Detection and recognition reagent, detection platform and detection method for pseudomonas aeruginosa

Abstract

The invention provides a detection and recognition reagent, a detection platform and a detection method for pseudomonas aeruginosa. The platform, the detection and recognition reagent and the detection method for rapidly detecting the pseudomonas aeruginosa PA through double-recognition surface-enhanced Raman spectroscopy SERS are mainly constructed on the basis of a nucleic acid aptamer Apt and a gold-silver core-shell double-layer reporter molecule tag structure. Wherein the SERS tag AuNRs-MBA (at) Ag-MBA-Van, the immunomagnetic SPION-PEI-AuNPs-Apt and PA can form a SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag (at) MBA-AuNRs sandwich structure, and qualitative and quantitative detection is carried out by measuring the Raman spectrum of the sandwich structure; meanwhile, a machine learning regression model and performance indexes thereof are combined, and an optimal PA quantitative detection standard curve is constructed by utilizing detected Raman signals and PA concentration. The high-sensitivity double-identification tag type SERS quantitative analysis system provided by the invention has the advantages of wide detection range, low detection limit, good specificity, high accuracy, short detection time and the like, thereby providing a new method and technology for prevention and clinical diagnosis of pseudomonas aeruginosa infection related diseases and food safety monitoring.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection, and in particular to a detection and identification reagent, a detection platform and a detection method for Pseudomonas aeruginosa. Background Art

[0002] Pseudomonas aeruginosa (PA), also known as Pseudomonas aeruginosa, is a Gram-negative bacterium and one of the three major pathogens of opportunistic infections. In the context of the development of drug-resistant bacteria caused by the abuse of antibiotics, PA was listed as a key pathogen by the World Health Organization in 2017, which can cause a variety of infections in immunocompromised people. It is widely present in nature and is the most common spoilage bacteria in aquatic products and meat. Although China does not require its detection in ordinary foods, it must be detected in drinking mineral water. In addition, it is also one of the important pathogens of nosocomial infections, introduced into hospitals through food and patients, and exists in many water bodies, such as disinfectants, respiratory equipment, food, sinks, faucets, and mops. A study on ICU patient infections found that PA infections accounted for 16.2% of patient infections. Therefore, rapid detection of PA is of great significance for hospital infection control, clinical diagnosis, and food safety monitoring.

[0003] There are many methods for detecting Pseudomonas aeruginosa. The traditional method of identifying Pseudomonas aeruginosa is based on the "National Clinical Laboratory Operation Procedures". Conventional isolation and culture of bacteria, microscopic observation, biochemical index identification, etc. are the "gold standard" for Pseudomonas aeruginosa detection. These detection methods not only require professional technicians and complex equipment and instruments, but also the identification process is time-consuming (48h), complicated to operate, and low in sensitivity. In addition, typical bacterial detection strategies include polymerase chain reaction (PCR) and immunoassay. Immunoassay is fast but has low sensitivity and high cost. Although the PCR method has high sensitivity, it requires cumbersome sample pretreatment. Therefore, it is particularly important to develop a fast, highly sensitive and low-cost PA detection strategy.

[0004] Raman scattering refers to when a laser of a certain frequency is irradiated onto the surface of a material, the molecules in the material transfer energy with photons, the vibrational state changes to varying degrees and in varying ways, and light of different frequencies is scattered. The change in frequency depends on the characteristics of the scattering material. Different types of atomic groups vibrate in unique ways, so they can produce scattered light with a specific difference from the frequency of the incident light. Its spectrum is called a "fingerprint spectrum," and different substances can be identified based on this principle. In addition, substances can be quantitatively analyzed based on the intensity of the fingerprint peak. Surface-enhanced Raman scattering (SERS) is an optical technology that adsorbs the object to be tested on the surface of a rough precious metal (gold, silver, copper, etc.) to significantly enhance its Raman spectrum. It has been applied to the detection of a variety of pathogens. In recent years, SERS tags have developed from simple metal nanospheres @ reporter molecules to more complex metal structures @ reporter molecules; for example, CN 117309840 A (Wuhan Boya Biotechnology Co., Ltd.) discloses a surface enhanced Raman scattering nanotag, including a gold-silver alloy nanobox, the gold-silver alloy nanobox is a rectangular or cubic structure, the gold-silver alloy nanobox is wrapped with a metal shell, Raman signal molecules are distributed between the gold-silver alloy nanobox and the metal shell, and the metal shell is one of a gold layer, a silver layer, and a copper layer; the surface enhanced Raman scattering nanotag is an anisotropic core-shell plasma nanostructure, using a cubic gold-silver alloy nanostructure with stronger electromagnetic field anisotropy than spherical nanoparticles as the core material, and after a silver layer is further deposited on the core surface to form a core-shell, the Raman signal is significantly amplified, which is conducive to accurately detecting clinical samples with low viral loads and improving the sensitivity of detection.

[0005] However, the surface enhanced Raman scattering nano-tags in the above patent application only contain one layer of Raman signal molecules, and the sensitivity still needs to be improved. In addition, the detection performance of the method described in the above patent in PA detection has not been verified. There are problems such as the lack of standard methods for PA detection using SERS technology. Summary of the invention

[0006] In view of this, the main purpose of the present invention is to provide a detection and identification reagent, a detection platform and a detection method for Pseudomonas aeruginosa based on the technology of dual recognition of PA by nucleic acid aptamer (Aptamer, Apt) and gold-silver core-shell double-layer reporter molecule label structure, which is beneficial to improve the sensitivity and specificity of PA detection, in order to provide new methods and technologies for PA control, clinical diagnosis and food safety monitoring.

[0007] To achieve the above-mentioned purpose, the technical scheme provided by the present invention is: a detection and identification reagent for PA, comprising immunomagnetic nanoparticles superparamagnetic iron oxide (SPION)-polyethyleneimine (PEI)-gold nanoparticles (AuNPs)-nucleic acid aptamer (Apt) complex SPION-PEI-AuNPs-Apt and SERS label gold nanorods (AuNRs)-mercaptobenzoic acid (MBA)@silver-MBA-vancomycin (Van) complex AuNRs-MBA@Ag-MBA-Van for identifying and capturing PA, wherein:

[0008] The immunomagnetic beads SPION-PEI-AuNPs-Apt are mainly formed by modifying the aptamer Apt to the surface of SPION-PEI-AuNPs particles through chemical bonds, wherein the SPION-PEI-AuNPs particles are mainly prepared by electrostatically adsorbing gold nanoparticles AuNPs using superparamagnetic iron oxide nanoparticles SPION-PEI, and the superparamagnetic iron oxide nanoparticles SPION-PEI are mainly synthesized by ferric chloride, anhydrous sodium acetate and polyethyleneimine using a hydrothermal method;

[0009] The SERS tag AuNRs-MBA@Ag-MBA-Van is mainly synthesized by modifying vancomycin Van on the surface of the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA through an amide condensation reaction; the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA is mainly prepared by first using a redox reaction to wrap a silver shell on the AuNRs-MBA nanoparticles, and then modifying the silver shell with a second layer of Raman reporter molecule mercaptobenzoic acid MBA; the AuNRs-MBA nanoparticles are mainly prepared by modifying the first layer of Raman reporter molecule MBA on gold nanorods AuNRs.

[0010] The aptamer Apt is mainly used to capture the target PA, which can ensure the detection specificity.

[0011] Based on the above, the immunomagnetic beads SPION-PEI-AuNPs-Apt use thiolated Apt (Apt-SH), magnesium-containing buffer and the SPION-PEI-AuNPs as raw materials, and the aptamer Apt is modified to the surface of the SPION-PEI-AuNPs by forming an Au-S bond between the sulfur atom in Apt-SH and the AuNPs in the SPION-PEI-AuNPs.

[0012] Another object of the present invention is to provide a PA detection platform, comprising the above-mentioned PA detection and identification reagent and a Raman spectrometer, wherein the immunomagnetic beads SPION-PEI-AuNPs-Apt can specifically identify PA and capture it to form a complex SPION-PEI-AuNPs-Apt / PA; Van in the SERS tag AuNRs-MBA@Ag-MBA-Van specifically interacts with D-Ala-D-Ala in the PA cell wall peptidoglycan in the complex SPION-PEI-AuNPs-Apt / PA through its own hydrogen bonds to form a sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs; the Raman spectrometer is used to detect the Raman spectrum of the sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs, and a PA quantitative detection standard curve is constructed according to the relationship between PA concentration and Raman signal.

[0013] The Raman signal used to construct the standard curve for quantitative detection of PA in the present invention refers to the Raman signal intensity of a characteristic peak. Preferably, the characteristic peak 1587cm in the Raman spectrum is -1 The relationship between the signal intensity at the PA site and the PA concentration was used to construct a standard curve for PA quantitative detection.

[0014] Based on the PA detection platform, a Raman signal data analysis module is also included. The module has a built-in machine learning algorithm. The machine learning algorithm establishes multiple regression models to process the Raman signal data, and the complex correlation coefficient R 2 The performance of the regression model was evaluated by the root mean square error, and after optimizing the parameters of each model, the optimal regression model was selected to construct the PA quantitative detection standard curve. The regression analysis models include XGBR, SVR, BRR, ANNR, PR and ENR models.

[0015] Another object of the present invention is to provide a SERS-based PA detection method, comprising the following steps:

[0016] Preparation of SPION-PEI-AuNPs: uniformly mixing superparamagnetic iron oxide nanoparticles SPION-PEI and gold nanoparticles AuNPs solution to form SPION-PEI-AuNPs particles;

[0017] Preparation of immunomagnetic beads: firstly subjecting Apt1-SH solution and tris(2-carboxyethyl)phosphine (TCEP) buffer to a room temperature reduction reaction, and then mixing with the SPION-PEI-AuNPs particles to a coupling reaction, thereby obtaining immunomagnetic beads SPION-PEI-AuNPs-Apt;

[0018] Preparation of AuNRs-MBA@Ag: silver nitrate and AuNRs-MBA particles are uniformly mixed, and the silver nitrate undergoes a redox reaction to coat the AuNRs-MBA nanoparticles with a silver shell to form AuNRs-MBA@Ag nanoparticles; wherein the AuNRs-MBA nanoparticles are mainly prepared by modifying the first Raman reporter molecule MBA on the gold nanorods AuNRs;

[0019] Preparation of SERS tag: firstly, the AuNRs-MBA@Ag nanoparticles and MBA solution are mixed and reacted, and the second Raman reporter molecule MBA is modified on the AuNRs-MBA@Ag nanoparticles to form a gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA; then, the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA and Van are subjected to room temperature oscillation reaction to form a SERS tag AuNRs-MBA@Ag-MBA-Van;

[0020] Capturing the target bacteria PA: the immunomagnetic beads SPION-PEI-AuNPs-Apt, the magnesium-containing solution and the target detection bacterial solution are shaken and incubated at room temperature, so that the immunomagnetic beads SPION-PEI-AuNPs-Apt can specifically recognize the PA therein and capture it, forming a complex SPION-PEI-AuNPs-Apt / PA, wherein the target detection bacterial solution contains PA;

[0021] Dual recognition of target bacteria PA: The composite SPION-PEI-AuNPs-Apt / PA and the SERS tag AuNRs-MBA@Ag-MBA-Van were incubated with shaking at room temperature to form a sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs;

[0022] Raman detection: A Raman spectrometer was used to detect the Raman signal of the sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs, and a PA quantitative detection standard curve was constructed based on the relationship between PA concentration and Raman signal.

[0023] The target bacterial liquid for detection is preferably derived from drinking water, fruit, hospital environment, blood, pleural effusion or sputum, etc.

