Colorimetric-Raman coding immunochromatography test strip based on 3D film-shaped SERS probe and multi-bacterium detection method of colorimetric-Raman coding immunochromatography test strip

Abstract

The invention relates to the technical field of bacterium detection, in particular to a colorimetric-Raman coding immunochromatography test strip based on a 3D film-shaped SERS probe and a multi-bacterium detection method thereof. According to the technical scheme, the kit comprises a sample pad, a nitrocellulose membrane, a water absorption pad, a 3D membrane-like SERS probe and an operation buffer solution. According to the present invention, the immunochromatography of the 3D film-like probe is provided, the MoDAu-coated Ag SERS nano-film is adopted as the probe in the system, such that the pathogenic bacteria can be highly specifically recognized and captured, the immune complex fluidity can be increased, the rapid quantitative detection of the three pathogenic bacteria can be achieved, and the flexible 3D film-like SERS probe capable of enhancing the colorimetric ability and the SERS activity and highly specifically capturing the pathogenic bacteria can be prepared; by combining with an antibody-modified ICA test strip, rapid, simultaneous and quantitative determination of various pathogenic bacteria is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial detection, and particularly to a colorimetric-Raman encoded immunochromatographic test strip based on a 3D membranous SERS probe and a multiplex bacterial detection method thereof. Background Art

[0002] Bacterial infections have long been one of the most important public health challenges, causing many serious diseases (such as pneumonia, sepsis, infectious diarrhea, meningitis), and resulting in high mortality. Timely and accurate pathogen identification is crucial for curbing disease transmission and guiding appropriate therapeutic interventions. Traditional clinical detection methods, including plate counting, polymerase chain reaction, and DNA sequencing, etc., face significant limitations, such as long processing times (3 - 24 hours), complex operations, high costs, and requirements for clean room facilities. Therefore, there is an urgent need to develop new diagnostic technologies that combine simplicity, rapidity, high sensitivity, and point-of-care capabilities as a powerful complement to existing early and accurate pathogen detection methods.

[0003] In the past three decades, lateral flow immunoassay (LFA) has become a leading point-of-care testing technology. This technology can rapidly, simply, and economically detect various biochemical targets, including proteins, toxins, biomarkers, and viruses, in different field environments. However, traditional LFA technology has obvious technical limitations in the detection of pathogenic microorganisms. First, traditional nanolabels (colloidal gold nanoparticles, latex beads, and fluorescent particles) exhibit insufficient performance, only generating moderate signal intensities (colorimetric or fluorescent), and having limited stability in complex matrices, resulting in unsatisfactory detection sensitivities. Second, the relatively large size of bacteria (0.5 - 4 μm) requires nitrocellulose membranes with large pores (>10 μm) to facilitate the flow of bacteria-nanolabel immunocomplexes. However, these large-pore nitrocellulose membranes impair antibody modification and immune recognition, further reducing the sensitivity and stability of this method. Therefore, developing an efficient LFA system with higher sensitivity and multi-target detection capabilities requires overcoming these inherent limitations of traditional nanolabels and large-pore NC membranes.

[0004] Surface-enhanced Raman scattering (SERS) labeling, as a highly sensitive detection technique, has attracted much attention in recent years. The SERS-based lateral flow assay (SERS-LFA) method has been successfully applied to the rapid quantitative detection of small molecules and proteins, with the sensitivity improved by 2-3 orders of magnitude. However, current SERS immunochromatography techniques still face great limitations in precise multiplex bacterial detection, especially in complex clinical samples, mainly for the following reasons: 1) Improving the colloidal stability of SERS tags in complex samples to ensure accuracy and avoid false positive results remains a challenge; ii) Increasing the reaction interface of SERS tags can effectively enhance their immune binding efficiency to bacteria and improve detection sensitivity, while traditional spherical tags cannot achieve this goal; iii) Integrating stable SERS hotspots and numerous active antibodies onto a single nanostructure to achieve highly sensitive and specific detection still has problems. Further efforts are needed to establish SERS immunochromatography as an effective tool for on-site pathogen screening.

