CN113552348B - Nanoparticle solution for detecting SARS-CoV-2 coronavirus S protein, preparation method, kit and application - Google Patents
Nanoparticle solution for detecting SARS-CoV-2 coronavirus S protein, preparation method, kit and application Download PDFInfo
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Abstract
The invention discloses the technical field of virus antibody detection. In particular to a reagent kit for detecting a novel coronavirus SARS-CoV-2 antibody, which is a reagent for rapidly detecting SARS-CoV-2 coronavirus Spike protein based on antigen-antibody immune recognition reaction, nanoparticle carrier and photosensitive hydrogel. The detection reagent for the novel coronavirus S protein provided by the invention is used for constructing an S protein nano antibody recognition complex aiming at 2019-nCoV by virtue of the existing S protein nucleic acid sequence, and can specifically recognize and identify the specific S protein of the novel coronavirus. The photosensitive hydrogel technology is combined with the immune recognition nanotechnology, and virus recognition signals are converted and presented as visible physicochemical phenomena, so that rapid screening and judgment are realized.
Description
Technical Field
The invention belongs to the technical field of virus antibody detection, in particular relates to a reagent for detecting novel coronavirus SARS-CoV-2, specifically relates to a nanoparticle for detecting novel coronavirus (SARS-CoV-2) Spike protein antigen, and more specifically relates to a rapid detection kit for SARS-CoV-2 coronavirus Spike protein based on antigen-antibody immune recognition reaction, nanoparticle carrier and photosensitive catalytic reaction.
Background
The SARS-CoV-2 coronavirus infection process is started by mutual recognition of Spike protein (S protein) on the surface of the coronavirus and angiotensin converting enzyme 2(ACE2) on the surface of a host cell, and the Spike protein is a kind of trimer transmembrane glycoprotein and forms a special corolla structure on the surface of the virus. Entry of coronaviruses into susceptible cells is a complex process requiring a synergistic interaction of receptor binding of the S protein and proteolytic processes to promote virus-cell fusion. Because S protein is exposed on the surface of virus and mediates the virus to enter host cells, it is the main target of neutralizing antibody after infection, can be used as the antigen of SARS-CoV-2 coronavirus, is the key target for the development of antiviral drugs, therapeutic antibody, diagnostic method, etc., and plays an irreplaceable role. However, the detection technology based on S protein immune recognition needs to further convert and present the recognition signal into visible physicochemical phenomena so as to realize rapid screening and judgment.
Researchers in various countries around the world are studying a rapid and accurate method for detecting coronavirus, and nucleic acid detection reagents based on PCR technology (molecular test) play an important role, but the detection method has several problems:
firstly, SARS-CoV-2 coronavirus is RNA virus, the detection needs to complete the complex processes of sample treatment, centralized inspection and the like in sequence, the total time consumption exceeds 1 hour, the detection requirement of rapidness and high flux cannot be met, if all medical personnel only adopt nucleic acid detection, and in some areas with incomplete medical systems, medical congestion is easily caused, so that some patients cannot be diagnosed late, treatment is delayed or the epidemic situation is expanded;
secondly, the nucleic acid detection needs professional personnel and higher laboratory conditions;
and thirdly, some asymptomatic infected persons have false negative results due to low toxic load or due to sample collection and treatment. Therefore, there is an urgent need to continue to develop detection techniques with higher sensitivity, shorter detection time, and more convenient methods of operation.
Disclosure of Invention
The invention aims to overcome the technical defects of long detection time, complex detection process and low detection sensitivity in the prior art, and provides a nanoparticle solution and a kit for detecting SARS-CoV-2 coronavirus S protein.
In order to achieve the above purpose, the invention provides the following technical scheme:
a nanoparticle solution for detecting SARS-CoV-2 coronavirus S protein, the nanoparticle solution comprising a nanoparticle support on which an S protein cloning antibody and a photosensitizing catalyst are supported.
According to the nano-particle solution disclosed by the invention, coupling sites can be provided on the surface of the nano-particles by virtue of the nano-particles as a carrier, and the S protein clone antibody capable of being specifically identified and the photosensitive catalyst are coupled on the nano-particles together, namely the photosensitive hydrogel technology and the immune identification nano-technology can be perfectly combined by taking the nano-particles as a medium. The specific recognition reaction of the novel coronavirus S protein and the S protein clone antibody is utilized to firmly attach the nanoparticles to a substance with the novel coronavirus S protein, and the characteristic that the photosensitive hydrogel can initiate gel curing is utilized to convert and present virus recognition signals into visible physicochemical phenomena. Thereby realizing rapid screening and judgment. The method is easier to operate, greatly improves the detection efficiency and reduces the risk of virus propagation.
