CN112675718B - Water-soluble gel film with virus enrichment function and preparation method and application thereof - Google Patents
Water-soluble gel film with virus enrichment function and preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The invention relates to a water-soluble gel film with a virus enrichment function, a preparation method and application thereof, and particularly discloses a preparation method of a chitosan water-soluble film, which comprises the following steps: 1) Preparation of chitosan salt solution: reacting chitosan with an acidic solution to obtain a filtrate, and drying to obtain chitosan salt powder; 2) Preparing a casting solution: mixing chitosan salt powder with a urea solution, and adding an additive A and an additive B to obtain a membrane casting solution; 3) Preparing the membrane casting solution into a membrane; 4) Fixing the membrane with a solidifying liquid; 5) Freeze-drying the fixed film to obtain a chitosan water-soluble film; wherein the additive A is a plasticizer, and the additive B is a surfactant; the coagulating liquid is selected from one or two of lower alcohol or acetone. The water-soluble gel film of the invention subverts the method in the field of enrichment detection in air in the prior art, and does not need filtration.
Description
Technical Field
The invention relates to a water-soluble gel film with a virus enrichment function.
Background
The pandemic of the virus has great influence on the physical health and the psychology of people, and some viruses have long latent period, for example, the new coronavirus is extremely infectious in latent period, can be transmitted by contacting and air, can survive for several days in the outside, has no medicine for treatment, has long course of disease, much severe disease, crowds medical resources and can overwhelm a regional medical system.
Viral RNA can be present on particles in the outdoor air, viruses can cluster with outdoor fine particles, and by reducing the diffusion coefficient, the persistence of viruses in the atmosphere is enhanced. The virus can be transmitted through fine particles in the air, and infected persons can transmit the virus under the conditions of cough, sneeze, speaking and even breathing, so that the detection of the virus in the air is particularly important for preventing and treating the virus.
The sampler for microorganisms in air can be classified into the following types: (1) an anderson solid impact sampler; (2) a centrifugal sampler; (3) a liquid impact sampler; (4) a cyclonic sampler; (5) a high-capacity electrostatic precipitation sampler; (6) a sampler of the filtering type. At present, solid impact type and filtration type are mostly adopted and are superior to other collection modes, however, a detection method combining filter collection and Polymerase Chain Reaction (PCR) is more advantageous than a traditional method combining impact type sampler and cell culture, the filtration type sampler can collect samples for a long time (> 30 h) and can carry out on-site multiple sampling, and the impact type sampler is not suitable for low-temperature and long-time collection and is not suitable for on-site multiple sampling.
The common filtering type filter membrane adopts a cellulose filter membrane which is not water-soluble, and because virus particles are small and are easy to clamp in membrane gaps, elution is difficult, so that the actual detection sensitivity is not high and the operation difficulty is high. Therefore, if a water-soluble membrane is adopted and is directly dissolved in water after the virus is trapped and enriched, the traditional filtration and elution mode can be overturned, and the trapped virus is completely used for detection, so that the detection sensitivity is greatly improved.
Disclosure of Invention
In view of the above, the present invention aims to provide a water-soluble gel membrane with a virus enrichment function, which has a 3D network structure, dense and permeable membrane pores, high virus enrichment capacity, and rapid dissolution in an aqueous solution.
One aspect of the present invention provides a method for preparing a chitosan water-soluble film, comprising the steps of:
1) Preparation of chitosan salt solution: reacting chitosan with an acidic solution to obtain a filtrate, and drying to obtain chitosan salt powder;
2) Preparing a casting solution: mixing chitosan salt powder with a urea solution, and adding an additive A and an additive B to obtain a membrane casting solution;
3) Preparing the membrane casting solution into a membrane;
4) Fixing the membrane with a solidifying liquid;
5) Freeze-drying the fixed film to obtain a chitosan water-soluble film;
wherein the additive A is a plasticizer, and the additive B is a surfactant;
the coagulating liquid is selected from one or two of lower alcohol or acetone, preferably, the lower alcohol is selected from one or more of methanol, glycerol, ethanol, ethylene glycol, isopropanol and tert-butanol.
In the technical scheme of the invention, the acidic solution is selected from a mixed solution of inorganic acid and lower alcohol, preferably, the inorganic acid is selected from hydrochloric acid, sulfuric acid or nitric acid; the lower alcohol is selected from one or more of methanol, glycerol, ethanol, ethylene glycol, isopropanol, and tert-butanol. In some preferred embodiments, the volume ratio of the inorganic acid to the lower alcohol is 1-2: 3-5. In a preferred embodiment, the acidic solution is selected from a mixture of hydrochloric acid and ethanol.
In the technical scheme of the invention, the additive A is one or a combination of more of glycerol, ethylene glycol, polyethylene glycol and propylene glycol.
In the technical scheme of the invention, the additive B is one or more of Cetyl Triethyl Ammonium Bromide (CTAB), stearic acid, sodium dodecyl benzene sulfonate, lecithin, span, tween, betaine and fatty glyceride.
In the solution according to the invention, the concentration of the urea solution is 0.1-2 wt.%, preferably 0.8-1.2 wt.%.
