new pathway for constructing a high-performance non-noble metal SERS platform.

[0021] The Ga-doped ZnO nanocrystals with a superlattice structure prepared in this invention possess excellent charge separation capability, interfacial polarization characteristics, and a resonant band gap matched with the excitation wavelength, thereby achieving a highly efficient SERS enhancement effect under non-plasmic conditions. The Ga-doped ZnO nanocrystals with a superlattice structure prepared in this invention exhibit high enhancement factor, good structural stability, and strong reproducibility, making them suitable for Raman detection, catalytic interface research, and photoelectric conversion systems, providing a new approach for constructing noble metal-alternative SERS substrates. Attached Figure Description

[0022] Figure 1 These are the characterization results of the Ga-doped ZnO nanocrystal material with a superlattice structure prepared in Example 1 of this invention. Figure 1 The top left image is a scanning transmission electron microscope image. Figure 1 The top right image shows the semi-quantitative elemental analysis results. Figure 1 The image in the lower left corner is a high-resolution transmission electron microscope image. Figure 1 The image in the lower right corner is an aberration-corrected scanning transmission electron microscope image in high-angle annular dark-field mode; Figure 2 Selected area electron diffraction image of the GZO superlattice material prepared in Example 1 of Test Example 1; Figure 3 The X-ray diffraction pattern of the GZO superlattice material prepared in Example 1 of Test Example 1; Figure 4 The X-ray photoelectron spectroscopy (XPS) spectrum of the GZO superlattice material prepared in Example 1 of Test Example 1 is shown below. Figure 5 This is a band structure analysis diagram of the GZO superlattice material prepared in Example 1 of Test Example 1; Figure 6 The enhanced Raman spectrum of the GZO superlattice material prepared in Example 1 of Test Example 1 for TCNQ molecules; Figure 7 The ultraviolet-visible absorption spectrum characterization of the SERS chip prepared in Example 2 of this invention; Figure 8 These are actual photographs of the SERS chip prepared in Embodiment 4 of the present invention; Figure 9 This is the energy level diagram of the Raman signal molecules used in the SERS chip prepared in Example 2 of this invention; Figure 10 This is a selective test of the SERS chip prepared in Embodiment 2 of the present invention; Figure 11 This is a linearity test of the SERS chip prepared in Embodiment 2 of the present invention; Figure 12This is a response time test of the SERS chip prepared in Embodiment 2 of the present invention; Figure 13 These are the pathogen test results of bronchoalveolar lavage fluid from 50 VAP patients using the SERS chip prepared in Example 2 of this invention; Figure 14 This refers to the subject operating characteristic curve of the SERS chip prepared in Example 2 of the present invention based on clinical test results; Figure 15 This refers to the long-term storage stability of the SERS chip prepared in Example 2 of this invention; Figure 16 This refers to the batch repeatability of the SERS chip prepared in Example 2 of this invention. Detailed Implementation

[0023] This invention provides a surface-enhanced Raman spectroscopy (SERS) chip, comprising a substrate and a plurality of detection sites disposed on the surface of the substrate. Each detection site comprises a reinforcing substrate material layer and an aptamer-Raman signal probe composite material layer stacked sequentially, wherein the reinforcing substrate material layer is in contact with the substrate; the reinforcing substrate material layer is a Ga-doped ZnO nanocrystal with a superlattice structure; the aptamer-Raman signal probe composite material layer comprises at least one aptamer-probe complex, wherein the aptamer-probe complex is a complex of a DNA aptamer and a Raman signal probe.

[0024] The SERS chip provided by this invention is used for rapid, culture-free detection of ventilator-associated pneumonia (VAP) pathogens. Based on Ga-doped ZnO superlattice semiconductor nanomaterials, and combining multi-site aptamer molecular recognition with Raman signal probe ratio analysis, this invention provides a highly selective, highly sensitive, low-cost, and portable SERS detection platform for rapidly identifying five major VAP pathogens in raw samples.

[0025] This invention is based on Ga-doped ZnO nanocrystals (GZO SL) with a superlattice structure. Utilizing the periodic alternating Ga-rich layers and Zn layers formed along the

[0001] crystal orientation, a superlattice structure is constructed, enabling precise control over photogenerated carrier migration, interfacial charge separation, and surface bandgap modulation. Simultaneously, the surface of this structure is enriched with oxygen vacancies and defect states, providing highly efficient charge coupling and photo-induced enhancement capabilities for Raman probe molecules. Building upon this, the invention employs DNA aptamers targeting typical VAP pathogens to label different Raman signal probe molecules, achieving simultaneous recognition of multiple targets. By fixing this recognition unit onto the surface of the GZO superlattice (GZO SL) and assembling it into a PDMS microcavity array chip structure, a point-of-care detection platform suitable for clinical airway samples and compatible with portable Raman spectrometers is constructed.

[0026] The SERS chip provided by this invention includes a substrate. In this invention, the substrate preferably includes a silicon oxide wafer, a glass wafer, or a single-crystal silicon wafer.

[0027] The SERS chip provided by this invention includes a plurality of detection sites disposed on the surface of a substrate. The detection sites are disposed on the upper surface of the substrate. Each detection site includes a reinforcing substrate material layer stacked sequentially. The reinforcing substrate material layer is in contact with the substrate. The reinforcing substrate material layer is disposed on the upper surface of the substrate. The reinforcing substrate material layer is a Ga-doped ZnO nanocrystal with a superlattice structure (GZO superlattice, GZO SL). In this invention, the superlattice structure of Ga-doped ZnO nanocrystals with a superlattice structure may include: the nanocrystals having a cubic morphology with a side length of 10-14 nm, and crystal planes extending along the

[100] and

[010] directions; alternating bright and dark intensity modulation stripes along the

[0001] direction, the modulation direction being consistent with the (002) crystal plane of ZnO, the spacing of the intensity modulation stripes being 1.3-1.6 nm, continuously arranged for no less than 5 modulation periods, the periodicity of the intensity modulation stripes along the

[0001] direction being 1.1-1.7 nm, and the lattice distortion being periodically repeated; the modulation region and the unmodulated region being alternately distributed, the thickness of each modulation region being 1-1.5 nm, and the length of the continuous modulation stripes being no less than 60% of the total length of the crystal; in the modulation region, the lattice has a slight expansion distortion, the crystal plane spacing being 0.26-0.28 nm, corresponding to the (002) crystal plane of ZnO; the Ga +3 Zn sites doped in the bulk phase of a crystal.

[0028] In this invention, the Ga-doped ZnO nanocrystals with a superlattice structure have a cubic superlattice structure with Ga-rich layers and Zn layers alternating periodically along the

[0001] direction. The average side length of the Ga-doped ZnO nanocrystals with the superlattice structure is 12.3 ± 2.7 nm, and they have obvious lattice fringes and interface potential distribution, which can effectively control the separation and transfer of photogenerated carriers and enhance surface charge coupling.

[0029] In this invention, the method for preparing the Ga-doped ZnO nanocrystals with a superlattice structure includes the following steps: A Zn source, a Ga source, and a solvent are first mixed to obtain a precursor solution; the molar ratio of Zn to Ga in the precursor solution is 1 to 9:1; the solvent includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol. An alkali metal hydroxide solution is added dropwise to the precursor solution to obtain a nanocrystal nucleus mixture; the solvent in the alkali metal hydroxide solution includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol. The nanocrystal nucleus mixture was mixed with polyvinyl alcohol in the second stage to construct a crystal lattice, thereby obtaining a GaZn lattice layered structure material. The GaZn lattice layered structure material was annealed in an air atmosphere to obtain Ga-doped ZnO nanocrystals with a superlattice structure.

[0030] This invention involves first mixing a Zn source, a Ga source, and a solvent to obtain a precursor solution. In this invention, the Zn source may include one or more of zinc nitrate, zinc chloride, zinc sulfate, and zinc acetate. In this invention, the Ga source may include one or more of gallium nitrate, gallium chloride, gallium sulfate, and gallium acetate. In this invention, the molar ratio of Zn to Ga in the precursor solution is 1-9:1, and may also be 2-8:1, specifically 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1. In this invention, the solvent includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol. In this invention, the total concentration of Zn and Ga in the precursor solution can be 0.3-0.7 mol / L, and may also be 0.4-0.6 mol / L, specifically 0.5 mol / L. In this invention, ethylene glycol, propylene glycol, glycerol, and diethylene glycol simultaneously act as solvents and reducing agents, exhibiting excellent coordination capabilities. This facilitates the stable coexistence of Zn and Ga metal ions in solution and controls subsequent reactivity, providing a good foundation for subsequent reactions. In this invention, the first mixing can be a stirring mixture; there are no special limitations on the first mixing method, as long as a transparent and homogeneous precursor solution is obtained; in specific embodiments of this invention, the temperature of the first mixing can be room temperature (18~30℃), and the mixing time can be 30 minutes.

