Raman immunoprobe and SERS-colloidal gold immunochromatographic test paper and their applications

Abstract

The present invention relates to the field of surface-enhanced Raman scattering and immunochromatographic detection technologies, and discloses a Raman immunoprobe and SERS-colloidal gold immunochromatographic test paper, as well as their applications. The Raman immunoprobe provided by the present invention has extremely high stability and sensitivity. The SERS-immunochromatographic test paper prepared based on the Raman immunoprobe is simple to prepare and use, easy to operate, and capable of highly precise and accurate on-site quantitative detection of samples. Furthermore, it requires relatively low professional expertise in large-scale detection instruments, experimental sites, and personnel, making it suitable for promotion and application at the grassroots level.

Description

Technical Field

[0001] The present invention relates to the technical field of surface enhanced Raman scattering and immunochromatographic detection, and in particular to a Raman immunoprobe and SERS-colloidal gold immunochromatographic test paper and applications thereof. Background Art

[0002] Colloidal gold rapid test strips are currently a common method for rapid on-site detection of pathogens and other targets in samples. They offer advantages such as ease of use and compact, portable test strips. However, this method typically only provides qualitative detection, and there is significant room for improvement in detection sensitivity. For on-site pathogen detection, achieving highly sensitive quantitative detection plays a crucial role in early detection and treatment of related diseases, as well as in mitigating economic losses.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a detection method that has developed rapidly in recent years. One of its main features is its extremely high detection sensitivity, with a detection limit as low as the single-molecule level. Currently, SERS technology is widely used in the fields of tumor target detection, pollutant monitoring, food hygiene, etc. If SERS technology is combined with colloidal gold immunochromatography technology, it can not only take advantage of the rapidity and on-site detection convenience of colloidal gold immunochromatography technology, but also greatly improve the detection sensitivity through SERS technology, and can achieve on-site quantitative detection through small and easy-to-carry devices such as portable Raman spectrometers, thereby making on-site detection of targets such as pathogens more accurate and efficient.

[0004] However, the nanomaterials commonly used in SERS detection, such as gold nanocones and gold nanoflowers, often require the addition of large amounts of surfactants during the preparation process to achieve structural control. This results in the surfactants attached to the nanomaterial surface being susceptible to etching, significantly weakening the SERS signal and causing poor material stability. SERS-colloidal gold immunochromatographic test strips made with these materials not only lack sensitivity gains but also suffer from long-term storage stability, seriously hindering their practical use. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above problems existing in the prior art and provide a Raman immunoprobe and SERS-colloidal gold immunochromatographic test paper and their applications. + The gold nanodimer prepared by welding method is used as a nano-labeling material for SERS detection, avoiding the adverse effects of surfactants, thereby making the Raman immunoprobe more stable and sensitive.

[0006] In order to achieve the above objectives, the present invention provides a Raman immunoprobe, which includes a gold nanoparticle dimer, a signal marker molecule modified on the gold nanoparticle dimer, and an antibody Ab1 that specifically binds to a target detection object.

[0007] The second aspect of the present invention provides a method for preparing the Raman immunoprobe according to the first aspect, the method comprising:

[0008] (1) Assembly of gold nanodimers;

[0009] (2) Modification of the signal marker molecule and the antibody Ab1 that specifically binds to the target detection substance.

[0010] The third aspect of the present invention provides a surface-enhanced Raman spectroscopy-colloidal gold immunochromatography test paper, which includes a sample addition area, a conjugate area, an observation area, and a water absorption area arranged in sequence on a bottom plate, wherein the conjugate area includes a conjugate pad, and the conjugate pad contains a Raman immunoprobe, which is the Raman immunoprobe described in the first aspect or a Raman immunoprobe prepared according to the method described in the second aspect.

[0011] The fourth aspect of the present invention provides the use of the Raman immune probe described in the first aspect, or the Raman immune probe prepared according to the method described in the second aspect, or the surface enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper described in the third aspect in the detection of pathogens in samples, especially in the detection of porcine epidemic diarrhea virus in samples.

[0012] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0013] (1) The gold nanodimer used in the Raman immunoprobe provided by the present invention does not require the use of a surfactant during the preparation process, thereby avoiding the adverse effects of surfactant attachment to the surface of the nanomaterial on the SERS detection signal and probe stability.

[0014] (2) The SERS-colloidal gold immunochromatographic test paper prepared using the Raman immunoprobe provided by the present invention has both the portability of colloidal gold immunochromatographic test paper and the high sensitivity of SERS detection. In addition, by using small equipment such as a portable Raman spectrometer, high-precision and high-accuracy on-site quantitative detection of samples can be achieved, effectively improving the efficiency of pathogen detection, which is of great significance for the prevention and control of diseases and public health events.

[0015] (3) Thanks to the high stability of the Raman immunoprobe provided by the present invention, the SERS-colloidal gold immunochromatographic test paper made with the probe can be stored stably for a long time. Experiments show that after being stored at 4°C for 6 months, the SERS signal intensity of the test paper can still basically maintain the original level.

[0016] (4) The Raman immunoprobe provided by the present invention has a wide range of uses and methods of application, and has a very broad application prospect. The SERS-colloidal gold immunochromatographic test paper prepared using the Raman immunoprobe is also simple to use and easy to operate, significantly reducing the professional requirements for detection instruments, laboratory sites, and personnel, and is suitable for promotion and application at the grassroots level. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the characterization and quality level test results of the gold nanodimer prepared in Preparation Example 1. Figure 1 -A is the result of agarose gel electrophoresis; Figure 1 -B is the purified gold nanodimer suspension; Figure 1 -C is the UV absorption spectrum of gold nanodimer suspension; Figure 1 -D is a transmission electron microscopy image of gold nanodimer.

[0018] Figure 2 This is a diagram showing the detection results of the Raman immunoassay optimization process in Example 1. Figure 2 -A is the UV spectra of the purified gold nanodimer suspension, gold nanodimer-4-MBA suspension, gold nanodimer-4-MBA-Ab1 suspension and Raman immunoprobe suspension; Figure 2 -B is the UV spectra of Raman immunoprobes prepared using 4-MBA solutions with different concentrations; Figure 2 -C is a comparison of the Raman signal intensities of the Raman immunoprobes prepared using 4-MBA solutions with different concentrations.

[0019] Figure 3 This is a graph showing the results of optimizing the concentration of Ab1 coated with T lines when preparing the SERS-immunochromatographic test paper in Example 1. Figure 3 -A is a comparison chart of the T line color development of the test paper in the experimental group; Figure 3 -B is the comparison of Raman signal intensity of the experimental group; Figure 3 -C is the comparison of Raman spectrum curves of the experimental group; Figure 3 -D is a comparison chart of the T line color development of the control group test paper; Figure 3 -E is the comparison chart of Raman signal intensity of the control group; Figure 3 -F is the comparison of Raman spectrum curves of the control group.

[0020] Figure 4 This is a graph showing the results of qualitative and quantitative detection of PEDV positive sample liquid standards with different virus loading amounts using SERS-immunochromatographic test paper in Example 2. Figure 4 -A is a comparison chart of the T-line color development of the test paper for qualitative detection of PEDV positive sample liquid standards with different virus loading amounts; Figure 4-B is a comparison of the Raman spectrum curves of the test paper T line region for quantitative detection of PEDV positive sample liquid standards with different drug loading amounts; Figure 4 -C is based on the Raman spectrum at 1586 cm -1 The linear fitting graph of the Raman signal intensity plotted against the signal intensity at the displacement point and the concentration of the positive sample solution standard.

[0021] Figure 5 This is a diagram showing the specificity detection results of the SERS-immunochromatographic test paper in Example 2. Figure 5 -A is a comparison chart of the T-line color development of the test paper for testing different pathogenic bacteria or virus samples; Figure 5 -B is a comparison of Raman spectrum curves obtained from the detection of each sample; Figure 5 -C is the quantitative detection of each sample, the Raman spectrum at 1586cm -1 Comparison of signal strength at displacement locations.

[0022] Figure 6 This is a graph showing the intra-batch repeatability test results of the SERS-immunochromatographic test paper in Example 2. Figure 6 -A is a comparison chart of the T-line color development of the qualitative test strips in the experimental group; Figure 6 -B is a comparison of the Raman spectra of the experimental group test strips for quantitative detection; Figure 6 -C is the Raman spectrum of the experimental group test paper quantitative detection at 1586cm -1 Comparison of signal strength at displacement locations; Figure 6 -D is a comparison chart of the T-line color development of the control group test strip qualitative detection; Figure 6 -E is the Raman spectrum comparison diagram of the control group test paper quantitative detection; Figure 6 -F is the Raman spectrum of the control group test paper quantitative detection at 1586cm -1 Comparison of signal strength at displacement locations.

