CN118067688A

CN118067688A - SERS substrate nanoparticle based on silicon core gold shell, SERS label and detection product

Info

Publication number: CN118067688A
Application number: CN202410315240.4A
Authority: CN
Inventors: 肖瑞; 沈皖珠
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2024-03-19
Filing date: 2024-03-19
Publication date: 2024-05-24

Abstract

The invention provides SERS substrate nanoparticles and SERS labels based on silicon core gold shells and detection products, and relates to the technical field of nanomaterials. The SERS substrate nanoparticle based on the silicon core gold shell sequentially comprises an SiO ₂ nanoparticle inner core, a electropositive polymer layer, a first Au nano layer, a PEI intermediate layer with the thickness of 0.5-2 nm and a second Au nano layer from inside to outside, has good activity and SERS stability, and the SERS label taking the SERS substrate nanoparticle or the SERS label as a substrate can excite stronger Raman signals, and has the advantages of simple preparation method and good sensitivity and specificity.

Description

SERS substrate nanoparticle based on silicon core gold shell, SERS label and detection product

Technical Field

The invention relates to the technical field of nano materials, in particular to a SERS substrate nano particle based on a silicon core gold shell, a SERS label and a detection product.

Background

Microorganisms responsible for acute respiratory infections mainly include respiratory viruses such as influenza a virus, human respiratory syncytial virus and human adenovirus; and common respiratory bacteria such as streptococcus pneumoniae and haemophilus influenzae. Given that respiratory viral and bacterial infections have similar clinical manifestations, such as cough, dyspnea and fever, accurate identification of pathogenic microorganisms from symptoms is difficult. Furthermore, respiratory viral infections are a common clinical manifestation in patients with secondary bacterial infections. Thus, the simultaneous accurate identification of viruses and bacteria is critical to avoiding misdiagnosis.

Currently, real-time fluorescent polymerase chain reaction and gene sequencing are the main methods of microbial assay. Although these nucleic acid amplification-based techniques have higher sensitivity and accuracy, they also have the following drawbacks: the operation steps are complex, and the requirement on the detection environment is high; the detection time is long, and usually, the detection can be completed within 2-12 hours; one reaction makes it difficult to detect a variety of pathogenic microorganisms; furthermore, most respiratory viruses are RNA viruses, and therefore, it is necessary to reverse transcribe the viral RNA genome into DNA as a template DNA for PCR amplification; when the detection target is bacteria, lysis is also required prior to detection. Thus, there is an urgent need to develop a method that supports rapid, accurate, and universal detection of viruses and bacteria.

The lateral flow immunochromatography (Lateral flow immunoassay, LFA) technology can directly detect structural proteins or particles of a target pathogen by using antigen-antibody immune reaction, and has the potential of realizing simultaneous detection of viruses and bacteria. However, currently available LFA kits rely mainly on colloidal gold nanoparticles to provide visual colorimetric signals, with lower sensitivity and insufficient quantification capability.

In recent years, a novel LFA technology based on surface enhanced raman effect (SERS) has attracted great interest because of its distinct advantages over other signal output modes such as colorimetric, magnetic and fluorescent signals. Firstly, the SERS nano label is utilized to replace the traditional colorimetric nano material, and the SERS-LFA can provide ultrasensitive (single molecule level), stable (no light bleaching) and specific (fingerprint characteristic) Raman signals for quantitative detection of targets. Second, the introduction of multiple raman molecules with different raman signals into the SERS tag can create encoding capability on the LFA strip, allowing multiple targets to be analyzed simultaneously, thus providing superior multiple detection capability of SERS-LFA over other methods.

Development of high-performance SERS nanotags is a key to improving the overall performance of SERS-LFA systems. Although tens of SERS materials, such as Au@Ag, si@Au, si@Ag and Fe ₃O₄ @ Au, are used as substrates for raman molecules in SERS nanotags in LFA methods, SERS nanotags using these materials as substrates generally have one or more drawbacks, including insufficient or unstable SERS activity, poor stability in actual biological samples, and complex synthesis processes. These drawbacks severely limit the practical use of SERS-LFA technology in clinical specimens. Therefore, developing a substrate material for a new SERS nanotag to obtain a SERS nanotag with better activity and stability is currently required in the market, which can help not only develop a SERS nanotag with better quality, but also develop a SERS-LFA product based on a SERS nanotag or other technologies or products based on detection using raman spectroscopy.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide SERS substrate nano particles based on silicon core-gold shells, which have good activity and SERS stability and can enhance the signal intensity of Raman molecules in SERS labels. The invention also aims to provide SERS labels and detection products based on the SERS substrate nanoparticles so as to improve detection methods and detection products based on Raman spectroscopy.

In order to solve the technical problems, the invention adopts the following technical scheme:

In a first aspect, a SERS substrate nanoparticle based on a silicon core-gold shell is provided, and the SERS substrate nanoparticle sequentially comprises an SiO ₂ nanoparticle inner core, an electropositive polymer layer, a first Au nano layer, a Polyacetylimine (PEI) intermediate layer and a second Au nano layer from inside to outside; the thickness of the PEI interlayer is 0.5-2 nm.

In a second aspect, there is also provided a method for preparing SERS base nanoparticles based on a silicon core gold shell of the first aspect, the method comprising: and enabling the electropositive polymer, the Au nano-particles, the PEI and the Au nano-particles to sequentially form the electropositive polymer layer, the first Au nano-layer, the PEI intermediate layer and the second Au nano-layer on the surface of the SiO ₂ nano-particles through electrostatic adsorption self-assembly.

In a third aspect, there is also provided a SERS tag comprising, in order from inside to outside, the SERS substrate nanoparticle of the first aspect and a raman molecular layer.

In a fourth aspect, there is also provided a SERS tag labelling detector comprising one of the members of a specific binding pair comprising an antigen and an antibody, an enzyme inhibitor and an enzyme, a complementary nucleotide sequence, biotin and avidin, or a cofactor and an enzyme, and a SERS tag of the third aspect labelling it.

In a fifth aspect, there is also provided a detection kit comprising the SERS substrate nanoparticle of the first haze prevention, the SERS tag of the third aspect, or the SERS tag labeled detector of the fourth aspect.

In a sixth aspect, there is also provided a kit for detecting influenza a virus and streptococcus pneumoniae, the kit comprising a reaction reagent and lateral flow assay paper;

The reaction reagent comprises a reaction buffer, a first detection antibody and a second detection antibody, wherein the first detection antibody and the second detection antibody respectively label SERS labels of the third aspect with different Raman molecules;

The first detection antibody specifically binds to influenza a virus antigen, and the second detection antibody specifically binds to streptococcus pneumoniae antigen;

The lateral flow chromatography test paper comprises a sample pad, a detection pad and an absorption pad which are sequentially arranged on a bottom plate along the chromatography direction; a detection line is arranged on the detection pad, and a first capture antibody and a second capture antibody are coated in the detection line area;

The first capture antibody and the first detection antibody form a first capture antibody-influenza a virus antigen-first detection antibody immune complex;

The second capture antibody and the second detection antibody form a second capture antibody-streptococcus pneumoniae antigen-second detection antibody immune complex.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides a self-assembly-based silicon-core-gold-shell-based SERS substrate nanoparticle Si@Au/Au with a nanogap, two layers of Au nanoparticles are self-assembled on the surface of SiO ₂ nanoparticles, and a PEI intermediate layer of 0.5-2 nm composed of a Polyethyleneimine (PEI) intermediate layer is constructed between the two layers of Au nanoparticles, so that the Si@Au/Au SERS substrate nanoparticle with the nanogap is formed, and the SERS substrate nanoparticle has good stability, dispersibility and excellent SERS activity from a plurality of Au-Au hot spots, and can provide higher surface enhanced Raman spectrum (sERS) strength.