[0031] After obtaining the precursor solution, this invention adds an alkali metal hydroxide solution dropwise to the precursor solution to obtain a nanocrystal nucleus mixture. In this invention, the solvent in the alkali metal hydroxide solution includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol; the concentration of the alkali metal hydroxide solution can be 0.5~2 mol / L, or 1~1.5 mol / L. In this invention, the ratio of the total molar amount of Zn and Ga elements in the precursor solution to the molar amount of alkali metal hydroxide in the alkali metal hydroxide solution is 1:0.5~2, or 1:1~1.5. In this invention, the dropping rate of the alkali metal hydroxide solution can be 0.5~1 drop / second, specifically 0.5 drops / second; the dropping is carried out under stirring conditions. This invention uses a dropwise addition of the alkali metal hydroxide solution to adjust the alkalinity of the reaction system and induce the formation of nanocrystal nuclei. It also ensures that local pH changes do not cause large-scale aggregation or abrupt precipitation changes, ensuring the uniformity of the reaction system and the controllability of particle size. This invention controls the (Zn+Ga) / alkali metal hydroxide molar ratio within the range of 1:0.5~2, which is beneficial for the gradual transformation of the Zn(OH)2 precursor phase into ZnO crystal nuclei, while suppressing the uneven precipitation of Ga(OH)3, ensuring the orderliness and controllability of the crystal formation process, and laying the morphological foundation for the subsequent formation of highly regular Ga-doped ZnO nanocrystals.

[0032] After obtaining the nanocrystalline nucleus mixture, this invention further mixes the nanocrystalline nucleus mixture with polyvinyl alcohol (PVA) to construct a crystal lattice, thereby obtaining a GaZn lattice layered structure material. In this invention, the mass ratio of the total Zn source and Ga source to the mass of PVA can be 10~30:1, or 11~20:1, specifically 10:1, 11.7:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 22:1, 25:1, 28:1, or 30:1. This invention, by adding PVA to the system, can control the product morphology and prevent excessive particle size growth; moreover, PVA, as a water-soluble polymer, possesses good film-forming properties and complexing ability, and can coat the particle surface through its molecular chains, inhibiting the aggregation between nanocrystals. Furthermore, the adsorption of PVA molecular chains onto the anisotropic growth surface of the crystal facilitates the orderly expansion of the crystal in a specific direction, thereby inducing the formation of periodically modulated nanostructures and creating physical conditions for superlattice arrangement. In this invention, the second mixing temperature can be room temperature; the second mixing time can be 30 min. In this invention, the lattice construction temperature can be 120~160℃, or 130~150℃, specifically 140℃; the lattice construction time can be 2~6 h, or 3~5 h, specifically 4 h. In this invention, during the lattice construction process, Zn...2+ and Ga 3+ Under the synergistic effect of ethylene glycol reduction and PVA coordination regulation, precipitation and crystallization gradually occur, forming a nanocrystalline structure dominated by ZnO and stably doped with Ga. Within the aforementioned temperature and time range, the Ga content in the reaction system... 3+ Zn can preferentially enter the ZnO lattice through heterogeneous nucleation mechanism. 2+ The site promotes the spontaneous formation of periodically distributed Ga-rich regions at the crystal growth and ordered interface, thereby inducing the crystal to self-assemble and arrange along the

[0001] direction, and initially establishing a periodically distributed superlattice modulation structure.

[0033] After the lattice is constructed, the present invention may further include: naturally cooling the system obtained by the lattice construction to room temperature, centrifuging, washing and drying the obtained solid components to obtain a GaZn lattice layered structure material. In the present invention, the centrifugation speed can be 6000~10000 r / min, or 7000~90000 r / min, or even 80000 r / min; the centrifugation time can be 10~20 min, or even 15 min. In the present invention, the washing solvent may include one or more of ethanol, dichloromethane, ethyl acetate and tetrahydrofuran; the washing solvent may be an anhydrous solvent; the purpose of washing is to remove residual organic matter, small molecule byproducts and free ions, which helps to improve the purity and structural stability of the final material, and at the same time eliminates impurity interference in the subsequent annealing process. In this invention, the drying temperature can be 60-100℃, 70-90℃, or even 80℃; the drying time can be 8-30 hours, 15-28 hours, or even 20-25 hours. The purpose of drying is to thoroughly remove residual solvent and adsorbed moisture. In this invention, drying helps prevent crystal agglomeration and maintain morphological integrity. The dried powder is light yellow or milky white, exhibiting preliminary crystallinity but not yet forming a complete superlattice arrangement.

[0034] After obtaining the GaZn lattice layered structure material, this invention anneals the GaZn lattice layered structure material in an air atmosphere to obtain Ga-doped ZnO nanocrystals with a superlattice structure. In this invention, the annealing temperature can be 500~650℃, or 550~620℃, or even 580~600℃; the annealing time can be 8~12h, or 9~11h, or even 10h; the annealing can be carried out in a muffle furnace. This invention, through annealing, promotes the Ga... 3+The ZnO lattice is fully doped by substitution and its periodic distribution within the crystal is stabilized. On the other hand, annealing can effectively release the residual stress inside the crystal and improve the integrity and order of the crystal. Based on this, the modulation structure arranged along the

[0001] direction is formally formed, thus completing the construction of the high-order superlattice. By improving the integrity of the crystal, enhancing the doping stability of metal ions, and activating the periodic arrangement between superlattice layers, Ga-doped ZnO nanocrystals with a high-order superlattice structure are finally obtained. The Ga-doped ZnO nanocrystals with a superlattice structure exhibit a cubic nanoparticle morphology with a side length of 10~14nm and good dispersion and lattice regularity.

[0035] This invention also provides Ga-doped ZnO nanocrystals with a superlattice structure prepared by the preparation method described above. 3+ The elements are doped in the ZnO lattice by substitution and arranged periodically along the

[0001] direction to form a regular element modulation structure.

[0036] In this invention, the detection site includes an aptamer-Raman signal probe composite material layer disposed on the upper surface of the reinforcing substrate material layer. The aptamer-Raman signal probe composite material layer includes at least one aptamer-probe complex, which is a complex of a DNA aptamer and a Raman signal probe. The aptamer-Raman signal probe composite material layer possesses both targeted recognition capability and Raman signal response capability, enabling it to synergistically achieve selective recognition and Raman signal amplification of ventilator-associated pneumonia pathogens with the Ga-doped ZnO superlattice reinforced substrate.

[0037] In this invention, the 3' end of the DNA aptamer is modified with an amino group, and the 5' end is modified with a carboxyl group. The DNA aptamer is a DNA aptamer capable of sequence recognition specificity with ventilator-associated pneumonia (VAP) pathogens. The VAP pathogens may include one or more of Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0038] In this invention, the DNA aptamer includes one or more of the following: DNA aptamers that have sequence recognition specificity with Streptococcus pneumoniae, DNA aptamers that have sequence recognition specificity with Escherichia coli, DNA aptamers that have sequence recognition specificity with Staphylococcus aureus, DNA aptamers that have sequence recognition specificity with Acinetobacter baumannii, and DNA aptamers that have sequence recognition specificity with Pseudomonas aeruginosa.

[0039] In this invention, the Raman signal probe preferably comprises one or more of 4-mercaptobenzoic acid (4-MBA), methylene blue (MB), copper phthalocyanine (CuPc), ferric ferrocyanide (PB), and tetracyanoquinoline (TCNQ). This invention introduces a Raman signal probe as a signal reporter unit onto a DNA aptamer, the Raman signal probe being linked to the 3' end of the DNA aptamer via an amide bond. In this invention, the LUMO energy level of the Raman signal probe molecule is preferably in the range of -2.5 eV to -3.2 eV, forming a good charge transfer path with the GZO SL conduction band position (approximately -2.3 eV) to enhance Raman scattering intensity.

[0040] In this invention, the aptamer-probe complex preferably includes one or more of a first aptamer-probe complex, a second aptamer-probe complex, a third aptamer-probe complex, a fourth aptamer-probe complex, and a fifth aptamer-probe complex; the first aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Streptococcus pneumoniae and 4-mercaptobenzoic acid (4-MBA); the second aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Escherichia coli and methylene blue (MB); the third aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Staphylococcus aureus and copper phthalocyanine (CuPc); the fourth aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Acinetobacter baumannii and ferric ferrocyanide (PB); and the fifth aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Pseudomonas aeruginosa and tetracyanoquinoline (TCNQ).

[0041] In this invention, the aptamer-Raman signal probe composite material layer of any one of the detection sites includes at least one aptamer-probe composite, specifically one, two, three, four or five of the above five aptamer-probe composites.

[0042] This invention labels each of the five DNA aptamers with a specific Raman signal probe, which is matched with the GZO energy level and used for photoinduced charge transfer to amplify the Raman signal.

[0043] In this invention, when there are multiple detection sites, the detection sites are arranged in an array on the upper surface of the substrate.

[0044] In this invention, the detection sites can be arranged in a 1×5 linear array or a 2×3 matrix arrangement to achieve simultaneous identification of five pathogens on a single chip. The diameter of the detection sites is 1-2 mm. When there are multiple detection sites, the spacing between adjacent detection sites is 2-5 mm. In this invention, the surface-enhanced Raman spectroscopy chip also includes a confining capping layer, which has several detection channels. Each detection channel corresponds one-to-one with a detection site, and each detection site is placed in one detection channel. The material of the confining capping layer preferably includes polydimethylsiloxane (PDMS). In this invention, the diameter of the detection channels is preferably 2-5 mm; the depth is preferably 0.5-1 mm, and in the embodiment, it can be 0.8 mm. The thickness of the confining capping layer is preferably 1.2 mm. In this invention, the detection sites located in a detection channel form a chip detection cavity. This invention forms a detection chip with confined space through the confining capping layer, which is suitable for small volume sample (<20 μL) reactions.

