CN113447467B - Method for detecting SARS-CoV-2 antigen of new coronavirus - Google Patents
Method for detecting SARS-CoV-2 antigen of new coronavirus Download PDFInfo
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a method for detecting a novel coronavirus SARS-CoV-2 antigen, belonging to the field of biotechnology. The invention captures nano particles and S protein through new coronavirus SARS-CoV-2S protein immunomagnetic beads to generate specific immunity, and then incubate with immuno-gold signal nano particles to construct a sandwich structure; then, SERS signals can be directly measured by a Raman spectrometer, the novel coronavirus SARS-CoV-2 protein can be specifically detected within 5 minutes, and the pseudovirus of SARS-COV-2, variant pseudovirus and pseudovirus diluted in human saliva can also be specifically detected. By the method, nucleic acid extraction or antibody-dependent detection is not required, and the method has the advantages of good stability, higher specificity and affinity, low immunogenicity, easiness in chemical modification, simplicity and convenience in operation and the like, can realize rapid detection of the SARS-CoV-2 antigen of the new coronavirus and the mutant virus within five minutes, and creates conditions for early detection of 2019SARS-CoV-2 pathogen.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for detecting a novel coronavirus SARS-CoV-2 antigen by surface enhanced Raman spectroscopy based on aptamer recognition.
Background
The etiology and serology examination in the detection method of the novel coronavirus SARS-CoV-2 mainly comprises nucleic acid detection, antibody detection and antigen detection. Among them, nucleic acid detection is the "gold standard" for SARS-CoV-2 detection, but has the disadvantages of long time consumption and incapability of on-site detection. Antibody detection requires a certain window period, and early diagnosis of infected persons is difficult to realize.
The existing detection methods are, for example:
CN202011431411.8 discloses a kit for detecting new coronavirus and its mutant, which is characterized by comprising the following parts: (1) a chain of pro-substrate A and a chain of pro-substrate B; (2) a Cas 13a protein; (3) crRNA or a DNA template thereof; (4) a nucleic acid ligase; (5) a light-up RNA aptamer or a DNA template thereof; (6) an in vitro transcription reagent.
CN202110353115.9 discloses a novel coronavirus neutralizing antibody detection kit prepared by adopting a Pt-Pd alloy nanoparticle-labeled immunochromatography method, and a detection method and application thereof. The kit for detecting the new coronavirus contains a Pt-Pd alloy nanoparticle test strip for a SARS-CoV-2 neutralizing antibody of the new coronavirus. The preparation method of the test paper strip for the Pt-Pd alloy nanoparticles of the neutralizing antibody of the new coronavirus SARS-CoV-2 comprises the steps of marking the antigen of the new coronavirus SARS-CoV-2 as a capture antibody nano marker by using the alloy nanoparticles, coating the purified A-type expression antigen and the antibody thereof on a cellulose membrane as a detection line T line and a quality control line C line respectively, and establishing the test paper strip for the Pt-Pd alloy nanoparticles of the neutralizing antibody of the new coronavirus through condition optimization.
Compared with nucleic acid and antibody detection, the detection of the SARS-CoV-2 antigen of the new coronavirus can be carried out at the early stage (1-10 days) of infection of the new coronavirus, has lower cost and higher speed, and is expected to obtain results within 15-20 minutes. Therefore, the antigen detection is more suitable for early large-scale screening, can be mutually verified with nucleic acid detection, antibody detection and CT detection, and becomes an important means for investigation of the new coronary pneumonia. And the new coronavirus SARS-CoV-2 is continuously mutated, which brings more pressure to the detection. However, the existing antigen detection kit has the problem of false negative caused by insufficient detection sensitivity, and an antigen detection means which is faster, has better detection sensitivity and can detect the mutated virus is urgently needed.
Disclosure of Invention
The invention mainly aims to provide a method for detecting a novel coronavirus SARS-CoV-2 antigen based on surface enhanced Raman spectroscopy identified by a nucleic acid aptamer. The invention utilizes the high affinity between biotin-streptavidin to modify a Aptamer SAptamer 1 on a magnetic microsphere to form a functional magnetic bead, realizes the targeted capture, separation and enrichment of SARS-CoV-2S protein of the new coronavirus, then specifically recognizes the virus through the Aptamer S Aptamer 2 modified with sulfydryl, combines metal nanoparticles with Raman signal expression to form a sandwich (sandwich type) structure, and utilizes a portable Raman spectrometer to realize the rapid detection of SARS-CoV-2 antigen of the new coronavirus within 5 minutes.
