CN111665356A - SERS-based virus detection method and device - Google Patents
SERS-based virus detection method and device Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a virus detection method and a device based on SERS (surface enhanced Raman scattering). A sol-gel substrate containing SERS active gold or silver is prepared and fixed in a channel; then injecting a predetermined concentration of antibody solution into the channel, functionalizing the gold or silver to mask the raman signal; and finally, obtaining a sample to be detected, injecting the sample into the channel, standing or incubating for 1-5 minutes, and detecting by using a Raman analyzer to obtain a spectrum to judge whether viruses exist. The invention not only can endow necessary specificity and sensitivity to the detection method, but also can effectively shorten the detection time, can quickly detect and diagnose the COVD-19 virus within 15 minutes, has relatively low cost, simultaneously reduces the false negative and false positive errors to the minimum, can help to cut off the infection source and reduce the quick spread of the virus.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a virus detection method and device based on SERS.
Background
Mandatory isolation measures are effective, but how to discriminate against the source of infection is a key to epidemic resistance. It is therefore necessary to detect samples that may carry viruses. The existing detection method judges whether a sample to be detected contains the virus or not by detecting a nucleic acid sequence, but the detection result is inaccurate and the time consumption is long due to low specificity and sensitivity, and further spreading of epidemic situation cannot be effectively controlled.
Disclosure of Invention
The technical problem to be solved by the invention is how to improve the specificity and sensitivity of the virus detection method, improve the detection accuracy, effectively shorten the detection time, quickly identify and block the infection source, and effectively control the further spread of the epidemic situation, but it needs to be stated that the detection method is not the purpose of diagnosis and treatment.
Therefore, the invention provides a virus detection method based on SERS, which comprises the following steps:
preparing a sol-gel substrate containing SERS active gold or silver, and fixing the sol-gel substrate in a channel;
injecting a predetermined concentration of antibody solution into the channel, functionalizing the gold or silver to mask raman signals;
and obtaining a sample to be detected, injecting the sample into a channel, standing or incubating for 1-5 minutes, and detecting by using a Raman analyzer to obtain a spectrum to judge whether viruses exist.
Further, the SERS-based virus detection method, wherein the step of functionalizing the gold or silver specifically comprises:
sucking a predetermined concentration of antibody solution sufficient to cover the surface of gold or silver metal particles into the glass capillary or channel, incubating at 22-30 ℃ for 0.5-24h, and washing with water or buffer solution to complete the gold or silver functionalization.
Further, the method for detecting the virus based on SERS comprises the following steps:
equal volume of 0.1-0.5mol/LHAuCl4.3H2Mixing a solution of O in 70% HNO3 with pure TMOS for 1-5 minutes, then pumping the liquid into the channel, sealing and gelling and curing at room temperature for 20-30 hours;
after the sol-gel is formed, the doped gold ions are reduced by 10-100 mu L of sodium borohydride with the concentration of 0.01-0.03 mol/L; and then, washing away excessive borohydride by using ultrapure water to obtain the sol-gel substrate containing SERS active gold.
Further, the method for detecting the virus based on SERS comprises the following steps of:
equal volumes of 0.5-2mol/L AgNO3, 28% ammonium hydroxide, and MeOH were mixed together, and then equal volumes of this solution and 5: 1: 1 MTMS: TMOS: ODS mixing, then pumping the liquid out of the channel, sealing and gelling and curing at room temperature for 20-30 hours;
after the sol-gel is formed, the doped silver ions are reduced by 10-100 mu L of dilute sodium borohydride with the concentration of 0.01-0.03mol/L, and then the excessive borohydride is washed away by ultrapure water, so that the sol-gel substrate containing SERS active silver is obtained.
Further, the SERS-based virus detection method includes: biological fluids, aerosol concentrates and swab solutions of the sample to be tested.
The invention also provides a virus detection device based on SERS, which comprises:
the sampler is used for collecting samples, and the sample loading port is connected with the output end of the sampler; the output end of the sample loading port is connected with a channel for loading the sol-gel substrate; the sample loading port is also provided with a sample loading syringe for injecting an antibody solution with a predetermined concentration into the channel, and functionalizing the gold or silver to shield a Raman signal; the passageway sets up in detecting the storehouse, it still is provided with the detection mouth that is used for inserting raman detector and detects to detect the storehouse.
