Raman enhanced nano-label and its preparation method and application, neutrophil elastase immunochromatographic detection system

Abstract

The present invention provides a Raman enhanced nano-tag, a preparation method and application thereof, and a neutrophil elastase immunochromatographic detection system, which belongs to the field of cell biology technology. The Raman enhanced nano-tag provided by the present invention comprises a gold core silver shell nano-star, a Raman signal molecule modified on the surface of the gold core silver shell nano-star, and a neutrophil elastase capture antibody coupled to the Raman signal molecule. The Raman enhanced nano-tag provided by the present invention combined with the immunochromatographic technology can realize accurate and rapid detection of neutrophil elastase.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to a Raman enhanced nano-label and a preparation method and application thereof, and a neutrophil elastase immunochromatographic detection system. Background Art

[0002] Neutrophil Elastase (ELANE) is a protease mainly secreted by neutrophils, which plays a key regulatory role in inflammation and immune response. ELANE belongs to the serine protease family. Its main function is to degrade extracellular matrix proteins and promote biological processes such as tissue repair, leukocyte migration and cell signal transduction during inflammation. At the same time, ELANE can also regulate the release of inflammatory mediators, degrade proinflammatory cytokines, and release soluble complement receptors that can inhibit complement activation, playing an important regulatory role in the continuation of the inflammatory process. However, excessive activation or abnormal release of ELANE is involved in the occurrence and development of many diseases, such as allergic diseases, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, cystic fibrosis, rheumatoid arthritis, vasculitis, acute respiratory distress syndrome, etc. In addition, studies have found that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness. In animal models, there are two phases of airway inflammation after secondary allergen stimulation, of which 6h is a neutrophil reaction, mainly manifested as an increase in ELANE, and 48h is an eosinophilic reaction. ELANE can be considered a molecular marker for the onset of allergic diseases, and the neutrophil reaction occurs at an earlier stage, which is conducive to the early diagnosis of the disease.

[0003] Currently, the main detection methods for ELANE include enzyme-linked immunosorbent assay (ELISA), gel electrophoresis, mass spectrometry analysis technology, etc., but these technologies often rely on large laboratory instruments and cannot meet the needs of real-time, accurate and rapid detection on site. Summary of the invention

[0004] The purpose of the present invention is to provide a Raman-enhanced nano-tag and a preparation method and application thereof, and a neutrophil elastase immunochromatographic detection system. The Raman-enhanced nano-tag provided by the present invention combined with immunochromatographic technology can realize real-time, on-site, accurate and rapid detection of neutrophil elastase.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a Raman enhanced nano-label, comprising a gold core silver shell nano-star, a Raman signal molecule modified on the surface of the gold core silver shell nano-star, and a neutrophil elastase capture antibody coupled to the Raman signal molecule.

[0007] Preferably, the gold core-silver shell nanostar comprises a gold nanocore, a gold nanocoating layer coated on the surface of the gold nanocore, and a silver nanoshell layer coated on the surface of the gold nanocoating layer.

[0008] Preferably, the average particle size of the gold nanocore is 18-22 nm, the thickness of the gold nanocoating layer is 40-60 nm, and the thickness of the silver nanoshell layer is 5-8 nm.

[0009] Preferably, the Raman signal molecule comprises 5,5-dithio-bis-(2-nitrobenzoic acid) and / or 4-mercaptobenzoic acid.

[0010] The present invention provides a method for preparing the Raman enhanced nano-label described in the above technical solution, comprising the following steps:

[0011] The gold core-silver shell nanostar, the Raman signal molecule and the first solvent are mixed and modified to obtain the gold core-silver shell nanostar with the Raman signal molecule modified on the surface;

[0012] The gold core-silver shell nanostar with the Raman signal molecule modified on the surface, the neutrophil elastase capture antibody and the second solvent are mixed to carry out coupling reaction to obtain the Raman enhanced nano-label.

[0013] Preferably, the modification treatment is carried out at a temperature of 20-26° C. for 10-14 h; the coupling reaction is carried out at a temperature of 35-37° C. for 1.5-3 h.

[0014] The present invention provides the use of the Raman enhanced nano-tag described in the above technical solution or the Raman enhanced nano-tag prepared by the preparation method described in the above technical solution in the preparation of a neutrophil elastase immunochromatographic detection system.

[0015] Preferably, samples applicable to the neutrophil elastase immunochromatographic detection system include biological samples.

[0016] Preferably, the biological sample comprises nasal secretions.

