Immunodetection kit based on surface enhanced Raman spectroscopy as well as preparation method and application of immunodetection kit

Abstract

The invention discloses an immunodetection kit based on surface enhanced Raman spectroscopy as well as a preparation method and application of the immunodetection kit. The kit comprises a surface enhanced Raman scattering active substrate, wherein the active substrate comprises cellulose paper loaded with first metal nanoparticles, carboxyl-sulfydryl polyethylene glycol and a capture antibody, wherein the carboxyl-sulfydryl polyethylene glycol and the capture antibody are used for modifying the surfaces of the first metal nanoparticles; the surface enhanced Raman scattering immunodetection probe comprises a second metal nanoparticle, a Raman signal molecule, methoxy-sulfydryl polyethylene glycol and a detection antibody, wherein the Raman signal molecule, the methoxy-sulfydryl polyethylene glycol and the detection antibody are used for modifying the surface of the second metal nanoparticle. The immunodetection kit disclosed by the invention is high in sensitivity, rapid, accurate and low in false positive.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and in particular relates to an immunoassay kit based on surface enhanced Raman spectroscopy and a preparation method and application thereof. Background Art

[0002] Paper-based lateral flow immunoassay (LFA) technology is a commonly used and inexpensive method for instant detection, but it still has problems such as high false positive rate, low sensitivity, and complex preparation procedures. SERS has the advantages of high sensitivity and rapid detection, and has been widely used in the field of LFA. However, SERS-based LFA detection still requires a chromatography process and a long detection time. At the same time, its sensitivity is restricted by the Raman enhancement effect of colloidal gold (Au NP) itself, that is, the lower the analyte concentration, the fewer Au NPs on the detection line, and the weaker the signal intensity. On the other hand, the surface of the gold nanoprobe is usually blocked by non-covalent bonding (such as BSA), and this unstable blocking method can easily cause BSA to fall off during the operation process, resulting in false positives.

[0003] Therefore, a sensitive, rapid and accurate immunoassay technology is urgently needed. Summary of the invention

[0004] The main purpose of the present invention is to provide a sensitive, rapid and accurate immunoassay kit based on surface enhanced Raman spectroscopy and a preparation method and application thereof in view of the problems and shortcomings of the prior art.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] The present invention provides an immunoassay kit based on surface enhanced Raman spectroscopy, the kit comprising:

[0007] A surface enhanced Raman scattering active substrate, the active substrate comprising cellulose paper loaded with first metal nanoparticles and carboxyl-thiol polyethylene glycol (PEG1) and capture antibodies modified on the surface of the first metal nanoparticles;

[0008] A surface enhanced Raman scattering immunodetection probe comprises a second metal nanoparticle and a Raman signal molecule modified on the surface of the second metal nanoparticle, methoxy-thiol polyethylene glycol (PEG2) and a detection antibody.

[0009] Preferably, the first metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, gold-silver nanoparticles or gold nanostars.

[0010] Preferably, the molecule of the carboxyl-thiol polyethylene glycol is HOOC(CH2)2-(OCH2CH2) n-SH, the molecular weight of which is 150 to 2000 Da1, preferably 680 Da1.

[0011] Preferably, the second metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, gold core-silver shell nanoparticles, with a particle size of 30 to 50 nm, more preferably 40 nm; the absorbance of the nanoparticle solution is 1 to 1.5, more preferably 1.3.

[0012] Preferably, it is characterized in that the molecule of the methoxy-mercapto polyethylene glycol is CH3O-(CH2CH2O) n -(CH2)2SH, with a molecular weight of 140 to 600 Da1, preferably 180 Da1.

[0013] The present invention also provides a method for preparing an immunoassay kit based on surface enhanced Raman spectroscopy, comprising the following steps:

[0014] S1: soaking the cellulose paper loaded with the first metal nanoparticles in a carboxyl-thiol polyethylene glycol aqueous solution, incubating with shaking at room temperature, washing, activating, then adding the capture antibody, incubating with shaking at room temperature, washing, and drying to obtain a surface-enhanced Raman scattering active substrate modified with carboxyl-thiol polyethylene glycol (PEG1) and capture antibody (Ab1);

[0015] S2: Raman signal molecules and methoxy-thiol polyethylene glycol (PEG2) are modified on the second metal nanoparticles, dispersed in a boric acid buffer solution after centrifugation and washing, activated, and then added with a detection antibody (Ab2) after incubation at room temperature. After further incubation at room temperature, the particles are centrifuged and washed, and dispersed in a preservation solution to obtain a surface-enhanced Raman scattering immunoassay probe modified with a Raman signal molecule, methoxy-thiol polyethylene glycol, and a detection antibody.

[0016] Preferably, the activation method in steps S1 and S2 is to add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution for activation.