As a preferable technical scheme, the nanoparticle carrier is formed by crosslinking a polymer A and a polymer B, wherein the polymer A is methoxy polyethylene glycol-polycaprolactone polymer (mPEG-PCL), and the polymer B is maleimide polyethylene glycol-polycaprolactone polymer (Mal-PEG-PCL).
The structural formula of mPEG-PCL is shown as follows:
the structural formula of Mal-PEG-PCL is shown as follows:
methoxy polyethylene glycol-polycaprolactone is an amphiphilic copolymer, namely monomethoxyl poly (ethylene glycol) -b-poly (epsilon-caprolactone), abbreviated as mPEG-PCL in English, is a diblock copolymer, one end of the diblock copolymer is hydrophilic chain segment methoxy polyethylene glycol, the other end of the diblock copolymer is lipophilic chain segment poly (epsilon-caprolactone), and the mPEG-PCL has biodegradability, biocompatibility and amphipathy, can be self-assembled into nanoparticles in aqueous solution, and is an ideal nano-drug carrier framework material.
The Maleimide-polyethylene glycol-polycaprolactone polymer is also an amphiphilic copolymer, the English name is Maleimide-poly (ethylene glycol) -b-poly (epsilon-caprolactone), which is called Mal-PEG-PCL for short, the Mal-PEG-PCL not only has biodegradability, biocompatibility and amphipathy, but also can be self-assembled into nanoparticles in aqueous solution, and can be covalently coupled with S protein antibody through Maleimide group, so that the Maleimide-polyethylene glycol-polycaprolactone polymer is an ideal nano-drug functionalized carrier framework material.
The polymer A and the polymer B form a nanoparticle through a crosslinking reaction and simultaneously provide a maleimide group (Mal-group) to provide a coupling site for S protein antibody and a photosensitive catalyst supported on the nanoparticle.
As a preferable technical scheme of the invention, the molecular weight range of the polymer A (mPEG-PCL) is 4000 Da-8000 Da, and in order to ensure that the skeleton structure of the prepared nanoparticle is uniform and prevent other influences caused by different molecular weights, the ratio of the molecular weights of the polymer A and the polymer B is 1: 1. The same molecular weight range of the polymer B (Mal-PEG-PCL) is 4000Da to 8000 Da.
As a preferred technical solution of the present invention, the present application provides a method for preparing the nanoparticle solution, specifically comprising the following steps:
s1, preparation of a nanoparticle carrier: weighing a polymer A and a polymer B according to a weight ratio, dissolving the polymer A and the polymer B by a solvent, performing rotary evaporation to form a film, dissolving the film in deionized water, and oscillating the film in a water bath at 50-70 ℃ to form a nanoparticle carrier solution; refrigerating at 2-8 deg.C; the nano-particle carrier solution is mPEG-PCL-Mal cationic polymer nano-particle solution;
s2, S protein-loaded clone antibody: adding the nanoparticle carrier solution prepared in S1 and the S protein cloned antibody into Hepes buffer solution, and reacting at 2-8 ℃ for more than 24h to obtain an intermediate product solution for later use; as mentioned above, in the preparation of the nanoparticle carrier, the required amount of the S protein clone antibody is only related to and corresponds to the content of the polymer B in the nanoparticle carrier;
s3, supporting a photocatalyst: and (2) carrying out sulfydryl modification on the photosensitive catalyst, adding the modified photosensitive catalyst into the intermediate product solution prepared in the S2, reacting for more than 24 hours at 3-8 ℃, putting reaction liquid into a dialysis bag for dialysis after the reaction is finished, wherein the dialysis liquid in the dialysis process is distilled water, and obtaining the nanoparticle solution after the dialysis is finished.
As a preferable technical scheme of the invention, the mass ratio of the polymer A to the polymer B is 1-2: 9-10. More preferably, the mass ratio between the polymer a and the polymer B is 1: 9.
When the S protein clone antibody and the photosensitive catalyst are coupled with the M-group, the coupling reaction of the S protein clone antibody is firstly carried out, and the S protein clone antibody coupling failure caused by the fact that excessive photosensitive catalyst occupies a coupling site when the photosensitive catalyst and the nanoparticle carrier are coupled is prevented. Affecting the final detection result. In the process of loading the S protein clone antibody and the photocatalyst by the nanoparticle carrier, a Mal-group on a polymer B (Mal-PEG-PCL) is coupled with the S protein clone antibody and the photocatalyst respectively according to the molar ratio of 1: 1; therefore, an excessive amount of either the S protein monoclonal antibody or the photocatalyst will affect the coupling of the partner to the Mal-group.
