A labeling SERS biosensor for novel coronavirus detection and its detection method

Abstract

The present invention relates to a labeled SERS biosensor for novel coronavirus detection and its detection method. The labeled SERS biosensor for novel coronavirus detection comprises: ACE2 (angiotensin-converting enzyme 2) modified magnetic beads, a SERS label solution, and a SERS substrate. The SERS label in the SERS label solution comprises, from the inside out, Au nanostars and a novel coronavirus S protein antibody labeled with a Raman reporter molecule modified on the surface of the Au nanostars.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a labeled SERS biosensor for detecting the new coronavirus and a detection method thereof. Background Art

[0002] Conventional testing methods for the novel coronavirus primarily target viral nucleic acids, viral antigens, and the antibodies they produce. Traditional SARS-CoV-2 detection methods primarily include pathogen testing, molecular biology testing, serology, and other spectroscopy and imaging methods. Nucleic acid testing, due to its high sensitivity and specificity, is considered the "gold standard" for early diagnosis of the novel coronavirus. Antigen / antibody testing is convenient and rapid, serving as an adjunct to nucleic acid diagnosis. Due to differences in sensitivity between different brands of antigen detection reagents, it is currently used only as an auxiliary screening method. "Gold standard" nucleic acid tests, such as real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), require skilled personnel and excellent laboratory conditions and take several hours to complete. In applications requiring rapid screening and when facing rapidly spreading viral variants, the development of a rapid and highly sensitive detection method is necessary to curb the spread of the virus.

[0003] Surface Enhanced Raman Scattering (SERS) refers to the phenomenon that the Raman signal adsorbed on the surface of a rough noble metal substrate is enhanced, and the Raman spectrum is considered to be the fingerprint spectrum of the molecule. The labeling SERS technology refers to converting the signal of the analyte into the Raman signal of a Raman reporter molecule, and reflecting the concentration information of the analyte by the intensity of the Raman molecule signal. The labeling SERS technology can be used for the detection of biological macromolecules. Compared with the direct detection of biological macromolecules, the signal stability, uniformity and repeatability of Raman reporter molecules are better, and they have better quantitative properties. In addition, combined with magnetic separation and enrichment, the rational design of SERS tags for labeling Raman reporter molecules can further simplify the detection process, shorten the detection time and improve the sensitivity of detection. Summary of the Invention

[0004] In response to the above problems, the present invention aims to provide a rapid and highly sensitive SERS biosensor for novel coronavirus detection and its detection method. The SERS biosensor elutes the "SERS tag-S protein-specific magnetic beads" structure and deposits the "SERS tag-S protein-ACE2" complex containing Raman reporter molecules on a SERS substrate to collect Raman signals. Compared with the macromolecular antibodies, S protein, and ACE2 in the complex, the Raman reporter molecules have a larger scattering cross-section, generate stronger Raman signals, and are not interfered with by the Raman signals of biological macromolecules, thereby effectively improving the speed and sensitivity of detection.

[0005] Specifically, in the first aspect, the present invention provides a labeling SERS biosensor for the detection of the new coronavirus, comprising: ACE2 (angiotensin-converting enzyme 2) modified magnetic beads, a SERS label solution, and a SERS substrate; the structure of the SERS label in the SERS label solution comprises, from the inside to the outside, Au nanostars, and a new coronavirus S protein antibody labeled with a Raman reporter molecule modified on the surface of the Au nanostars.

[0006] Preferably, the ACE2-modified magnetic beads include: amino-Fe3O4 magnetic particles, and ACE2 modified on the surface of the amino-Fe3O4 magnetic particles; preferably, the shape of the amino-Fe3O4 magnetic particles is spherical, and the particle size is 20 to 50 nm.

[0007] Preferably, the preparation method of the aminated Fe3O4 magnetic particles comprises the following steps: adding sodium acetate and polyetherimide to a FeCl3·6H2O solution, and obtaining the aminated Fe3O4 magnetic particles through a hydrothermal reaction; wherein the mass ratio of the FeCl3·6H2O, sodium acetate and polyetherimide is (0.5-2):(2.0-8):(2-4).

[0008] Preferably, the loading condition of ACE2 in the ACE2-modified magnetic beads is saturated adsorption, and the saturated adsorbed ACE2 accounts for 4 to 7 wt % of the total mass of the ACE2-modified magnetic beads.

[0009] Preferably, the preparation process of the ACE2-modified magnetic beads is: mixing the magnetic beads and ACE2 in a solution, and incubating at 4 to 25° C. for 2 to 5 hours.