[0017] Preferably, the Raman signal molecule in step S2 is one or more of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-nitrothiophenol (4-NTP), p-carboxythiophenol (4-MBA), p-aminothiophenol (4-ATP), and thiophenol (TP), and DTNB is more preferably used. Its solution is an ethanol solution of 1 to 100 mmol / L, and its concentration is more preferably 10 mmol / L; the volume is 10 to 30 μL, and more preferably 20 μL.

[0018] Preferably, in step S1, the concentration of PEG1 solution is 5-15 mmol / L, more preferably 10 mmol / L; the volume of the solution is 1-100 μL, more preferably 20 μL; and the incubation time is 1.5-2.5 h, more preferably 2 h.

[0019] Preferably, the concentration of PEG2 solution in step S2 is 5-15 mmol / L, more preferably 10 mmol / L; the volume is 1-100 μL, more preferably 20 μL; the incubation time is 1.5-2.5 h, more preferably 2 h.

[0020] Preferably, the concentrations of EDC and NHS in steps S1 and S2 are both 5-15 mmol / L, more preferably 10 mmol / L; the solution volume is 1-100 μL, more preferably 20 μL; and the activation time is 10-50 min, more preferably 30 min.

[0021] Preferably, the concentration of the boric acid buffer solution in step S2 is 5-20 mmol / L, more preferably 10 mmol / L; the pH is 7-9, more preferably 8.5; the volume of the boric acid buffer solution used during the dispersion is 0.5-1.5 mL, more preferably 1 mL.

[0022] Preferably, in step S1, the concentration of the capture antibody is 1.5 to 10 μg / mL, more preferably 2.3 μg / mL; the volume is 1 to 30 μL, more preferably 20 μL; and the incubation time is 2 to 6 h, more preferably 4 h.

[0023] Preferably, in step S2, the concentration of the detection antibody is 2.5 to 10 μg / mL, more preferably 5 μg / mL; the volume is 1 to 3 μL, more preferably 2 μL; and the incubation time is 2 to 6 h, more preferably 4 h.

[0024] Preferably, the washing in step S1 is performed using PBS buffer, which is prepared from Na2HPO4 and NaH2PO4, wherein the concentration of Tween 20 is 0.01-0.5% (wt%), more preferably 0.05%, and the pH is 7.2-7.6, more preferably 7.4.

[0025] Preferably, the number of washing times in step S1 is 3 to 5 times, more preferably 3 times; the drying temperature is 20 to 50° C., more preferably 37° C.; and the drying time is 10 to 40 min, more preferably 30 min.

[0026] Preferably, the gold-silver paper in step S1 is prepared by the following steps: multiple sheets (preferably 5 sheets) of cellulose paper are placed in a mixed solution of HAuCl4 and AgNO3, kept in a drying oven for a period of time, and then immersed in a NaBH4 solution, taken out, washed with ultrapure water, and dried to obtain cellulose paper loaded with gold and silver nanoparticles, i.e., gold-silver paper.

[0027] Further preferably, in the preparation of the gold-silver paper in step S1, the size of the cellulose paper is: 0.5-2 cm in length, 0.5-2 cm in width, and more preferably 1 cm in length and 1 cm in width; the volume and concentration of the HAuCl4 solution are 0.5-3 mL and 25-150 mmol / L, respectively, and more preferably 1.5 mL and 75 mmol / L, respectively; the volume and concentration of the AgNO3 solution are 0.5-3 mL and 5-100 mmol / L, respectively, and more preferably 1.5 mL and 25 mmol / L, respectively; the temperature maintained in the drying oven after the HAuCl4 and AgNO3 solutions are mixed is 20-40°C, more preferably 30°C, and the holding time is 5-20 min, more preferably 10 min; the volume and concentration of the NaBH4 solution are 2-10 mL and 10-500 mmol / L, respectively, and more preferably 5 mL and 100 mmol / L, respectively; the number of ultrapure water washings is 2-5 times, and more preferably 3 times; the drying temperature is 35-75°C, and more preferably 55°C.

[0028] Preferably, the Venus paper in step S1 is prepared by the following steps: adding cellulose paper to a solution of mercaptosilane, oscillating at room temperature, washing with an organic solvent and distilled water for several times after the reaction, and drying at room temperature to obtain thiolated cellulose paper; then, adding HAuCl4 solution to a HEPES buffer solution, oscillating and standing to obtain a gold nanostar solution; finally, immersing the thiolated cellulose paper in the gold nanostar solution, oscillating, reacting, rinsing with ultrapure water, and drying to obtain the Venus paper.