[0005] Membrane-like (ML) tags constructed from ultrathin two-dimensional (2D) materials such as graphene oxide and molybdenum disulfide show obvious advantages over traditional spherical particles on the LFA platform. These advantages include superior stability in complex solutions, excellent optoelectronic properties, and abundant surface-active functional groups. In particular, the large reaction interface and high signal loading capacity of 2D ML tags can effectively overcome the adverse effects of large-pore nitrocellulose membranes on the sensitivity of the LFA method, thereby improving the accuracy and stability of immunochromatographic detection of pathogenic bacteria. Inspired by these works, this patent introduces MoS2 as a nanocarrier into the immunochromatography system, achieving ultrasensitive, multiplex, and rapid simultaneous detection of multiple pathogenic bacteria.

[0006] In summary, it is highly necessary to prepare a membrane-like probe with the ability to capture multiple pathogenic bacteria commonly, strong SERS activity, strong colorimetric performance, and SERS encoding, and to establish a general immunochromatography technique for rapid, accurate, and simultaneous detection of multiple pathogenic bacteria. This application proposes a colorimetric-Raman encoded immunochromatographic test strip based on a 3D membrane-like SERS probe and its multiplex bacterial detection method. Summary of the Invention

[0007] The object of the present invention is to address the problem in the background art that there is no general immunochromatography technique for rapid, accurate, and simultaneous detection of multiple pathogenic bacteria, and to propose a colorimetric-Raman encoded immunochromatographic test strip based on a 3D membrane-like SERS probe and its multiplex bacterial detection method.

[0008] In the first aspect, this application provides a colorimetric-Raman encoded immunochromatographic test strip based on a 3D membrane-like SERS probe, comprising:

[0009] Sample pad for loading the sample solution to be tested;

[0010] Nitrocellulose membrane, comprising a mixed detection line and a quality control line, wherein the mixed detection line is sprayed with a mixture of antibodies against Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli O157:H7, and the quality control line is sprayed with goat anti-mouse IgG;

[0011] Absorbent pad;

[0012] 3D membranous SERS probe (MoDAu@Ag), which sequentially includes ultrathin molybdenum disulfide (MoS2) nanosheets, two precisely arranged gold core-silver shell (Au@Ag) nanoparticle layers, a nanogap layer loaded with Raman molecules, and detection antibodies modified by electrostatic adsorption from the inside to the outside; the Raman molecules of the 3D membranous SERS probe are selected from at least three of DTNB, MPY or MBA;

[0013] Running buffer;

[0014] The test strip generates colorimetric signals and SERS signals on the detection line through the 3D membranous SERS probe, and is used for the simultaneous detection and quantitative analysis of three pathogenic bacteria.

[0015] Optionally, the particle size of the MoS2 nanosheets of the 3D membranous SERS probe is 100 - 1000 nm, and the particle size of the Au@Ag nanoparticle layer is 10 - 40 nm.

[0016] Optionally, the particle size of the Au@Ag nanoparticle layer is 24 nm.

[0017] Optionally, the spraying concentrations of the antibodies against Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli O157:H7 on the mixed detection line are all 0.1 - 2.5 mg / mL.

[0018] Optionally, the detection antibodies of the 3D membranous SERS probe are modified by electrostatic adsorption, and the colorimetric signal detection limit of the probe is 500 cells / mL, and the SERS coding detection sensitivity is 30 - 40 cells / m.

[0019] Optionally, the preparation method of the 3D membranous SERS probe comprises the following steps:

[0020] (1) Centrifuge the monolayer MoS2 nanosheets and resuspend them in deionized water;

[0021] (2) Synthesize Au@Ag nanoparticles: Heat the gold nanoparticle solution to boiling, add trisodium citrate and AgNO3, and stir and cool;

[0022] (3) Mix the MoS2 nanosheets with a polyethyleneimine (PEI) solution and sonicate to obtain PEI-modified MoS2 nanosheets; add the Au@Ag solution thereto and sonicate to obtain monolayer MoAu@Ag composites;

[0023] (4) Sonicate the MoAu@Ag with the PEI solution to form a PEI-coated nanofilm; then sonicate it with an ethanol solution of DTNB, MPY, or MBA to obtain Raman molecule-modified nanosheets;

[0024] (5) Add the Au@Ag solution and the PEI solution to the nanofilm solution in step (4) and sonicate to form a 3D nanofilm loaded with bilayer Au@Ag;

[0025] (6) Modify the surface of the 3D nanofilm with specific bacterial antibodies by electrostatic adsorption to obtain a 3D film-shaped SERS probe.