[0024] The steps of preparing SPION-PEI-AuNPs include:

[0025] The superparamagnetic iron oxide nanoparticles SPION-PEI are synthesized by a hydrothermal method using ferric chloride, anhydrous sodium acetate and polyethyleneimine PEI as main raw materials;

[0026] Using chloroauric acid and sodium citrate as main raw materials, the gold nanoparticles AuNPs are synthesized by a seed growth method to obtain the gold nanoparticles AuNPs solution;

[0027] The superparamagnetic iron oxide nanoparticles SPION-PEI are added to the gold nanoparticles AuNPs solution and ultrasonically stirred, so that the gold nanoparticles AuNPs therein are modified onto the superparamagnetic iron oxide nanoparticles SPION-PEI by electrostatic adsorption, magnetically separated, washed with water and then resuspended in water to obtain a SPION-PEI-AuNPs resuspension.

[0028] The steps of preparing the immunomagnetic beads include:

[0029] Reducing thiol groups: mixing Apt1-SH solution and TCEP buffer, and performing reduction reaction at room temperature to obtain Apt thiol reduction solution;

[0030] Coupling: adding the SPION-PEI-AuNPs resuspension to the Apt thiol reduction solution, incubating overnight, so that the aptamer Apt is modified to the surface of the SPION-PEI-AuNPs particles through chemical bonds, magnetically separated, and the immunomagnetic beads SPION-PEI-AuNPs-Apt are obtained; then the immunomagnetic beads SPION-PEI-AuNPs-Apt are blocked and magnetically separated in sequence, and resuspended in PBS buffer to obtain the immunomagnetic beads resuspension.

[0031] The steps of preparing AuNRs-MBA@Ag include:

[0032] Forming an AuNRs-MBA resuspension: firstly mixing a first MBA solution and a first TCEP buffer solution at room temperature, then adding the gold nanorods AuNRs for ultrasonic mixing, modifying the first Raman reporter molecule MBA on the gold nanorods AuNRs to form a first reaction solution; performing gradient centrifugation purification on the first reaction solution to obtain the AuNRs-MBA nanoparticles; resuspending the AuNRs-MBA nanoparticles in a CTAB buffer solution to form an AuNRs-MBA resuspension;

[0033] Forming AuNRs-MBA@Ag heavy suspension: firstly, hexadecyl trimethylammonium bromide (CTAB) and the AuNRs-MBA heavy suspension are mixed and dissolved in a water bath; then, under stirring, ascorbic acid (AA) solution and silver nitrate solution are added in sequence, and a silver ion reduction reaction is carried out at a pH value of 9.5-10.5 so that the generated silver is wrapped on the AuNRs-MBA nanoparticles to form the silver shell. After the reaction is completed, gradient centrifugation purification treatment is carried out, and the precipitate is resuspended in CTAB buffer to form AuNRs-MBA@Ag heavy suspension.

[0034] The steps of preparing the SERS tag include:

[0035] Modifying the reporter molecule: firstly, the second MBA solution and the second TCEP buffer solution are mixed and reacted at room temperature, and then the AuNRs-MBA@Ag resuspension solution is subjected to ultrasonic treatment to modify the second Raman reporter molecule MBA on the AuNRs-MBA@Ag nanoparticles to form a second reaction solution; the second reaction solution is subjected to gradient centrifugation purification treatment to obtain the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA; the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA is resuspended in a buffer solution of MES and CTAB to form a AuNRs-MBA@Ag-MBA resuspension solution;

[0036] One-step coupling of Van: first, the AuNRs-MBA@Ag-MBA resuspension is activated; then Van is added and shaken at room temperature to produce an amide condensation reaction, and vancomycin Van is modified on the surface of the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA to form a SERS tag AuNRs-MBA@Ag-MBA-Van; then the SERS tag AuNRs-MBA@Ag-MBA-Van is blocked and magnetically separated in turn, and resuspended in a buffer solution of PBS and CTAB to obtain a SERS tag resuspension.

[0037] The step of capturing the target bacteria PA comprises: shaking and incubating the immunomagnetic bead resuspension, magnesium-containing PBS buffer and the target detection bacterial solution at room temperature, wherein Apt can specifically recognize PA in the target detection bacterial solution and capture it, magnetic separation, and PBST buffer washing to obtain the composite SPION-PEI-AuNPs-Apt / PA.

[0038] The step of dual identification of the target bacteria PA includes: adding the SERS tag resuspension to the composite SPION-PEI-AuNPs-Apt / PA and incubating with shaking at room temperature, wherein Van specifically interacts with D-Ala-D-Ala in the PA cell wall peptidoglycan through its own hydrogen bond to form the sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs; magnetic separation, washing to obtain a sandwich structure precipitate, and resuspending the sandwich structure in water to obtain a sandwich structure resuspension.

[0039] The Raman detection step comprises: firstly dropping the sandwich structure resuspended liquid onto a glass slide and drying it, then detecting it with a Raman spectrometer, firstly dropping the sandwich structure resuspended liquid onto a glass slide and drying it, then detecting it with a Raman spectrometer, and using a machine learning algorithm to establish multiple regression models based on the relationship between the detected Raman signal and the concentration of Pseudomonas aeruginosa, with a complex correlation coefficient R 2 The performance of the regression model was evaluated by the root mean square error and after optimizing the model parameters, the optimal regression model was selected to construct the standard curve for PA quantitative detection.

[0040] Therefore, in the detection and identification reagent, detection platform and detection method of Pseudomonas aeruginosa provided by the present invention, the capture efficiency of the immunomagnetic SPION-PEI-AuNPs-Apt for Pseudomonas aeruginosa PA can reach 80.1%; the SERS label AuNRs-MBA@Ag-MBA-Van, the immunomagnetic SPION-PEI-AuNPs-Apt and the Pseudomonas aeruginosa PA can form a SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs sandwich structure, and qualitative and quantitative detection is performed by measuring the Raman spectrum of the sandwich structure; at the same time, combined with the machine learning regression model and its performance indicators, the detected Raman signal and PA concentration are used to construct the optimal PA quantitative detection standard curve.

[0041] After experiments, the highly sensitive dual-recognition tag-type SERS quantitative analysis system established by combining the nucleic acid aptamer Apt and the gold-silver core-shell double-layer reporter molecule tag structure provided by the present invention has the advantages of a wide detection range, a low detection limit, good specificity, high accuracy and a short detection time, thereby providing new methods and technologies for the prevention and clinical diagnosis of diseases related to Pseudomonas aeruginosa infection and food safety monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The principle diagram of quantitative detection of Pseudomonas aeruginosa based on tag-type SERS combined with machine learning provided by the present invention;

[0043] Figure 2Transmission electron microscopy images of AuNPs, SPION-PEI and SPION-PEI-AuNPs; Note: (A) Transmission electron microscopy image of AuNPs at 100nm scale; (B) Transmission electron microscopy image of SPION-PEI at 100nm scale; (C) Transmission electron microscopy image of SPION-PEI-AuNPs at 50nm scale;

[0044] Figure 3 EDS spectra of SPION-PEI-AuNPs, including: (A) electronic image of SPION-PEI-AuNPs; (B) Au atomic distribution map; (C) Fe atomic distribution map; (D) fusion image;

[0045] Figure 4 is the XPS spectrum of SPION-PEI-AuNPs;

[0046] Figure 5 is the absorption spectrum of immunomagnetic beads;

[0047] Figure 6 This is a characterization diagram of the Zeta potential of the immunomagnetic beads;

[0048] Figure 7 Magnetic characterization diagram of immunomagnetic beads, including (A) hysteresis loops of SPION-PEI, SPION-PEI-AuNPs and SPION-PEI-AuNPs-Apt; (B) magnetic separation characterization (a: before magnetic separation; b: after magnetic separation);

[0049] Figure 8 TEM characterization images of AuNRs and AuNRs-MBA@Ag, including (A) TEM image of AuNRs at 2nm scale; (B) TEM image of AuNRs-MBA@Ag at 50nm scale;

[0050] Fig. 9 EDS characterization images of AuNRs and AuNRs-MBA@Ag, where (AE) from left to right are the electron image, Au atomic distribution map, Ag atomic distribution map, fusion image and EDS point scan element ratio of AuNRs; (FJ) from left to right are the electron image, Au atomic distribution map, Ag atomic distribution map, fusion image and EDS point scan element ratio of AuNRs-MBA@Ag;

[0051] Fig.10 XPS spectra of AuNRs-MBA@Ag and AuNRs-MBA@Ag-MBA-Van;

[0052] Fig.11Raman spectra of SERS tags, including (A) Raman spectra of AuNRs and AuNRs-MBA; (B) Raman spectra of AuNRs@Ag and AuNRs@Ag-MBA;

[0053] Fig.12 is the UV-visible absorption spectrum of the SERS tag;

[0054] Fig.13 Zeta potential characterization diagram of SERS tag;

[0055] Fig.14 UV-visible absorption spectra of SERS tags after 1, 15 and 30 days of storage;

[0056] Fig.15 The results of the performance test of immunomagnetic beads binding to target bacteria are shown in Figure 1. (A) The capture efficiency of SPION-PEI-AuNPs-Apt and SPION-PEI-AuNPs is significantly different, *P < 0.01; (B) Raman spectra of SPION-PEI-AuNPs-Apt and SPION-PEI-AuNPs in the complete experiment;

[0057] Fig.16 This is the Raman spectrum of the double-identified target bacteria;

[0058] Fig.17 This is the result of aptamer optimization, among which Apt1 and Apt2 have statistically significant differences in binding to target bacteria, *P < 0.01; Apt1 and Apt3 have statistically significant differences in binding to target bacteria, *P < 0.01;

[0059] Fig.18 Schematic diagram of aptamer, antibody and vancomycin modified on magnetic beads;

[0060] Fig.19 This is the result of immunomagnetic bead optimization. The results of aptamer magnetic bead group and antibody magnetic bead group were statistically different, *P < 0.01; the results of vancomycin magnetic bead group and antibody magnetic bead group were statistically different, *P < 0.01;

[0061] Fig. 20 The results of the optimization of the dosage of immunomagnetic beads and the incubation time are shown in Figure 1, where (A) the dosage of immunomagnetic beads is optimized; (B) the incubation time of immunomagnetic beads is optimized;

[0062] Fig.21 The results of the optimization of the reporter molecule MBA modification step are shown in Figure 1; (A) Raman spectra of the reporter molecule MBA modified with or without TCEP reduction; (B) Histogram results of the reporter molecule MBA modified with or without TCEP reduction; *P<0.05;

[0063] Fig. 22 The results of AuNRs length optimization are shown in Figure 1, including (A) UV-visible absorption spectra of AuNRs generated under 0.1, 0.2, 0.3, 0.4, and 0.5 mL 0.01 mol / L AgNO3 conditions; (B) Raman signals of AuNRs-MBA with different lengths;

[0064] Fig.23 The graph shows the optimization results of the silver shell thickness, including (A) UV-visible absorption spectra of AuNRs@Ag generated under the conditions of 30, 50, 70, 80 and 100 μL 0.01 mol / L AgNO3; (B) Raman signals of AuNRs@Ag-MBA with different Ag shell thicknesses;

[0065] Fig.24 This is the SERS substrate optimization result diagram;

[0066] Fig.25 Optimization results of SERS tag incubation time (A) and dosage (B);

[0067] Fig.26 The standard curve (A) and the corresponding Raman spectrum (B) were established for the general linear regression model;

[0068] Fig. 27 Create a standard curve graph for a machine learning regression model;

[0069] Fig.28 This is a specificity survey diagram. DETAILED DESCRIPTION

[0070] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0071] Unless otherwise specified, the terms used in the present invention are commonly used in the field. The technical means used in the examples, such as preparation processes, test methods, etc., are all conventional means well known to those skilled in the art, and the reagents and products used are all commercially available. The sources and trade names of the reagents used, as well as the components thereof, are indicated when they first appear.

[0072] in, Nucleic acid names and base sequences used in the embodiments of the present invention As shown in Table 1.