The purpose of the invention is realized by the following technical scheme, which comprises the following steps:
a method for detecting SARS-CoV-2 antigen of new coronavirus includes the following steps:
s1: modifying a first aptamer or aptamer combination of a new coronavirus antigen on a magnetic microsphere through a biotin-streptavidin interaction to form an immunomagnetic bead capture nanoparticle;
s2: preparing metal nano sol;
s3: mixing the metal nano sol prepared in the step S2 with Raman reporter molecules, and mixing with a second aptamer or aptamer combination of the sulfhydryl-modified new coronavirus antigen; and blocking the active sites to obtain immune metal signal nanoparticles;
s4: mixing the immunomagnetic bead capture nanoparticles prepared in the step S1 with a sample to be detected, and mixing the immunomagnetic bead capture nanoparticles with the immunometal signal nanoparticles in the step S3 to obtain a sandwich structure;
step S5: and carrying out Raman detection on the obtained sandwich structure by using a Raman spectrometer to obtain a Raman spectrum result of the new coronavirus antigen.
The invention has the beneficial effects that: the method takes the aptamer as a specific recognition element, specifically binds SARS-CoV-2 antigen, does not need to carry out nucleic acid extraction and amplification, and can stabilize the dispersion state of the nano material in a detection system on the one hand compared with the method of taking the antibody as the recognition element in an immunoassay method; on the other hand, the method can identify the target protein with high specificity and high affinity, has good stability and low preparation cost, improves the detection accuracy to a great extent, and has higher detection speed.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a schematic diagram of the detection principle of the present invention, in which immunomagnetic beads capture nanoparticles to perform specific immunoreaction with SARS-CoV-2S protein, and form a sandwich structure by incubating with immuno-gold signal nanoparticles. Then, performing rapid magnetic separation, and realizing rapid detection of new coronavirus through a portable Raman spectrometer;
FIG. 2 is a diagram of the ultraviolet absorption spectrum of an immunogold signaling nanoparticle;
FIG. 3 is an SEM image of a sandwich (sandwich) structure;
FIG. 4 is a graph of surface enhanced Raman spectra of different SARS-CoV-2S protein concentrations;
FIG. 5 is a surface enhanced Raman spectrum for detecting SARS-CoV-2 pseudovirus;
FIG. 6 is a surface enhanced Raman spectrum of saliva showing the detection of different SARS-CoV-2 pseudovirus concentrations.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The detection method of the invention utilizes the principle of the specific recognition of the aptamer of the SARS-CoV-2S protein of the new coronavirus (preferably, the aptamer of the SARS-CoV-2S protein is adopted, but not limited to the aptamer of the S protein, and the aptamer of other antigen components of SARS-CoV-2 can also be used), and after the specific recognition of the captured nanoparticles and the S protein (or other antigen components) of the immune magnetic beads of the SARS-CoV-2 antigen is carried out, the immune metal signal nanoparticles which modify the aptamer of the SARS-CoV-2 antigen are incubated to form a sandwich structure.
The new coronavirus SARS-CoV-2 antigen immunomagnetic bead capture nanoparticle has magnetism and is modified with an aptamer or an aptamer combination (preferably, a SARS-CoV-2S protein aptamer is adopted, but not limited to the S protein aptamer, and aptamers of other antigen components of SARS-CoV-2 can also be adopted); the immune gold signal nano particle has Raman signal expression and aptamer or aptamer combination modification of SARS-CoV-2 antigen.
Preferably, the new coronavirus SARS-CoV-2 antigen immunomagnetic bead capture nanoparticle is prepared by the following method: taking magnetic beads modified with streptavidin, and adding a B & W washing buffer (preferably, but not limited to, the buffer) for washing; the magnetic beads are resuspended in B & W washing buffer, a biotinylated Aptamer S Aptamer1 or a biotinylated Aptamer combination is added, the mixture is incubated at room temperature, the mixture is washed by the washing buffer, the mixture is resuspended in PBS buffer, BSA solution (bovine serum albumin, preferably, but not limited to, the blocking agent) is added, the reaction is carried out at room temperature, the residual binding sites on the surfaces of the magnetic beads are blocked, the magnetic separation is carried out, the mixture is washed by the PBS buffer, and finally the mixture is redispersed in the PBS buffer, so that the immunomagnetic bead capture nanoparticles are prepared.