In the technical scheme of the invention, the concentration of the additive A in the casting solution is 0.1-10wt%.
In the technical scheme of the invention, the concentration of the additive B in the casting solution is 0.1-8wt%.
In the technical scheme of the invention, the concentration of the chitosan salt in the casting solution is 0.1-10wt%.
In the technical scheme of the invention, the method for preparing the film in the step 3) is selected from blade coating, spraying or casting.
The invention also provides the chitosan water-soluble film obtained by the preparation method.
In the technical scheme of the invention, the chitosan water-soluble film has a structure that film holes are through and are stacked layer by layer.
In a further aspect of the invention, there is provided an air filtration device or component comprising as a filtration material a chitosan water-soluble film as described herein.
In a further aspect the invention provides the use of a water-soluble film of chitosan as a filter material, preferably as a filter material for airborne microorganisms.
In still another aspect, the present invention provides a method for separating microorganisms by filtration, comprising the steps of:
i) The chitosan water-soluble film is used as a filtering material for filtering microorganisms in air;
ii) dissolving the chitosan water-soluble film and then obtaining a solution of the microorganism.
A method of detecting airborne microorganisms comprising the steps of:
i) Filtering microorganisms in the air by using the chitosan water-soluble film as claimed in claim 6 as a filtering material;
ii) dissolving the chitosan water-soluble film and then obtaining an enriched microbial solution;
iii) The microbial solution in step ii) is used for qualitative or quantitative detection.
In the technical scheme of the invention, the microorganism is selected from viruses, bacteria or fungi.
In the technical scheme of the invention, the qualitative or quantitative detection comprises culturing the microorganisms or directly detecting the nucleic acid of the microorganisms.
Advantageous effects
1) Traditional filtration filter membrane adopts cellulose filter membrane water-insoluble, because the viral particle is less presss from both sides in the membrane gap easily, causes the elution difficulty, leads to actual detection sensitivity not high and the operation degree of difficulty big. The water-soluble gel film is directly dissolved in water after intercepting the enriched viruses, so that the traditional filtration and elution mode can be reversed, the intercepted viruses are all used for detection, and the detection sensitivity is greatly improved. Moreover, the chitosan membrane is applied to the field of virus (including but not limited to new coronavirus) enrichment detection for the first time, and has a good application prospect.
2) The chitosan filter membrane disclosed by the invention is simple in preparation process, has a 3D network structure, compact and transparent membrane pores, has high-efficiency virus enrichment capacity, and can be quickly dissolved in an aqueous solution.
Drawings
FIG. 1 is a photograph of a water-soluble gel film having a virus-enriching function according to a first embodiment of the present invention; it can be seen from the figure that the surface of the hydrogel film is relatively flat.
FIG. 2 is an electron micrograph of a water-soluble gel film with virus enrichment function according to a first embodiment of the present invention; it can be seen from the figure that the hydrogel film has three-dimensional network structure pores on both sides, and the pores of the film are through.
FIG. 3 is a comparison of a water-soluble gel film with virus enrichment function in deionized water for two minutes and before and after water dissolution; it can be seen from the figure that the hydrogel film was completely dissolved in deionized water.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.
EXAMPLE 1 preparation of Water-soluble gel film
1) 196g of chitosan powder and 784mL of hydrochloric acid ethanol solution (the volume ratio of the hydrochloric acid solution to the ethanol is 1.5: 4) are uniformly mixed, stirred and reacted for 2 hours, filtered to obtain 575mL of filtrate and 262.5g of filter residue, the filter residue is washed by 500mL of ethanol, the filter residue is dried in a vacuum drying oven at 65 ℃ after being combined, and the mixture is crushed to 80 meshes to obtain 219g of chitosan hydrochloride.
2) Dissolving the chitosan hydrochloride prepared in the above step into 1% of urea aqueous solution to form a mixed solution with the chitosan hydrochloride concentration of 5wt%, then adding a proper amount of additive A and additive B to make the additive A concentration of 5wt% and the additive B concentration of 4wt%, and strongly stirring for 0.5-3 h to obtain a casting solution; the additive A is glycerol; the additive B is CTAB.
3) Pouring the casting solution on a glass plate and scraping to form a film;
4) Placing the glass plate containing the gel film into a solidification solution for replacement (> 24 h) to obtain a chitosan wet film; the coagulating liquid is an alcohol solvent;
5) And (3) freeze-drying the obtained chitosan wet membrane (for more than 24 h) to obtain the water-soluble gel membrane with the virus enrichment function.
EXAMPLE 2 preparation of Water-soluble gel film
The same procedure as in example 1 was followed except that the volume ratio of the hydrochloric acid solution to ethanol was 1: 5. Water-soluble gel films can also be prepared.
EXAMPLE 3 preparation of Water-soluble gel film
The same procedure as in example 1 was followed except that the additive A was polyethylene glycol, the concentration of the additive A was 2% by weight, the additive B was stearic acid, and the concentration of the additive B was 6% by weight. Water-soluble gel films can also be prepared.