[0017] The present invention provides a neutrophil elastase immunochromatographic detection system, comprising a Raman enhanced nano-label, an immunochromatographic test strip and a sample solution; the Raman enhanced nano-label is the Raman enhanced nano-label described in the above technical solution or the Raman enhanced nano-label prepared by the preparation method described in the above technical solution; the immunochromatographic test strip is provided with a detection line and a quality control line, the detection line is loaded with a neutrophil elastase detection antibody, and the quality control line is loaded with goat anti-rabbit IgG.

[0018] Beneficial effects: The Raman enhanced nano-tag provided by the present invention comprises a gold core silver shell nano-star, a Raman signal molecule modified on the surface of the gold core silver shell nano-star, and a neutrophil elastase capture antibody coupled to the Raman signal molecule. The present invention uses a gold core silver shell nano-star, that is, a silver shell-wrapped gold nano-star as a surface enhanced Raman spectroscopy (SERS) substrate, which has both abundant SERS "hot spots" and the advantages of silver-based nanomaterials. After modification with Raman signal molecules and coupling with ELANE capture antibodies, it is applied as a SERS tag to immunochromatography, and the formation of an antigen-antibody complex causes a change in the signal intensity of the Raman signal molecule to achieve ELANE qualitative and quantitative analysis. It has the advantages of simple and fast immunochromatography and low detection limit and accurate quantification of SERS detection. The results of the test example show that the Raman enhanced nano-tag combined with the immunochromatography technology of the present invention can realize the real-time on-site rapid and accurate detection of the ELANE level in human nasal secretions, and the detection limit is low, which has a reference value for early detection of acute attacks and aggravation of allergic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 TEM and SEM images of Au NS and Au@Ag NS;

[0020] Figure 2 The particle size distribution diagram of Au NS and Au@Ag NS and the Zeta potential test results of Au NS, Au@Ag NS and Au@Ag@Anti-ELANE;

[0021] Figure 3 Raman spectra of Au@Ag NS and Au@Ag@DTNB NS;

[0022] Figure 4 This is the result diagram of the sensitivity of ELANE detected by Raman immunochromatography;

[0023] Figure 5 This is the result diagram of specificity verification of Raman immunochromatography detection;

[0024] Figure 6 This is a photo of the immunochromatographic test strip for detecting ELANE in nasal secretions. DETAILED DESCRIPTION

[0025] The invention provides a Raman enhanced nano-label, comprising a gold core silver shell nano-star, a Raman signal molecule modified on the surface of the gold core silver shell nano-star, and a neutrophil elastase capture antibody coupled to the Raman signal molecule.

[0026] The Raman enhanced nano-tag of the present invention comprises a gold core silver shell nanostar, and the gold core silver shell nanostar preferably comprises a gold nanocore, a gold nanocoating layer coated on the surface of the gold nanocore, and a silver nanoshell layer coated on the surface of the gold nanocoating layer. In the present invention, the average particle size of the gold nanocore is preferably 18-22nm, more preferably 20nm; the thickness of the gold nanocoating layer is preferably 40-60nm, more preferably 55nm; the thickness of the silver nanoshell layer is preferably 5-8nm, more preferably 6nm.

[0027] The Raman enhanced nano-tag of the present invention comprises a Raman signal molecule modified on the surface of the gold core silver shell nanostar. In the present invention, the Raman signal molecule preferably comprises 5,5-disulfide-bis-(2-nitrobenzoic acid) and / or 4-mercaptobenzoic acid, more preferably 5,5-disulfide-bis-(2-nitrobenzoic acid). The gold core silver shell nanostar and the Raman signal molecule of the present invention are specifically modified on the gold core silver shell nanostar through an Ag-S bond.

[0028] The Raman enhanced nano-tag of the present invention comprises a neutrophil elastase capture antibody coupled to the Raman signal molecule. In the present invention, the neutrophil elastase capture antibody is specifically coupled to the Raman signal molecule by an amide reaction.

[0029] The present invention provides a method for preparing the Raman enhanced nano-label described in the above technical solution, comprising the following steps:

[0030] The gold core-silver shell nanostar, the Raman signal molecule and the first solvent are mixed and modified to obtain the gold core-silver shell nanostar with the Raman signal molecule modified on the surface;

[0031] The gold core-silver shell nanostar with the Raman signal molecule modified on the surface, the neutrophil elastase capture antibody and the second solvent are mixed to carry out coupling reaction to obtain the Raman enhanced nano-label.

[0032] In the present invention, unless otherwise specified, the reagents or raw materials used are purchased from commercial channels or prepared by methods well known to those skilled in the art. The method of the present invention is described in detail below.

