Fe3O4 (at) Au-WGA nano particle and preparation method thereof, SERS label and detection product

Abstract

The invention provides Fe3O4 (at) Au-WGA nanoparticles, a preparation method thereof, an SERS tag and a detection product, and particularly relates to the technical field of nano materials. The Fe3O4 (at) Au-WGA nano particle sequentially comprises a Fe3O4 inner core, a polymer layer, a nano Au layer, a first Raman molecular layer, an Au shell layer, a second Raman molecular layer and a WGA molecular layer from inside to outside, and has relatively strong paramagnetism, a stable structure and monodispersity. The two Raman molecular layers can generate stable and strong SERS signals, the outer Raman molecular layer is connected with WGA molecules, and influenza A virus and streptococcus pneumoniae can be captured at the same time. The invention further aims to provide an SERS label and a detection product based on the Fe3O4 (at) Au-WGA nano particle, so as to improve a detection method and a detection product based on a Raman spectrum.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to Fe3O4@Au-WGA nanoparticles and a preparation method thereof, a SERS label and a detection product. Background Art

[0002] Coinfections with respiratory viruses and bacteria pose a serious threat to human health worldwide, especially influenza A (H1N1) virus and Streptococcus pneumoniae (Latin abbreviation S. pneumoniae ) are co-infected with influenza A virus, which causes pneumonia with a very high mortality rate. Influenza A virus is a common respiratory virus with the characteristics of strong pathogenicity, short incubation period, high infectivity and high mortality rate. After infection with influenza A virus (abbreviated as Flu A), the human immune system may be disturbed, causing Streptococcus pneumoniae that was originally asymptomatically colonized in the nasopharynx to spread to the lower respiratory tract, causing secondary pneumonia. Therefore, rapid, accurate and highly sensitive detection of influenza A virus and Streptococcus pneumoniae is crucial for the diagnosis of the disease and the rational use of antibiotics.

[0003] At present, the detection methods of influenza A virus and Streptococcus pneumoniae mainly include virus / bacteria isolation and culture, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). However, these methods have some limitations: the culture method is time-consuming, PCR requires sophisticated instruments and professional technicians, and ELISA is cumbersome to operate and has low sensitivity. Therefore, it is urgent to develop a simple, rapid, highly sensitive and universal detection platform to protect human life and health.

[0004] Surface enhanced Raman scattering (SERS)-lateral flow chromatography (LFA) is a rapid detection technology developed in recent years. This technology uses gold or silver (Au / Ag)-based nanoparticles (NPs), such as Au@Ag NPs, hollow gold NPs, multi-branched gold nanostars (AuNS), silicon core-gold shell NPs (SiO2@Au NPs) and Au@SiO2, to replace traditional colloidal gold to prepare SERS nanoprobes. SERS-LFA has the advantages of simplicity, rapidity, high sensitivity, quantification, and multi-channel detection. It has been widely used in clinical disease diagnosis, foodborne bacteria detection, and toxin detection.

[0005] Au / Ag-based magnetic NPs, such as gold-shelled magnetic beads (Fe3O4@Au MNPs) and silver-shelled magnetic beads (Fe3O4@Ag MNPs), have been developed for complex samples (such as blood samples, nasopharyngeal swab samples, and various food samples). These magnetic NPs have good magnetic enrichment ability and SERS activity. Magnetic SERS nanoprobes are prepared by modifying Raman molecules and biorecognition molecules on magnetic NPs. These nanoprobes can capture targets and form probe-target complexes, which are separated from samples by magnetic enrichment and then captured by antibodies on the detection line (T line) by LFA capillary action. The SERS-LFA technology based on magnetic SERS nanoprobes can eliminate the pretreatment process of complex samples and has achieved rapid, specific, and highly sensitive detection of various markers in blood samples, pathogens in respiratory samples, and bacteria in food samples.

[0006] The biorecognition molecules modified on magnetic SERS nanoprobes are usually specific antibodies, aptamers, antibiotics or antimicrobial peptides, but they can only recognize and capture one or a few targets and cannot achieve universal detection of bacteria and viruses. Lectins are a class of non-immune proteins that can recognize and bind to special molecular structures of carbohydrates. Wheat germ agglutinin (WGA) can specifically bind to n-acetyl-d-glucosamine (GlcNAc) and its derivatives on the surface of microorganisms and is a broad-spectrum recognition molecule for pathogens. WGA has the advantages of high stability, easy modification and low cost, and has been used to prepare broad-spectrum nanoprobes for the detection of a variety of bacteria. In addition, it has been confirmed that WGA has antiviral activity (such as SARS-CoV-2) by binding to viral envelope glycoproteins. However, there is no research reporting the application of WGA as a broad-spectrum recognition molecule in the detection of influenza A virus and Streptococcus pneumoniae, which provides a new direction for future research.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a Fe3O4@Au-WGA nanoparticle with strong paramagnetism, stable structure and monodispersity. The two Raman molecular layers can generate stable and strong SERS signals, and the outer Raman molecular layer is connected to the WGA molecule, which can capture influenza A virus and Streptococcus pneumoniae at the same time. The purpose of the present invention is also to provide a SERS label and detection product based on the Fe3O4@Au-WGA nanoparticle to improve the detection method and detection product based on Raman spectroscopy.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The first aspect of the present invention provides a Fe3O4@Au-WGA nanoparticle, which comprises, from the inside to the outside, a Fe3O4 core, a polymer layer, a nano Au layer, a first Raman molecular layer, an Au shell layer, a second Raman molecular layer and a WGA molecular layer.

[0010] Furthermore, the material of the polymer layer is polyethyleneimine (PEI).