[0045] In this invention, the confined capping layer is preferably made of polydimethylsiloxane (PDMS) and prepared by template casting to form a microcavity assembly with multiple detection channels. The detection channels of the confined capping layer are aligned with the detection sites and attached to the upper surface of the substrate to form a closed microreaction unit.

[0046] This invention provides a method for preparing the surface-enhanced Raman spectroscopy chip described above, comprising the following steps: A dispersion of Ga-doped ZnO nanocrystals with a superlattice structure was coated onto the surface of a substrate, and then subjected to plasma treatment, silane coupling agent activation treatment and heat treatment in sequence to obtain an activated semi-finished product. The aptamer-Raman signal probe complex solution is coated onto the surface of the activated semi-finished product and covalently coupled to obtain the surface-enhanced Raman spectroscopy chip; the aptamer-Raman signal probe complex solution contains at least one aptamer-probe complex, which is a complex of a DNA aptamer and a Raman signal probe.

[0047] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0048] This invention involves coating a dispersion of Ga-doped ZnO nanocrystals with a superlattice structure onto a substrate surface, followed by sequential plasma treatment, silane coupling agent activation, and heat treatment to obtain an activated semi-finished product. In this invention, the dispersion of Ga-doped ZnO nanocrystals with a superlattice structure comprises Ga-doped ZnO nanocrystals with a superlattice structure and an organic solvent, such as ethanol. The coating can be drop-coating, which can be performed using a mask template or an automated spotting device to sequentially spot-coat different predetermined areas on the same substrate surface, forming a multi-channel or multi-site array structure. After coating, the substrate is preferably air-dried to form a reinforcing substrate material layer on the substrate surface. The plasma treatment is preferably performed in a plasma cleaner. The plasma treatment conditions preferably include: a power of 50-150 W (100 W in the example); and an oxygen atmosphere. This invention enhances the hydrophilicity of the reinforcing substrate material layer through plasma treatment. The silane coupling activation treatment employs a silane coupling agent for modification, preferably 3-aminopropyltriethoxysilane (APTES). The silane coupling activation treatment preferably involves immersing the plasma-treated product in a silane coupling agent solution for activation. The solvent in the silane coupling agent solution is preferably ethanol. The activation time is preferably 1-2 hours. After the silane coupling agent activation treatment, the product is preferably naturally dried at room temperature before undergoing the heat treatment. The heat treatment is preferably drying. The heat treatment temperature is preferably 100-110°C, and the time is preferably 1-1.5 hours. Through this heat treatment, the silane layer is cured, and amino groups are bonded to the reinforcing substrate material layer.

[0049] The present invention involves coating an aptamer-Raman signal probe complex solution onto the surface of an activated semi-finished product for covalent coupling to obtain the surface-enhanced Raman spectroscopy chip; the aptamer-Raman signal probe complex solution contains at least one aptamer-probe complex, wherein the aptamer-probe complex is a complex of a DNA aptamer and a Raman signal probe.

[0050] In this invention, the aptamer-probe complex can be purchased directly or prepared using the following method: The present invention involves mixing a Raman signal probe solution and a DNA aptamer solution for reaction to obtain the aptamer-Raman signal probe complex. Preferably, the mixing process includes: dissolving the Raman signal probe in MES buffer (pH 5.5) to obtain an initial Raman signal probe solution; then pre-mixing the initial Raman signal probe solution with an activator solution for carboxyl activation to obtain a Raman signal probe solution; the concentration of the Raman signal probe in the initial Raman signal probe solution is preferably 2 mM. The activator solution is preferably a mixture of EDC and NHS. The concentration of EDC in the EDC and NHS mixture is preferably 100 mM. The concentration of NHS is preferably 100 mM. The volume ratio of the initial Raman signal probe solution to the activator solution is preferably 1:1. The carboxyl activation is performed at room temperature and in the dark. The carboxyl activation time is preferably 20-30 min. The DNA aptamer solution preferably includes a DNA aptamer and PBS buffer (pH 7.4). The concentration of DNA aptamer in the DNA aptamer solution is preferably 10 μM. The molar ratio of the Raman signal probe to the DNA aptamer can be 200:1. The reaction is carried out under light-protected conditions, and the reaction temperature can be 2-4 °C. The reaction time is preferably 10-12 h. In this invention, after the reaction is completed, the obtained reaction solution is preferably subjected to ultrafiltration purification sequentially, and finally resuspended in PBS buffer (pH 7.4). The molecular weight cutoff of the ultrafiltration purification can be 3 kDa.

[0051] In this invention, the aptamer-Raman signal probe complex solution is a PBS solution of the aptamer-Raman signal probe complex, wherein the concentration of the aptamer-Raman signal probe complex is preferably 10-50 μM. The solvent in the aptamer-Raman signal probe complex solution is PBS buffer. Before coating, this invention preferably activates the aptamer-Raman signal probe complex solution with an EDC / NHS solution. The EDC / NHS solution is preferably a 0.1 M MES buffer containing 0.2 MEDC and 0.05 M NHS, pH 5.5. The activation time is preferably 30 min. After the activation treatment, this invention preferably further includes centrifugation desalting to remove excess EDC / NHS.

[0052] Immediately after the activation treatment, coating is performed. The coating is preferably drop-coating. The covalent coupling temperature is preferably 2-4°C, and the time is preferably 10-24 h. The covalent coupling involves the reaction of the 5' carboxyl group of the aptamer-Raman signal probe complex with the amino group on the surface of the reinforced substrate material layer of the activated semi-finished product.

[0053] The present invention preferably uses a mask template or an automatic spotting device to sequentially spot the aptamer-Raman signal probe complex solution corresponding to different pathogens onto different preset areas on the same chip surface to form a multi-channel or multi-site array structure, which facilitates the simultaneous identification and analysis of multiple pathogens.

[0054] In this invention, after the covalent coupling reaction is completed, the product obtained from the covalent coupling reaction is preferably washed and then blocked with bovine serum albumin (BSA) solution to avoid non-specific adsorption. Finally, it is washed with PBS and then soaked in PBS to maintain a moist state, and stored at 4 °C in the dark. The washing reagent is PBST (PBS containing 0.05% Tween-20). The concentration of mercaptoethanol in the mercaptoethanol solution can be 1 mM. The mass content of bovine serum albumin in the bovine serum albumin solution is 1%.

[0055] In this invention, the surface-enhanced Raman spectroscopy chip has a multi-site SERS detection array structure with target recognition capability.

[0056] In this invention, the preferred method for preparing the confined capping layer includes: mixing polydimethylsiloxane (PDMS) prepolymer and curing agent at a mass ratio of 10:1, degassing for 30 min, pouring into a SU-8 silicone mold with several circular protrusions, curing at 70°C for 2 h, and peeling off to obtain a confined capping layer with multiple circular perforations.

[0057] The present invention preferably further includes aligning the detection channels of the confinement capping layer with the detection sites and attaching them to the upper surface of the substrate for encapsulation. After encapsulation, the present invention preferably further includes a drying process, wherein the drying temperature is preferably 30-40°C and the drying time is preferably 1-2 hours. The present invention enhances the bonding strength between the confinement capping layer and the substrate through the drying process.

[0058] In this invention, the packaged chip is preferably reinforced with a PET frame and cut to the size of a standard microscope slide (e.g., 25 mm × 75 mm) for easy use later.

[0059] Preferably, the surface-enhanced Raman spectroscopy chip is placed in an aluminum foil bag and vacuum-sealed or nitrogen-filled for stable storage at room temperature for at least 6 months with a Raman signal drift of less than 10%. The preferred storage conditions include: humidity not exceeding 40% and temperature controlled within the range of 15~30℃.

[0060] In the present invention, the SERS chip has good long-term stability and batch-to-batch repeatability. The prepared SERS chip of the present invention is sealed away from light under normal temperature and humidity air conditions, and samples are taken at 0, 7, 14, 30, 60, 90, and 180 days respectively to test the standard Raman response signals of Raman signal probes such as 4-MBA and TCNQ. Calculate the change rate of the ratio of the characteristic peak of the Raman signal probe to the internal standard peak (Iprobe / I437). If the change rate is less than 10%, it is determined to be qualified; it is accelerated aging for 7 days in an environment of 37°C constant temperature and 80% relative humidity to simulate the storage effect for 3 to 6 months, and the Raman response deviation does not exceed 15%.

[0061] The present invention continuously prepares no less than 3 batches of SERS chips with the same formula and conditions. 10 chips are taken from each batch to measure the response signals of the same probe molecule at the same concentration, and the relative standard deviation (RSD) of the Raman intensity ratio is calculated, which should be less than 10%; if RSD < 5%, it is a superior batch.

[0062] The SERS chip of the present invention is stored by vacuum or inert gas packaging. Each SERS chip is sealed with an aluminum-plastic composite bag or a moisture-proof bag, and can be stored at room temperature for more than 6 months; for long-distance transportation, the storage and transportation temperature can be controlled not to exceed 35°C, and the relative humidity is lower than 60%.

[0063] The present invention provides the application of the surface-enhanced Raman spectroscopy chip described in the above technical solution or the surface-enhanced Raman spectroscopy chip prepared by the preparation method described in the above technical solution in the detection of bacteria.

[0064] The present invention provides the application of the surface-enhanced Raman spectroscopy chip described in the above technical solution or the surface-enhanced Raman spectroscopy chip prepared by the preparation method described in the above technical solution in the non-culture detection of pathogens related to ventilator-associated pneumonia.

