CN111896520A - Raman substrate for respiratory virus detection and preparation method and application thereof - Google Patents
Raman substrate for respiratory virus detection and preparation method and application thereof Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D7/00—Processes, other than flocking, specially adapted for applying liquids or other fluent materials to particular surfaces or for applying particular liquids or other fluent materials
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D7/00—Processes, other than flocking, specially adapted for applying liquids or other fluent materials to particular surfaces or for applying particular liquids or other fluent materials
- B05D7/24—Processes, other than flocking, specially adapted for applying liquids or other fluent materials to particular surfaces or for applying particular liquids or other fluent materials for applying particular liquids or other fluent materials
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B22—CASTING; POWDER METALLURGY
- B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
- B22F9/00—Making metallic powder or suspensions thereof
- B22F9/16—Making metallic powder or suspensions thereof using chemical processes
- B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
- B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
Abstract
The invention discloses a surface enhanced Raman substrate for rapid detection of respiratory viruses, which comprises: the flexible substrate layer is made of flexible substrate materials, and noble metal particles are loaded on the surface of the flexible substrate materials; the preparation process comprises the following steps: preparing a seed particle dispersion, preparing AgNPs, preparing Au growth liquid, preparing Ag @ AuNPs, and dripping the Ag @ AuNPs on a flexible substrate to obtain the surface enhanced Raman substrate.
Description
Technical Field
The invention relates to the technical field of virus detection and nano materials, in particular to a Raman substrate for detecting respiratory viruses and a preparation method and application thereof.
Background
Respiratory viruses are a general term of viruses which take respiratory tracts as entry portals and cause local respiratory tract infection or pathological changes of tissues and organs except the respiratory tracts due to proliferation of skin cells on the respiratory tracts, are mainly transmitted through the respiratory tracts and have high prevalence rate in people. Common respiratory viruses comprise influenza virus, parainfluenza virus, respiratory syncytial virus, adenovirus, rhinovirus, coronavirus and the like, and 90-95% of clinical acute respiratory infections are caused by the viruses.
Pathogens causing respiratory tract infection are various in types and different in harm degree, but initial clinical symptoms of most patients after infection are similar, the pathogens mainly harm the health of middle-aged and old people, infants and partial people with chronic diseases, and even can endanger life safety. Therefore, the diagnosis and timely treatment of pathogens can be made as soon as possible, and the method has important significance for avoiding cross infection, controlling epidemic outbreak and quickly recovering patients.
The current clinical respiratory virus diagnosis method mainly comprises RT-PCR or/and NGS etiology examination, IgM/IgG antibody serum examination, chest imaging examination and other general clinical index analysis. The diagnosis method can basically realize the diagnosis of pathogens, but has the defects of long detection time consumption, large sampling infection risk, low accuracy, high technical requirement on analysts, invasive detection and the like.
Therefore, how to provide a substrate material capable of rapidly detecting respiratory viruses is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a raman substrate for rapidly screening respiratory viruses, and the raman substrate with high sensitivity is prepared by combining noble metal particles and a porous flexible substrate.
In order to achieve the purpose, the invention adopts the following technical scheme:
a raman substrate for respiratory virus detection, comprising: the flexible substrate layer is composed of a flexible substrate material, and the surface of the flexible substrate material is loaded with noble metal particles; wherein, the flexible substrate material includes but is not limited to any one of a nitrocellulose membrane, a cellulose acetate membrane or a polyvinylidene fluoride membrane.