[0026] Optionally, the concentration of the PEI solution is 0.01 - 1 mg / mL, and the sonication reaction time is 10 - 60 min.

[0027] In a second aspect, the present application provides a multiplex bacterial detection method based on the 3D film-shaped SERS probe, including the following steps:

[0028] a. Load the test sample solution onto the sample pad of the immunochromatographic test strip according to any one of claims 1 - 7;

[0029] b. Drive the sample solution to migrate on the test strip through a running buffer, so that the target pathogenic bacteria bind to the 3D film-shaped SERS probe to form a complex;

[0030] c. The complex is captured by the specific antibody on the mixed detection line, generating a visible black colorimetric signal;

[0031] d. Read the SERS signal on the detection line through a Raman spectrometer, and judge the type of pathogenic bacteria and perform quantitative analysis according to the displacement and intensity of the characteristic signal peaks.

[0032] Optionally, the pathogenic bacteria include at least one of Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7.

[0033] Optionally, the quantitative analysis of the SERS signal is judged by the Raman characteristic peak intensities of DTNB, MPY, and MBA.

[0034] Compared with the prior art, the present application includes at least one of the following beneficial technical effects:

[0035] The present invention proposes a 3D film-shaped SERS probe to provide a strong SERS signal with two layers of Au@Ag particles with controllable density, and successfully synthesizes a SERS nanofilm.

[0036] The preparation method of the 3D film-like SERS probe proposed by the present invention adopts the PEI-mediated layer-by-layer self-assembly method, and a multilayer Au@Ag shell is assembled on the surface of the MoS2 nanofilm to provide a large number of stable SERS hot spots.

[0037] The 3D film-like SERS probe proposed by the present invention is modified with bacterial antibodies to provide high-specificity capture ability for a variety of pathogenic bacteria.

[0038] The immunochromatography based on the 3D film-like SERS probe proposed by the present invention includes a nitrocellulose membrane with a mixed detection line (antibodies against Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7) and a quality control line (goat anti-mouse IgG), a water absorption pad, and a sample pad for loading the sample solution, which solves the problem of the complex production process of existing multiplex chromatographic detection.

[0039] The present invention proposes immunochromatography of 3D film-like probes. This system uses the MoDAu@Ag SERS nanofilm as a probe, which can not only specifically recognize and capture pathogenic bacteria with high specificity, but also increase the fluidity of immune complexes, and realize the simultaneous and rapid quantitative detection of three pathogenic bacteria on the immunochromatography test strip. Using monolayer MoS2 as the substrate, two layers of Au@Ag nanoparticles are loaded and Raman dye molecules are filled in the PEI interlayer, and bacterial antibodies are modified to prepare a flexible 3D film-like SERS probe with enhanced colorimetric ability and SERS activity and high-specificity capture of pathogenic bacteria. Combined with the antibody-modified ICA test strip, the rapid, simultaneous, and quantitative determination of a variety of pathogenic bacteria is realized. Brief Description of the Drawings

[0040] Figure 1 It is a schematic diagram of the preparation method of the 3D film-like SERS probe of Example 1 of the present invention.

[0041] Figure 2 It is an electron microscopy characterization diagram of the 3D film-like SERS probe of Example 1 of the present invention.

[0042] Figure 3 It is an XPS characterization diagram of elemental analysis of the 3D film-like SERS probe of Example 1 of the present invention.

[0043] Figure 4 It is a working principle diagram of the immunochromatography of the 3D film-like SERS probe of Example 2 of the present invention for Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7.

[0044] Figure 5 It is the feasibility verification result of the immunochromatography of the 3D film-like SERS probe of Example 2 of the present invention Figure 1 。

[0045] Figure 6 Feasibility verification results of immunochromatography of the 3D film-like SERS probe in Example 2 of the present invention Figure 2 。

[0046] Figure 7 Optimization results of the membrane coating concentration of immunochromatography of the 3D film-like SERS probe in Example 2 of the present invention.