[0033] The present invention first prepares a gold core-silver shell nanostar, and the preparation method of the gold core-silver shell nanostar preferably comprises the following steps:

[0034] The HAuCl4 solution is mixed with water and heated to boiling, and the resulting material is mixed with a sodium citrate solution for reduction reaction to obtain a gold nanoparticle dispersion;

[0035] The gold nanoparticle dispersion, the hydroquinone solution, the sodium dodecyl sulfate solution and water are mixed to grow the gold nanostar dispersion to obtain the gold nanostar dispersion;

[0036] The gold nanostar dispersion, cetyltrimethylammonium bromide solution, ascorbic acid solution and AgNO3 solution are mixed, and then the pH value of the obtained mixed solution is adjusted to alkaline to obtain the gold core silver shell nanostar.

[0037] The present invention mixes HAuCl4 solution with water and heats it to boiling, mixes the obtained material with sodium citrate solution for reduction reaction, and obtains a gold nanoparticle dispersion. In the present invention, the concentration of the HAuCl4 solution is preferably 0.009~0.011g / mL, more preferably 0.01g / mL; the water is preferably deionized water; the concentration of the sodium citrate solution is preferably 0.009~0.011g / mL, more preferably 0.01g / mL; the volume ratio of the water, HAuCl4 solution and sodium citrate solution is preferably 50:0.45~0.55:14~16, more preferably 50:0.5:15. In the present invention, the time of the reduction reaction is preferably 13~16min, more preferably 15min; the reduction reaction is preferably carried out under stirring conditions, and the stirring speed is preferably 500~650rpm, more preferably 600rpm.

[0038] After obtaining the gold nanoparticle dispersion, the present invention mixes the gold nanoparticle dispersion, hydroquinone solution, sodium dodecyl sulfate solution and water to grow and obtain a gold nanostar dispersion. In the present invention, the concentration of the hydroquinone solution is preferably 20-40 mM, more preferably 30 mM; the concentration of the sodium dodecyl sulfate solution is preferably 0.008-0.015 mg / mL, more preferably 0.01 mg / mL; the volume ratio of the gold nanoparticle dispersion, hydroquinone solution, sodium dodecyl sulfate solution and water is preferably 0.14-0.16:0.9-1:0.9-1:10, more preferably 0.15:1:1:10. In the present invention, the growth temperature is preferably 20-26°C, more preferably 25°C; the time is preferably 25-35 min, more preferably 30 min.

[0039] After obtaining the gold nanostar dispersion, the present invention mixes the gold nanostar dispersion, hexadecyltrimethylammonium bromide solution, ascorbic acid solution and AgNO3 solution, and then adjusts the pH value of the obtained mixed liquid to alkaline to obtain the gold core silver shell nanostar. In the present invention, the concentration of the hexadecyltrimethylammonium bromide solution is preferably 80-120mM, more preferably 100mM; the concentration of the ascorbic acid solution is preferably 0.08-0.12M, more preferably 0.1M; the concentration of the AgNO3 solution is preferably 9-11mM, more preferably 10mM; the volume ratio of the gold nanostar dispersion, hexadecyltrimethylammonium bromide solution, ascorbic acid solution and AgNO3 solution is preferably 15mL:7-9mL:240-260μL:40-50μL, more preferably 15mL:8mL:260μL:50μL. In the present invention, the reagent used to adjust the pH value is preferably a NaOH solution; the alkalinity specifically refers to a pH value of 8.5 to 9.0, preferably 8.5 to 8.7. In the present invention, after the pH value of the obtained mixed liquid is adjusted to alkaline, the system quickly turns yellow to obtain a gold core silver shell nanostar dispersion, which can be directly used in subsequent steps without any post-treatment.

[0040] After obtaining the gold core silver shell nanostar, the present invention mixes the gold core silver shell nanostar, the Raman signal molecule and the first solvent, and performs a modification treatment to obtain a gold core silver shell nanostar with a surface modified with a Raman signal molecule. In the present invention, the first solvent is preferably water and ethanol; the volume ratio of water to ethanol is preferably 1:0.01~0.02, and more preferably 1:0.015. The present invention preferably mixes the gold core silver shell nanostar dispersion (i.e., water as a dispersant) prepared by the above technical solution with a Raman signal molecule solution (the solvent is preferably ethanol, more preferably anhydrous ethanol), and performs a modification treatment to obtain a gold core silver shell nanostar with a surface modified with a Raman signal molecule. In the present invention, the concentration of the Raman signal molecule solution is preferably 8~12mM, and more preferably 10mM. In the present invention, the temperature of the modification treatment is 20-26°C, more preferably 25°C; the time is preferably 10-14h, more preferably 12h; the modification treatment is preferably carried out under stirring conditions, and the stirring speed is preferably 500-650rpm, more preferably 600rpm.