The invention has the beneficial effects that: the invention provides a virus detection method and a device based on SERS, wherein the method comprises the steps of preparing a sol-gel substrate containing SERS active gold or silver and fixing the sol-gel substrate in a channel; then injecting a predetermined concentration of antibody solution into the channel, functionalizing the gold or silver to mask the raman signal; and finally, obtaining a sample to be detected, injecting the sample into a channel, standing or incubating for 1-5 minutes, detecting by using a Raman analyzer to obtain a spectrum to judge whether viruses exist, so that the necessary specificity and sensitivity of the detection method can be endowed, the detection time can be effectively shortened, the COVD-19 viruses can be quickly detected and diagnosed within 15 minutes, the cost is relatively low, meanwhile, the false negative and false positive errors are minimized, the infection source can be cut off, and the quick spread of the viruses is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without any creative work.
FIG. 1 is a flow chart of a virus detection method based on SERS according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the structure of an antibody;
FIG. 3 is a schematic representation of the specificity of binding of an antibody used in the present invention to a virus;
FIG. 4 is a schematic diagram of the principle of SERS-active gold or silver-containing sol gel substrate before and after virus recognition after antibody functionalization;
FIG. 5 is a surface enhanced Raman spectrum of Bacillus cereus, Escherichia coli and Staphylococcus aureus, with the conditions: 10to the 4thSpores or cells/mL water, silver-doped sol, 80mW of 785nm excitation, and the collection time of each time is 40 seconds;
FIG. 6 is a surface enhanced Raman spectrum of Listeria monocytogenes on gold and silver SERS active sol-gel under the action of 80mW 1min and 785nm laser;
FIG. 7 is a graph of gold pairs functionalized with peptides 105Surface enhanced raman spectra of cfu/mL (300 cells) of listeria monocytogenes (top) and salmonella typhimurium (bottom);
FIG. 8 is a graph of gold pairs functionalized with antibodies 105Surface enhanced Raman spectra of cfu/mL (300 cells) of Listeria monocytogenes (top) and Salmonella typhimurium (bottom);
FIG. 9 is a schematic structural diagram of a SERS-based virus detection apparatus according to an embodiment of the present invention;
FIG. 10 is a partial schematic view of A in FIG. 9;
FIG. 11 is a sectional view in the plane of the detection chamber B-B of FIG. 10, partially schematically showing the structure thereof.
Reference numerals
10. A sampler; 20. a sample loading port; 21. a sample loading syringe; 30. a detection bin; 31. a channel; 32. a detection port; 40. a Raman detector.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to better understand the detection steps of the method of the invention and the corresponding detection mechanism, some concepts are first explained and illustrated below.
Raman Spectroscopy (RS), similar to infrared spectroscopy, is used to analyze the band wavelength distribution composition of sample specific molecular vibrations, identified by frequency, and quantified by peak intensity. Specifically, a laser is focused on a sample, inelastic scattered radiation (Raman) is collected and then directed into a spectrometer, which provides wavelength dispersion, and a detector converts photon energy into electrical signal intensity. During the past decade, significant technological advances in stabilized diode lasers, notch filters and high quantum efficiency detectors have made raman spectrometers the standard equipment for analytical laboratories, as have portable systems. In addition, the detection method does not need to extract or prepare a sample, and chemical analysis can be carried out only by aligning the optical fiber probe to the sample. Although RS can provide detailed highly specific molecular information that is listed as a potential method for detecting biological species, the sensitivity of RS is limited to only around 0.1% due to the extremely low conversion rate of incident radiation to inelastically scattered radiation.
Fortunately, the disadvantage of low sensitivity of Raman spectroscopy is overcome thanks to surface-enhanced Raman spectroscopy (SERS). This is because in the excitation area on the surface or in the sol of some specially prepared metal benign conductors, the enhancement of the electromagnetic field on the surface or near the surface of the sample leads to the great enhancement of the Raman scattering signal of the adsorbed molecules compared with the ordinary Raman scattering (NRS) signal, so that the structural information which is not easily obtained by the conventional Raman spectroscopy can be obtained. The invention is based on the high sensitivity provided by SERS, and can provide an antibody aiming at the virus by combination, so as to detect the sample to be detected.