The method comprises the steps of preparing a high-efficiency surface-enhanced Raman substrate by combining noble metal particles with a porous flexible substrate, detecting droplets or secretions which are discharged through a respiratory tract and remain in the mask by combining a medical mask commonly used in virus protection by using a Raman technology, and determining whether a disease is caused and the type of a pathogen by judging and analyzing Raman signals of the obtained secretions; the Surface Enhanced Raman Scattering (SERS) technology can provide a characteristic fingerprint spectrogram of a molecule based on molecular vibration information, and greatly improve a Raman signal of the molecule to be detected by utilizing an electromagnetic field enhancement principle, so that the detection of a substance with extremely low concentration can be carried out, and even the resolution of a single base or amino acid can be realized; aiming at different viruses, the nucleic acid, protein and lipid components contained in the viruses are different, so different Raman signals can be obtained by detecting different viruses, and the diagnosis and the differentiation of the viruses can be realized;
the transmission routes of respiratory infectious diseases are mainly respiratory droplet transmission and close contact transmission. The size of the virus is small, usually about 10 to 100nm, but the virus usually cannot exist independently, and the propagation path mainly includes secretion and droplets during sneezing, and the size of the droplets is about 5 μm. The nitrocellulose membrane, the cellulose acetate membrane or the polyvinylidene fluoride membrane can be used as a transfer medium for western blotting, has strong binding capacity to protein, and therefore has strong binding capacity to a protein receptor on the surface of a virus, and simultaneously, the microstructure of the membrane is a fiber mesh porous structure, the pore size distribution is dozens to hundreds of microns, the specific surface area is large, the arrangement is compact and high, the filtering performance is good, and the nitrocellulose membrane has good filtering and intercepting effects on virus-containing droplets or aerogel.
As a preferred embodiment of the present invention, the noble metal particles include: gold and silver, and the particle size of the noble metal particles is 20-300 nm.
The invention adopts silver particle material, the size of the silver particle can be adjusted and controlled in a large range according to the concentration of the reactant, the silver particle is used for matching the required detection laser wavelength and the detection object to obtain the strongest plasma excited resonance effect, and the silver particle generally has better SERS enhancement effect compared with the gold particle, but on the other hand, the silver particle is not easy to store and is easy to oxidize, so that the invention wraps a layer of gold shell on the periphery of the silver particle, thereby ensuring the detection sensitivity and simultaneously improving the stability of the substrate.
A preparation method of a Raman substrate for respiratory virus detection comprises the following steps:
1) preparing bright yellow silver seed particle dispersoid by using a sodium citrate aqueous solution, a silver nitrate aqueous solution, a sodium chloride aqueous solution and an ascorbic acid aqueous solution as raw materials;
2) at room temperature, taking the bright yellow silver seed particle dispersoid, and adding the dispersoid into pure water under the stirring condition to obtain a silver seed solution; sequentially adding the aqueous solution of the silver-ammonia complex and the aqueous solution of ascorbic acid into the silver seed solution, and stirring to prepare AgNPs;
3) mixing pure water, a tetrachloroauric acid aqueous solution, a sodium hydroxide aqueous solution and a sulfite aqueous solution, and standing for 12 hours to obtain an Au growth solution;
4) sequentially adding Ag NPs, PVP K30, an ascorbic acid aqueous solution, a sodium hydroxide aqueous solution and a sodium sulfite aqueous solution into a reaction vessel, adding an Au growth solution, slightly oscillating, standing at 60 ℃ for reaction for 1h, centrifuging, and taking a precipitate to obtain Ag @ AuNPs;
5) and uniformly dripping Ag @ AuNPs on the flexible substrate material to enable the particles to be self-assembled and uniformly and densely dispersed, so as to obtain the Raman substrate.
As a preferred technical solution of the present invention, step 1) specifically includes:
11) sequentially adding a sodium citrate aqueous solution, a silver nitrate aqueous solution and a sodium chloride aqueous solution into pure water, stirring and premixing at room temperature to obtain a citrate-silver-sodium chloride premix;
12) adding ascorbic acid water solution into boiling water to obtain boiling water mixture, maintaining for 1-2min, adding citrate-silver-sodium chloride premix into the boiling water mixture within 10s, heating and stirring under boiling condition to obtain solution, and cooling to room temperature to obtain bright yellow silver seed particle dispersion.