[0041] After obtaining the gold core silver shell nanostar with a Raman signal molecule modified on the surface, the present invention mixes the gold core silver shell nanostar with a Raman signal molecule modified on the surface, the neutrophil elastase capture antibody and a second solvent, and performs a coupling reaction to obtain the Raman enhanced nanotag. In the present invention, the second solvent is preferably water. In the present invention, the modification treatment preferably further includes an activation treatment; the reagent used for the activation treatment preferably includes carbodiimide and N-hydroxysuccinimide. Specifically, after the modification treatment, a dispersion of gold core silver shell nanostars with a Raman signal molecule modified on the surface is obtained, and then the dispersion of gold core silver shell nanostars with a Raman signal molecule modified on the surface is mixed with a carbodiimide solution (the solvent is preferably water) and an N-hydroxysuccinimide solution (the solvent is preferably water) for activation treatment. In the present invention, the concentration of the carbodiimide solution is preferably 8-10mM, more preferably 9-10mM; the concentration of the N-hydroxysuccinimide solution is preferably 8-10mM, more preferably 9-10mM; based on the volume of the gold core silver shell nanostar dispersion, the volume ratio of the gold core silver shell nanostar dispersion, the carbodiimide solution and the N-hydroxysuccinimide solution is preferably 1mL:2-4μL:4-8μL, more preferably 1mL:3μL:6μL. In the present invention, the temperature of the activation treatment is preferably 35-37°C, more preferably 36-37°C; the time is preferably 12-16min, more preferably 15min; the activation treatment is preferably carried out under shaking conditions.

[0042] After the activation treatment, the present invention preferably mixes the obtained activation liquid with the neutrophil elastase capture antibody to carry out a coupling reaction. In the present invention, based on the volume of the gold core silver shell nanostar dispersion, the dosage ratio of the gold core silver shell nanostar dispersion to the neutrophil elastase capture antibody is preferably 1mL: 8~10μg, more preferably 1mL: 9~10μg. In the present invention, the temperature of the coupling reaction is preferably 35~37°C, more preferably 36~37°C; the time is preferably 1.5~3h, more preferably 2h; the coupling reaction is preferably carried out under shaking conditions. After the coupling reaction, the present invention preferably centrifuges the obtained liquid, collects the precipitate (i.e., the Raman enhanced nanotag) and resuspends it in the dispersion medium to obtain a Raman enhanced nanotag dispersion. In the present invention, the centrifugal speed is preferably 3500~4500rpm, more preferably 4000rpm; the centrifugal time is preferably 5~7min, more preferably 6min. In the present invention, the dispersion medium is preferably a PBS buffer containing BSA, and the concentration of BSA in the dispersion medium is preferably 0.01 mg / mL. The Raman enhanced nano-tag dispersion of the present invention is preferably stored at 4°C for future use.

[0043] The Raman-enhanced nano-tag provided by the present invention is combined with immunochromatography technology, which can achieve efficient capture of ELANE antigen and can be used for quantitative detection of ELANE in complex biological samples. In the embodiment of the present invention, the gold core silver shell nanostar (Au@Ag NS) is prepared by seed growth method, Raman signal molecules such as 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) are firmly bound and adsorbed on the surface of the silver shell through Ag-S bonds, and the DTNB carboxyl group is coupled to the ELANE capture antibody at the end of the DTNB by amide reaction to form a Raman-enhanced nano-tag; based on the principle of antibody sandwich method, the Raman-enhanced nano-tag is combined with immunochromatography technology, which can detect low-concentration ELANE antigen in nasal secretion samples without any treatment, and the detection sensitivity is high and the detection time is short.

[0044] The present invention provides the use of the Raman enhanced nano-tag described in the above technical solution or the Raman enhanced nano-tag prepared by the preparation method described in the above technical solution in the preparation of a neutrophil elastase immunochromatographic detection system. In the present invention, the neutrophil elastase immunochromatographic detection system preferably includes a Raman enhanced nano-label, an immunochromatographic test strip and a sample solution; the immunochromatographic test strip is preferably provided with a detection line and a quality control line; the detection line is preferably loaded with a neutrophil elastase detection antibody, specifically, a neutrophil elastase detection antibody solution is coated at the detection line (preferably obtained by diluting the neutrophil elastase detection antibody stock solution with PBS buffer), and the concentration of the neutrophil elastase detection antibody solution is preferably 0.55~0.6mg / mL, more preferably 0.58~0.6mg / mL; the quality control line is preferably loaded with goat anti-rabbit IgG, specifically, a goat anti-rabbit IgG solution is coated at the quality control line (preferably obtained by diluting the goat anti-rabbit IgG stock solution with PBS buffer), and the concentration of the goat anti-rabbit IgG solution is preferably 0.7~0.9mg / mL, more preferably 0.8mg / mL. In the present invention, the loading solution is preferably a PBS buffer solution containing BSA and TW20; the concentration of BSA in the loading solution is preferably 0.01 mg / mL, and the concentration of TW20 is preferably 0.01 mg / mL.