[0023] Figure 7 This is a graph showing the batch reproducibility test results of the SERS-immunochromatographic test paper in Example 2. Figure 7 -A is a comparison chart of the T-line color development of the qualitative test paper in experimental group I; Figure 7 -B is a comparison of the Raman spectra of the test paper quantitative detection in experimental group I; Figure 7 -C is the Raman spectrum of the test paper in experimental group I detected quantitatively at 1586 cm -1 Comparison of signal strength at displacement locations; Figure 7 -D is a comparison chart of the T line color development of the qualitative test paper of the control group I; Figure 7 -E is a comparison of the Raman spectra of the control group I test paper quantitative detection; Figure 7 -F is the Raman spectrum of the control group I test paper quantitative detection at 1586cm -1Comparison of signal strength at displacement locations.

[0024] Figure 8 This is a graph showing the batch reproducibility test results of the SERS-immunochromatographic test paper in Example 2. Figure 8 -A is a comparison chart of the T-line color development of the qualitative test paper in experimental group II; Figure 8 -B is a comparison of the Raman spectra of the quantitative detection of the test paper in experimental group II; Figure 8 -C is the Raman spectrum of the experimental group II test paper quantitative detection at 1586cm -1 Comparison of signal strength at displacement locations; Figure 8 -D is a comparison chart of the T line color development of the qualitative test paper of the control group II; Figure 8 -E is a comparison of the Raman spectra of the control group II test paper quantitative detection; Figure 8 -F is the Raman spectrum of the control group II test paper quantitative detection at 1586cm -1 Comparison of signal strength at displacement locations.

[0025] Figure 9 This is a graph showing the stability test results of the SERS-immunochromatographic test paper in Example 2. Figure 9 -A is a comparison chart of T-line color development of the test strips in the experimental groups stored at different times for qualitative detection; Figure 9 -B is a comparison of the Raman spectra of the test strips in the experimental group stored at different times for quantitative detection; Figure 9 -C is the Raman spectrum of the test paper quantitative detection of the experimental group stored at different times at 1586cm -1 Comparison of signal strength at displacement locations; Figure 9 -D is a comparison chart of T-line color development of the control group test strips stored at different times; Figure 9 -E is a comparison of the Raman spectra of the control group test strips stored at different times; Figure 9 -F is the Raman spectrum of the control group test paper stored at different times at 1586cm -1 Comparison of signal strength at displacement locations.

[0026] Figure 10 The figure is a schematic diagram of the process and principle of using the SERS-immunochromatographic test paper of the present invention to detect PEDV. DETAILED DESCRIPTION

[0027] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0028] In the present invention, "target detection object" refers to any substance that can be detected by immunoassay (chromatography), which can be pathogens such as viruses and pathogenic bacteria, or other substances that need to be detected, such as toxins, proteins, genes (fragments), biomarkers, drug molecules, etc.

[0029] In the present invention, "Raman immunoprobe" can also be referred to as "Raman immunolabel," or simply "immunoprobe" or "immunolabel," which have the same meaning and can be used interchangeably. It refers to an immunolabel that can specifically bind to a target analyte and produce a specific SERS signal.

[0030] In this invention, "surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper" is referred to as "SERS-immunochromatographic test paper" for short. They are synonymous and can be used interchangeably. This refers to a novel immunochromatographic test paper that combines SERS technology with (colloidal gold) immunochromatographic technology, resulting in a novel immunochromatographic test paper that combines the portability and ease of detection of immunochromatographic technology with the sensitivity, accuracy, and ease of (on-site) quantitative detection of SERS technology.

[0031] In the present invention, operation numbers, such as "first contact," "second contact," and "third contact," are primarily used to facilitate the distinction of operations within different steps in the description and do not limit the specific processing methods, conditions, or sequence corresponding thereto. For example, "first contact," "second contact," and "third contact" may employ the same processing method and conditions, or different processing methods and conditions.

[0032] In the present invention, the numbers of the reagents used, such as "antibody Ab1", "antibody Ab2", etc., are mainly used to facilitate the distinction of the antibodies used in different parts of the test paper in the description, and have no limiting effect on the specific types and sources of the antibodies used. For example. Antibody Ab1 is an antibody that can specifically bind to the target detection object, and antibody Ab2 is an antibody that can specifically bind to Ab1. The above antibodies can be mouse monoclonal antibodies with corresponding functions, or rabbit monoclonal antibodies or sheep monoclonal antibodies with corresponding functions, etc., and Ab1 and Ab2 can both be antibodies from the same source, or can also be antibodies from different sources (for example, both are mouse monoclonal antibodies, or one is a mouse monoclonal antibody and the other is a rabbit or sheep monoclonal antibody, etc.).

[0033] The inventors of the present invention discovered in their research that by assembling gold nanoparticles into a special structure capable of amplifying Raman signals and then modifying their surfaces with modifying groups such as signal marker molecules and target detection antibodies, a SERS tag suitable for use in immunochromatographic test strips can be obtained, thereby obtaining a SERS-immunochromatographic test strip that has the advantages of being easy to carry and use, and can be used for on-site direct quantitative detection using small, easy-to-carry, and easy-to-operate equipment such as Raman spectrometers.

[0034] Typically, SERS enhancement relies on nanometal markers with a certain curvature, which requires them to have a number of peaks or valleys. However, to obtain nanometal materials with the desired structure, large amounts of surfactants are often required. While the addition of surfactants allows the nanomaterial structure to meet the desired requirements, the presence of surfactants also significantly reduces the sensitivity of SERS-immunochromatographic test strips made with such markers and has a significant negative impact on the stability and storage conditions of the test strips.

[0035] After extensive research, the inventors of the present invention have cleverly discovered that the use of Ag + The welding method assembles gold nanoparticles into gold nanodimers, eliminating the need for surfactants and yielding a gold nanostructure that significantly enhances the SERS signal. Furthermore, the SERS tag, created by combining this gold nanodimer with a specific signal marker molecule and an antibody against the target, exhibits not only extremely high sensitivity, specificity, and accuracy, but also excellent reproducibility and long-term stability. This makes the SERS tag ideal for use in the preparation of SERS-immunochromatographic test strips, which combine SERS technology with immunochromatographic techniques.

[0036] Based on the above findings, the present invention provides a Raman immunoprobe, which includes a gold nanoparticle dimer, a signal marker molecule modified on the gold nanoparticle dimer, and an antibody Ab1 that specifically binds to a target detection object.

[0037] In the present invention, "gold nanodimer" refers to a gold nanostructure assembled from two gold nanoparticles of similar particle size (for example, two gold nanoparticles with a particle size ratio of no more than 1:1.2), with a subnanometer gap between the two gold nanoparticles, thereby having excellent SERS signal enhancement capabilities. Any gold nanodimer with the above structure can be applied to the Raman immunoprobe provided by the present invention. In order to further improve the SERS signal enhancement capability of the gold nanodimer and ensure its colloidal stability during the modification process of signal molecules and antibodies, according to a preferred embodiment of the present invention, the gold nanodimer is assembled from two gold nanoparticles with a particle size of no more than 30nm, with a spacing of no more than 1nm.

[0038] Preferably, the gold nanodimer is formed by assembling two gold nanoparticles with a particle size of 20-25 nm.

[0039] In the present invention, "signal marker molecule" refers to a compound with a clear SERS characteristic signal, and the qualitative and quantitative detection of the target object can be achieved by analyzing the signal peak position and intensity in the SERS detection results. Any compound with the above characteristics can be used as a signal marker molecule and is suitable for the Raman immunoprobe provided by the present invention. According to some preferred embodiments of the present invention, the signal marker molecule is selected from 4-nitrobenzenethiol (4-NTP, with a characteristic peak at 1330 cm -1 ), 4-mercaptopyridine (4-MPY, with a characteristic peak at 1105 cm -1 ), 4-mercaptobenzonitrile (4-MBN, with a characteristic peak at 2246 cm -1 ) and 4-mercaptobenzoic acid (4-MBA, with a characteristic peak at 1080 cm -1 Any one of the above.

[0040] According to a preferred embodiment of the present invention, the Raman immunoprobe further comprises a blocking agent. The function of the blocking agent is to prevent nonspecific adsorption of the Raman immunoprobe on the test strip from interfering with the measurement results. Any substance having this function and having no adverse effect on the accuracy, sensitivity, stability, etc. of the Raman immunoprobe provided by the present invention can be used as a blocking agent and is suitable for use in the Raman immunoprobe provided by the present invention.