As a preferred embodiment of the present invention, the molar ratio of the S protein cloned antibody to the polymer B is 1: 2-10; the molar ratio of the photosensitive catalyst to the polymer B is 8-16: 1.
as a preferred embodiment of the present invention, the concentration of S protein of SARS-CoV-2 coronavirus is at least 50. mu.g/mL. The applicant used SARS-CoV-2 coronavirus solution in the course of the study; the concentration of the S protein antigen is far lower than the antigen content of the S protein antigen existing in the pharynx of a patient infected with the novel coronavirus, which shows that in the technical scheme of the application, the antigen-nanoparticle carrier can be firmly combined through the specific recognition of the antigen and the antibody at the concentration, and the detection accuracy is ensured.
As a preferred technical scheme of the invention, the photosensitive catalyst is Irgacure 2959. The photosensitive catalyst can rapidly absorb energy under the action of blue light (with the wavelength of 405nm) to generate free radicals, cations and the like, so that the photosensitive hydrogel is initiated to be cured.
As a preferred technical scheme of the invention, the modification of the photosensitive catalyst is specifically carried out as follows:
under the protection of nitrogen, dissolving the Irgacure2959, thioglycollic acid and p-toluenesulfonic acid in a dichloromethane solution according to a proportion, carrying out reflux reaction for 5-8h, and carrying out column chromatography separation after the reaction is finished to obtain a target modified product: HS-Irgacure 2959.
As the preferred technical scheme of the invention, the invention also discloses a kit for detecting SARS-CoV-2 coronavirus S protein, which comprises the nanoparticle solution, a second detection solution and a third detection solution; the second detection solution is a photosensitive hydrogel; and the third detection solution is a cleaning solution.
After the transfer carrier loaded with the novel coronavirus S protein enters a nanoparticle solution, the nanoparticle carrier in the nanoparticle solution can be bound on the transfer carrier through an antigen-antibody recognition reaction, so that sufficient photosensitive catalyst and photosensitive hydrogel generate a crosslinking curing reaction in a blue light wave band in the second detection solution; thereby determining the presence or absence of the novel coronavirus.
Wherein in the kit, the second detection solution is regulated to be 1 mL. In the present application, the amount of each material is based on the minimum amount of the photocatalyst used for initiating the photosensitive gelation reaction, in which the minimum concentration of the photocatalyst for initiating the gelation reaction is 0.25% (g/mL), that is, 0.0025g/mL, and when the specification of the second detection solution is 1mL, it is at least required that the mass of the photocatalyst Irgacure2959 is 2.5mg, and the corresponding molar amount is 0.01 mmoL.
More specifically, the molar ratio of the photosensitive catalyst to the polymer B is 8-16: 1. the mass range of the Irgacure2959 is 5-8 mg. Further preferably, the molar ratio of the photosensitive catalyst to the polymer B is 8-10: 1, the proportion can ensure that enough of the photosensitive catalyst is loaded on the nanoparticle carrier, and ensure that enough of the photosensitive catalyst can generate cross-linking curing reaction with gel when a throat swab is dipped in a protein antibody solution.
In a preferred embodiment of the present invention, in the step 1, the solvent is any one of dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, diethyl ether, pentane, ethyl acetate, or cyclohexane.
In a preferred embodiment of the present invention, the photosensitive hydrogel in the second detection solution is methacrylated gelatin. Methacrylated gelatin (GelMA) is a double bond modified gelatin that can be cross-linked and cured to a gel by ultraviolet and visible light under the action of a photoinitiator. The GelMA photo-cured hydrogel has the characteristics of natural and synthetic biomaterials, has a three-dimensional structure suitable for cell growth and differentiation, and has excellent biocompatibility and cell reaction characteristics. Irgacure2959 is a blue light initiator, and under the action of blue light (with the wavelength of 405nm), Irgacure2959 can rapidly absorb energy to generate free radicals, cations and the like, so that the photosensitive hydrogel is initiated to be cured.
Specifically, the second detection solution is prepared by the following steps:
as a preferable technical scheme of the invention, the concentration range of the GelMA hydrogel solution is 5-30 mg/mL, and the GelMA hydrogel solution can be solidified under the irradiation of blue light (with the wavelength of 405nm) under the action of a photocatalyst.
In a preferred embodiment of the present invention, the cleaning liquid is ultrapure water or physiological saline. 2mL dose packs.
A method for applying the kit specifically comprises the following steps:
step 1, inserting the sampled throat swab into the nanoparticle solution, stirring for 3-5 seconds, and taking out;
step 2, taking the throat swab out of the nanoparticle solution, inserting the throat swab into a third detection solution, and fully stirring and washing;
step 3, taking the throat swab out of the third detection solution, inserting the throat swab into the second detection solution, stirring for 3-5 seconds, and taking out;
and 4, holding the blue light source by hand, continuously irradiating the second detection solution for 10 seconds at the wavelength of 405nm, inverting the second detection solution to enable the bottle mouth to incline downwards, and judging whether the throat swab carries the novel coronavirus or not by observing whether the second detection solution is solidified or not.
