An immunological detection kit based on surface-enhanced Raman spectroscopy and a preparation method and application thereof

Abstract

The application discloses an immunological detection kit based on surface-enhanced Raman spectroscopy and a preparation method and application thereof. The kit comprises a surface-enhanced Raman scattering active substrate, the active substrate comprises cellulose paper loaded with first metal nanoparticles and carboxyl-mercapto polyethylene glycol and a capture antibody modified on the surface of the first metal nanoparticles; and a surface-enhanced Raman scattering immunological detection probe, the detection probe comprises second metal nanoparticles and a Raman signal molecule, methoxy-mercapto polyethylene glycol and a detection antibody modified on the surface of the second metal nanoparticles. The immunological detection kit is high in sensitivity, fast and accurate, and low in false positive.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to an immunoassay kit based on surface-enhanced Raman spectroscopy, its preparation method, and its application. Background Technology

[0002] Paper-based side-flow immunoassay (LFA) is a commonly used and inexpensive method for point-of-care testing, but it still suffers from problems such as high false positive rates, low sensitivity, and complex preparation procedures. Sequencing-based immunoassay (SERS) offers advantages such as high sensitivity and rapid detection, and has been widely applied in the LFA field. However, SERS-based LFA detection still requires a chromatography process, resulting in a long detection time. Furthermore, its sensitivity is limited by the Raman enhancement effect of colloidal gold (Au NPs) themselves; 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 gold nanoprobes is usually blocked by non-covalent linkages (such as BSA), and this unstable blocking method makes it easy for BSA to detach during operation, resulting in false positives.

[0003] Therefore, there is an urgent need for a sensitive, rapid and accurate immune detection technology. Summary of the Invention

[0004] The main objective of this invention is to address the problems and shortcomings of existing technologies by providing a sensitive, rapid, and accurate immunoassay kit based on surface-enhanced Raman spectroscopy, along with its preparation method and applications.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] This 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 antibody modified on the surface of the first metal nanoparticles;

[0008] A surface-enhanced Raman scattering immunoassay probe, the probe comprising a second metal nanoparticle and Raman signaling molecules, methoxy-thiol polyethylene glycol (PEG2), and a detection antibody modified on the surface of the second metal nanoparticle.

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

[0010] Preferably, the molecule of the carboxyl-mercaptopolyethylene glycol is HOOC(CH2)2-(OCH2CH2). n-SH, with a molecular weight of 150-2000 Da, preferably 680 Da.

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

[0012] Preferably, the methoxy-mercaptopolyethylene glycol has a molecular structure of CH3O-(CH2CH2O). n -(CH2)2SH, with a molecular weight of 140-600 Da, preferably 180 Da.

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

[0014] S1: Cellulose paper loaded with the first metal nanoparticles was soaked in carboxyl-mercaptopolyethylene glycol aqueous solution, incubated with shaking at room temperature, washed and activated, and then the capture antibody was added and incubated with shaking at room temperature, washed and dried to obtain a surface-enhanced Raman scattering active substrate modified with carboxyl-mercaptopolyethylene glycol (PEG1) and capture antibody (Ab1).

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

[0016] Preferably, the activation method used in steps S1 and S2 is to activate the product by adding a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[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), more preferably DTNB, and its solution is an ethanol solution of 1-100 mmol / L, with a concentration more preferably 10 mmol / L; the volume is 10-30 μL, 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 solution volume 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; and 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; and the volume of the boric acid buffer solution used for dispersion is 0.5-1.5 mL, more preferably 1 mL.

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

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

[0024] Preferably, the washing in step S1 is performed using PBS buffer. The PBS buffer 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 cleaning cycles in step S1 is 3 to 5, more preferably 3; the drying temperature is 20 to 50°C, more preferably 37°C; and the drying time is 10 to 40 minutes, more preferably 30 minutes.

[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 soaked in NaBH4 solution. After being taken out, washed and dried with ultrapure water, the cellulose paper loaded with gold and silver nanoparticles is obtained, i.e., gold-silver paper.