[0011] The Raman molecules in the first Raman molecular layer and / or the second Raman molecular layer include DTNB.

[0012] Furthermore, the thickness of the nano-Au layer is 5-8 nm.

[0013] The thickness of the Au shell layer is 7-15 nm.

[0014] The second aspect of the present invention provides a method for preparing the Fe3O4@Au-WGA nanoparticles, comprising the following steps: A. Fe3O4 nanoparticle solution and PEI solution were mixed and ultrasonically reacted at a volume ratio of 1:30-50 to obtain Fe3O4 / PEI; B. Under ultrasonic conditions, nano-gold is added to the Fe3O4 / PEI for a first reaction to obtain a first nano-Au layer, and an ethanol solution of Raman molecules is continuously added for a second reaction to obtain the first Raman molecule layer; then a stabilizer, a reducing agent and a chloroauric acid solution are added for a third reaction to obtain an Au shell layer; and an ethanol solution of Raman molecules is continuously added for a fourth reaction to obtain double-layer Raman molecule-modified Fe3O4@Au MNPs; C. Using an activator solution to activate the double-layer Raman molecule-modified Fe3O4@Au MNPs, adding WGA after activation to carry out the fifth reaction, and finally adding a blocking agent solution to block the unreacted carboxyl sites on the outer Raman molecules of the Fe3O4@Au MNPs to obtain the Fe3O4@Au-WGA nanoparticles.

[0015] Furthermore, the concentration of the Fe3O4 nanoparticle solution is 5-15 mg / mL.

[0016] The concentration of the PEI solution is 0.1-10 mg / mL.

[0017] The ultrasonic reaction time is 20 to 40 minutes.

[0018] In step B, the particle size of the nanogold is 1-5 nm.

[0019] The stabilizer includes PVP.

[0020] The reducing agent includes hydroxylamine hydrochloride.

[0021] The concentration of the chloroauric acid solution is 0.5-1.5wt%.

[0022] The activator of the activator solution includes EDC and NHS.

[0023] In the activator solution, the concentration of the activator is 100-300 mM.

[0024] The blocking agent in the blocking agent solution includes BSA.

[0025] The concentration of the sealing agent solution is 8-12wt%.

[0026] Furthermore, the first reaction time is 20 to 40 minutes.

[0027] The second reaction is carried out under ultrasound, and the time of the second reaction is 1 to 2 hours.

[0028] The third reaction is carried out under ultrasound, and the time of the third reaction is 10 to 20 minutes.

[0029] The fourth reaction is carried out under ultrasound, and the time of the fourth reaction is 1 to 2 hours.

[0030] The fifth reaction time is 1 to 3 hours.

[0031] The third aspect of the present invention provides a SERS tag, comprising the Fe3O4@Au-WGA nanoparticles.

[0032] The fourth aspect of the present invention provides a SERS tag-labeled detection object, comprising one of the members of the specific binding pair and the SERS tag labeling the member.

[0033] The specific binding pairs include antigens and antibodies, enzyme inhibitors and enzymes, complementary nucleotide sequences, biotin and avidin, or cofactors and enzymes.

[0034] The fifth aspect of the present invention provides a detection kit, comprising the Fe3O4@Au-WGA nanoparticles, or the SERS tag, or the SERS tag-labeled detection object.

[0035] A sixth aspect of the present invention provides a use of the detection kit for simultaneously detecting influenza A virus and Streptococcus pneumoniae.

[0036] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention provides a Fe3O4@Au-WGA nanoparticle, wherein the Fe3O4 core provides good magnetism, so that the nanoparticle is manipulated by an external magnetic field, and the movement of the nanoparticle is controlled. The polymer layer enhances the binding performance between the Fe3O4 core and the nanogold layer. The gold nanolayer (Au) not only enhances the Raman scattering signal and improves the detection sensitivity, but also plays a role in magnetic resonance imaging and optical imaging due to its photothermal properties. The addition of the Raman molecular layer enables the nanoparticle to have higher sensitivity and specificity in SERS detection. As a broad-spectrum recognition molecule, the WGA particle layer can identify a variety of pathogens, which makes the Fe3O4@Au-WGA nanoparticle have potential application value in detecting a variety of bacteria and viruses. The high stability, easy modification and low cost of WGA further enhance the practical application prospects of this nanoparticle.