When the SARS-CoV-2 coronavirus S protein detection kit is used, sampling is firstly carried out: a clean throat swab is taken and immersed in SARS-CoV-2 coronavirus S protein solution with the concentration of more than 0.1mmoL for about one centimeter, and the swab head is stirred for 5 seconds after being completely immersed in the solution.
Inserting the sampled throat swab into the nanoparticle solution, completely immersing the swab head in the solution, and stirring the nanoparticle solution with the throat swab for 3-5 seconds to ensure that the swab head is fully contacted with the solution; then taking the pharyngeal swab out of the nanoparticle solution, inserting a third detection solution to enable the swab head to be completely immersed in the solution, and stirring for 3-5 seconds to carry out full washing; taking out the pharyngeal swab from the third detection solution, inserting the second detection solution into the pharyngeal swab so that the swab head is completely immersed in the solution, stirring for 3-5 seconds to fully and uniformly mix the pharyngeal swab with the solution, and taking out the pharyngeal swab; using a 405nm blue light source, enabling the light source to be close to the pipe wall on the premise that the second detection solution is vertical to the horizontal plane, and continuously irradiating the second detection solution for 10 seconds at an angle of 45 degrees; inverting the second detection solution to make the bottle mouth face downwards, making the bottle body and the horizontal plane be 45 degrees, judging whether the solution in the tube is solidified or not is related to whether the SARS-CoV-2 coronavirus Spike protein is contained, namely if the SARS-CoV-2 coronavirus Spike protein is contained, the solution in the tube can be changed into a solid state from a liquid state after being irradiated, and the liquid level of the solution can not be inclined, thus judging the solution to be positive; otherwise, the solution is still liquid, the solution can flow, the liquid level is inclined, and the result is negative.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a novel mode for detecting whether a novel coronavirus exists, which utilizes three properties of antigen-antibody immune recognition reaction, a nanoparticle carrier and a photosensitive catalytic reaction, and respectively loads an S protein clone antibody and a photosensitive catalyst on the nanoparticle carrier by constructing the nanoparticle carrier, utilizes the specific recognition reaction of the novel coronavirus S protein and the S protein clone antibody and the characteristic that the photosensitive catalyst can initiate photosensitive hydrogel to generate gel curing, and converts and presents a virus recognition signal into a visible physical and chemical phenomenon because the photosensitive catalyst is rapid in the curing process and obvious in the solution state before and after the curing reaction. Thereby realizing rapid screening and judgment. The method is easier to operate, greatly improves the detection efficiency and reduces the risk of virus propagation.
The kit for detecting the S protein of the SARS-CoV-2 coronavirus is simple to operate, has obvious change before and after reaction, can be operated by ordinary people according to the using steps under the critical condition so as to self-check whether the novel coronavirus is infected, make protection in time, adopt a protection mode in time and avoid mass spread of the virus.
Description of the drawings:
FIG. 1 is a control chart showing that the second detection solution is solidified after detecting the SARS-CoV-2 coronavirus S protein-containing solution and is still liquid after detecting the blank control solution without SARS-CoV-2 coronavirus S protein under the irradiation of the blue-light flashlight in the embodiment of the present invention;
Detailed Description
Selecting materials:
photocatalyst (Irgacure 2959): 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone, molecular weight: 224.25; purchased from MERCK corporation; item number 410896;
s protein clone antibody: from a positive organism, Human anti-SARS-CoV-2Spike RBD antibody (hFC), cat # S209906;
s protein virus: purchased from orignee corporation, CAT No.: TP 701142;
mPEG-PCL: purchased from sienna millennium biotechnology limited, molecular weight 2000 Da;
Mal-PEG-PCL: purchased from sienna millennium biotechnology limited, molecular weight 2000 Da;
hepes buffer: purchased from Guangzhou Sai Biotechnology Ltd, molecular weight 238.31, prepared as a buffer solution with concentration of 5moL/L, and the main component is hydroxyethyl piperazine ethanethiosulfonic acid;
methacrylated gelatin: from Engineering For Life, Inc.;
according to the invention, through a specific recognition mode of S protein antigen-S protein clone antibody, nanoparticles loaded with a photosensitive catalyst are removed from a nanoparticle solution through a pharynx swab, then interference binding is washed away in a third detection solution, and the pharynx swab is inserted into a second detection solution, because enough photosensitive catalyst is adhered on the pharynx swab, after the pharynx swab is mixed with the second detection solution, energy is rapidly absorbed under the action of blue light (with the wavelength of 405nm), and radicals, cations and the like are generated, so that photosensitive hydrogel solidification is initiated. Realize the detection of the new coronavirus antigen.