[0041] Preferably, the blocking agent is selected from bovine serum albumin (BSA).

[0042] In the present invention, there is no particular limitation on the specific selection of antibody Ab1 that specifically binds to the target detection object. Any antibody that can specifically bind to the corresponding target detection object is suitable for use in the present invention. In the present invention, there is no particular limitation on the source of antibody Ab1. It can be a finished product obtained through commercial or custom channels, or it can be a related product prepared according to existing technologies.

[0043] According to a particularly preferred embodiment of the present invention, the Raman immunoprobe is a probe that uses porcine epidemic diarrhea virus (PEDV) as a target detection object.

[0044] Preferably, the antibody Ab1 that specifically binds to PEDV selected in the Raman immunoprobe is at least one selected from PEDV mouse monoclonal antibody, PEDV rabbit monoclonal antibody, and PEDV sheep monoclonal antibody.

[0045] The second aspect of the present invention provides a method for preparing the Raman immunoprobe according to the first aspect, the method comprising:

[0046] (1) Assembly of gold nanodimers;

[0047] (2) Modification of signal marker molecules and antibody Ab1 that specifically binds to the target substance ( Figure 10 A preferred preparation process is shown exemplarily in FIG.

[0048] In the present invention, the purpose of step (1) is to assemble the gold nanoparticles into a gold nanodimer structure capable of enhancing the SERS signal for use in a Raman immunoprobe. In order to prevent the adverse effects of reagents such as surfactants on the SERS signal enhancement effect of the Raman immunoprobe, the present invention preferably uses a method in step (1) that does not require the use of reagents having the above-mentioned functions and effects (such as surfactants, etc.) to assemble the gold nanodimer.

[0049] According to a preferred embodiment of the present invention, in step (1), Ag is used + The assembly of gold nanodimers was performed by welding. + Welding method refers to the use of Ag + The interaction between the gold nanoparticles and the ligand molecules on the surface of the gold nanoparticles induces the aggregation of the gold nanoparticles, and then the aggregation reaction is terminated by fish sperm DNA, promoting the formation of stable gold nanoparticle oligomers.

[0050] Preferably, step (1) comprises: fish sperm DNA (FsDNA), Ag + The solution is mixed with a colloidal gold solution in a buffer system to induce an assembly reaction.

[0051] In the present invention, the role of the buffer system is to +The welding reaction system provides a buffered environment to ensure good solubility of fish sperm DNA in solution and good adsorption to the gold nanoparticle surface. Any buffer known in the art that can fulfill these functions can be used in the method provided herein. According to a preferred embodiment of the present invention, the buffer system comprises TBE buffer, preferably at a concentration of 0.3x to 1x.

[0052] In the present invention, the role of fish sperm DNA is to quickly terminate the aggregation of gold nanoparticles and adsorb on the surface of the aggregated nanostructure to ensure its colloidal stability, facilitating subsequent separation and application. The present invention does not particularly limit the amount of fish sperm DNA used in step (1), as long as it can achieve the above-mentioned purpose. According to some preferred embodiments of the present invention, the amount of fish sperm DNA used is such that its final concentration in the reaction system is 1-5 μg / μL. Preferably, it is 1-3 μg / μL.

[0053] In the present invention, Ag + The role of the solution is to provide Ag for the reaction system + , thereby promoting the assembly of gold nanoparticles into gold nanodimers. + , and any aqueous solution of any reagent that has no adverse effect on the reaction can be used in the method provided by the present invention. + The solution may be an aqueous solution of silver nitrate.

[0054] In order to improve the yield of preparing gold nano dimers, more preferably, the Ag + The amount of solution used is such that Ag + The final concentration in the reaction system is 10-20 mM.

[0055] More preferably, the amount of colloidal gold solution used is such that the final concentration of the gold nanoparticles in the reaction system is not less than 30 nM, preferably 30-50 nM.

[0056] The inventors of the present invention have also discovered in their research that the particle size of the gold nanoparticles that constitute the gold nanodimer has a significant impact on the colloidal stability and SERS signal enhancement ability of the dimer. Moreover, the particle size ratio of the two gold nanoparticles that constitute the gold nanodimer can also affect the separation and purification effect of the gold nanodimer. Therefore, selecting a colloidal gold solution with an appropriate particle size and a relatively uniform particle size distribution for the preparation of gold nanodimers is very important for obtaining a Raman immunoprobe with high sensitivity, high accuracy and high stability.

[0057] Preferably, in the colloidal gold solution, nano-gold particles with a particle size not exceeding 30 nm account for more than 90% of the total amount of nano-gold particles, and preferably, nano-gold particles with a particle size of 20-25 nm account for more than 80% of the total amount of nano-gold particles.

[0058] In the method provided by the present invention, in step (1), the final concentration of each component in the reaction system can be adjusted by mixing the stock solutions of each component at a certain concentration and then adding additional water (e.g., sterile water, deionized water, ultrapure water, etc.). Alternatively, the amount of each component can be calculated according to the total volume of the required reaction system, and the components can be directly mixed after being prepared into solutions of appropriate concentrations.

[0059] In order to avoid the adverse effects of slight differences in the degree of mixing during the addition of samples on the assembly effect, according to a preferred embodiment of the present invention, the reagents used in the reaction system are mixed in a step-by-step mixing manner in step (1). Preferably, fish sperm DNA, a buffer system (such as TBE buffer, etc.) and Ag are first mixed. + The solution is first mixed, and then the first mixed product is second mixed with the colloidal gold solution to obtain a mixed reaction system. In order to fully mix the components in the reaction system, it is preferred that the first mixing and the second mixing are both carried out under vigorous stirring conditions.

[0060] Preferably, the first mixing conditions include: temperature 20-30° C., stirring speed 1500-2500 rpm, and time 10-40 s.

[0061] Preferably, the second mixing conditions include: temperature 20-30° C., stirring speed 1500-2500 rpm, and time 10-40 s.

[0062] In order to fully mix the reaction system, the second mixing time is preferably slightly longer than the first mixing time (for example, 10-20 seconds longer than the first mixing time).

[0063] According to a preferred embodiment of the present invention, the method of inducing the assembly reaction includes: allowing the mixed reaction system to stand at 20-30° C. for 40-80 minutes.

[0064] Preferably, step (1) further comprises the step of purifying the gold nanoparticle dimers in the product of the induced assembly reaction. Preferably, the purification comprises screening the uniform gold nanoparticle dimers by gel electrophoresis and recovering the uniform gold nanoparticle dimers.

[0065] Specifically, the mode that the gold nanometer dimer is carried out to purification can comprise adopting agarose gel (0.8-1.5 weight %) that reaction product is carried out electrophoresis, then the gel block with the gold nanometer dimer band is cut after pulverizing, then the gel after pulverizing is immersed in TBE buffer for 10-20h. Carry out centrifugal after immersion finishing, the precipitation that obtains is the gold nanometer dimer after purification. Usually, can adopt the color of the gold nanometer dimer suspension that direct observation adopts TBE buffer to resuspend, adopt the ultraviolet absorption spectrum of ultraviolet spectrophotometer scanning gold nanometer dimer suspension and adopt transmission electron microscope to observe the particle size of gold nanometer dimer and homogeneity and the mode such as uniformity to determine the quality and the homogeneity of the gold nanometer dimer after purification.

[0066] In the present invention, the purpose of step (2) is to modify the signal labeling molecule having a SERS characteristic signal peak and the antibody Ab1 that specifically binds to the target detection object on the surface of the gold nanodimer, thereby forming a Raman immune label.

[0067] Any method in the art that can modify the signal marker molecule and the antibody Ab1 on the surface of the gold nanodimer can be applied to the present invention, and the present invention has no particular restrictions on the specific modification method, conditions, and order. In order to enable the signal marker molecule and the antibody Ab1 to be more tightly bound to the surface of the gold nanodimer, according to a preferred embodiment of the present invention, step (2) includes first contacting the signal marker molecule with the gold nanodimer to obtain a gold nanodimer-signal marker molecule, and then contacting the antibody Ab1 that specifically binds to the target detection object with the gold nanodimer-signal marker molecule for a second time to obtain a gold nanodimer-signal marker molecule-antibody Ab1 conjugate.

[0068] During their research, the inventors discovered that the concentration of the signal labeling molecule in the first-contact reaction system is crucial for the preparation of Raman immunoprobes. If the concentration is too low, the Raman immunolabeled detection signal will be poor and sensitivity will decrease. If the concentration is too high, the gold nanoparticles will precipitate or aggregate.