When a plurality of SERS labels with different specific signal Raman molecules are introduced into the same detection system, simultaneous detection of multiple pathogens in the same detection reaction can be realized. Si@Au/Au and SERS labels obtained based on the Si@Au/Au have great potential in the aspect of instant diagnosis of pathogenic microorganisms. Based on the concept, the invention also provides a detection kit based on SERS-LFA to detect influenza A virus and streptococcus pneumoniae, which can rapidly determine the existence of target respiratory pathogens and has good sensitivity and specificity; and the test proves that the kit can be used for detecting clinical and environmental samples and can be used as POCT detection products. In a preferred scheme, the sensitivity of the detection of the H1N1 influenza A virus can reach 29pfu/mL, and the sensitivity of the detection of the streptococcus pneumoniae can reach 16 cells/mL.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the construction principle and flow of an embodiment of the present invention;

FIG. 2 is a TEM or HRTEM image of the nanoparticle of example 1, where a is a TEM image of SiO ₂ (-170 nm), b is a TEM image of AuNPs (-20 nm), c is a TEM image of Si@Au, d is a HRTEM image of Si@Au local morphology, e is a HRTEM image of Si@Au@PEI local morphology, f is a TEM image of Si@Au/Au, g is a HRTEM image of Si@Au, h is a Si@Au/Au TEM image, i is a HADDF-STEM image of Si@Au/Au;

FIG. 3 is the EDS element line scan results of the Si@Au/Au NPs in example 1;

FIG. 4 is the elemental mapping results for Si@Au/Au NPs in example 1;

FIG. 5 shows Zeta potentials of different nanoparticles in example 1;

FIG. 6 is a schematic diagram of the relation between the thickness of the PEI interlayer on the Si@Au surface and the PEI assembly reaction time in example 1, a is a schematic diagram of the formation of an accurate PEI interlayer on the Si@Au surface, b is a TEM image of the Si@Au@PEI NP, c, d and e are High Resolution Transmission Electron Microscopy (HRTEM) images of the Si@Au@PEI of the PEI interlayers of 0.5nm, 1nm and 2nm, respectively;

FIG. 7 is a surface enhanced Raman spectrum of Si@Au/Au of a PEI interlayer of different nanocomposites (0.5 nm, 1nm and 2 nm) for DTNB in example 1;

FIG. 8 is a three-dimensional model of (i) Si@Au NPs and (ii) Si@Au/Au NPs simulated using FDTD in example 1;

FIG. 9 shows the electromagnetic distribution of (i) Si@Au NPs, (ii) Si@Au/Au NPs with a PEI interlayer thickness of 0.5nm, (iii) Si@Au/Au NPs with a PEI interlayer thickness of 1nm, and (iv) Si@Au/Au NPs with a PEI interlayer thickness of 2nm in example 1;

FIG. 10 is a HRTEM image of the local topography of (a) Si@Au/Au-DTNB NPs and (b) Si@Au/Au-MBA NPs in example 2;

FIG. 11 is a SERS spectrum of Si@Au/Au-MBA (blue line) and Si@Au/Au-DTNB (red line) of example 2, wherein Si@Au/Au-MBA is based on peak intensity of 1587cm ^-1 and Si@Au/Au-DTNB is based on peak intensity of 1331cm ^-1;

FIG. 12 is a photograph of a chromatograph of samples 1-4 of example 4 on a Si@Au/Au-LFA strip after reaction;

FIG. 13 is the corresponding Surface Enhanced Raman Scattering (SERS) signal on the T line after chromatography on the Si@Au/Au-LFA strip after reaction of samples 1-4 in example 4;

FIG. 14 is a TEM image of (i) Streptococcus pneumoniae antibody modified Si@Au/Au tags, (ii) Streptococcus pneumoniae and (iii) Si@Au/Au-Streptococcus pneumoniae immune complexes formed in example 4;

FIG. 15 is an SEM image of the T-line in example 4 of (i) 1X 10 ⁶ pfu/mL H1N1 influenza A virus, (ii) 1X 10 ⁶ cells/mL Streptococcus pneumoniae and (iii) a blank;

FIG. 16 shows the variation of the Raman signal intensity with T-line coated antibody concentration in example 5;

FIG. 17 is a graph showing the variation of Raman signal intensity with SERS label dosage for example 5;

FIG. 18 is a photograph of T-line and corresponding SERS mapping image of the SERS-LFA detection system constructed in example 6 for simultaneous detection of different concentrations of Streptococcus pneumoniae/H1N 1 influenza A virus;

FIG. 19 is a SERS spectrum on T-line of different concentrations of Streptococcus pneumoniae/H1N 1 influenza A virus in example 6;

FIG. 20 is a calibration curve plotted against log concentration of Streptococcus pneumoniae at 1587cm ^-1 and the corresponding 4-MBA signal intensity on the Si@Au/Au-LFA band in example 6;

FIG. 21 is a calibration curve plotted against logarithmic concentration of H1N1 influenza A virus at 1331cm ^-1 and corresponding signal intensity of DTNB on the Si@Au/Au-LFA band in example 6;

FIG. 22 is a graph showing the results of detection of samples of Streptococcus pneumoniae and H1N1 influenza A virus using the Au NP-based colorimetric LFA test strips of example 6;

FIG. 23 is a result of the specificity verification of the detection method provided in example 6, which shows the detection of four common respiratory viruses (influenza B virus, novel coronavirus, human adenovirus and respiratory syncytial virus) and three bacteria (Staphylococcus aureus, klebsiella pneumoniae and Escherichia coli) using the detection method provided in example 6;

FIG. 24 is a result of the reproducibility of the detection method provided in example 6;

FIG. 25 is a graph showing the use of the SERS-LFA method of example 7 for simultaneous detection of H1N1 influenza A virus and Streptococcus pneumoniae in authentic clinical and environmental samples of saliva;

FIG. 26 is a graph showing the use of the SERS-LFA method of example 7 for simultaneous detection of H1N1 influenza A virus and Streptococcus pneumoniae in real clinical and environmental samples on an object surface.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In a first aspect, a SERS substrate nanoparticle based on a silicon core-gold shell is provided, the SERS substrate nanoparticle comprising, in order from inside to outside, an SiO ₂ nanoparticle core, an electropositive polymer layer, a first Au nanolayer, a Polyethyleneimine (PEI) intermediate layer, and a second Au nanolayer. Wherein the thickness of the PEI interlayer is 0.5-2 nm, such as but not limited to 0.5, 1, 1.5 or 2nm. The SERS substrate nanoparticle based on the silicon core-gold shell is named as Si@Au/Au.