[0073] Table 1 Nucleic acid names and base sequences

[0074]

[0075] in,Apt1 and Apt1-SH originate from Kubiczek D, Raber H, Bodenberger N, et al. TheDiversity of a Polyclonal FluCell-SELEX Library Outperforms IndividualAptamers as Emerging Diagnostic Tools for the Identification of CarbapenemResistant Pseudomonas aeruginosa[J].Chem-Eur J,2020,26(64):14536-14545.

[0076] Apt2 originated from Zhong Z, Gao R, Chen Q, et al. Dual-aptamers labeled polydopamine-polyethyleneimine copolymer dots assisted engineering afluorescence biosensor for sensitive detection of Pseudomonas aeruginosa infood samples [J]. Spectrochim Acta A Mol Biomol Spectrosc, 2020, 224: 117417.

[0077] Apt3 originated from Hu J, Fu K, Bohn P W. Whole-cell Pseudomonas aeruginosa localized surface plasmon resonance aptasensor[J]. Anal Chem, 2018, 90(3): 2326-2332.

[0078] The main solutions used in the following implementations are as follows:

[0079] (1) 0.01 mol / L phosphate buffer solution (PBS): Dissolve commercially available PBS powder in 2 L of Milli-Q water, mix well, and sterilize by high pressure before use.

[0080] (2) Binding buffer (PBSM): Weigh 0.0102 g MgCl2·6H2O and dissolve it in PBS, make up to 50 mL, and store at 4°C.

[0081] (3) 10 mmol / L Tris(2-carboxyethyl)phosphine (TCEP) buffer: Weigh 2.8665 g TCEP and dissolve it in Milli-Q water, make up to 1 L, and store at 4°C.

[0082] (4) 0.01 mol / L 4-Morpholineethanesulfonic acid (MES) buffer: Weigh 1.0 g of MES monohydrate and dissolve it in water to make up to 500 mL. Adjust the pH to 6 with potassium hydroxide.

[0083] (5) Mixed buffer of MES and hexadecyl trimethyl ammonium bromide (CTAB) (MES+CTAB buffer): Weigh 91.1 mg of CTAB and dissolve it in MES buffer, make up to 50 mL, and store at 4°C.

[0084] (6) Mixed buffer of PBS and CTAB (PBS+CTAB buffer): Weigh 91.1 mg of CTAB and dissolve it in PBS buffer, make up to 50 mL, and store at 4°C.

[0085] (7) EDC solution: Weigh 10 mg of EDC and dissolve it in 1 mL of MES buffer. Prepare the solution before use.

[0086] (8) Phosphate-Tween buffer (PBST, pH 7.4): Use a pipette to slowly draw out 25 μL of Tween-20, add it to 50 mL of PBS solution, mix thoroughly, and store at 4°C.

[0087] The bacterial culture used in the following examples :

[0088] Pseudomonas aeruginosa, Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), Klebsiella pneumoniae (KP), Acinetobacter baumannii (AB), and Haemophilus influenzae (HI) provided by the Laboratory Department of Henan Provincial People's Hospital were cultured in LB medium (Luria-Bertani, LB) at 37°C for 24 hours, then cryopreservation solution was added and stored in a -80°C refrigerator.

[0089] Bacterial recovery: Take the bacteria stored in a -80℃ refrigerator and transfer them on ice to a biosafety laboratory; heat the inoculation loop with an alcohol lamp for 30 seconds, and after the inoculation loop cools down, pick out an appropriate amount of bacteria and place it in LB medium, incubate at 37℃ for 24 hours, and set aside.

[0090] Instruments for characterization of immunomagnetic beads and SERS tags : Transmission electron microscope TEM, energy dispersive spectrometer EDS, X-ray electron spectrometer XPS, magnetic measurement system, and Zeta potential analyzer, UV-visible spectrophotometer.

[0091] in, Characterization method of magnetic bead coupling efficiency : The coupling of SPION-PEI-AuNPs with Apt was characterized using a UV-visible spectrophotometer, and the coupling efficiency of Apt with SPION-PEI-AuNPs was calculated according to the following formula (1).

[0092]

[0093] The coupling efficiency of antibody and SPION-PEI-AuNPs was investigated and characterized using Bradford kit.

[0094] Capture efficiency: The supernatant wash solution after magnetic separation of the corresponding magnetic beads was plate counted, the supernatant was spread on agar medium, and counted after incubation at 37°C overnight. The capture efficiency was calculated according to the following formula (2).

[0095]

[0096] Actual samples

[0097] Water samples: Tap water from the teaching building of Zhengzhou University and bottled drinking water from a drinking water company in Zhengzhou were collected. 10mL of water sample was enriched to 500μL by centrifugation and then detected by SERS platform. In addition, the standard culture method was used as the gold standard for comparison and verification. The detection process was referenced to GB 8538, as follows: The water sample was filtered with a filter membrane with a pore size of 0.45μm by the membrane method, and the filter membrane was moved to the CN agar medium and cultured in a 36±1℃ incubator for 48h. The growth and production of pyocyanin on CN agar selective medium were identified as positive; other non-blue and non-green colonies were further identified by fluorescence test, 42℃ growth test and ammonia production test, and the above three tests were all positive to define the colony as positive. Fluorescence test: The colonies grown on CN agar medium fluoresce under ultraviolet irradiation, that is, a positive result, otherwise it is negative. 42℃ growth test: Take an appropriate amount of bacterial solution and dilute it, then apply it on LB solid culture medium. If there is colony growth at 42℃ for 24-48h, it is a positive result, otherwise it is negative. Ammonia production test: Take a suspicious colony and inoculate it in a nitrate peptone water culture medium containing a small tube, and culture it at 37℃ for 24h. If there is gas produced in the small tube, it is a positive result, otherwise it is negative. If one of the above two conditions is met, the bacteria can be identified as PA.

[0098] Food samples: Fruit samples (including grapes, pears, and oranges) were purchased from local markets and local supermarkets in Zhengzhou, China. The samples were squeezed into a sterile Eppendorf tube to extract juice, and the juice was transferred to a disposable culture tube and incubated at 36±1℃ for 8h. 500μL of juice sample was used for SERS detection. In addition, the standard culture method was used as a gold standard for comparison and verification. After the juice sample was appropriately diluted, 100μL was evenly spread on CN agar selective culture medium and incubated in a 36±1℃ incubator for 48h. Identification of PA was the same as that of water samples.

[0099] Hospital environmental samples: Environmental samples (including sinks, mops, and faucets) were collected from a hospital in Zhengzhou. Liquid Amies transport medium was used for sampling and transported to the laboratory at 4°C. After the transport medium was shaken, an equal amount of LB medium was added for enrichment for 4 to 8 hours. 500 μL of the enriched environmental samples were used for SERS detection, standard culture method, and identification of PA and food samples.

[0100] Clinical samples: Blood, pleural effusion, and sputum were collected from the Department of Respiratory Medicine, the First Affiliated Hospital of Zhengzhou University and the Henan Provincial Chest Hospital. Patient inclusion criteria were as follows. Patients with new or persistent pulmonary infiltration accompanied by the following two points were eligible for inclusion: (1) body temperature less than 36 degrees or greater than 38 degrees; (2) white blood cell count greater than 12×10 9 / L or less than 4×10 9 / L; (3) Purulent respiratory secretions. Patients aged over 18 years with a simplified clinical cor pulmonale infection score (CPIS) greater than 6 points. Patients with severe immunosuppression (including those after chemotherapy and HIV infection, etc.) were excluded.

[0101] After blood samples are collected in blood collection tubes, they are transported to the laboratory at 4°C within 2 hours; pleural effusions are collected in 50mL sterile tubes and transported to the laboratory at 4°C within 2 hours. For SERS detection, the blood is first diluted 2 times, and 500μL is taken for detection; 500μL of pleural effusion samples are directly taken for detection. For the standard culture method, blood and pleural effusions are first enriched in blood culture bottles (36±1°C) for 2 days, and suspicious colonies are picked for further identification and counting. If there is no bacterial growth, the culture time is extended to 7 days, and if there is still no sterile growth, it is judged to be negative. The specific identification method is the same as that of water samples.

[0102] Sputum samples were stored in liquid Amies transport medium and transported to the laboratory at 4°C. The mixture of sputum and transport medium was dispensed into sterile Eppendorf tubes and mixed with freshly prepared 0.1% DTT at a volume ratio of 4:1. The samples were then left at room temperature for 30 min, with the liquid pipetted up and down every 15 min until the samples were completely liquefied. 500 μL of the sample was used for SERS detection; the standard culture method was the same as for water samples.

[0103] The software used for statistical analysis in each embodiment of the present invention is SPSS21.0. The comparison of the results in the optimization of experimental conditions was performed using the Wilcoxon rank sum test. The correlation analysis between the established method and the results of the culture method was performed using the Bland-Altman scatter plot. The test level α=0.05.

[0104] See also Figure 1 Similar to the immunoassay platform with antibody / antigen / antibody sandwich structure, the SERS platform for detecting Pseudomonas aeruginosa provided by the present invention adopts a sandwich structure formed by the interaction of Apt / target / Van. First, AuNPs are bound to SPION-PEI nanoparticles by electrostatic adsorption, and then the aptamer Apt is modified to the surface of SPION-PEI-AuNPs by Au-S bond to synthesize immunomagnetic beads SPION-PEI-AuNPs-Apt. Apt can specifically recognize PA and capture it. Due to the binding of Mg in the buffer solution PBSM, the aptamer Apt is bound to the surface of SPION-PEI-AuNPs. 2+The secondary structure of the aptamer Apt can be stabilized. When the immunomagnetic beads capture the target bacteria PA, PBSM is used as the reaction buffer solution. AuNRs were prepared by the seed growth method, and the Raman reporter MBA was modified on its surface. The silver shell was wrapped on the AuNRs-MBA by redox reaction, and the silver shell was labeled with MBA to form a gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA. Van was modified on its surface by amide condensation reaction to synthesize the SERS tag AuNRs-MBA@Ag-MBA-Van. Van on the SERS tag can bind bacteria through its own hydrogen bond and D-Ala-D-Ala in the cell wall peptidoglycan to form an Apt / target bacteria PA / Van sandwich structure. After magnetic separation, the unbound SERS tag was washed, the complex was resuspended in water, dropped on a glass slide and dried, and its Raman spectrum was measured using a Raman spectrometer. A highly sensitive dual-recognition tag-type SERS quantitative analysis method for detecting PA was established, and the general linear regression and machine learning regression model analysis were compared to compare R 2 The optimal model is selected by RMSE and then the standard curve is established. The technical solution to be protected by the present invention is further described in detail with specific embodiments below.

[0105] Embodiment 1

[0106] The embodiment of the present invention provides a detection and identification reagent for PA, including immunomagnetic beads SPION-PEI-AuNPs-Apt for identifying and capturing PA and a SERS label AuNRs-MBA@Ag-MBA-Van.

[0107] 1.1 Preparation and characterization of immunomagnetic beads SPION-PEI-AuNPs-Apt

[0108] 1.1.1 Preparation of SPION-PEI

[0109] Take a clean 250mL beaker and add 200mL (222.6g) of ethylene glycol; weigh 6.80g of ferric chloride hexahydrate with an electronic balance, add it to ethylene glycol, and use an ultrasonic cleaner to shake it to disperse it evenly; weigh 18.00g of anhydrous sodium acetate with an electronic balance, add it to the above mixed solution, and place it on a magnetic stirrer to stir evenly; weigh 7.5g of PEI with an electronic balance, add it to the above mixed solution, place it on a magnetic stirrer and heat and stir at 60°C for 20min; transfer the above mixed solution to a tetrafluoroethylene inner tank, place it in a reactor, and then place it in a forced air drying oven at 220°C for reaction for 2h. After the reaction is completed, naturally cool to room temperature to obtain SPION-PEI; transfer the reaction product to a clean 1000mL beaker, magnetically wash it with 200mL of anhydrous ethanol and 200mL of deionized water, respectively, repeat it three times, disperse it with 100mL of deionized water, and transfer the solution to a new bottle for standby use. Centrifuge at 1000r / min for 5min and save the supernatant for later use.