EXAMPLE 4 preparation of Water-soluble gel film
The same procedure as in example 1 was followed except that the additive A was ethylene glycol and the concentration of the additive A was 7wt%, the additive B was betaine and the concentration of the additive B was 1wt%. Water-soluble gel films can also be prepared.
Example 5 Virus enrichment experiment
1) Sterilizing, before the suction filtration experiment, sterilizing and drying experimental instruments (the surface of a suction pump, an atomizer and a beaker) by using 75% alcohol;
2) Selecting pseudovirus samples (5-10 mL) with different concentrations according to experimental requirements, putting the pseudovirus samples into an atomizer, and fixing a water-soluble gel membrane selected from any one of the embodiments 1-4 on a suction pump;
3) Placing a suction pump in an atomization system, closing a top cover to form a closed space, starting an atomizer, and working for 1-5 min to ensure uniform water mist distribution in the atomization system;
4) Switching on a power supply of a suction pump, and performing suction filtration for different durations (1-60 min) according to different requirements;
5) After the suction filtration is finished, taking out the suction pump, unloading the water-soluble film and dissolving the water-soluble film in deionized water;
6) Nucleic acid detection an aqueous solution in which a water-soluble membrane is dissolved. And (3) extracting the virus nucleic acid in the aqueous solution by using nano magnetic beads, and detecting the nucleic acid in the aqueous solution dissolving the water-soluble film by performing automatic amplification on the extracted product of the virus nucleic acid based on an RT-PCR (reverse transcription-polymerase chain reaction) technology.
Claims (16)
1. A preparation method of a chitosan water-soluble film comprises the following steps:
1) Preparation of chitosan salt powder: reacting chitosan with an acidic solution to obtain a filtrate, and drying to obtain chitosan salt powder;
2) Preparing a casting solution: mixing chitosan salt powder with a urea solution, and adding an additive A and an additive B to obtain a membrane casting solution;
3) Preparing the membrane casting solution into a membrane;
4) Fixing the membrane with a solidifying liquid;
5) Freeze-drying the fixed film to obtain a chitosan water-soluble film;
wherein, the additive A is one or a combination of more of glycerol, glycol, polyethylene glycol and propylene glycol, and the additive B is a surfactant; the coagulating liquid is selected from one or two of lower alcohol or acetone.
2. The production method according to claim 1, wherein the concentration of the chitosan salt in the casting solution is 0.1 to 10wt%.
3. The production method according to claim 2, wherein the concentration of the additive A in the dope solution is 0.1 to 10wt%.
4. The method according to claim 1, wherein the additive B is one or more selected from Cetyl Triethyl Ammonium Bromide (CTAB), stearic acid, sodium dodecyl benzene sulfonate, lecithin, span, tween, betaine and fatty glyceride.
5. The production method according to claim 1, wherein the concentration of the additive B in the dope solution is 0.1 to 8wt%.
6. The method according to claim 1, wherein the acidic solution is a mixed solution of an inorganic acid and a lower alcohol.
7. The method according to claim 6, wherein the inorganic acid is selected from hydrochloric acid, sulfuric acid or nitric acid; the lower alcohol is selected from one or more of methanol, glycerol, ethanol, ethylene glycol, isopropanol, and tert-butanol.
8. The method according to claim 7, wherein the volume ratio of the inorganic acid to the lower alcohol is 1-2: 3-5.
9. The method of claim 1, wherein the film is formed in step 3) by a method selected from the group consisting of knife coating, spray coating, and casting.
10. The water-soluble chitosan film obtained by the preparation method according to any one of claims 1 to 9, wherein the interior of the water-soluble chitosan film has a structure that membrane pores are penetrated and stacked layer by layer.
11. An air filtration device or component comprising the chitosan water-soluble film of claim 10 as a filtration material.
12. Use of the chitosan water-soluble film of claim 10 as an air filter material.
13. A method for the filtration separation of microorganisms in air, comprising the steps of:
i) Filtering microorganisms in the air by using the chitosan water-soluble film as claimed in claim 10 as a filtering material;
ii) dissolving the chitosan water-soluble film and then obtaining a solution enriched in microorganisms.
14. The method of claim 13, wherein the microorganism is selected from the group consisting of a virus, a bacterium, and a fungus.
15. A method of detecting airborne microorganisms comprising the steps of:
i) Filtering microorganisms in the air by using the chitosan water-soluble film as claimed in claim 10 as a filtering material;
ii) dissolving the chitosan water-soluble film and then obtaining an enriched microbial solution;
iii) The microbial solution in step ii) is used for qualitative or quantitative detection.
16. The method of claim 15, wherein the microorganism is selected from the group consisting of a virus, a bacterium, and a fungus.
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| JPS56112937A (en) * | 1980-02-08 | 1981-09-05 | Mitsubishi Rayon Co Ltd | Preparation of chitosan molded product |
| KR20010016131A (en) * | 2000-11-10 | 2001-03-05 | 손태원 | Amorphous Water-soluble chitosan films |
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| CN109022560A (en) * | 2018-09-07 | 2018-12-18 | 天津市武清区疾病预防控制中心 | The detection method of pathogenic microorganism is carried in a kind of atmospheric haze |
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| CN112675718A (en) | 2021-04-20 |
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