[0045] In the present invention, the samples applicable to the neutrophil elastase immunochromatographic detection system preferably include biological samples. In the present invention, the biological samples preferably include nasal secretions.

[0046] In the present invention, taking the detection of nasal secretions as an example, the method for detecting neutrophil elastase in nasal secretions using the neutrophil elastase immunochromatographic detection system preferably includes the following steps:

[0047] The Raman enhanced nano-label dispersion prepared by the above technical scheme is mixed with the nasal secretion sample for vibration reaction to form an Au@Ag NS / ELANE antigen complex. Then, the sample solution is added to the obtained system and the immunochromatographic test strip is inserted. After a predetermined time, the Raman signal at the detection line of the immunochromatographic test strip is read, thereby realizing the accurate quantitative detection of ELANE in the nasal secretion sample.

[0048] In the present invention, the nasal secretion sample is preferably not pretreated; the volume ratio of the Raman enhanced nano-tag dispersion, the nasal secretion sample and the loading liquid is preferably 2:7~8:70~75, more preferably 2:8:70. In the present invention, the temperature of the oscillation reaction is preferably 35~37°C, more preferably 36~37°C; the time is preferably 8~12min, more preferably 10min. In the present invention, the predetermined time is preferably 12~16min, more preferably 15min.

[0049] The present invention provides a neutrophil elastase immunochromatographic detection system, comprising a Raman-enhanced nano-label, an immunochromatographic test strip and a loading solution; the Raman-enhanced nano-label is the Raman-enhanced nano-label described in the above technical solution or the Raman-enhanced nano-label prepared by the preparation method described in the above technical solution; the immunochromatographic test strip is provided with a detection line and a quality control line, the detection line is loaded with a neutrophil elastase detection antibody, and the quality control line is loaded with goat anti-rabbit IgG. The immunochromatographic test strip and the loading solution in the neutrophil elastase immunochromatographic detection system of the present invention are preferably consistent with the above technical solution, which will not be repeated here; the method for detecting neutrophil elastase using the neutrophil elastase immunochromatographic detection system is preferably consistent with the above technical solution, which will not be repeated here.

[0050] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] The main materials and sources used in the following examples are as follows:

[0052] Anhydrous ethanol: Sinopharm Chemical Reagent Co., Ltd., catalog number 10009218;

[0053] Chloroauric acid tetrahydrate (HAuCl4·4H2O): Sinopharm Chemical Reagent Co., Ltd., catalog number 10010711;

[0054] Sodium citrate: Sinopharm Chemical Reagent Co., Ltd., catalog number 10019408;

[0055] Hydroquinone: Merck, Germany, product number H17902;

[0056] Sodium dodecyl sulfate (SDS): Sinopharm Chemical Reagent Co., Ltd., catalog number 30166627;

[0057] Hexadecyltrimethylammonium bromide (CTAB): Merck, Germany, product number H5882;

[0058] Ascorbic acid (AA): Merck, Germany, product number 1043003;

[0059] Silver nitrate (AgNO3): Sinopharm Chemical Reagent Co., Ltd., catalog number 10018464;

[0060] 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB): Merck, Germany, product number D218200;

[0061] EDC: Merck, Germany, product number 39391;

[0062] N-hydroxysuccinimide (NHS): Merck, Germany, catalog number 130672;

[0063] Sodium hydroxide (NaOH): Merck, Germany, product number S5881;

[0064] ELANE antigen: Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., catalog number HY-P72179;

[0065] Anti-ELANE capture antibody: Wuhan Proteintech Biotechnology Co., Ltd., catalog number 27642-1-AP;

[0066] Anti-ELANE detection antibody: Shanghai Abcam Trading Co., Ltd., catalog number EPR7479;

[0067] Bovine serum albumin (BSA): Beijing Solebow Technology Co., Ltd., catalog number: A8020;

[0068] PBS buffer: Thermo Fisher Scientific, Cat. No. 10010002;

[0069] Tween 20 (TW20): Merck, Germany, catalog number P9416;

[0070] Goat anti-rabbit IgG: Shanghai Sangon Biotechnology Co., Ltd., catalog number: D111018;

[0071] Immunochromatographic test strip components: Shanghai Jinbiao Biotechnology Co., Ltd.