[0027] More preferably, in step S1, the preparation of the gold-silver paper involves cellulose paper with dimensions of 0.5–2 cm in length and 0.5–2 cm in width, 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, 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, more preferably 1.5 mL and 25 mmol / L, respectively; the temperature maintained in the drying oven after mixing the HAuCl4 and AgNO3 solutions 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, more preferably 5 mL and 100 mmol / L, respectively; the number of washings with ultrapure water is 2–5 times, more preferably 3 times; and the drying temperature is 35–75 °C, more preferably 55 °C.

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

[0029] More preferably, in step S1, the preparation of the Venus paper involves cellulose with dimensions of 0.5–2 cm in length and 0.5–2 cm in width, more preferably 1 cm in length and 1 cm in width; the mercaptosilane solution is an ethanol solution of (3-mercaptopropyl)trimethoxysilane with a concentration of 2%–50%, more preferably 10%; the shaking reaction time at room temperature is 2–6 h, more preferably 4 h; the organic solvents used for washing are acetone and ethanol in sequence, and the number of washings is 2–5 times, more preferably 3 times; the drying time at room temperature is 4–12 h, more preferably 8 hours. The volume and concentration of the HAuCl4 solution are 0.1–1 mL and 1–10 mmol / L, respectively, 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, more preferably 5 mL, 50 mmol / L, and 7.5, respectively; the shaking and standing times are 0.5–3 min and 20–50 min, respectively, more preferably 1 min and 30 min, respectively. The soaking time of the thiolized cellulose paper in the gold nanostar solution is 8–16 h, more preferably 12 h; the drying time at room temperature is 2–5 h, more preferably 3 h.

[0030] Preferably, the preservation solution in step S2 comprises PBS buffer solution, BSA, sucrose, and Tween 20, with concentrations of 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 nanoparticle is a gold-core, silver-shell nanoparticle, the Au@DTNB@Ag SERS immunoassay probe is prepared by the following steps:

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

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

[0034] Au@DTNB@Ag crude solution;

[0035] c. Take the above Au@DTNB@Ag solution, centrifuge and wash with ultrapure water, and disperse it in borate 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. After activating the above Au@DTNB@Ag-PEG1 with DEC / NHS, it is linked to Ab1 to obtain the Au@DTNB@Ag SERS probe.

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

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

[0041] The present invention also provides an application of the surface-enhanced Raman spectroscopy-based immunoassay kit, wherein the application is as follows: the antigen to be tested is added to the surface-enhanced Raman scattering immunoassay probe solution, mixed and incubated, then dropped onto the surface-enhanced Raman scattering active substrate, allowed to react fully, washed, and then Raman detection is performed.

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

[0043] Preferably, the volume of the antigen solution is 1–10 μL, more preferably 5 μL; the volume of the detection probe is 5–20 μL, more preferably 10 μL; and the incubation time is 3–7 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, or a handheld Raman spectrometer, with an excitation wavelength of 633 nm.

[0046] The principle of this invention is as follows: This invention utilizes HOOC(CH2)2-(OCH2CH2) n -SH molecules (PEG1) are covalently modified and linked to the SERS substrate and the capturing antibody Ab1 to form a gold (or gold-silver) paper-PEG1-Ab1 SERS substrate. DTNB is then used as a Raman signal reporter molecule, modified on the surface of Au NPs or in the middle of the shell of gold-core silver-shell nanoparticles (Au@DTNB@Ag). Finally, a detection antibody Ab2 is covalently linked to prepare a SERS immunoassay probe. During detection, antigen is added to the probe solution, and the specific recognition of the antigen by the detection antibody Ab2 forms an immune complex. This complex is then dropped onto the SERS substrate, where it is captured by antibody Ab1, forming an immune sandwich structure of "SERS substrate-antigen-SERS probe". Raman spectroscopy enables ultrasensitive, stable, and rapid detection of the antigen within 5 minutes.

[0047] Compared with the prior art, the beneficial effects of the present invention are as follows:

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

[0049] 2. This invention directly mixes the antigen analyte with the nanoprobe and drops it onto the substrate for Raman signal detection, eliminating the side-flow chromatography process in LFA and greatly reducing the detection time.