[0037] Introducing multiple detection lines into a single test strip detection system can achieve simultaneous detection of multiple pathogens. Based on this concept, the present invention provides a SERS-LFA-based detection kit for detecting influenza A virus and Streptococcus pneumoniae, which can quickly detect the presence of respiratory pathogens with good sensitivity and specificity. In the preferred embodiment, the detection limit for influenza A virus is 14 copies / mL, and the detection limit for Streptococcus pneumoniae is 10 cells / mL, and the sensitivity is about 100 times that of the visual signal. It has strong clinical application potential in the rapid, accurate, highly sensitive and universal detection of influenza A virus and Streptococcus pneumoniae. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 a in it is the synthesis principle of Fe3O4@Au-WGA nanoparticles; Figure 1 b is a schematic diagram of the dual-channel SERS-LFA test strip based on Fe3O4@Au-WGA nanoparticles for simultaneous detection of influenza A virus and Streptococcus pneumoniae; Figure 2 HRTEM images of (i) Fe3O4-Au seed MNPs and (ii) Fe3O4@Au MNPs in Example 1; Figure 3 This is the EDS elemental analysis diagram of Fe3O4@Au MNP in Example 1; Figure 4is the XRD pattern of Fe3O4 and Fe3O4@Au MNPs in Example 1; Figure 5 is the Zeta potential of the products at different stages in Example 1; Figure 6 The Raman spectra of Fe3O4-Au seed, Fe3O4-Au seed / DTNB, Fe3O4@Au / single-layer DTNB, and Fe3O4@Au / double-layer DTNB in ​​Example 1; Figure 7 The Fe3O4@Au MNPs of Example 1 have a -1 The Raman signal intensity at Figure 8 Figure 2. Colonies of Streptococcus pneumoniae on MacConkey agar plates before and after capture (supernatant) (10-10 5 cells / mL); Fig. 9 TEM image of Fe3O4@Au-WGA-Streptococcus pneumoniae composite; Fig.10 is the capture efficiency of Fe3O4@Au-WGA nanoprobe for Streptococcus pneumoniae; Fig.11 is the capture efficiency of the Fe3O4@Au-WGA nanoprobe for influenza A virus; Fig.12 (a) Photographs of SERS-LFA test strips for detecting influenza A virus and Streptococcus pneumoniae (i), Streptococcus pneumoniae (ii), influenza A virus (iii), Staphylococcus aureus (iv), Pseudomonas aeruginosa (v), Salmonella typhimurium (vi), influenza B virus (vi), respiratory syncytial virus (viii), monkeypox virus (ix), and blank (x), with a concentration of 10 5 cells / mL (copies / mL); (b) Flu A (influenza A virus) T1 line and Streptococcus pneumoniae T2 line 1331 cm -1 The SERS signal intensity; Fig.13 (a) Simultaneous detection of 10 5 SERS-lfa band photos of Streptococcus pneumoniae and Flu A at cells / mL (copy number / mL), and Fig.12 The corresponding (b) is 1331 cm on the T line. -1 SERS signal intensity at , the error bars represent the standard deviation of five measurements; Fig.14 (a) Simultaneous detection of 10 4 SERS-LFA band photos of Streptococcus pneumoniae and Flu A at 10 cells / mL (copy number / mL), and Fig.14The corresponding (b) T line is 1331 cm -1 SERS signal intensity at , the error bars represent the standard deviation of five measurements; Fig.15 (a) Dual channel detection of different concentrations of influenza A virus (10 6 -0 copies / mL) and Streptococcus pneumoniae (10 6 -0 cells / mL) of SERS-LFA test strip; Fig.15 (b) Raman spectrum of influenza A virus on line T1; Fig.15 (c) 1331 cm −1 Calibration curve between the logarithm of influenza A virus concentration and SERS signal intensity; Fig.15 (d) is the Raman spectrum of Streptococcus pneumoniae on line T2; Fig.15 (e) is 1331 cm −1 Calibration curve between the logarithm of Streptococcus pneumoniae concentration and SERS signal intensity, and the error bars are the standard deviation of five measurements; Fig.16 (a) Photo of the SERS-LFA test strip used for dual-channel detection of 80 positive samples of influenza A virus and Streptococcus pneumoniae and 30 negative samples; Fig.16 (b) is T1 line (influenza A virus) and T2 line (Streptococcus pneumoniae) 1331 cm -1 The SERS signal intensity of the positive group and the negative group of influenza A virus is Fig.16 (c) and Streptococcus pneumoniae Fig.16 Statistical analysis results in (e), ***p<0.0001; Fig.16 (d) in the figure is the ROC curve of influenza A virus; Fig.16 (f) in the figure is the ROC curve of Streptococcus pneumoniae; Fig.17 A photo of a commercial colloidal gold-LFA strip used to test 110 clinical samples; Fig.18 The results are from the commercial H1N1 colloidal gold-LFA test strips on 110 clinical samples. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0041] Hereinafter, the terms "including", "having" and their cognates, which may be used in various embodiments of the present invention, are intended only to indicate specific features, numbers, steps, operations, elements, components or combinations of the foregoing items, and should not be understood as first excluding the existence of one or more other features, numbers, steps, operations, elements, components or combinations of the foregoing items or adding the possibility of one or more features, numbers, steps, operations, elements, components or combinations of the foregoing items.

[0042] The first aspect of the present invention provides a Fe3O4@Au-WGA nanoparticle, which comprises, from the inside to the outside, a Fe3O4 core, a polymer layer, a nano Au layer, a first Raman molecular layer, an Au shell layer, a second Raman molecular layer and a WGA molecular layer.

[0043] Furthermore, the material of the polymer layer is polyethyleneimine (PEI). Polyethyleneimine, referred to as PEI, is a water-soluble polymer. PEI provides a stable water environment for Fe3O4 nanoparticles through its numerous amino groups, and gives the surface positive charge, thereby enhancing the dispersibility and stability of the nanoparticles in water.

[0044] The Raman molecules in the first Raman molecule layer and / or the second Raman molecule layer include DTNB. DTNB is a Raman molecule, namely 5,5'-dithiobis(2-nitrobenzoic acid). It can enhance Raman signals and improve detection sensitivity by combining with the metal surface of gold nanoparticles.

[0045] WGA is wheat germ agglutinin, which can specifically bind to n-acetyl-d-glucosamine (GlcNAc) and its derivatives on the surface of microorganisms and is a broad-spectrum recognition molecule for pathogens.

[0046] Furthermore, the thickness of the nano-Au layer is 5-8 nm.

[0047] Typically but not restrictively, the thickness of the nano-Au layer may be 5 nm, 6 nm, 7 nm or 8 nm, or any value within the range of 5 nm to 8 nm.

[0048] The thickness of the Au shell layer is 7-15 nm.

[0049] Typically but not limiting, the thickness of the Au shell layer can be 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm or 15 nm, or any value within the range of 7 nm to 15 nm.