[0069] Preferably, in the reaction system of the first contact, the final concentration of the signal marker molecule is 5-15 μM, preferably 5-10 μM.

[0070] Preferably, in the reaction system of the first contact, the final concentration of the gold nanodimer is 30-50 nM.

[0071] More preferably, in the reaction system of the first contact, the final concentration ratio of the signal marker molecule to the gold nanodimer is 3000-5000:1.

[0072] To ensure a sufficient reaction of the first contact system, preferably, the first contact conditions include: a temperature of 20-30°C and a duration of 1-5 hours. Preferably, the first contact is performed under an oscillation speed of 1000-1500 rpm to allow the signal marker molecules to more evenly bind to each gold nanoparticle dimer in the reaction system.

[0073] Preferably, in the reaction system of the second contact, the final concentration of the antibody Ab1 that specifically binds to the target detection substance is 1-10 μg / mL.

[0074] Preferably, in the reaction system of the second contact, the final concentration of the gold nanodimer-signal marker molecule is 0.5-2 nM.

[0075] More preferably, relative to 1 nM of the gold nanodimer-signal marker molecule, the amount of the antibody Ab1 that specifically binds to the target detection object is 2-20 μg / mL.

[0076] Preferably, the second contacting is performed at a temperature of 20-30° C. for 1-5 hours and preferably at an oscillation speed of 1000-1500 rpm to allow the signal marker molecules to bind more evenly to each gold nanoparticle dimer in the reaction system.

[0077] In order to improve the binding rate of antibody Ab1 modified to the surface of gold nanodimer, preferably, in step (2), before the antibody Ab1 that specifically binds to the target detection object is brought into second contact with the gold nanodimer-signal marker molecule, the gold nanodimer-signal marker molecule is first immunoconjugated and activated using EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to obtain an activated gold nanodimer-signal marker molecule, and then the activated gold nanodimer-signal marker molecule is brought into second contact with the antibody Ab1 that specifically binds to the target detection object.

[0078] More preferably, in the immunoconjugate activation reaction system, the final concentration of EDC is 20-30 μM, the final concentration of NHS is 20-30 μM, and preferably the final concentration ratio of EDC to NHS is 1:0.8-1.2.

[0079] More preferably, in the immunoconjugate activation reaction system, the final concentration of the gold nanodimer-signal marker molecule is 0.5-3 μM. Preferably, the amount of EDC used is 20-30 μM relative to 1 nM of the gold nanodimer-signal marker molecule.

[0080] According to a preferred embodiment of the present invention, the method further comprises step (3) Raman probe sealing.

[0081] The purpose of step (3) is to prevent the non-specific adsorption of the Raman immunoprobe on the test strip from interfering with the measurement results. The present invention has no particular restrictions on the specific blocking method or conditions, and any blocking method that can achieve the above purpose in the art can be applied to the present invention.

[0082] Preferably, step (3) comprises contacting the gold nanodimer-signal marker molecule-antibody Ab1 conjugate with a blocking agent for a third time.

[0083] Preferably, in the reaction system of the third contact, the final concentration of the blocking agent (preferably BSA) is 0.5-1.5 wt %.

[0084] Preferably, in the reaction system of the third contact, the final concentration of the gold nanodimer-signal marker molecule-antibody Ab1 conjugate is 0.5-2 nM.

[0085] Preferably, the amount of the blocking agent is 0.5-1.5 wt % relative to 1 nM of the gold nanoparticle dimer-signal marker molecule-antibody Ab1 conjugate.

[0086] The present invention further provides the use of the Raman immunoprobe described in the first aspect, or the Raman immunoprobe prepared by the method described in the second aspect, in detecting target detection objects in samples, especially in detecting target detection objects such as pathogens that have high requirements for detection speed, sensitivity and accuracy.

[0087] In the present invention, there is no particular limitation on the specific form of the above-mentioned application. Any method in the art that can achieve (qualitative and / or quantitative) detection of the target detection object in the sample by Raman spectroscopy-immunoconjugation method can be applied to the present invention. For example, the Raman immunoprobe provided by the present invention can be directly contacted with the sample and subjected to Raman spectroscopy detection and immunoassay (such as enzyme-linked immunosorbent assay, etc.). The Raman immunoprobe provided by the present invention can also be made into surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper for detection.

[0088] The third aspect of the present invention provides a surface-enhanced Raman spectroscopy-colloidal gold immunochromatography test paper, which includes a sample addition area, a conjugate area, an observation area, and a water absorption area arranged in sequence on a bottom plate, wherein the conjugate area includes a conjugate pad, and the conjugate pad contains a Raman immunoprobe, which is the Raman immunoprobe described in the first aspect or a Raman immunoprobe prepared according to the method described in the second aspect.

[0089] In the present invention, there is no particular limitation on the specific method of fixing the Raman immunoprobe on the conjugate pad, and any method commonly used in the art for preparing immunochromatographic test paper conjugate pads can be applied to the present invention. For example, the conjugate pad can be prepared by the dipping method, that is, the Raman immunoprobe is dispersed in a buffer solution (such as TEB buffer, etc.) to prepare a dispersion of a certain concentration, and then the material used for the conjugate pad (such as nitrocellulose membrane, etc.) is immersed in the dispersion for a period of time and then taken out and dried. For another example, the conjugate pad can also be prepared by the spraying method, that is, the Raman immunoprobe dispersion is directly sprayed on the surface of the material used for the conjugate pad by the spraying method.

[0090] Preferably, when the conjugate pad is prepared by the dipping method, the concentration of the Raman immunoassay probe in the Raman immunoassay dispersion is 0.5-2 nM.

[0091] Preferably, when the conjugate pad is prepared by a spraying method, the concentration of the Raman immunoassay probe in the Raman immunoassay dispersion used is 0.5-2 nM.

[0092] According to a preferred embodiment of the present invention, the sample addition area includes a sample pad. Preferably, the sample pad is made of glass fiber membrane and / or polyester fiber membrane.

[0093] According to a preferred embodiment of the present invention, the observation area includes a chromatographic membrane, on which a detection line (T line) and a control line (C line) are fixed. The detection line is coated with an antibody Ab1 that specifically binds to the target detection object, and the control line is coated with an antibody Ab2 that specifically binds to antibody Ab1. Preferably, the chromatographic membrane is made of nitrocellulose membrane. More preferably, at least one type of nitrocellulose membrane is used: HFO135, HF180 (manufactured by Milipore), and CN140 (manufactured by Sartorius).

[0094] In the present invention, there are no particular limitations on the specific methods and conditions for immobilizing the antibodies used for the T and C lines on the chromatographic membrane. Any method for immobilizing the T and C lines used in the art for preparing immunochromatographic test strips can be applied to the present invention. For example, a T and C line can be formed by spraying a predetermined concentration of antibody Ab1 solution and antibody Ab2 solution onto the surface of the chromatographic membrane.

[0095] Preferably, the concentration of the antibody Ab1 solution is not less than 0.25 mg / mL, preferably 0.5-1 mg / mL.

[0096] Preferably, the concentration of the antibody Ab2 solution is not less than 0.25 mg / mL, preferably 0.25-1 mg / mL.

[0097] According to a preferred embodiment of the present invention, the water absorbing area comprises absorbent paper.

[0098] Preferably, the absorbent paper has a gram weight of 90-150 g / m 2 , thickness 0.35-0.60mm, water creep speed 3-5cm / s, water storage capacity 400-600g / m 2 .

[0099] For the convenience of carrying and storage, preferably, the surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper also includes a shell, and the shell is provided with a sample addition hole at the sample addition area position, and an observation window is provided at the observation area position. The size of the observation window is such that the T line and the C line can be fully observed through the observation window (that is, when a positive sample containing the target detection object is added, the T line and the C line can be observed simultaneously through the observation window).

[0100] The fourth aspect of the present invention provides the use of the Raman immunoprobe described in the first aspect, or the Raman immunoprobe prepared according to the method described in the second aspect, or the surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper described in the third aspect in the detection of pathogens in samples (preferably on-site detection). In particular, the use of the Raman immunoprobe described in the first aspect, or the Raman immunoprobe prepared according to the method described in the second aspect, in the detection of pathogens in samples (preferably on-site detection).

[0101] The present invention further provides a method for detecting porcine epidemic diarrhea virus (PEDV) in a sample (the detection process and principle can be referred to Figure 10 ), the method comprises using the surface enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper described in the third aspect to detect the sample.