In alternative embodiments, the PEI in the PEI interlayer has a number average molecular weight of 1 to 50kDa, such as but not limited to 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50kDa, preferably 25kDa.

In alternative embodiments, the SiO ₂ nanoparticle core has a particle size of 150 to 220nm, which may be, for example, but not limited to, 150, 160, 170, 180, 190, 200, 210, or 220nm, preferably 170nm.

In an alternative embodiment, the particle size of the Au nanoparticles in the first Au nanolayer is 15 to 30nm, for example, but not limited to 15, 17, 20, 22, 25, 28 or 30nm, preferably 20nm.

In an alternative embodiment, the particle size of the Au nanoparticles in the second Au nanolayer is 15 to 30nm, for example, but not limited to 15, 17, 20, 22, 25, 28 or 30nm, preferably 20nm.

In alternative embodiments, the electropositive polymer in the electropositive polymer layer comprises PEI having a number average molecular weight of 1 to 50kDa, such as but not limited to 1,2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50kDa, preferably 25kDa.

In alternative embodiments, the electropositive polymer layer has a thickness of 1 to 8nm, such as but not limited to 1, 2, 3, 4, 5, 6, 7, or 8nm.

In a second aspect, a preparation method of the SERS substrate nanoparticle of the first aspect is further provided, including forming the electropositive polymer layer, the first Au nano layer, the PEI intermediate layer and the second Au nano layer sequentially on the surface of the SiO ₂ nano particle by electrostatic adsorption self-assembly. Electrostatic adsorption self-assembly may be performed using any method, reagents and reaction medium known in the art and is not limited in this regard.

Because the electropositive polymer is positively charged, it can self-assemble to the negatively charged SiO ₂ nanoparticle core, and the resulting nanoparticle is positively charged, so that the negatively charged Au nanoparticle can continue to assemble to the surface of the electropositive polymer layer, and the assembled first Au nanoparticle is negatively charged, so that the positively charged PEI continues to self-assemble to the surface of the first Au nanoparticle, and the resulting nanoparticle surface is positively charged, and further, the negatively charged Au nanoparticle continues to assemble to obtain the second Au nanoparticle, see fig. 5.

In an alternative embodiment, the self-assembly includes: the components to be assembled are assembled to the nanoparticle surface by ultrasonic treatment by mixing the nanoparticles into a liquid system containing the components.

Illustratively, the self-assembly includes mixing the SiO ₂ nanoparticle core in a liquid system containing an electropositive polymer, and ultrasonically treating to assemble the electropositive polymer to the surface of the SiO ₂ nanoparticle core to provide an electropositive polymer layer.

Illustratively, the nanoparticles modified with the electropositive polymer layer are mixed in a liquid system containing Au nanoparticles, and the Au nanoparticles are assembled to the surface of the electropositive polymer layer by ultrasonic treatment, thereby obtaining the first Au nanolayer.

Illustratively, the nano particles modified with the first Au nano layer are mixed in a liquid system containing PEI, and PEI is assembled on the surface of the first Au nano layer through ultrasonic treatment, so that the PEI intermediate layer is obtained. Optionally, in the step of forming the PEI interlayer, the time of the ultrasonic treatment is 10-30 min, for example, but not limited to 10, 15, 20, 25 or 30min, and the concentration of PEI in the system is 0.2v/v%, and the number average molecular weight of PEI is 25kDa. Experiments show that the thickness of the PEI interlayer under the conditions is related to the ultrasonic treatment time, and the thickness of the PEI interlayer after 10 minutes of ultrasonic treatment is 0.5nm; the thickness of the PEI interlayer after 20 minutes of ultrasonic treatment is 1nm; the thickness of the PEI interlayer was 2nm after 30min of ultrasound.

The nano particles of the PEI intermediate layer are mixed in a liquid system containing the Au nano particles, and the Au nano particles are assembled on the surface of the PEI intermediate layer through ultrasonic treatment, so that a second Au nano layer is obtained.

In alternative embodiments, the raman molecule comprises DTNB or 4-MBA. DTNB and 4-MBA contain sulfhydryl groups and terminal carboxyl groups and thus can form strong covalent bonds (Au-S) on the SERS tag surface, providing active sites for conjugation to other molecules.

In a fourth aspect, there is also provided a SERS tag label detector comprising one of the members of a specific binding pair and a SERS tag of the third aspect labeled therewith. Specific binding pairs refer to two substances having a specific binding relationship, which may be of the immune or non-immune type. The SERS label is marked on one of the specific binding pairs, and the purpose of targeting the target substance can be achieved through the specific binding capacity of the specific binding pair. Such specific binding pairs include, but are not limited to, antigens and antibodies, enzyme inhibitors and enzymes, complementary nucleotide sequences, biotin and avidin, or cofactors and enzymes.

In a fifth aspect, there is also provided a detection kit comprising the SERS base nanoparticle of the first aspect, or the SERS tag of the third aspect, or the SERS tag-labeled detector of the fourth aspect.

In an alternative embodiment, the detection kit comprises a detection unit and a capture unit:

the detection unit contains detection antibody, and the SERS label is marked on the detection antibody.

The capture unit contains a capture antibody, the capture antibody and a detection antibody forming a detection antibody-antigen-capture antibody immune complex. The detection antibody and the capture antibody may be the same or different. An antigen may comprise a plurality of identical or non-identical epitopes. When the antigen has a plurality of identical epitopes and is capable of accommodating binding of at least two antibodies thereto, e.g. the antigen is a whole virus or bacterium, the detection antibody and the capture antibody may be identical; the detection antibody and the capture antibody may also be different for binding to different epitopes on the same antigen to form the above-described immune complex.

In alternative embodiments, the kit comprises at least two detection antibodies, each binding a different antigen, and the raman molecules in the SERS tags labeled by each detection antibody are different to enable detection of multiple targets in a single assay. Different raman molecules refer to raman molecules having different raman spectra. The capture antibodies may contain one or more capture antibodies. In alternative embodiments, a plurality of capture antibodies are included, each capture antibody capturing a respective corresponding target antigen. In alternative embodiments, the capture antibodies present in the capture antibodies have capture antibodies that capture multiple target antigens, e.g., where different target antigens contain epitopes that bind to the same antibody, e.g., antibodies that bind to different antigens but are of the same type Fc-terminal, the same capture antibody may be used for capture.

The detection means to which the detection kit is applicable is not limited to the present invention, and the detection kit is applicable to any detection means based on an antigen-antibody immune reaction known in the art, including but not limited to immunochromatography, immunoblotting, immunohistochemistry, ELISA or immunomagnetic beads.

Exemplary detection units are for example: a binding pad in the immunochromatographic test paper, wherein the antibody marked with the SERS label is coated on the binding pad so as to be combined with the antigen flowing through the binding pad; or a reaction solution containing the SERS-tagged antibody, for example, and judging the reaction result by capturing the raman signal after the antibody is captured after the reaction is sufficiently performed.