[0110] 1.1.2 Preparation of AuNPs

[0111] Add 40 μL 2.943 mol / L chloroauric acid and 100 mL Milli-Q water to a round-bottom flask. Heat the oil bath at 150°C, and immediately add 3.5 mL 11.41 mg / mL sodium citrate after the water boils. After reacting for 30 minutes, continue stirring at room temperature until cooled, and the reaction will produce a rose-red AuNPs solution. To enhance the dispersibility of AuNPs, add 200 μL K2CO3 (1.38 g / mL) to 50 mL AuNPs, and the pH range after mixing is 8-9.

[0112] 1.1.3 Preparation of SPION-PEI-AuNPs: 1.53 mL of SPION-PEI was added to 5.1 mL of AuNPs solution and ultrasonicated for 1 h. Magnetic separation was performed, and the mixture was washed with water 3 times and resuspended in 1 mL of water (5 mg / L) and stored at 4°C.

[0113] 1.1.4 Preparation of SPION-PEI-AuNPs-Apt

[0114] (1) Reduction of thiol groups: Mix 200 μL of 5 μmol / L Apt1-SH (take another portion of Apt1-SH with the same concentration, marked as pre) with TCEP buffer and reduce at room temperature for 45 min before use.

[0115] (2) Coupling: Add 1 mL of SPION-PEI-AuNPs to the above mixture and incubate at 37°C overnight. Magnetic separation, save the supernatant (labeled as post). Wash the magnetic beads twice with PBS, 1.2 mL each time, and save the supernatant (labeled as wash1 and wash2, respectively).

[0116] (3) Blocking: Add 100 μL of ethanolamine solution and block for 30 min.

[0117] (4) Magnetic separation, washing three times with PBS, resuspending in 500 μL PBS to obtain SPION-PEI-AuNPs-Apt, and storing at 4°C.

[0118] The morphologies of AuNPs, SPION-PEI and SPION-PEI-AuNPs were characterized by TEM. Figure 2 As shown in the figure, AuNPs are approximately spherical, with a diameter of about 17nm, uniform in size, and well dispersed. SPION-PEI is also approximately spherical, with a diameter of about 150nm, uniform in size, and well dispersed. Figure 2 C clearly shows that AuNPs are successfully bound to the SPION-PEI surface.

[0119] EDS was used to characterize the elemental composition and distribution of SPION-PEI-AuNPs. Figure 3 As shown. The synthesized SPION-PEI-AuNPs contain Fe and Au elements. The Fe element (red) is mainly distributed in the core area, that is, the SPION area, and the Au element (green) is evenly distributed around the SPION. The above results further illustrate that AuNPs are bound to the SPION-PEI surface.

[0120] XPS was used to characterize the elemental composition and content of SPION-PEI-AuNPs. Figure 4 and as shown in Table 2. The C, N, O, and Fe elements are derived from SPION-PEI. The Au element peak indicates that AuNPs are successfully coupled to the magnetic beads.

[0121] Table 2 Relative content of each element in SPION-PEI-AuNPs

[0122]

[0123] The absorption spectra of SPION-PEI-AuNPs and SPION-PEI-AuNPs-Apt were measured in the range of 400-1100 nm. Figure 5 .Depend on Figure 5 It can be seen that SPION-PEI-AuNPs has a characteristic absorption peak at 704nm. After coupling with the aptamer Apt, the absorption peak blue-shifted to 656nm, indicating that Apt and SPION-PEI-AuNPs were successfully coupled.

[0124] Using the characteristic absorption peak of nucleic acid at 260 nm, the absorbance of pre, post, wash1 and wash2 at 260 nm was measured respectively. The binding efficiency in Table 3 was calculated by the formula, indicating that a considerable amount of Apt was successfully coupled to the surface of SPION-PEI-AuNPs.

[0125] Table 3 Binding efficiency of Apt and SPION-PEI-AuNPs

[0126] Number of experiments Pre post Wash1 Wash2 Binding efficiency (%) 1 1.8 1.3 0.001 0.003 27.8 2 1.6 1.2 0.000 0.006 25.0

[0127] To characterize whether AuNPs and Apt were successfully coupled to SPION-PEI, the Zeta potential of AuNPs, SPION-PEI, SPION-PEI-AuNPs, and SPION-PEI-AuNPs-Apt was measured. Figure 6 As shown. The Zeta potentials of AuNPs, SPION-PEI, SPION-PEI-AuNPs, and SPION-PEI-AuNPs-Apt were -18mV, 32mV, 14mV, and -30mV, respectively. After the negatively charged AuNPs and Apt were coupled to the positively charged SPION-PEI, the potential of the magnetic beads shifted to the negative direction, indicating that AuNPs and Apt were successfully bound to the SPION-PEI surface.

[0128] In order to characterize whether the coupling of AuNPs and aptamers would affect the magnetic properties of magnetic beads, the magnetic properties of SPION-PEI, SPION-PEI-AuNPs and SPION-PEI-AuNPs-Apt were characterized by a magnetic measurement system. Figure 7 As shown in A, the saturation magnetization intensities of SPION-PEI, SPION-PEI-AuNPs and SPION-PEI-AuNPs-Apt are 71.9emu / g, 53.3emu / g and 52.9emu / g, respectively, indicating that the magnetism of the magnetic beads is reduced after coupling with AuNPs, and the coupling with the aptamer has a weak effect on the magnetism, but the magnetism of SPION-PEI-AuNPs-Apt is still good. In addition, their hysteresis loops all pass through the origin, indicating that SPION-PEI, SPION-PEI-AuNPs and SPION-PEI-AuNPs-Apt have no remanence and coercive force at 300K and have superparamagnetism. Figure 7 B. It can be directly observed that when there is no external magnetic field, the immunomagnetic beads are uniformly brown-black in the solution (a); under the action of the external magnetic field, the immunomagnetic beads aggregate rapidly in a short period of time, and the supernatant is clear and transparent (b). This shows that the immunomagnetic beads have strong magnetic separation performance and can be used for subsequent experiments.

[0129] 1.2 Preparation and characterization of SERS label AuNRs-MBA@Ag-MBA-Van

[0130] 1.2.1 Preparation of AuNRs

[0131] AuNRs were synthesized by the seed growth method. The specific steps are as follows:

[0132] (1) Seed solution: Add 2.5 mL of 0.5 mmol / L chloroauric acid to 2.5 mL of 0.2 mol / L CTAB solution, place in a light-proof 29°C water bath, and stir at 500 r / min to mix evenly. Then, quickly add 0.3 mL of 0.01 mol / L NaBH4 prepared with ice water to the above reaction system. After stirring at 1000 r / min for 1 min, the solution turns brown-yellow and is allowed to stand at 29°C for 1 h.

[0133] (2) Growth solution: Add 20 mL of 1 mmol / L chloroauric acid to 20 mL of 0.2 mol / L CTAB, keep in a light-proof 29°C water bath, and stir at 200 r / min to mix evenly; add 0.2 mL of 0.01 mol / L AgNO3, 0.38 mL of 2 mol / L HCl, and 0.33 mL of 0.1 mol / L ascorbic acid (AA), and stir at 200 r / min to mix evenly. The growth solution changes from yellow to colorless.

[0134] (3) The seed solution was stirred at 400 r / min before use; 0.13 mL of the seed solution was added to the growth solution and kept in a light-proof 29°C water bath overnight. (4) After the mixture changed from colorless to purple, it was centrifuged three times at 10,000 r / min for 14 min to purify, the supernatant was removed, and the precipitate was resuspended in 40 mL of 5 mmol / L CTAB.

[0135] 1.2.2 Preparation of AuNRs-MBA

[0136] (1) 1 mL of 10 mmol / L 4-Mercaptobenzoic acid (MBA) solution and 1 mL of 10 mmol / L TCEP buffer were mixed and reacted at room temperature for 40 min.

[0137] (2) Take 10 μL of the above mixture and add it to 10 mL of AuNRs. Ultrasonicate at 20 °C for 4 h.

[0138] (3) Gradient centrifugation purification: The reaction solution was centrifuged three times at 9000 r / min for 14 min, and the precipitate was retained; the supernatant was centrifuged three times at 10000 r / min for 14 min, and the precipitate was retained. Finally, the combined precipitates were resuspended in 10 mL of 5 mmol / L CTAB solution.

[0139] 1.2.3 Preparation of AuNRs-MBA@Ag

[0140] Take 72.8mg CTAB and 6mL AuNRs-MBA and add them to a brown conical flask in turn, dissolve them in a water bath at 29℃. Stir at 9℃, 700r / min, add 54μL 0.1mol / L AA, 70μL 0.01mol / L AgNO3, 240μL 0.1mol / LNaOH in turn, the pH value of the reaction system is 10 to trigger silver ion reduction, the reaction ends after 2min, and the solution changes from purple to blue. Purify by gradient centrifugation, the steps are shown in 1.2.2.

[0141] 1.2.4 Preparation of AuNRs-MBA@Ag-MBA-Van

[0142] (1) Modification of reporter molecules: First, 1 mL of 10 mmol / L MBA solution and 1 mL of 10 mmol / L TCEP buffer were mixed and reacted at room temperature for 40 min; 60 μL of the above mixture was added to 6 mL of AuNRs-MBA@Ag and ultrasonicated at 20°C for 4 h. Gradient centrifugation purification: The reaction solution was centrifuged three times at 7000 r / min for 14 min, and the precipitate was retained; the supernatant was centrifuged three times at 9000 r / min for 14 min, and the precipitate was retained. Finally, all the precipitates (AuNRs-MBA@Ag-MBA) were resuspended in 2 mL of MES+CTAB buffer.

[0143] (2) One-step coupling of Van.

[0144] ① Activation: First, add 50 μL of EDC (10 mg / mL) solution to 2 mL of AuNRs-MBA@Ag-MBA resuspended in MES+CTAB buffer and shake for 30 min.

[0145] ② Add Van: Add 1.5 mg Van, shake and react at room temperature overnight. Centrifuge at 5000 r / min for 14 min, and wash three times with 2.05 mL MES+CTAB buffer.

[0146] ③ Blocking: Add 100 μL ethanolamine (5%) to block for 1 h.

[0147] ④ Gradient centrifugation purification: The reaction solution was centrifuged three times at 5000rpm for 14min, and the precipitate was retained; the first supernatant was centrifuged three times at 7000r / min for 14min, and the precipitate was retained; the second supernatant was centrifuged three times at 9000r / min for 14min, and the precipitate was retained. Finally, all the precipitates (AuNRs-MBA@Ag-MBA-Van) were resuspended in 500μL PBS+CTAB buffer and stored at 4℃.

[0148] The morphology of AuNRs and AuNRs-MBA@Ag was characterized by TEM. Figure 8 As shown in the figure, the width of AuNRs is 11nm and the length is 29nm; the morphology of AuNRs-MBA@Ag is roughly the same as that of AuNRs.

[0149] The elemental composition and content of AuNRs and AuNRs-MBA@Ag were characterized by EDS. Fig. 9 As shown in the figure, AuNRs only contain gold (purple), while AuNRs-MBA@Ag contains gold (green) and silver (red), with a content ratio of about 86:13, and the silver element is evenly coated on the surface of AuNRs. This indicates that the silver shell is successfully coated on AuNRs.

[0150] To characterize the successful coupling of Van to the SERS tag and further verify the coating of the silver shell, XPS was used to analyze the elemental composition and content of AuNRs-MBA@Ag and AuNRs-MBA@Ag-MBA-Van. Fig.10 As shown in Table 4, the silver element in AuNRs-MBA@Ag indicates that the silver shell is successfully coated. The content of nitrogen and oxygen elements increases after coupling with Van, which may be due to the presence of N and O elements in Van. The results suggest that Van is successfully coupled to the SERS tag.