[0050] The second aspect of the present invention provides a method for preparing the Fe3O4@Au-WGA nanoparticles, comprising the following steps: A. Fe3O4 nanoparticle solution and PEI solution were mixed at a volume ratio of 1:30-50 and ultrasonically reacted to obtain Fe3O4 / PEI; PEI is a cationic polymer that can interact with the negative charges on the surface of Fe3O4 nanoparticles to form a stable Fe3O4 / PEI complex.

[0051] B. Under ultrasonic conditions, nano-gold is added to the Fe3O4 / PEI for a first reaction to obtain a nano-Au layer, and an ethanol solution of Raman molecules is continuously added for a second reaction to obtain the first Raman molecule layer; then a stabilizer, a reducing agent and a chloroauric acid solution are added for a third reaction to obtain an Au shell layer; and an ethanol solution of Raman molecules is continuously added for a fourth reaction to obtain double-layer Raman molecule-modified Fe3O4@Au MNPs; C. Using an activator solution to activate the double-layer Raman molecule-modified Fe3O4@Au MNPs, adding WGA after activation to carry out the fifth reaction, and finally adding a blocking agent solution to block the unreacted carboxyl sites on the outer Raman molecules of the Fe3O4@Au MNPs to obtain the Fe3O4@Au-WGA nanoparticles.

[0052] Typically but not limiting, the volume ratio of the Fe3O4 nanoparticle solution to the PEI solution can be 1:30, 1:35, 1:40, 1:45 or 1:50, or any value within the range of 1:30-50.

[0053] Furthermore, the concentration of the Fe3O4 nanoparticle solution is 5-15 mg / mL.

[0054] Typically but not limiting, the concentration of the Fe3O4 nanoparticle solution can be 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL or 15 mg / mL, or any value within the range of 5 mg / mL to 15 mg / mL.

[0055] The concentration of the PEI solution is 0.1-10 mg / mL.

[0056] Typically but not limiting, the concentration of the PEI solution can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1.0 mg / mL, or any value within the range of 0.1 mg / mL to 10 mg / mL.

[0057] The ultrasonic reaction time is 20 to 40 minutes, and the ultrasonic reaction is used to enhance the combination of PEI and Fe3O4 nanoparticles. Typically but not restrictively, the ultrasonic reaction time can be 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes or 40 minutes, or any value within the range of 20 minutes to 40 minutes.

[0058] In step B, the particle size of the nano-gold is 1-5 nm. Typically but not restrictively, in step B, the particle size of the nano-gold can be 1 nm, 2 nm, 3 nm, 4 nm or 5 nm, or any value within the range of 1 nm to 5 nm.

[0059] The stabilizer includes PVP. The reducing agent includes hydroxylamine hydrochloride. By adding the stabilizer and the reducing agent, a growth environment for the gold seeds is formed.

[0060] The concentration of the chloroauric acid solution is 0.5-1.5wt%, and a larger gold layer is grown on the surface of the existing particles by reducing the chloroauric acid. Typically but not limiting, the concentration of the chloroauric acid solution can be 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt% or 1.5wt%, or any value within the range of 0.5wt% to 1.5wt%.

[0061] The activation is to enhance the activity of the carboxyl groups on the particle surface. The activators of the activator solution include EDC and NHS. EDC is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and NHS is N-hydroxysuccinimide. Then, WGA forms a stable covalent bond with the amino groups on the particle surface through the carboxyl groups.

[0062] In the activator solution, the concentration of the activator is 100-300 mM. Typically but not limiting, the concentration of the activator in the activator solution can be 100 mM, 150 mM, 200 mM, 250 mM or 300 mM, or any value within the range of 100 mM-300 mM.

[0063] The blocking agent in the blocking agent solution includes BSA, which is bovine serum albumin, and blocks unreacted carboxyl sites to prevent non-specific binding.

[0064] The concentration of the sealing agent solution is 8-12 wt %. Typically but not limiting, the concentration of the sealing agent solution can be 8 wt %, 9 wt %, 10 wt %, 11 wt % or 12 wt %, or any value within the range of 8 wt % to 12 wt %.

[0065] Further, the time of the first reaction is 20 to 40 min. Typically but not limiting, the time of the first reaction can be 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min or 40 min, or any value within the range of 20 min to 40 min.

[0066] The second reaction is carried out under ultrasound, and the time of the second reaction is 1 to 2 hours. Typically but not limiting, the second reaction is carried out under ultrasound, and the time of the second reaction can be 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours or 2 hours, or any value within the range of 1 hour to 2 hours.

[0067] The third reaction is carried out under ultrasound, and the time of the third reaction is 10 to 20 minutes. Typically but not limiting, the time of the third reaction can be 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes or 20 minutes, or any value within the range of 10 minutes to 20 minutes.

[0068] The fourth reaction is carried out under ultrasound, and the time of the fourth reaction is 1 to 2 hours. Typically but not limiting, the time of the fourth reaction can be 1 hour, 1.2 hours, 1.4 hours, 1.8 hours or 2 hours, or any value within the range of 1 hour to 2 hours.

[0069] The fifth reaction time is 1 to 3 hours.

[0070] Typically but not limiting, the time of the fifth reaction can be 1 h, 1.5 h, 2.0 h, 2.5 h or 3 h, or any value within the range of 1 h to 3 h.

[0071] The third aspect of the present invention provides a SERS tag, comprising the Fe3O4@Au-WGA nanoparticles.

[0072] The fourth aspect of the present invention provides a SERS tag-labeled detection object, comprising one of the members of the specific binding pair and the SERS tag labeling the member.

[0073] The specific binding pairs include antigens and antibodies, enzyme inhibitors and enzymes, complementary nucleotide sequences, biotin and avidin, or cofactors and enzymes.