[0072] TEM images were taken with a JEM-2100 microscope; SEM images were taken with a Hitachi S-4800 microscope; the Zeta potential of nanomaterials was detected using a Malvern Nano-ZS90 Zetasizer; Raman spectra were collected by B&W TEK i-Ramanplus; and antibody streaking was achieved using a GoldBio HM3035 gold-spraying streaking instrument.

[0073] The nasal secretion samples came from Tongren Hospital affiliated to Capital Medical University. The volunteers who provided the nasal secretions followed the principle of informed consent, and the acquisition of the nasal secretions was reviewed and approved by the Ethics Committee of Capital Medical University.

[0074] Example 1

[0075] The preparation of Raman enhanced nano-labels comprises the following steps:

[0076] (1) 50 mL of deionized water was mixed with 0.5 mL of 0.01 g / mL HAuCl4 solution, and the resulting mixed solution was heated to boiling. Then, 1.5 mL of 0.01 g / mL sodium citrate solution was added under constant stirring at 600 rpm, and the constant stirring was continued at 600 rpm for 15 min to obtain a dispersion of gold nanoparticles (average particle size of 20 nm); 0.15 mL of the gold nanoparticle dispersion was taken as a seed dispersion and mixed with 1 mL of 30 mM hydroquinone solution, 1 mL of 0.01 mg / mL SDS solution, and 10 mL of deionized water, and the mixture was stirred at 600 rpm for 30 min at 25 °C to obtain a dispersion of gold nanostars (Au NS);

[0077] (2) 15 mL of Au NS dispersion was mixed with 8 mL of 100 mM cetyltrimethylammonium bromide solution, and then 260 μL of 0.1 M ascorbic acid solution and 50 μL of 10 mM AgNO3 solution were added in sequence. The components were completely mixed by ultrasonic treatment, and then NaOH solution was added to adjust the pH value of the mixed solution to 8.5. The system quickly turned yellow to obtain a gold core silver shell nanostar (Au@Ag NS) dispersion.

[0078] (3) 1 mL of Au@Ag NS dispersion was mixed with 15 μL of 10 mM 5,5'-dithiobis(2-nitrobenzoic acid) solution (solvent: anhydrous ethanol) under constant stirring at 600 rpm, and the mixture was stirred at 600 rpm for 12 h at 25 °C to obtain Au@Ag NS modified with 5,5'-dithiobis(2-nitrobenzoic acid) (Au@Ag@DTNB NS). Then, 3 μL of 10 mM carbodiimide solution and 6 μL of 10 mM N-hydroxysuccinimide solution were added, and the mixture was shaken at 25 °C for 15 min. Then, 10 μg Anti-ELANE capture antibody was incubated at 37°C with shaking for 2 h. After the incubation, the resulting solution was centrifuged at 4000 rpm for 6 min, and the precipitate (i.e., Raman-enhanced nanotag) was collected and resuspended in PBS buffer containing BSA (BSA concentration was 0.01 mg / mL) to obtain a Raman-enhanced nanotag dispersion, which was stored at 4°C for later use.

[0079] Figure 1 TEM images and SEM images of Au NS and Au@Ag NS, where a is the TEM image of Au NS, b is the SEM image of Au NS, c is the TEM image of Au@Ag NS, and d is the SEM image of Au@Ag NS. The results show that the surfaces of Au NS and Au@Ag NS prepared in Example 1 have multiple irregular branches and sharp tips, and the branches of Au NS are sharper. After the surface of Au NS is coated with a silver shell by reduction reaction, the branches of the obtained Au@Ag NS are significantly blunted, proving that the silver shell is successfully coated.

[0080] Figure 2 The particle size distribution diagram of Au NS and Au@Ag NS and the Zeta potential test results of Au NS, Au@Ag NS and Au@Ag@Anti-ELANE are shown in Figure 1, where a is the particle size distribution diagram of Au NS and Au@Ag NS, and b is the Zeta potential test results of Au NS, Au@Ag NS and Au@Ag@Anti-ELANE. Figure 2 From a in the figure, we can see that the Au NS particle size distribution is uniform, with an average particle size of 88.78 nm. As the silver shell grows on the surface of the Au NS, the average particle size of the resulting Au@Ag NS increases to 90.07 nm. Figure 2 As shown in b, the Zeta potential of Au NS is -37.1 mV; after the silver shell is deposited on the surface of Au NS, the Zeta potential of Au@Ag NS increases sharply to 47.1 mV; after coupling with ELANE antibody, the Zeta potential of the obtained Au@Ag@Anti-ELANE drops to -33.6 mV, proving the successful encapsulation of the silver shell and the successful coupling of ELANE.