As the preferable technical scheme of the invention, the mass fractions of the sodium citrate and the silver nitrate are both 1 percent, and the concentration of the sodium chloride is 10-30 mM; the volume ratio of the sodium citrate aqueous solution to the silver nitrate aqueous solution to the sodium chloride aqueous solution to the pure water is 20:5:4: 21; in the step 12), the concentration of the ascorbic acid is 0.1M, and the volume ratio of the ascorbic acid aqueous solution to the boiling water is 4: 2375.
As a preferable technical scheme of the invention, in the step 2), the volume ratio of the bright yellow silver seed particle dispersion, the pure water, the silver-ammonia complex aqueous solution and the ascorbic acid aqueous solution is 20:473:7: 200;
wherein, each 560 μ L of silver-ammonia complex aqueous solution comprises 160 μ L of mixed solution of silver nitrate aqueous solution with mass fraction of 1% and 400 μ L of ammonia water with mass fraction of 25-28%;
the concentration of the ascorbic acid aqueous solution is 1-4 mM.
In a preferred embodiment of the present invention, in step 3), the concentration of tetrachloroauric acid is 0.5M, the concentration of sodium hydroxide is 1.0M, the concentration of sodium sulfite is 0.5M, and the volume ratio of pure water, the aqueous tetrachloroauric acid solution, the aqueous sodium hydroxide solution, and the aqueous sodium sulfite solution is 487:2:5: 6.
As a preferable embodiment of the present invention, in step 4), the mass fraction of PVP K30 is 5%, the concentration of ascorbic acid is 0.1M, the concentration of sodium hydroxide is 1.0M, and the concentration of sodium sulfite is 0.1M; the volume ratio of the Ag NPs, PVP K30, ascorbic acid aqueous solution, sodium hydroxide aqueous solution, sodium sulfite aqueous solution and Au growth solution is (60-1100):300:300:45:21 (12-120); the diameter of the Ag @ AuNPs silver core is 20-300nm, and the thickness of the gold shell is 1-10 nm.
As a preferable technical scheme, in the step 5), the concentration of Ag @ Au NPs dripped on the flexible substrate material is 1E + 12-1E +13NPs/mL, and the dripping volume is 1-20 mu L; when the particles are self-assembled, the environment temperature is controlled to be 20-50 ℃, and the humidity is controlled to be 50-70%.
The dropping volume influences the amount and the distribution compactness of particles, the more metal particles are, the tighter the distribution is, and the better the SERS enhancement effect is; temperature and humidity affect the rate of droplet evaporation and the rate of diffusion across the flexible material, with slower evaporation and diffusion, more uniform particle distribution.
The Raman substrate prepared by the preparation method is applied to preparation of respiratory tract virus detection products.
In the application process, the film substrate can be attached to the inner side of the mask, and droplets and secretions generated when a user breathes, speaks or coughs can be adsorbed on the surface of the substrate, so that the sampling infection risk can be greatly reduced. Meanwhile, in order to improve the detection accuracy, a culture medium sample collected after sampling a pharyngeal swab or a nasopharyngeal swab can be directly dripped on the surface of a film substrate, droplets or secretions which are discharged through a respiratory tract and remain in a mask are detected by combining a Raman technology, and the respiratory tract viruses are detected by distinguishing and analyzing Raman signals of the obtained secretions.
According to the technical scheme, compared with the prior art, the invention has the following technical effects:
firstly, the Raman detection operation is simple, the sample pretreatment requirement is low, the rapid detection of the sample can be realized by matching with the portable Raman spectrum, and meanwhile, the infection risk of sampling personnel can be greatly reduced; secondly, a high-sensitivity SERS enhanced substrate is prepared and synthesized, and the whole detection method is low in cost and is combined with a protective mask which is most commonly used for epidemic prevention, so that an additional biological reagent is not needed. Therefore, the method provides the surface enhanced Raman substrate which can provide rapid and convenient identification and screening for respiratory tract infection diseases, and can provide an effective supplement for the conventional respiratory tract virus detection method under the condition of shortage of medical resources caused by large-scale epidemic outbreak.