The invention provides a SARS-CoV-2 coronavirus S protein detection kit, which comprises a nanoparticle solution, a second detection solution and a third detection solution, wherein the nanoparticle solution is a nanoparticle carrier solution, and an S protein clone antibody and a photosensitive catalyst are loaded on a nanoparticle carrier; the second detection solution is a photosensitive hydrogel solution; after the transfer carrier loaded with the novel coronavirus S protein enters a nanoparticle solution, the nanoparticle carrier in the nanoparticle solution can be bonded on the transfer carrier through antigen-antibody specific recognition reaction, so that the photosensitive catalyst enters the second detection solution to generate crosslinking curing reaction with the photosensitive hydrogel in a blue light wave band; the minimum concentration of the photo-catalyst to initiate the cross-linking cure reaction is 0.0025 (g/mL).
Specifically, the detection kit further comprises a third detection solution, after the transfer carrier is dipped in the nanoparticle solution, the interference binding is washed away by the third detection solution, and then the transfer carrier enters the second detection solution for reaction.
Further, the nano-carrier particles are formed by cross-linking a polymer A and a polymer B, wherein the polymer A is a methoxy polyethylene glycol-polycaprolactone polymer, and the polymer B is a maleimide polyethylene glycol-polycaprolactone polymer. The polymer A and the polymer B are amphiphilic copolymers, have biodegradability, biocompatibility and amphipathy, can be self-assembled into nanoparticles in aqueous solution, and are ideal nano-drug carrier framework materials. There is some literature support for linking other substances to the carrier via a coupling reaction to achieve substance delivery. The kit for rapidly detecting the new coronavirus is developed based on three performances of coupling reaction on a framework material, specific recognition of an antigen and an antibody and crosslinking and curing of a photosensitive catalyst and photosensitive hydrogel. The result is rapid, very big improvement detection efficiency, and this kit convenient operation, simple, more be applicable to the ordinary personnel and examine by oneself, can very big reduction medical personnel's work load.
The polymer A and the polymer B can form a nanoparticle through a crosslinking reaction, and simultaneously can provide a maleimide group (Mal-group) to provide a coupling site loaded on the nanoparticle for an S protein antibody and a photosensitive catalyst.
Furthermore, the nanoparticles used in the present invention are determined by the previous exploration experiments that the mass ratio between the polymer a and the polymer B is 1-5:9, and further screening determines that the nanoparticles have a better polymerization effect when the mass ratio between the polymer a and the polymer B is 1-2: 9.
Furthermore, the S protein clone antibody and the photosensitive catalyst are coupled through the Mal-group on the polymer B, the minimum concentration of the photosensitive catalyst for initiating the crosslinking and curing reaction is 0.25(g/mL), the molar mass of the photosensitive catalyst can be calculated to be 0.01mmol according to the molecular weight of the photosensitive catalyst, at least 2.5mg of the photosensitive catalyst is needed in the specified detection kit when the second detection solution contains 1mL of photosensitive hydrogel, and the mass of the photosensitive catalyst is preferably controlled to be about 2.5mg-10mg in the general feeding process in order to ensure that enough photosensitive catalyst is loaded on the nanoparticles.
The molar ratio of the photocatalyst to polymer B is in the range of 1-2:1, and polymer B may be in a suitable excess to ensure sufficient Mal-groups thereon to effect coupling of the photocatalyst; since the S protein antibody is also coupled to the Mal-group on the nanoparticle carrier, the molar ratio of S protein antibody to the nanoparticle carrier is 1-1.5: 1.
specifically, in the preparation process of the nanoparticle solution, the S protein antibody is loaded first, and then the photocatalyst is loaded.
Further, the photosensitive catalyst is Irgacure2959, and the chemical name is 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-acetone; before loading, the photosensitive catalyst needs to be modified, namely, the modification process is to connect sulfydryl on the photosensitive catalyst, and the coupling with the Mal-group can be realized through the sulfydryl. Specifically, the photosensitive hydrogel in the second detection solution is methacrylated gelatin, and the optimal concentration range after screening is 5-30% (g/mL).
The present invention will be described in further detail with reference to test examples and specific embodiments. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting.
Example 1Preparation of nanoparticle solutions
1. Preparation of nanoparticle support
Mixing 10mg of polymer A (mPEG-PCL) (molecular weight 4000Da, PEG-PCL 2000Da-2000Da) and 90mg of polymer B (Mal-PEG-PCL), wherein the polymer B (molecular weight 4000Da, PEG-PCL 2000Da-2000 Da); the mixture was dissolved in dichloromethane and then placed on a rotary evaporator to be rotary evaporated at 60 ℃ for 45min, followed by film formation. Taking out the formed membrane, dissolving the membrane in deionized water, oscillating the membrane for 5min under the condition of water bath at the temperature of 60 ℃ to obtain MP-Mal cationic polymer nanoparticle solution, and storing the MP-Mal cationic polymer nanoparticle solution in a refrigerator at the temperature of 4 ℃ for later use.