[0102] Specifically, the method may include the following steps:

[0103] (a) preparing a sample into a sample solution, and then adding the sample solution into the sample addition hole of the surface enhanced Raman spectroscopy-colloidal gold immunochromatography test paper;

[0104] (b) After adding the sample solution, let the test paper stand for at least 10 minutes before performing qualitative and / or quantitative analysis of the test results.

[0105] Preferably, in step (a), the concentration of PEDV in the sample solution is not less than 1 TCID 50 / mL. Preferably not less than 100TCID 50 / mL. More preferably, 1×10 2 -1×10 6 TCID 50 / mL. 1×10 n TCID 50 / mL means 1 ml of virus solution diluted 1×10 n After doubling, it can infect just half of the cells.

[0106] Preferably, the amount of the sample solution added to the sample well is preferably 40-80 μL.

[0107] Preferably, the standing time in step (b) is 10-20 minutes.

[0108] Preferably, in step (b), the sample is qualitatively analyzed by directly observing the color development of the T line and C line of the test paper. Preferably, the qualitative analysis method includes: when both the T line and the C line are colored, it indicates that the sample contains the target detection substance (positive sample); when the C line is not colored, regardless of whether the T line is colored, it is considered an invalid test; when the T line is not colored and only the C line is colored, it indicates that the sample does not contain the target detection substance (negative sample), or the target detection substance content is too low to be detected.

[0109] Preferably, in step (b), the sample is quantitatively analyzed by performing Raman spectroscopy on the T-line region. Preferably, 3-10 points are randomly selected in the T-line region for Raman spectroscopy analysis, and the average value is taken.

[0110] Since the detection limit of the qualitative detection of the SERS-immunochromatographic test paper provided by the present invention is higher than the detection limit of the quantitative detection, when the T line does not develop color but the C line develops color, further detection can be performed in the following manner to determine whether the sample contains the target detection substance:

[0111] (i) performing quantitative detection on the test paper, that is, performing Raman spectroscopy detection on the T-line region;

[0112] (ii) Increase the concentration of the sample solution and repeat the qualitative test (if necessary, perform the quantitative test on this basis).

[0113] If further testing still shows a negative result, it can be determined as a negative sample.

[0114] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0115] In the following examples, mouse anti-PEDV antibody (Ab1) was purchased from Beijing Jinnuo Biotech Co., Ltd. under the designation JN1401, and goat anti-mouse anti-PEDV antibody (Ab2) was purchased from Beijing Solebao Technology Co., Ltd. under the designation SPA131. Unless otherwise specified, all other reagents used were commercially available from reputable chemical or biological reagent suppliers and were of analytical grade.

[0116] The sample diluent used in the following examples comprises the following components: BB buffer (pH=8) supplemented with 1% by volume of Tween-20.

[0117] Preparation Example 1

[0118] This preparation example is used to illustrate the preparation of the Raman immunolabel provided by the present invention.

[0119] (1) Assembly of gold nanodimers

[0120] Place a rotor in the test tube and rotate the test tube at high speed (about 1500 rpm). Add 50 μL of ultrapure water, 100 μL of TBE buffer (1×), 400 μL of 5 μg / μL FsDNA solution and 150 μL of 100 mM AgNO3 solution in sequence. After rapid mixing for 20 seconds, add 300 μL of colloidal gold solution (prepared by trisodium citrate method, with a concentration of 30 nM, of which nanogold particles with a particle size of ≤30 μm account for about 90%, and nanogold particles with a particle size of 20-25 μm account for about 80%), mix for 30 seconds, stop stirring, and let it stand for 1 hour to wait for the reaction.

[0121] After the reaction, add 25 μL of 75% glycerol while rotating vigorously (approximately 1500 rpm) and mix thoroughly. Adjust the voltage to 120 V and the time to 60 minutes in the electrophoresis apparatus. After the run on a 1% agarose gel, cut and mince the gel with the dimer band. Soak overnight in TBE buffer (use enough TBE buffer to completely cover the gel fragments). Aliquot into centrifuge tubes and centrifuge at 5000 rpm / min for 15 minutes. Discard the supernatant and resuspend in a small amount of TBE buffer to prepare a gold nanodimer suspension with a concentration of approximately 1 nM.

[0122] The color of the gold nanodimer suspension was observed, the UV absorption spectrum was scanned using a UV spectrophotometer, and the uniformity and particle size of the gold nanodimer were observed using a transmission electron microscope to determine the quality level of the prepared gold nanodimer. Figure 1 .

[0123] Figure 1 -A shows the result of agarose gel electrophoresis. It can be seen from the figure that the bands of gold nanodimers are clear without any blurring or dragging, indicating that the gold nanodimers are not aggregated and are evenly distributed.

[0124] Figure 1 -B shows the purified gold nanodimer suspension. As can be seen from the figure, the gold nanodimer suspension is light purple, clear and transparent without impurities, indicating that the purification is successful.

[0125] Figure 1 -C shows the ultraviolet absorption spectrum of the gold nanodimer suspension, from which it can be seen that the gold nanodimer is relatively stable.

[0126] Figure 1-D shows a transmission electron microscopy image of the gold nanodimers. It can be seen from the image that the gold nanodimers have uniform shape, consistent size, and uniform distribution.

[0127] It can be seen from the above pictures that the quality of the gold nanodimers prepared by the above method is relatively high.

[0128] (2) Modification of the signal marker molecule and the antibody Ab1 that specifically binds to the target detection substance.

[0129] Place 1 mL of the gold nanodimer suspension in a clean, transparent glass bottle. Add 20 μL of a 4-MBA solution of a desired concentration. Mix thoroughly at room temperature and shake at 1000 rpm for 3 hours. After the reaction, centrifuge at 4000 rpm for 15 minutes, discard the supernatant, and resuspend the precipitate in 1 mL of ultrapure water. This yields a gold nanodimer-4-MBA suspension.

[0130] Add 2.5 μL of a 10 mM EDC solution and 2.5 μL of a 10 mM NHS solution to the gold nanodimer-4-MBA suspension, mix thoroughly, and let it react for 15 minutes. After the reaction, centrifuge at 4000 rpm for 15 minutes, discard the supernatant, and resuspend in 1 mL of ultrapure water to obtain an activated gold nanodimer-4-MBA suspension.

[0131] Add 5 μL of 1 mg / mL mouse anti-PEDV antibody (Ab1) to the activated gold nanodimer-4-MBA suspension. Mix thoroughly and shake at 1000 rpm for 2 hours. After the reaction, centrifuge at 4000 rpm for 15 minutes, discard the supernatant, and resuspend in 1 mL of ultrapure water to obtain a gold nanodimer-4-MBA-Ab1 suspension.

[0132] 100 μL of 10 wt% BSA solution was added to the gold nanodimer-4-MBA-Ab1 suspension, mixed, and then blocked at 1000 rpm for 1 hour to obtain a Raman immunoprobe suspension.

[0133] Preparation Example 2

[0134] This preparation example is used to illustrate the preparation of the SERS-immunochromatographic test paper provided by the present invention.

[0135] Cut the NC membrane, glass fiber membrane and absorbent paper into rectangular strips for later use.

[0136] A specific concentration of Ab1 solution is sprayed onto the NC membrane perpendicular to the long edge of the membrane strip to form a T-line. A specific concentration of Ab2 solution is also sprayed onto the NC membrane strip parallel to the T-line to form a C-line. This serves as a chromatography membrane. During spraying, the distance between the T-line and the C-line should be controlled so that they can be observed simultaneously through the observation window in the housing.

[0137] Ultrapure water and the Raman probe prepared in Preparation Example 1 were used to prepare a Raman probe suspension with a concentration of about 1 nM. The Raman probe suspension was fixed on a new rectangular NC membrane strip by spraying to serve as a conjugate pad.

[0138] Assembly of SERS-immunochromatographic test paper: Take the plastic chromatography membrane shell, and install the absorbent paper, binding pad, chromatography membrane and sample pad (glass fiber membrane) into it from bottom to top, adjust the position of the membrane strip, and place the sample loading port reserved on the shell at the sample pad position, so that the T line and C line can be observed at the same time at the observation window position reserved on the shell.

[0139] Example 1

[0140] This example is used to illustrate the optimization of Raman signal intensity of the SERS-immunochromatographic test paper provided by the present invention.

[0141] (1) Optimization of Raman immunoprobes

[0142] Prepare 4-MBA solutions with different concentration gradients, and prepare Raman immunoprobes according to the method of Preparation Example 1. The concentration gradient of 4-MBA solution is set to: 2×10 -4 M, 4×10 -4 M, 6×10 -4 M, 8×10 -4 M, 10×10 -4 M, 12×10 -4 M, 14×10 -4 M.