Coronaviruses belong to four genera: alpha coronavirus, beta coronavirus, gamma coronavirus, and coronavirus. The coronavirus SARS-CoV-2 of COVID-19 belongs to the genus Betacononavirus, which originates from Bat. Coronaviruses can infect mammals, are pathogens of zoonosis, and can cause severe respiratory diseases in humans. Other viruses in this family are the SARS coronavirus and MERS coronavirus. SARS-CoV-2 has about 79% sequence identity to SARS-CoV and about 50% sequence identity to MERS-CoV. Furthermore, homology modeling shows that SARS-CoV-2 has a similar receptor binding domain structure to SARS-CoV, and there has been relevant evidence to date that SARS-CoV-2 is infected by the ACE2 receptor in humans.
Among them, ACE2 is also called achh, and is called angiotensin converting enzyme 2. The protein coded by the gene belongs to an angiotensin converting enzyme family of dipeptidyl carboxyl dipeptidase and has considerable homology with human angiotensin converting enzyme 1. This secreted protein catalyzes the cleavage of angiotensin I to angiotensin 1-9 and angiotensin II to the vasodilator angiotensin 1-7. ACE2 has strong affinity for Ang type II type 1 and type 2 receptors, and regulates blood pressure, fluid balance, inflammation, cell proliferation, hypertrophy, and fibrosis. Meanwhile, the specific expression of organs and cells of the gene suggests that the gene may play a role in regulating cardiovascular and renal functions and fertility. In addition, the gene encoded protein is a functional receptor for SARS and HCoV-NL63 human coronavirus S glycoprotein.
Referring to fig. 1, a flowchart of a SERS-based virus detection method disclosed in this embodiment is shown, which in one embodiment includes the steps of:
step S100: a sol-gel substrate containing SERS-active gold or silver is prepared and immobilized in a channel.
In specific implementations, silver or gold metal particles are encapsulated in a porous glass structure, the immobilized glass sol-gel (attached to the inner walls of the channel) is optically transparent, porous, comprising gold or silver metal nanoparticles dispersed therein, the nanoparticles being fractal aggregate clusters having an average size between 10 and 200 nm; and the liquid sample can flow in the sol-gel substrate by means of external force, so as to carry out detection and identification, wherein the external force is the force pushed or flowed by a connected sample loading syringe type device or a dropper.
Step S200: a solution of a predetermined concentration of antibody is injected into the channel, functionalizing the gold or silver to mask the raman signal.
The present invention functionalizes metal nanoparticles in sol-gels using antibodies (molecular recognition elements, MREs). Referring to fig. 2, an antibody, also known as immunoglobulin (Ig), is a large Y-shaped protein produced primarily by plasma cells and used by the immune system to neutralize pathogens, such as pathogenic bacteria and viruses. The Fc portion of the antibody will bind electrostatically to the SERS substrate and the antigen binding site without the antibody will be exposed, allowing it to bind to and act as a capture for the virus in the surrounding solution. Therefore, the antibody can be functionalized on the surface through static electricity or directional conjugate combination with metal particles and used for capturing viruses, and SERS active gold or silver exposed in the capturing process is exposed, so that a spectrum signal appears at a specific position. The prepared sol-gel substrate can be stored in a sealed manner for 60 days, and the application range of the detection substrate is widened.
Step S300: obtaining a sample to be detected, injecting the sample into a channel, standing or incubating for 1-5 minutes, and detecting by a Raman analyzer to obtain a spectrum to judge whether viruses exist or not; the chip is mounted on a raman instrument, then laser irradiates the SERS-active capillary or channel at a predetermined location, and the raman probe optics collects the scattered radiation back into the raman instrument for analysis. The channel is placed in a Raman analyzer for detection to obtain a spectrum, and a virus RNA nucleotide biological signal can be observed to indicate that the virus is captured.