[0010] Preferably, the gold nanostar has a synaptic structure with a length of 20 to 50 nm and a width of 10 to 20 nm, and an overall particle size of 30 to 100 nm; the Raman reporter molecule is one of 4-mercaptobenzoic acid (4-MBA) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0011] Preferably, the preparation method of the SERS label solution is: adding S protein antibody labeled with Raman reporter molecules to the gold nanostar solution, incubating at 4-25°C for 6-12 hours; collecting the product after centrifugation, redissolving it, adding bovine serum albumin (BSA) solution, and incubating at 4-25°C for 2-6 hours.

[0012] Preferably, in the SERS label solution, the concentration of gold nanostars is 1-3 mg / mL; the concentration of the reporter molecule is 0.1-1 mM; and the concentration of the novel coronavirus S protein antibody is 10-50 μg / mL.

[0013] Preferably, the SERS substrate is a gold nanoparticle array, a gold nanocone array or a SERS solid-state chip.

[0014] Preferably, the biosensor further comprises an elution buffer; the elution buffer is an aqueous HCl solution with a pH of 4 to 5.5.

[0015] In a second aspect, the present invention provides a detection method for non-diagnostic and therapeutic purposes of the aforementioned labeled SERS biosensor for novel coronavirus detection, comprising the following steps:

[0016] (1) collecting a test sample and placing it in a virus lysis solution to release and lyse the virus to obtain solution 1;

[0017] (2) Take ACE2-modified magnetic beads, add them to solution 1, and mix to obtain solution 2;

[0018] (3) Enriching and washing the magnetic beads in solution 2;

[0019] (4) adding the SERS label solution to the enriched magnetic beads and mixing to obtain solution 3;

[0020] (5) Enriching and washing the magnetic beads in solution 3;

[0021] (6) adding elution buffer solution to the enriched magnetic beads and mixing to obtain solution 4;

[0022] (7) Apply 4 drops of the solution on the SERS substrate and collect and analyze the Raman signal.

[0023] Preferably, deionized water is used for cleaning.

[0024] Beneficial effects

[0025] (1) The amino magnetic beads used in the magnetic bead-based labeling SERS biosensor designed by the present invention can quickly and specifically bind to the novel coronavirus S protein within 15 minutes. At the same time, the enrichment ability of the magnetic beads can improve the detection sensitivity and simplify the detection process;

[0026] (2) The SERS tag designed in the present invention can simultaneously label the Raman reporter molecule and the antibody on the gold nanostar in a one-step process. Since the antibody and the Raman reporter molecule are covalently bonded by amide, it is more stable than the conventional electrostatic binding between the gold nanostar and the Raman reporter molecule. In addition, the preparation method of the SERS tag is simple and the performance is excellent and stable.

[0027] (3) The present invention constructs a "sandwich structure" of "SERS tag-S protein-specific magnetic beads". This structure has high specificity. After elution, the Raman signal of the "SERS tag-S protein-ACE2" complex can be collected on the SERS substrate. The concentration information of the S protein is obtained by analyzing the Raman signal of the reporter molecule. It has excellent sensitivity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the sandwich structure of "SERS tag-S protein-specific magnetic beads" described in the present invention;

[0029] Figure 2 The microscopic structure of gold nanostars;

[0030] Figure 3 This is the Raman test result of the new coronavirus S protein, where A is the SERS tag with gold nanostar@antibody-4MBA at a concentration of 10 -6 -10 -12 Figure 2 shows the Raman test spectrum of the SARS-CoV-2 virus S protein solution with a concentration of 10 μg / mL; B shows the Raman test spectrum of the gold nanostar@antibody-DTNB SERS tag with a concentration of 10 μg / mL. -6 -10 -9 Raman test spectrum of SARS-CoV-2 virus S protein solution at 100 μg / mL;

[0031] Figure 4 This is a flow chart of the detection of novel coronavirus using a SERS biosensor based on specific magnetic beads;

[0032] Figure 5 Schematic diagram of Raman detection results of negative samples and positive simulation samples. DETAILED DESCRIPTION

[0033] The present invention is further described below through embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0034] The present invention provides a labeling SERS biosensor for detecting the new coronavirus and a detection method thereof. The SERS biosensor includes: ACE2 (angiotensin-converting enzyme 2) modified magnetic beads, a SERS label solution and a SERS substrate.

[0035] The present invention converts the signal of the new coronavirus into a more stable Raman reporter molecule signal through the SERS method. The entire detection process mainly includes the following steps: virus lysis, magnetic bead specific capture, magnetic enrichment and washing, SERS tag binding, elution, Raman detection, etc. Due to the dual binding of ACE2 modified on the surface of the magnetic beads and the S protein antibody in the SERS tag, the labeling method SERS biosensor has a high specificity for the S protein on the surface of the new coronavirus, avoiding the interference of some viruses that can bind to ACE2 on the test results. The magnetic guidance of the magnetic beads can achieve a fast and simple operation process. The nanometer-level gap between the SERS tag and the SERS substrate can produce a strong electromagnetic field coupling enhancement, which cooperates with the electromagnetic field enhancement of the gold nanostars and the SERS substrate itself, which is beneficial to improve the Raman signal intensity of the reporter molecule and can achieve high-sensitivity detection of the new coronavirus. Combined with the automated high-throughput detection design, a detection throughput of not less than 120 samples / h can be achieved.