[0047] Figure 8 Optimization results of the composition of the running buffer for immunochromatography of the 3D film-like SERS probe in Example 2 of the present invention.

[0048] Figure 9 Optimization results of the chromatographic reaction time of immunochromatography of the 3D film-like SERS probe in Example 2 of the present invention.

[0049] Figure 10 Results graph of simultaneous detection of Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 by immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention.

[0050] Figure 11 Results graph of simultaneous detection of Escherichia coli O157:H7 by immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention.

[0051] Figure 12 Results graph of simultaneous detection of Pseudomonas aeruginosa by immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention.

[0052] Figure 13 Results graph of simultaneous detection of Salmonella typhimurium by immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention.

[0053] Figure 14 Repeatability results of immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention.

[0054] Figure 15 Specificity results of immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention.

[0055] Figure 16 Analysis results of three pathogenic bacteria in food and environmental simulation samples by immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention.

[0056] Figure 17 Correlation analysis between the detection results of immunochromatography of the 3D film-like SERS probe in Example 3 of the present invention for detecting samples of clinical urinary tract infection patients and the ELISA method. Detailed implementation manners

[0057] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0058] The present invention provides a preparation method of a 3D film-like SERS probe. By using MoS2 nanosheets to load two layers of Au@Ag nanoparticles, a film-like substrate (MoDAu@Ag) with a large number of stable SERS hot spots is prepared; through ultrasonic action, different Raman dye molecules are filled in the PEI interlayer between the two layers of Au@Ag to provide a strong SERS signal, and a 3D film-like SERS tag is prepared; a bacterial antibody is modified on the surface of MoDA@Ag to provide a high-specificity ability to capture pathogenic bacteria.

[0059] Among them, the MoS2 nanofilm provides a large reaction interface and a flexible structure. The MoS2 nanofilm is preferably a single-layer MoS2 nanosheet, and the sheet size of the MoS2 nanosheet is preferably 100 - 1000 nm, more preferably 200 - 500 nm.

[0060] In addition, the film-like SERS nanomaterial includes two layers of Au@Ag shells loaded on the surface of the MoS2 nanosheets and two layers of cationic polymer interlayers; both of the two layers of Au@Ag shells use the cationic polymer as a linker, and the two layers of the cationic polymer self-assembly layers are in contact with the surface of the MoS2 nanofilm and the Au@Ag shell respectively. In the present invention, the arrangement of the two layers of Au@Ag shells and the two layers of cationic polymer interlayers is specifically MoS2 nanofilm, the first layer of cationic polymer, the first layer of Au@Ag shell, the second layer of cationic polymer, and the second layer of Au@Ag shell. In the present invention, the thickness of the single-layer cationic polymer self-assembly layer is preferably 0 - 2 nm, and the reaction time is preferably 10 - 60 min. The cationic polymer interlayer is preferably a polyethyleneimine (PEI) layer, and the molecular weight of the polyethyleneimine is preferably 5000 - 8000, more preferably 6000 - 70000. The obtained 3D film-like SERS nanomaterial is freeze-dried and stored. The 3D film-like SERS probe provides a strong SERS signal with two layers of Au@Ag particles with controllable density, and the SERS nanofilm is successfully synthesized.

[0061] It is worth noting that based on the electrostatic self-assembly method mediated by cationic polymers, two layers of Au@Ag shells are assembled layer by layer on the surface of MoS2 nanosheets. The preparation process is efficient, simple, and repeatable, and is suitable for large-scale production.

[0062] The present invention provides a 3D film-like SERS tag, which is suitable for immunochromatographic detection. It is composed of the above-mentioned 3D film-like SERS nanofilm (providing a large surface area, strong SERS activity, and strong colorimetric signal) and a bacterial antibody (specifically binding to pathogenic bacteria) modification combination.

[0063] The 3D film-like SERS probe provided by the present invention can efficiently and specifically bind to the surface of bacteria. After adding the running buffer, a chromatographic reaction is carried out to improve the detection sensitivity.