Detection sensitivity is improved by optimizing antibody function, target capture and SERS reaction. The schematic representation of figure 3 is presented graphically, and to improve the capture of antigen (target), the optimal antibody concentration is determined experimentally to ensure that the target species has sufficient binding sites. By adjusting the silver or gold concentration, the optimal concentration value is found to provide a sufficient SERS response. The binding scheme of sol-gel substrate, antibody, virus is shown in FIG. 4.
The detection method of the invention not only can endow necessary specificity and sensitivity to the detection method, but also can effectively shorten the detection time, can help to cut off the infection source, reduce the rapid spread of the virus and effectively control the frequent epidemic situation. However, it should be noted that this determination method is not a method for diagnosing and treating diseases, not only because the detection method provided by the present invention is to obtain a biological fluid separated from a living organism for detection, but also it cannot be directly determined whether the person to be detected who provides a sample has the new coronavirus pneumonia through the detection result, and the detection result is only an intermediate result, for example, the detection result is virus-containing, and it may be that the person to be detected is a carrier of the virus.
In one embodiment, the specific step of functionalizing said gold or silver comprises:
sucking a predetermined concentration of antibody solution sufficient to cover the surface of gold or silver metal particles into the glass capillary or channel, incubating at 22-30 ℃ for 0.5-24h, and washing with water or buffer solution to complete the gold or silver functionalization.
Wherein the concentration of the antibody solution is sufficient to cover the surface of the metal particles, the antibody-functionalized SERS-active substrate is stable for several months and can be stored until an assay can be performed. Note that successful functionalization will be confirmed by the unique spectral peak at 630cm-1 that occurs with cys-Metal interaction. Complete surface coverage can be tested by running a test chemical analyte, which if covered by an antibody, does not produce a SERS response.
The antibody is modified at its ends with a thiol-containing linker group, which will coordinate the antibody with the antibody-active metal nanoparticles incorporated into the sol-gel matrix (gold or silver). This thiol bond anchors the antibody antenna (antennae) to the gold or silver nanoparticles of the antibody, thereby orienting and orienting the antibody perpendicular to the metal surface, so that complete monolayer surface coverage can be obtained.
Successful thiol-linked antibody functionalization will be 630cm in SERS spectra-1A distinct peak appears in and near the vicinity. Functionalization of gold or silver metal surfaces can also be successfully achieved without terminal thiol modification, and the antibodies are non-covalently bound to the metal surface without the additional modifications described above. The successful functionalization here is from 1200--1The peaks in between are indicative that these peaks are due to antibody protein patterns and are weakly attributable to SERS zero backgroundThe sign intensity gain contributes.
It should be noted that binding events with specific pathogens (bacteria or viruses) are expected to produce a biosignal imprint from unit-forming colonies consisting of 650 to 750cm-1The nucleobase characteristics of the region. This characteristic was observed in both gram-positive and gram-negative bacteria, with an adenine peak at 675cm-1Nearby. Data for more than 20 species and strains have been obtained to confirm this and the results observed in the literature. SERS of DNA and RNA based viruses has been reported and it is clear that similar nucleotypic characteristics are observed in this spectral region. In the present invention, the spectral region allows (simultaneously) confirmation of successful antibody binding to the metal site, as well as specific binding (capture) events of the pathogen to the functionalized antibody on the metal site. The bound pathogen was in close proximity and interacted spatially with the SERS substrate, thereby reaching 650--1The region produces an enhanced biometric signal.
In addition, the SERS signal can be enhanced by an order of magnitude by adding a SERS enhancing agent in step S200. The SERS enhancer may be a reagent such as a SERS-active colloid in the range of 50-250nm, or a mild DNA/RNA extraction chemical such as chloroform, which increases the concentration of nucleotide molecules on the surface of the pathogen.
Referring to fig. 5 and 6, the results of bacterial SERS obtained are shown. Wherein each pathogen produces a unique set of peaks. Experiments prove that biological signals of spores (dipyridyl formic acid) and bacteria (adenine and adenosine) can be obtained, and similarly, biological signals of viruses can also be obtained to obtain corresponding specific surface enhanced Raman spectrograms.