[0050] 3. In this invention, the capture antibody and the detection antibody are covalently linked to the SERS substrate and the nanoprobe, respectively, and the exposed gold surface is sealed with PEG molecules in the form of chemical bonds, thereby reducing the false positive problem caused by non-covalent modification (such as BSA backfill) that exposes the gold surface. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the detection principle of the present invention.

[0052] Figure 2 (a) Schematic diagram of the detection of novel coronavirus antigen using a gold paper SERS substrate and gold nanoparticle SERS probes; (b) SERS spectra of different concentrations of novel coronavirus antigen detected; and (c) SERS spectra at 1328 cm⁻¹. -1Linear fitting plot of SERS intensity at the location;

[0053] Figure 3 (a) Schematic diagram of the detection of cardiac troponin using a gold paper SERS substrate and gold nanoparticle SERS probes; (b) SERS spectra of cardiac troponin at different concentrations; and (c) SERS spectra of cardiac troponin at 1328 cm⁻¹. -1 Linear fitting plot of SERS intensity at the location;

[0054] Figure 4 (a) Schematic diagram of the detection of novel coronavirus antigen using a gold-silver paper SERS substrate and gold nanoparticle SERS probes; (b) SERS spectra of different concentrations of novel coronavirus antigen; and (c) SERS spectra at 1328 cm⁻¹. -1 Linear fitting plot of SERS intensity at the location;

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

[0056] Figure 6 (a) Schematic diagram of detecting novel coronavirus antigen using gold-flecked SERS substrate and Au@DTNB@Ag SERS probe, (b) SERS spectra of detecting novel coronavirus antigen at different concentrations, and (c) SERS spectra at 1328 cm⁻¹. -1 Linear fitting plot of SERS intensity at the location;

[0057] Figure 7 (a) Schematic diagram of (a) 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 spectra at 1328 cm⁻¹. -1 Linear fitting plot of SERS intensity at the location;

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

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

[0060] Figure 10 (a) Schematic diagram of the detection of novel coronavirus antigen using a gold-flecked paper SERS substrate and gold nanoparticle SERS probes; (b) SERS spectra of different concentrations of novel coronavirus antigen detected; and (c) SERS spectra at 1328 cm⁻¹. -1 Linear fitting plot of SERS intensity at the location;

[0061] Figure 11 The SERS spectra of (a) gold paper SERS substrate + gold nanoparticle SERS probe for detecting different concentrations of novel coronavirus antigen in nasal swabs and (b) at 1328 cm⁻¹ -1 Linear fitting plot of SERS intensity at the location;

[0062] Figure 12 The SERS spectra of (a) gold paper SERS substrate + gold nanoparticle SERS probe for detecting different concentrations of novel coronavirus antigen in pharyngeal swabs and (b) at 1328 cm⁻¹ -1 Linear fitting plot of SERS intensity at the location. Detailed Implementation

[0063] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0064] This invention utilizes HOOC(CH2)2-(OCH2CH2) n -SH molecule (PEG1) is covalently modified to link the SERS substrate and the capture antibody Ab1, forming a gold (-silver) paper-PEG1-Ab1 detection substrate. Then, DTNB molecule is covalently modified to link gold nanoparticles and the detection antibody Ab2, and CH3O-(CH2CH2O) is used as the substrate. n Excess gold surface is capped with -(CH2)2SH(PEG2) to form an Au-PEG2 / DTNB-Ab2 detection probe (or Au@DTNB@Ag-PEG1-Ab2 SERS probe). During detection, the antigen is added, and the detection antibody Ab2 specifically recognizes the antigen to form an immune complex. This complex is then dropped onto the SERS substrate, where it is captured by the capture antibody Ab1, forming an immune sandwich structure of "SERS substrate-antigen-detection probe". Raman spectroscopy enables ultrasensitive, stable, and rapid detection of the antigen 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 with the novel coronavirus antigen and incubated to form a "double antibody" immune complex. This complex is then dropped onto a gold paper-PEG1-Ab1 detection substrate to further form an immune sandwich complex. After washing with PBS buffer, Raman signal detection is performed. The principle is similar if a gold-silver paper SERS substrate or an Au@DTNB@Ag SERS probe is used.