[0029] Further preferably, in the preparation of Venus paper in step S1, the size of cellulose is: 0.5-2 cm in length, 0.5-2 cm in width, and more preferably 1 cm in length and 1 cm in width; the mercaptosilane solution is an ethanol solution of (3-mercaptopropyl)trimethoxysilane, and the concentration is 2% to 50%, and more preferably 10%; the oscillation reaction time at room temperature is 2 to 6 h, and more preferably 4 h; the organic solvents used for washing are acetone and ethanol, respectively, and the number of washing times is 2 to 5 times, and more preferably 3 times; the drying time at room temperature is 4 to 12 h, and more preferably 8 hours. The volume and concentration of the HAuCl4 solution are 0.1-1 mL and 1-10 mmol / L, respectively, and more preferably 0.5 mL and 5 mmol / L, respectively; the volume, concentration and pH of the HEPES buffer solution are 1-10 mL, 10-100 mmol / L and 7.0-8.0, respectively, and more preferably 5 mL, 50 mmol / L and 7.5, respectively; the oscillation and standing time are 0.5-3 min and 20-50 min, respectively, and more preferably 1 min and 30 min, respectively. The immersion time of the thiolated cellulose paper in the gold nanostar solution is 8-16 h, and more preferably 12 h; the drying time at room temperature is 2-5 h, and more preferably 3 h.

[0030] Preferably, the storage solution in step S2 comprises PBS buffer solution, BSA, sucrose, and Tween 20, and their concentrations are 5-15 mmol / L, 0.5-1.5%, 0.5-2%, and 0.01-0.5% (wt%), respectively, and more preferably 10 mmol / L, 1%, 1%, and 0.05%, respectively.

[0031] Preferably, when the second metal nanoparticles are gold core-silver shell nanoparticles, the Au@DTNB@Ag SERS immunodetection probe is prepared by the following steps:

[0032] a. After diluting the colloidal gold with MES buffer solution at pH = 6.0, add DTNB solution and incubate for a period of time to obtain Au@DTNB solution;

[0033] b. Add ascorbic acid (AA) to the Au@DTNB solution, and then add AgNO3 to react to obtain

[0034] Au@DTNB@Ag crude solution;

[0035] c. Take the above Au@DTNB@Ag solution and wash it by centrifugation with ultrapure water, and disperse it in a boric acid buffer solution to obtain

[0036] Au@DTNB@Ag;

[0037] d. Add PEG1 molecules to the above Au@DTNB@Ag solution to prepare Au@DTNB@Ag-PEG1;

[0038] e. The Au@DTNB@Ag-PEG1 was activated with DEC / NHS and then connected to Ab1 to obtain the Au@DTNB@Ag SERS probe.

[0039] Further preferably, in the preparation process of Au@DTNB@Ag SERS probe, the size of the colloidal gold is 10-30nm, more preferably 18nm; the volume and concentration of the DTNB are 10-100μL and 1-50mmol / L, more preferably 50μL and 10mmol / L; the incubation condition is 20-60min at room temperature, more preferably 30min; the volume and concentration of AA are 100-1500μL and 50-500mmol / L, more preferably 750μL and 100mmol / L; the volume and concentration of AgNO3 are 1-5mL and 0.1-3mmol / L, more preferably 1.75mL and 1mmol / L, the centrifugal washing condition is 5min, 2 times; the pH of the boric acid buffer solution is 8.5, and the volume is 1mL. The PEG1 connected to the Au@DTNB@Ag is HOOC(CH2)2-(OCH2CH2) n -SH, the molecular weight is 150 to 2000 dal, more preferably 680 dal.

[0040] The modification steps and storage method of Au@DTNB@Ag-PEG1 connected to Ab2 are the same as those of Au-PEG2 / DTNB-Ab2.

[0041] The present invention also provides an application of the immunoassay kit based on surface enhanced Raman spectroscopy, which comprises adding the antigen to be detected into a surface enhanced Raman scattering immunoassay probe solution, mixing and incubating, and then dropping the solution onto a surface enhanced Raman scattering active substrate, reacting the solution sufficiently, and performing Raman detection after washing.

[0042] Preferably, the capture antibody and the detection antibody are both novel coronavirus N protein or cardiac troponin.

[0043] Preferably, the volume of the antigen solution is 1 to 10 μL, more preferably 5 μL; the volume of the detection probe is 5 to 20 μL, more preferably 10 μL; and the incubation time is 3 to 7 min, more preferably 5 min.

[0044] Preferably, the reaction time of the SERS immunodetection probe on the SERS active substrate is 1 to 5 minutes, more preferably 3 minutes.

[0045] Preferably, the Raman instrument is one of a portable Raman spectrometer, a confocal Raman spectrometer, and a handheld Raman spectrometer, and the excitation wavelength is 633 nm.

[0046] The principle of the present invention is: the present invention utilizes HOOC(CH2)2-(OCH2CH2) n -SH molecules (PEG1) are covalently modified to connect the SERS substrate and the capture antibody Ab1 to form a SERS substrate of gold (or gold-silver) paper-PEG1-Ab1. Then DTNB is used as a Raman signal reporter molecule to modify the surface of Au NPs or the middle of the shell of gold core silver shell nanoparticles (Au@DTNB@Ag), and finally covalently linked to the detection antibody Ab2 to prepare a SERS immune detection probe. During detection, the antigen is added to the probe solution, and the specific recognition of the antigen by the detection antibody Ab2 is used to form an immune complex. Then it is dropped on the SERS substrate, and the immune complex is captured by the antibody Ab1 to form an immune sandwich structure of "SERS substrate-antigen-SERS probe". The Raman spectrometer achieves ultra-sensitive, stable and rapid detection of the antigen within 5 minutes.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. The present invention uses cellulose paper modified with in situ prepared gold (or gold-silver) nanoparticles as a SERS enhancement substrate, and the detection process forms an immune sandwich structure of "SERS substrate-antigen-detection probe", and a "hot spot" is formed between the SERS substrate and the SERS immune detection probe. Its sensitivity is improved by nearly four orders of magnitude compared with the traditional LFA method.