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, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a transmission electron microscope characterization diagram of AgNPs particles in example 6 of the present invention;
FIG. 2 is a transmission electron microscopy characterization of Ag @ AuNPs particles in example 6 of the present invention;
FIG. 3A is a scanning image of transmission electron microscopy EDS energy spectra of Ag element in Ag @ AuNPs particles in example 6 of the present invention;
FIG. 3B is a scanning image of EDS (Electron Spectroscopy) of an Au element in Ag @ AuNPs particles in example 6 of the present invention;
FIG. 3C is an EDS energy spectrum linear scan of Ag @ AuNPs particles in example 6 of the present invention;
FIG. 4 is a schematic representation of a scanning electron microscope for field emission scanning of a cellulose nitrate film substrate loaded with Ag @ AuNPs particles in example 6 of the present invention;
FIG. 5A is a SERS graph showing the detection of different viral proteins and BSA proteins when a Raman substrate prepared in example 6 is tested;
FIG. 5B is a graph showing the components of SERS spectra of different viral proteins when a Raman substrate prepared in example 6 is tested;
FIG. 6 is a graph of comparative data for stability of different substrate materials;
FIG. 7A is a field emission scanning electron microscope characterization of Ag @ AuNPs particles prepared under low concentration sodium hydroxide conditions;
FIG. 7B is a field emission scanning electron microscope characterization of Ag @ AuNPs particles prepared under high concentration sodium hydroxide conditions.
FIG. 8 is a schematic view of the detection scheme of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
Example 1
A raman substrate for respiratory virus detection comprising: the flexible substrate layer is composed of a nitrocellulose membrane, silver and gold are loaded on the surface of the flexible substrate material, and the particle diameter of the silver and the gold is 140 nm.
Example 2
A raman substrate for respiratory virus detection comprising: the flexible substrate layer is composed of cellulose acetate membranes, silver and gold are loaded on the surface of the flexible substrate material, and the particle diameters of the silver and the gold are 20 nm.
Example 3
A raman substrate for respiratory virus detection comprising: the flexible substrate layer is composed of a polyvinylidene fluoride film, silver and gold are loaded on the surface of the flexible substrate material, and the particle diameters of the silver and the gold are 300 nm.
Example 4
Taking example 1 as an example, the preparation of the raman substrate comprises the following specific steps:
1) sequentially adding 1mL of sodium citrate aqueous solution (1 wt%), 0.25mL of silver nitrate aqueous solution (1 wt%) and 0.2mL of sodium chloride aqueous solution (20mM) into 1.05mL of pure water, and stirring and premixing at room temperature to obtain a citrate-silver-sodium chloride premix; adding 80 μ L of ascorbic acid aqueous solution (0.1M) to 47.5mL of boiling water to obtain a boiling water mixture, after 1min, rapidly adding the citrate-silver-sodium chloride premix to the boiling water mixture, continuing to heat and stir for 1h, cooling the obtained solution to room temperature, and finally obtaining the silver seed particle dispersion containing brilliant yellow color.