Exemplary capture units are, for example: a detection line in the immunochromatographic test paper, wherein a capture antibody is coated in the region of the detection line to capture antigens flowing through the detection line, so that SERS labels are gathered in the region of the detection line; or an ELISA plate coated with a capture antibody, for example, to allow the SERS tag to aggregate in the reaction well; or for example, magnetic beads coated with capture antibodies, allowing the SERS tags to aggregate on the magnetic beads.

The detection kit also optionally includes reagents and/or consumables well known to those skilled in the art for detecting reactions, including, but not limited to, one or more of buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, wash solutions, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality controls, and labels, which can be selected by those skilled in the art depending on the particular detection means of the kit, without limitation.

In a sixth aspect, a kit for detecting H1N1 influenza a virus and streptococcus pneumoniae is also provided, the kit detects pathogens based on the SERS-LFA method of si@au/Au SERS substrate nanoparticles combined with two different raman molecules, two SERS tags are introduced into the SERS-LFA system to be combined with the corresponding pathogens respectively, different raman peaks are provided for simultaneous analysis of two pathogens on the same detection line, simultaneous detection of respiratory bacteria (streptococcus pneumoniae) and viruses (influenza a virus) in a sample is achieved, and a signal reading process is simplified. And if SERS labels are gathered on the detection line of the lateral flow chromatography test paper, the detection line can be in a dark band, so that the detection line can be used for rapidly and qualitatively analyzing whether the sample to be detected contains influenza A virus and/or streptococcus pneumoniae or not based on colorimetric signals, and can be used for accurately and quantitatively detecting target pathogenic microorganisms in the sample by detecting Raman scattering signals. Specifically, the kit comprises a reaction reagent and lateral flow chromatography (LFA) test paper:

the reaction reagent comprises a first detection antibody and a second detection antibody, each of which labels a SERS tag of the third aspect having a different raman molecule. The first detection antibody specifically binds to influenza A H1N1 virus antigen and the second detection antibody specifically binds to Streptococcus pneumoniae antigen.

In an alternative embodiment, the reaction reagents further comprise a reaction buffer, exemplary reaction buffers comprising 10mM PBS,5% v/v Tween 20, and 10% w/v BSA.

The lateral flow chromatography test paper comprises a sample pad, a detection pad and an absorption pad which are sequentially arranged on a bottom plate along the chromatography direction; and a detection line is arranged on the detection pad, and a first capture antibody and a second capture antibody are coated in the detection line area.

The first capture antibody and the first detection antibody form a first capture antibody-influenza a virus antigen-first detection antibody immune complex; the first detection antibody and the first capture antibody may bind to the same or different epitopes; when the first detection antibody and the first capture antibody bind to the same epitope, the first detection antibody and the first capture antibody may be the same or different. In alternative embodiments, the first detection antibody and the first capture antibody bind to influenza a virus particles.

The second capture antibody and the second detection antibody form a second capture antibody-streptococcus pneumoniae antigen-second detection antibody immune complex. The second detection antibody and the second capture antibody may bind to the same or different epitopes; when the second detection antibody and the second capture antibody bind to the same epitope, the second detection antibody and the second capture antibody may be the same or different. In an alternative embodiment, the second detection antibody and the second capture antibody bind to Streptococcus pneumoniae cells.

In an alternative embodiment, the detection pad is further provided with a quality control line. The quality control line can be provided with components to be coated according to the general principle of immunochromatography detection, and can be selected by a person skilled in the art according to a well-known and conventional manner, and the invention is not limited thereto. The components of the control line coating illustratively include antibodies capable of specifically binding to the first detection antibody and antibodies that specifically bind to the second detection antibody. For example, when the first detection antibody is a murine antibody, the quality control line is coated with an anti-murine antibody; when the second detection antibody is a rabbit antibody, the quality control line is coated with an anti-rabbit antibody. The antibodies on the quality control line specifically bind the rest of the antigens which are not bound with the antigens so as to judge whether the sample is effectively chromatographed.

The materials comprising the various portions of the lateral flow assay paper may be selected from materials acceptable in the art and well known. Sample pad materials illustratively include, but are not limited to, materials selected from the group consisting of glass cellulose films, polyester fiber films, and nonwoven fabrics, preferably glass cellulose films. Exemplary detection pad materials include, but are not limited to, nitrocellulose membranes. Absorbent pad materials illustratively include, but are not limited to, absorbent paper.

In an alternative embodiment, the lateral flow assay test strip further comprises a housing. The housing may be selected from those conventional in the art, and the present invention is not limited thereto.

In an optional embodiment, a window for observing the detection line and the quality control line is arranged on the shell; the housing is further provided with a through hole for applying a sample to the sample pad.

In an alternative embodiment, the raman molecule of the SERS tag labeled with the first detection antibody is DTNB and the raman molecule of the SERS tag labeled with the second detection antibody is 4-MBA.

In alternative embodiments, the coating concentration of the first capture antibody is 0.4 to 1.2mg/mL, such as but not limited to 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4mg/mL, preferably 0.8mg/mL; the second capture antibody may be coated at a concentration of 0.4 to 1.2mg/mL, for example, but not limited to, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4mg/mL, preferably 1mg/mL. The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.

The main reagents used in the following examples were as follows:

Murine monoclonal anti-H1N 1 antibody (fepeng, guangzhou); rabbit monoclonal anti-streptococcus pneumoniae antibodies (middle mei, new); sheep anti-rabbit and sheep anti-mouse IgG (Shanghai worker); ethyl orthosilicate, branched polyethylenimine, DTNB, MBA, EDC, NHS, bovine serum albumin, fetal bovine serum was purchased from Sigma-Aldrich; chloroauric acid tetrahydrate and sodium citrate are purchased from the national drug group; nitrocellulose membranes were purchased from Sartorius; the absorbent pad, sample pad, plastic back plate were purchased from Shanghai Jie one.

Measurement conditions: raman signal SERS mapping images of the test line SERS tags were recorded using a Renishaw confocal microscopy raman spectrometer with a 785nm laser source. A 20-fold objective, 5% laser power, 0.5s acquisition time was used to obtain SERS map images of the test lines. A selected area (150 μm x 150 μm area) containing 225 pixels of 10 μm x 10 μm size was scanned using a computer controlled x-y translation stage. SERS data were processed using RENISHAW WIRE 4.2 software. The average SERS spectrum of the test zone is used to generate a repeatable SERS signal for target quantification.

In the following examples, the ultrasonic power is 100 to 1000W.

The particle size of SiO ₂ nano particles is 170nm, and the preparation method is as follows:

(1) 3.5 mL-28% aqueous ammonia solution was added to the ethanol/deionized water (100 mL/6 mL) mixture and stirred vigorously at room temperature.

(2) 4ML of ethyl orthosilicate was then added quickly and stirring was continued for 3 hours.

(3) The SiO ₂ nanoparticles were centrifuged at 6000 rpm for 6 minutes and the supernatant was discarded.

(4) The precipitate is washed three times with absolute ethyl alcohol and finally dried in an oven at 60 ℃ for standby.

The particle size of the Au nano-particles is 20nm, and the preparation method is as follows:

(1) 1mL of 1% chloroauric acid was mixed with 100mL of deionized water and heated to boiling.

(2) 1.7ML of 1% aqueous sodium citrate solution was rapidly added to the boiling solution and vigorously stirred at 600 rpm for 15 minutes.