Preferably, the magnetic beads have a particle size of 0.5-5 microns, more preferably 1-2 microns.
The B & W washing buffer solution is prepared by using 1-15mM Tris-HCl (pH 4-8, 1-10mM EDTA and 2-10M NaCl).
Preferably, the immunomagnetic bead capture nanoparticles are prepared by mixing magnetic beads with a solution of biotinylated Aptamer S Aptamer1 (or a combination of aptamers) and incubating for 5-30 minutes at room temperature.
Preferably, the preferably biotinylated Aptamer S Aptamer1 (or other neocorona antigen biotinylated Aptamer) is labeled with a terminally modified biotin label.
Preferably, the biotinylation Aptamer S Aptamer1 has the sequence
5-biotin-ATCCAGAGTGACGCAGCATTTCATCGGGTCCAAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT-3。
The immune metal nano-particles comprise gold nano-particles, silver nano-particles and the like.
Preferably, the immuometal signal nanoparticle is prepared by the following method: mixing gold nanoparticles with Raman reporter molecules (preferably Nile blue A (NBA for short) or other Raman reporter molecules), incubating at room temperature, centrifuging, discarding supernatant, redispersing in PBS-T solution, mixing with sulfhydryl modified Aptamer S Aptamer 2 (or Aptamer combination), freezing at-20 deg.C for 1-5h, thawing again, washing with PBS-T, adding BSA solution to seal residual active sites, incubating at room temperature, centrifuging, washing with PBS-T solution, discarding supernatant, redispersing in PBS-T solution, and obtaining the immuno-gold signal nanoparticles.
Preferably, in the preparation of the immuno-gold signaling nanoparticles, the gold nanoparticles are prepared by the following method: heating 200mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; and then 2-4mL of sodium citrate solution with the mass fraction of 1% is taken and added into boiling chloroauric acid solution, and after the reaction is finished after boiling for 20-30min, the solution is cooled to room temperature to prepare nano gold sol.
Preferably, in the preparation of the immuno-gold signal nanoparticle, the particle size of the gold nanoparticle is preferably 30-35nm, and the ultraviolet absorption wavelength is 526 nm.
Preferably, the immune gold signal nano-particle is prepared by mixing 50-500ul gold nano-particle with 1-30ul 30uM NBA solution and incubating for 1-30 minutes at room temperature.
In the preparation of the immune gold signal nano-particle, the preparation of washing solution PBS-T: adding Tween-20 with the concentration of 0.01% -1% into the PBS solution.
Preferably, the thiol-modified Aptamer S Aptamer 2 is
5-SH-CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3
Preferably, said preferably biotinylated Aptamer S Aptamer 2 (or other neocorona antigen biotinylated Aptamer) is characterized in that said Aptamer is thiol-labeled, preferably terminally modified.
Preferably, the method for detecting the new coronavirus SARS-CoV-2 antigen based on aptamer recognition surface enhanced Raman spectroscopy comprises the steps of mixing the prepared immunomagnetic bead capture nanoparticles with the new coronavirus SARS-CoV-2 antigen or virus sample, mixing with the immuno-gold signal nanoparticles (or other mixing sequences), incubating at room temperature, washing with PBS-T water after magnetic separation, performing magnetic separation, directly performing Raman detection, and realizing rapid detection of the new coronavirus SARS-CoV-2 antigen within 1-30 minutes (preferably 5 minutes).
Preferably, the excitation wavelength is 785nm for a portable raman spectrometer or other type of raman spectrometer.
Preferably, after magnetic separation, the precipitate is taken out and dropped on a silicon wafer plated with a gold film for SERS detection.