[0074] In one embodiment of the invention, one of the members of the specific binding pair is an antibody (immobilized on the test strip test line), and a broad-spectrum recognition molecule WGA modified on the SERS tag, which can recognize and bind to influenza A virus (H1N1) and Streptococcus pneumoniae (Pneumococcus). When a sample containing these pathogens comes into contact with the SERS tag, the pathogens in the sample will bind to the WGA on the SERS tag; then, the pathogens are captured by the specific antibodies on the test line, and the results are usually indicated by color changes. If the test line changes color, it indicates that H1N1 or Pneumococcus is present in the sample; if there is no color change, it indicates that these pathogens are not detected in the sample.

[0075] The fifth aspect of the present invention provides a detection kit, comprising the Fe3O4@Au-WGA nanoparticles, or the SERS tag, or the SERS tag-labeled detection object.

[0076] A sixth aspect of the present invention provides a use of the detection kit for simultaneously detecting influenza A virus and Streptococcus pneumoniae.

[0077] The main reagents used in the following examples are as follows: Magnesium chloride (MgCl2), manganese chloride (MnCl2) and calcium chloride (CaCl2) were produced by Shanghai Aladdin Biochemical Technology Co., Ltd.; hydroxylamine hydrochloride, trisodium citrate (TSC) and chloroauric acid tetrahydrate (HAuCl4·4H2O) were supplied by Sinopharm Chemical Reagent Shanghai Co., Ltd.; phosphate buffered saline (PBS), bovine serum albumin (BSA), fetal bovine serum (FBS), 2-(N-morpholino)ethanesulfonic acid (MES), tween-205, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB), PEI branched chain (MW 25 kDa), polyvinylpyrrolidone (PVP, kDa), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS).

[0078] Blood agar plates were purchased from BeNa Culture Collection, Beijing, China. Nitrocellulose (NC) membrane (CN95) with a pore size of 15 mm was provided by Sartorius, Spain. Other LFA components (plastic backing card, absorbent pad, and sample loading pad) were provided by Jieyi Biotechnology Co., Ltd. (Shanghai, China).

[0079] WGA (Cat. No. L9640) was provided by Sigma-Aldrich (USA); mouse monoclonal anti-influenza A antibody (Cat. No. FLUA-REAB-G1-006) was provided by Feipeng Biotechnology Co., Ltd. (China); rabbit monoclonal anti-Streptococcus pneumoniae antibody (Cat. No. Strpcpnu-001) was provided by Changzhou Sino-US Xinxin Biotechnology Co., Ltd. (Guangzhou, China); Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus typhi, influenza A (H1N1 2009 / A) and influenza B viruses were provided by our institution; respiratory syncytial virus (RSV) was from Nanjing Baikang Biological Co., Ltd.; monkeypox virus (MPXV) was provided by our institution. Commercial colloidal gold-LFA test strips were provided by Beijing Runbofude Biotechnology Development Co., Ltd.

[0080] The main instruments used in this paper include: a transmission electron microscope (TEM) using a Philips Tecnai G2 F20 microscope, a Nano-ZS90 Zeta Sizer from Malvern, UK, and a portable Raman system with a laser spot diameter of 100 μm (B&W Tek, i-Raman Plus BWS465-785H spectrometer).

[0081] Figure 1 a in it is the synthesis principle of Fe3O4@Au-WGA nanoparticles; Figure 1 Figure b is a schematic diagram of the dual-channel SERS-LFA test strip based on Fe3O4@Au-WGA nanoparticles for simultaneous detection of influenza A virus and Streptococcus pneumoniae. Fe3O4@Au-WGA nanoparticles capture influenza A virus and Streptococcus pneumoniae in throat swab / sputum samples, and then the Fe3O4@Au-WGA-influenza A virus / Streptococcus pneumoniae complex is magnetically enriched and separated from the sample solution. The complex precipitate is resuspended with loading buffer and then subjected to capillary reaction on the LFA test strip. Finally, Fe3O4@Au-WGA-influenza A virus / Streptococcus pneumoniae are captured by influenza A-specific monoclonal antibodies on the T1 line or Streptococcus pneumoniae-specific monoclonal antibodies on the T2 line, and then the SERS signal on the T line is detected by a Raman instrument.

[0082] Example 1 This embodiment provides a Fe3O4@Au-WGA nanoparticle, and the synthesis process is as follows: 1. Mix 1 mL of Fe3O4MNP solution (10 mg / mL) with 40 mL of PEI solution (0.5 mg / mL) and react with ultrasound for 30 min to obtain Fe3O4 / PEI. Perform magnetic enrichment and separation of Fe3O4 / PEI twice with deionized water.

[0083] 2. Add 40 mL of 3 nm Au NPs under ultrasonic conditions and react for 30 min to obtain Fe3O4-Au seed. Wash the Fe3O4-Au seed twice with ethanol solution, then react with 40 μL of DTNB ethanol solution (100 mM) under ultrasonic conditions for 1.5 h to obtain Fe3O4-Au seed / DTNB, which was resuspended with 5 mL of ethanol solution.

[0084] 3. Ultrasonic mix 1 mL of Fe3O4-Au seed / DTNB with 40 mL of deionized water containing 120 mg of PVP and 20 mg of hydroxylamine hydrochloride for 15 min, add 180 μL of 1% HAuCl4 solution and ultrasonically react for 15 min to obtain Fe3O4@Au, which is resuspended in 5 mL of ethanol solution. The second layer of DTNB is modified under the same conditions to obtain double-layer DTNB-modified Fe3O4@Au MNPs.