2) Taking 4mL of silver seeds synthesized in the first step in advance, concentrating the silver seeds to make the volume of the silver seeds be 1.6mL, adding the silver seeds into 37.84mL of pure water under the stirring condition to obtain a silver seed solution, then adding 560 μ L of an aqueous solution of silver-ammonia complex (a mixed solution of 1mL of a silver nitrate aqueous solution (1 wt%) and 400 μ L of ammonia water (25%)) and 16mL of an ascorbic acid aqueous solution (2.5mM) into the silver seed solution in sequence, and stirring for 2h to obtain large-particle AgNPs;
3) mixing 4.87mL of pure water, 20. mu.L of an aqueous tetrachloroauric acid solution (0.5M), 50. mu.L of an aqueous sodium hydroxide solution (1.0M), and 60. mu.L of an aqueous sodium sulfite solution (0.5M), and allowing the mixture to stand for 12 hours to obtain an Au growth solution;
4) sequentially adding 3mL of AgNPs particles synthesized in step 2), 15mL of 5% PVP K30, 15mL of 0.1M ascorbic acid aqueous solution, 2.25mL of 1.0M sodium hydroxide aqueous solution and 1.05mL of 0.1M sodium sulfite aqueous solution into a reaction vessel, then adding 0.6mL of pre-synthesized gold precursor solution to start the growth of a gold shell, slightly shaking and standing at 60 ℃ for 1h to obtain Ag @ AuNPs, and further cleaning the particles by centrifugation; the diameter of the silver core of the Ag @ AuNPs is 20nm, and the thickness of the gold shell is 1 nm;
5) coating Ag @ AuNPs on a pre-cut square nitric acid cellulose membrane with the side length of 0.5cm in a dripping mode, slowly evaporating the liquid drops, and performing self-assembly to enable particles to be uniformly and densely dispersed in pores of the membrane, so that a surface enhanced Raman substrate is obtained; wherein the concentration of Ag @ Au NPs dripped on the cellulose nitrate membrane is 1E +13NPs/mL, the dripping volume is 1 mu L, and the environmental temperature is controlled to be 20 ℃ and the humidity is 50 percent when the particles are self-assembled.
Example 5
Taking example 1 as an example, the preparation of the raman substrate comprises the following specific steps:
1) sequentially adding 1mL of sodium citrate aqueous solution (1 wt%), 0.25mL of silver nitrate aqueous solution (1 wt%) and 0.2mL of sodium chloride aqueous solution (20mM) into 1.05mL of pure water, and stirring and premixing at room temperature to obtain a citrate-silver-sodium chloride premix; 80 μ L of an aqueous ascorbic acid solution (0.1M) was added to 47.5mL of boiling water and after 1min the citrate-silver-sodium chloride premix was added quickly. Stirring was continued for 1h and the resulting solution was cooled to room temperature to finally obtain a silver seed particle dispersion containing a bright yellow color.
2) At room temperature, 40. mu.L of the silver seeds previously synthesized in step one, the volume of the original silver seeds obtained by dilution control was 1.6mL, and added to 37.84mL of pure water under stirring to obtain a silver seed solution, and then 560. mu.L of an aqueous solution of silver-ammonia complex (a mixture of 1mL of an aqueous silver nitrate solution (1 wt%) and 400. mu.L of aqueous ammonia (28%)), and 16mL of an aqueous ascorbic acid solution (2.5mM) were sequentially added to the silver seed solution. Stirring for 2 hours to obtain large-particle AgNP;
3) mixing 4.87mL of pure water, 20. mu.L of an aqueous tetrachloroauric acid solution (0.5M), 50. mu.L of an aqueous sodium hydroxide solution (1.0M), and 60. mu.L of an aqueous sodium sulfite solution (0.5M), and allowing the mixture to stand for 12 hours to obtain an Au growth solution;
4) adding 55mL of AgNP synthesized in step 2), 15mL of 5% PVP K30, 15mL of 0.1M ascorbic acid aqueous solution, 2.25mL of 1.0M sodium hydroxide aqueous solution and 1.05mL of 0.1M sodium sulfite aqueous solution into a reaction vessel in this order, then adding 6mL of pre-synthesized gold precursor solution to start the growth of the gold shell, slightly shaking and standing at 60 ℃ for 1h to obtain @ Ag AuNPs, and further cleaning the particles by centrifugation; the diameter of the silver core of the Ag @ AuNPs is 300nm, and the thickness of the gold shell is 10 nm;
5) coating Ag @ AuNPs on a pre-cut square nitric acid cellulose membrane with the side length of 0.5cm in a dripping mode, slowly evaporating the liquid drops, and performing self-assembly to enable particles to be uniformly and densely dispersed in pores of the membrane, so that a surface enhanced Raman substrate is obtained; wherein the concentration of Ag @ AuNPs dripped on the cellulose nitrate membrane is 1E +12NPs/mL, the dripping volume is 20 mu L, and when the particles are self-assembled, the environmental temperature is controlled to be 50 ℃ and the humidity is 70 percent
Example 6
Taking example 1 as an example, the preparation of the raman substrate comprises the following specific steps:
1) sequentially adding 1mL of sodium citrate aqueous solution (1 wt%), 0.25mL of silver nitrate aqueous solution (1 wt%) and 0.2mL of sodium chloride aqueous solution (20mM) into 1.05mL of pure water, and stirring and premixing at room temperature to obtain a citrate-silver-sodium chloride premix; 80 μ L of an aqueous ascorbic acid solution (0.1M) was added to 47.5mL of boiling water and after 1min the citrate-silver-sodium chloride premix was added quickly. Stirring was continued for 1h and the resulting solution was cooled to room temperature to finally obtain a silver seed particle dispersion containing a bright yellow color.