[0143] During the preparation process, the purified gold nanodimer suspension, gold nanodimer-4-MBA suspension, gold nanodimer-4-MBA-Ab1 suspension and Raman immunoprobe suspension were subjected to UV spectroscopic detection. Figure 2 -A (shown 4×10 -4 The UV spectra of the Raman immunoprobe prepared with a 4-MBA solution containing 100 μM of MBA were plotted. The results of Raman immunoprobes prepared with other 4-MBA concentrations were similar. As can be seen from the figure, the UV absorption curve of the dimer prepared after modification with the signal molecule MBA, linking with the PEDV antibody, and blocking with BSA to form the Raman signal molecule exhibits a significant red shift compared to the original dimer, demonstrating that both the signal molecule and the antibody were successfully modified onto the dimer.

[0144] After the concentration of the Raman immunoprobe suspension was adjusted to 1 nM, Raman spectroscopy was performed. Figure 2 -B and Figure 2 -C. From Figure 2-C, it can be seen that the Raman signal intensities of the Raman immunoprobes prepared with different concentrations of 4-MBA solutions are different. -4 The signal was strongest when the final concentration of 4-MBA solution was 7.84 μM (corresponding to the final concentration of 4-MBA solution in the reaction system). Then, the signal gradually weakened with the increase of the concentration of 4-MBA solution. -4 After 4 × 10 -4 After M, the gold nanoparticles began to aggregate, and the aggregation became more serious with the increase of concentration. Figure 2 -B, where the significant decrease in the coupling peak at 630 nm also indicates that the gold nanodimers have aggregated.

[0145] (II) Optimization of Ab1 concentration in T-line

[0146] The concentration was 4×10 -4 M 4-MBA solution, Raman immunoprobe prepared according to the method of Preparation Example 1, and SERS-immunochromatographic test paper prepared according to the method of Preparation Example 2. During the test paper preparation process, Ab1 solutions with different concentration gradients were prepared and used to coat T lines to obtain SERS-immunochromatographic test paper with different antibody concentrations. The concentrations of Ab1 solutions were set to 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1 mg / mL.

[0147] The PEDV was prepared with sample diluent to a drug loading of 1×10 2 TCID 50 / mL of PEDV positive sample fluid.

[0148] Two SERS-immunochromatographic test strips of each antibody concentration were taken and marked as experimental and control groups, respectively. 60 μL of PEDV-positive sample solution was dripped into the sample port of the experimental group test strips, while 60 μL of sample diluent was dripped into the sample port of the control group test strips.

[0149] After 15 minutes of rest, the T-line color of each test paper was observed, and Raman spectroscopy was performed on the T-line area of ​​each test paper (using a Raman spectrometer RTS2 from Zhuoli Hanguang Company. Five points were selected in the T-line area of ​​each test paper for detection, with a spacing of 100 μm between each detection point, and the average value was taken). The detection conditions included an excitation time of 5000 ms and an excitation power of 50%. For detailed results, see Figure 3 .

[0150] Figure 3 -A to Figure 3-C shows the color development of the T line of the test paper in the experimental group, the Raman signal intensity and the Raman spectrum curve. It can be seen that the color development is lighter when the concentration of the antibody Ab1 coated with the T line is 0.25 mg / mL, and the color development of the other three concentrations is relatively similar. The Raman signal intensity also shows similar results. However, no matter how the concentration of the antibody Ab1 coated with the T line changes, it shows a peak at 1586 cm in the Raman spectrum. -1 Characteristic peaks at displacement.

[0151] Figure 3 -D to Figure 3 Figures 1 and 2 show the T-line color development, Raman signal intensity, and Raman spectrum curves of the control group test paper. The figures show that the T-line test papers with different coating concentrations gave good negative results, with no false positives observed in either T-line color development or Raman spectrum detection.

[0152] Example 2

[0153] This example is used to illustrate the detection of the sensitivity, specificity, repeatability and stability of the SERS-immunochromatographic test paper provided by the present invention.

[0154] The test paper used in this example is a 4×10 -4 M of 4-MBA solution, a Raman immunoprobe prepared according to the method of Preparation Example 1, and then a SERS-immunochromatographic test paper was prepared according to the method of Preparation Example 2, wherein the concentration of the Ab1 solution used for coating the T line was 0.5 mg / mL.

[0155] (1) Sensitivity detection

[0156] The PEDV virus load was prepared with sample diluent to be 1×10 6 TCID 50 / mL, 1×10 5 TCID 50 / mL, 1×10 4 TCID 50 / mL, 1×10 3 TCID 50 / mL, 1×10 2 TCID 50 / mL, 1×10 1 TCID 50 / mL and 1×10 0 TCID 50 / mL, 1×10 -1 TCID 50 / mL of positive sample solution standard.

[0157] According to the method of test (II) in Example 1, the above-mentioned PEDV positive sample liquid standards with different drug loading amounts were qualitatively and quantitatively tested. Figure 4 .

[0158] Figure 4 -A shows the color development of the T line in the qualitative detection of positive sample solution standards with different concentrations from high to low from left to right. As can be seen from the figure, as the PEDV load in the positive sample solution standard decreases, the color of the T line gradually becomes lighter, and the detection limit of the qualitative detection is 1×10 2 TCID 50 / mL (No. 5 test paper, the T line of the test paper after it does not show color).

[0159] Figure 4 -B shows the Figure 4 -A is the Raman spectrum of the T-line area in each test paper when quantitative detection is performed. As can be seen from the figure, the detection limit of quantitative detection is 1×10 0 TCID 50 / mL, and the characteristic signal peak of 4-MBA (1586cm -1 The signal intensity of the positive sample solution (at the displacement point) gradually increases with the increase of the PEDV virus load in the positive sample solution standard.

[0160] Figure 4 -C shows the Figure 4 -B medium 1586cm -1 The linear fitting diagram of the signal intensity at the displacement point and the concentration of the positive sample solution standard is shown in Figure 1. As can be seen from the figure, the signal intensity of the 4-MBA characteristic signal peak has a good linear relationship with the PEDV load in the positive sample solution standard. The linear equation is y = 3560.0098x + 1843.52691, R 2 =0.9916.

[0161] (2) Specificity detection

[0162] According to the method in Experiment (II) of Example 1, the sample diluent (negative control, No. 1), rotavirus solution (No. 2), porcine circovirus solution (No. 3), pseudorabies virus solution (No. 4), transmissible gastroenteritis virus solution (No. 5), Staphylococcus aureus solution (No. 6), Salmonella typhimurium solution (No. 7), Vibrio alginolyticus solution (No. 8), Salmonella enteritidis solution (No. 9), Vibrio parahaemolyticus solution (No. 10), Escherichia coli solution (No. 11) and porcine epidemic diarrhea virus solution (No. 12) were qualitatively and quantitatively detected using SERS-immunochromatographic test paper. The virus solution and bacterial solution were both prepared using the sample diluent, and the virus loading capacity of the virus solution was 1×10 3 TCID 50 / mL, the concentration of the bacterial solution is 1×10 3 TCID 50 / mL. Figure 5 .

[0163] Figure 5 -A shows, from left to right, the qualitative detection results of the SERS-immunochromatographic test strips on the aforementioned virus or bacterial dilutions. As can be seen from the figure, except for the test strip on the far right that detects porcine epidemic diarrhea virus solution, the T lines of the other test strips do not show color.

[0164] Figure 5 -B shows the quantitative detection results of the above virus or bacteria dilutions by SERS-immunochromatographic test paper from bottom to top. As can be seen from the figure, except for curve No. 12, the characteristic peak of 4-MBA does not appear in the detection spectrum of the other samples.

[0165] Figure 5 Figure C shows the Raman spectra of each sample at 1586 cm -1 As can be seen from the figure, except for sample No. 12, the negative control and other samples did not show false positives.

[0166] (3) Repeatability test

[0167] (1) Intra-batch repeatability

[0168] 12 test strips were randomly selected from the same batch and divided into two groups, 6 in each of the experimental group and the control group. 4 TCID 50 / mL of PEDV-positive sample solution (experimental group) and sample dilution without PEDV added (control group) were tested. Figure 6 .