[0036] The ACE2-modified magnetic beads include: amino-Fe3O4 magnetic particles, and ACE2 modified on the surface of the amino-Fe3O4 magnetic particles; preferably, the shape of the amino-Fe3O4 magnetic particles is spherical, and the particle size is 20 to 50 nm.

[0037] The preparation method of the aminated Fe3O4 magnetic particles may include the following steps: adding 0.5-2 g of FeCl3·6H2O to 50 mL of ethylene glycol and dissolving it by ultrasonication to obtain solution 1; then, adding 2.0-8 g of sodium acetate and 2-4 g of polyetherimide (PEI) to the solution 1, stirring at 40-70° C. for 20 minutes to obtain solution 2; transferring solution 2 to a 100 mL reactor, and reacting at 220-250° C. for 2-3 hours; washing the resulting black product twice with anhydrous ethanol and deionized water, and drying it in a vacuum oven at 45-60° C. for 8-12 hours to obtain the aminated Fe3O4 magnetic particles.

[0038] The loading condition of the ACE2 is saturated adsorption, and the saturated adsorbed ACE2 accounts for 4 to 7 wt % of the total mass of the ACE2-modified magnetic beads.

[0039] In some optional embodiments, the preparation process of the ACE2-modified magnetic beads can be as follows: add 30 to 50 μL of a 2 to 3 mg / mL magnetic bead solution and 20 to 40 μg of ACE2 to 950 to 970 μL of phosphate buffer solution DPBS, shake and mix for 2 to 6 minutes (e.g., 5 minutes), and then incubate at 4 to 25°C (e.g., 4°C) for 2 to 5 hours (e.g., 5 hours). After incubation, the magnetic beads are enriched under the action of an external magnetic field, and the excess unadsorbed ACE2 is washed away with 200 to 500 μL of phosphate buffer solution DPBS; after washing, the enriched magnetic beads are dispersed in 0.5 to 1.5 mL of DPBS for use.

[0040] Specific magnetic beads are modified with ACE2, which is electrostatically attached to the amino-encapsulated surface of the beads. ACE2-modified magnetic beads can rapidly and specifically bind to the novel coronavirus S protein.

[0041] The structure of the SERS tag includes: Au nanostars, Raman reporter molecules (abbreviated as "reporter molecules") labeled with the novel coronavirus S protein antibody, and the connection method is: Au nanostar@antibody-Raman reporter molecules. Among them, the Au nanostars in the SERS tag solution have a synaptic structure of 20-50nm in length and 10-20nm in width, with an overall particle size of 30-100nm and a concentration of 1-3mg / mL. Its microstructure is shown in Figure 2. Figure 2 The reporter molecule can be one of 4-mercaptobenzoic acid (4-MBA) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), with a concentration of 0.1 to 1 mM. The concentration of the novel coronavirus S protein antibody is 10 to 50 μg / mL.

[0042] The reporter molecule is directly labeled on the antibody of the new coronavirus S protein. Compared with the two-step process of separately labeling the reporter molecule and the antibody on the nanostar, the technical solution disclosed in the present invention only requires one step to modify both the antibody and the reporter molecule on the surface of the nanostar. Compared with the electrostatic binding of the reporter molecule to the nanostar, the reporter molecule directly labeled on the antibody is more stable, and the concentration of the reporter molecule is the initial concentration used during labeling.

[0043] The SERS-labeled reporter molecule performs three primary functions: specifically binding to the S protein, indirectly indicating S protein concentration through the reporter molecule, and assisting in enhancing the Raman reporter signal. Compared to traditional gold nanoparticles, the surface plasmon absorption peak of gold nanostars is red-shifted, making them more conducive to resonance with the 785nm excitation wavelength, resulting in stronger signal enhancement.

[0044] The Raman reporter molecules described in the present invention are 4-MBA or DTNB, which contain carboxyl functional groups, facilitating strong covalent binding to antibodies via amide bonds. Furthermore, 4-MBA and DTNB have fewer distinct characteristic peaks, which do not affect the Raman test results of the sample.