(3) After 15 minutes, the gold nanoparticle solution turns dark red in color, slowly cools to room temperature and is stored in a dark place.

NPs are short for nanoparticles (nanoparticles) in the examples below.

The following embodiments are designed according to the concept and principle shown in fig. 1: sequentially assembling PEI, 20nm AuNP, PEI and 20nm AuNP on the surface of 170nm SiO ₂, sequentially adding an electropositive Polymer (PEI) layer, a first Au nano layer, a PEI intermediate layer and a second Au nano layer, and sequentially forming Si@PEI NP, si@Au NP and Si@Au@PEI NP in each step to finally obtain Si@Au/Au.

Preparing two SERS labels Si@Au/Au-DTNB and Si@Au/Au-MBA which are respectively wrapped with DTNB and 4-MBA by taking Si@Au/Au as a substrate; si@Au/Au-DTNB was then conjugated with murine H1N1 influenza A virus antibodies, si@Au/Au-MBA was conjugated with rabbit Streptococcus pneumoniae antibodies and mixed into the reaction buffer for binding to H1N1 influenza A virus and Streptococcus pneumoniae in the sample.

The lateral flow analytical test paper is also constructed, a detection line is arranged on the lateral flow analytical test paper, and the detection line area is coated with an H1N1 influenza A virus antibody and a streptococcus pneumoniae antibody and is used for capturing pathogens in a sample to be tested, so that SERS labels are gathered on the detection line. If the corresponding pathogen exists in the sample to be detected, the corresponding Raman signal can be detected on the detection line. If H1N1 influenza A virus and streptococcus pneumoniae coexist in the sample to be detected, the detection line can detect two Raman signals.

The lateral flow chromatography test paper is also provided with a quality control line, and an anti-mouse antibody and an anti-rabbit antibody are coated on the quality control line, and can be combined with a mouse-source H1N1 influenza A virus antibody and a rabbit-source streptococcus pneumoniae antibody on a SERS label so as to verify whether the sample is subjected to effective chromatography.

Example 1

The preparation method comprises the following steps:

1. The preparation method of the SERS substrate nanoparticle Si@Au/Au based on the silicon core gold shell comprises the following steps:

1.1 preparation of Si@Au: the Si@Au core-shell nanocomposite is prepared by coating compact 20nm AuNPs on the surface of SiO ₂ modified by PEI, and comprises the following steps: 2mg of SiO ₂ NPs were dispersed in 20mL of PEI solution (0.1%, v/v, PEI number average molecular weight 25 kDa). The resulting mixture was sonicated for 30min to give an electropositive polymer layer with a thickness of 2 nm. The SiO ₂ @PEI NPs were collected by centrifugation (6000 rpm. Times.6 min) and redispersed in 10mL deionized water. 1mL of SiO ₂ @ PEI NPs was then mixed with a sufficient amount of 20nm AuNPs (40 mL, water as the medium) under intense sonication. After 15min of reaction the Si@Au NPs formed were separated and washed twice by centrifuging (4500 rpm. Times.6 min) the Si@Au NPs and then dispersing in 10mL deionized water.

1.2 Preparation of Si@Au/Au of PEI interlayers of different thicknesses:

1.2.1 forming an exact PEI interlayer: 10mL of Si@Au NPs were incubated with 10mL of PEI solution (0.2%, v/v, PEI number average molecular weight 25 kDa) and sonicated for different durations (10, 20 or 30 min), during which the surface of the Si@Au nanoparticles was coated with a PEI layer, and the thickness of the PEI layer was dependent on the duration of the reaction, yielding Si@Au@PEI NPs.

1.2.2 Adsorption of second layer AuNPs: the Si@Au@PEI NPs were centrifuged, rinsed twice with deionized water, and then reacted with 40mL of 20nm AuNPs. After 30min of sonication, the Si@Au/Au NPs formed were collected by centrifugation and dispersed in 10mL ethanol for use in the subsequent step.

The preparation method of the SERS label comprises the following steps:

Two Si@Au/Au SERS tags covering the Raman molecule DTNB and the Raman molecule 4-MBA, respectively, were prepared and 10. Mu.L of DTNB ethanol (10 mM) and 10. Mu.L of 4-MBA ethanol (10 mM) were added to 1mL of Si@Au/Au solution, respectively. The mixture was then sonicated vigorously for 1 hour, then the Si@Au/Au-DTNB and Si@Au/Au-MBA were separated by centrifugation (4000 rpm,6 minutes).

(II) experimental results:

1. TEM images of a, b and c in FIG. 2 illustrate typical morphologies of 170nm SiO ₂, 20nm AuNPs and Si@Au NPs, respectively. The positively charged polymer PEI can be easily coated on the silica surface and provides a strong positive charge for electrostatic adsorption of small gold nanoparticles. Example 1 using 20mM AuNPs ultrasound, a first Au nanolayer was formed that provided good SERS, colorimetric activity and a large negative charge, the si@au nanoparticles formed with a pronounced core-satellite structure and a rough surface (d and g in fig. 2). The diameter of Si@Au is about 210nm. The zeta potential results provided in FIG. 5 show that the surface potential of SiO ₂ increased greatly from-35.4 mV to 39.2mV after PEI modification, and then decreased significantly to-9.5 mV after coating the first layer of Au NPs. These changes indicate that the driving force for si@au preparation is electrostatic interactions. The PEI interlayer can be assembled on the surface of the Si@Au nanoparticle through ultrasonic reaction.

High Resolution Transmission Electron Microscopy (HRTEM) images (e in fig. 2) and high potential values (fig. 5) of si@au NPs confirm successful assembly of PEI at the surface of si@au NPs. The Si@Au/Au NPs obtained after coating the second layer Au NPs retained good dispersibility and exhibited a rough surface morphology (f in FIG. 2). Their enlarged TEM images (h in fig. 2) and HADDF-STEM images (i in fig. 2) clearly show that dense AuNPs are strongly integrated into Au/Au shells with many nanogaps, which is an effective SERS active region for loading raman molecules. The UV-vis spectrum of the formed si@au/Au nanostructure shows a strong and broad plasmonic peak at about 534nm due to Mie plasmon resonance from the aggregated AuNPs. In addition, EDS element line scan (fig. 3) and mapping analysis results (fig. 4) verify that the SiO ₂ core (Si and O elements) is covered by a strong Au element signal, reflecting the typical core-shell structure of si@au/Au NPs.

2. Previous studies have shown that a 1-3 nm size nanogap formed in the nanostructure or between two raman molecules is the strongest and most stable electromagnetic hotspot for raman enhancement. The use of a PEI layer as a nanogap has two distinct advantages: the PEI layer is amorphous and porous, thus allowing small Raman molecules to freely enter the PEI nanogap and hold these molecules on the surface of its core, a property of PEI that facilitates modification of Raman molecules and preparation of SERS tags. (ii) by controlling the reaction time, the thickness of the PEI layer can be conveniently adjusted. Thus, by adjusting the width of the PEI nanogap, the strongest SERS signal can be obtained on one SERS tag. Based on this, example 1 designed si@au/Au nanogap particles with ultra-narrow PEI interlayers to ensure high SERS activity and stability of the subsequent LFA system.