[0169] Figure 6 -A to 6-C are the test results of the experimental group. Figure 6 -A shows the color development of the T lines of the 6 test strips in the experimental group. As can be seen from the figure, the color development is relatively consistent. Figure 6 -B shows the Raman spectra of three repeated tests on each test strip in the experimental group (numbers 1-3 on the y-axis correspond to test strip T-1 in the experimental group, and so on). As can be seen from the figure, whether it is the repeated test spectra of the same test strip or the test spectra of different test strips, the peak positions and signal intensities in the Raman spectra show high consistency. Figure 6 -C shows the Raman detection spectrum of the five detection points on each test paper of the experimental group at 1586 cm -1The Raman signal intensity at the displacement point (numbers 1-5 on the x-axis correspond to test paper T-1 in the experimental group, and so on) can be seen from the figure. Its standard deviation is only 3.34%, and the signal intensity obtained at different detection points of each test paper is also relatively consistent.

[0170] Figure 6 -D to 6-F are the test results of the control group. Figure 6 -D shows the color development of the T lines of the 6 test strips in the control group. As can be seen from the figure, the T lines of the test strips in the control group did not show any color. Figure 6 -E is the Raman spectrum of three repeated tests on each test paper in the control group (numbers 1-3 on the y-axis correspond to test paper D-1 in the control group, and so on). It can be seen from the figure that the Raman detection spectra of the test papers in the control group do not show the characteristic peaks of 4-MBA. Figure 6 -F is the Raman detection spectrum of the five detection points on each test strip of the control group at 1586 cm -1 The Raman signal intensity at the displacement point (numbers 1-5 on the x-axis correspond to the D-1 test paper in the control group, and so on). It can be seen from the figure that the different detection points of each test paper are at 1586cm -1 No obvious signals were shown at the displacement locations.

[0171] The above results indicate that the SERS-immunochromatographic test paper provided by the present invention has good intra-batch reproducibility.

[0172] (2) Batch-to-batch reproducibility

[0173] Four test strips were randomly selected from each of the three batches of test strips and divided into four groups, with three test strips from different batches in each group. 4 TCID 50 / mL of PEDV positive sample solution (experimental group I), the virus load was 1×10 2 TCID 50 / mL of PEDV-positive sample solution (experimental group II) and sample dilutions without PEDV added (control group I and control group II) were tested. Figure 7 and Figure 8 .

[0174] Figure 7 -A to 7-C are the test results of the experimental group. Figure 7 -A shows the color development of the T lines of the three test strips in experimental group I. As can be seen from the figure, the color development is relatively consistent. Figure 7-B shows the Raman spectra of five repeated tests on each test paper in experimental group I (numbers 1-5 on the y-axis correspond to test paper T-1 in experimental group I, and so on). It can be seen from the figure that whether it is the repeated test spectra of the same test paper or the test spectra of different test papers, the peak positions and signal intensities in the Raman spectra show high consistency. Figure 7 -C shows the Raman detection spectrum of the five detection points on each test paper in experimental group I at 1586 cm -1 The Raman signal intensity at the displacement points (numbers 1-5 on the x-axis correspond to test paper T-1 in experimental group I, and so on) can be seen from the figure. Its standard deviation is only 6.39%, and the signal intensity obtained at different detection points of each test paper is also relatively consistent.

[0175] Figure 7 -D to 7-F are the test results of control group I. Figure 7 -D shows the color development of the T lines of the three test papers in the control group I. As can be seen from the figure, the T lines of the test papers in the control group I did not develop color. Figure 7 -E is the Raman spectrum of each test paper in the control group I repeated five times (the numbers 1-5 on the y-axis correspond to the test paper D-1 in the control group I, and so on). It can be seen from the figure that the Raman detection spectrum of the test paper in the control group I does not show the characteristic peak of 4-MBA. Figure 7 -F is the Raman detection spectrum of the five detection points on each test strip of the control group I at 1586cm -1 The Raman signal intensity at the displacement point (numbers 1-5 on the x-axis correspond to the D-1 test paper in the control group I, and so on). It can be seen from the figure that the different detection points of each test paper are at 1586 cm -1 No obvious signals were shown at the displacement locations.

[0176] Figure 8 -A to 8-C are the test results of experimental group II. Figure 8 -A shows the color development of the T lines of the three test strips in experimental group II. As can be seen from the figure, the color development is relatively consistent. Figure 8 -B shows the Raman spectra of five repeated tests on each test paper in experimental group II (numbers 1-5 on the y-axis correspond to test paper T-1 in experimental group II, and so on). It can be seen from the figure that whether it is the repeated test spectra of the same test paper or the test spectra of different test papers, the peak positions and signal intensities in the Raman spectra show high consistency. Figure 8 -C shows the Raman detection spectrum of the five detection points on each test paper in Experimental Group II at 1586 cm -1The Raman signal intensity at the displacement points (numbers 1-5 on the x-axis correspond to test paper T-1 in experimental group II, and so on) can be seen from the figure. Its standard deviation is only 5.27%, and the signal intensity obtained at different detection points of each test paper is also relatively consistent.

[0177] Figure 8 -D to 8-F are the test results of control group I. Figure 8 -D shows the color development of the T lines of the three test strips in the control group II. As can be seen from the figure, the T lines of the test strips in the control group II did not develop color. Figure 8 -E is the Raman spectrum of each test paper in the control group II repeated for five times (the numbers 1-5 on the y-axis correspond to the test paper D-1 in the control group II, and so on). It can be seen from the figure that the Raman detection spectrum of the test paper in the control group II does not show the characteristic peak of 4-MBA. Figure 8 -F is the Raman detection spectrum of the five detection points on each test strip of the control group II at 1586 cm -1 The Raman signal intensity at the displacement point (numbers 1-5 on the x-axis correspond to the D-1 test paper in the control group II, and so on). It can be seen from the figure that the different detection points of each test paper are at 1586cm -1 No obvious signals were shown at the displacement locations.

[0178] The above results indicate that the SERS-immunochromatographic test paper provided by the present invention has good intra-batch reproducibility.

[0179] (4) Stability

[0180] Take 12 test strips from the same batch and store them at 4°C. Take out 2 strips each at the first month (30 days), second month (60 days), third month (90 days), fourth month (120 days), fifth month (150 days) and sixth month (180 days) of storage. One of the strips is used to detect the virus load of 1×10 5 TCID 50 / mL of PEDV positive sample solution (experimental group), and 1 was used to test the sample dilution solution (control group). Qualitative and quantitative detection were performed according to the method in Example 1, Test (II). Figure 9 .

[0181] Figure 9 -A shows the T line color development of the test strips of the experimental groups after storage for 1 to 6 months from left to right. It can be seen from the figure that the T line color development of each test strip is relatively consistent. Figure 9 -B is the Raman spectrum of the quantitative detection of the test strips in the experimental group after storage for 1-6 months. It can be seen from the figure that each test strip shows the characteristic peak of 4-MBA, and the signal intensity is relatively consistent. Figure 9 -C is the 1586cm in the Raman spectrum of each test paper in the experimental group-1 As can be seen from the figure, the standard deviation of the detection signal intensity of the test strips with different storage time is only 1.18%, indicating that the Raman signal intensity does not change significantly with the extension of storage time.

[0182] Figure 9 -D shows the color development of the T line of the control group test strips after storage for 1 to 6 months from left to right. It can be seen from the figure that the T line of each test strip did not develop color. Figure 9 -E is the Raman spectrum of the control group test strips after storage for 1-6 months. It can be seen from the figure that none of the test strips showed the characteristic peak of 4-MBA. Figure 9 -F is the 1586cm in the Raman spectrum of each test paper in the control group -1 The Raman signal intensity at the displacement point can be seen from the figure. No false positive results occurred in the test strips at different storage times, indicating that as the storage time increases, the negative detection results of the test strips are better and no obvious changes occur.

[0183] These results demonstrate that the SERS-immunochromatographic test strips provided by the present invention possess extremely high sensitivity, with quantitative detection exhibiting higher sensitivity than qualitative detection. The strips also possess good specificity and demonstrate excellent reproducibility both within and between batches. Furthermore, the strips exhibit exceptional stability, maintaining their T-line color development and Raman signal intensity even after six months of storage at 4°C.

[0184] Example 3

[0185] This example is used to illustrate the effect of the SERS-immunochromatographic test paper provided by the present invention in actual sample detection.

[0186] The test paper used in this example is a 4×10 -4 M of 4-MBA solution, a Raman immunoprobe prepared according to the method of Preparation Example 1, and then a SERS-immunochromatographic test paper was prepared according to the method of Preparation Example 2, wherein the concentration of the Ab2 solution used for coating the T line was 0.5 mg / mL.

[0187] Take seven test tubes and add 1g of fresh healthy pig feces to each tube. 50 The amount of PEDV virus added was calculated using the unit [unit / mL] as the unit. 0 (negative control, sample diluent only), 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 units of PEDV were added to the test tubes, mixed, and used as simulated positive samples for quantitative detection using SERS-immunochromatographic test strips. The detection method was the same as that of Experiment (II) in Example 1. The specific amount of PEDV-positive sample solution added and the detection results are detailed in Table 1.