[0045] In an optional embodiment, the preparation method of the SERS label solution can be as follows: add 20 to 50 μL of a 10 to 30 μg / mL 4-MBA or other reporter molecule labeled S protein antibody to 0.5 to 1 mL of a gold nanostar solution (1 to 3 mg / mL) to obtain solution 1; after oscillation mixing, incubate at 4 to 25 ° C for 6 to 12 hours. After the incubation, centrifuge at 10,000 rpm for 1 minute, collect the centrifuged product and disperse it into 1 mL of PBS solution to obtain solution 2; then, add 20 to 50 μL of a 5 to 20 wt% bovine serum albumin (BSA) solution to solution 2 to block the surface of the gold nanostars that are not bound to the antibody to prevent non-specific binding of the gold nanostars to the magnetic beads-ACE2; after oscillation mixing, incubate at 4 to 25 ° C for 2 to 6 hours to obtain solution 3; solution 3 is centrifuged at 10,000 rpm for 1 minute, collect the centrifuged product and disperse it into 1 mL of PBS solution for use. The preparation method of the S protein antibody labeled with a Raman reporter molecule is as follows: adding the S protein antibody to a Raman reporter molecule solution and maintaining the solution at 4 to 25° C. for 4 to 8 hours to obtain the antibody.

[0046] Taking 4-MBA as an example, the following illustrates a method for preparing a reporter molecule-labeled S protein antibody: 10 to 30 μL of 15 μg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is added to 0.5 to 1.5 mL of a 0.1 to 1 mM 4-MBA solution, and the mixture is shaken to mix. After that, 10 to 30 μL of 15 μg / mL N-hydroxysulfosuccinimide (NHS) is added, and the mixture is shaken to mix to obtain Solution 1.

[0047] Among them, EDC can activate the carboxyl group in the 4-MBA molecule to form an O-acylurea intermediate, and the primary amine of the antibody can then react with the intermediate formed by the carboxyl group. However, this intermediate is not stable and is easily hydrolyzed to reform the carboxyl group, so it is necessary to add NHS to stabilize the reaction intermediate to form an NHS activated lipid. The activated lipid is easily replaced by the amino group of the protein under certain conditions to increase the efficiency of the EDC-mediated reaction, so that the antibody is coupled to the carboxyl group on the reporter molecule to form a stable amide bond. Excessive EDC will cause aggregation of the antibody protein and affect the activity of the antibody. The content ratio of EDC to NHS has a fixed range, and the optimal ratio is around 1. In addition, too low EDC and NHS content will affect the efficiency of amide bond formation.

[0048] Next, 10-30 μg of novel coronavirus S protein antibody is added to solution 1, shaken and mixed, and incubated at 4-25°C for 4-8 hours to obtain the reporter molecule-labeled S protein antibody.

[0049] Among them, the new coronavirus S protein antibody and ACE2 have different binding sites for the S protein, so there is no competitive adsorption between the magnetic bead-ACE2-S protein complex and ACE2 during the binding of the SERS tag.

[0050] The SERS active substrate mainly plays the role of enhancing the Raman signal. In some embodiments, the SERS substrate can be a gold nanoparticle array, a gold nanocone array, or a SERS solid-state chip.

[0051] The biosensor further includes an elution buffer solution, which is an aqueous HCl solution with a pH of 4 to 5.5.

[0052] The following combination Figure 4 The non-diagnostic and therapeutic detection method of the magnetic bead-based SERS biosensor for detecting the new coronavirus provided by the present invention is exemplified. The detection process includes the following steps.

[0053] (1) Collect the sample and place it in a virus lysis solution to release and lyse the virus. The virus is lysed in the lysis solution and loses its infectivity. The resulting solution is recorded as solution 1.

[0054] The principle of selecting a viral lysis buffer is to have a small number of Raman peaks and the peak position will not interfere with the Raman signal of the Raman reporter molecule. In some embodiments, a lysis buffer with Tween as the main component can be selected. The formula of the lysis buffer can be: 0.1 M Tris, 2wt% polysorbate-20 (tween-20), 1wt% polyvinylpyrrolidone (PVP), 0.05M ethylenediaminetetraacetic acid (EDTA).

[0055] (2) Take 20-50 μL of ACE2-modified magnetic beads at a concentration of 2-3 mg / mL, add them to solution 1, and shake and mix for 1-2 minutes to allow the S protein on the virus surface to fully bind to the specific magnetic beads to obtain solution 2.

[0056] (3) Enrich the magnetic beads in solution 2 under the action of an external magnetic field, and remove the lysate containing other components of viral lysis; add 200 μL of deionized water, shake and mix for 20-60 seconds, enrich the magnetic beads again using an external magnetic field, and remove the supernatant.

[0057] (4) Add 50-150 μL of SERS tag solution to the enriched magnetic beads, and shake and mix for 1-2 minutes to allow the antibody in the SERS tag to fully mix with the S protein to obtain solution 3.

[0058] (5) The magnetic beads in solution 3 were enriched under the action of an external magnetic field to remove excess unabsorbed SERS tags; after adding 200 μL of deionized water, the mixture was shaken and mixed for 20-60 seconds, and the magnetic beads were enriched again using an external magnetic field, and the supernatant was removed.