Referring to fig. 7 and 8, two graphs showing SERS MRE measurements of bacteria are shown, measuring SERS of listeria monocytogenes and salmonella typhimurium to minimum concentrations. Gold-doped and silver-doped sol gels functionalized with antibodies and peptides, which can be at 105Excellent surface enhanced Raman spectra were obtained for both pathogens cfu/mL, the concentration indicating detection in the 10. mu.L sample volume measuredAbout 103Individual cells (about 300 cells in the laser focus), SERS sensitivity using antibody and silver decreased by 2-3 orders of magnitude.
In the above scheme, gold or silver is used for preparing the SERS active material, because the following conditions need to be satisfied to realize SERS detection:
1) the material prepared is particles much smaller than the incident wavelength of the laser (Rayleigh regime, surface defects of similar size can also be used);
2) the material prepared has suitable optical properties to couple light (extinction);
3) the free electrons available upon excitation are limited by the particle size forming surface modes or plasmons;
4) the molecules have matched optical properties (absorption), coupling to plasmonic fields.
These very specific conditions therefore limit SERS to metals Ag, Au and Cu with diameters of 5 to 200 nm. Meanwhile, in order to provide a widely used SERS-active material, a wet chemical method in which metal particles of silver or gold are encapsulated in a porous glass structure has been developed. The SERS-active sol-gel substrate consists of a two-component system of a silane oxide precursor (e.g., tetramethyl orthosilicate (TMOS), methyltrimethoxysilane (MTMS), and/or Octadecyltrimethoxysilane (ODS)) and a noble metal salt (e.g., a precursor of gold chloride or silver nitrate).
In one embodiment, the preparation of the SERS-active gold-containing sol-gel substrate comprises the steps of:
adding equal volume of 0.1-0.5mol/L HAuCl4.3H2O at 70% HNO3Mixing the solution with pure TMOS for 1-5 min, pumping the liquid into the channel, sealing, and gelatinizing and curing at room temperature for 20-30 hr; after the sol-gel is formed, the doped gold ions are reduced by 10-100 mu L of sodium borohydride with the concentration of 0.01-0.03 mol/L; and then, washing away excessive borohydride by using ultrapure water to obtain the sol-gel substrate containing SERS active gold.
Wherein, equal volume of HAuCl4.3H2The concentration of O may be 0.1mol/L, 0.2mol/L, 0.5mol/L, whereinPreferably 0.25 mol/L; the sol-gel substrate is sealed and gelated at room temperature, and the curing time can be 20h, 25h and 30 h; the concentration of the sodium borohydride solution for reducing the doped gold ions is 0.01mol/L, 0.02mol/L and 0.03mol/L, wherein the concentration is preferably 0.0132 mol/L. When washing with ultrapure water, it is preferable to wash twice to remove the excess borohydride therefrom and to increase the aggregation of the nanogold particles, which may have an effect on the particle size.
In the specific implementation process, the preferable scheme is as follows:
an equal volume of 0.25mol/L HAuCl4.3H2O at 70% HNO3The solution in (a) was mixed with pure TMOS for 2 minutes, then the liquid was pumped out into the channel, sealed and gelled and cured at 23 ℃ for 24 hours; after the sol-gel is formed, the doped gold ions are reduced by 60 mu L of sodium borohydride with the concentration of 0.0132 mol/L; and then, washing away excessive borohydride by using ultrapure water to obtain the sol-gel substrate containing SERS active gold.
In one embodiment, the preparation of the SERS-active silver-containing sol-gel substrate comprises the steps of:
equal volume of 0.5-2mol/L AgNO328% ammonium hydroxide and MeOH were mixed together, then equal volumes of this solution and 5: 1: 1 MTMS: TMOS: ODS mixing, then pumping the liquid out of the channel, sealing and gelling and curing at room temperature for 20-30 hours;
after the sol-gel is formed, the doped silver ions are reduced by 10-100 mu L of dilute sodium borohydride with the concentration of 0.01-0.03mol/L, and then the excessive borohydride is washed away by ultrapure water, so that the sol-gel substrate containing SERS active silver is obtained.
Wherein the equal volume of AgNO3The concentration can be 0.5mol/L, 1mol/L and 2mol/L, wherein 1mol/L is preferred; the sol-gel substrate is sealed and gelated at room temperature and the curing time can be 20h, 25h and 30 h. When washing with ultrapure water, it is preferable to wash twice to remove the excess borohydride therefrom and to increase aggregation of the nano-silver particles, which may have an effect on the particle size.