This example investigated the relationship between the thickness of the PEI interlayer on the si@au surface and the PEI assembly reaction time (a and b in fig. 6). HRTEM images (c, d and e in fig. 6) demonstrate that the PEI layer thickness of si@au@pei can be precisely adjusted to 0.5, 1 and 2nm according to ultrasonic times of 10, 20 and 30min, respectively. In addition, due to the strong positive charge and rich primary amine groups of the PEI interlayer, the high-density AuNPs can be firmly attached to the surface of Si@Au@PEI even though the PEI layer is only 0.5nm thick.

Three Si@Au/AuNPs (0.5 nm-PEI nanogap, 1nm-PEI nanogap and 2nm-PEI nanogap) were evaluated for SERS activity using a Raman molecule DTNB. Fig. 7 shows that the si@au/Au nanogap label has a higher DTNB signal than the si@au label with only one layer of AuNPs. This result shows that the second layer of gold nanoparticles increases the Surface Enhanced Raman Scattering (SERS) activity of the SiO ₂ -based nanomaterial. The Si@Au/Au tag with 0.5nm PEI nanogap showed stronger SERS signal than the other Si@Au/Au tags. The results show that the SERS activity of Si@Au/Au (0.5 nm-PEI nanogap) is 1.58 times and 2.26 times higher than that of Si@Au/Au (1 nm-PEI nanogap) and Si@Au/Au (2 nm-PEI nanogap), respectively. Thus the thinner the PEI interlayer, the more SERS-active the Si@Au/Au tag.

The different SERS capabilities of the si@au/Au tag can be explained by the results of time domain finite difference (FDTD) simulations, as shown in fig. 9. We found that the electromagnetic hot spot of the si@au structure is mainly distributed between the gaps of adjacent 20nm Au NPs on the SiO ₂ surface (i in fig. 9). At the same time, many additional hot spots appear between the gaps of adjacent Au NPs on the second layer Au NPs and the gaps between the two AuNP layers of the si@au/Au nanostructure (ii, iii and iv in fig. 9). The maximum electromagnetic enhancement (|E/E0|2) values for these hot spots were 3156.81, 1338.63, and 1273.53, respectively, for PEI-nanogaps at 0.5, 1, and 2 nm. The FDTD model is shown in fig. 8.

The SERS Enhancement Factors (EF) of the DTNB molecules Si@Au/Au (0.5 nm-PEI nanogap) were further calculated, and the EF values of the Si@Au and Si@Au/Au (0.5 nm-PEI nanogap) nanostructures were 4.38X10 ⁷ and 1.11X10 ⁸, respectively. Therefore, the subsequent preference for Si@Au/Au (0.5 nm-PEI nanogap) as the SERS label for LFA applications, experimental and FDTD simulation results verify that Si@Au/Au (0.5 nm-PEI nanogap) has the best SERS performance. The stability of the Si@Au/Au tag was further verified. The Si@Au/Au NPs modified by the Raman molecules (DTNB/4-MBA) in aqueous solutions with different pH values (3-13) maintain good dispersibility and quite stable surface enhanced Raman spectrum (sERS) strength.

Example 2

The present example provides a method for preparing SERS tags of conjugated antibodies:

The experimental method comprises the following steps:

1.1 Si@Au/Au-DTNB and Si@Au/Au-MBA were dispersed in 0.5mL MES buffer (10 mM, pH 5.5), respectively, and reacted with a mixture of EDC (5. Mu.L, 0.1M)/sulfo-NHS (10. Mu.L, 0.1M) for 15 minutes. The activated SERS tags were centrifuged and dispersed in 200. Mu.L PBS solution (10 mM, pH 7.4) and then Si@Au/Au-DTNB incubated with 10. Mu. g H1N1 antibody and Si@Au/Au-MBA incubated with Streptococcus pneumoniae antibody. The raman molecules DTNB and 4-MBA contain thiol and carboxyl groups, which can form strong covalent bonds (Au-S) at the surface of AuNP and provide active sites for antibody conjugation.

1.2 The dispersion was shaken for 2 hours, then 80. Mu.L BSA (1%, w/v) was added to block unreacted sites on the Si@Au/Au tag. Finally, the antibody conjugated Si@Au/Au tags (designated Immuno-Si@Au/Au DTNB and Immuno-Si@Au/Au MBA, respectively) were centrifuged at 4500rpm for 6 minutes, washed with 10mM PBST buffer (pH 7.4,0.05% Tween), then lyophilized by vacuum and stored in a refrigerator at 4 ℃.

1.3 For subsequent steps of SERS tags for preparation of conjugated antibodies to SERS-LFA, 2mg of the immunoSi@Au/Au tag was weighed, resuspended in 200. Mu.L of preservation solution containing 10mM PBS,1% BSA (w/v), 1% sucrose (w/v) and 0.02% NaN ₃ (w/v) and stored in a refrigerator at 4 ℃.

Experimental results:

TEM images in FIG. 10 confirm that the modification of DTNB and 4-MBA does not affect the surface morphology of Si@Au/Au NPs. Typical SERS spectra of Si@Au/Au-DTNB and Si@Au/Au-MBA in FIG. 11 show that DTNB and MBA have specific Raman peaks at 1331cm ^-1 and 1587cm ^-1, respectively. Thus, the two non-intersecting peaks are suitable for detecting two target substances in the same detection process and constructing a calibration curve for the respective target substances. The detection result also shows that the SERS signal of Si@Au/Au-DTNB is stronger than that of Si@Au/Au-MBA. This phenomenon may be due to the DTNB molecules having a larger raman scattering cross section. In addition, zeta potential results confirm successful conjugation of the antibody to the si@au/Au tag.

Example 3

The embodiment provides a kit for simultaneously detecting H1N1 influenza A virus and streptococcus pneumoniae, which comprises a reaction reagent and an immunity lateral chromatography test paper.

1. Preparation of kit for simultaneously detecting H1N1 influenza A and streptococcus pneumoniae

1.1. The reaction reagents contained the Immuno-Si@Au/Au DTNB reagent prepared in step 1.3 of example 2 and the Immuno-Si@Au/Au MBA reagent and a reaction buffer containing 10mM PBS,5% v/v Tween 20 and 10% w/v BSA.

1.2. The immunity lateral chromatography test paper comprises a sample pad, a detection pad and an absorption pad which are sequentially fixed on the bottom plate along the chromatography direction; the sample pad is made of a glass cellulose film, the detection pad is made of a nitrocellulose film (NC film), and a detection line (T) and a quality control line (C) are arranged on the detection pad.

T-wire was coated with a mixture of the same concentration of anti-H1N 1 influenza A virus antibody (final concentration 1 mg/mL) and anti-Streptococcus pneumoniae antibody (final concentration 1 mg/mL) mixed in equal amounts. Line C was coated with a mixture of goat anti-mouse IgG (final concentration 1 mg/mL) and goat anti-rabbit IgG (final concentration 1 mg/mL) mixed in equal amounts. The spray amount of the T line and the C line is 1 mu L/cm. NC membrane with antibody was thoroughly dried with a 37℃oven and then attached to a bottom plate for immunolateral chromatography test paper (LFA) assembly. Finally, the assembled LFA strips are cut into strips with the width of 3.5mm, and the strips are placed in a vacuum cabinet for storage.

