CN115508329A - Method for detecting novel coronavirus N protein based on surface enhanced Raman spectroscopy - Google Patents
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Abstract
The invention discloses a method for detecting novel coronavirus N protein based on surface enhanced Raman spectroscopy, which comprises the following steps: s1, preparing immune Raman signal gold nanoparticles of an Au @ IR808@ antibody; s2, preparing a biotinylated antibody Ab2; s3, preparing an immune sandwich compound; s4, magnetically attracting and washing the washing solution, and finally concentrating to a small volume; s5, dropping the small-volume liquid on the surface enhanced Raman chip for Raman detection. The invention utilizes the high binding efficiency of immune Raman signal gold nanoparticles, SARS-CoV-2N protein and biotinylation antibody Ab2 in liquid phase to realize the specific recognition and capture of the N protein to form a double-antibody immune complex, and then utilizes the high affinity of streptavidin and biotin to capture the immune complex formed in the last step by magnetic beads modified with streptavidin to form an immune sandwich complex, thus realizing the ultrasensitive stable and rapid detection of the SARS-CoV-2 virus N-protein within 15 min.
Description
Technical Field
The invention belongs to the field of biological immunoassay detection, and particularly relates to a method for detecting novel coronavirus N protein based on surface enhanced Raman spectroscopy, which is used for detecting nucleocapsid protein (N) of the novel coronavirus.
Background
The etiology and serological examination in the method for detecting 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. The antibody detection cannot be used for diagnosing new coronavirus alone, infection or infection state cannot be eliminated, the antigen detection is an important supplement for nucleic acid detection, positive infected persons can be found earlier through the antigen detection, isolation and nucleic acid rechecking are carried out as soon as possible, and early discovery, early reporting and early isolation are realized to the maximum extent.
The existing detection methods are, for example:
CN202011431411.8 discloses a kit for detecting a 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 its DNA template; (4) a nucleic acid ligase; (5) a light-up RNA aptamer or a DNA template thereof; (6) in vitro transcription reagents.
CN202110353115.9 discloses a novel coronavirus neutralizing antibody detection kit prepared by immunochromatography labeled with Pt-Pd alloy nanoparticles, 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 detection, the method has the advantages of higher antigen detection speed and more convenient operation, can be used for screening specific people as a supplementary means, and is favorable for improving the early discovery capability. And the cost is lower, the speed is faster, and the result can be obtained within 10-20 min. 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. However, the existing rapid antigen detection kit has the problems of false negative caused by insufficient detection sensitivity, low detectable rate and the like, so that a rapid antigen detection means which is rapid, high in detection sensitivity and stable is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, make up the defects of the prior art for detecting novel coronavirus, and provide a method for quickly and sensitively detecting novel coronavirus N protein based on surface-enhanced Raman spectroscopy. The invention utilizes the high binding efficiency of immune Raman signal gold nanoparticles, SARS-CoV-2N protein and biotinylated antibody Ab2 in liquid phase to realize the specificity recognition capture of N protein to form a 'double-antibody' immune complex, then utilizes the high affinity of streptavidin and biotin, captures the immune complex formed in the last step by magnetic beads modified with streptavidin to form an immune sandwich complex with the structure of 'immune Raman signal gold nanoparticles-N protein-biotinylated antibody-streptavidin magnetic beads', and a Raman spectrometer realizes the ultrasensitive stable rapid detection of SARS-CoV-2 virus N-protein within 15 min.
In order to achieve the above purpose, one of the technical solutions of the present invention is: a method for detecting novel coronavirus N protein based on surface enhanced Raman spectroscopy specifically comprises the following steps:
s1: respectively coupling Raman signal molecules and an antibody Ab1 of an N protein of SARS-CoV-2 virus to the gold nanoparticles to prepare the immune Raman signal gold nanoparticles of the Au @ IR808@ antibody;
s2: biotin is coupled with an antibody Ab2 to prepare a biotinylated antibody Ab2 (Bio-Ab 2);
s3: mixing and incubating immune Raman signal gold nanoparticles prepared in the step S1, biotinylated antibody Ab2 prepared in the step S2 and novel coronavirus N protein diluent, and mixing and incubating the mixture with streptavidin magnetic beads to obtain an immune sandwich compound;
s4: magnetically attracting and washing the immune sandwich compound in the step S3 for a plurality of times by using washing liquid, and finally concentrating and fixing the volume to a small volume;
s5: and dripping the small-volume liquid in the S4 on a surface enhanced Raman chip for Raman detection.