[0049] 2. The present invention directly mixes the antigen to be detected with the nanoprobe and then drips it onto the substrate for Raman signal detection, thereby eliminating the lateral flow chromatography process in LFA and greatly reducing the detection time.

[0050] 3. The present invention connects the capture antibody and the detection antibody to the SERS substrate and the nanoprobe respectively through covalent modification, and uses PEG molecules to seal the exposed gold surface in the form of chemical bonds, thereby reducing the false positive problem caused by non-covalent modification (such as BSA backfilling) causing the gold surface to be exposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the detection principle diagram of the present invention.

[0052] Figure 2 (a) Schematic diagram of the gold paper SERS substrate and gold nanoparticle SERS probe for detecting novel coronavirus antigens, (b) SERS spectra of novel coronavirus antigens at different concentrations, and (c) SERS spectra at 1328 cm -1Linear fitting diagram of SERS intensity at ;

[0053] Figure 3 (a) Schematic diagram of the gold paper SERS substrate and gold nanoparticle SERS probe for detecting cardiac troponin, (b) SERS spectra of cardiac troponin at different concentrations and (c) SERS at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0054] Figure 4 (a) Schematic diagram of the gold-silver paper SERS substrate and gold nanoparticle SERS probe for detecting novel coronavirus antigens, (b) SERS spectra of novel coronavirus antigens at different concentrations, and (c) SERS at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0055] Figure 5 (a) Schematic diagram of the gold-silver paper SERS substrate and gold nanoparticle SERS probe for detecting cardiac troponin, (b) SERS spectra of cardiac troponin at different concentrations and (c) SERS at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0056] Figure 6 (a) Schematic diagram of the gold paper SERS substrate and Au@DTNB@Ag SERS probe for detecting novel coronavirus antigens, (b) SERS spectra of novel coronavirus antigens at different concentrations, and (c) SERS spectra at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0057] Figure 7 (a) Schematic diagram of the gold paper SERS substrate and Au@DTNB@Ag SERS probe for detecting cardiac troponin, (b) SERS spectra of cardiac troponin at different concentrations and (c) SERS at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0058] Figure 8 (a) Schematic diagram of the gold-silver paper SERS substrate and Au@DTNB@Ag SERS probe for detecting novel coronavirus antigens, (b) SERS spectra of novel coronavirus antigens at different concentrations, and (c) SERS at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0059] Fig. 9 (a) Schematic diagram of the gold-silver paper SERS substrate and Au@DTNB@Ag SERS probe for detecting cardiac troponin, (b) SERS spectra of cardiac troponin at different concentrations and (c) SERS at 1328 cm -1Linear fitting diagram of SERS intensity at ;

[0060] Fig.10 (a) Schematic diagram of the detection of novel coronavirus antigens by the Venus paper SERS substrate and gold nanoparticle SERS probe, (b) SERS spectra of novel coronavirus antigens at different concentrations, and (c) SERS spectra at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0061] Fig.11 (a) SERS spectra of gold paper SERS substrate + gold nanoparticle SERS probe detecting different concentrations of novel coronavirus antigens in nasal swabs and (b) SERS spectra at 1328 cm -1 Linear fitting diagram of SERS intensity at ;

[0062] Fig.12 (a) SERS spectra of gold paper SERS substrate + gold nanoparticle SERS probe detecting different concentrations of novel coronavirus antigens in throat swabs and (b) SERS spectra at 1328 cm -1 Linear fitting diagram of SERS intensity at . DETAILED DESCRIPTION

[0063] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0064] The present invention utilizes HOOC(CH2)2-(OCH2CH2) n -SH molecules (PEG1) are covalently modified to connect the SERS substrate and the capture antibody Ab1 to form a detection substrate of gold (-silver) paper-PEG1-Ab1. Then, DTNB molecules are covalently modified to connect the gold nanoparticles and the detection antibody Ab2, and CH3O-(CH2CH2O) is used to n -(CH2)2SH(PEG2) caps the excess gold surface to form a detection probe of Au-PEG2 / DTNB-Ab2 (or Au@DTNB@Ag-PEG1-Ab2 SERS probe). Antigens are added during detection, and the specific recognition of the antigen by the detection antibody Ab2 is used to form an immune complex. Then it is dropped onto the SERS substrate, and the immune complex is captured by the capture antibody Ab1 to form an immune sandwich structure of "SERS substrate-antigen-detection probe". The Raman spectrometer achieves ultra-sensitive, stable and rapid detection of antigens within 5 minutes.