2. Preparation of S protein antibody-MP-Mal nanoparticles
Taking 100mg of the prepared MP-Mal nano-particles, dissolving the prepared MP-Mal nano-particles and 100mg of SARS-CoV-2 coronavirus S protein antibody in Hepes buffer solution, mixing uniformly, and reacting overnight at 4 ℃. And after the reaction is finished, the obtained solution is the S protein antibody-MP-Mal nanoparticle aqueous solution, and is stored in a refrigerator at 4 ℃ for later use.
3. Preparation of protein antibody-MP-Irgacure 2959 nano-particle
Firstly, under the protection of nitrogen, dissolving an Irgacure 29591.12 g (5mmoL), thioglycolic acid 0.24mL (3.4mmoL) and p-toluenesulfonic acid 0.16g (0.83mmoL) in 50mL of dichloromethane solution, heating and refluxing, stopping the reaction after 5h of reaction, directly spin-drying the reaction solvent, and finally performing column chromatography separation to obtain a target modified product (2-thioglycolic acid 2- (4- (2-hydroxy-2-methylpropanoyl) phenoxy) ethyl ester), HS-Irgacure2959 for short.
And dissolving the prepared S protein antibody-MP-Mal nano-particle aqueous solution into Hepes buffer according to 100mg of S protein antibody-MP-Mal nano-particle and 29596 mg of the modified product HS-Irgacure, uniformly mixing, and reacting at 4 ℃ overnight. After the reaction is finished, the mixed solution is put into a dialysis bag with the molecular weight of 2000Da, and the dialysis solution is distilled water and dialyzed overnight at the temperature of 4 ℃. The solution obtained after dialysis is S protein antibody-MP-Irgacure 2959 nanoparticle aqueous solution, namely nanoparticle solution is obtained, and the nanoparticle solution is stored in a refrigerator at 4 ℃ for later use.
In order to ensure that virus-carrying pharyngeal swabs can adequately bind nanoparticles during the preparation of nanoparticle solutions, the inventors conducted the following exploratory experiments:
in this example 1, throat swabs were dipped with the new coronavirus at a concentration of 50. mu.g/mL during the series of experiments. The volume of the fixed photosensitive hydrogel is 1mL, the concentration is 25% (w/v), and nano particle solutions with different concentrations are prepared according to the conditions of the photosensitive gel catalytic reaction. And the volume of the nanoparticle solution taken each time is controlled to be fixed. As shown in table 1 below:
table 1 is a data statistical table for different contents of the photocatalyst
In table 1 above, the data in each set of examples are data values of 10 replicates. The control group was an experiment performed by directly inserting a freshly-unpacked pharyngeal swab into the nanoparticle solution.
As can be seen from Table 1, in examples 1-1 to 1-5, the increase of the content of the photocatalyst ensures that the nanoparticles carried out from the nanoparticle solution by the pharyngeal swab in the case of carrying the virus can be washed away from the interfering bonds by the washing solution, and the lowest molar ratio for curing in the photosensitive hydrogel is 8:1 in examples 1-3, and when the molar ratio exceeds 8:1, the experimental result is the same, so the molar ratio between the photocatalyst and the polymer B in the nanoparticle solution is selected to be 8: 1. The interference combination specifically refers to other substances attached to the pharyngeal swab besides nanoparticles combined on the pharyngeal swab through antigen-antibody reaction
Further, since the theoretical molar ratio between the S protein clone antibody and the polymer B is 1:1, the S protein clone antibody and the photosensitive catalyst are in a mutual competition relationship, in order to ensure that the S protein clone antibody can be successfully coupled with the Mal-group, the S protein clone antibody is generally fed in an excessive amount, in order to prevent the excessive S protein clone antibody from occupying the Mal-group of the photosensitive catalyst to cause insufficient coupling of the photosensitive catalyst on the nano-particles, the proportion of the S protein clone antibody in the coupling process is explored, and the specific formula is shown in Table 2:
table 2 is a table of data of detection experiments for different S protein antibody contents
When the ratio of protein S antibody/polymer B addition is too small, for example 1: 12, because the amount of the S protein antibody is too small, the nanoparticles cannot be connected with enough S protein antibody, so that insufficient nanoparticles are brought into the second detection solution, and the gelation reaction cannot be caused; when the ratio of protein S antibody/polymer B addition is too large, for example 1:1, the S protein antibody can invade most nanoparticle connection sites, so that the photosensitive catalyst cannot be connected to the nanoparticles, and thus, insufficient photosensitive catalyst is brought into the second detection solution, and a gelling reaction cannot be caused. In conclusion, the molar ratio of the S protein clone antibody to the polymer B is 1: 2-10.