[0059] (6) Add 10-20 μL of HCl elution buffer solution with a pH of 4-5.5 to the enriched magnetic beads, and shake and mix for 20-60 seconds to dissociate ACE2 from the magnetic bead surface to obtain solution 4.

[0060] The negatively charged imidazole group of histidine in ACE2 binds to the positively charged amino-labeled magnetic beads through electrostatic interactions. Adding HCl to the elution buffer changes the pH, protonating the imidazole group and ultimately dissociating it from the surface of the amino-labeled magnetic beads.

[0061] (7) Apply 4 drops of the solution on the SERS substrate, dry it at 45°C, collect the Raman signal, and analyze and judge the collected signal.

[0062] The virus sample collected by the present invention will release S protein, N protein, nucleic acid and other components after being lysed by the virus lysis solution. The detection method designed by the present invention mainly detects the S protein. Specifically, the ACE2 modified magnetic beads provided by the present invention are used to achieve the specific capture of the new coronavirus S protein, and the interference of the lysis solution and other components in the lysed virus on the Raman signal of the S protein is removed by enrichment and washing. The SERS tag can form a "sandwich structure" of "SERS tag-S protein-specific magnetic beads" with the specific magnetic beads by binding to the S protein, such as Figure 1 The structure above, through the elution process, disrupts the electrostatic binding between ACE2 and the magnetic beads, causing the "SERS tag-S protein-ACE2" complex to dissociate from the bead surface. By collecting the Raman signal of the reporter molecule in the SERS tag, the concentration of the S protein and the new coronavirus is indirectly reflected.

[0063] Among them, the main function of the eluent is to destroy the electrostatic binding between the histidine in ACE2 and the magnetic beads, and will not affect the binding of the S protein with ACE2 and the antibody in the SERS tag. In addition, the antibody and the gold nanostar are covalently bound by an amide bond, so elution will not affect the binding of the SERS tag-S protein part.

[0064] The amount of the eluent needs to be controlled within the range of 10 to 20 μL. If the amount of eluent is too small, the volume of the solution in which the complex is dispersed is too small and cannot be fully dispersed, resulting in reduced elution efficiency. If the amount of eluent is too large, the enrichment effect will be weakened because not all the eluent can be transferred to the SERS substrate, resulting in a decrease in the SERS signal. The pH of the eluent should be controlled within the range of 4 to 5.5. If the pH of the eluent is too low, it will cause changes in the protein structure and affect the binding of the protein to ACE2 and the antibody. If the pH value is too high, the protonation reaction of the imidazole group in ACE2 cannot be completed, affecting the elution efficiency.

[0065] The following examples are further cited to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are only an example within a suitable range, that is, those skilled in the art can make a selection within a suitable range based on the description herein, and are not limited to the specific values ​​​​exemplified below. It is worth noting that in order to protect the safety of experimental operators, during the implementation of the present invention, a pseudovirus of a determined concentration is mixed with a negative nasal swab solution to simulate a positive nasal swab solution for detection.

[0066] Example 1

[0067] ACE2-modified magnetic beads were prepared by adding 50 μL of a 2.5 mg / mL magnetic bead solution and 30 μg of ACE2 to 950 μL of phosphate-buffered saline (DPBS). The mixture was shaken for 5 minutes and incubated at 4°C for 2 hours. After incubation, the beads were concentrated in an external magnetic field and washed with 400 μL of phosphate-buffered saline (DPBS) to remove excess unadsorbed ACE2. After washing, the concentrated beads were dispersed in 1 mL of DPBS for later use.

[0068] The SERS tag preparation process is as follows: to 1 mL of a 2 mg / mL gold nanostar solution, 50 μL of 30 μg / mL 4-MBA-labeled S protein antibody was added to obtain solution 1. After vortexing, the solution was incubated at 4°C for 12 hours. After centrifugation at 10,000 rpm for 1 minute, the product was collected and dispersed in 1 mL of PBS to obtain solution 2. Next, 50 μL of a 10 wt% bovine serum albumin (BSA) solution was added to solution 2, vortexed, and incubated at 4°C for 2 hours to obtain solution 3. Solution 3 was centrifuged at 10,000 rpm for 1 minute, the product was collected and dispersed in 1 mL of PBS for later use. The resulting SERS tag solution contained 2 mg / mL gold nanostar particles, 0.1 mM reporter molecule, and 10 μg / mL of S protein antibody.

[0069] The SERS substrate is a gold nanoparticle array.

[0070] The process of using specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection:

[0071] Step 1: Take a nasal swab from a healthy individual and place it in 250 μL of lysis buffer. Then, add 200 μL of the novel coronavirus pseudovirus, and the virus will be lysed in the lysis buffer. The resulting solution is recorded as Solution 1.

[0072] Step 2: Take 50 μL of 2.5 mg / mL ACE2-modified magnetic beads, add them to Solution 1, and oscillate for 90 seconds to allow the S protein on the virus surface to fully bind to the specific magnetic beads to obtain Solution 2.