Preferably, the immuno-raman signaling gold nanoparticles in step S1 are prepared by the following method: mixing gold nanoparticles and Raman signal molecules under stirring, incubating at room temperature, centrifuging, discarding supernatant, washing with buffer solution once and fixing volume, adding EDC ((1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution for activation, washing after centrifuging, fixing volume with buffer solution, adding SARS-CoV-2 virus N protein antibody Ab1, incubating and combining overnight, centrifuging, discarding supernatant, adding confining liquid for incubation, washing and centrifuging, discarding supernatant, and finally redispersing in preservation solution to obtain the immune Raman signal gold nanoparticles.
Furthermore, the buffer solution is BB buffer solution which is prepared from borax and sodium hydroxide, and the pH value is 7.2-7.6.
Further, the particle size of the gold nanoparticles is 30-80nm.
Furthermore, the EDC and the NHS are added for reaction, the activation time is 10-60min, the activation temperature is room temperature, the antibody is added for incubation, the combination time is 10-24h, and the incubation temperature is 2-8 ℃.
Furthermore, the sealing liquid comprises PBS salt solution, bovine serum albumin, casein and skimmed milk powder, and the preservation liquid comprises PBS salt solution, bovine serum albumin, casein, glycine and skimmed milk powder.
Furthermore, the sealing time for adding the sealing liquid is 2-12h, and the sealing temperature is room temperature.
Preferably, the gold nanoparticles for immune Raman signals in the step S1 have Raman signal expression and specific recognition antibodies of novel coronavirus SARS-CoV-2N proteins.
Preferably, the SARS-CoV-2 virus N protein antibody Ab1 in step S1 is a novel coronavirus N protein antibody SARS-CoV-2NP-mAb.
Preferably, the raman signal molecule in step S1 is near-infrared indole cyanine dye IR808, but not limited to this signal molecule, other raman molecules such as nile blue a, indocyanine green (ICG) and the like are also in the range, and the incubation time with gold nanoparticles is 5-30min.
Preferably, the biotinylated antibody in step S2 is prepared by mixing the antibody with biotin for 2-6h, and dialyzing in PBS for 15-30h.
Preferably, the antibody Ab2 added to the antibody that is bio-ized in step S2 is a novel coronavirus neutralizing antibody SARS-CoV-2NP-mAb.
Preferably, the dilution of the N protein of the novel coronavirus in step S2 is composed of 10mM PBS, 0.01% -1% Triton X-100, 0.05% -5% EDTA, 0.05-5% Tween-20.
Preferably, the biotinylated antibodies in steps S2 and S3 have biotin-modified antibodies.
Preferably, the streptavidin magnetic beads in step S3 are magnetic and modified with streptavidin.
The method for detecting the novel coronavirus N protein based on the surface enhanced Raman spectroscopy comprises the following steps: mixing and incubating immune Raman signal gold nanoparticles for preparing Au @ IR808@ antibody, novel coronavirus SARS-CoV-2N protein and biological antibody Ab2 to form a 'double-antibody' immune complex, adding streptavidin magnetic bead capture complex to further form an immune sandwich complex, washing PBS-T for several times after magnetic separation, concentrating to a small volume, directly carrying out Raman detection, and realizing the Raman spectrum result of rapid detection of the novel coronavirus N protein within 15 min.
Preferably, in the step S4, the washing solution is PBS-T, the solution is PBS, and Tween-20 with the concentration of 0.01% -1% is added into the PBS solution to prepare the PBS-T.
Preferably, the number of washing in step S4 is 1-5 times, and the small volume is 3-30ul.