[0066] Example 1

[0067] This embodiment confirms the feasibility of detecting novel coronavirus antigens using a gold paper SERS substrate and gold nanoparticle SERS probes (e.g., Figure 2 a) The specific experimental method is as follows:

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

[0069] 2) Preparation of SERS probe using gold nanoparticles: 1 mL of 40 nm gold nanoparticles with an absorbance of 1.3 was mixed with 20 μL of 10 mmol / L LTNB and 20 μL of 10 mmol / L CH3O-(CH2CH2O)2-(CH2)2SH (PEG2) molecules. The mixture was incubated at room temperature for 2 h, centrifuged, and the supernatant was discarded. The nanoparticles were 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. The mixture was incubated at room temperature for 30 min, and then 2 μL of 5 μg / mL antibody 11F2 (Ab2) was added. The mixture was incubated at room temperature for 4 h, centrifuged, washed three times with PBS buffer, and dispersed in storage solution (10 mmol / L PBS buffer, 1% BSA, 1% sucrose, 0.05% Tween). 20) was stored at 4℃ to prepare the Au-PEG2 / DTNB-Ab2 immunoRaman signal detection probe.

[0070] 3) SERS intensity detection: Add 5 μL of the test solution containing 10 ng / mL of novel coronavirus antigen and PBS buffer to 10 μL of the detection probe solution, mix well, incubate for 5 min, drop onto the detection substrate, react fully for 3 min, wash three times with different volumes of PBS buffer solution (pH=7.4, 0.05% Tween 20) and then perform Raman detection.

[0071] Results analysis: such as Figure 2 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 2 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 695.5X + 10579, and R0 2 = 0.9948, indicating a good linear relationship. The limit of detection formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 29.71 fg / mL.

[0072] Example 2

[0073] This embodiment confirms the feasibility of detecting cardiac troponin using a gold paper SERS substrate and gold nanoparticle SERS probes (e.g., Figure 3 a) The specific experimental method is as follows:

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

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

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

[0077] Results analysis: such as Figure 3 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 3 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 1037.1X + 13069, and R0 2 =0.9935, indicating a good linear relationship. The limit of detection formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 657.6 fg / mL.

[0078] Example 3

[0079] This embodiment confirms the feasibility of detecting novel coronavirus antigens using a gold-silver paper SERS substrate and gold nanoparticle SERS probes (e.g., Figure 4 a) The specific experimental method is as follows:

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

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

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

[0083] Results analysis: such as Figure 4 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 4 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 507.6X + 6513, and R0 2 = 0.9894, indicating a good linear relationship. The limit of detection formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 721.6 fg / mL.

[0084] Example 4

[0085] This embodiment confirms the feasibility of detecting cardiac troponin using a gold-silver paper SERS substrate and gold nanoparticle SERS probes (e.g., Figure 5 a) The specific experimental method is as follows:

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

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

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

[0089] Results analysis: such as Figure 5 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 5 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 509.4X + 5979, and R0 2 =0.9934, indicating a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blankThis represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 8.24 pg / mL.

[0090] Example 5

[0091] This embodiment confirms the feasibility of detecting novel coronavirus antigens using a gold-paper SERS substrate + Au@DTNB@Ag SERS probe (e.g., Figure 6 a) The specific experimental method is as follows:

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

[0093] 2) Preparation of Au@DTNB@Ag SERS probe: The 18nm colloidal gold solution was diluted with MES buffer (pH=6) to make its absorbance 0.5; then 50μL of 10mmol / L DTNB solution was added, and after incubation at room temperature for 30min, 750μL of 100mmol / L AA was added and the solution was fully dispersed; then 1.75mL of 1mmol / L AgNO3 solution was added, and after the reaction was fully completed, the solution was centrifuged for 5min, the supernatant was removed, and the solution was washed twice with ultrapure water. Finally, the solution was dispersed in 1mL of borate buffer solution at pH=8.5 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. After centrifugation, the sample was washed three times with PBS buffer and dispersed in storage solution (10 mmol / L PBS buffer, 1% BSA, 1% sucrose, 0.05% Tween 20) and stored at 4 °C to obtain the Au@DTNB@Ag-PEG1-Ab2 SERS immunoassay probe.

