CN113173576A - Graphene aerogel, preparation method and application thereof, and elution method of food-borne pathogenic microorganisms on graphene aerogel - Google Patents
Graphene aerogel, preparation method and application thereof, and elution method of food-borne pathogenic microorganisms on graphene aerogel Download PDFInfo
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Abstract
The invention provides a graphene aerogel, a preparation method and application thereof, and an elution method of food-borne pathogenic microorganisms on the graphene aerogel, and belongs to the technical field of food-borne pathogenic microorganism detection. The graphene aerogel provided by the invention is large in specific surface area, has a three-dimensional network structure, is rich in nitrogen-containing active groups, and can efficiently adsorb and enrich food-borne pathogenic microorganisms in a sample. Meanwhile, the elution method provided by the invention can efficiently elute food-borne pathogenic microorganisms adsorbed on the graphene aerogel, so that the food-borne pathogenic microorganisms can be effectively enriched and detected.
Description
Technical Field
The invention relates to the technical field of food-borne pathogenic microorganism detection, in particular to a graphene aerogel, a preparation method and application thereof, and an elution method of food-borne pathogenic microorganisms on the graphene aerogel.
Background
The threat of food-borne pathogenic microorganisms to the health of people has attracted great attention, but the content of pathogenic microorganisms in food is lower than that of clinical samples, and the components of the food samples are complex and easy to interfere, so that the enrichment of pathogens becomes an important link for restricting the detection of the food-borne pathogenic microorganisms. The conventional methods for enriching pathogens include a culture method, a precipitation method, an ultracentrifugation method, an immunomagnetic bead method and the like, but the methods have the disadvantages of complex operation, high equipment requirement, low efficiency, high price and the like, and have certain limitations on food samples.
Disclosure of Invention
In view of the above, the present invention provides a graphene aerogel, a preparation method and an application thereof, and an elution method of food-borne pathogenic microorganisms on the graphene aerogel. The graphene aerogel provided by the invention has the advantages of high specific surface area, three-dimensional network structure, rich nitrogen-containing active groups, capability of efficiently adsorbing and enriching food-borne pathogenic microorganisms in a sample, simplicity in operation and low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a graphene aerogel, which has a three-dimensional network structure and is loaded with active groups, wherein the active groups comprise carboxyl, carbonyl, hydroxyl, epoxy and nitrogen-containing groups.
The invention also provides a preparation method of the graphene aerogel, which comprises the following steps:
mixing graphene oxide with water, and performing ultrasonic treatment to obtain a graphene oxide dispersion liquid;
mixing the graphene oxide dispersion liquid, ammonia water, triethylene tetramine and boric acid, and carrying out redox reaction to obtain graphene hydrogel;
and washing, freezing and drying the graphene hydrogel to obtain the graphene aerogel.
Preferably, the mass concentration of the ammonia water is 25-28%, and the concentration of the boric acid is 2 mg/mL; the dosage ratio of the graphene oxide, the ammonia water, the triethylene tetramine and the boric acid is 10 mg: 10-40 μ L: 16-40 μ L: 36-160 μ L.
Preferably, the temperature of the oxidation-reduction reaction is 120-125 ℃ and the time is 6-7 h.
Preferably, the washing comprises water washing and ethanol washing which are carried out in sequence; the washing mode is washing; the ethanol washing mode is soaking, and the soaking time is 8-16 h; the reagent for washing with ethanol is an ethanol water solution, and the volume concentration of the ethanol water solution is 5-20%.
Preferably, the freeze-drying comprises the steps of: pre-freezing at-80 deg.C for 8 hr, and vacuum drying for 24 hr.
The invention also provides the application of the graphene aerogel in the technical scheme or the application of the graphene aerogel prepared by the preparation method in the technical scheme in enriching food-borne pathogenic microorganisms.
Preferably, the graphene aerogel, when enriched in food-borne pathogenic microorganisms, comprises the following steps:
and (3) passing the solution to be detected through the graphene aerogel, and adsorbing the food-borne pathogenic microorganisms in the solution to be detected on the graphene aerogel.