[0151] Table 4 Relative contents of elements in AuNRs-MBA@Ag and AuNRs-MBA@Ag-MBA-Van

[0152]

[0153]

[0154] Raman spectroscopy was used to characterize whether the reporter molecule MBA was modified on the surface of AuNRs and AuNRs@Ag, and the Raman spectra of AuNRs, AuNRs-MBA, AuNRs@Ag and AuNRs@Ag-MBA were measured respectively. Fig.11 As shown, when there is only substrate AuNRs@Ag or AuNRs, the Raman spectrometer detects no Raman signal; when MBA is modified on the surface of AuNRs and AuNRs@Ag, the Raman spectrum shows the characteristic spectrum of MBA, and the maximum signal characteristic peak is obtained at 1587cm-1. This result suggests that MBA can be modified on the surface of AuNRs and AuNRs@Ag, so the gold-silver core-shell double-layer Raman reporter molecular complex structure is successfully synthesized.

[0155] The UV-visible absorption spectra of AuNRs, AuNRs-MBA, AuNRs-MBA@Ag, AuNRs-MBA@Ag-MBA and AuNRs-MBA@Ag-MBA-Van were measured in the range of 400-800 nm. Fig.12 As shown. Gold nanorods have a transverse plasmon resonance absorption peak and a longitudinal plasmon resonance absorption peak. The transverse peak is located near 520nm, and the longitudinal peak is located in the range of 550-1550nm. After MBA was modified on AuNRs, its longitudinal peak red-shifted from 680nm to 683nm, indicating that MBA was successfully modified on the surface of AuNRs. The silver shell coated on AuNRs-MBA caused its longitudinal peak to blue-shift from 683nm to 634nm, indicating that the silver shell was successfully coated on AuNRs-MBA. The modification of MBA by AuNRs-MBA@Ag caused its longitudinal peak to red-shift from 634nm to 653nm, indicating that the second layer of MBA was successfully modified on the surface of AuNRs-MBA@Ag. After AuNRs-MBA@Ag-MBA was coupled with Van, its longitudinal peak red-shifted from 653nm to 661nm, indicating that Van was coupled to AuNRs-MBA@Ag-MBA, and the target SERS tag was successfully synthesized.

[0156] The zeta potentials of AuNRs, AuNRs-MBA, AuNRs-MBA@Ag, AuNRs-MBA@Ag-MBA and AuNRs-MBA@Ag-MBA-Van were measured respectively. Fig.13 As shown, the Zeta potentials of AuNRs, AuNRs-MBA, AuNRs-MBA@Ag, AuNRs-MBA@Ag-MBA and AuNRs-MBA@Ag-MBA-Van are 26mV, 9mV, 32mV, 9mV and 27mV respectively. Modification of MBA causes the potential of the material to shift to negative charge, which is attributed to the negative charge of the carboxyl group on MBA. Coupling Van causes the potential of the material to shift to positive charge, which is attributed to the positive charge of the amino group on Van. The results suggest that MBA and Van are successfully modified on the SERS tag.

[0157] The UV-visible absorption spectra of the SERS tags were measured after being stored at 4°C for 1, 15, and 30 days. Fig.14 As time goes by, the width of the vertical peak of the SERS tag becomes slightly larger. However, the overall position of its horizontal and vertical peaks shifts slightly, and the height ratio of the vertical and horizontal peaks does not change much. The results suggest that this SERS tag has good stability.

[0158] Embodiment 2

[0159] The embodiment of the present invention provides a PA detection platform, including the PA detection and identification reagent and Raman spectrometer provided in Embodiment 1, wherein the immunomagnetic beads SPION-PEI-AuNPs-Apt can specifically identify PA and capture it to form a complex SPION-PEI-AuNPs-Apt / PA; Van in the SERS tag AuNRs-MBA@Ag-MBA-Van specifically interacts with D-Ala-D-Ala in the PA cell wall peptidoglycan in the complex SPION-PEI-AuNPs-Apt / PA through its own hydrogen bonds to form a sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs; the Raman spectrometer is used to detect the Raman spectrum of the sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs, and a PA quantitative detection standard curve is constructed according to the relationship between PA concentration and Raman signal.

[0160] 2.1 Performance evaluation of immunomagnetic beads SPION-PEI-AuNPs-Apt combined with target bacteria PA

[0161] The overnight cultured PA at 37°C was centrifuged at 4500 r / min for 5 min and resuspended in PBS. The bacterial concentration was determined by plate counting and the bacteria were diluted to 10 4 CFU / mL. Two groups of magnetic beads were prepared from the same batch: SPION-PEI-AuNPs and SPION-PEI-AuNPs-Apt. The capture efficiency and Raman spectroscopy detection were calculated according to the following steps.

[0162] (1) Add 15 μL of SPION-PEI-AuNPs and SPION-PEI-AuNPs-Apt to 500 μL of 10 4 The cells were incubated in a 40-min shaking bacterial solution at room temperature, separated by magnetic separation, and washed three times with PBST to remove uncaptured bacteria.

[0163] (2) Add 30 μL SERS tags and 150 μL PBS to the two groups of precipitates after magnetic separation, and continue to incubate at room temperature with shaking for 20 min. After incubation, magnetic separation was performed and PBST was washed three times to remove unbound SERS tags.

[0164] (3) Finally, the complex was resuspended in 20 μL of water, dropped onto a glass slide and dried, and two sets of SERS spectra were measured. Fig.15 shown.

[0165] Fig.15A shows that the capture efficiency of SPION-PEI-AuNPs-Apt for the target bacteria PA is 80.1±6.3%; the capture efficiency of SPION-PEI-AuNPs for the target bacteria PA is 18.3±2.3%, which may be due to the non-specific adsorption of SPION-PEI-AuNPs to the target bacteria PA. This result suggests that the high capture efficiency of SPION-PEI-AuNPs-Apt is due to Apt. When SPION-PEI-AuNPs was used to capture PA for subsequent experiments, the Raman signal intensity was weak; when SPION-PEI-AuNPs-Apt was used to capture PA for subsequent experiments, the Raman signal was significantly stronger than the control group, such as Fig.15 As shown in B. This further suggests that the immunomagnetic beads modified with Apt can effectively capture PA.

[0166] 2.2 Feasibility study of dual recognition of target bacteria PA

[0167] 15 μL SPION-PEI-AuNPs-Apt and 50 μL PBSM were added to 500 μL PBS buffer and 500 μL 10 4 CFU / mL bacterial solution, incubate at room temperature with shaking for 40 minutes, and after incubation, separate by magnetic separation and wash three times with PBST to remove uncaptured bacteria. The remaining steps are the same as (3) and (4) in the above "2.1". Fig.16 shown.

[0168] When there was no bacterial PA, the Raman signal intensity was weak; when PA was present, the Raman signal was significantly stronger than that of the control group. Fig.16 This indicates that after the immunomagnetic beads SPION-PEI-AuNPs-Apt capture PA, the SERS tag AuNRs-MBA@Ag-MBA-Van can still effectively bind to bacterial PA to form a SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs complex. Therefore, the dual recognition strategy is feasible.

[0169] The PA detection platform provided in this embodiment also includes a Raman signal data analysis module, which has a built-in machine learning algorithm. The machine learning algorithm establishes multiple regression models to process the Raman signal data. 2 The performance of the regression model was evaluated by the root mean square error, and after optimizing the parameters of each model, the optimal regression model was selected to construct the PA quantitative detection standard curve. The regression analysis models include XGBR, SVR, BRR, ANNR, PR and ENR models.

[0170] Embodiment 3

[0171] This embodiment provides a method for detecting PA using the detection platform provided in Embodiment 2, comprising the following steps:

[0172] Preparation of SPION-PEI-AuNPs: uniformly mixing superparamagnetic iron oxide nanoparticles SPION-PEI and gold nanoparticles AuNPs solution to form SPION-PEI-AuNPs particles;

[0173] Preparation of immunomagnetic beads: firstly subjecting Apt1-SH solution and tris(2-carboxyethyl)phosphine (TCEP) buffer to a room temperature reduction reaction, and then mixing with the SPION-PEI-AuNPs particles to a coupling reaction, thereby obtaining immunomagnetic beads SPION-PEI-AuNPs-Apt;

[0174] Preparation of AuNRs-MBA@Ag: silver nitrate and AuNRs-MBA particles are uniformly mixed, and the silver nitrate undergoes a redox reaction to coat the AuNRs-MBA nanoparticles with a silver shell to form AuNRs-MBA@Ag nanoparticles; wherein the AuNRs-MBA nanoparticles are mainly prepared by modifying the first Raman reporter molecule MBA on the gold nanorods AuNRs;

[0175] Preparation of SERS tag: firstly, the AuNRs-MBA@Ag nanoparticles and MBA solution are mixed and reacted, and the second Raman reporter molecule MBA is modified on the AuNRs-MBA@Ag nanoparticles to form a gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA; then, the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA and Van are subjected to room temperature oscillation reaction to form a SERS tag AuNRs-MBA@Ag-MBA-Van;

[0176] Capturing the target bacteria PA: the immunomagnetic beads SPION-PEI-AuNPs-Apt, the magnesium-containing solution and the target detection bacterial solution are shaken and incubated at room temperature, so that the immunomagnetic beads SPION-PEI-AuNPs-Apt can specifically recognize the PA therein and capture it, forming a complex SPION-PEI-AuNPs-Apt / PA, wherein the target detection bacterial solution contains PA;

[0177] Dual recognition of target bacteria PA: The composite SPION-PEI-AuNPs-Apt / PA and the SERS tag AuNRs-MBA@Ag-MBA-Van were incubated with shaking at room temperature to form a sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs;

[0178] Raman detection: A Raman spectrometer was used to detect the Raman signal of the sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@MBA-AuNRs, and a PA quantitative detection standard curve was constructed based on the relationship between PA concentration and Raman signal.

[0179] The main factors affecting the sensitivity and accuracy of PA quantitative detection in the above detection method provided in this embodiment include immunomagnetic beads SPION-PEI-AuNPs-Apt, SERS tags AuNRs-MBA@Ag-MBA-Van, regression model, etc. Each influencing factor is further described below.

[0180] 3.1 Aptamer optimization

[0181] The affinity of Apt and PA on the immunomagnetic beads SPION-PEI-AuNPs-Apt will directly affect its capture efficiency of the target bacterial PA, so it is very important to select the aptamer with the best affinity for the target bacterial PA. Three aptamers with different sequences modified by different fluorescent groups, Apt1, Apt2, and Apt3, were incubated with bacterial PA, and the binding of the aptamer to the bacterial PA was determined based on the change in fluorescence intensity. The specific steps are as follows:

[0182] (1) PA cultured overnight at 37°C was centrifuged at 4500 r / min for 5 min and resuspended in PBS. The bacterial concentration was determined by plate counting and the bacteria were diluted to 10 5 CFU / mL.

[0183] (2) 100 μL of 1 μmol / L Apt1, Apt2, and Apt3 labeled with fluorescent groups (with the initial fluorescence intensity marked as Pre) were added to 100 μL of bacteria, respectively, and the mixture was centrifuged at 4500 r / min for 5 min. The fluorescence intensity of the supernatant was measured by a fluorescence spectrophotometer and recorded as Post. The excitation wavelengths of Cy5, FAM, and TAMRA were 649 nm, 494 nm, and 560 nm, respectively, and the emission wavelengths were 670 nm, 520 nm, and 584 nm, respectively.

[0184] (3) The affinity of the aptamer was measured by the change in fluorescence intensity (Pre-Post) / Pre. The results are as follows Fig.17 shown. Fig.17 The results showed that Apt1 had the strongest affinity to the target bacteria PA compared with Apt2 and Apt3. Therefore, Apt1 was used in subsequent experiments.