2) At room temperature, 1mL of the silver seeds previously synthesized in step one was taken, and the volume of the original silver seeds obtained by dilution control was 1.6mL, and added to 37.84mL of pure water under stirring to obtain a silver seed solution, and then 560. mu.L of an aqueous solution of silver-ammonia complex (a mixture of 1mL of an aqueous silver nitrate solution (1 wt%) and 400. mu.L of aqueous ammonia (26%)), and 16mL of an aqueous ascorbic acid solution (2.5mM) were sequentially added to the silver seed solution. Stirring for 1.5h to obtain large-particle AgNPs, wherein a transmission electron microscope image of the large-particle AgNPs is shown in figure 1;
3) mixing 4.87mL of pure water, 20. mu.L of an aqueous tetrachloroauric acid solution (0.5M), 50. mu.L of an aqueous sodium hydroxide solution (1.0M), and 60. mu.L of an aqueous sodium sulfite solution (0.5M), and allowing the mixture to stand for 12 hours to obtain an Au growth solution;
4) adding 45mL of AgNP synthesized in step 2), 15mL of 5% PVP K30, 15mL of 0.1M ascorbic acid aqueous solution, 2.25mL of 1.0M sodium hydroxide aqueous solution and 1.05mL of 0.1M sodium sulfite aqueous solution into a reaction vessel in this order, then adding 1.3mL of pre-synthesized gold precursor solution to start the growth of the gold shell, after slightly shaking and standing at 60 ℃ for 1h, obtaining Ag @ AuNPs, and further cleaning the particles by centrifugation; the diameter of the silver core of the Ag @ AuNPs is 140nm, the thickness of the gold shell is 1.5nm, and a transmission electron microscope image is shown in FIG. 2; successful synthesis of the structure of the silver-core gold shell was demonstrated by elemental analysis, see fig. 3A, 3B and 3C;
5) coating Ag @ AuNPs on a pre-cut square nitric acid cellulose membrane with the side length of 0.5cm in a dripping mode, slowly evaporating the liquid drops, and performing self-assembly to enable particles to be uniformly and densely dispersed in pores of the membrane, so that a surface enhanced Raman substrate is obtained; as shown in fig. 4; wherein the concentration of Ag @ Au NPs dripped on the cellulose nitrate membrane is 5.1E +12NPs/mL L, the dripping volume is 10 mu L, and the environment temperature is controlled to be 35 ℃ and the humidity is 60% during the self-assembly of the particles.
The SERS substrate prepared by the invention can be used for respiratory virus detection, and specifically can be used for detecting influenza virus, parainfluenza virus, respiratory syncytial virus, adenovirus, rhinovirus, coronavirus and combination of any two or more of the respiratory viruses. When the mask is used specifically, the film substrate loaded with the noble metal particles can be attached to the inner side of a common medical mask, droplets and secretions generated when a user breathes, speaks or coughs can be adsorbed on the surface of the substrate, and respiratory viruses possibly contained in the droplets can be detected after the substrate is taken down. Meanwhile, in order to improve the detection accuracy, a culture medium sample collected after sampling the nasopharyngeal swab or the nasopharyngeal swab can be directly dripped on the surface of the film substrate for subsequent Raman detection.