[0064] The present invention provides an immunochromatography of a 3D film-like SERS probe, including a sample pad for loading a sample solution, a nitrocellulose membrane having a mixed detection line (taking antibodies against Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 as examples) and a quality control line (goat anti-mouse IgG), a water-absorbing pad, a running buffer, and a 3D film-like SERS probe for detection. The preparation method of the 3D film-like SERS probe proposed by the present invention adopts the PEI-mediated layer-by-layer self-assembly method, and a multi-layer Au@Ag shell is assembled on the surface of the MoS2 nanofilm to provide a large number of stable SERS hot spots. The proposed 3D film-like SERS probe is modified with bacterial antibodies to provide a high-specificity capture ability for a variety of pathogenic bacteria.

[0065] In the present invention, the sample pad is used for loading the sample solution to be detected, and the water-absorbing pad is used to provide capillary force. The material of the bottom plate is preferably PVC, and the bottom plate serves as the backing card of the immunochromatographic test strip. On the nitrocellulose membrane, a detection line loaded with three kinds of bacterial antibodies is used to simultaneously quantitatively detect Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7. In the present invention, the quality control line modifier is preferably goat anti-mouse IgG.

[0066] The present invention provides a modification method for the nitrocellulose membrane in the immunochromatographic test strip of the 3D film-like SERS probe, which specifically includes the following steps:

[0067] Antibodies against Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 and goat anti-mouse IgG are respectively sprayed on the surface of the nitrocellulose membrane, and then the modified nitrocellulose membrane is placed in a constant temperature drying oven to obtain the nitrocellulose membrane in the immunochromatographic test strip of the 3D film-like SERS probe. The concentration of the antibody against Pseudomonas aeruginosa is 0.1 - 2.5 mg / mL, more preferably 0.8 mg / mL; the concentration of the antibody against Salmonella typhimurium is 0.1 - 2.5 mg / mL, more preferably 1.0 mg / mL; the concentration of the antibody against Escherichia coli O157:H7 is 0.1 - 2.5 mg / mL, more preferably 0.8 mg / mL; the concentration of the goat anti-mouse IgG antibody is 0.1 - 2.5 mg / mL, more preferably 0.8 mg / mL. The temperature of the constant temperature drying oven is preferably set at 37°C.

[0068] The immunochromatography of the 3D film-like SERS probe proposed by the present invention provides SERS quantitative detection, and high-sensitivity quantitative analysis is achieved by detecting the change of the SERS signal on the detection line.

[0069] The present invention provides a method for immunochromatographic detection using a 3D membranous SERS probe: incubate the sample to be detected with the 3D membranous SERS probe, add 10× running buffer, mix well and load it onto the sample pad of the test strip, and contact the test line on the test strip for chromatographic reaction. After the reaction is completed, use a portable Raman spectrometer to read the SERS signal for quantitative analysis.

[0070] In the present invention, the components of the running buffer are preferably Tween 20, FBS and PBS buffer. The content of Tween 20 is preferably 10%, the content of FBS is preferably 40%, and the concentration of PBS buffer is preferably 10 mM.

[0071] The immunochromatography based on the 3D membranous SERS probe proposed by the present invention includes a nitrocellulose membrane with a mixed test line (antibodies against Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli O157:H7) and a control line (goat anti-mouse IgG), a water absorption pad and a sample pad for loading the sample solution, which solves the problem of the complex production process of existing multiplex chromatographic detection.

[0072] The present invention is further illustrated by the following examples, but is not limited thereto.

[0073] Example 1

[0074] The 3D membranous SERS probe prepared in this example is based on the PEI-mediated electrostatic adsorption method, and two layers of Au@Ag shells are continuously adsorbed to prepare a membranous SERS probe with a large reaction interface, strong SERS activity and excellent colorimetric performance. A variety of bacterial antibodies are used to modify the 3D SERS nanofilm to endow it with the ability to capture multiple pathogenic bacteria.

[0075] A preparation method of the 3D membranous SERS probe of this example is as Figure 1 shown, including the following steps:

[0076] (1) Mix the MoS2 aqueous solution with the cationic polymer PEI aqueous solution, and react under vigorous ultrasonic treatment to obtain MoS2 nanosheets coated with PEI (MoS2-PEI).