[0073] Step 3: Enrich the magnetic beads in solution 2 under the action of an external magnetic field, and remove the lysate containing other components of viral lysis; add 200 μL of deionized water, shake and mix for 40 seconds, enrich the magnetic beads again using an external magnetic field, and remove the supernatant.

[0074] Step 4: Add 100 μL of SERS tag solution to the enriched magnetic beads, and oscillate for 90 seconds to allow the antibody in the SERS tag to be fully mixed with the S protein to obtain solution 3.

[0075] Step 5: Enrich the magnetic beads in solution 3 under the action of an external magnetic field to remove excess unabsorbed SERS tags; add 200 μL of deionized water, shake and mix for 40 seconds, enrich the magnetic beads again using an external magnetic field, and remove the supernatant.

[0076] Step 6: Add 10 μL of HCl elution solution with a pH of 4.5 to the enriched magnetic beads, shake and mix for 40 seconds to dissociate ACE2 from the magnetic bead surface to obtain solution 4.

[0077] Step 7: Apply 4 drops of the solution on the SERS substrate, dry it at 45°C, and then collect and analyze the Raman signal.

[0078] The above experimental process can generate a stable 4-MBA Raman signal, verifying the feasibility of the detection process. In addition, the process can detect pseudoviruses with a virus titer of 1000 copies / mL.

[0079] Figure 5 Schematic diagram of Raman analysis results for negative and positive simulated samples. As can be seen from the figure, due to nonspecific adsorption between the SERS tag and the magnetic beads, the negative sample also has a 4-MBA signal. However, the 4-MBA signal in the positive sample is much higher than that in the negative sample, allowing the positive and negative samples to be distinguished based on the distinct signal intensity difference.

[0080] Example 2

[0081] The process of using the specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection in this Example 2 is similar to that in Example 1, with the only difference being that: in step 6, 10 μL of HCl elution solution with a pH of 4 is added to the enriched magnetic beads, and the mixture is shaken and mixed for 40 seconds to dissociate ACE2 from the magnetic bead surface to obtain Solution 4.

[0082] Following the above experimental process, a stable 4-MBA Raman signal was still obtained using an HCl elution solution with a pH of 4, verifying the excellent elution efficiency of this eluent. Furthermore, this process can also detect pseudoviruses with a viral titer of 1000 copies / mL.

[0083] Example 3

[0084] The process of using the specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection in this Example 3 is similar to that in Example 1, with the only difference being that: in step 6, 10 μL of HCl elution solution with a pH of 5.5 is added to the enriched magnetic beads, and the mixture is shaken and mixed for 40 seconds to dissociate ACE2 from the magnetic bead surface to obtain solution 4.

[0085] Following the above experimental process, a stable 4-MBA Raman signal was still obtained using an elution solution of HCl at pH 5.5, validating the excellent elution efficiency of this eluent. Furthermore, this process can also detect pseudoviruses with a titer of 1000 copies / mL.

[0086] Example 4

[0087] The process of using the specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection in this Example 4 is similar to that in Example 1, with the only difference being that: in step 6, 20 μL of HCl elution solution with a pH of 4.5 is added to the enriched magnetic beads, and the mixture is shaken and mixed for 40 seconds to dissociate ACE2 from the magnetic bead surface to obtain Solution 4.

[0088] Following the above experimental process, a stable 4-MBA Raman signal was still obtained using 20 μL of HCl elution solution at pH 4.5, verifying that a 20 μL eluent volume can meet the detection requirements. Furthermore, this process can also detect pseudoviruses with a viral titer of 1000 copies / mL.

[0089] Example 5

[0090] The process of using the SERS biosensor based on the specific magnetic bead labeling method for the detection of the new coronavirus in this Example 5 is similar to that in Example 1, with the only difference being that the SERS substrate in this Example is a gold nanocone array.

[0091] After the above experimental process, using the gold nanocone array as the SERS substrate, a stable 4-MBA Raman signal can still be obtained, and the detection of pseudoviruses with a virus titer of 1000 copies / mL can also be achieved.

[0092] Example 6

[0093] The process of using the specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection in Example 6 is similar to that in Example 1, with the only difference being that the SERS substrate in this example is a SERS solid-state chip.

[0094] After the above experimental process, a stable 4-MBA Raman signal can still be obtained using the SERS solid-state chip as the substrate, and the detection of pseudoviruses with a virus titer of 1000 copies / mL can also be achieved.

[0095] Example 7

[0096] The process of using the specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection in this Example 7 is similar to that in Example 1, with the only difference being that the Raman reporter molecule used in the SERS tag in this Example is DTNB.