Preferably, the surface-enhanced raman chip in step S5 is a silicon wafer plated with a gold film.
Preferably, the raman detection device in step S5 is a portable raman spectrometer, a confocal raman spectrometer, a handheld raman spectrometer, or the like, and the excitation wavelength is 785nm.
Preferably, the method for detecting the novel coronavirus N protein based on the surface enhanced Raman spectroscopy specifically comprises the following steps:
(1) Preparing immune Raman signal gold nanoparticles: mixing 2-20ml of gold nano particles and 10-100ul of 0.5mM of IR808 Raman signal molecules under stirring, incubating at room temperature for 2-60min, centrifuging at 5000-8000rpm, discarding supernatant, washing with 2-20ml of BB buffer solution once and fixing volume, adding 50-100ul of 5-50mM EDC ((1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution respectively for activation, washing with 2-20ml of BB buffer solution after centrifugation, fixing volume with 1-20ml of BB buffer solution, adding 1-100ug of SARS-CoV-2 virus N protein antibody 1, incubating and combining at 2-8 ℃ overnight, centrifuging, discarding supernatant, adding 1-20ml of confining liquid for incubation for 1-12h, washing and centrifuging, discarding supernatant, finally re-dispersing in 2-10ml of Ab solution again to prepare the immune Raman signal Ab gold nano particles.
(2) Preparation of biotinylated antibody: mixing 10-500ul 1-10mg/ml N protein antibody Ab2 with 1-20ml 50mM biotin for 1-10h, dialyzing in PBS (pH7.2) solution through a dialysis membrane with molecular weight of 1000-10000 for 10-24h, and diluting to 1-50mg/ml with PBS solution.
(3) Preparation of immune sandwich complex structure: mixing 10-100ul of immune Raman signal gold nanoparticles for preparing an Au @ IR808@ antibody, 100-1000ul of novel coronavirus SARS-CoV-2N protein (0.1 pg/ml, 1pg/ml, 5pg/ml and 50 pg/ml) prepared by different concentrations of diluents and 1-50ul of a biochemical antibody Ab2, incubating for 1-10min to form a 'double-antibody' immune complex, adding 10-100ul of a 1-20mg/ml streptavidin magnetic bead capture complex, incubating for 1-10min to construct an immune sandwich complex, washing for 1-5 times by PBS-T after magnetic separation, and concentrating to a constant volume of 3-30ul.
(4) Detection of novel coronavirus SARS-CoV-2N protein: taking out the formed small-volume immune sandwich compound solution, dripping the solution 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 novel coronavirus N protein within 15 min.
Compared with the prior art, the invention has the beneficial effects that:
1. the immune Raman signal gold nanoparticle of the Au @ IR808@ antibody is prepared, wherein the IR808 Raman signal molecule contains a-COOH group, and can be directly connected with the antibody after being activated by EDC and NHS, namely the Raman signal group is also a connector combined with the antibody, so that the complex modification process of the antibody on the surface of the gold nanoparticle is avoided, and the IR808 is in a resonance state under 785nm laser, has higher signal intensity compared with other Raman signal molecules, and greatly improves the detection sensitivity.
2. The invention utilizes the high combination efficiency of immune Raman signal gold nanoparticles, SARS-CoV-2N protein and biotinylated antibody Ab2 in liquid phase to realize the specificity recognition capture of N protein to form a 'double-antibody' immune complex, and then utilizes the high affinity of streptavidin and biotin to capture the immune complex formed in the last step by the magnetic bead modified with streptavidin to form an immune sandwich complex with the structure of 'immune Raman signal gold nanoparticles-N protein-biotinylated antibody-streptavidin magnetic bead', compared with the sandwich complex of 'Raman signal gold-antigen-magnetic bead' formed by simple reaction, the reaction mode can improve the sensitivity of nearly one order of magnitude.