The preferred method of the invention is:
(1) preparing immunomagnetic bead capture nanoparticles: taking 100-; and (2) resuspending the magnetic beads in a B & W washing buffer solution to half of the final concentration, adding 10-200ul of 100uM biotinylated aptamer SAptamer 1, incubating at room temperature for 5-30min, magnetically attracting to remove supernatant, washing with the B & W washing buffer solution, resuspending in a PBS solution, adding 100-5000ul of 1% BSA solution, reacting at room temperature for 1-5h to seal the residual binding sites on the surfaces of the magnetic beads, magnetically separating, washing with the PBS solution, and finally redispersing in the PBS solution to obtain the immunomagnetic bead capture nanoparticles.
(2) Preparing nano gold sol: heating 200mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; and then adding a 1% sodium citrate solution in mass fraction into a boiling chloroauric acid solution, after the reaction is finished after the solution is boiled for 20-30min, cooling to room temperature to prepare the nano gold sol.
(3) Preparing immune gold signal nanoparticles: mixing 100-1000ul of ion-doped gold nanoparticles with 1-10ul of 10-100uM Raman reporter NBA (Nile blue A), incubating at room temperature for 1-15min, centrifuging, removing supernatant, re-dispersing in PBS-T solution, mixing with thiol-modified Aptamer S Aptamer 2, freezing at-20 ℃ for 1-10h, thawing, washing with PBS-T for several times, adding 100-5000ul of 1% BSA solution, reacting at room temperature for 1-5h to seal residual active sites, centrifuging, washing with PBS-T solution, finally removing supernatant, re-dispersing in 100-1000ul of PBS-T solution to obtain the immuno-gold signal nanoparticles.
(4) Preparation of sandwich (sandwich) structure: mixing the prepared immunomagnetic bead capture nanoparticles with new coronavirus SARS-CoV-2S proteins (10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml and 1ng/ml) with different concentrations, incubating at room temperature for 0.5-5h, washing with PBS-T solution after magnetic separation, mixing with immunogold signal nanoparticles, incubating at room temperature for 0.5-5h, washing with PBS-T solution after magnetic separation, and constructing into a sandwich structure.
(5) Detection of the novel coronavirus SARS-CoV-2S protein: taking out the precipitate with the sandwich structure, dripping the precipitate on a silicon chip plated with a gold film, directly carrying out Raman detection by using a portable Raman spectrometer, and realizing the rapid detection of the Raman spectrum result of the S protein within 3 minutes.
Detection of new corona pseudovirus-2019-nCoV pseudovirus and variant pseudovirus: mixing the prepared immunomagnetic bead capture nanoparticles with new crown pseudovirus-2019-nCoV pseudovirus/variant pseudovirus and immunogold signal nanoparticles with different concentrations, incubating at room temperature for 1-10min, washing with PBS-T solution after magnetic separation, performing magnetic separation again to construct a sandwich structure, dripping the sandwich structure on a silicon wafer plated with a gold film, directly performing Raman detection by using a portable Raman spectrometer, and realizing the rapid detection of the new crown pseudovirus-2019-nCoV pseudovirus within 2 min.
Example 1
1) Preparing nano gold sol: heating 200mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; and then adding 4.5mL of 1% sodium citrate solution into boiling chloroauric acid solution, after the reaction is finished after boiling for 20-30min, cooling to room temperature to prepare nano gold sol.
2) Preparing immune gold signal nanoparticles: mixing 100ul gold nanoparticles with 10ul 30uM Raman reporter NBA (Nile blue A), incubating at room temperature for 10 minutes, centrifuging, discarding the supernatant, redispersing in PBS-T solution, mixing with thiol-modified Aptamer S Aptamer 2 (sequence as described above), freezing at-20 ℃ for 2 hours, thawing, washing with PBS-T for 3 times, adding 500ul 1% BSA solution to seal the residual active sites, incubating at room temperature for 2 hours, centrifuging, washing with PBS-T solution for 3 times, discarding the supernatant, and redispersing in 500ul PBS-T solution to obtain the immuno-gold signal nanoparticles.
3) Preparing immunomagnetic bead capture nanoparticles: taking 200ul of streptavidin-modified magnetic microspheres with the concentration of 10ug/ul, adding 1ml of B & W washing buffer solution, uniformly mixing by vortex to fully suspend the magnetic beads, magnetically attracting to remove supernatant, and washing twice by 200ul of B & W; the magnetic beads are resuspended to half of the final concentration in B & W washing buffer, 200ul10uM biotinylated Aptamer S Aptamer1 (sequence as described above) is added, incubated at room temperature for 20 minutes, magnetically attracted to remove supernatant, washed twice with B & W washing buffer, resuspended in 500ul PBS solution, 3000ul 1% BSA solution is added, reacted at room temperature for 1.5h to block the remaining binding sites on the surface of the magnetic beads, magnetically separated, washed with PBS solution, and finally redispersed in 500ul PBS solution to obtain the immunomagnetic bead capture nanoparticles.