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

[0095] Results analysis: such as Figure 6 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 6 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 359.2X + 6591, and R0 2 =0.9803, indicating a good linear relationship. The limit of detection formula is LOD = Y blank +3×SDblank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 2.19 pg / mL.

[0096] Example 6

[0097] This embodiment confirms the feasibility of detecting cardiac troponin using a gold paper SERS substrate and Au@DTNB@Ag SERS probe (e.g., Figure 7 a) The specific experimental method is as follows:

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

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

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

[0101] Results analysis: such as Figure 7 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 7 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 98.69X + 1475, and R0 2 = 0.9798, indicating a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 8.19 pg / mL.

[0102] Example 7

[0103] This embodiment confirms the feasibility of detecting novel coronavirus antigens using a gold-silver paper SERS substrate + Au@DTNB@Ag SERS probe (e.g., Figure 8 a) The specific experimental method is as follows:

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

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

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

[0107] Results analysis: such as Figure 8 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 8 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 179.2X + 2722, and R0 2 =0.9761, indicating a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 244.33 fg / mL.

[0108] Example 8

[0109] This embodiment confirms the feasibility of detecting cardiac troponin using a gold-silver paper SERS substrate and Au@DTNB@Ag SERS probe (e.g., Figure 9 a) The specific experimental method is as follows:

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

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

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

[0113] Results analysis: such as Figure 9 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 9 As shown in (c), the linear relationship for fitting the signal intensity at different concentrations is Y = 156.5X + 2371, and R0 2 = 0.9861, indicating a good linear relationship. The detection limit formula is LOD = Yblank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 9.55 fg / mL.

[0114] Example 9

[0115] This embodiment confirms the feasibility of detecting novel coronavirus antigens using a gold-flecked paper SERS substrate and gold nanoparticle SERS probes (e.g., Figure 10 a) The specific experimental method is as follows:

[0116] 1) Preparation of the gold-star paper SERS substrate: The preparation method is the same as that of the gold-paper SERS substrate in Example 1, except that the cellulose paper modified with nanoparticles is gold-star paper. The preparation steps of the gold-star paper are as follows: cellulose paper with a length and width of 1 cm is immersed in a 10% ethanol solution of (3-mercaptopropyl)trimethoxysilane and reacted with shaking at room temperature for 4 h. Then, it is washed three times with acetone, ethanol and distilled water respectively, and dried at room temperature for 8 h to obtain thiolized cellulose paper. Then, 0.5 mL of 5 mmol / L HAuCl4 solution is added to 5 mL of 50 mmol / L HEPES buffer solution with pH = 7.5, shaken for 1 min and allowed to stand for 30 min to obtain gold nanostar solution. Finally, the above thiolized cellulose paper is immersed in gold nanostar solution for 12 h, rinsed three times with ultrapure water, and dried at room temperature for 3 h to obtain gold-star paper.

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

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

[0119] Results analysis: such as Figure 10 As shown in (b), signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 10 As shown in (c), the linear relationship between the signal intensities at different concentrations is Y = 1504X + 15986, and R0 2 = 0.9910, indicating a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 63.9 pg / mL.

[0120] Example 10

[0121] To further verify the authenticity of the experiment, this embodiment used a gold paper SERS substrate with gold nanoparticle SERS probes to detect different concentrations of novel coronavirus antigen in nasal swab samples (e.g., Figure 11 a) The specific method is as follows:

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

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

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

[0125] Results analysis: such as Figure 11 As shown in Figure a, signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 11 As shown in b, the linear relationship for fitting the signal intensity at different concentrations is Y = 156.5X + 2371, and R0 2 = 0.9861, indicating a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 29.90 fg / mL.