[0065] The present invention provides a portable point-of-care testing (POCT) platform, including the surface-enhanced Raman spectroscopy chip described in the above technical solution or the surface-enhanced Raman spectroscopy chip prepared by the preparation method described in the above technical solution, and a readout module, and the readout module includes a Raman spectrometer.

[0066] The portable point-of-care testing (POCT) platform provided by the present invention is a multi-pathogen rapid detection system suitable for the point-of-care testing (POCT) scenario. The portable point-of-care testing (POCT) platform provided by the present invention has the characteristics of rapid response, simple operation, intelligent data analysis, and instant result output.

[0067] In this invention, the SERS chip is adapted for use in a portable point-of-care testing (POCT) platform. The SERS chip is housed in a replaceable chip cartridge, which is slot-mounted into the detection chamber at the bottom of the Raman spectrometer. The detection window is sealed with a high-transmittance quartz sheet to prevent external contamination and signal interference. The chip cartridge can be made of plastic or glass.

[0068] In this invention, the Raman spectrometer can be a handheld Raman spectrometer, which integrates a 633nm laser source, a CCD detector, and a high-sensitivity Raman signal acquisition module (i.e., a low-noise optical system), and has the capability to complete a full-spectrum scan within 10 seconds. The Raman spectrometer has dimensions not exceeding 200×120×80 mm and a weight not exceeding 1.2 kg, more preferably less than 1 kg, making it suitable for bedside deployment. The Raman spectrometer can be powered by a built-in lithium battery or a USB interface, enabling it to operate independently in environments without a laboratory.

[0069] The present invention preferably pre-writes the Raman peak positions of each probe and their signal ratio at different concentrations as a concentration standard curve model into the system database of the Raman spectrometer; the instrument software supports automatic multi-peak identification, automatic extraction of internal standard ratios, quantitative curve fitting and pathogen type concentration correspondence analysis, and can simultaneously output analysis reports.

[0070] In this invention, the Raman spectrometer is equipped with an adjustable sample holder to ensure that the laser is aligned with the center of each array point on the chip, thereby improving data consistency and repeatability.

[0071] In this invention, the preferred method of using the portable point-of-care testing (POCT) platform includes: the user inserts the sample to be tested into the chip and then into the instrument, clicks the "Start Detection" button, and the system automatically begins acquiring signals at each site; the total detection time for each sample does not exceed 30 minutes. The system automatically outputs a result file containing the following content: (1) Pathogen type identification results (represented by text or icons); (2) Concentration values ​​of each pathogen (CFU / mL); (3) Determination of whether the result is positive; (4) Recommended treatment suggestions or linkage to clinical databases (optional).

[0072] The results can be sent to a PC terminal, doctor's workstation, or mobile APP via USB, Bluetooth, or WiFi.

[0073] In one specific implementation, the portable Raman spectrometer is model Metrohm MIRA XTR, with a power setting of 3.5mW, an excitation wavelength of 633 nm, and an automatic acquisition interval of 8 s. The instrument is equipped with a mobile APP that can automatically generate pathogen distribution maps and detection trend curves, enabling rapid tracking and tracing of nosocomial infections.

[0074] This invention preloads the ratio-bacterial concentration standard curve data into the Raman spectrometer's memory or a mobile terminal application. Users can then connect to a smart device via Bluetooth or a wired interface to upload data, automatically identify peak values, calculate concentrations, generate quantitative results, and receive risk alerts.

[0075] In this invention, the portable instant detection platform is equipped with an optical window or adapter module that can accommodate the PDMS chip in this invention, ensuring accurate alignment of the laser and the detection site (deviation <0.1 mm) and avoiding a decrease in signal acquisition efficiency due to chip structure mismatch.

[0076] In this invention, the POCT platform supports batch pre-installation of multi-site SERS chip modules, with each chip simultaneously detecting 5 pathogens. The manufacturing cost of a single chip is controlled within RMB 1, and the cost of consumables for each test is approximately USD 0.15. It is suitable for rapid pathogen screening and dynamic monitoring in ICU, emergency, infectious disease wards and community healthcare scenarios.

[0077] This invention provides a method for rapid detection of bacteria, comprising the following steps: The sample to be tested is applied to the detection site of the surface-enhanced Raman spectroscopy chip described in the above technical solution or the surface-enhanced Raman spectroscopy chip prepared by the preparation method described in the above technical solution, and incubated. After the incubation is completed, Raman signals are acquired to obtain the probe peak intensity of the Raman signal probe. The ratio of the probe peak intensity of the Raman signal probe to the internal standard peak intensity of the Ga-doped ZnO nanocrystal with a superlattice structure is calculated. The ratio is substituted into the standard curve or standard equation to obtain the detection result of bacteria.

[0078] This invention provides a method for culture-free detection of pathogens causing ventilator-associated pneumonia using the surface-enhanced Raman spectroscopy chip described above.

[0079] In this invention, the sample to be tested can be sputum, tracheal aspirate, endotracheal tube rinsing fluid, or bronchoalveolar lavage fluid (BALF), preferably clinical airway secretions or BALF samples. In the embodiments, it can be BALF or deep sputum from the lower respiratory tract of a ventilator-inserted patient, filtered to remove large particulate impurities. The sample to be tested is preferably diluted before use. The dilution is performed using PBS buffer (pH 7.4), and the dilution factor can be 1 to 10 times. During the dilution process, a nonionic surfactant is preferably added to prevent cell aggregation. The nonionic surfactant can be Tween-20. The mass content of the added nonionic surfactant can be 0.01% to 0.1%.

[0080] In a specific embodiment of the present invention, for high-viscosity samples (such as sputum or tracheal aspirate), an equal volume of 0.1% dithiothreitol (DTT) solution or 4% NaOH solution is added for liquefaction, and the mixture is incubated at room temperature with shaking for 5-10 minutes. After liquefaction, the sample is coarsely filtered through a 100 μm filter membrane or a 0.22 μm needle filter to remove large particulate impurities and mucus clumps, obtaining a clear liquid for subsequent reactions. For samples with sparse colonies, centrifugation at 3000-5000 rpm for 5-10 minutes can be performed, the supernatant discarded, and the sample resuspended in PBS, with a concentration factor of 2-10 times, to improve detection sensitivity. Preferably, the liquefied sample is added to Tween-20 at a final concentration of 0.01-0.1% and BSA to block the non-specific binding of host proteins or mucopolysaccharides to the chip surface, ensuring the exposure of specific recognition sites. This invention verifies the interference resistance of various pathogen signals by adding 10%, 20%, and 50% human sputum or BALF background to standardized bacterial solutions. The results show that the method of this invention has high anti-interference ability against complex sample backgrounds, and the SERS signal deviation is less than ±15%.

[0081] In this invention, the volume of the sample applied to each detection site can be 10 μL, ensuring sufficient coverage of each detection site area. The incubation temperature can be 37 °C. The incubation time is preferably 5–15 min, more preferably 5–10 min, and in this example, it can be 8 min to enhance binding efficiency. During the incubation process, the target bacteria specifically bind to the DNA aptamer, inducing the Raman signal probe to approach the reinforcing substrate material layer and achieve Raman signal activation. The relative humidity of the incubation environment is 60%–80% to prevent the sample from drying out.

[0082] This invention preferably uses a portable 633 nm Raman laser source (in the embodiments, this can be a handheld or portable Raman spectrometer under 633 nm laser excitation) for Raman signal acquisition. The acquisition time for each detection site does not exceed 10 seconds, preferably 5~10 s. The preferred conditions for Raman signal acquisition include: laser power set to 3~5 mW, which can be 4mW in the embodiments. The spot diameter is controlled at 1~2 μm to avoid damaging the biological probe structure.

[0083] The internal standard peak position of the Ga-doped ZnO nanocrystals with the superlattice structure is 437 cm⁻¹. -1 The detection result of the bacteria is the concentration of bacteria in the sample (CFU / mL). This invention uses a threshold value set based on the bacterial concentration to determine whether a result is positive or negative (e.g., a positive result is defined as when the I probe / I437 ratio exceeds the mean of the blank sample plus three times the standard deviation). The characteristic peak position of the Raman signal probe is: the characteristic peak position of 4-MBA is 1099 cm⁻¹. -1 The characteristic peak position of MB is 1392 cm⁻¹. -1 The characteristic peak of CuPc is located at 1529 cm⁻¹. -1 The characteristic peak position of PB is 2155 cm⁻¹. -1 The characteristic peak position of TCNQ is 2228 cm⁻¹. -1 .

[0084] This invention is based on the Raman fingerprint peak positions (1099, 1392, 1529, 2155, 2228 cm⁻¹) corresponding to different pathogenic bacteria. -1 This allows for multiple identification and differentiation, with a detection limit of up to 1.27 CFU / mL and a signal linear range covering 10... 0 ~10 7 CFU / mL, with a single detection time of less than 30 minutes. This invention preferably uses a mobile app or computing terminal to automatically output the pathogen type and concentration. The linear interval of the standard equation is 10. 0 ~10 7 CFU / mL, goodness of fit R 2 ≥0.99.