4) Preparation of sandwich (sandwich) structure: mixing the prepared 20ul immunomagnetic bead capture nanoparticles with 50ul new coronavirus SARS-CoV-2S protein (100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml and 10pg/ml), incubating at room temperature for 1.5h, washing with PBS-T solution for 4 times after magnetic separation, mixing with 50ul immunogold signal nanoparticles, incubating at room temperature for 1.5h, washing with PBS-T solution for 4 times after magnetic separation, and magnetically separating to construct a sandwich structure.
5) Detection of the novel coronavirus SARS-CoV-2S protein: taking out the precipitate with sandwich structure, dropping the precipitate onto silicon chip coated with gold film, drying, Raman detecting with portable Raman spectrometer (laser wavelength 785nm), and rapidly detecting Raman spectrum result of S protein within 5 min.
Detection of novel corona pseudovirus-2019-nCOV pseudoviruses and variant viruses: mixing the prepared immunomagnetic bead capture nanoparticles with new crown pseudovirus-2019-nCoV pseudoviruses with different concentrations and immunogold signal nanoparticles, incubating at room temperature for 1min, washing with PBS-T solution for 4 times after magnetic separation, performing magnetic separation again to construct a sandwich structure, dripping the sandwich structure on a silicon chip plated with a gold film, directly performing Raman detection (laser wavelength 785nm) by using a portable Raman spectrometer, and realizing the rapid detection of the Raman spectrum result of the new crown pseudovirus-2019-nCoV pseudoviruses within 5 minutes.
The results are shown in FIGS. 2-6.
Example 2
1) Preparing nano gold sol: heating 200mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; and then 4.5mL of 1% sodium citrate solution with mass fraction is added into boiling chloroauric acid solution, and after the reaction is finished after boiling for 20-30min, the solution is cooled to room temperature to prepare nano gold sol.
2) Preparing immune gold signal nanoparticles: mixing 100ul gold nanoparticles with 10ul 30uM Raman reporter NBA (Nile blue A), incubating for 10 minutes at room temperature, centrifuging, discarding supernatant, redispersing in PBS-T solution, mixing with sulfhydryl modified Aptamer combination S Aptamer1 and S Aptamer 2, freezing for 2 hours at-20 ℃, thawing, washing for 3 times with PBS-T, adding 500ul 1% BSA solution to seal the residual active sites, incubating for 1 hour at room temperature, centrifuging, washing for 3 times with PBS-T solution, discarding supernatant, redispersing in 500ul PBS-T solution to obtain the immuno-gold signal nanoparticles.
3) Preparing immunomagnetic bead capture nanoparticles: taking 200ul of streptavidin-modified magnetic microspheres with the concentration of 10ug/ul, adding 1ml of B & W washing buffer solution, uniformly mixing by vortex to fully suspend magnetic beads, magnetically sucking to remove supernatant, and washing twice by 100ul of B & W; and (2) resuspending the magnetic beads in a B & W washing buffer solution to half of the final concentration, adding 200ul of 10uM biotinylation Aptamer combination S Aptamer1 and S Aptamer 2, incubating for 15 minutes at room temperature, magnetically attracting to remove supernatant, washing twice with the B & W washing buffer solution, resuspending in 500ul of PBS solution, adding 5000ul of 1% BSA solution, reacting for 1.5 hours at room temperature to seal the residual binding sites on the surfaces of the magnetic beads, magnetically separating, washing with the PBS solution, and finally redispersing in 1000ul of PBS solution to prepare the immunomagnetic bead capture nanoparticles.
4) Preparation of sandwich (sandwich) structure: mixing the prepared 20ul immunomagnetic bead capture nanoparticles with 50ul new coronavirus SARS-CoV-2S protein (100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml and 10pg/ml), incubating at room temperature for 1.5h, washing with PBS-T solution for 4 times after magnetic separation, mixing with 50ul immunogold signal nanoparticles, incubating at room temperature for 1.5h, washing with PBS-T solution for 4 times after magnetic separation, and magnetically separating to construct a sandwich structure.