[0077] (2) Add the Au@Ag aqueous solution to the MoS2-PEI nanofilm aqueous solution, mix well and ultrasonicate vigorously to obtain a 2D nanofilm MoAu@Ag adsorbed with Au@Ag.

[0078] (3) Mix the MoAu@Ag nanofilm with an ethanol solution of Raman dye molecules (2-MPY, 4-MBA, DTNB) and ultrasonicate to obtain the nanofilm MoAu@Ag MPY 、MoAu@Ag MBA and MoAu@AgDTNB 。

[0079] (4) Mix the aforementioned MoAu@Ag MPY , MoAu@Ag MBA and MoAu@Ag DTNB nanofilm with the aqueous solution of cationic polymer PEI again, and react under intense ultrasonic waves to obtain MoAu@Ag MPY -PEI, MoAu@Ag MBA -PEI and MoAu@Ag DTNB -PEI.

[0080] (5) Add the Au@Ag aqueous solution to the aqueous solution of the MoAu@Ag-PEI nanofilm, mix well and then react under intense ultrasonic waves to obtain the 3D nanofilm MoDAu@Ag adsorbed with a second layer of Au@Ag.

[0081] (6) Modify the aforementioned 3D film-like SERS probe (MoDAu@Ag) with various specific bacterial antibodies through electrostatic adsorption to obtain a 3D film-like SERS probe with strong SERS activity and colorimetric ability.

[0082] Figure 2 are the high-resolution transmission electron microscopy (HRTEM) images of the MoS2 nanosheets in step (1) of this example, the MoAu@Ag nanosheets prepared in step (2), the MoDAu@Ag composite nanofilm prepared in step (5) and its local details. It is shown by Figure 2 that the synthesized MoDAu@Ag composite nanofilm is a film-like structure wrapped by two layers of dense Au@Ag nanoparticles. Figure 3 shows the elemental analysis results of the prepared MoDAu@Ag composite nanofilm, and each element is evenly and correctly distributed in the nanostructure, indicating that the film-like label is successfully prepared.

[0083] Example 2

[0084] Figure 4 is the working principle diagram of the immunochromatography based on the 3D film-like SERS probe for Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli O157:H7.

[0085] Figures 5 - 6For the feasibility verification of simultaneously detecting three pathogenic bacteria by immunochromatography based on 3D membranous SERS probes, it is proved that there is no cross-reaction among the three pathogenic bacteria detected by SERS immunochromatography and with other pathogens. The inside of the test strip was observed by scanning electron microscope, and it was proved that the labeled captured bacteria could aggregate at the test line of the test strip. The above results verify that the 3D membranous SERS probes and the bacterial immune complexes formed by them flow smoothly on the NC membrane and work stably in immunochromatography. In this example, the operating conditions of SERS immunochromatography were optimized to achieve the best performance of the multi-reusable platform.

[0086] Figure 7 The optimized results for the concentration of the captured antibody on the test line show that when the concentrations of the anti-Pseudomonas aeruginosa antibody, anti-Salmonella typhimurium antibody, and anti-Escherichia coli O157:H7 antibody on the test line reach 0.8 mg / mL, 1.0 mg / mL, and 0.8 mg / mL respectively, the highest signal-to-noise ratio is obtained and the detection result is the best.

[0087] Figure 8 The optimized results for the running buffer show that when the composition of the loading buffer is 10 mM PBS, 10% Tween 20, and 40% FBS, the highest signal-to-noise ratio is obtained and the detection result is the best.

[0088] Figure 9 The optimized results for the immunochromatography reaction time based on 3D membranous SERS probes show that when the reaction time is 14 min, the highest signal-to-noise ratio is obtained and the detection result is the best.