[0085] In this invention, the quantitative analysis of the Raman signal employs a ratio calibration method based on the internal standard peak, combined with a triple identification mechanism to improve the selectivity and accuracy of detection. Specifically, this invention utilizes the inherent E2 (high) mode Raman peak (437 cm⁻¹) in the Ga-doped ZnO superlattice. -1 As an internal standard for intensity, the target probe peak (e.g., 1099 cm⁻¹ of 4-MBA) is recorded synchronously during each Raman signal acquisition. -1 Or TCNQ's 2228 cm -1The ratio of the intensity of the probe peak to the internal standard peak is calculated as Iprobe / Iinternal standard. This eliminates the influence of non-specific factors such as sample concentration differences, laser drift, and optical path fluctuations, thus achieving signal standardization. Different Raman signal probe molecules in this invention possess unique Raman characteristic peak positions, serving as identification tags for different pathogens. The probes corresponding to the five types of pathogens are: Streptococcus pneumoniae (4-MBA, 1099 cm2). -1 ), Escherichia coli (MB, 1392 cm) -1 Staphylococcus aureus (CuPc, 1529 cm) -1 Acinetobacter baumannii (PB, 2155cm) -1 Pseudomonas aeruginosa (TCNQ, 2228 cm) -1 In this invention, the LUMO energy level of the Raman signal probe matches the conduction band of the GZO SL superlattice, enabling directional charge transfer (LICT / LIHET) from GZO to the Raman signal probe molecule in the excited state. Significant enhancement occurs only when the probe approaches the surface due to target binding, forming a signal activation window and suppressing background interference signals. Simultaneously, the specific binding conformation of the aptamer to the surface protein or polysaccharide of the target pathogen alters its spatial structure, changing from an unbound state to a hairpin-closed state, significantly shortening the distance between the Raman signal probe and the GZO SL substrate, achieving energy coupling, and completing the dual functions of physical localization and signal amplification.

[0086] In this invention, the bacteria include pathogens causing ventilator-associated pneumonia, other bacterial lower respiratory tract infections, urinary tract infections, wound infections, blood infections, bacteria on the surface of medical equipment, bacteria in the medical facility environment, bacteria in the food production environment, or bacteria in the pharmaceutical production environment; the other bacterial lower respiratory tract infections include pathogens causing non-ventilator-associated community-acquired pneumonia, hospital-acquired pneumonia, or bacterial infections during acute exacerbations of chronic obstructive pulmonary disease; the urinary tract infections include pathogens causing cystitis or pyelonephritis; the wound infections include pathogens causing burn wound infections; and the blood infections include pathogens causing bacterial sepsis.

[0087] In this invention, pathogens causing urinary tract infections, wound infections, and blood infections include Gram-negative bacilli and Gram-positive cocci. Bacteria from food production environments or pharmaceutical production environments include Staphylococcus aureus and Escherichia coli.

[0088] The method for rapid bacterial detection provided by this invention has the characteristics of being culture-free, rapid, high-throughput, and capable of visual analysis.

[0089] The SERS chip provided by this invention combines specific aptamer recognition and Raman signal internal standard ratio calibration mechanism to achieve rapid, culture-free, and highly specific detection of common VAP pathogens, and has good portability and clinical applicability.

[0090] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0091] Example 1: Fabrication of Ga-doped ZnO superlattice SERS substrate 0.495 g of zinc nitrate (Zn(NO3)2·6H2O), 0.090 g of gallium nitrate (Ga(NO3)3·8H2O), and 25 mL of anhydrous ethylene glycol were stirred and mixed at room temperature for 30 min to obtain a precursor solution. Under stirring conditions, a 1 mol / L NaOH ethylene glycol solution (total addition volume 25 mL) was added dropwise to the precursor solution at a rate of 1 drop / 2 seconds to obtain a nanocrystalline nucleus mixture. 0.05 g of polyvinyl alcohol was added to the nanocrystalline nucleus mixture, and the mixture was stirred and mixed for 30 min. The mixture was then transferred to a reaction vessel connected to a reflux condenser, heated to 140 °C, and reacted at this temperature for 4 h. After naturally cooling to room temperature, the resulting solid component (grayish-white loose powder) was washed three times with anhydrous ethanol at 8000 r / min for 15 min. During each wash, 20 mL of ethanol was added and the mixture was shaken thoroughly for 5 min. The washed and moistened powder was then dried in a vacuum drying oven at 80℃ for 24 h. After drying, the powder was placed in a muffle furnace and annealed at 600℃ in air for 10 h to obtain Ga-doped ZnO nanocrystals with a superlattice structure, i.e., Ga-doped ZnO superlattice SERS substrate.

[0092] Structure and performance characterization of Ga-doped ZnO nanocrystals with superlattice structure prepared in Example 1 (1) Observation of surface morphology and particle size Surface observation of GZO superlattice materials was performed using field emission scanning transmission electron microscopy. Figure 1 The top left image shows that the test results indicate the sample consists of regular cubic or near-spherical nanoparticles with a particle size distribution ranging from 10 to 14 nm. The particles are uniformly distributed without obvious agglomeration, indicating that the material possesses good crystallinity and dispersibility. The dense and smooth surface with clear boundary edges demonstrates that the annealing process effectively controlled the grain growth direction and size.

[0093] (2) Elemental composition and periodic doping analysis Semi-quantitative elemental analysis was performed using an energy dispersive spectroscopy (EDX, X-Max 150T), and the results are shown in the figure. Figure 1The upper right corner of the image shows that Ga, Zn, and O are evenly distributed in the sample, and the Ga content is close to the theoretical molar ratio, indicating good doping efficiency. Further line scan analysis revealed that Ga elements exhibit a periodic alternating distribution along the axial direction (

[0001] ) within the nanocrystals, which is inversely correlated with the Zn concentration, indicating that Ga has successfully substituted for doping and formed an ordered modulation layer, verifying the intrinsic characteristics of the superlattice structure.

[0094] (3) Observation of internal crystal structure and superlattice fringes The internal microstructure of the material was analyzed in detail using transmission electron microscopy (TEM, JEOL JEM-2100F) and high-resolution transmission electron microscopy (HR-TEM). HR-TEM images ( Figure 1 In the lower left corner image, periodic lattice fringes distributed along the

[0001] direction are clearly visible, with a stripe spacing of approximately 0.48 ± 0.05 nm. At least five consecutive modulation layers are observed, proving the formation of a typical periodic Ga-doped modulation structure, i.e., a superlattice morphology, within the material. The strong contrast and clear boundaries of the fringes indicate a highly ordered structure. This precise superlattice structure was further characterized using a typical aberration-corrected high-angle annular dark-field scanning transmission electron microscope (AC HAADF-STEM). Figure 1 As shown in the lower right corner of the image, stripes composed of alternating bright and dark spots are clearly visible, with a periodicity of 0.48 nm. Heavier Ga atoms (presenting bright spots) and Zn atoms (presenting dark spots) are clearly distinguishable, fully demonstrating the existence of highly coherent interfaces between the alternating layers. Selected area electron diffraction (SAED) patterns show a series of diffraction spots with crystal plane indices labeled (101̅0) and (112̅0), corresponding to a hexagonal wurtzite ZnO crystal with space group P63mc. These diffraction patterns consist of principal diffraction spots, accompanied by satellite spots distributed along continuous straight lines in their vicinity, thus exhibiting characteristics of a superlattice structure. Figure 2 ).

[0095] (4) Identification of crystal phase structure The samples were diffraction tests were performed using an X-ray diffractometer (XRD, Bruker D8 ADVANCE), and the results are shown in the figure. Figure 3 The scanning range was 10~80° with a step size of 0.02°. The diffraction peaks corresponded to the characteristic diffraction planes of hexagonal wurtzite ZnO, including (100), (002), and (101). Among them, the diffraction angle of the (101) plane was slightly shifted and broadened, and its interplanar spacing was broadened from 0.248 nm in conventional ZnO to about 0.475 nm. Combined with the HR-TEM results, the superlattice modulation effect was verified.

[0096] (5) Analysis of chemical valence state and defect state The valence state and chemical environment of the elements were determined using X-ray photoelectron spectroscopy (XPS, PHI Quantera II ESCA System), and the results are shown in the figure. Figure 4 The Ga 2p spectrum shows that it is mainly in the +3 valence (Ga 3+ The presence of oxygen vacancies (Vo) in the O 1s peak, combined with the Zn 2p and O 1s spectra, indicates that the superlattice formation process is accompanied by lattice defect reconstruction. These defects help to enhance charge trapping and transfer capabilities and are one of the important auxiliary mechanisms for SERS performance.

[0097] (6) Optical bandgap testing and absorption performance analysis The absorption edge of the material was measured using a UV-Vis diffuse reflectance spectrometer (UV-Vis, HITACHI UH-5300), and the results are shown in [Figure number missing]. Figure 5 The results showed a significant redshift at the absorption edge, with the superlattice sample exhibiting absorption extending into the visible light region compared to the blue edge of the conventional ZnO sample. Bandgap estimation based on the Tauc plot yielded a value of approximately 1.93 eV (the exact value depends on the doping concentration and modulation period), indicating bandgap contraction. This bandgap is beneficial for achieving energy resonance with the 633 nm excitation wavelength, thereby enhancing the photoexcited carrier generation efficiency.