To evaluate the virus detection performance of the material, experiments were performed using the raman substrate prepared in example 6, and the receptor binding region RBD of the SARS-CoV-2 surface protein N, the receptor binding protein S1, and the S1 protein were selected for detection, while Bovine Serum Albumin (BSA) was also tested for comparative reference. The specific steps are that 5 mu L of the protein aqueous solution (10ng/mL) is respectively absorbed and dripped on the surface of the substrate metal coating, and after complete absorption and drying, Raman testing is carried out. The protein SERS detection result is shown in FIG. 5A, because the difference of direct observation of the Raman peak of the protein is not obvious, the main component analysis (PCA) is carried out on the result, as shown in FIG. 5B, different types of proteins can be well distinguished, and the Raman detection has good potential in distinguishing different types of viruses.
Comparative example 1
The difference from the example 6 is that the gold shell is not generated by continuous reaction after the silver particles are synthesized, and the concentrated solution is directly dripped on the cellulose nitrate membrane.
When the Raman substrate is tested according to the method, because the naked silver particles are unstable and are easily oxidized, the Raman performance is further influenced, and the detection performances of the substrate prepared from AgNPs and Ag @ Au NPs after the substrate is synthesized for the first day and placed in the environment for seven days are respectively tested and compared. The SARS-CoV-2 surface receptor binding protein S1 protein was selected, and S1 protein 1320cm was compared as shown in FIG. 6-1The change in peak intensity. It can be seen that the signal intensity of the bare silver particles decreased by about 30% after being left for seven days, and the signal intensity of the substrate coated with the gold shell was not substantially changed. Indicating that the substrate of the present invention has better stability.
Comparative example 2
Different from the embodiment 6, the concentration of the sodium hydroxide aqueous solution in the step (4) is changed to 0.5M, the high pH condition is favorable for reducing the replacement reaction of gold ions and silver, and is favorable for forming a complete gold shell, and FIGS. 7A and 7B show that the field emission scanning electron microscope characterization graphs of the Ag @ Au particles prepared under the conditions of low-concentration sodium hydroxide and high-concentration sodium hydroxide have obvious defects on the surfaces of the particles generated under the condition of low-concentration sodium hydroxide and have no obvious defects on the surfaces of the particles under the condition of high concentration.
A schematic of the detection scheme of the present invention is shown in FIG. 8.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A raman substrate for respiratory virus detection, comprising: the flexible substrate layer is composed of a flexible substrate material, and the surface of the flexible substrate material is loaded with noble metal particles; wherein, the flexible substrate material includes but is not limited to any one of a nitrocellulose membrane, a cellulose acetate membrane or a polyvinylidene fluoride membrane.
2. A Raman substrate for respiratory virus detection according to claim 1, wherein the noble metal particles are selected from silver and gold and have a particle size of 20-300 nm.
3. A method of preparing a raman substrate for detection of respiratory viruses according to any one of claims 1 to 2, comprising the steps of:
1) preparing bright yellow silver seed particle dispersoid by using a sodium citrate aqueous solution, a silver nitrate aqueous solution, a sodium chloride aqueous solution and an ascorbic acid aqueous solution as raw materials;
2) at room temperature, taking the bright yellow silver seed particle dispersoid, and adding the dispersoid into pure water under the stirring condition to obtain a silver seed solution; sequentially adding the aqueous solution of the silver-ammonia complex and the aqueous solution of ascorbic acid into the silver seed solution, and stirring to prepare AgNPs;
3) mixing pure water, a tetrachloroauric acid aqueous solution, a sodium hydroxide aqueous solution and a sulfite aqueous solution, and standing for 12 hours to obtain an Au growth solution;
4) sequentially adding Ag NPs, PVP K30, an ascorbic acid aqueous solution, a sodium hydroxide aqueous solution and a sodium sulfite aqueous solution into a reaction vessel, adding an Au growth solution, slightly oscillating, standing at 60 ℃ for reaction for 1h, centrifuging, and taking a precipitate to obtain Ag @ AuNPs;
5) and uniformly dripping Ag @ AuNPs on the flexible substrate material to enable the particles to be self-assembled and uniformly and densely dispersed, so as to obtain the Raman substrate.