[0065] Figure 1This is a schematic diagram of the detection principle of the present invention. The prepared Au-PEG2 / DTNB-Ab2 detection probe is mixed and incubated with the novel coronavirus antigen to form a "double-antibody" immune complex, which is then dropped onto the gold paper-PEG1-Ab1 detection substrate to further form an immune sandwich complex. After washing with PBS buffer, the Raman signal is detected. If a gold-silver paper SERS substrate or Au@DTNB@Ag SERS probe is used, the principle is similar.

[0066] Example 1

[0067] This example determines the feasibility of gold paper SERS substrate + gold nanoparticle SERS probe to detect novel coronavirus antigens (such as Figure 2 a), the specific experimental methods are as follows:

[0068] 1) Preparation of gold paper SERS substrate: 20 pieces of 10mm×10mm cellulose paper were added to 5mL 40mmol / L chloroauric acid aqueous solution, soaked for 2min, then taken out, and then placed in 10mL 40mmol / L NaBH4 aqueous solution, soaked for 10min and taken out to prepare gold seed paper. Then, 1.8mL 0.5mmol / L HAuCl4 solution was added to a solution containing 9.0mL 0.2mol / L CTAB and ultrasonicated for 6min, and then 1.0mL 0.5mmol / L ascorbic acid solution was added. When the solution changed from yellow to colorless, the gold seed paper was immediately added and soaked for 20min, then taken out and rinsed with deionized water 8 times, and dried in a vacuum drying oven at 60℃ for 2h to obtain the in-situ prepared gold paper. Then 20μL 10mmol / L HOOC(CH2)2-(OCH2CH2) 13 -SH was added to 2 mL of ultrapure water and then the 10 pieces of gold paper prepared in situ were added, and incubated at 30 rpm for 2 h. The excess HOOC(CH2)2-(OCH2CH2) was removed by washing three times with ultrapure water. 13 -SH, use filter paper to absorb excess water from the paper base. Then soak the above materials in 2mL ultrapure water, add 20μL 10mmol / L EDC and NHS solution respectively to react for 30min, then add 20μL 2.3μg / mL SARS-CoV-2 labeled antibody 15C3 (Ab1) 0~30μL, and incubate at room temperature for 4h. Wash with PBS buffer solution 3 times and dry at 37℃ for 30min to obtain the SERS substrate of gold paper-PEG1-Ab1.

[0069] 2) Preparation of gold nanoparticle SERS probe: 1 mL of 40 nm gold nanoparticles with an absorbance of 1.3 was mixed with 20 μL of 10 mmol / L DTNB molecules and 20 μL of 10 mmol / L CH3O-(CH2CH2O)2-(CH2)2SH(PEG2) molecules, incubated at room temperature for 2 h, centrifuged, discarded the supernatant, washed three times with borate buffer (pH=8.5), and dispersed in 1 mL of 10 mM borate buffer. Then, 20 μL of 10 mmol / L EDC and 20 μL of 10 mmol / L NHS were added to the above solution for activation, incubated at room temperature for 30 min, 2 μL of 5 μg / mL antibody 11F2 (Ab2) was added, incubated at room temperature for 4 h, centrifuged, washed three times with PBS buffer solution, and dispersed in storage solution (10 mmol / L PBS buffer solution, 1% BSA, 1% sucrose, 0.05% Tween 20) and stored at 4°C to prepare the Au-PEG2 / DTNB-Ab2 immune Raman signal detection probe.

[0070] 3) SERS intensity detection: 5 μL of the test solution containing 10 ng / mL of the new coronavirus antigen and PBS buffer were added to 10 μL of the detection probe solution, mixed, incubated for 5 min, added dropwise to the detection substrate, reacted for 3 min, washed three times with different volumes of PBS buffer solution (pH=7.4, 0.05% Tween 20) and then Raman detection was performed.

[0071] Result analysis: Figure 2 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Figure 2 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 695.5X + 10579, and R 2 =0.9948, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 29.71fg / mL.

[0072] Example 2

[0073] This example determines the feasibility of gold paper SERS substrate + gold nanoparticle SERS probe to detect cardiac troponin (e.g. Figure 3 a), the specific experimental methods are as follows:

[0074] 1) Preparation of gold paper SERS substrate: The preparation method of the gold paper SERS substrate is the same as that in Example 1, except that the antibody Ab1 is 20C6.

[0075] 2) Preparation of gold nanoparticle SERS probe: The preparation method of the gold nanoparticle SERS probe is the same as that in Example 1, except that the antibody Ab2 is 560CC.

[0076] 3) SERS intensity detection: The SERS intensity detection method is the same as that in Example 1, except that the detection substance is cardiac troponin.

[0077] Result analysis: Figure 3 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Figure 3 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 1037.1X + 13069, and R 2 =0.9935, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 657.6fg / mL.