5) Detection of the novel coronavirus SARS-CoV-2S protein: taking out the precipitate with the sandwich structure, dripping the precipitate on a silicon chip plated with a gold film, directly carrying out Raman detection (laser wavelength of 785nm) by a portable Raman spectrometer after the precipitate is dried, and realizing the rapid detection of the Raman spectrum result of the S protein within 5 minutes.
Detection of novel corona pseudovirus-2019-nCOV pseudoviruses and variant viruses: mixing the prepared immunomagnetic bead capture nanoparticles with new crown pseudovirus-2019-nCoV pseudoviruses with different concentrations and immunogold signal nanoparticles, incubating at room temperature for 5min, washing with PBS-T solution for 4 times after magnetic separation, performing magnetic separation again to construct a sandwich structure, dripping the sandwich structure on a silicon chip plated with a gold film, directly performing Raman detection (laser wavelength 785nm) by using a portable Raman spectrometer, and realizing the rapid detection of the Raman spectrum result of the new crown pseudovirus-2019-nCoV pseudoviruses within 5 min.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
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Claims (9)
1. A method for detecting SARS-CoV-2 antigen of new coronavirus includes the following steps:
s1: modifying a first aptamer or aptamer combination of a new coronavirus antigen on a magnetic microsphere through a biotin-streptavidin interaction to form an immunomagnetic bead capture nanoparticle;
s2: preparing metal nano sol;
s3: mixing the metal nano sol prepared in the step S2 with Raman reporter molecules, and mixing with a second aptamer or aptamer combination of the sulfhydryl-modified new coronavirus antigen; and blocking the active sites to obtain immune metal signal nanoparticles; the step S3 includes: mixing gold nanoparticles with Raman reporter molecules, incubating at room temperature, centrifuging, discarding supernatant, redispersing in PBS-T solution, mixing with sulfhydryl-modified aptamer or aptamer, freezing at-20 deg.C for 1-5h, thawing, washing with PBS-T, adding BSA solution to seal residual active sites, incubating at room temperature, centrifuging, washing with PBS-T solution, discarding supernatant, redispersing in PBS-T solution to obtain immunemetal signal nanoparticles;
s4: mixing the immunomagnetic bead capture nanoparticles prepared in the step S1 with a sample to be detected, and mixing the immunomagnetic bead capture nanoparticles with the immunometal signal nanoparticles in the step S3 to obtain a sandwich structure;
step S5: and carrying out Raman detection on the obtained sandwich structure by using a Raman spectrometer to obtain a Raman spectrum result of the new coronavirus antigen.
2. The detection method according to claim 1, characterized in that: the aptamer is an aptamer of the S protein of the novel coronavirus.
3. The detection method according to claim 1, wherein step S1 includes:
taking magnetic beads modified with streptavidin, and adding a buffer solution for washing; and (2) resuspending the magnetic beads in a buffer solution, adding a biotinylated aptamer or biotinylated aptamer combination, incubating at room temperature, washing with the buffer solution, resuspending in a PBS solution, adding a sealant, sealing the residual binding sites on the surfaces of the magnetic beads, carrying out magnetic separation, washing with the PBS solution, and finally redispersing in the PBS solution to obtain the immunomagnetic bead capture nanoparticles.
4. The detection method of claim 3, wherein the magnetic beads have a particle size of 0.5 to 5 μm.
5. The assay of claim 3 wherein the buffer is a B & W wash buffer formulated with 1-50mM Tris-HCl pH 4-8, 1-10mM EDTA and 2-10M NaCl.
6. The assay of claim 3 wherein the blocking agent is a BSA solution.
7. The detection method according to claim 1, wherein the first nucleic acid aptamer of step S1 is:
5-biotin-ATCCAGAGTGACGCAGCATTTCATCGGGTCCAAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT-3。
8. the detection method according to claim 1, characterized in that: the sulfhydryl-modified second aptamer of step S3 is
5-SH- CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3。
9. The detection method according to claim 1, characterized in that: in step S5, after magnetic separation, the precipitate is taken out and dropped on a silicon wafer plated with a gold film for SERS detection.
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