3. The detection method provided by the invention is used for detecting the novel coronavirus SARS-CoV-2N protein, the detection limit for detecting the novel coronavirus recombinant N protein under the standard buffer solution is 0.1pg/ml, the detection limit of a simulated throat swab sample is 1pg/ml, the sensitivity is improved by 20-50 times compared with a novel coronavirus antigen detection kit (a gold nanoparticle method) (the detection limit of the N protein is 10-100 pg/ml), the problem of insufficient sensitivity of the conventional rapid antigen detection method is well solved, the detection method can also specifically detect the novel coronavirus SARS-CoV-2N protein in the nasopharynx swab, and the requirement of field detection can be met only within 15min by adopting a portable Raman spectrometer.
Drawings
FIG. 1 is a schematic view of the detection principle of the present invention;
FIG. 2 is an SEM photograph of an immune sandwich complex structure in example 1 of the present invention;
FIG. 3 is a surface enhanced Raman spectrum of different SARS-CoV-2N protein concentrations in a dilution;
FIG. 4 is a surface enhanced Raman spectrum of different SARS-CoV-2N protein concentrations in pharyngeal swab collection;
FIG. 5 is a schematic diagram showing the structure of an immunological sandwich complex in the "Raman signal gold-antigen-magnetic bead" reaction mode in comparative example 1 of the present invention;
FIG. 6 is a surface enhanced Raman spectrum of the "Raman signal gold-antigen-magnetic bead" reaction method in comparative example 1 for detecting the concentration of different SARS-CoV-2N proteins in the diluted solution;
FIG. 7 is the surface enhanced Raman spectrum of the gold-antigen-magnetic bead reaction method for detecting different SARS-CoV-2N protein concentrations in the collected liquid of throat swab in comparative example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of the present invention is not limited to these embodiments.
The detection method of the invention utilizes the high combination efficiency of immune Raman signal gold nanoparticles, SARS-CoV-2N protein and biotinylated antibody Ab2 in liquid phase to realize the specific recognition and capture of the N protein to form a double-antibody immune complex, and then utilizes the high affinity of streptavidin and biotin to capture the immune complex formed in the last step by magnetic beads modified with streptavidin, thereby forming the immune sandwich complex with the structure of immune Raman signal gold nanoparticles-N protein-biotinylated antibody-streptavidin magnetic beads.
A method for detecting novel coronavirus N protein based on surface enhanced Raman spectroscopy specifically comprises the following steps:
s1: respectively coupling Raman signal molecules and an antibody Ab1 of an N protein of SARS-CoV-2 virus to the gold nanoparticles to prepare immune Raman signal gold nanoparticles of the Au @ IR808@ antibody;
s2: biotin was coupled to the antibody Ab2 to prepare a biotinylated antibody Ab2 (Bio-Ab 2);
s3: mixing and incubating immune Raman signal gold nanoparticles prepared in the step S1, biotinylated antibody Ab2 prepared in the step S2 and novel coronavirus N protein diluent, and mixing and incubating the mixture with streptavidin magnetic beads to obtain an immune sandwich compound;
s4: magnetically attracting and washing the immune sandwich compound in the step S3 for several times by using washing liquid, and finally concentrating and fixing the volume to a small volume;
s5: and dropping the small-volume liquid in the S4 on a surface enhanced Raman chip for Raman detection.
FIG. 1 is a schematic diagram of the detection principle of the present invention, in which an immune Raman signal gold nanoparticle for preparing an Au @ IR808@ antibody, a novel coronavirus SARS-CoV-2N protein and a biochemical antibody Ab2 are mixed and incubated to form a double-antibody immune complex, and then a streptavidin-based magnetic bead capture complex is added to further form an immune sandwich complex.
The nano-particles used in the following examples are ultra-high concentration gold nano-particle solution with the particle size of 40-60nm, which is developed by Zhengzhou rabdosia biotechnology and Limited liability company; the SARS-CoV-2 virus N protein antibody Ab1 is a novel coronavirus N protein antibody SARS-CoV-2NP-mAb with the product number of C00154 developed by Jiangsu Dongzhao anti-biological medicine science and technology Limited; the antibody Ab2 added in the biological antibody is a novel coronavirus neutralizing antibody SARS-CoV-2NP-mAb with the product number of C00172 developed by Jiangsu Dongzhong biological medicine science and technology Limited.