[0185] 3.2 Optimization of immunomagnetic beads

[0186] In addition to aptamers, Anti-Pseudomonas aeruginosa antibody [B11] (from Abcam) and vancomycin are also commonly used to capture bacteria. The aptamer, antibody, and vancomycin were coupled to SPION-PEI-AuNPs to prepare three different capture magnetic beads. See the schematic diagram. Fig.18 By comparing the capture efficiency of the three magnetic beads for PA, the optimal immunomagnetic beads were selected. The preparation process of the aptamer magnetic beads SPION-PEI-AuNPs-Apt is shown in the above-mentioned Example 1 "1.1" section. The preparation process of the antibody magnetic beads and vancomycin magnetic beads is as follows:

[0187] (1) The preparation method of SPION-PEI-AuNPs is consistent with the preparation method of “SPION-PEI-AuNPs” in the aptamer magnetic beads SPION-PEI-AuNPs-Apt.

[0188] (2) 5 mL of 20 μmol / L 11-mercaptoundecanoic acid and 5 mL of 10 mmol / L TCEP buffer were mixed and reacted at room temperature for 40 min; 1 mL of SPION-PEI-AuNPs was added to the above mixture, ultrasonicated for 2 h, magnetically separated, and washed once with anhydrous ethanol; 5 mL of MES buffer was added; 10 mg / mL EDC solution and 20 mg / mL sulfo-NHS solution were prepared using MES buffer; 200 μL of sulfo-NHS and 200 μL of EDC were added to the reaction system in sequence, and shaken at room temperature for 30 min.

[0189] (3) Magnetic separation: the preparation steps of the two magnetic beads are as follows: antibody magnetic beads: add 8 μL antibody stock solution and 192 μL PBS antibody, shake and react at room temperature for 2 h; vancomycin magnetic beads: add 500 μL 5 mg / mL Van, the stock solution is marked as Pre, shake and react at room temperature for 2 h.

[0190] (4) Blocking: Add 100 μL ethanolamine for 30 min, separate by magnetic separation, and label the supernatant as Post. Wash twice with water, label them as Wash1 and Wash2, resuspend in PBS, and store at 4°C for later use.

[0191] Prepare 3 sets of 500 μL 10 4 CFU / mL PA bacterial suspension, 5 μL of the above three magnetic beads and 50 μL PBSM were added respectively, and the mixture was shaken at room temperature for 20 min. After magnetic separation, the capture efficiency of each group was calculated.

[0192] Taking advantage of the characteristic absorption peak of Van solution at 280nm, the absorbance of pre, post, wash1 and wash2 at 280nm was measured respectively. The binding efficiency in Table 5 was calculated by the formula, indicating that a considerable amount of Van was successfully coupled to SPION-PEI-AuNPs. Taking advantage of the property that the maximum absorption peak of Coomassie Brilliant Blue G-250 dye changes from 465nm to 595nm after binding to protein, the coupling effect of antibody and SPION-PEI-AuNPs was evaluated based on the absorbance of pre, post, wash1 and wash2 at 595nm before and after coupling. As shown in Table 6, the OD of the pre group 595(pre) It was significantly higher than the solvent blank, while the signal intensities of post, wash1 and wash2 were comparable to those of the solvent blank, indicating that almost all antibodies were coupled to the surface of SPION-PEI-AuNPs.

[0193] Table 5 Binding efficiency of Van and SPION-PEI-AuNPs

[0194] Number of experiments Pre post Wash1 Wash2 Binding efficiency (%) 1 2.8 2.1 0.00 0.03 25.0 2 3.4 2.4 0.08 0.00 29.4 3 2.4 1.2 0.02 0.05 41.7

[0195] Table 6 Coupling characterization of antibodies and SPION-PEI-AuNPs

[0196] <![CDATA[OD 595(pre) ]]> <![CDATA[OD 595(post) ]]> <![CDATA[OD 595(wash1) ]]> <![CDATA[OD 595(wash2) ]]> <![CDATA[OD 595(溶剂空白) ]]> 0.5453 0.516 0.5134 0.5142 0.5139

[0197] Fig.19 The results show that the capture efficiency of antibody magnetic beads is significantly lower than that of aptamer magnetic beads and vancomycin magnetic beads. Therefore, Apt and Van were selected in the subsequent experiments, and the two affinity molecules Apt and Van were used in the dual recognition SERS platform. In view of the consistency of Apt and Van in capture efficiency and the good specificity of Apt, in the subsequent experiments, Apt was combined with SPION-PEI-AuNPs to synthesize immunomagnetic beads for the separation and enrichment of PA, and Van was combined with AuNRs-MBA@Ag-MBA to synthesize SERS tags, which were combined with PA for dual recognition detection.

[0198] 3.3 Optimization of immunomagnetic bead dosage and incubation time

[0199] The amount of immunomagnetic beads and the incubation time will directly affect the capture effect of the target PA. Insufficient amount will reduce the capture efficiency, while excessive amount will lead to poor dispersion. Therefore, it is necessary to optimize it. The results are as follows Fig. 20 First, PA was captured using magnetic beads of different volumes, and the optimal dosage was obtained and applied to the incubation time experiment.

[0200] The steps for optimizing the amount of immunomagnetic beads include: adding 500 μL of 10 450 μL PBSM was added to the CFU / mL PA bacterial suspension, and then 10, 15, 20, 25, and 30 μL of SPION-PEI-AuNPs-Apt were added, respectively, and incubated with shaking for 30 minutes; after magnetic separation, the capture efficiency of each group was calculated.

[0201] The steps for optimizing the incubation time of immunomagnetic beads include: 5 groups of 500 μL 10 4 50 μL PBSM was added to the CFU / mL PA bacterial suspension, and the optimal amount of magnetic beads was added, and the mixture was shaken and incubated for 20, 30, 40, 50, and 60 min, respectively; after magnetic separation, the capture efficiency of each group was calculated.

[0202] Fig. 20 It shows that when the amount of magnetic beads increases from 10μL to 15μL, the capture efficiency increases; however, when the amount of magnetic beads continues to increase, the capture efficiency does not increase significantly. Considering the cost-effectiveness, 15μL magnetic beads were selected for subsequent experiments. When the incubation time increases from 20min to 40min, the capture efficiency increases; however, if the incubation time is further extended, the capture efficiency even decreases, which may be due to the decrease in PA activity over a long period of time, resulting in the shedding of immunomagnetic beads. In summary, the subsequent experimental conditions are set as: 15μL of magnetic beads and 40min of incubation time.

[0203] 3.4 Optimization of reporter molecule modification steps

[0204] The amount of Raman reporter molecules bound will affect the SERS tag signal intensity, so the modification steps were optimized. By changing the reaction solution when modifying MBA, the reaction conditions with better modification effect of MBA were selected. Fig.21 shown.

[0205] (1) Grouping: ① 1mL 10mmol / L MBA solution and 1mL 10mmol / L TCEP buffer were mixed and reacted at room temperature for 40min; ② 1mL 10mmol / L MBA solution and 1mL anhydrous ethanol were mixed and reacted at room temperature for 40min. (2) Take 10μL of the above mixed solution and add it to 10mL AuNRs respectively, and ultrasonicate at 20℃ for 4h. (3) Gradient centrifugation purification: centrifuge three times at 9000r / min and 14min, and keep the precipitate; centrifuge the supernatant three times at 10000r / min and 14min, and keep the precipitate. Finally, the combined precipitate was resuspended in 10mL 5mmol / LCTAB solution. (4) Take 2.5μL of the above reaction solution and drop it on a glass slide to dry, and measure and compare the Raman spectra.

[0206] Fig.21The results show that the Raman signal is significantly enhanced when TCEP is added before modifying MBA, which indicates that more MBA is modified to SERS tag. Therefore, adding TCEP reduction step can effectively enhance the performance of SERS tag.

[0207] 3.5 Optimization of AuNRs SERS substrate material

[0208] 3.5.1 Length optimization of AuNRs

[0209] The length of AuNRs can be adjusted by changing the amount of AgNO3. The steps for preparing AuNRs of different morphologies are as follows:

[0210] (1) Seed solution: Add 2.5 mL of 0.5 mmol / L chloroauric acid to 2.5 mL of 0.2 mol / L CTAB solution, place in a light-proof 29°C water bath, and stir at 500 r / min to mix. Then quickly add 0.3 mL of 0.01 mol / L NaBH4 prepared with ice water to the reaction system, stir at 1000 r / min for 1 min, and the solution turns brown. Let stand at 29°C for 1 h.

[0211] (2) Prepare 5 groups of growth solutions (except for the different amounts of AgNO3, the other steps are the same): add 20 mL of 1 mmol / L chloroauric acid to 20 mL of 0.2 mol / L CTAB, keep in a light-proof 29°C water bath, and stir at 200 r / min to mix evenly; add 0.1, 0.2, 0.3, 0.4, and 0.5 mL of 0.01 mol / L AgNO3, 0.38 mL of 2 mol / L HCl, and 0.33 mL of 0.1 mol / L AA respectively; stir at 200 r / min to mix until the growth solution changes from yellow to colorless.

[0212] (3) Stir the seed solution at 400 r / min before use; add 0.13 mL of the seed solution to the growth solution and place in a water bath at 29°C overnight in the dark. (4) The mixture changes from colorless to different colors. Centrifuge for purification three times at 10,000 r / min for 14 min, remove the supernatant, and resuspend the precipitate in 40 mL of 5 mmol / L CTAB.

[0213] (5) Five groups of AuNRs modified with different forms of MBA: 1 mL of 10 mmol / L MBA solution and 1 mL of 10 mmol / L TCEP buffer were mixed and reacted at room temperature for 40 min; 10 μL of the above mixture was added to 10 mL of AuNRs and ultrasonicated at 20°C for 4 h. Gradient centrifugation purification: centrifuged three times at 9000 r / min for 14 min, and the precipitate was retained; the supernatant was centrifuged three times at 10000 r / min for 14 min, and the precipitate was retained. Finally, the combined precipitates were resuspended in 10 mL of 5 mmol / L CTAB solution.

[0214] (6) Take 2.5 μL of each solution and drop it onto a glass slide to dry, then measure and compare the Raman spectra.

[0215] The length of AuNRs was adjusted by changing the amount of AgNO3, and its UV-visible absorption spectrum was measured for characterization. Fig. 22 A. With the increase of AgNO3 dosage, the vertical peak of AuNRs red-shifts and becomes sharper. Five groups of AuNRs were modified with MBA and their Raman spectra were measured. The characteristic peak at 1587 cm -1 The signal intensity was used to evaluate the SERS performance. Fig. 22 B, The SERS performance of AuNRs-MBA generated by 0.2mL AgNO3 group is the best.

[0216] 3.5.2 Silver shell thickness optimization

[0217] In order to optimize the effect of the thickness of the silver shell coated on the AuNRs on the Raman signal, the content of AgNO3 in the reaction system during silver ion reduction was also changed, resulting in different silver shell thicknesses. Fig.23 The specific steps are as follows:

[0218] (1) Synthesize AuNRs of optimal length by referring to the above steps.

[0219] (2) Preparation of 5 groups of AuNRs@Ag (except for the amount of AgNO3, the rest of the steps are the same): 72.8 mg CTAB and 6 mL AuNRs were added to a brown conical flask in sequence and dissolved in a 29°C water bath; 54 μL 0.1 mol / L AA, 30, 50, 70, 80, 100 μL 0.01 mol / L AgNO3, and 240 μL 0.1 mol / L NaOH were added in sequence at 29°C and 700 r / min with stirring; the pH value of the reaction system was 10 to induce silver ion reduction, and the reaction was completed after 2 min. Gradient centrifugation purification, the steps are shown in 3.5.1.

[0220] (3) The steps for modifying MBA with 5 different groups of AuNRs@Ag were the same as above.

[0221] (4) Take 2.5 μL of each solution and drop it onto a glass slide to dry, then measure and compare the Raman spectra.