2. The detection method comprises the following steps:

2.1. mu.L of the Immuno-Si@Au/Au DTNB reagent prepared in step 1.3 of example 2 and 2. Mu.L of the Immuno-Si@Au/Au MBA reagent and 10. Mu.L of the reaction buffer were added in a mix to each sample (100. Mu.L) and vortexed vigorously for 10 seconds to ensure complete immunological binding between the SERS tag and its target.

2.2. The mixture was dropped onto a sample pad of an immunochromatographic test strip for chromatographic separation and detection. After 15 minutes, SERS signals of the T region were measured with a Raman spectrometer, and H1N1 influenza A virus detection was based on peak intensity of 1331cm ^-1, and Streptococcus pneumoniae detection was based on peak intensity of 1587cm ^-1, for quantitative analysis of H1N1 influenza A virus and Streptococcus pneumoniae. For labeled sample detection, the measured SERS signal values of the T-line were replaced into an established calibration curve to determine the concentrations of H1N1 influenza a virus and streptococcus pneumoniae.

The detection principle is as follows:

The detection principle of the SERS-LFA method is based on the formation of sandwich-like SERS tag-target pathogen-antibody immune complexes in the T region. In one exemplary procedure, a mixture of Si@Au/Au tag and 10. Mu.L of reaction buffer was added to 100. Mu.L of test sample. The mixture was then added drop wise to the sample pad of the immunochromatographic strip. Under capillary forces, the sample and SERS tags migrate towards the T-line and C-line. If the sample contains the target bacteria or virus, the Si@Au/Au-pathogen immunocomplexes formed are immobilized by a capture antibody pre-coated on the T line, creating a distinct dark band in the T region that can be observed with the naked eye.

SERS signals on the T line were then measured using a Raman spectrometer to quantitatively analyze influenza A H1N1 virus and Streptococcus pneumoniae based on signal intensities of 1331cm ^-1 (DTNB) and 1587cm ^-1 (4-MBA), respectively. For negative samples, no visible band and corresponding SERS signal was detected in the T-zone, since the free si@au/Au tag did not immunoreact with the capture antibody on the T-line. The secondary antibody on the control line binds to the conjugated antibody on the SERS tag that does not bind to the antigen, creating a distinct colorimetric band for quality control.

Example 4

The following four samples were tested using the kit and detection method of example 3 to verify the selectivity of antibodies on the T-line, and the concentrations of bacterial and viral samples were determined using plate counting and plaque assays, respectively.

Sample 1: streptococcus pneumoniae (1×10 ⁵ cells/mL) and H1N1 influenza a virus (1×10 ⁵ pfu/mL);

sample 2: streptococcus pneumoniae (1×10 ⁵ cells/mL) and H1N1 influenza a virus (0 pfu/mL);

Sample 3: streptococcus pneumoniae (0 cells/mL) and H1N1 influenza a virus (1 x 10 ⁵ pfu/mL);

Sample 4: blank control-Streptococcus pneumoniae (0 cells/mL) and H1N1 (0 pfu/mL).

FIG. 12 shows that when the sample contains one or two pathogens of interest (lanes 1,2 and 3), the Si@Au/Au-LFA lanes show a distinct dark band under natural light. When a negative sample (band 4) was detected, no distinct dark band was observed in the T region. These results indicate that Si@Au/Au-LFA can rapidly screen target respiratory pathogens. However, merely visually observing the T line does not distinguish between influenza a H1N1 virus and streptococcus pneumoniae. The SERS signal measured from the T-line of the test strip is shown in fig. 13. The presence of the DTNB signal (1331 cm ^-1) and the 4-MBA signal (1557 cm ^-1) on the T region is consistent with and specific for the presence of H1N1 influenza A virus and Streptococcus pneumoniae in the sample. The TEM image in fig. 14 shows that many of the imuno-si@au/AuMBA tags can bind rapidly to the streptococcus pneumoniae surface, clearly indicating the high activity and affinity of the surface modified antibodies for their targets.

After detection, the internal structure of the immune lateral chromatography test paper along the T area is observed by using a scanning electron microscope, as shown in FIG. 15, a plurality of Si@Au/Au labels and Si@Au/Au bacterial immune complexes are respectively captured on T lines of the H1N1 influenza A virus sample and the streptococcus pneumoniae sample, and the Si@Au/Au labels are not observed in the T area of a blank control. These results confirm that the dark bands and SERS signals on the T-line originate from si@au/Au tags immobilized by specific immunological recognition.

Example 5

The detection line antibody coating concentration and SERS tag dosage of the immunolateral chromatography test paper in the kit prepared in example 3 were optimized:

The anti-influenza a H1N1 antibodies were used at the following concentrations, respectively: 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL of anti-streptococcus pneumoniae antibodies were used at the following concentrations, respectively: 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL, antibodies were coated on the detection lines at 1:1 volumes as above concentrations, respectively, and an immunolateral chromatographic test paper was prepared as described in example 3. The detection results of the detection line coated with antibodies with different concentrations are shown in FIG. 16, and the result shows that the optimal concentration of the anti-Streptococcus pneumoniae antibody is 1.0mg/mL, and the optimal concentration of the anti-H1N 1 influenza A virus antibody is 0.8mg/mL.

The following doses were used for the Si@Au/Au-DTNB tag and the Si@Au/Au-MBA tag, respectively: 1. Mu.L, 2. Mu.L and 3. Mu.L, influenza A virus H1N1 and Streptococcus pneumoniae negative positive groups were tested, respectively, with the best dose of SERS tags being 2. Mu.L as shown in FIG. 17.

Example 6

This example evaluates the analytical performance of a SERS-LFA system for simultaneous analysis of H1N1 influenza a virus and streptococcus pneumoniae under optimal conditions.

The optimal conditions are as follows:

The concentration of the anti-influenza A H1N1 virus antibody in the detection line is 0.8mg/mL, and the concentration of the anti-streptococcus pneumoniae antibody is 1.0mg/mL; the SERS tag dose was 2 μl.

Preparation method and detection method of the kit referring to example 3, a SERS-LFA detection system was obtained.

A mixed sample containing H1N1 influenza A virus (1X 10 ⁶ -10 pfu/mL) and streptococcus pneumoniae (1X 10 ⁶ -10 cells/mL) with different concentrations is prepared by adopting a gradient dilution method, and is detected by using the SERS-LFA detection system established in the embodiment. FIG. 18 shows the chromatographic results of the Si@Au/Au-LFA bands and SERS mapping images of the corresponding T lines.