[0081] Figure 3 The Raman spectra of Au@Ag NS and Au@Ag@DTNB NS show that after the Raman characteristic molecule DTNB is used to modify Au@Ag NS, the DTNB can be seen at 1331cm -1 The characteristic signal peak at , and the Raman signal intensity is high, proving that the Au@Ag NS has a good Raman scattering enhancement effect.

[0082] Example 2

[0083] The construction of ELANE Raman immunochromatography includes the following steps:

[0084] (1) Preparation of immunochromatographic test strips: Goat anti-rabbit IgG and Anti-ELANE detection antibody were coated on the control line (C) and the detection line (T) of a blank test strip, respectively, to obtain an immunochromatographic test strip, wherein the concentration of Anti-ELANE detection antibody coated on the immunochromatographic test strip was 0.6 mg / mL, and the concentration of goat anti-rabbit IgG was 0.8 mg / mL;

[0085] (2) 8 μL of the Raman enhanced nano-label dispersion prepared in Example 1 was added to different concentrations of ELANE antigen standard solutions (concentrations of 1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, and 0.01 ng / mL; volume of 2 μL), and the mixture was shaken and reacted at 37°C for 10 min to form an Au@Ag NS / ELANE antigen complex; then 70 μL of the loading solution (specifically PBS buffer containing BSA and TW20, wherein the concentrations of BSA and TW20 were both 0.01 mg / mL) was added and inserted into the immunochromatographic test strip obtained in step (1); after 15 min, the Raman signal at the detection line of the immunochromatographic test strip was read, thereby achieving accurate quantitative detection of ELANE;

[0086] (3) 8 μL of the Raman enhanced nano-tag dispersion prepared in Example 1 was added to the ELANE antigen standard solution, CA19-9 standard solution, CEA standard solution, P-Tau217 standard solution and P-Tau231 standard solution (the concentration of each standard solution was 1 μg / mL and the volume was 2 μL), respectively, and the reaction was shaken at 37°C for 10 min. Then, 70 μL of the sample solution was added and the immunochromatographic test strip obtained in step (1) was inserted. After 15 minutes, the Raman signal at the detection line of the immunochromatographic test strip was read to verify the specificity of the detection system.

[0087] Figure 4 This is the sensitivity result diagram of ELANE detected by Raman immunochromatography. Figure 4Figure a is a photograph of an immunochromatographic test strip using Raman immunochromatography to detect different concentrations of ELANE antigen standard solutions. The results show that the color depth of the Raman enhanced nanolabel in the T-line area decreases as the ELANE concentration decreases. Figure 4 Figure b is the Raman spectrum detection result of the T line area of ​​the immunochromatographic test strip. It can be seen that as the ELANE concentration decreases, the Raman signal intensity gradually weakens. When the ELANE concentration drops to 0.01 ng / mL, DTNB can still be clearly observed at 1331 cm -1 The characteristic signal peak at . Figure 4 The c in the equation is based on 1331cm -1 The detection limit was calculated to be 0.045 pg / mL by fitting the Raman signal intensity to the ELANE standard curve.

[0088] Figure 5 This is the result diagram of specificity verification of Raman immunochromatography detection. Figure 5 Figure a is a photograph of the immunochromatographic test strips for detecting ELANE, CA19-9, CEA, P-Tau217 and P-Tau231. The results show that only the T-line area of ​​ELANE has obvious black bands, while the T-line areas of the other five immunochromatographic test strips (including the blank control) have no obvious changes. Figure 5 b is the corresponding Raman spectrum detection result of the T line area, and c is the corresponding Raman spectrum of the T line area at 1331cm -1 The average Raman signal intensity at , proves that the detection of human ELANE by the method of the present invention is not easily interfered by other proteins.

[0089] Example 3

[0090] The detection of ELANE in nasal secretions based on Raman immunochromatography includes the following steps:

[0091] 2 μL of the Raman enhanced nano-label dispersion prepared in Example 1 was added to 8 μL of nasal secretions containing different concentrations of ELANE (100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL), and the mixture was shaken and reacted at 37°C for 10 min to form an Au@Ag NS / ELANE antigen complex. Then, 70 μL of the sample solution was added and the immunochromatographic test strip obtained in step (1) of Example 2 was inserted. After 15 min, the Raman signal at the detection line of the immunochromatographic test strip was read, thereby achieving accurate quantitative detection of ELANE in nasal secretions.