[0222] Subsequent AuNRs were prepared under the reaction conditions of 0.2 mL AgNO3. To explore the effect of the thickness of the coated silver shell on the SERS performance of the substrate, five groups of AuNRs@Ag with different amounts of AgNO3 were prepared and their UV-visible absorption spectra were measured for characterization. The results are shown in Fig.23A. As the amount of AgNO3 increases, the vertical peak of AuNRs@Ag shifts to the blue. The Raman spectra of the five groups of AuNRs@Ag modified with MBA were measured. The characteristic peak at 1587 cm -1 The signal intensity was used to evaluate the SERS performance. Fig.23 B, AuNRs@Ag-MBA prepared with 80μL AgNO3 has the best SERS performance.

[0223] 3.5.3 Investigation of the performance of different SERS substrates

[0224] The performance of different SERS substrates varies greatly. Compared with the existing AuNPs and AuNRs as substrates, the SERS tag performance after AuNRs wrapped in Ag shell is compared. To ensure that the three different substrates have the same quality, AuNPs are diluted 2.35 times for use. The synthesis steps of AuNPs-MBA, AuNRs-MBA and AuNRs-MBA@Ag-MBA are shown in 1.2. Take 2.5μL of the three different SERS substrates and drop them on the glass slide, dry them and measure the Raman spectrum. The results are shown in Fig.24 shown. Fig.24 The results show that the Raman signal of AuNRs-MBA@Ag-MBA is the strongest, followed by AuNRs-MBA, and AuNPs-MBA is the weakest. The high Raman signal of AuNRs-MBA@Ag-MBA is due to its superior SERS performance, so it was selected as the SERS tag in the subsequent experiments.

[0225] 3.6 Optimization of SERS label dosage and incubation time

[0226] The amount of SERS label and the incubation time will affect the binding efficiency to PA, which will directly affect the measured Raman signal. Therefore, they are optimized. The results are as follows: Fig.25 shown.

[0227] The steps for optimizing the amount of SERS tag include: adding 15 μL SPION-PEI-AuNPs-Apt and 50 μL PBSM to 500 μL 10 4 CFU / mL PA bacterial suspension, incubate at room temperature with shaking for 40 minutes; after magnetic separation, wash three times with PBST; add 150μL PBS, 10, 20, 30, 40, 50μL SERS label, incubate at room temperature with shaking for 5 minutes; after magnetic separation and water washing three times, resuspend in 20μL water; drop onto a glass slide, measure and compare Raman spectra.

[0228] The SERS tag incubation time optimization steps include: adding 15 μL SPION-PEI-AuNPs-Apt and 50 μL PBSM to 500 μL 10 4CFU / mL PA bacterial suspension, incubate at room temperature with shaking for 40 minutes; after magnetic separation, wash three times with PBST; add 150μL PBS and 30μL SERS label, incubate at room temperature with shaking for 5, 10, 15, 20, and 25 minutes; after magnetic separation and water washing three times, resuspend in 20μL water; drop onto a glass slide, and measure and compare Raman spectra.

[0229] from Fig.25 It can be seen that as the incubation time increases from 5min to 20min, the Raman signal increases from low to high; the signal changes no longer significantly when the incubation time continues to increase. When the amount of SERS tag increases from 10μL to 30μL, the Raman signal increases from low to high; the signal changes no longer significantly when the amount continues to increase. In summary, considering the cost-effectiveness, the subsequent experiments use a SERS tag amount of 30μL and an incubation time of 20min.

[0230] 3.7 Construction of standard curve

[0231] Based on the above optimized conditions, 50, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 CFU / mL and other PA bacterial solutions with different concentrations were used for SERS detection. The logarithm of bacterial concentration was used as the independent variable and the corresponding Raman characteristic peak (1587 cm -1 ) signal value was used as the dependent variable, and standard curves were established using general linear regression and machine learning regression (XGBR, SVR, BRR, ANNR, PR and ENR), and the standard curve under the optimal model was selected after comparative analysis.

[0232] The specific SERS detection steps include:

[0233] (1) Immunomagnetic separation and enrichment of target bacteria PA: 15 μL SPION-PEI-AuNPs-Apt and 50 μL PBSM were added to each concentration of bacterial solution and incubated at room temperature for 40 min. After incubation, magnetic separation was performed and PBST was washed three times to remove uncaptured bacteria.

[0234] (2) SERS tag recognition and binding: 30 μL SERS tag and 150 μL PBS were added and incubated at room temperature for 20 min. After incubation, magnetic separation was performed and PBST was washed three times to remove unbound SERS tag.

[0235] (3) Detection: The immunomagnetic beads / PA / SERS tag complex was resuspended in 20 μL of water and dropped onto a glass slide to dry. Three points were taken for each sample to measure the SERS signal, and the average was taken as the final result.

[0236] 3.7.1 General Linear Regression Model

[0237] The results of establishing the standard curve using the general linear regression model are as follows Fig.26 , the linear regression equation is Y = -2462.8 + 2046.9X, where R 2 = 0.978, X is the logarithmic value of bacterial concentration, Y is the Raman signal value, see Fig. 27 A; Fig. 27 B is the Raman spectrum corresponding to the detection of PA at various concentrations.

[0238] 3.7.2 Machine Learning Regression Model

[0239] According to the optimized machine learning regression models support vector machine regression (SVR), Bayesian ridge regression (BRR), elastic net regression (ENR), artificial neural network regression (ANNR), extreme gradient boosting regression (XGBR), and polynomial regression (PR).

[0240] The data set comes from the SERS spectra of 8 different concentrations of Pseudomonas aeruginosa. The program code is written in PyCharm2021.1.1 (×64) and runs on Python3.10 (64-bit). Import related modules, such as sklearn, numpy, pandas, etc. Import data and divide the coordinate axes. Logarithmically transform the value of bacterial concentration and record it as the X-axis; the corresponding Raman spectrum is 1587cm -1 The signal strength is recorded as the Y axis. 80% of the data is randomly selected as the training set, and the remaining 20% ​​of the data is used as the test set. The grid search method is used to adjust the parameters. SVR adjusts kernel, C, epsilon, degree, coef0 and other parameters; BRR adjusts n_iter, alpha_1, etc.; ENR adjusts alpha, l1_ratio, etc.; ANNR adjusts max_iter, etc.; XGBR adjusts booster, n_estimators, learning_rate, objective, etc.; PR adjusts degree. After all regression models are adjusted, use R2 The performance of the model was evaluated by two indicators, RMSE, and the best model was selected for subsequent quantitative analysis. The model was saved and called using joblib. The results are shown in the figure below. Fig. 27 shown.

[0241] from Fig. 27 It can be seen that the predicted values ​​of the non-linear models SVR and PR are closer to the true values, among which the SVR model R 2 =0.998, RMSE=159, the best effect, so the SVR model was used to construct the standard curve for the system, and its applicable PA concentration range was 50~10 8 CFU / mL.

[0242] 3.8 Methodological evaluation

[0243] 3.8.1 Detection limit

[0244] At the same time, six standard blank samples were tested and the experiment was carried out according to step 3.7. The characteristic peak 1587cm in the SERS spectrum was taken. -1 Calculate the signal intensity of the blank solution. Substituting this value into the SVR model standard curve, the detection limit was calculated to be 15 CFU / mL.

[0245] 3.8.2 Precision and spike recovery

[0246] To verify that the SERS detection strategy can detect target bacteria well in different matrices, the concentration of 10 2 , 10 4 , 10 6 CFU / mL of PA bacterial liquid, three parallels were set for each sample. Milk came from the local market, blood and sputum came from healthy people, and pleural effusion came from the First Affiliated Hospital of Zhengzhou University. All spiked matrices were verified to be sterile by plate counting method before use. Perform the experiment according to step 3.7, record the signal intensity of the Raman characteristic peak, and substitute this value into the advantage model and general linear regression model to obtain the corresponding concentration. The relative standard deviation (RSD) and spike recovery rate were calculated by the formula. The advantage model and the general linear regression model were compared to verify the advantages of the machine learning regression model in the quantitative analysis of SERS spectra. The results are shown in Table 7.

[0247] As can be seen from Table 7, the RSD of the SVR model is 3.0% to 9.3%, and the spike recovery is 89.7% to 106.3%; the RSD of the general linear regression model is 3.0% to 12.3%, and the spike recovery is 29.4% to 125.2%. The results show that the SERS platform combined with the machine learning regression model has better precision and accuracy, and this method can also effectively quantify PA in complex matrices.

[0248] Table 7 Precision and spike recovery under different solution environments (n=3)

[0249] 3.8.3 Specificity

[0250] Since actual samples usually contain various bacteria and other impurities, the detection method is required to have good specificity. In order to investigate the specificity of the SERS method, 10 4 CFU / mL target bacteria, 10 6 CFU / mL of five interfering bacteria, including A. baumannii, H. influenzae, K. pneumoniae, S. aureus and S. pneumoniae, and PBS blank control. The experiment was carried out according to step 3.7, and three parallels were set for each sample. The results are shown in Fig.28 As shown. Fig.28 It can be seen that the signal of the target bacteria is significantly stronger than that of the interfering bacteria and the blank control, which proves that the SERS detection strategy has good specificity.

[0251] 3.9 Actual Sample Testing

[0252] In order to study the potential value of the SERS platform in hospital infection control, clinical diagnosis, water and food safety monitoring, a total of 68 samples were collected in this experiment, including 34 community-source samples (drinking water and fruit) and 34 hospital-source samples (hospital environment, blood, pleural effusion and sputum). The analytical performance of this method was compared with the culture-based "gold standard" method, and the results are shown in Table 8. The detection results of the SERS method: 33 samples did not detect the target bacteria, and 35 samples detected the target bacteria. This result is consistent with the culture-based method. Therefore, there is no difference in classification between the two methods. In terms of quantification, the results of three samples (1 tap water, 1 fruit and 1 sputum) differed by an order of magnitude from the culture method.

[0253] Table 8 Actual sample testing

[0254]

[0255]

[0256] Note: For the SERS method, the results below the detection limit are considered as undetected; for the culture method, the results without the growth of target colonies are considered as undetected.

[0257] Undetected data were excluded and other data were logarithmically transformed. The Bland-Altman scatter plot results showed that most points fell within the 95% consistency limit. Overall, the SERS detection method and the culture method had good consistency in results.

[0258] The SERS detection method constructed in the embodiment of the present invention uses vancomycin and aptamers as recognition elements of PA bacteria, AuNRs@Ag as substrate, with a detection limit of 15 CFU / mL and a detection and analysis time of 1.5 hours. It can be used for the detection of samples such as water, fruit, blood, pleural effusion, sputum and hospital environment; therefore, compared with other existing label-based SERS methods, the various performances of the above-mentioned SERS detection method provided in the embodiment of the present invention can be comparable to the existing results, and it also has the advantages of wide practicality and accuracy. Compared with other non-SERS-based bacterial detection methods, the above-mentioned SERS detection method provided in the embodiment of the present invention has the advantages of high sensitivity, good accuracy, short detection time, low detection limit, no fluorophore bleaching and flashing. In addition, the SERS detection system does not require complicated and cumbersome sample pretreatment.

[0259] Therefore, compared with other existing bacterial detection methods, the SERS detection strategy provided by the embodiment of the present invention has the advantages of low cost, high sensitivity, high accuracy, short detection time, low detection limit, and no need for complicated sample pretreatment. The above shows that the method has application value. The established method can effectively detect target bacteria in different matrices, further verifying the practicality of the method.