The invention also provides an elution method of food-borne pathogenic microorganisms on the graphene aerogel, which comprises the following steps:
placing the graphene aerogel adsorbed with the food-borne pathogenic microorganisms at the bottom of an injector, eluting by using an eluent, and collecting an elution solution;
mixing the elution solution with a treatment reagent to obtain a solution to be treated;
standing and centrifuging the solution to be treated, fixing the volume of the obtained centrifugal precipitate by using a PBS buffer solution, and taking supernatant to perform bacterial culture or polymerase chain reaction;
the pH value of the eluent is 9;
the eluent is an aqueous solution comprising the following components in mass concentration: 15g/L of sodium chloride, 20g/L of sodium hydroxide, 38.5g/L of glycine, 15g/L of beef powder, 30g/L of peptone, 34g/L of Tris-Base and 58.7g/L of MOPS;
the treatment reagent is PEG-6000 or PEG-8000, and the dosage ratio of the elution solution to the treatment reagent is 10 mL: 1g of the total weight of the composition.
The invention provides a graphene aerogel, which has a three-dimensional network structure and is loaded with active groups, wherein the active groups comprise carboxyl, carbonyl, hydroxyl, epoxy and nitrogen-containing groups. The graphene aerogel provided by the invention has a three-dimensional network structure, is loaded with a large number of active groups, greatly improves the adsorption enrichment property of the graphene aerogel on food-borne pathogenic microorganisms, and is simple to operate and high in enrichment rate when the graphene aerogel is used for enriching the food-borne pathogenic microorganisms.
The invention also provides a preparation method of the graphene aerogel in the technical scheme, ammonia water and triethylene tetramine are used as reducing agents, and nitrogen-containing groups are introduced at the same time, so that the graphene aerogel is rich in nitrogen-containing active groups; boric acid is used as a cross-linking agent to endow the graphene aerogel with a three-dimensional network structure; and (4) freeze-drying to endow the graphene aerogel with a large number of pores.
The invention also provides the application of the graphene aerogel in the technical scheme in the enrichment of food-borne pathogenic microorganisms, and the graphene aerogel has high enrichment efficiency which can reach more than 99% when being used for enriching the food-borne pathogenic microorganisms, and is simple to operate.
The invention also provides an elution method of the food-borne pathogenic microorganisms on the graphene aerogel, the elution method can efficiently elute the food-borne pathogenic microorganisms adsorbed on the graphene aerogel for detection, and the elution recovery rate is over 90%.
Drawings
Fig. 1 is a photograph of a graphene hydrogel;
fig. 2 is a photograph of a graphene aerogel;
FIGS. 3 to 6 are scanning electron micrographs of the graphene oxide used at different magnifications;
fig. 7 to 10 are scanning electron micrographs of the graphene aerogel obtained in example 1 at different magnifications;
FIGS. 11 to 12 are transmission electron microscope photographs of graphene oxide at different magnifications;
FIGS. 13 to 14 are transmission electron micrographs of the graphene aerogel obtained in example 1 at different magnifications;
fig. 15 is a graph of energy spectrum analysis of graphene aerogel;
fig. 16 is a C1s spectrum of graphene oxide;
fig. 17 is a C1s spectrum of graphene aerogel;
fig. 18 is an O1s spectrum of graphene oxide;
fig. 19 is an O1s spectrum of graphene aerogel;
fig. 20 is an N1s spectrum of graphene aerogel;
fig. 21 is an infrared spectrum of graphene oxide;
fig. 22 is an infrared spectrum of a graphene aerogel;
fig. 23 is a raman spectrum of graphene oxide and graphene aerogel;
fig. 24 is an XRD pattern of graphene oxide and graphene aerogel;
fig. 25 is a schematic contact angle diagram of a graphene aerogel;
fig. 26 is a negative staining electron micrograph of graphene aerogel enriched hepatitis a virus.
Detailed Description
The invention provides a graphene aerogel, which has a three-dimensional network structure and is loaded with active groups, wherein the active groups comprise carboxyl, carbonyl, hydroxyl, epoxy and nitrogen-containing groups.