As can be seen from fig. 18, the colorimetric signal on the T line gradually deepens as the concentrations of H1N1 influenza a virus and streptococcus pneumoniae increase in the specimen. The black T-line is still visible at pathogen concentrations as low as 5X 10 ² pfu/mL (H1N 1 influenza A virus) and 5X 10 ² cells/mL (Streptococcus pneumoniae), respectively. Blue and red SERS map images (15 pixels x 15 pixels) of the T region were generated using raman intensities of 1587cm ^-1 and 1331cm ^-1, respectively. The results indicate that SERS intensity across the scan area decreases with decreasing pathogen concentration. The SERS intensities of all 225 pixels on the mapped image are averaged to produce a reproducible SERS signal for quantitative detection. The corresponding SERS spectra from the T-line of the si@au/Au-LFA band are shown in fig. 19 and used to construct a calibration curve. FIG. 20 is a calibration curve plotted against log concentration of Streptococcus pneumoniae at 1587cm ^-1 and the corresponding 4-MBA signal intensity on the Si@Au/Au-LFA band; FIG. 21 is a calibration curve plotted at 1331cm ^-1 for logarithmic concentration of H1N1 influenza A virus and corresponding signal intensity of DTNB on Si@Au/Au-LFA band.

The correlation coefficient (R ²) of the streptococcus pneumoniae calibration curve is 0.997, the correlation coefficient (R ²) of the H1N1 influenza A virus calibration curve is 0.995, and the detection orders of the two pathogens to be detected cover 5 orders of magnitude. The limit of detection (LOD) for the system of this example was 16 cells/mL for streptococcus pneumoniae and 29pfu/mL for H1N1 influenza a virus, calculated according to standard IUPAC method (LOD = mean SERS intensity of blank samples plus 3 times standard deviation of blank measurements).

The analysis performance of the traditional colorimetric LFA test strip based on Au NP in the detection of the same streptococcus pneumoniae and H1N1 influenza A virus samples is compared with that of the method of the embodiment. FIG. 22 shows that the LOD of the AuNP-based LFA method determined by using colorimetric signals was about 5X 10 ³ cells/mL for Streptococcus pneumoniae and about 5X 10 ³ pfu/mL for H1N1 influenza A virus. Thus, the method established in this example is approximately 312 and 172 times more sensitive to streptococcus pneumoniae and H1N1 influenza a viruses than current AuNP-based LFA methods.

The sensitivity and sensing range of SERS-LFA to a single target bacterium/virus is substantially consistent with the sensitivity and sensing range of simultaneous detection of two target pathogens. The specific results of the method of this example are shown in FIG. 23, which shows that the kit and method of this example specifically detect Streptococcus pneumoniae and H1N1 influenza A virus, but do not detect influenza B virus (Flu B), staphylococcus aureus (S.aureus), novel coronavirus (SARS-CoV-2), human Adenovirus (HADV), respiratory Syncytial Virus (RSV), klebsiella pneumoniae (K.pneumoniae) and Escherichia coli (E.coli). The reproducibility results are shown in fig. 24, and the results show that the relative standard deviation RSD of the method of this example is not more than 15%, which meets the methodological requirements.

Example 7

A labeled recovery experiment was performed to evaluate the accuracy of the SERS-LFA method established in example 6 in detecting real clinical samples (saliva) and environmental samples (object surfaces). Different concentrations of influenza A H1N1 virus (1X 10 ⁴,1×10³ and 1X 10 ² pfu/mL) and Streptococcus pneumoniae (1X 10 ⁴,1×10³ and 1X 10 ² cells/mL) were added to the real samples and then assayed in triplicate. Fig. 25 and 26 show the visualization results of the immunolateral chromatographic strip. These results indicate that the colorimetric intensity on the T-line of the non-labeled authentic sample Si@Au/Au-LFA band is substantially identical to the colorimetric intensity of the PBS sample. The corresponding SERS signal on the T line was measured and the average recovery calculated to be between 84.3% and 120.2% and RSD value between 2.73% and 16.24%. These results demonstrate that the Si@Au/Au-based SERS-LFA has good accuracy and reliability when used for actual complex sample detection.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. The SERS substrate nanoparticle based on the silicon core and the gold shell is characterized by sequentially comprising an SiO ₂ nanoparticle core, an electropositive polymer layer, a first Au nano-layer, a PEI intermediate layer and a second Au nano-layer from inside to outside;

the thickness of the PEI interlayer is 0.5-2 nm.

2. The SERS substrate nanoparticle according to claim 1 wherein the PEI in the PEI interlayer has a number average molecular weight of 1 to 50kDa.

3. The SERS substrate nanoparticle according to claim 1 wherein the SiO ₂ nanoparticle cores have a particle size of 150 to 220nm; the particle sizes of the Au nano particles in the first Au nano layer and the second Au nano layer are respectively and independently 15-30 nm;

optionally, the electropositive polymer in the electropositive polymer layer comprises PEI, the number average molecular weight of PEI in the electropositive polymer layer is 1-50 kDa, and the thickness of the electropositive polymer layer is 1-8 nm.

4. A method for preparing SERS substrate nanoparticles according to any one of claims 1 to 3, comprising: and enabling the electropositive polymer, the Au nano-particles, the PEI and the Au nano-particles to sequentially form the electropositive polymer layer, the first Au nano-layer, the PEI intermediate layer and the second Au nano-layer on the surface of the SiO ₂ nano-particles through electrostatic adsorption self-assembly.

5. The method of preparing according to claim 4, wherein the self-assembling comprises: assembling the components to the surfaces of the nano-particles through ultrasonic treatment by mixing the nano-particles into a liquid system containing the components to be assembled;

optionally, in the step of forming the PEI interlayer, the ultrasonic treatment time is 10-30 min, the concentration of PEI in the system is 0.2v/v%, and the number average molecular weight of PEI is 25kDa.

A SERS tag comprising, in order from the inside to the outside, a SERS substrate nanoparticle according to any one of claims 1 to 3 and a raman molecular layer.

7. The SERS tag according to claim 6 wherein the raman molecule comprises DTNB or 4-MBA.

A SERS tag labelling detector comprising one of the members of a specific binding pair comprising an antigen and an antibody, an enzyme inhibitor and an enzyme, a complementary nucleotide sequence, biotin and avidin, or a cofactor and an enzyme, and the SERS tag of claim 6 or 7 labelled therewith.

9. A detection kit comprising the SERS substrate nanoparticle of any one of claims 1 to 3, the SERS tag of claim 6 or 7, or the SERS tag-labeled detector of claim 8.

10. The detection kit according to claim 9, comprising a detection unit and a capture unit;

the detection unit contains a detection antibody, and the SERS label is marked on the detection antibody;

the capture unit contains a capture antibody, the capture antibody and the detection antibody forming a detection antibody-antigen-capture antibody immune complex;

Optionally, the kit comprises at least two detection antibodies, each detection antibody binding a different antigen, and the raman molecules in the SERS tags labeled by each detection antibody are different.

11. The kit for detecting influenza A virus and streptococcus pneumoniae is characterized by comprising a reaction reagent and lateral flow chromatography test paper;

The reaction reagent comprises a first detection antibody and a second detection antibody, which respectively label the SERS tag of claim 8 or 9 having different raman molecules;

12. The kit of claim 11, wherein the raman molecule of the SERS tag labeled with the first detection antibody is DTNB and the raman molecule of the SERS tag labeled with the second detection antibody is 4-MBA;

Alternatively, the coating concentration of the first capture antibody is 0.4-1.2 mg/mL and the coating concentration of the second capture antibody is 0.4-1.2 mg/mL.