Example 2Preparation of the second detection solution
1. Preparation of GelMA hydrogel matrix
10g of Gelatin was weighed out into 100mL of PBS, stirred at 60 ℃ until dissolved, and 8mL of MA (methacrylic anhydride) was slowly dropped into the Gelatin solution using a dropping funnel, followed by reaction at 50 ℃ for 3 hours. The reaction was terminated by diluting the gelatin solution 5-fold with PBS preheated at 40 ℃. Dialyzing with 12-14kD dialysis bag at 40 deg.C and 300rmp under stirring for one week, freeze drying for one week to give white foam, and storing at 4 deg.C.
2. Preparation of GelMA hydrogel solution
Dissolving the white foam GelMA prepared in the above steps with PBS to a concentration of 25% (w/v), subpackaging with 1ml of dosage per bottle, sealing at 4 ℃ and keeping out of the sun.
The optimum concentration of the photosensitive hydrogel was searched by a single variable method according to the crosslinking curing reaction of the photosensitive catalyst and the photosensitive hydrogel, the concentration of the fixed photosensitive catalyst was 0.25% (w/v), the photosensitive hydrogel having a gradient concentration as shown in table 1 below was prepared, and then the change of the gel was observed under irradiation of a blue light (405nm) light source.
TABLE 3 recording table of the conditions of the coagulation of the photosensitive hydrogel at different concentrations
| Item | 2-1 | 2-2 | 2-3 | 2-4 | 2-5 |
| Concentration of photosensitive hydrogel | 1％ | 2.5％ | 5％ | 25％ | 30％ |
| Photoinitiator concentration | 0.25％ | 0.25％ | 0.25％ | 0.25％ | 0.25％ |
| Whether or not to solidify | Whether or not | Is that | Is that | Is that | The gel is insoluble |
| Setting time | / | 15s | 3s | 3s | / |
As can be seen from the entries in Table 3, the cross-linking cure is best when the concentration of the photosensitive hydrogel is between 5 and 25% by combining the set time and the set. In the subsequent examples, 25% of the photosensitive hydrogels of examples 2-4 were selected for detection tests.
Example 3Preparation of third test solution
The MiliQ water sterilized at 121 ℃ for 40 minutes was dispensed in 2mL doses per bottle and stored in a sealed state.
Test example 1
1. Firstly, preparing SARS-CoV-2 coronavirus S protein solution, when in use, taking out the throat swab, removing the package, immersing the throat swab into the SARS-CoV-2 coronavirus S protein solution with the concentration of more than 0.1mmoL for about one centimeter, and stirring for 5 seconds after completely immersing the swab head in the solution.
2. The nanoparticle solution of the example is taken out, a pharyngeal swab after sampling is inserted into the nanoparticle solution to ensure that the swab head is completely immersed in the solution, and the pharyngeal swab is used for stirring the detection reagent A for 3-5 seconds to ensure that the swab head is fully contacted with the solution;
3. taking out the third detection solution, taking out the pharyngeal swab from the nanoparticle solution, inserting the third detection solution to enable the swab head to be completely immersed in the solution, and stirring for 3-5 seconds to carry out full washing;
4. taking out the second detection solution, taking out the pharyngeal swab from the third detection solution, inserting the second detection solution to enable the swab head to be completely immersed in the solution, stirring for 3-5 seconds to enable the pharyngeal swab head to be fully and uniformly mixed with the solution, and taking out;
5. taking out a 405nm blue light source, enabling the light source to be close to the pipe wall on the premise that the second detection solution is vertical to the horizontal plane, and continuously irradiating the second detection solution for 10 seconds at an angle of 45 degrees;
6. inverting the second detection solution to make the bottle mouth face downwards, making the bottle body and the horizontal plane be 45 degrees, judging whether the solution in the tube is solidified or not is related to whether the SARS-CoV-2 coronavirus Spike protein is contained, namely if the SARS-CoV-2 coronavirus Spike protein is contained, the solution in the tube can be changed into a solid state from a liquid state after being irradiated, and the liquid level of the solution can not be inclined, thus judging the solution to be positive; otherwise, the solution is still liquid, the solution can flow, the liquid level is inclined, and the result is negative.
Test example 2
Experimental example 2 the procedure was identical to that of Experimental example 1, except that the freshly unsealed pharyngeal swab was inserted directly into the nanoparticle solution and did not carry the SARS-CoV-2 coronavirus S protein.