[0085] 4. The prepared Fe3O4@Au MNPs were resuspended in 500 μL MES buffer (100 mM, pH 5.5) and activated with EDC (100 mM) and NHS (100 mM). The activated MNPs were resuspended in 200 μL 0.05% PBST, oscillated with 100 μg of WGA for 2 h, and then 100 μL (10%) BSA was added and oscillated for 1 h to block the unreacted carboxyl sites. After magnetic enrichment and separation, the prepared Fe3O4@Au-WGA nanoparticles were washed twice with PBST and stored at 4°C.

[0086] Experimental results: Figure 2 As shown in the high-resolution transmission electron microscopy (HRTEM) image (i) in the figure, 200 nm Fe3O4 / PFI adsorbed 3 nm Au NPs to prepare Fe3O4-Au seeds with a diameter of about 215 nm. After adding chloroauric acid, a layer of gold shell was reduced on the surface of Fe3O4-Auseed to prepare Fe3O4@Au MNPs with a diameter of about 240 nm. Figure 2 As shown in (ii) in . Figure 3 X-ray energy spectrum elemental analysis (EDS) shows that Fe3O4@Au is composed of three elements: Fe (i) red, O (ii) green and Au (iii) yellow. (iv) is a fusion image of the three elements, and the three elements are evenly distributed. Figure 4The X-ray photoelectron spectroscopy (XRD) results of Fe3O4@Au MNPs are shown. The diffraction peaks of Fe3O4@Au MNPs can be retrieved from the powder diffraction file (PDF) database (PDF of Fe3O4 is 79-0416, PDF of Au is 99-0056). The curve peaks at 2θ values ​​of 44.7, 52.0 and 76.7 correspond to the 200, 311 and 333 crystal plane reflections of Fe3O4, and the peaks at 24.6, 41.4 and 67.2 correspond to the 111, 200 and 220 crystal plane reflections of the Au shell, which once again proves that the synthesis of Fe3O4@Au MNPs is successful. Zeta potential analysis of samples at different stages of the synthesis of Fe3O4@Au MNPs was carried out, such as Figure 5 As shown, the PEI layer with a strong positive charge increases the potential, while the negatively charged Au shell decreases the potential.

[0087] Figure 6 The Raman spectrum of Fe3O4@Au MNPs is shown in Figure 2. The Fe3O4-Au seed modified with DTNB has no Raman peak, while the Fe3O4-Au seed modified with DTNB has a Raman peak at 1331 cm −1 The SERS intensity at is lower than that of single-layer DTNB modified Fe3O4@AuMNPs, and the SERS signal intensity of double-layer DTNB modified Fe3O4@Au MNPs is about 2.5 times that of single-layer DTNB modified materials, indicating that the SERS signal of double-layer DTNB modified Fe3O4@Au MNPs is the strongest.

[0088] In addition, four different batches of materials were tested 20 times each to study the SERS signal intensity of double-layer DTNB-modified Fe3O4@Au MNPs at 1331 cm−1. Figure 7 As shown, the relative standard deviation of 80 detection signals is 3.01%, indicating good inter-batch and intra-batch reproducibility and stability. Monodisperse and uniformly sized gold-shelled magnetic beads (Fe3O4@AuMNPs) were successfully prepared and modified with broad-spectrum molecule WGA to prepare universal magnetic SERS nanoprobes for capturing influenza A virus and Streptococcus pneumoniae.

[0089] The prepared Fe3O4@Au-WGA nanoparticles were used to verify the capture efficiency of Streptococcus pneumoniae / influenza A virus.

[0090] Different concentrations (10 5 -10 cells / mL) of Streptococcus pneumoniae were incubated with 10 μL of Fe3O4@Au-WGA MNPs (10 mg / mL) in a saline buffer solution (containing 10 mM PBS, 0.05% Tween 20, 1 mM Ca 2+ , 1 mM Mg2+ , 1 mMMn 2+ ) for 30 minutes. Magnetic separation of pneumococcal complexes, 100 μL of supernatant was spread on MacConkey agar plates (three plates in duplicate), placed in anaerobic bags, and cultured at 37°C for 16 h. The capture efficiency was calculated by comparing the difference between the number of pneumococci in the supernatant after culture and the number of pneumococci before culture.

[0091] The capture efficiency of Fe3O4@Au-WGA nanoprobe for Flu A was verified by droplet digital PCR (ddPCR). 5 -10 copies / mL) of Flu A was incubated with Fe3O4@Au-WGA MNPs in a salt ion buffer for 30 minutes. After magnetic separation of the Fe3O4@Au-WGA-Flu A complex, the supernatant of each group was quantified by ddPCR. The capture efficiency can be calculated by comparing the difference between the number of Flu A viruses in the supernatant after incubation and the number of original Flu A viruses before incubation.

[0092] Culture at different concentrations (10-10 5 The image of MacConkey agar plate of Streptococcus pneumoniae (cells / mL) shows that the number of colonies increases with the increase of Streptococcus pneumoniae concentration, such as Figure 8 The plate image of the culture supernatant (Streptococcus pneumoniae captured and magnetically separated by Fe3O4@Au-WGA nanoparticles) shows that at a concentration of 10 cells / mL-10 4 Very few colonies are formed on a plate with cells / mL, such as Figure 8 As shown in (ii) in the figure, the capture efficiency of Fe3O4@Au-WGA nanoparticles for Streptococcus pneumoniae was 10 cells / mL-10 4 cells / mL, reaching 99%-100%. 5 cells / mL) still reached 95%. Fig.10 As shown. Fig. 9 In the TEM image, Streptococcus pneumoniae is bound and surrounded by Fe3O4@Au-WGA nanoparticles, which once again verifies the nanoparticles' ability to capture Streptococcus pneumoniae. Since influenza A virus is only about 80 nm, the Fe3O4@Au-WGA-influenza A virus complex cannot be observed under TEM. ddPCR was used to detect the original number of influenza A viruses before capture and the number of remaining viruses in the supernatant after capture. The results are shown in Table 1.