[0260] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A detection and identification reagent for Pseudomonas aeruginosa, It is characterized in that It includes immunomagnetic beads SPION-PEI-AuNPs-Apt and SERS tags AuNRs-MBA@Ag-MBA-Van for identifying and capturing Pseudomonas aeruginosa; The immunomagnetic beads SPION-PEI-AuNPs-Apt are mainly formed by modifying the aptamer Apt of Pseudomonas aeruginosa to the surface of SPION-PEI-AuNPs particles through chemical bonds, wherein the SPION-PEI-AuNPs particles are mainly prepared by electrostatically adsorbing gold nanoparticles AuNPs using superparamagnetic iron oxide nanoparticles, and the superparamagnetic iron oxide nanoparticles are mainly synthesized by ferric chloride, anhydrous sodium acetate and polyethyleneimine using a hydrothermal method; The SERS tag AuNRs-MBA@Ag-MBA-Van is mainly synthesized by modifying vancomycin Van on the surface of the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA through an amide condensation reaction; the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA is mainly prepared by first wrapping a silver shell on the AuNRs-MBA nanoparticles through a redox reaction, and then modifying the second layer of Raman reporter molecules on the silver shell; the AuNRs-MBA nanoparticles are mainly prepared by modifying the first layer of Raman reporter molecules on gold nanorods AuNRs, and the first layer of Raman reporter molecules and the second layer of Raman reporter molecules are both mercaptobenzoic acid; the SERS tag AuNRs-MBA@Ag-MBA-Van can form a sandwich structure SPION-PEI-AuNPs-Apt / with the immunomagnetic beads SPION-PEI-AuNPs-Apt PA / Van-MBA-Ag@ MBA-AuNRs; Preferably, the immunomagnetic beads SPION-PEI-AuNPs-Apt use thiolated aptamer Apt, magnesium-containing buffer and the SPION-PEI-AuNPs as raw materials, and the aptamer Apt is modified to the surface of the SPION-PEI-AuNPs by forming Au-S bonds between the sulfur atoms in the thiolated aptamer Apt and the AuNPs in the SPION-PEI-AuNPs.

2. A detection platform for Pseudomonas aeruginosa, It is characterized in that The method comprises the detection and identification reagent and the Raman spectrometer according to claim 1, wherein the immunomagnetic beads SPION-PEI-AuNPs-Apt can specifically identify Pseudomonas aeruginosa and capture it to form a composite SPION-PEI-AuNPs-Apt / PA The vancomycin Van in the SERS tag AuNRs-MBA@Ag-MBA-Van binds to the composite SPION-PEI-AuNPs-Apt / PA The D-Ala-D-Ala in the cell wall peptidoglycan of Pseudomonas aeruginosa interacts specifically to form a sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@ MBA-AuNRs; using the Raman spectrometer to detect the sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@ MBA-AuNRs Raman spectrum, based on the relationship between the concentration of Pseudomonas aeruginosa and the Raman signal, a standard curve for quantitative detection of Pseudomonas aeruginosa was constructed; wherein the Raman signal is the signal intensity of a certain characteristic peak; Preferably, the detection platform includes a Raman signal data analysis module, which has a built-in machine learning algorithm. The machine learning algorithm establishes multiple regression models to process the Raman signal data, and uses the complex correlation coefficient R 2 and RMS error RMSE After evaluating the performance of the regression model and optimizing the parameters of each model, the best regression model was selected to construct the standard curve for quantitative detection of Pseudomonas aeruginosa; More preferably, the regression analysis model includes an XGBR model, an SVR model, a BRR model, an ANNR model, a PR model and an ENR model.

3. A method for detecting Pseudomonas aeruginosa based on the detection platform according to claim 2, The following steps are involved: Preparation of SPION-PEI-AuNPs: uniformly mixing the superparamagnetic iron oxide nanoparticles and the gold nanoparticle AuNPs solution to form SPION-PEI-AuNPs particles; Preparation of immunomagnetic beads: firstly subjecting the thiolated aptamer Apt solution and tris(2-carboxyethyl)phosphine buffer to a room temperature reduction reaction, and then mixing with the SPION-PEI-AuNPs particles for a coupling reaction to obtain immunomagnetic beads SPION-PEI-AuNPs-Apt; Preparation of AuNRs-MBA@Ag: uniformly mixing silver nitrate and the AuNRs-MBA particles, and causing the silver nitrate to undergo an oxidation-reduction reaction to coat the AuNRs-MBA nanoparticles with a silver shell to form the AuNRs-MBA@Ag nanoparticles; wherein the AuNRs-MBA nanoparticles are mainly prepared by modifying mercaptobenzoic acid on gold nanorods AuNRs; Preparation of SERS tag: firstly, the AuNRs-MBA@Ag nanoparticles and mercaptobenzoic acid solution are mixed and reacted, and mercaptobenzoic acid is modified on the AuNRs-MBA@Ag nanoparticles again to form a gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA; then, the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA and vancomycin Van are subjected to room temperature oscillation reaction to form a SERS tag AuNRs-MBA@Ag-MBA-Van; Capturing target bacteria: The immunomagnetic beads SPION-PEI-AuNPs-Apt, the magnesium-containing solution and the target detection bacterial solution are incubated at room temperature with shaking, so that the immunomagnetic beads SPION-PEI-AuNPs-Apt can specifically identify the Pseudomonas aeruginosa therein and capture it, forming a composite SPION-PEI-AuNPs-Apt / PA , wherein the target detection bacterial solution contains Pseudomonas aeruginosa; Dual recognition of target bacteria: the composite SPION-PEI-AuNPs-Apt / PA and the SERS tag AuNRs-MBA@Ag-MBA-Van were incubated at room temperature to form a sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@ MBA-AuNRs; Raman detection: Raman spectrometer was used to detect the sandwich structure SPION-PEI-AuNPs-Apt / PA The Raman signal of / Van-MBA-Ag@MBA-AuNRs was used to construct a standard curve for quantitative detection of Pseudomonas aeruginosa based on the relationship between the concentration of Pseudomonas aeruginosa and the Raman signal.

4. The detection method according to claim 3, It is characterized in that The steps of preparing SPION-PEI-AuNPs include: Using chloroauric acid and sodium citrate as main raw materials, the gold nanoparticles AuNPs are synthesized by a seed growth method to obtain the gold nanoparticles AuNPs solution; The superparamagnetic iron oxide nanoparticles are added to the gold nanoparticle AuNPs solution and ultrasonically stirred so that the gold nanoparticles AuNPs therein are modified onto the superparamagnetic iron oxide nanoparticles by electrostatic adsorption, magnetically separated, washed with water and then resuspended in water to obtain a SPION-PEI-AuNPs resuspension.

5. The detection method according to claim 4, It is characterized in that The steps of preparing the immunomagnetic beads include: Reducing the thiol group: mixing the thiol-modified aptamer Apt solution and the first tris(2-carboxyethyl)phosphine buffer, and performing a reduction reaction at room temperature to obtain an Apt thiol-reduced solution; Coupling: adding the SPION-PEI-AuNPs resuspension to the Apt thiol reduction solution, incubating overnight, so that the aptamer Apt is modified to the surface of the SPION-PEI-AuNPs particles through chemical bonds, magnetically separated, and the immunomagnetic beads SPION-PEI-AuNPs-Apt are obtained; then the immunomagnetic beads SPION-PEI-AuNPs-Apt are blocked and magnetically separated in sequence, and resuspended in PBS buffer to obtain the immunomagnetic beads resuspension.

6. The detection method according to claim 5, It is characterized in that The steps of preparing AuNRs-MBA@Ag include: Forming an AuNRs-MBA resuspension: firstly subjecting a first mercaptobenzoic acid solution and a second tri(2-carboxyethyl)phosphine buffer to a mixing reaction at room temperature, then adding the gold nanorods AuNRs to ultrasonic mixing, modifying the gold nanorods AuNRs with mercaptobenzoic acid, and forming a first reaction solution; subjecting the first reaction solution to a gradient centrifugation purification treatment to obtain the AuNRs-MBA nanoparticles; and resuspending the AuNRs-MBA nanoparticles in a hexadecyltrimethylammonium bromide buffer to form an AuNRs-MBA resuspension; Forming AuNRs-MBA@Ag heavy suspension: firstly, hexadecyltrimethylammonium bromide and the AuNRs-MBA heavy suspension are mixed and dissolved in a water bath; then, under stirring, ascorbic acid solution and silver nitrate solution are added in sequence, and a silver ion reduction reaction is carried out at a pH value of 9.5-10.5 so that the generated silver is wrapped on the AuNRs-MBA nanoparticles to form the silver shell. After the reaction is completed, gradient centrifugation purification treatment is carried out, and the precipitate is resuspended in hexadecyltrimethylammonium bromide buffer to form AuNRs-MBA@Ag heavy suspension.

7. The detection method according to claim 6, It is characterized in that The steps of preparing the SERS tag include: Modifying the reporter molecule: firstly, the second mercaptobenzoic acid solution and the third tris(2-carboxyethyl)phosphine buffer solution are mixed and reacted at room temperature, and then the AuNRs-MBA@Ag resuspension solution is ultrasonically treated, and the mercaptobenzoic acid is modified again on the AuNRs-MBA@Ag nanoparticles to form a second reaction solution; the second reaction solution is subjected to gradient centrifugation purification to obtain the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA; the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA is resuspended in a mixed buffer solution of MES and hexadecyltrimethylammonium bromide to form an AuNRs-MBA@Ag-MBA resuspension solution; One-step coupling of Van: first, the AuNRs-MBA@Ag-MBA resuspension is activated; then, vancomycin Van is added and shaken at room temperature to produce an amide condensation reaction, and the vancomycin Van is modified on the surface of the gold-silver core-shell double-layer Raman reporter molecule AuNRs-MBA@Ag-MBA to form a SERS tag AuNRs-MBA@Ag-MBA-Van; then, the SERS tag AuNRs-MBA@Ag-MBA-Van is blocked and magnetically separated in turn, and resuspended in a mixed buffer of PBS and hexadecyltrimethylammonium bromide to obtain a SERS tag resuspension.

8. The detection method according to claim 7, It is characterized in that The step of capturing the target bacteria comprises: incubating the immunomagnetic bead resuspension, the magnesium-containing PBS buffer and the target detection bacterial solution at room temperature, wherein the aptamer Apt can specifically recognize the Pseudomonas aeruginosa in the target detection bacterial solution and capture it, magnetic separation, and PBST buffer washing to obtain the composite SPION-PEI-AuNPs-Apt / PA .

9. The detection method according to claim 8, It is characterized in that The step of dual recognition of target bacteria comprises: adding the composite SPION-PEI-AuNPs-Apt / PA The SERS tag resuspension is added to the mixture and incubated at room temperature under shaking, wherein the vancomycin Van interacts specifically with the D-Ala-D-Ala in the cell wall peptidoglycan of Pseudomonas aeruginosa through its own hydrogen bond to form the sandwich structure SPION-PEI-AuNPs-Apt / PA / Van-MBA-Ag@ MBA-AuNRs; magnetic separation, washing to obtain a sandwich structure precipitate, and resuspending the sandwich structure in water to prepare a sandwich structure resuspension.

10. The detection method according to claim 9, It is characterized in that The Raman detection step includes: first dropping the sandwich structure resuspended liquid onto a glass slide and drying it, then using a Raman spectrometer for detection, and using a machine learning algorithm to establish multiple regression models based on the relationship between the detected Raman signal and the concentration of Pseudomonas aeruginosa, and then using a complex correlation coefficient to determine the correlation coefficient between the detected Raman signal and the concentration of Pseudomonas aeruginosa. R 2 The performance of the regression model was evaluated by the root mean square error and after optimizing the parameters of each model, the optimal regression model was selected to construct the standard curve for the quantitative detection of Pseudomonas aeruginosa. Preferably, the characteristic peak 1587 cm in the Raman spectrum -1 The relationship between the signal intensity at 100 μg / cm2 and the concentration of Pseudomonas aeruginosa was used to construct a standard curve for quantitative detection of Pseudomonas aeruginosa. The concentration range of Pseudomonas aeruginosa was 50-10 8 CFU / mL, SVR regression model performed best, and the multiple correlation coefficient R 2 =0.998, RMS error RMSE =159, detection limit was 15 CFU / mL; Preferably, the target bacterial liquid for detection is derived from drinking water, fruit, hospital environment, blood, pleural effusion or sputum.

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