[0098] (7) SERS performance testing and enhancement factor evaluation Surface-enhanced Raman scattering (SERS) performance was tested using a Renishaw inVia Raman spectrometer. The excitation wavelength was 633 nm, the power was 0.5 mW, the exposure time was 10 s, and the scans were repeated 5 times and averaged. Tetracyanoquinoline (TCNQ) was used as the Raman probe. -9 A mol / L TCNQ ethanol solution was added dropwise to the material surface, dried, and then tested. Results are shown below. Figure 6 The results showed that at 1455cm -1 The C=C stretching vibration peak signal at the location is clearly identifiable, and its intensity is more than 100 times stronger than that of ZnO signal.

[0099] By calculating the SERS performance factor (SPF), the GZO superlattice material still achieved a SERS performance factor of 1.5 × 10⁻⁶ under conditions without the involvement of noble metals. 6 The above enhancement factor is approximately 10% higher than that of undoped Ga ZnO material. 5 This represents an improvement of over 10 times compared to GZO non-superlattice materials. 5The SERS performance is more than six orders of magnitude higher than that of the Si substrate, with good repeatability (RSD < 10%), indicating that its SERS performance is stable and reliable, making it suitable for applications such as trace analysis and molecular recognition. Compared with other control materials (such as Si substrate), this superlattice substrate exhibits high sensitivity and low detection limit performance under non-plasmic conditions, and shows consistent enhancement effects in various organic molecule systems (including 4-MBA, MB, CuPc, PB, TCNQ, etc.).

[0100] Example 2: Construction of the aptamer probe recognition unit Step (1) Covalent ligation of DNA aptamers and Raman signal probes First, DNA aptamers are selected for different target bacteria. The DNA aptamers have an amino group (-NH2) introduced at the 3' end and an amino group (-COOH) introduced at the 5' end. For *Streptococcus pneumoniae*, the nucleotide sequence of the DNA aptamer is shown in SEQ ID NO.1, which is: 5'-COOH-TGACGAGCCCAAGTTACCTGCCCCCGAACCATACCACACGATGCCCCGTACCCCAGCCACCAGAATCTCCGCTGCCTACA-NH2-3'; For E. coli, the complex of DNA aptamer and methylene blue (MB) is readily available for purchase. The nucleotide sequence of the DNA aptamer in the complex is shown in SEQ ID NO.2, where SEQ ID NO.2 is: 5'-COOH-GCAATGGTACGGTACTTCCCCATGAGTGTTGTGAAATGTTGGGACACTAGGTGGCATAGAGCCGCAAAAGTGCACGCTACTTTGCTAA-MB-3'; For Staphylococcus aureus, the nucleotide sequence of the DNA aptamer is shown in SEQ ID NO.3, which is: 5'-COOH-GCAATGGTACGGTACTTCCTCCCACGATCTCATTAGTCTGTGGATAAGCGTGGGACGTCTATGACAAAAGTGCACGCTACTTTGCTA-NH2-3'; For Acinetobacter baumannii, the nucleotide sequence of the DNA aptamer is shown in SEQ ID NO.4, which is: 5'-COOH-ACAGCACCACAGACCACATATCACATGCTGTCGCCTTGCGATATCAATTCCAGTGATGTTTGTCTTCCTGCC-NH2-3'; For Pseudomonas aeruginosa, the nucleotide sequence of the DNA aptamer is shown in SEQ ID NO.5, which is: 5'-COOH-CCCCCGTTGCTTTCGCTTTTCCTTTCGCTTTTGTTCGTTTCGTCCCTGCTTCCTTTCTTG-NH2-3'.

[0101] ①4-MBA: 4-Mercaptobenzoic acid itself contains a carboxyl group (-COOH) and is used directly as is without any additional modification.

[0102] ②TCNQ: A carboxyl-functionalized TCNQ derivative (i.e., 7,7,8,8-tetracyano-p-benzodiquinone-2,5-diyl-(3-propionic acid)) was purchased from the market and used directly as is.

[0103] ③MB: A methylene blue (MB)-labeled DNA aptamer was purchased commercially available (Shanghai Dina Biotechnology Co., Ltd.); the MB label was covalently linked to the aptamer via an NHS ester activation coupling reaction. No additional chemical functionalization was performed.

[0104] ④CuPc: 4-Carboxyphthalic anhydride (CPA, 6.725 g, 35 mmol), urea (12.15 g, 0.20 mol), CuCl2 (1.175 g, 8.75 mmol), NH4Cl (0.94 g, 17.5 mmol), and (NH4)2Mo2O7 (0.107 g, 0.087 mmol) were thoroughly mixed and ground, then placed in a muffle furnace and calcined at 220 °C for 6 hours. After naturally cooling to room temperature, the resulting dark powder was successively soaked in 1.0 mol·L⁻¹ solution. -1 HCl solution and 1.0 mol·L -1 Each was soaked in NaOH solution for 8 hours. Then, the resulting tetraamide copper phthalocyanine (CuPc-CONH2) was placed in a three-necked flask and incubated in a solution containing 2.0 mol·L⁻¹ NaOH. -1 The solution was refluxed in a saturated NaCl solution (100 mL) for 6 hours. After filtration, the pH of the filtrate was adjusted to approximately 2.0 with concentrated HCl. The mixture was allowed to stand overnight to allow precipitation of the product; the precipitate was then collected and washed repeatedly with deionized water and methanol to finally obtain a blue solid product.

[0105] ⑤PB: Carboxylated Prussian blue (PB) was synthesized using a typical aqueous phase procedure, with citric acid used as an in-situ capping agent. Specifically, citric acid (0.5 mmol, 98 mg) was added to an aqueous FeCl4 solution (20 mL, 1.0 mM) under stirring. The reaction was carried out at 60 °C. Subsequently, while maintaining stirring at 60 °C, an aqueous K4[Fe(CN)6] solution (20 mL, 1.0 mM) containing an equal volume of citric acid was added dropwise. A clear, bright blue dispersion was formed immediately upon mixing. The pH of the resulting dispersion was measured to be approximately 2.8. After stirring at 60 °C for 1 minute, the reaction mixture was cooled to room temperature and stirred for another 5 minutes. An equal volume of acetone was then added to induce precipitation. The resulting product was collected after centrifugation at 10,000 rpm for approximately 15 minutes. The precipitate was redispersed in approximately 20 mL of distilled water by ultrasonication, followed by the addition of an equal volume of acetone for recrystallization and centrifugation. Repeat the purification step twice to finally obtain purified carboxylated Prussian blue (PB).

[0106] In this embodiment, the aptamer-probe complex formed by MB and the DNA aptamer was a commercially available product. The remaining four carboxyl-containing Raman probes (4-MBA, CuPc, PB, and TCNQ) were coupled to their corresponding 3'-amino-modified aptamers via a standard EDC / NHS amide coupling reaction. Specifically, each probe (2 mM) was first placed in MES buffer (pH 5.5) and activated for 30 minutes using EDC (5 mM) and NHS (5 mM); then, it was immediately reacted with the corresponding 3'-amino-modified DNA aptamer (10 μM) overnight at 4 °C, with a reaction molar ratio (Raman signal probe: DNA aptamer) set at 200:1. The reaction products were purified by ultrafiltration (molecular weight cutoff 3 kDa) and resuspended in PBS buffer.

[0107] Step (2): Coupling of the aptamer labeled with the signal probe to the substrate A 5×5 mm monocrystalline silicon wafer was rinsed with deionized water and ethanol and dried under a nitrogen atmosphere. The GZO SL prepared in Example 1 was then dispersed in ethanol to form a uniform slurry, which was drop-coated onto the surface of a 5×5 mm silica slide and allowed to dry naturally. It was then treated with oxygen plasma (100 W, 5 min) to increase the number of hydroxyl groups on the GZO SL coating surface. Subsequently, the GZO SL-loaded monocrystalline silicon wafer was immersed in anhydrous ethanol-APTES solution (2% by volume) and reacted for 2 hours under gentle shaking to perform silanization modification. After silanization, the GZO SL-loaded monocrystalline silicon wafer was thoroughly cleaned with ethanol, dried under nitrogen, and then thermosetting at 110 °C for 60 minutes to achieve covalent immobilization.

[0108] The PBS solutions of the five Raman signal probe-conjugated 5'-terminal carboxylated DNA aptamers were activated for 30 minutes at room temperature using freshly prepared EDC / NHS solution (0.1 M MES buffer containing 0.2 M EDC and 0.05 M NHS, pH 5.5). After activation, the Raman signal probe-conjugated DNA aptamers were desalted by centrifugation and ultrafiltration to remove excess EDC / NHS. Immediately afterwards, 50 μL of the activated Raman signal probe-conjugated DNA aptamer solution was added dropwise to the surface of each heat-cured substrate and incubated overnight in a humidified chamber at 4 °C to prevent evaporation. After incubation, the substrates were washed with PBST (PBS containing 0.05% Tween-20) to remove physically adsorbed aptamers. To minimize nonspecific adsorption, the aptamer-probe complex-modified samples were then immersed in a PBS solution containing 1% (w / v) BSA and blocked at room temperature for 30–60 minutes, followed by washing with PBS. After washing, the prepared SERS chip was kept moist by immersing it in PBS and stored at 4 °C in the dark to maintain the conformation of the aptamer for subsequent bacterial detection.

[0109] Example 3: SERS Detection Procedure and Clinical Sample Application This embodiment provides a method for detecting pathogens of ventilator-associated pneumonia based on a GZO SL-SERS chip and an aptamer probe recognition unit. It is suitable for the simultaneous identification and Raman response analysis of multiple target bacteria, and features high sensitivity, strong specificity, and short response time, which can meet the needs of rapid clinical diagnosis and point-of-care testing (POCT).