Example 1
(1) Preparing immune Raman signal gold nanoparticles: mixing 10ml of gold nanoparticles with 20ul of 0.5mM of IR808 Raman signal molecules under stirring, incubating at room temperature for 10min, centrifuging at 6000rpm, discarding a supernatant, washing once with 10ml of BB buffer solution and fixing the volume, adding 50ul of 5mM EDC ((1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution into each solution for activation, centrifuging, washing with 10ml of BB buffer solution, fixing the volume with 5ml of BB buffer solution, adding 50ug of SARS-CoV-2 virus N protein antibody Ab1, incubating and combining at 4 ℃ overnight, centrifuging, discarding the supernatant, adding 10ml of confining liquid for incubating for 1h, washing and centrifuging, discarding the supernatant, and finally redispersing in 10ml of preservation solution to prepare the immune Raman signal gold nanoparticles.
(2) Preparation of biotinylated antibody: 250ul of 5mg/ml N protein antibody Ab2 was mixed with 2ml of 50mM biotin for 5 hours, and then dialyzed through a 5000 molecular weight dialysis membrane in a PBS (pH 7.2) solution for 20 hours, and then made to a volume of 1mg/ml with the PBS solution.
(3) Preparation of an immune sandwich complex structure: 20ul of immune Raman signal gold nanoparticles for preparing Au @ IR808@ antibody, 300ul of novel coronavirus SARS-CoV-2N protein (0.1 pg/ml, 1pg/ml, 5pg/ml and 50 pg/ml) prepared from diluents with different concentrations and 10ul of biochemical antibody Ab2 are mixed and incubated for 4min to form a 'double-antibody' immune complex, then 40ul of SEM 1mg/ml streptavidin magnetic bead capture complex is added to incubate for 4min to construct an immune sandwich complex, PBS-T is washed for 2 times after magnetic separation, and the volume is determined to be 10ul after concentration, and FIG. 2 is a structural diagram of the immune sandwich complex. .
(4) Detection of novel coronavirus SARS-CoV-2N protein: taking out the immune sandwich compound solution with small volume, dripping the immune sandwich compound solution on a silicon chip plated with a gold film, directly carrying out Raman detection by a portable Raman spectrometer, and rapidly detecting a Raman spectrum result of the novel coronavirus N protein within 15min, wherein the Raman spectrum result is shown in figure 3.
As can be seen from fig. 3: the detection limit of the recombinant N protein of the novel coronavirus under a standard buffer solution (standard system) is 0.1pg/ml, and compared with a novel coronavirus antigen detection kit (gold nanoparticle method) (the detection limit of the N protein is 10-100 pg/ml), the sensitivity is improved by 100-1000 times.
Example 2
(1) Preparing immune Raman signal gold nanoparticles: mixing 10ml of gold nanoparticles with 20ul 0.5mM of IR808 Raman signal molecules under stirring, incubating at room temperature for 10min, centrifuging at 6000rpm, discarding supernatant, washing once with 10ml of BB buffer solution and fixing volume, adding 50ul of 5mM EDC ((1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution for activation, centrifuging, washing with 10ml of BB buffer solution, fixing volume with 5ml of BB buffer solution, adding 50ug of SARS-CoV-2 virus N protein antibody Ab1, incubating and combining at 4 ℃ overnight, centrifuging, discarding supernatant, adding 10ml of confining liquid for incubation for 1h, washing and centrifuging, discarding supernatant, and finally redispersing in 10ml of gold preservation solution to prepare the immune Raman signal nanoparticles.
(2) Preparation of biotinylated antibody: 250ul of 5mg/ml N protein antibody Ab2 was mixed with 2ml of 50mM biotin for 5 hours, and then dialyzed through a 5000 molecular weight dialysis membrane in a PBS (pH 7.2) solution for 20 hours, and then made to a volume of 1mg/ml with the PBS solution.