[0078] Example 3

[0079] This example determines the feasibility of gold-silver paper SERS substrate + gold nanoparticle SERS probe to detect novel coronavirus antigens (such as Figure 4 a), the specific experimental methods are as follows:

[0080] 1) Preparation of gold-silver paper SERS substrate: The preparation of gold paper SERS substrate is the same as that of Example 1, except that the cellulose paper modified with nanoparticles is gold-silver nanopaper. The preparation steps of gold-silver nanopaper are as follows: 5 pieces of 1×1 cm cellulose paper are placed in a mixed solution of 1.5 mL 75 mmol / L HAuCl4 and 1.5 mL 25 mmol / L AgNO3, shaken for 5 min, added in a 30°C drying oven for 10 min, then added to 5 mL 100 mmol / L NaBH4 solution and soaked for 10 min, then washed with ultrapure water 3 times, and then dried in a 55°C drying oven to obtain gold-silver nanopaper.

[0081] 2) Preparation of gold nanoparticle SERS probe: The preparation method of the gold nanoparticle SERS probe is the same as that in Example 1.

[0082] 3) SERS intensity detection: the same as the SERS intensity detection method in Example 1.

[0083] Result analysis: Figure 4 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Figure 4 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 507.6X + 6513, and R 2 =0.9894, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 721.6fg / mL.

[0084] Example 4

[0085] This example determines the feasibility of gold-silver paper SERS substrate + gold nanoparticle SERS probe to detect cardiac troponin (such as Figure 5 a), the specific experimental methods are as follows:

[0086] 1) Preparation of gold-silver paper SERS substrate: The preparation method of the gold-silver paper SERS substrate is the same as that in Example 3, except that the antibody Ab1 is 20C6.

[0087] 2) Preparation of gold nanoparticle SERS probe: The preparation method of the gold nanoparticle SERS probe is the same as that in Example 2.

[0088] 3) SERS intensity detection: the same as the SERS intensity detection method in Example 2.

[0089] Result analysis: Figure 5 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Figure 5 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 509.4X + 5979, and R 2 =0.9934, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blankIt represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 8.24pg / mL.

[0090] Example 5

[0091] This example determines the feasibility of gold paper SERS substrate + Au@DTNB@Ag SERS probe to detect novel coronavirus antigens (such as Figure 6 a), the specific experimental methods are as follows:

[0092] 1) Preparation of gold paper SERS substrate: The preparation method of the gold paper SERS substrate is the same as that in Example 1.

[0093] 2) Preparation of Au@DTNB@Ag SERS probe: 18 nm colloidal gold solution was diluted with MES buffer solution (pH = 6) to make its absorbance 0.5; then 50 μL of 10 mmol / L DTNB solution was added, incubated at room temperature for 30 min, and then 750 μL of 100 mmol / L AA was added to fully disperse; then 1.75 mL of 1 mmol / L AgNO3 solution was added, and after sufficient reaction, centrifuged for 5 min, the supernatant was removed, and washed twice with ultrapure water, and finally dispersed in 1 mL of pH = 8.5 boric acid buffer solution to obtain Au@DTNB@Ag. Then, 200 μL of 10 mmol / L PEG1 was added to the above Au@DTNB@Ag solution and incubated at room temperature for 4 h. Then, 20 μL of 10 mmol / L EDC and 20 μL of 10 mmol / L NHS were added to the above solution for activation and incubated at room temperature for 30 min. Then, 2 μL of 5 μg / mL antibody 11F2 (Ab2) was added and incubated at room temperature for 4 h. The solution was centrifuged and washed three times with PBS buffer solution, dispersed in a storage solution (10 mmol / L PBS buffer solution, 1% BSA, 1% sucrose, 0.05% Tween 20), and stored at 4°C to obtain the SERS immunodetection probe of Au@DTNB@Ag-PEG1-Ab2.

[0094] 3) SERS intensity detection: the same as the SERS intensity detection method in Example 1.

[0095] Result analysis: Figure 6 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Figure 6 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 359.2X + 6591, and R 2 =0.9803, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SDblank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 2.19pg / mL

[0096] Example 6

[0097] This example determines the feasibility of gold paper SERS substrate + Au@DTNB@Ag SERS probe to detect cardiac troponin (e.g. Figure 7 a), the specific experimental methods are as follows:

[0098] 1) Preparation of gold paper SERS substrate: The preparation method of the gold paper SERS substrate is the same as that in Example 2.

[0099] 2) Preparation of Au@DTNB@Ag SERS probe: The preparation method of Au@DTNB@Ag SERS immunodetection probe was the same as that in Example 5, except that Ab2 was 560CC.

[0100] 3) SERS intensity detection: the same as the SERS intensity detection method in Example 2.

[0101] Result analysis: Figure 7 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Figure 7 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 98.69X + 1475, and R 2 =0.9798, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 8.19pg / mL.