Test example 3
Experimental example 3 was identical to the procedure of Experimental example 1, except that 300-500 microliters of SARS-CoV-2 coronavirus S protein solution with a concentration of 50. mu.g/mL was first mixed with a volume of 1mL of nanoparticle solution, then a freshly unsealed pharyngeal swab was inserted into the nanoparticle solution to completely immerse the swab head in the solution, and the pharyngeal swab was used to agitate the detection reagent A for 3-5 seconds to ensure that the swab head was in full contact with the solution.
Test example 1 shows that: the photosensitive hydrogel solution is changed from a liquid state to a solid state after being irradiated by blue light, and the liquid level of the solution is not inclined;
test example 2 shows that: the photosensitive hydrogel solution still shows a liquid state within 5-10 seconds after the blue light irradiation, and the liquid level of the solution can be inclined;
test example 3 shows that: the photosensitive hydrogel solution still shows a liquid state within 5-10 seconds after the blue light irradiation, and the liquid level of the solution can be inclined;
experimental example 3 shows that the nanoparticle solution contaminated with the S protein antibody is immersed in a clean pharyngeal swab and then does not undergo a crosslinking and curing reaction with the photosensitive hydrogel solution after washing, which proves that the nanoparticle solution bound to the pharyngeal swab is insufficient to initiate curing of the photosensitive hydrogel solution, and that the binding firmness of the S protein virus on the pharyngeal swab can ensure that the S protein virus cannot be separated from the pharyngeal swab after being mixed with the nanoparticle solution, thereby ensuring that a sufficient amount of the photosensitive catalyst undergoes a crosslinking and curing reaction. The S protein virus was demonstrated to be from pharyngeal swabs, rather than misjudged due to contamination of the nanoparticle solution. The technical scheme of the invention can quickly judge whether the patient carries the new coronavirus or not, and the result has high accuracy.
FIG. 1 is a graph showing the results of detection with and without SARS-CoV-2 coronavirus S protein. As shown in FIG. 1, the left side 1 shows the detection result without SARS-CoV-2 coronavirus S protein, wherein the gel is in a fluid state, and the left side 2 shows the second detection solution carrying SARS-CoV-2 coronavirus S protein, and the gel is still not inclined even when the bottle body is turned over. Is always in a solidified state.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (4)
1. A kit for detecting SARS-CoV-2 coronavirus S protein is characterized by comprising a nanoparticle solution, a second detection solution and a third detection solution;
the second detection solution is a photosensitive hydrogel; the third detection solution is a cleaning solution;
the nanoparticle solution comprises a nanoparticle carrier, and an S protein clone antibody and a photosensitive catalyst are loaded on the nanoparticle carrier; the photosensitive catalyst is Irgacure 2959;
the nano-particle carrier is formed by crosslinking a polymer A and a polymer B, wherein the polymer A is a methoxy polyethylene glycol-polycaprolactone polymer, and the polymer B is a maleimide-polyethylene glycol-polycaprolactone polymer;
the nanoparticle solution was prepared as follows:
s1, preparation of a nanoparticle carrier: dissolving a polymer A and a polymer B in a solvent, and performing rotary evaporation to form a film; dissolving in deionized water, and oscillating in water bath at 50-70 deg.C to obtain nanoparticle carrier solution; refrigerating at 2-8 deg.C;
s2, S protein-loaded clone antibody: adding the nanoparticle carrier solution prepared in S1 and the S protein cloned antibody into Hepes buffer solution, and reacting at 2-8 ℃ for more than 24h to obtain an intermediate product solution for later use;
s3, supporting a photocatalyst: carrying out sulfydryl modification on the photosensitive catalyst, adding the modified photosensitive catalyst into the intermediate product solution prepared in S2, reacting for more than 24h at 3-8 ℃, putting reaction liquid into a dialysis bag for dialysis after the reaction is finished, wherein the dialysis liquid is distilled water, and obtaining the nanoparticle solution after the dialysis is finished;
the mass ratio of the polymer A to the polymer B is 1: 9;
the molar ratio of the S protein clone antibody to the polymer B is 1: 2-10;
the molar ratio of the photosensitive catalyst to the polymer B is 8-16: 1.
2. the kit for detecting SARS-CoV-2 coronavirus S protein according to claim 1, wherein the modification of the photosensitizing catalyst is specifically as follows: under the protection of nitrogen, dissolving the Irgacure2959, thioglycollic acid and p-toluenesulfonic acid in a dichloromethane solution, carrying out reflux reaction for 5-8h, and carrying out column chromatography separation after the reaction is finished to obtain a target modified product: HS-Irgacure 2959.
3. The kit for detecting SARS-CoV-2 coronavirus S protein according to claim 1, wherein the photosensitive hydrogel is methacrylated gelatin.
4. The kit for detecting SARS-CoV-2 coronavirus S protein according to claim 3, wherein the concentration of the photosensitive hydrogel is in the range of 5% w/v-25% w/v.
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