(3) Preparation of an immune sandwich complex structure: the pharynx swab sampling rod is adopted to wipe the pharynx of the oral cavity fully, then the pharynx of the oral cavity is stirred in 3ml of N protein diluent, the pharynx swab sampling liquid is prepared, and N protein is added to prepare different novel coronavirus SARS-CoV-2N protein (0.1 pg/ml, 1pg/ml, 5pg/ml and 50 pg/ml) pharynx swab sample liquids. 20ul of immune Raman signal gold nanoparticles for preparing Au @ IR808@ antibody, 300ul of novel coronavirus SARS-CoV-2N protein (1 pg/ml, 5pg/ml and 50 pg/ml) prepared from throat swab sampling solutions with different concentrations and 10ul of biological antibody Ab2 are mixed and incubated for 5min to form a 'double-antibody' immune complex, 40ul of 1mg/ml streptavidin magnetic bead capture complex is added for incubation for 5min to form an immune sandwich complex, PBS-T is washed for 3 times after magnetic separation, and the volume is concentrated to 10ul.
(4) Detection of novel coronavirus SARS-CoV-2N protein throat swab simulation sample: taking out the formed small-volume immune sandwich compound solution, dripping the solution 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 novel coronavirus N protein in 15min to obtain a Raman spectrum result, as shown in figure 4.
As can be seen from fig. 4: the detection limit of the novel simulated sample of the coronavirus swab is 1pg/ml, and compared with a novel coronavirus antigen detection kit (gold nanoparticle method) (the detection limit of the N protein is 10-100 pg/ml), the sensitivity is improved by 10-100 times.
Comparative example 1
(1) Preparing immune Raman signal gold nanoparticles: mixing 10ml of gold nanoparticles with 20ul of 0.5mM of IR808 Raman signal molecules under stirring, incubating at room temperature for 10min, centrifuging at 6000rpm, discarding a supernatant, washing once with 10ml of BB buffer solution and fixing the volume, adding 50ul of 5mM EDC ((1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution into each solution for activation, centrifuging, washing with 10ml of BB buffer solution, fixing the volume with 5ml of BB buffer solution, adding 50ug of SARS-CoV-2 virus N protein antibody Ab1, incubating and combining at 4 ℃ overnight, centrifuging, discarding the supernatant, adding 10ml of confining liquid for incubating for 1h, washing and centrifuging, discarding the supernatant, and finally redispersing in 10ml of preservation solution to prepare the immune Raman signal gold nanoparticles.
(2) Preparing immunomagnetic beads: incubating and combining 100ul 10mg/ml carboxylated magnetic beads and 50ug SARS-CoV-2 virus N protein antibody Ab2 at 4 ℃ overnight, centrifuging, discarding supernatant, adding 10ml confining liquid, incubating for 1h, washing, centrifuging, discarding supernatant, and finally dispersing in 1ml of preservation solution again to obtain the immune Raman magnetic beads.
(3) Preparation of an immune sandwich complex structure: 20ul of immune Raman signal gold nanoparticles for preparing Au @ IR808@ antibody, 300ul of novel coronavirus SARS-CoV-2N protein (0.1 pg/ml, 1pg/ml, 5pg/ml and 50 pg/ml) prepared from different concentrations of diluents and immunomagnetic beads are mixed together and incubated for 5min to form a Raman signal gold-antigen-magnetic bead immune complex, PBS-T is washed for 2 times after magnetic separation, the volume is determined to be 10ul by concentration, and FIG. 5 is a structural schematic diagram of the immune sandwich complex in a Raman signal gold-antigen-magnetic bead reaction mode.
(4) Detection of novel coronavirus SARS-CoV-2N protein: taking out the solution of the formed small-volume immune sandwich compound, dripping the solution on a silicon chip plated with a gold film, directly carrying out Raman detection by a portable Raman spectrometer, and quickly detecting a Raman spectrum result of the novel coronavirus N protein within about 15min, as shown in figure 6.