[0089] Example 3

[0090] Figures 10 - 13 The performance of the colorimetric-SERS encoded immunochromatography technology mediated by 3D membranous probes was evaluated by detecting samples of Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 at different concentrations (106 - 0 cells / mL). Figure 10 The SERS immunochromatography strip photos for simultaneously detecting three pathogenic bacteria, the SERS mapping results corresponding to the entire ICA strip, the detailed SERS signals of a single test line, and the corresponding S-type calibration curves are shown. In the detection range of 5×10 2 -10 6 cells / mL, as the concentrations of the Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 samples increase, the SERS intensity gradually increases. Under the laser detection of the Raman spectrometer, the visual limit of the colorimetric signal of the test line is 5×10 2 cells / mL. The results of the S-type calibration curve show that the dynamic detection range of SERS immunochromatography for the 3 pathogenic bacteria is 5×10 2 -10 cells / mL, and the correlation coefficient (R2 ) are all greater than 0.98.

[0091] Figure 14 Three groups of Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 with different concentrations (10 6 / 10 4 / 10 2 cells / mL) were used to verify the repeatability of the 3D membranous probe-mediated colorimetric-SERS encoded immunochromatography. As shown in the figure, the SERS signal changes on the test line were small, and the RSD values of each test group were all less than 10.46%, indicating that the established SERS immunochromatography technology has good repeatability.

[0092] Figure 15 Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli O157:H7, Shigella, Klebsiella pneumoniae, Staphylococcus epidermidis, Acinetobacter baumannii, Helicobacter pylori, Enterococcus faecium, Listeria, Campylobacter jejuni, and Staphylococcus aureus at a concentration of 10 5 cells / mL were used to verify the specificity of the 3D membranous probe-mediated colorimetric-SERS encoded immunochromatography. As shown in the figure, the SERS signals of the Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 groups on the test line were strong, and there were no obvious colorimetric signals and SERS signals in the other groups, indicating that the established SERS immunochromatography technology has good specificity.

[0093] In this example, contaminated food and environmental samples were simulated by adding pathogenic bacteria to milk samples and lake water samples to evaluate the performance of immunochromatography based on 3D membranous SERS probes during actual detection. Different concentrations (10 6 , 10 4 , 10 2 and 0 cells / mL) of Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli O157:H7 were added to milk and lake water samples, and an immunochromatography system was used for detection. As Figure 16 The results showed that the recovery rates of the three different pathogenic bacteria were calculated based on the SERS signal intensity of the test line. The detection recovery rates were 83.4% - 118.8%, and the RSD values were 2.71% - 9.4%, indicating that this method has good accuracy and reliability for the detection of milk samples and lake water samples.

[0094] In addition, in this example, clinically urine specimens infected with Pseudomonas aeruginosa were used to further study the clinical application potential of immunochromatography based on 3D membranous SERS probes. Twenty-one positive specimens and twenty-four negative specimens of Pseudomonas aeruginosa were collected for standard urine culture identification, and the immunochromatography effects of the proposed 3D membranous SERS probes were compared. Figure 17The results showed that immunochromatography based on 3D membranous SERS probes could accurately identify Pseudomonas aeruginosa in urine samples, and the detection accuracy for clinical respiratory samples infected with Pseudomonas aeruginosa was 100%. Deming regression analysis showed that for 21 positive samples, the proposed immunochromatography based on 3D membranous SERS probes was highly correlated with standard urine culture. The slope of the regression equation for Pseudomonas aeruginosa positive specimens was 1.066 (95% confidence interval was 0.996 - 0.999), and R 2 was 0.999. Therefore, MoDAu@Ag immunochromatography has the potential to replace standard urine culture for the detection of clinical bacterial infection samples, can provide higher sensitivity and a wider detection range, and can shorten the detection time to 16 min.

[0095] The above specific embodiments are merely several alternative embodiments of the present invention. Based on the technical solution of the present invention and the relevant revelations of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A colorimetric-Raman coded immunochromatographic test strip based on a 3D membrane-like SERS probe, characterized in that: include: A sample pad, used for loading the sample solution to be tested; A nitrocellulose membrane comprising a mixed test line and a quality control line, wherein the mixed test line is sprayed with a mixture of anti-Pseudomonas aeruginosa antibodies, anti-Salmonella typhimurium antibodies and anti-Escherichia coli O157:H7 antibodies, and the quality control line is sprayed with goat anti-mouse IgG; Absorbent pads; The 3D membrane SERS probe comprises, from inside to outside, an ultrathin MoS2 nanosheet, two precisely arranged gold core-silver shell nanoparticle layers, a nanogap layer loaded with Raman molecules, and a detection antibody modified by electrostatic adsorption; the Raman molecules of the 3D membrane SERS probe are selected from at least three of DTNB, MPY or MBA; Running buffer; The test strip generates a colorimetric signal and a SERS signal on the detection line through the 3D film-shaped SERS probe, which is used for simultaneous detection and quantitative analysis of three pathogens.