[0097] Through the above experimental process, using DTNB as a Raman reporter molecule, it is possible to detect pseudoviruses with a virus titer of 10,000 copies / mL.

[0098] Figure 3This is the Raman test result of the new coronavirus S protein, where A is the SERS tag with gold nanostar@antibody-4MBA at a concentration of 10 -6 -10 -12 g / mL SARS-CoV-2 virus S protein solution Raman test spectrum; B is the gold nanostar @ antibody-DTNB as SERS label, the concentration of 10 -6 -10 -9 Figure 2 shows the Raman spectrum of a SARS-CoV-2 S protein solution at 10000 copies / mL. As can be seen from the figure, gold nanostars are not as effective as 4-MBA in enhancing DTNB, but can still detect the novel coronavirus at a titer of 10,000 copies / mL.

[0099] Comparative Example 1

[0100] The ACE2-modified magnetic beads were prepared by adding 50 μL of a 2.5 mg / mL magnetic bead solution and 30 μg of ACE2 to 950 μL of phosphate-buffered saline (DPBS). The mixture was shaken for 5 minutes and incubated at 4°C for 2 hours. After incubation, the beads were concentrated in an external magnetic field and washed with 400 μL of phosphate-buffered saline (DPBS) to remove excess unadsorbed ACE2. After washing, the concentrated beads were dispersed in 1 mL of DPBS for later use.

[0101] The SERS tag preparation process is as follows: To 1 mL of a 2 mg / mL gold nanoparticle solution, 50 μL of 30 μg / mL 4-MBA-labeled S protein antibody was added to obtain Solution 1. After vortexing, the solution was incubated at 4°C for 12 hours. After centrifugation at 10,000 rpm for 1 minute, the product was collected and dispersed in 1 mL of PBS to obtain Solution 2. Next, 50 μL of a 10 wt% bovine serum albumin (BSA) solution was added to Solution 2. After vortexing, the product was incubated at 4°C for 2 hours to obtain Solution 3. Solution 3 was centrifuged at 10,000 rpm for 1 minute, the product was collected and dispersed in 1 mL of PBS for later use. The gold nanoparticles are spherical with a particle size of 30 to 50 nm. The resulting SERS tag solution contains 2 mg / mL of gold nanoparticles, 0.1 mM of the reporter molecule, and 10 μg / mL of the novel coronavirus S protein antibody.

[0102] The SERS substrate is a gold nanoparticle array.

[0103] The process of using specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection:

[0104] Step 1: Take a nasal swab from a healthy individual and place it in 250 μL of lysis buffer. Then, add 200 μL of the novel coronavirus pseudovirus, and the virus will be lysed in the lysis buffer. The resulting solution is recorded as Solution 1.

[0105] Step 2: Take 50 μL of 2.5 mg / mL ACE2-modified magnetic beads, add them to Solution 1, and oscillate for 90 seconds to allow the S protein on the virus surface to fully bind to the specific magnetic beads to obtain Solution 2.

[0106] Step 3: Enrich the magnetic beads in solution 2 under the action of an external magnetic field, and remove the lysate containing other components of viral lysis; add 200 μL of deionized water, shake and mix for 40 seconds, enrich the magnetic beads again using an external magnetic field, and remove the supernatant.

[0107] Step 4: Add 100 μL of SERS tag solution to the enriched magnetic beads, and oscillate for 90 seconds to allow the antibody in the SERS tag to be fully mixed with the S protein to obtain solution 3.

[0108] Step 5: Enrich the magnetic beads in solution 3 under the action of an external magnetic field to remove excess unabsorbed SERS tags; add 200 μL of deionized water, shake and mix for 40 seconds, enrich the magnetic beads again using an external magnetic field, and remove the supernatant.

[0109] Step 6: Add 10 μL of HCl elution solution with a pH of 4.5 to the enriched magnetic beads, shake and mix for 40 seconds to dissociate ACE2 from the magnetic bead surface to obtain solution 4.

[0110] Step 7: Apply 4 drops of the solution on the SERS substrate, dry it at 45°C, and then collect and analyze the Raman signal.

[0111] Due to the poor enhancement effect of gold nanoparticles on the dye molecule 4-MBA, the new coronavirus detection process using the SERS label prepared by the same method has poor detection effect on the virus and can only achieve a titer of 10 5 Pseudovirus detection at 10 copies / mL.

[0112] Comparative Example 2

[0113] The process of using the specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection in Comparative Example 2 is similar to that in Example 1, with the only difference being that: in step 6, 10 μL of HCl elution solution with a pH of 6 is added to the enriched magnetic beads, and the mixture is shaken and mixed for 40 seconds to dissociate ACE2 from the magnetic bead surface to obtain Solution 4.

[0114] After the above experimental process, the protonation process of the histidine imidazole group in ACE2 could not occur using an HCl elution solution with a pH of 6, resulting in a significant reduction in the elution efficiency of the elution step. It was impossible to obtain a strong and stable 4-MBA Raman signal in the eluate, and it was impossible to detect the new coronavirus pseudovirus.