As can be seen from fig. 6: the limit of detection of the novel coronavirus recombinant N protein in a Raman signal gold-antigen-magnetic bead reaction mode under a standard buffer solution (standard system) is 1pg/m, and compared with the result of example 1, the limit of detection in example 1 is 0.1pg/ml, and the limit of detection in comparative example 1 is 1pg/ml, so that the example 1 is improved by one order of magnitude compared with the example 1.
Comparative example 2
(1) Preparing immune Raman signal gold nanoparticles: mixing 10ml of gold nanoparticles with 20ul of 0.5mM of IR808 Raman signal molecules under stirring, incubating at room temperature for 10min, centrifuging at 6000rpm, discarding a supernatant, washing once with 10ml of BB buffer solution and fixing the volume, adding 50ul of 5mM EDC ((1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution into each solution for activation, centrifuging, washing with 10ml of BB buffer solution, fixing the volume with 5ml of BB buffer solution, adding 50ug of SARS-CoV-2 virus N protein antibody Ab1, incubating and combining at 4 ℃ overnight, centrifuging, discarding the supernatant, adding 10ml of confining liquid for incubating for 1h, washing and centrifuging, discarding the supernatant, and finally redispersing in 10ml of preservation solution to prepare the immune Raman signal gold nanoparticles.
(2) Preparation of immunomagnetic beads: incubating and combining 100ul 10mg/ml carboxylated magnetic beads and 50ug SARS-CoV-2 virus N protein antibody Ab2 at 4 ℃ overnight, centrifuging, discarding supernatant, adding 10ml confining liquid, incubating for 1h, washing, centrifuging, discarding supernatant, and finally dispersing in 1ml of preservation solution again to obtain the immune Raman magnetic beads.
(3) Preparation of immune sandwich complex structure: mixing 20ul immune Raman signal gold nanoparticles for preparing Au @ IR808@ antibody, novel coronavirus SARS-CoV-2N protein (0 pg/ml, 10pg/ml and 100 pg/ml) prepared from 300ul pharyngeal swab sampling solutions with different concentrations and immunomagnetic beads, incubating for 5min to form a Raman signal gold-antigen-magnetic bead immune complex, washing for 2 times by PBS-T after magnetic separation, concentrating and fixing the volume to 10ul in small volume.
(4) Detection of novel coronavirus SARS-CoV-2N protein throat swab simulation sample: taking out the immune sandwich complex solution with small volume, dripping the immune sandwich complex solution on a silicon chip plated with a gold film, directly carrying out Raman detection by a portable Raman spectrometer, and rapidly detecting a Raman spectrum result of the novel coronavirus N protein within 15min, as shown in figure 7.
As can be seen from fig. 7: the detection limit of the 'Raman signal gold-antigen-magnetic bead' reaction mode for detecting the novel coronavirus recombinant N protein under the condition of detecting the novel coronavirus throat swab simulation sample is 10pg/ml, and compared with the result of the example 2, the detection limit of the example 2 (throat swab simulation sample) is 1pg/ml, and the detection is more sensitive by one order of magnitude than that of the comparison ratio 2.
The above embodiments are merely preferred embodiments of the present invention, which are provided for illustrating the principles and effects of the present invention and not for limiting the present invention. It should be noted that modifications to the above-described embodiments can be made by persons skilled in the art without departing from the spirit and scope of the present invention and such modifications should also be considered as within the scope of the present invention.
Claims (10)
1. A method for detecting novel coronavirus N protein based on surface enhanced Raman spectroscopy is characterized by comprising the following steps:
s1: respectively coupling Raman signal molecules and an antibody Ab1 of an N protein of SARS-CoV-2 virus to the gold nanoparticles to prepare the immune Raman signal gold nanoparticles of the Au @ IR808@ antibody;
s2: biotin is coupled with an antibody Ab2 to prepare a biotinylated antibody Ab2;
s3: mixing and incubating immune Raman signal gold nanoparticles prepared in the step S1, biotinylated antibody Ab2 prepared in the step S2 and novel coronavirus N protein diluent, and mixing and incubating the mixture with streptavidin magnetic beads to obtain an immune sandwich compound;
s4: magnetically attracting and washing the immune sandwich compound in the step S3 by using a washing solution, and finally concentrating and fixing the volume to a small volume;
s5: and dropping the small-volume liquid in the S4 on a surface enhanced Raman chip for Raman detection.