2. The colorimetric-Raman coded immunochromatographic test strip based on 3D membrane-like SERS probe according to claim 1, characterized in that: The particle size of the MoS2 nanosheet of the 3D film-like SERS probe is 100-1000nm, and the particle size of the Au@Ag nanoparticle layer is 10-40nm.

3. The colorimetric-Raman coded immunochromatographic test strip based on 3D membrane-like SERS probe according to claim 1, characterized in that: The particle size of the Au@Ag nanoparticle layer is 24 nm.

4. The colorimetric-Raman coded immunochromatographic test strip based on 3D membrane-like SERS probe according to claim 1, characterized in that: The spraying concentrations of the anti-Pseudomonas aeruginosa antibody, the anti-Salmonella typhimurium antibody and the anti-Escherichia coli O157:H7 antibody of the mixed test line are all 0.1-2.5 mg / mL.

5. The colorimetric-Raman coded immunochromatographic test strip based on 3D membrane-like SERS probe according to claim 1, characterized in that: The detection antibody of the 3D film-shaped SERS probe is modified by electrostatic adsorption, and the colorimetric signal detection limit of the probe is 500 cells / mL, and the SERS coding detection sensitivity is 30-40 cells / m.

6. The colorimetric-Raman coded immunochromatographic test strip based on 3D membrane-like SERS probe according to claim 1, characterized in that: The preparation method of the 3D film-shaped SERS probe comprises the following steps: (1) Centrifuge the monolayer MoS2 nanosheets and resuspend them in deionized water; (2) Synthesis of Au@Ag nanoparticles: Heat the gold nanoparticle solution to boiling, add trisodium citrate and AgNO3, stir and cool; (3) mixing the MoS2 nanosheets with the polyethyleneimine solution and ultrasonically obtaining the PEI-modified MoS2 nanosheets; adding the Au@Ag solution thereto and ultrasonically obtaining the monolayer MoAu@Ag composite; (4) ultrasonically reacting the MoAu@Ag with a PEI solution to form a PEI-wrapped nanofilm; and then ultrasonically reacting the MoAu@Ag with an ethanol solution of DTNB, MPY or MBA to obtain a Raman molecule-modified nanosheet; (5) adding the Au@Ag solution and the PEI solution to the nanomembrane solution of step (4), and ultrasonically forming a 3D nanomembrane loaded with a double layer of Au@Ag; (6) The specific bacterial antibodies are modified onto the surface of the 3D nanomembrane by electrostatic adsorption to obtain a 3D membrane-like SERS probe.

7. The colorimetric-Raman coded immunochromatographic test strip based on 3D membrane-like SERS probe according to claim 6, characterized in that: The concentration of the PEI solution is 0.01-1 mg / mL, and the ultrasonic reaction time is 10-60 min.

8. A multiple bacteria detection method based on 3D membrane SERS probes, characterized in that: The following steps are involved: a. Loading the sample solution to be tested onto the sample pad of the immunochromatographic test strip according to any one of claims 1 to 7; b. Using the running buffer to drive the sample solution to migrate on the test strip, the target pathogens bind to the 3D membrane-like SERS probe to form a complex; c. The complex is captured by the specific antibody on the mixed detection line, generating a black colorimetric signal visible to the naked eye; d. The SERS signal on the detection line is read by a Raman spectrometer, and the type of pathogen is determined and quantitatively analyzed based on the displacement and intensity of the characteristic signal peak.

9. The method for multiple bacteria detection based on 3D membrane-like SERS probe according to claim 8, characterized in that: The pathogenic bacteria include at least one of Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli O157:H7.

10. The method for multiple bacteria detection based on 3D membrane-like SERS probe according to claim 9, characterized in that: The quantitative analysis of the SERS signal is determined by the Raman characteristic peak intensities of DTNB, MPY and MBA.

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