[0115] Comparative Example 3

[0116] The process of using the specific magnetic bead-based labeling SERS biosensor for novel coronavirus detection in Comparative Example 3 is similar to that in Example 1, with the only difference being that: in step 6, 10 μL of HCl elution solution with a pH of 3 is added to the enriched magnetic beads, and the mixture is shaken and mixed for 40 seconds to dissociate ACE2 from the magnetic bead surface to obtain Solution 4.

[0117] Following the above experimental process, the protein structure was disrupted at an excessively low pH, resulting in a Raman signal that could not be attributed to the S protein. During the machine learning phase, this peak could not be identified as positive, and therefore the HCl elution solution based on this pH value could not be used in this process.

[0118] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A labeling-based SERS biosensor for detecting novel coronavirus, characterized in that: include: Angiotensin-converting enzyme 2 (ACE2)-modified magnetic beads, SERS labeling solution, elution buffer, and SERS substrate; The ACE2-modified magnetic beads include: aminated Fe3O4 magnetic particles and ACE2 modified on the surface of the aminated Fe3O4 magnetic particles; the aminated Fe3O4 magnetic particles are spherical in shape and have a particle size of 20 to 50 nm; The structure of the SERS tag in the SERS tag solution includes, from the inside out, Au nanostars and a novel coronavirus S protein antibody labeled with a Raman reporter molecule modified on the surface of the Au nanostars; The elution buffer is an aqueous solution of HCl at pH 4 to 5.5; The non-diagnostic and therapeutic detection method of the labeled SERS biosensor for novel coronavirus detection comprises the following steps: (1) Collect the test sample and place it in a virus lysis solution to release and lyse the virus to obtain solution 1; (2) Take ACE2-modified magnetic beads, add them to solution 1, and mix to obtain solution 2; (3) Enriching and washing the magnetic beads in solution 2; (4) Adding the SERS label solution to the enriched magnetic beads and mixing to obtain solution 3; (5) Enriching and washing the magnetic beads in solution 3; (6) Add elution buffer solution to the enriched magnetic beads and mix to obtain solution 4; (7) Apply 4 drops of the solution on the SERS substrate to collect and analyze the Raman signal.

2. The SERS biosensor for novel coronavirus detection according to claim 1, characterized in that: The preparation method of the amination Fe3O4 magnetic particles comprises the following steps: adding sodium acetate and polyetherimide to a FeCl3·6H2O solution, and performing a hydrothermal reaction to obtain the amination Fe3O4 magnetic particles; The mass ratio of FeCl3·6H2O, sodium acetate and polyetherimide is (0.5-2):(2.0-8):(2-4).

3. The SERS biosensor for detecting novel coronavirus according to claim 1, wherein: The loading condition of ACE2 in the ACE2-modified magnetic beads is saturated adsorption, and the saturated adsorbed ACE2 accounts for 4 to 7 wt % of the total mass of the ACE2-modified magnetic beads.

4. The SERS biosensor for novel coronavirus detection according to claim 1, characterized in that: The preparation process of the ACE2-modified magnetic beads is as follows: mixing the magnetic beads and ACE2 in a solution and incubating at 4-25° C. for 2-5 hours.

5. The SERS biosensor for detecting novel coronavirus according to claim 1, wherein: The Au nanostar has a synaptic structure with a length of 20 to 50 nm and a width of 10 to 20 nm, and an overall particle size of 30 to 100 nm; the Raman reporter molecule is one of 4-mercaptobenzoic acid 4-MBA and 5,5'-dithiobis(2-nitrobenzoic acid) DTNB.

6. The labeling SERS biosensor for novel coronavirus detection according to claim 1, characterized in that: The preparation method of the SERS label solution is as follows: adding S protein antibody labeled with Raman reporter molecules to Au nanostar solution, incubating at 4-25°C for 6-12 hours; collecting the product after centrifugation, redissolving it, adding bovine serum albumin (BSA) solution, and incubating at 4-25°C for 2-6 hours to obtain the SERS label solution.

7. The SERS biosensor for detecting novel coronavirus according to claim 1, wherein: In the SERS label solution, the concentration of Au nanostars is 1-3 mg / mL, the concentration of the reporter molecule is 0.1-1 mM, and the concentration of the novel coronavirus S protein antibody is 10-50 μg / mL.

8. The labeling SERS biosensor for novel coronavirus detection according to claim 1, characterized in that: The SERS substrate is a gold nanoparticle array, a gold nanocone array or a SERS solid-state chip.

9. The labeling SERS biosensor for novel coronavirus detection according to claim 1, characterized in that: In step (3) and step (5) of the detection method, deionized water is used for cleaning.

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