[0093] Table 1 Concentration of influenza virus before and after capture detected by ddPCR

[0094] Influenza A virus is between 10 copies / mL and 10 4 The capture efficiency was 100% at a concentration of 10 copies / mL. 5 The capture efficiency at the concentration of copies / mL was 98%. Fig.11 The above results show that the Fe3O4@Au-WGA nanoprobe has a high capture efficiency for both influenza A virus and Streptococcus pneumoniae.

[0095] Example 2 This embodiment provides a dual-channel LFA test strip for influenza and Streptococcus pneumoniae, and the preparation process is as follows: mouse monoclonal anti-influenza A antibody and rabbit monoclonal anti-Streptococcus pneumoniae antibody are sprayed on the NC membrane, respectively, and the T1 detection line and T2 detection line are established respectively, and then dried at 37°C for 3 h. The prepared NC membrane is assembled with the sample pad and the absorption pad on the bottom plate to form an LFA test paper. The assembled LFA board is then cut into 3 mm wide test strips and stored in a vacuum dryer for further use.

[0096] The standard strains used below (Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus typhi) were cultured and preserved by Xiao's group. The bacteria were quantified using the classic plate count method. Culture on blood agar plates for 16 hours (37°C). Scrape the colonies on the plates and resuspend them in PBS buffer. Take 100 μL of the bacterial solution and spread it evenly on the blood agar plate, culture it for 16 hours, and calculate the concentration of the bacterial solution by counting colony forming units (cfu). The remaining bacteria were washed twice with PBS (centrifuged at 5000 rpm for 5 minutes). Finally, according to the calculation results, dilute the bacterial solution to the required concentration.

[0097] 1. Detect influenza A virus, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, influenza B virus, respiratory syncytial virus and monkeypox virus, and verify the specific selectivity of the prepared SERS-LFA test strips for influenza A virus and Streptococcus pneumoniae. Fig.12 In a (i), when the concentration is 10 5 When the number of copies / mL (cells / mL) of influenza A virus and Streptococcus pneumoniae is less than 100, two gray stripes appear on the test strip (T1 line represents influenza A virus, T2 line represents Streptococcus pneumoniae). T1 and T2 lines are 1331cm -1 The corresponding SERS signal intensity is stronger, such as Fig.12 When influenza A virus or pneumococcus is detected, only a gray band appears, and only the T1 or T2 line has a strong SERS signal, as shown in (i) of (b). Fig.12 a(ⅱ-ⅲ) and Fig.12The results show that there is no cross reaction between influenza A virus and Streptococcus pneumoniae, which is suitable for dual-channel detection using SERS-LFA test strips. When detecting Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, influenza B virus, respiratory syncytial virus and monkeypox virus, no gray bands appear on the T line, and the SERS signal is very weak, such as Fig.12 a(ⅳ-ⅸ) and Fig.12 As shown in b (ⅳ-ⅸ) in the blank group, no gray band appeared on the T line, and the SERS signal was very weak. Fig.12 a(ⅹ) and Fig.12 The results showed that the prepared SERS-LFA strips had good specific selectivity for influenza A virus and Streptococcus pneumoniae.

[0098] Repeatability test is an indicator to verify the stability and accuracy of SERS-LFA test strips. 5 The gray bands of the T1 / T2 line are uniform in color, such as Fig.13 As shown in a. The SERS signal intensity on the T line is Fig.13 As shown in b, the relative standard deviations (RSDs) of influenza A virus and Streptococcus pneumoniae were 2.24% and 3.34%, respectively. 4 When the RSD of influenza A virus was 3.39%, the RSD of Streptococcus pneumoniae was 2.09%. Fig.14 The results show that the prepared SERS-LFA test strips have good repeatability and stability for influenza A virus and Streptococcus pneumoniae.

[0099] 2. Dual-channel SERS-LFA test strips for detection of influenza A virus and Streptococcus pneumoniae Fig.15 Figure a shows the simultaneous detection of different concentrations of influenza A virus (10 6 -0 copies / mL) and Streptococcus pneumoniae (10 6 -0 cells / mL) of SERS-LFA test strip. As the concentration of influenza A virus and Streptococcus pneumoniae decreases, the gray bands of T1 and T2 lines become weaker. When the concentration is lower than 10 3 When the number of copies / mL (cells / mL) is less than 1, the gray band disappears. The Raman spectra of the T1 line of influenza A virus and the T2 line of Streptococcus pneumoniae are as follows: Fig.15 As shown in b and d, 1331cm -1 The SERS signal intensity at is positively correlated with the concentration. A calibration curve between the logarithm of the concentration and the SERS signal intensity is drawn as Fig.15As shown in c and e in Figure 1, there is a good correlation coefficient (R 2 The R of Streptococcus pneumoniae is 0.987. 2 The limits of detection (LOD) of influenza A virus and Streptococcus pneumoniae were 14 copies / mL and 10 cells / mL, respectively, calculated using the IUPAC scheme: LOD = y 空白 (Average SERS signal intensity of blank group) + 3×SD 空白 (Standard deviation of the blank group).