[0092] Figure 6 Photo of the immunochromatographic test strip for ELANE in nasal secretions, reading 1331 cm -1The Raman signal intensity at the position was substituted into the ELANE standard curve established in Example 2 to calculate the detection concentration and recovery rate, and the results are shown in Table 1. As can be seen from Table 1, the recovery rate of ELANE detected in nasal secretions is between 92.34% and 105.26%, and the relative standard deviation values ​​are all less than 3.77%, indicating that the method of the present invention has good accuracy and applicability for detecting ELANE in nasal secretions.

[0093] Table 1 Recovery and relative standard deviation of ELANE in nasal secretions

[0094]

[0095] It can be seen from the above embodiments and test examples that the present invention has at least the following beneficial effects:

[0096] (1) The Raman-enhanced nano-tag provided by the present invention has superior performance. From a structural point of view, it has a silver nano-shell layer and has extremely strong Raman enhancement ability. In addition, the Raman-enhanced nano-tag has good biocompatibility, good dispersibility, strong stability, and easy storage. The carboxylated nano-shell provides a site for ELANE antibody coupling, and has excellent comprehensive performance.

[0097] (2) The present invention prepares Raman enhanced nano-labels by combining the seed growth method with the ascorbic acid reduction method. The preparation method is convenient, has good repeatability, and can be mass-produced.

[0098] (3) In the neutrophil elastase immunochromatographic detection system provided by the present invention, the Raman enhanced nano-tag has a silver nano-shell layer, which can detect extremely low Raman signal values, realize trace detection of ELANE in samples, have good specificity, and can detect low concentrations of ELANE through weak Raman signals, thereby effectively improving the sensitivity of SERS immunochromatographic detection, being suitable for rapid quantitative detection of low concentration samples, and being beneficial to the application of clinical detection.

[0099] (4) The neutrophil elastase immunochromatographic detection system provided by the present invention has a short detection time and can be used for direct detection of nasal secretion samples, avoiding the complicated pre-treatment process of the samples and can be applied to various scenarios; the neutrophil elastase immunochromatographic detection system has good quantitative ability and does not need to rely on large laboratory instruments and professionals, which has great advantages in real-time on-site detection.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Raman enhanced nanotag, comprising a gold core silver shell nanostar, a Raman signal molecule modified on the surface of the gold core silver shell nanostar, and a neutrophil elastase capture antibody coupled to the Raman signal molecule; The gold core-silver shell nanostar is composed of a gold nanocore, a gold nanocoating layer coated on the surface of the gold nanocore, and a silver nanoshell layer coated on the surface of the gold nanocoating layer; The Raman signal molecule is 5,5-dithio-bis-(2-nitrobenzoic acid).

2. The Raman enhanced nano-tag according to claim 1, characterized in that: The average particle size of the gold nanocore is 18-22 nm, the thickness of the gold nanocoating layer is 40-60 nm, and the thickness of the silver nanoshell layer is 5-8 nm.

3. The method for preparing the Raman enhanced nano-tag according to claim 1 or 2, comprising the following steps: The gold core-silver shell nanostar, the Raman signal molecule and the first solvent are mixed and modified to obtain the gold core-silver shell nanostar with the Raman signal molecule modified on the surface; The gold core-silver shell nanostar with the Raman signal molecule modified on the surface, the neutrophil elastase capture antibody and the second solvent are mixed to carry out coupling reaction to obtain the Raman enhanced nano-label.

4. The preparation method according to claim 3, characterized in that: The temperature of the modification treatment is 20-26° C., and the time is 10-14 h; the temperature of the coupling reaction is 35-37° C., and the time is 1.5-3 h.

5. Use of the Raman-enhanced nanotag according to claim 1 or 2 or the Raman-enhanced nanotag prepared by the preparation method according to claim 3 or 4 in preparing a neutrophil elastase immunochromatographic detection system.

6. The use according to claim 5, characterized in that: The samples applicable to the neutrophil elastase immunochromatographic detection system include biological samples.

7. The use according to claim 6, characterized in that: The biological sample includes nasal secretions.

8. A neutrophil elastase immunochromatographic detection system, comprising a Raman-enhanced nanotag, an immunochromatographic test strip and a sample solution; the Raman-enhanced nanotag is the Raman-enhanced nanotag described in claim 1 or 2 or the Raman-enhanced nanotag prepared by the preparation method described in claim 3 or 4; the immunochromatographic test strip is provided with a detection line and a quality control line, the detection line is loaded with a neutrophil elastase detection antibody, and the quality control line is loaded with goat anti-rabbit IgG.

Images