[0126] Table 1

[0127]

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

[0129] Example 11

[0130] To further verify the authenticity of the experiment, this embodiment used a gold paper SERS substrate with gold nanoparticle SERS probes to detect different concentrations of novel coronavirus antigen in pharyngeal swab samples (e.g., Figure 12 a) The specific preparation method is as follows:

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

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

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

[0134] Results analysis: such as Figure 12 As shown in Figure a, signal maps of different intensities were obtained for different samples, and the Raman signal spectrum was at 1328 cm⁻¹. -1 The value changes with concentration. For example... Figure 12 As shown in b, the linear relationship for fitting the signal intensity at different concentrations is Y = 156.5X + 2371, and R0 2 = 0.9861, indicating a good linear relationship. The detection limit formula is LOD = Y blank +3×SD blank , where Y blank The average signal intensity when the concentration is zero, SD blank This represents the standard deviation of the measurements in the blank area. The calculated detection limit for this method is 34.95 fg / mL.

[0135] Table 2

[0136]

[0137] As shown in Table 2, the results indicate that the sample recovery rate is between 80% and 120%, demonstrating the feasibility of the highly sensitive novel coronavirus antigen detection device in pharyngeal swab samples.

[0138] It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An immunoassay kit based on surface-enhanced Raman spectroscopy, characterized in that, The kit includes: A surface-enhanced Raman scattering active substrate, the active substrate comprising cellulose paper loaded with first metal nanoparticles and carboxyl-thiol and capture antibody modified on the surface of the first metal nanoparticles; A surface-enhanced Raman scattering immunoassay probe, comprising a second metal nanoparticle and Raman signal molecules, methoxy-mercaptopolyethylene glycol, and a detection antibody modified on the surface of the second metal nanoparticle, wherein the methoxy-mercaptopolyethylene glycol molecule is CH3O-(CH2CH2O). n -(CH2)2SH, with a molecular weight of 140 ~ 600 Dal.

2. The immunoassay kit as described in claim 1, characterized in that, The first metal nanoparticle is one or more of gold nanoparticles, silver nanoparticles, or gold-silver nanoparticles.

3. The immunoassay kit as described in claim 1, characterized in that, The gold nanoparticles are gold nanostars.

4. The immunoassay kit as described in claim 1, characterized in that, The molecule of the carboxyl-mercapto-polyethylene glycol is HOOC(CH2)2-(OCH2CH2). n -SH, with a molecular weight of 150 ~ 2000 Dal.

5. The immunoassay kit as described in claim 1, characterized in that, The second metal nanoparticle is one or more of gold nanoparticles, silver nanoparticles, and gold-core silver-shell nanoparticles, with a particle size of 30-50 nm.

6. The immunoassay kit as described in claim 1, 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 as described in any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Cellulose paper loaded with the first metal nanoparticles was soaked in a carboxyl-mercaptopolyethylene glycol solution, incubated with shaking at room temperature, washed and activated, and then the capture antibody was added and incubated with shaking at room temperature, washed and dried to obtain a surface-enhanced Raman scattering active substrate modified with carboxyl-mercaptopolyethylene glycol and capture antibody. S2: Raman signal molecules and methoxy-mercapto-polyethylene glycol were modified on the second metal nanoparticles. After centrifugation and washing, the particles were dispersed in borate buffer solution, activated, incubated at room temperature, and then the detection antibody was added. After further incubation at room temperature, the particles were centrifuged, washed, and dispersed in storage solution to obtain a surface-enhanced Raman scattering immunoassay probe modified with Raman signal molecules, methoxy-mercapto-polyethylene glycol, and detection antibody.

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

9. The application of the immunoassay kit based on surface-enhanced Raman spectroscopy as described in any one of claims 1 to 6, characterized in that, The application involves adding the antigen to be tested into a surface-enhanced Raman scattering immunoassay probe solution, mixing and incubating, then dropping the solution onto a surface-enhanced Raman scattering active substrate, allowing it to react fully, washing it, and then performing Raman detection.

10. The application as described in claim 9, characterized in that, The capture antibody and detection antibody are both novel coronavirus N protein or cardiac troponin.

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