In the specific implementation process, the preferable scheme is as follows:
equal volume of 1mol/L AgNO328% ammonium hydroxide and MeOH were mixed together and then equal volumes of this solution were mixed with 5: 1: 1 MTMS: TMOS: ODS mixing, then drawing the liquid out into the channel, sealing and gelling and curing at 23 ℃ for 24 hours; after the sol-gel is formed, the doped silver ions are reduced by 60 mu L of dilute sodium borohydride with the concentration of 0.0132mol/L, and then the excessive borohydride is washed away by ultrapure water, so that the sol-gel substrate containing SERS active silver is obtained.
In the two schemes, the sol-gel substrate containing SERS active gold is preferable, so that the preparation is easier, the stability and the specificity are stronger, and more accurate detection results can be brought.
In one embodiment, the sample to be tested is a biological fluid, an aerosol concentrate, and a swab solution. Wherein the biological fluid includes, but is not limited to, sputum, saliva, nasal mucus, and the like; the aerosol concentrated solution is mainly obtained from the air by a specific means and is mainly used for detecting whether the air contains viruses or not so as to facilitate disinfection or take corresponding isolation measures; the swab solution is collected from the surface of an object by adopting a special means and prepared into liquid for detection. Because the key point of the invention is based on the sensitivity provided by SERS, SERS active gold or silver is functionalized by using an antibody, and the sensitivity and specificity required by virus detection are endowed, the obtaining mode of the sample is not repeated.
The present invention employs sol-gel-surface enhanced raman spectroscopy to specifically detect viruses using SERS capture assay, and when used to detect coronavirus covi-19, can detect, identify and quantify the presence of coronavirus covi-19 with a desired specificity (no false positives or negatives) and a desired sensitivity (e.g., 1000 virus units per ml of sputum) within 15 minutes.
On the other hand, referring to fig. 9 to 11, in an embodiment of the present invention, there is further provided a SERS-based virus detection apparatus, including:
a sampler 10 for collecting a sample, a sample loading port 20 connected to an output of the sampler 10; the output end of the sample loading port 20 is connected with a channel 31 for loading a sol-gel substrate; the sample loading port 20 is further provided with a sample loading syringe 21 for injecting an antibody solution of a predetermined concentration into the channel 31 to functionalize the gold or silver to mask a raman signal; the channel 31 is arranged in the detection chamber 30, and the detection chamber 30 is further provided with a detection port 32 for accessing the raman detector 40 for detection. Wherein the sample is to be obtained from the person being sampled, may be a biological fluid obtained directly from the nasopharynx or lungs of the person being sampled, and is collected and stored in a container such as a vial, centrifuge tube, or the like. Antibodies may be used as Anti-SARS-CoV-2 antibodies (Anti-SARS-CoV-2 antibodies), which have been shown to be highly specific for a particular virus (e.g., COVID19) and to not cross-react with SARS or MERS. Available from commercial companies as bio vision (BioVision).
The website is as follows: https:// www.biovision.com/anti-ncovid-19-anti-clone-6 f10. html.
In particular embodiments, the sampler 10 may use a screw-on cup to collect sputum or a bubbler and face mask to sample exhaled air. The virus detection apparatus of the present invention also has a waste reservoir that will contain any spills due to injection. The channel 31 may be made of a closed plastic card or may be, for example, a glass capillary tube embedded in a plastic card. One chip is now tested once, possibly after multiple passes if it is desired to detect different viruses using different detection methods for each target virus.
The virus detection device based on SERS has the working principle that: referring to fig. 9, a prepared channel of a sol-gel substrate containing SERS active gold or silver is installed in the detection bin, then an antibody solution with a predetermined concentration is injected from a loading port through a sample loading injector to functionalize the gold or silver, a raman signal of the gold or silver is shielded, then the sample to be detected in the sampler is pushed to flow through the sol-gel substrate through the sample loading injector, after 1-5 minutes, a detector of the raman detector is inserted through the detection port, and then the detection is performed by electrifying to obtain a spectrum, and whether a virus exists or not is determined by observing whether a signal peak exists at a specific position on the spectrum.