[0100] 3. Dual-channel SERS-LFA test strips for clinical samples The prepared SERS-LFA test strips were used to detect 80 positive throat swab / sputum samples of influenza A virus and Streptococcus pneumoniae and 30 negative samples. Fig.16 As shown in a in the figure. Two gray bands appeared on the T1 and T2 lines of the positive group samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 26, 30, 31, 32, and 36. No gray band appeared on the T line of the negative group. Fig.16 The b in Figure 1 shows the 1331 cm −1 The SERS signal intensity of line T1 (influenza A virus) and line T2 (Streptococcus pneumoniae) at the positive group was significantly higher than that of the negative group, with significant statistical significance (***p<0.0001). Fig.16 The SERS signal intensities of the T1 line (influenza A virus) and the T2 line (Streptococcus pneumoniae) of all samples were analyzed and the ROC curve was drawn, as shown in FIG. Fig.16 As shown in d and f, the area under the ROC curve (AUS) is 1, indicating that the established SERS-LFA test strip has a high accuracy in diagnosing influenza A virus and Streptococcus pneumoniae. According to the ROC curve of influenza A virus, the best sensitivity and specificity are 100%, and the cutoff value is 1936; the SERS signal intensity of the T1 line (influenza A virus) of 80 positive samples is higher than 1936, and the SERS signal intensity of 30 negative samples is lower than 1936. According to the ROC curve of Streptococcus pneumoniae, the best sensitivity and specificity are 100%, and the cutoff value is 1613. The SERS signal intensity of the T2 line (Streptococcus pneumoniae) of 80 positive samples is greater than 1613, and the SERS signal intensity of 30 negative samples is lower than 1613. The results show that the dual-channel SERS-LFA test strip based on Fe3O4@Au-WGA nanoprobe has a 100% diagnostic coincidence rate for 110 clinical influenza A virus and Streptococcus pneumoniae. In addition, 110 clinical samples were tested using commercial colloidal gold-LFA test strips, with a diagnostic rate of 81%, e.g. Fig.17 and Fig.18The above results show that the diagnostic rate of SERS-LFA test strips for influenza A virus and Streptococcus pneumoniae is 19% higher than that of commercial colloidal gold-LFA test strips.

[0101] Finally, it should be noted that the above-described embodiments are only specific implementations of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above-described embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the above-described embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A Fe3O4@Au-WGA nanoparticle, characterized in that: From the inside to the outside, it includes a Fe3O4 core, a polymer layer, a nano-Au layer, a first Raman molecular layer, an Au shell layer, a second Raman molecular layer and a WGA molecular layer.

2. The Fe3O4@Au-WGA nanoparticles according to claim 1, characterized in that: The material of the polymer layer is polyethyleneimine; The Raman molecules in the first Raman molecular layer and / or the second Raman molecular layer include DTNB.

3. The Fe3O4@Au-WGA nanoparticles according to claim 1, characterized in that: The thickness of the nano Au layer is 5-8 nm; The thickness of the Au shell layer is 7-15 nm.

4. A method for preparing Fe3O4@Au-WGA nanoparticles according to any one of claims 1 to 3, characterized in that: The following steps are involved: A. Fe3O4 nanoparticle solution and PEI solution were mixed and ultrasonically reacted at a volume ratio of 1:30-50 to obtain Fe3O4 / PEI; B. Under ultrasonic conditions, nano-gold is added to the Fe3O4 / PEI for a first reaction to obtain a nano-Au layer, and an ethanol solution of Raman molecules is continuously added for a second reaction to obtain the first Raman molecule layer; then a stabilizer, a reducing agent and a chloroauric acid solution are added for a third reaction to obtain an Au shell layer; and an ethanol solution of Raman molecules is continuously added for a fourth reaction to obtain double-layer Raman molecule-modified Fe3O4@Au MNPs; C. Using an activator solution to activate the carboxyl groups on the second layer of Raman molecules on the Fe3O4@Au MNPs, adding WGA after activation to carry out the fifth reaction, and finally adding a blocking agent solution to block the unreacted carboxyl sites on the second layer of Raman molecules on the Fe3O4@Au MNPs to obtain the Fe3O4@Au-WGA nanoparticles.

5. The preparation method according to claim 4, characterized in that: The concentration of the Fe3O4 nanoparticle solution is 5-15 mg / mL; The concentration of the PEI solution is 0.1-10 mg / mL; The ultrasonic reaction time is 20 to 40 minutes; In step B, the particle size of the nanogold is 1-5 nm; The stabilizer includes PVP; The reducing agent includes hydroxylamine hydrochloride; The concentration of the chloroauric acid solution is 0.5-1.5wt%; The activator of the activator solution includes EDC and NHS; In the activator solution, the concentration of the activator is 100-300 mM; The blocking agent in the blocking agent solution includes BSA; The concentration of the sealing agent solution is 8-12wt%.

6. The preparation method according to claim 4, characterized in that: The time of the first reaction is 20 to 40 minutes; The second reaction is carried out under ultrasound, and the time of the second reaction is 1 to 2 hours; The third reaction is carried out under ultrasound, and the time of the third reaction is 10 to 20 minutes; The fourth reaction is carried out under ultrasound, and the time of the fourth reaction is 1 to 2 hours; The fifth reaction time is 1 to 3 hours.

7. A SERS tag, characterized in that: It comprises the Fe3O4@Au-WGA nanoparticles as described in any one of claims 1 to 3.

8. A SERS tag-labeled detection object, characterized in that: A SERS tag comprising one of the members of the specific binding pair and labeling the SERS tag of claim 7; The specific binding pairs include antigens and antibodies, enzyme inhibitors and enzymes, complementary nucleotide sequences, biotin and avidin, or cofactors and enzymes.

9. A detection kit, characterized in that: The method comprises the Fe3O4@Au-WGA nanoparticles according to any one of claims 1 to 3, or the SERS tag according to claim 7, or the SERS tag-labeled detection object according to claim 8.

10. Use of the detection kit according to claim 9 in the simultaneous detection of influenza A virus and Streptococcus pneumoniae.

Images