[0110] Step (1): Bacterial culture Streptococcus pneumoniae (ATCC 49619), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Acinetobacter baumannii (ATCC 19606), and Pseudomonas aeruginosa (ATCC 27853) were all purchased commercially and stored as glycerol cultures at -80°C. For culturing, the frozen cultures were thawed at room temperature and inoculated into 5 mL of liquid culture medium: Streptococcus pneumoniae was inoculated into Brain Heart Infusion (BHI) medium, while Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were inoculated into LB (Luria–Bertani) medium. The cultures were then incubated at 37°C and 180 rpm on a shaker until they reached mid-logarithmic growth phase (OD600 ≈ 0.5).

[0111] Bacterial cells were collected by centrifugation at 5000×g for 5 minutes at room temperature; they were then washed twice with sterile phosphate-buffered saline (PBS, pH 7.4) and resuspended in PBS to adjust to the desired concentration. Bacterial concentration (CFU / mL) was determined using the plate count method: tenfold serial dilutions of the bacterial suspension were prepared, and appropriate amounts of each dilution were spread onto agar plates. After incubation (18–24 hours at 37°C, depending on the strain), colonies were counted. The CFU / mL value used for Raman spectroscopy was calculated based on the colony count, dilution factor, and spread volume. For each concentration gradient, three independent plates were counted, and the average value was used as the final CFU / mL report value.

[0112] Step (2): Incubation reaction between chip and sample In the bacterial capture experiment, a freshly prepared bacterial suspension was adjusted to a predetermined concentration (determined by plate counting) and added dropwise to the surface of the SERS chip prepared in Example 2 (typically 10 μL per 5 × 5 mm substrate). The substrate was then placed in a humidified chamber and incubated at room temperature for 10 minutes to induce specific recognition and binding between the hairpin aptamers and the target bacteria. After incubation, the substrate was gently rinsed with PBS, followed by washing with PBS solution containing 0.05% Tween-20 (PBST) to remove unbound or loosely attached bacteria. The captured bacteria were placed in PBS to prevent deformation due to drying, and Raman measurements were subsequently performed.

[0113] Step (3): Raman signal acquisition and data recording All handheld Raman measurements were performed using a Metrohm MIRA XTR handheld Raman spectrometer equipped with a 633 nm laser excitation source. The laser power at the sample was set to 50 mW. The exposure time for spectral acquisition was set to 1.0 s per scan, and each spectrum was obtained by averaging 10 scans. According to the manufacturer, the instrument's acquisition optics system provides a working distance of approximately 5 mm and a nominal spot size of approximately 0.04 mm (40 μm); its built-in orbital grating scanning function achieves an effective grating scan range of approximately 2.5 mm, thereby improving the representativeness of the sampling. Raman shift calibration was performed according to the manufacturer's standard procedures using Metrohm calibration standards; in addition, a 520.7 cm⁻¹ calibrator was used before the formal measurements. -1 The silicon peak at the location further validated the calibration results. For each experimental condition, spectral data were collected from ≥3 randomly selected sites on different chips, and the final results are presented in the form of "mean ± standard deviation (SD)".

[0114] Step (4): Validation of detection sensitivity and specificity In 1×10 7 Up to 1×10 0 Five pathogens were detected within a concentration range of CFU / mL. The results showed that characteristic peaks could still be detected stably below 10 CFU / mL, and the limit of detection (LOD) was approximately 1.27 CFU / mL.

[0115] To verify specificity, non-target bacteria (such as Salmonella, Haemophilus influenzae, etc.) and a single probe were used for detection. No target peak response was observed, indicating that the probe-chip system has good specific recognition ability and low cross-reactivity.

[0116] Step (5): Analysis of clinical respiratory samples Clinical bronchoalveolar lavage fluid (BALF) samples were collected from patients suspected of having ventilator-associated pneumonia (VAP) according to an approved institutional protocol. 50 μL of BALF sample was used for each test. The sample was gently vortexed for 5 seconds and then centrifuged at 3000×g for 5 minutes to remove mucus and large debris; the supernatant was then collected and used directly for subsequent tests. Next, 10 μL of the pretreated BALF sample was added to the aptamer-functionalized GZO SL sensing area and incubated at room temperature for 10 minutes. After incubation, the chip was washed with PBS solution (3×100 μL) to remove unbound components. All tests were performed within 2 hours of sample preparation, avoiding repeated freeze-thaw cycles. Clinical samples were added to the functionalized chip, incubated, washed, and Raman signals were acquired as described above.

[0117] Figure 7 This is the ultraviolet-visible absorption spectrum characterization of the SERS chip prepared in Example 2 of the present invention. Figure 9 This is the energy level diagram of the Raman signal molecules selected for the SERS chip prepared in Embodiment 2 of the present invention. Figure 10 This is a selective test of the SERS chip prepared in Embodiment 2 of the present invention. Figure 11 This is a linearity test of the SERS chip prepared in Embodiment 2 of the present invention. Figure 12 This is a response time test of the SERS chip prepared in Embodiment 2 of the present invention. Figure 13 The results are from the pathogen test of bronchoalveolar lavage fluid from 50 VAP patients using the SERS chip prepared in Example 2 of this invention. Figure 14 This is the subject operating characteristic curve of the SERS chip prepared in Example 2 of the present invention based on clinical test results. Figure 15 This refers to the long-term storage stability of the SERS chip prepared in Example 2 of this invention. Figure 16 This refers to the batch repeatability of the SERS chip prepared in Example 2 of this invention.

[0118] By comparing the signal intensity with a standard curve, quantitative detection of target pathogens (i.e., pathogenic bacteria) in clinical samples can be achieved. Comparison with results from routine hospital culture methods shows a concordance rate exceeding 92%, indicating that the detection platform constructed in this invention possesses good clinical applicability. The prepared SERS chip exhibits excellent selectivity for the simultaneous analysis of five VAP pathogens. Potential interfering substances in mixed BALF samples include other pathogens (Klebsiella pneumoniae, Haemophilus influenzae, etc.), cytokines (IL-1, TNF-α, etc.), proteins (CRP, SPD, etc.), and metal ions (Na+). + K + Ca 2+ The response caused by these factors is negligible. Under optimal conditions (pH 7.4, 37°C, 10 mM PBS buffer), the ratio of the specificity intensity to the logarithm of the VAP pathogen concentration is between 10 and 10^-10. 7 A good linear relationship was observed within the range of CFU / mL (R0). 2 = 0.999), and exhibited linear concentration dependence of VAP pathogens in the low range of 0–500 CFU / mL, consistent with the Langmuir isotherm binding model. The limits of detection (LODs) for Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were 5.0, 2.0, 1.0, 4.0, and 5.0 CFU / mL (3σ / k, n=5, standard deviation), respectively, which are 1 to 3 orders of magnitude lower than previously reported methods. The Raman intensity ratio reached its maximum within approximately 10 minutes, highlighting the platform's rapid response capability. Such a fast detection speed is unprecedented for a simple technique using handheld devices. Furthermore, the GZO SL-based SERS chip retained over 95% of its signal after six months of storage in the dark at 4°C and demonstrated excellent manufacturing consistency, with batch-to-batch variability remaining below 5%. Finally, the point-of-care diagnostic capability of the SERS platform was validated using 50 bronchoalveolar lavage fluid (BALF) clinical samples collected from patients with ventilator-associated pneumonia (VAP). The SERS platform showed a significant positive correlation with standard quantitative sputum culture methods for all five pathogens (Pearson correlation coefficient r > 0.85, p < 0.001), validating the clinical application value of the developed SERS platform. Notably, the SERS platform achieved perfect identification of all five pathogens, with an area under the curve (AUC) of 1.00, providing extremely high sensitivity and specificity for pathogen identification in clinical samples. Furthermore, the total testing time was reduced from 48–72 hours to 10 minutes, with an estimated cost of $0.15 per test, highlighting the potential of the SERS chip as a rapid, low-cost, and accurate point-of-care diagnostic tool in ICUs and resource-constrained environments.

[0119] Example 4: Chip Array Integration and Portable Testing Platform Construction This embodiment provides a method for constructing a multi-channel array integration and portable detection platform based on a GZO SL superlattice SERS chip. Figure 8 This is a photograph of the GZO superlattice SERS chip prepared in Example 4 of this invention. By standardizing the chip array module, differentiating and coding the identification probes through multiple channels, and combining it with a portable Raman spectrometer, on-site, rapid, and accurate identification and detection of ventilator-associated pneumonia pathogens can be achieved.

[0120] Step (1): Construction of the array chip module GZO SL dispersion was prepared according to the method in Example 2. Using a mask template or an automated spotting device, the GZO SL dispersion was sequentially spotted onto different preset areas on the surface of the same monocrystalline silicon wafer to form a multi-channel or multi-site array structure, facilitating the simultaneous identification and analysis of multiple pathogens. After natural drying, a uniform reinforcing substrate material layer was formed. Then, it was placed in a plasma cleaner (100 W, O2 atmosphere) for 10 minutes to enhance the surface hydrophilicity. Subsequently, it was immersed in a 2% APTES ethanol solution for 2 hours, naturally dried at room temperature, and then baked at 110°C for 1 hour to solidify the silane layer, obtaining a chip with amino groups on the surface.