2. The method for detecting the N protein of the novel coronavirus based on the surface-enhanced Raman spectroscopy as claimed in claim 1, wherein the step S1 comprises: mixing gold nanoparticles and Raman signal molecules under stirring, incubating at room temperature, centrifuging, discarding supernatant, washing with buffer solution once and fixing the volume, adding EDC and NHS solution for reaction and activation, centrifuging, washing, fixing the volume with buffer solution, adding SARS-CoV-2 virus N protein antibody Ab1, incubating and combining overnight, centrifuging, discarding supernatant, adding confining liquid for incubation, washing and centrifuging, discarding supernatant, and finally redispersing in preservation solution to obtain the immune Raman signal gold nanoparticles.
3. The method for detecting the N protein of the novel coronavirus based on the surface-enhanced Raman spectroscopy as claimed in claim 2, wherein the buffer solution is prepared from borax and sodium hydroxide, and has the pH value of 7.2-7.6; the particle size of the gold nanoparticles is 30-80nm.
4. The method for detecting the N protein of the novel coronavirus based on the surface-enhanced Raman spectroscopy of claim 2, wherein EDC and NHS react for 10-60min at room temperature for 10-24h at 2-8 ℃; the confining liquid comprises PBS salt solution, bovine serum albumin, casein and skimmed milk powder; the preserving fluid comprises PBS salt solution, bovine serum albumin, casein, glycine and skimmed milk powder; the sealing time of the sealing liquid is 2-12h, and the sealing temperature is room temperature.
5. The method for detecting the N protein of the novel coronavirus based on the surface-enhanced Raman spectroscopy as claimed in claim 1, wherein the gold nanoparticles for the immune Raman signal in the step S1 have Raman signal expression and specific recognition antibodies for the N protein of the novel coronavirus SARS-CoV-2; in the step S1, the Raman signal molecule is one of near-infrared indole cyanine dye IR808, nile blue A dye and indocyanine green (ICG), and the incubation time with the gold nanoparticles is 5-30min.
6. The method for detecting N protein of novel coronavirus according to claim 1, wherein the antibody Ab1 for N protein of SARS-CoV-2 virus and the antibody SARS-CoV-2NP-mAb for N protein of novel coronavirus in step S1 are the antibodies Ab2 for neutralizing SARS-CoV-2NP-mAb for novel coronavirus in step S2.
7. The method for detecting the N protein of the novel coronavirus based on the surface-enhanced Raman spectroscopy of claim 1, wherein the biotinylated antibody in the step S2 is prepared by mixing the antibody and biotin for 2-6h, and then dialyzing in a PBS solution for 5-30h; the composition of the N protein diluent of the novel coronavirus in the step S2 is 10mM PBS (pH7.2-7.4), 0.01% -1% Triton X-100, 0.05% -5% EDTA, 0.05-5% Tween-20.
8. The method for detecting the N protein of the novel coronavirus based on surface-enhanced Raman spectroscopy as claimed in claim 1, wherein the biotinylated antibody in steps S2 and S3 is an antibody with a function of modifying biotin; and in the step S3, the streptavidin magnetic beads have magnetism and modify streptavidin.
9. The method for detecting N protein of novel coronavirus according to claim 1, wherein the solution in step S4 is PBS, and the washing solution is PBS-T prepared by adding 0.01% -1% Tween-20 into the PBS solution; in the step S4, the washing times are 1-5 times, and the small volume is 3-30ul.
10. The method for detecting N protein of coronavirus based on surface enhanced Raman spectroscopy as claimed in claim 1, wherein the surface enhanced Raman chip in step S5 is a silicon wafer plated with a gold film; the Raman detection instrument in the step S5 is one of a portable Raman spectrometer, a confocal Raman spectrometer and a handheld Raman spectrometer, and the excitation wavelength is 785nm.
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