[0188] Table 1 PEDV addition amount in simulated positive samples and test results

[0189]

[0190] It can be seen from the data in Table 1 that the SERS-immunochromatographic test paper provided by the present invention has extremely high accuracy when testing actual samples, and can meet the needs of obtaining rapid and accurate test results for PEDV on-site testing.

[0191] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A Raman immunoprobe, characterized in that: The Raman immunoprobe includes a gold nanoparticle dimer, a signal marker molecule modified on the gold nanoparticle dimer, and an antibody Ab1 that specifically binds to a target detection object; The gold nanodimer is formed by assembling two gold nanoparticles with a particle size not exceeding 30 nm and a distance between them not exceeding 1 nm.

2. The Raman immunoprobe according to claim 1, wherein The gold nanodimer is formed by assembling two gold nanoparticles with a particle size of 20-25 nm; And / or, the signal marker molecule is selected from any one of 4-nitrobenzenethiol, 4-mercaptopyridine, 4-mercaptobenzonitrile and 4-mercaptobenzoic acid; And / or, the Raman immunoprobe further comprises a blocking agent.

3. The method for preparing the Raman immunoprobe according to claim 1 or 2, characterized in that: The method comprises: (1) Assembly of gold nanodimers; (2) Modification of the signal marker molecule and the antibody Ab1 that specifically binds to the target detection substance.

4. The method according to claim 3, wherein: In step (1), Ag + The gold nanodimers were assembled by welding method.

5. The method according to claim 4, wherein Step (1) includes: fish sperm DNA, Ag + The solution is mixed with a colloidal gold solution in a buffer system to induce an assembly reaction.

6. The method according to claim 5, wherein: The buffer system includes TBE buffer.

7. The method according to claim 6, wherein: The buffer system includes TBE buffer with a concentration of 0.3 -1 .

8. The method according to claim 5, wherein The amount of fish sperm DNA is such that the final concentration in the reaction system is 1-5 μg / μL; and / or, the Ag + The amount of solution used is such that Ag + The final concentration in the reaction system is 10-20mM; and / or, the colloidal gold solution is used in an amount such that the final concentration of the gold nanoparticles in the reaction system is not less than 30 nM; and / or, in the colloidal gold solution, nano-gold particles with a particle size not exceeding 30 nm account for more than 90% of the total amount of nano-gold particles; And / or, the method of inducing the assembly reaction includes: allowing the mixed reaction system to stand at 20-30° C. for 40-80 minutes.

9. The method according to claim 8, wherein The amount of colloidal gold solution used is such that the final concentration of gold nanoparticles in the reaction system is 30-50 nM; And / or, in the colloidal gold solution, nano-gold particles with a particle size of 20-25 nm account for more than 80% of the total amount of nano-gold particles.

10. The method according to any one of claims 3 to 9, wherein: Step (1) also includes the operation of purifying the gold nanodimer in the product of the induced assembly reaction.

11. The method according to claim 10, wherein: The purification comprises screening uniform gold nanometer dimers by gel electrophoresis and recovering the uniform gold nanometer dimers.

12. The method according to claim 3, wherein: Step (2) includes first bringing the signal marker molecule into contact with the gold nanodimer to obtain a gold nanodimer-signal marker molecule, and then bringing the antibody Ab1 that specifically binds to the target detection object into contact with the gold nanodimer-signal marker molecule to obtain a gold nanodimer-signal marker molecule-antibody Ab1 conjugate.

13. The method according to claim 12, wherein: In the first contact reaction system, the final concentration of the signal marker molecule is 5-15 μM; and / or, in the reaction system of the first contact, the final concentration of the gold nanodimer is 1-5 nM; And / or, the conditions of the first contact include: temperature 20-30° C., time 1-5 h; and / or, in the reaction system of the second contact, the final concentration of the antibody Ab1 that specifically binds to the target detection substance is 1-10 μg / mL; and / or, in the reaction system of the second contact, the final concentration of the gold nanodimer-signal marker molecule is 1-5 nM; And / or, the conditions for the second contact include: temperature 20-30° C., time 1-5 h.

14. The method according to claim 13, wherein: In the first contact reaction system, the final concentration ratio of the signal marker molecule to the gold nanodimer is 3000:1-5000:1; and / or, performing the first contacting under an oscillation condition of 1000-1500 rpm; and / or, relative to 1 nM of the gold nanodimer-signal marker molecule, the amount of the antibody Ab1 that specifically binds to the target detection object is 200-2000 μg / mL; And / or, the second contacting is performed under shaking conditions of 1000-1500 rpm.

15. The method according to any one of claims 12 to 14, wherein: In step (2), before the antibody Ab1 that specifically binds to the target detection object is brought into contact with the gold nanodimer-signal marker molecule for the second time, the gold nanodimer-signal marker molecule is first immunoconjugated and activated using EDC and NHS to obtain an activated gold nanodimer-signal marker molecule, and then the activated gold nanodimer-signal marker molecule is brought into contact with the antibody Ab1 that specifically binds to the target detection object for the second time.

16. The method according to claim 15, wherein In the immunoconjugate activation reaction system, the final concentration of EDC is 20-30 μM, and the final concentration of NHS is 20-30 μM; And / or, in the immunoconjugate activation reaction system, the final concentration of the gold nanodimer-signal marker molecule is 0.5-3 nM.

17. The method according to claim 16, wherein In the immunoconjugate activation reaction system, the final concentration ratio of EDC and NHS is 1:0.8-1:1.2; And / or, in the immunoconjugate activation reaction system, the amount of EDC used is 20-30 μM relative to 1 nM of the gold nanodimer-signal marker molecule.

18. The method according to claim 3, wherein The method further comprises step (3) Raman probe sealing.

19. The method according to claim 18, wherein Step (3) includes bringing the gold nanodimer-signal marker molecule-antibody Ab1 conjugate into a third contact with a blocking agent.

20. The method according to claim 19, wherein In the reaction system of the third contact, the final concentration of the blocking agent is 0.5-1.5% by weight; And / or, in the reaction system of the third contact, the final concentration of the gold nanoparticle dimer-signal marker molecule-antibody Ab1 conjugate is 0.5-2 nM.

21. The method according to claim 20, wherein The amount of the blocking agent used is 0.5-1.5 wt % relative to 1 nM of the gold nanoparticle dimer-signal marker molecule-antibody Ab1 conjugate.

22. A surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper, comprising a sample addition area, a conjugate area, an observation area, and a water absorption area sequentially arranged on a bottom plate, wherein: The conjugate area includes a conjugate pad, characterized in that the conjugate pad contains a Raman immunoprobe, and the Raman immunoprobe is the Raman immunoprobe according to claim 1 or 2 or a Raman immunoprobe prepared according to the method of any one of claims 3-21.

23. The surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper according to claim 22, wherein: The conjugate pad is made of nitrocellulose membrane; and / or, the sample application area includes a sample pad; And / or, the observation area includes a chromatographic membrane, on which a detection line and a control line are fixed, wherein the detection line is coated with an antibody Ab1 that specifically binds to the target detection object, and the control line is coated with an antibody Ab2 that specifically binds to the antibody Ab1; And / or, the water absorbing area includes absorbent paper.

24. The surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper according to claim 23, wherein: The sample pad is made of glass fiber membrane and / or polyester fiber membrane; And / or, the chromatography membrane is made of nitrocellulose membrane; And / or, the absorbent paper has a gram weight of 90-150 g / m 2 , thickness 0.35-0.60mm, water creep speed 3-5cm / s, water storage capacity 400-600g / m 2 .

25. The surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper according to any one of claims 22 to 24, wherein: The surface enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper also includes a shell, which is provided with a sample addition hole at the sample addition area and an observation window at the observation area. The size of the observation window allows the detection line and the control line to be fully observed through the observation window.

26. Use of the Raman immunoprobe according to claim 1 or 2, or the Raman immunoprobe prepared according to the method according to any one of claims 3 to 21, or the surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper according to any one of claims 22 to 25 in the detection of pathogens in a sample, wherein: The applications described are non-diagnostic applications.

27. Use of the Raman immunoprobe according to claim 1 or 2, or the Raman immunoprobe prepared according to the method according to any one of claims 3 to 21, or the surface-enhanced Raman spectroscopy-colloidal gold immunochromatographic test paper according to any one of claims 22 to 25 in the detection of porcine epidemic diarrhea virus in a sample, wherein: The applications described are non-diagnostic applications.

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