[0102] Example 7

[0103] This example determines the feasibility of gold-silver paper SERS substrate + Au@DTNB@Ag SERS probe to detect novel coronavirus antigens (such as Figure 8 a), the specific experimental methods are as follows:

[0104] 1) Preparation of gold-silver paper SERS substrate: The preparation method of the gold-silver paper SERS substrate is the same as that in Example 3.

[0105] 2) Preparation of Au@DTNB@Ag SERS probe: The preparation method of Au@DTNB@Ag SERS immunodetection probe is the same as that in Example 5.

[0106] 3) SERS intensity detection: the same as the SERS intensity detection method in Example 1.

[0107] Result analysis: Figure 8 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Figure 8 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 179.2X + 2722, and R 2 =0.9761, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 244.33fg / mL.

[0108] Example 8

[0109] This example determines the feasibility of gold-silver paper SERS substrate + Au@DTNB@Ag SERS probe to detect cardiac troponin (e.g. Fig. 9 a), the specific experimental methods are as follows:

[0110] 1) Preparation of gold-silver paper SERS substrate: The preparation method of the gold-silver paper SERS substrate is the same as that in Example 4.

[0111] 2) Preparation of Au@DTNB@Ag SERS probe: The preparation method of Au@DTNB@Ag SERS immunodetection probe is the same as that in Example 6.

[0112] 3) SERS intensity detection: the same as the SERS intensity detection method in Example 2.

[0113] Result analysis: Fig. 9 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Fig. 9 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 156.5X + 2371, and R 2 =0.9861, indicating that the results have a good linear relationship. The detection limit formula is LOD = Yblank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 9.55fg / mL.

[0114] Example 9

[0115] This example determines the feasibility of Venus paper SERS substrate + gold nanoparticle SERS probe to detect novel coronavirus antigens (such as Fig.10 a), the specific experimental methods are as follows:

[0116] 1) Preparation of Venus Paper SERS Substrate: The preparation of the gold paper SERS substrate is the same as that of Example 1, except that the cellulose paper modified with nanoparticles is Venus Paper. The steps for preparing Venus Paper are as follows: 1 cm long and 1 cm wide cellulose paper is soaked in 10% (3-mercaptopropyl) trimethoxysilane ethanol solution, shaken for 4 hours at room temperature, then washed with acetone, ethanol and distilled water for 3 times, and dried at room temperature for 8 hours to obtain thiolated cellulose paper; then, 0.5 mL 5 mmol / L HAuCl4 solution is added to 5 mL, 50 mmol / L pH=7.5 HEPES buffer solution, shaken for 1 minute and then allowed to stand for 30 minutes to obtain a gold nanostar solution; finally, the thiolated cellulose paper is soaked in the gold nanostar solution for 12 hours, then rinsed with ultrapure water for 3 times, and dried at room temperature for 3 hours to obtain Venus Paper.

[0117] 2) Preparation of gold nanoparticle SERS probe: The preparation method of the gold nanoparticle SERS probe is the same as that in Example 1.

[0118] 3) SERS intensity detection: the same as the SERS intensity detection method in Example 1.

[0119] Result analysis: Fig.10 As shown in (b), different signal intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm -1 The concentration varies with the change of concentration. Fig.10 As shown in (c), the signal intensities of different concentrations were fitted, and the linear relationship was Y = 1504X + 15986, and R 2 =0.9910, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 63.9pg / mL.

[0120] Example 10

[0121] In order to further verify the authenticity of the experiment, this example uses a gold paper SERS substrate + gold nanoparticle SERS probe to detect different concentrations of novel coronavirus antigens in nasal swab samples (such as Fig.11 a), the specific method is as follows:

[0122] 1) Preparation of gold paper SERS substrate: The preparation method of the gold paper SERS substrate is the same as that in Example 1.

[0123] 2) Preparation of gold nanoparticle SERS probe: The preparation method of the gold nanoparticle SERS probe is the same as that in Example 1.

[0124] 3) SERS intensity detection: Add 5 μL of the test liquid containing different concentrations of the new coronavirus antigen in the nasal swab to 10 μL of the detection probe solution, mix well, incubate for 5 minutes, drop it onto the detection substrate, react for 3 minutes, wash three times with 300 μL PBS buffer solution (pH=7.4, 0.05% Tween 20) and perform Raman detection.

[0125] Result analysis: Fig.11 As shown in a, signal graphs of different intensities were obtained according to different samples, and the Raman signal spectrum was at 1328cm -1 The concentration varies with the change of concentration. Fig.11 As shown in b, the signal intensity of different concentrations was fitted, and the linear relationship was Y = 156.5X + 2371, and R 2 =0.9861, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 29.90fg / mL.

[0126] Table 1

[0127]

[0128] As shown in Table 1, the results showed that the sample recovery rate was between 80% and 120%, indicating the feasibility of a highly sensitive novel coronavirus antigen detection device in nasal swab samples.