[0121] A 1 μM aptamer-Raman signal probe complex solution was prepared according to the method in Example 2. Using a mask template or an automated spotting device, 50 μL of the 1 μM aptamer-Raman signal probe complex solution obtained in step (2) was added dropwise to the surface of the modified chip prepared in step (3), and placed in a humidified dark box for incubation at 4°C in the dark for 12 hours to achieve covalent coupling. After the reaction was completed, the chip was thoroughly washed three times with 100 μL of PBS buffer each time to ensure the removal of unbound probe molecules. Through this strategy, Raman signal molecules and recognition DNA aptamers are stably anchored on the chip surface, constructing a functionalized SERS chip with high specific recognition capability.

[0122] Step (2): Multiple probe distribution and spatial segmentation To achieve simultaneous detection of multiple pathogens, chips modified with corresponding aptamer probes are arranged in different regions of the array. For example, the first row is used to detect Streptococcus pneumoniae, the second row is used to detect Staphylococcus aureus, and so on. Each row has at least 8 repeat units to ensure that the detection data are statistically significant.

[0123] The separation and fixation of probes are achieved through a programmed drop and mask micro-tipping system, which can automatically complete the distribution and modification of multiple probes at different sites, avoiding cross-contamination caused by manual operation.

[0124] Step (3): Sample processing and array incubation reaction After resuspending respiratory samples or clinical cultures in PBS and adjusting their concentration, 50 μL of sample solution was added to the entire surface of the array module using a multichannel pipette. The module was then covered with a glass cover and incubated in a 37°C incubator for 1 hour, during which time pathogens recognized and bound to their respective probe chips.

[0125] After incubation, remove the array module, gently rinse it three times with PBS buffer, and allow it to air dry in preparation for subsequent Raman scanning.

[0126] Step (4): Construction of Portable Raman Platform and Data Acquisition A compact, portable Raman spectrometer with an integrated scanning platform (such as a handheld Raman system equipped with a CCD detector and a micro laser module) was selected, along with a high-precision two-dimensional scanning robotic arm and a preset array positioning program. The Raman excitation source wavelength was set to 633 nm, the laser power did not exceed 50 mW, and the integration time was 5–10 s.

[0127] The platform automatically identifies the encoding information of the array chips, sequentially collects the Raman signal of each chip, and transmits the data to the data analysis system on the host computer in real time.

[0128] Step (5): Result recognition and visualization output The host computer system has a pre-set standard peak position and template recognition program that compares the detection signal with the database to quickly identify positive targets and perform quantitative analysis. The system automatically generates a two-dimensional array heat map, displaying the Raman intensity level and positive status of each detection unit, and finally outputs a PDF detection report.

[0129] The entire testing process (from sample addition to result interpretation) is controlled within 90 minutes, and the operation process is highly modular, making it suitable for point-of-care testing (POCT) scenarios, such as ICU wards, infectious disease clinics, or mobile medical vehicles.

[0130] As demonstrated by the above embodiments, this invention provides a SERS chip detection platform based on a Ga-doped ZnO superlattice. Combining photogenerated charge modulation and aptamer recognition, it enables rapid and quantitative detection of five types of pathogens associated with ventilator-associated pneumonia (VAP): Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. This platform utilizes the built-in electric field and quantum confinement effect formed by the GZO superlattice to enhance the Raman signal. Through a triple recognition mechanism (selective charge transfer between GZO and probe molecules, Raman characteristic peak recognition, and pathogen aptamer binding), pathogen detection is achieved without culture, with a detection limit as low as 1.27 CFU / mL. The entire process is completed within 30 minutes, with a detection cost of approximately US$0.15. The chip is stably stored at room temperature for over 6 months, making it suitable for rapid bedside screening and guidance on anti-infective medication in ICUs.

[0131] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A surface-enhanced Raman spectroscopy chip, characterized in that, The device includes a substrate and a plurality of detection sites disposed on the surface of the substrate. Each detection site includes a reinforcing substrate material layer and an aptamer-Raman signal probe composite material layer stacked sequentially, wherein the reinforcing substrate material layer is in contact with the substrate. The reinforcing substrate material layer is a Ga-doped ZnO nanocrystal with a superlattice structure; the aptamer-Raman signal probe composite material layer includes at least one aptamer-probe complex, which is a complex of a DNA aptamer and a Raman signal probe.

2. The surface-enhanced Raman spectroscopy chip according to claim 1, characterized in that, The preparation method of the Ga-doped ZnO nanocrystals with the superlattice structure includes the following steps: A Zn source, a Ga source, and a solvent are first mixed to obtain a precursor solution; the molar ratio of Zn to Ga in the precursor solution is 1 to 9:1; the solvent includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol. An alkali metal hydroxide solution is added dropwise to the precursor solution to obtain a nanocrystalline nucleus mixture; the ratio of the total molar amount of Zn and Ga elements in the precursor solution to the molar amount of alkali metal hydroxide in the alkali metal hydroxide solution is 1:0.5~2; the solvent in the alkali metal hydroxide solution includes one or more of ethylene glycol, propylene glycol, glycerol, and diethylene glycol; The nanocrystal nucleus mixture was mixed with polyvinyl alcohol in the second stage to construct a crystal lattice, thereby obtaining a GaZn lattice layered structure material. The GaZn lattice layered structure material was annealed in an air atmosphere to obtain Ga-doped ZnO nanocrystals with a superlattice structure.

3. The surface-enhanced Raman spectroscopy chip according to claim 1, characterized in that, The DNA aptamer is a DNA aptamer with sequence recognition specificity for ventilator-associated pneumonia (VAP) pathogens, including one or more of Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa; the Raman signal probe includes one or more of 4-mercaptobenzoic acid, methylene blue, copper phthalocyanine, ferric ferrocyanide, and tetracyanoquinoline.

4. The surface-enhanced Raman spectroscopy chip according to claim 1 or 3, characterized in that, The aptamer-probe complex includes one or more of a first aptamer-probe complex, a second aptamer-probe complex, a third aptamer-probe complex, a fourth aptamer-probe complex, and a fifth aptamer-probe complex; the first aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Streptococcus pneumoniae and 4-mercaptobenzoic acid; the second aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Escherichia coli and methylene blue; the third aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Staphylococcus aureus and copper phthalocyanine; the fourth aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Acinetobacter baumannii and ferric ferrocyanide; and the fifth aptamer-probe complex is a complex of a DNA aptamer with sequence recognition specificity for Pseudomonas aeruginosa and tetracyanoquinoline.

5. The surface-enhanced Raman spectroscopy chip according to claim 1, characterized in that, The diameter of the detection site is 1~2mm; when there are multiple detection sites, the spacing between adjacent detection sites is 2~5mm; the surface-enhanced Raman spectroscopy chip also includes a confining capping layer, which has a plurality of detection channels, each detection channel corresponding to a detection site, and each detection site is placed in a detection channel.

6. The method for preparing the surface-enhanced Raman spectroscopy chip according to any one of claims 1 to 5, characterized in that, Includes the following steps: A dispersion of Ga-doped ZnO nanocrystals with a superlattice structure was coated onto the surface of a substrate, and then subjected to plasma treatment, silane coupling agent activation treatment and heat treatment in sequence to obtain an activated semi-finished product. The aptamer-Raman signal probe complex solution is coated onto the surface of the activated semi-finished product and covalently coupled to obtain the surface-enhanced Raman spectroscopy chip; the aptamer-Raman signal probe complex solution contains at least one aptamer-probe complex, which is a complex of a DNA aptamer and a Raman signal probe.

7. The application of the surface-enhanced Raman spectroscopy chip according to any one of claims 1 to 5 or the surface-enhanced Raman spectroscopy chip prepared by the preparation method according to claim 6 in the detection of bacteria.

8. A portable instant detection platform, comprising a surface-enhanced Raman spectroscopy chip as described in any one of claims 1 to 5 or a surface-enhanced Raman spectroscopy chip prepared by the preparation method described in claim 6, and a readout module, wherein the readout module comprises a Raman spectrometer.

9. A method for rapid detection of bacteria, characterized in that, Includes the following steps: The sample to be tested is applied to the detection site of the surface-enhanced Raman spectroscopy chip according to any one of claims 1 to 5 or the surface-enhanced Raman spectroscopy chip prepared by the preparation method according to claim 6, and incubated. After the incubation, Raman signal is acquired to obtain the probe peak intensity of the Raman signal probe, and the ratio of the probe peak intensity of the Raman signal probe to the internal standard peak intensity of the Ga-doped ZnO nanocrystal with a superlattice structure is calculated. The ratio is substituted into the standard curve or standard equation to obtain the detection result of bacteria.

10. The method for rapid bacterial detection according to claim 9, characterized in that, The bacteria include pathogens causing ventilator-associated pneumonia, other bacterial lower respiratory tract infections, urinary tract infections, wound infections, blood infections, bacteria on the surface of medical equipment, bacteria from the medical facility environment, bacteria from the food production environment, or bacteria from the pharmaceutical production environment; the other bacterial lower respiratory tract infections include pathogens causing non-ventilator-associated community-acquired pneumonia, hospital-acquired pneumonia, or bacterial infections during acute exacerbations of chronic obstructive pulmonary disease; the urinary tract infections include pathogens causing cystitis or pyelonephritis; the wound infections include pathogens causing burn wound infections; and the blood infections include pathogens causing bacterial sepsis.

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