4. The method for preparing a Raman substrate for detecting respiratory viruses according to claim 3, wherein the step 1) specifically comprises:
11) sequentially adding a sodium citrate aqueous solution, a silver nitrate aqueous solution and a sodium chloride aqueous solution into pure water, stirring and premixing at room temperature to obtain a citrate-silver-sodium chloride premix;
12) adding ascorbic acid water solution into boiling water to obtain boiling water mixture, maintaining for 1-2min, adding citrate-silver-sodium chloride premix into the boiling water mixture within 10s, heating and stirring under boiling condition to obtain solution, and cooling to room temperature to obtain bright yellow silver seed particle dispersion.
5. A method for preparing a Raman substrate for detecting respiratory viruses as claimed in claim 4, wherein in the step 11), the mass fractions of the sodium citrate and the silver nitrate are both 1%, and the concentration of the sodium chloride is 10-30 mM; the volume ratio of the sodium citrate aqueous solution to the silver nitrate aqueous solution to the sodium chloride aqueous solution to the pure water is 20:5:4: 21; in the step 12), the concentration of the ascorbic acid is 0.1M, and the volume ratio of the ascorbic acid aqueous solution to the boiling water is 4: 2375.
6. A method for preparing a Raman substrate for detecting respiratory viruses according to claim 3, wherein in the step 2), the volume ratio of the bright yellow silver seed particle dispersion, the pure water, the silver-ammonia complex aqueous solution and the ascorbic acid aqueous solution is 20:473:7:200
Wherein, each 560 μ L of silver-ammonia complex aqueous solution comprises 160 μ L of mixed solution of silver nitrate aqueous solution with mass fraction of 1% and 400 μ L of ammonia water with mass fraction of 25-28%;
the concentration of the ascorbic acid aqueous solution is 1-4 mM.
7. A method for preparing a raman substrate for respiratory virus detection according to claim 3, wherein in step 3), the concentration of tetrachloroauric acid is 0.5M, the concentration of sodium hydroxide is 1.0M, the concentration of sodium sulfite is 0.5M, and the volume ratio of pure water, the aqueous tetrachloroauric acid solution, the aqueous sodium hydroxide solution, and the aqueous sodium sulfite solution is 487:2:5: 6.
8. A method for preparing a raman substrate for respiratory tract virus detection according to claim 3, wherein in step 4), the mass fraction of PVP K30 is 5%, the concentration of ascorbic acid is 0.1M, the concentration of sodium hydroxide is 1.0M, and the concentration of sodium sulfite is 0.1M; the volume ratio of the Ag NPs, PVP K30, ascorbic acid aqueous solution, sodium hydroxide aqueous solution, sodium sulfite aqueous solution and Au growth solution is (60-1100):300:300:45:21 (12-120); the diameter of the Ag @ Au NPs silver core is 20-300nm, and the thickness of the gold shell is 1-10 nm.
9. The method for preparing a Raman substrate for detecting respiratory viruses according to claim 3, wherein in the step 5), the concentration of Ag @ AuNPs dripped on the flexible substrate material is 1E + 12-1E +13NPs/mL, and the dripping volume is 1-20 μ L; when the particles are self-assembled, the environment temperature is controlled to be 20-50 ℃, and the humidity is controlled to be 50-70%.
10. The application of the Raman substrate prepared according to the preparation method of any one of claims 3-9 in preparation of a product for detecting respiratory viruses.
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