[0129] Embodiment 11

[0130] In order to further verify the authenticity of the experiment, this example uses a gold paper SERS substrate + gold nanoparticle SERS probe to detect different concentrations of novel coronavirus antigens in throat swab samples (such as Fig.12 a), the specific preparation method is as follows:

[0131] 1) Preparation of gold paper SERS substrate: The preparation method of the gold paper SERS substrate is the same as that in Example 1.

[0132] 2) Preparation of gold nanoparticle SERS probe: The preparation method of the gold nanoparticle SERS probe is the same as that in Example 1.

[0133] 3) SERS intensity detection: Add 5 μL of the test liquid containing different concentrations of the new coronavirus antigen in the throat swab to 10 μL of the detection probe solution, mix well, incubate for 5 minutes, drop it onto the detection substrate, react for 3 minutes, wash three times with 300 μL PBS buffer solution (pH=7.4, 0.05% Tween 20) and perform Raman detection.

[0134] Result analysis: Fig.12 As shown in a, signal graphs of different intensities were obtained according to different samples, and the Raman signal spectrum was at 1328cm -1 The concentration varies with the change of concentration. Fig.12 As shown in b, the signal intensity of different concentrations was fitted, and the linear relationship was Y = 156.5X + 2371, and R 2 =0.9861, indicating that the results have a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank is the average signal intensity when the concentration is zero, SD blank It represents the standard deviation of the blank area measurement value. The detection limit of this detection method is calculated to be 34.95fg / mL.

[0135] Table 2

[0136]

[0137] As shown in Table 2, the results showed that the sample recovery rate was between 80% and 120%, indicating the feasibility of a highly sensitive novel coronavirus antigen detection device in throat swab samples.

[0138] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An immunoassay kit based on surface enhanced Raman spectroscopy, It is characterized in that The kit comprises: A surface enhanced Raman scattering active substrate, the active substrate comprising cellulose paper loaded with first metal nanoparticles and carboxyl-thiol polyethylene glycol and capture antibodies modified on the surface of the first metal nanoparticles; A surface enhanced Raman scattering immune detection probe comprises a second metal nanoparticle and a Raman signal molecule, methoxy-thiol polyethylene glycol and a detection antibody modified on the surface of the second metal nanoparticle.

2. The immunoassay kit according to claim 1, It is characterized in that The first metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, gold-silver nanoparticles or gold nanostars.

3. The immunoassay kit according to claim 1, It is characterized in that The molecule of the carboxyl-thiol polyethylene glycol is HOOC (CH 2 ) 2 -(OCH 2 CH 2 ) n -SH, its molecular weight is 150~2000Dal.

4. The immunoassay kit according to claim 1, It is characterized in that The second metal nanoparticles are one or more of gold nanoparticles, silver nanoparticles, gold core-silver shell nanoparticles, and have a particle size of 30 to 50 nm.

5. The immunoassay kit according to claim 1, It is characterized in that, The methoxy-mercapto polyethylene glycol molecule is CH 3 O-(CH 2 CH 2 O) n -(CH 2 ) 2 SH, its molecular weight is 140~600Dal.

6. The immunoassay kit according to claim 1, It is characterized in that The Raman signal molecule is one or more of 5,5'-dithiobis(2-nitrobenzoic acid), p-nitrothiophenol, p-carboxythiophenol, p-aminothiophenol or thiophenol.

7. The method for preparing the immunoassay kit based on surface enhanced Raman spectroscopy according to any one of claims 1 to 6, It is characterized in that The following steps are involved: S1: soaking the cellulose paper loaded with the first metal nanoparticles in a carboxyl-thiol polyethylene glycol solution, incubating with shaking at room temperature, washing, activating, then adding the capture antibody, incubating with shaking at room temperature, washing, and drying to obtain a surface-enhanced Raman scattering active substrate modified with carboxyl-thiol polyethylene glycol and capture antibody; S2: Raman signal molecules and methoxy-thiol polyethylene glycol are modified on the second metal nanoparticles, which are dispersed in a boric acid buffer solution after centrifugation and washing, activated, and then added with detection antibodies after incubation at room temperature. The particles are further incubated at room temperature, centrifuged and washed, and then dispersed in a preservation solution to obtain a surface-enhanced Raman scattering immunoassay probe modified with Raman signal molecules, methoxy-thiol polyethylene glycol, and detection antibodies.

8. The preparation method according to claim 7, It is characterized in that The storage solution comprises 5-15 mmol / L PBS buffer solution, 0.5-1.5 wt% BSA, 0.5-2 wt% sucrose, and 0.01-0.5 wt% Tween 20.

9. Use of the immunoassay kit based on surface enhanced Raman spectroscopy according to any one of claims 1 to 6, It is characterized in that The application is: adding the antigen to be tested into the surface enhanced Raman scattering immunoassay probe solution, mixing and incubating, then dropping it onto the surface enhanced Raman scattering active substrate, reacting fully, and performing Raman detection after washing.

10. The use according to claim 9, It is characterized in that The capture antibody and detection antibody are both novel coronavirus N protein or cardiac troponin.

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