The invention detects and identifies viruses such as COVID-19 and the like by the SERS active chip and the Raman analyzer, not only can provide higher sensitivity for the detection of the viruses, but also can specifically identify the viruses by the application of the antibody, detects whether the viruses exist in the sample to be detected or not by SERS on the basis of sensitivity and specificity, can improve the detection accuracy and effectively shorten the detection time.
The method comprises the steps of preparing a sol-gel substrate containing SERS active gold or silver, and fixing the sol-gel substrate in a channel; then injecting a predetermined concentration of antibody solution into the channel, functionalizing the gold or silver to mask the raman signal; and finally, obtaining a sample to be detected, injecting the sample into a channel, standing or incubating for 1-5 minutes, detecting by using a Raman analyzer to obtain a spectrum to judge whether viruses exist, so that the necessary specificity and sensitivity of the detection method can be endowed, the detection time can be effectively shortened, the COVD-19 viruses can be rapidly detected and diagnosed within 15 minutes, the cost is relatively low, meanwhile, the false negative and false positive errors are minimized, the infection source can be cut off, and the rapid spread of the viruses is reduced.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that variations and modifications can be made by those skilled in the art without departing from the structure of the present invention. These should also be construed as the scope of the present invention, and they should not be construed as affecting the effectiveness of the practice of the present invention or the applicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (6)
1. A virus detection method based on SERS is characterized by comprising the following steps:
preparing a sol-gel substrate containing SERS active gold or silver, and fixing the sol-gel substrate in a channel;
injecting a predetermined concentration of antibody solution into the channel, functionalizing the gold or silver to mask raman signals;
and obtaining a sample to be detected, injecting the sample into a channel, standing or incubating for 1-5 minutes, and detecting by using a Raman analyzer to obtain a spectrum to judge whether viruses exist.
2. The SERS-based virus detection method according to claim 1, wherein the step of functionalizing the gold or silver comprises:
the gold or silver functionalization is accomplished after sucking a predetermined concentration of antibody solution sufficient to cover the surface of gold or silver metal particles into the glass capillary or channel, incubating at 22-30 ℃ for 0.5-24h, washing with water or buffer.
3. The SERS-based virus detection method according to claim 2, wherein the preparation of the SERS-active gold-containing sol-gel substrate comprises the steps of:
adding equal volume of 0.1-0.5mol/L HAuCl4.3H2O at 70% HNO3Mixing the solution with pure TMOS for 1-5 min, pumping the liquid into the channel, sealing, and gelatinizing and curing at room temperature for 20-30 hr;
after the sol-gel is formed, the doped gold ions are reduced by 10-100 mu L of sodium borohydride with the concentration of 0.01-0.03 mol/L; and then, washing away excessive borohydride by using ultrapure water to obtain the sol-gel substrate containing SERS active gold.
4. The SERS-based virus detection method according to claim 2, wherein the preparation of the SERS-active silver-containing sol-gel substrate comprises the steps of:
equal volumes of 0.5-2mol/L AgNO3, 28% ammonium hydroxide, and MeOH were mixed together, and then equal volumes of this solution and 5: 1: 1 MTMS: TMOS: ODS mixing, then pumping the liquid out of the channel, sealing and gelling and curing at room temperature for 20-30 hours;
after the sol-gel is formed, the doped silver ions are reduced by 10-100 mu L of dilute sodium borohydride with the concentration of 0.01-0.03mol/L, and then the excessive borohydride is washed away by ultrapure water, so that the sol-gel substrate containing SERS active silver is obtained.
5. The SERS-based virus detection method according to claim 1, wherein the sample comprises: biological fluids, aerosol concentrates and swab solutions of the sample to be tested.
6. A SERS-based virus detection apparatus, comprising:
the sampler is used for collecting samples, and the sample loading port is connected with the output end of the sampler; the output end of the sample loading port is connected with a channel for loading the sol-gel substrate; the sample loading port is also provided with a sample loading injector for injecting an antibody solution with a predetermined concentration into the channel and functionalizing the gold or silver to mask a Raman signal; the passageway sets up in detecting the storehouse, it still is provided with the detection mouth that is used for inserting raman detector and detects to detect the storehouse.
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