The graphene aerogel provided by the invention has a three-dimensional network structure, is loaded with a large number of active groups, greatly improves the adsorption enrichment property of the graphene aerogel on food-borne pathogenic microorganisms, and is simple to operate and high in enrichment rate when the graphene aerogel is used for enriching the food-borne pathogenic microorganisms.
The invention also provides a preparation method of the graphene aerogel, which comprises the following steps:
mixing graphene oxide with water, and performing ultrasonic treatment to obtain a graphene oxide dispersion liquid;
mixing the graphene oxide dispersion liquid, ammonia water, triethylene tetramine and boric acid, and carrying out redox reaction to obtain graphene hydrogel;
and washing, freezing and drying the graphene hydrogel to obtain the graphene aerogel.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The graphene oxide dispersion liquid is obtained by mixing and ultrasonically treating graphene oxide and water.
In the invention, the concentration of the graphene oxide dispersion liquid is preferably 2.5-3.0 mg/mL. In the present invention, the time of the sonication is preferably 1 h. In the present invention, the ultrasound enables graphene oxide to be uniformly dispersed in water, forming a graphene oxide dispersion.
After the graphene oxide dispersion liquid is obtained, the graphene oxide dispersion liquid, ammonia water, triethylene tetramine and boric acid are mixed for oxidation-reduction reaction, and graphene hydrogel is obtained.
In the present invention, the mass concentration of the ammonia water is preferably 25 to 28%. In the present invention, the concentration of boric acid is preferably 2 mg/mL. In the present invention, the dosage ratio of the graphene oxide, the ammonia water, the triethylene tetramine and the boric acid is preferably 10 mg: 10-40 μ L: 16-40 μ L: 36-160 μ L, more preferably 10 mg: 40 μ L of: 16 μ L of: 36 μ L.
In the invention, the temperature of the oxidation-reduction reaction is preferably 120-125 ℃, and more preferably 120-123 ℃; the time is preferably 6 to 7 hours, and more preferably 6.5 hours.
According to the invention, graphene oxide is used as an oxidant, ammonia water and triethylene tetramine are used as reducing agents to provide nitrogen-containing groups, boric acid is used as a cross-linking agent, and an oxidation-reduction reaction is carried out, so that the obtained graphene hydrogel is rich in nitrogen-containing active groups and has a three-dimensional network structure.
After the graphene hydrogel is obtained, the graphene hydrogel is washed, frozen and dried to obtain the graphene aerogel.
In the present invention, the washing preferably comprises: washing with water and washing with ethanol in sequence, wherein the washing mode is preferably washing; the dosage ratio of the water for washing to the graphene hydrogel is preferably 300 mL: 5cm3. In the present invention, the ethanol washing is preferably performed by soaking, and the soaking time is preferably 8 hours. In the invention, the ethanol washing reagent is preferably an ethanol aqueous solution, and the volume concentration of the ethanol aqueous solution is preferably 5-20%; the dosage ratio of the ethanol aqueous solution to the graphene hydrogel is preferably 100 mL: 5cm3。
In the present invention, the freeze-drying preferably comprises the steps of: pre-freezing at-80 deg.C for 8 hr, and vacuum drying for 24 hr.
The invention also provides the application of the graphene aerogel in the technical scheme or the application of the graphene aerogel prepared by the preparation method in the technical scheme in enriching food-borne pathogenic microorganisms. In the present invention, the food-borne pathogenic microorganism includes hepatitis A virus, norovirus, hepatitis E virus, rotavirus, astrovirus, enterovirus, adenovirus, salmonella, Vibrio parahaemolyticus, pathogenic Escherichia coli, Clostridium botulinum, Brucella, Campylobacter jejuni, Shigella, Bacillus cereus, Staphylococcus aureus, Listeria, Vibrio cholerae, Aspergillus, Penicillium, Giardia, or Cryptosporidium.
In the present invention, when the graphene aerogel is used for enriching food-borne pathogenic microorganisms, the method preferably comprises the following steps:
and (3) passing the solution to be detected through the graphene aerogel, and adsorbing the food-borne pathogenic microorganisms in the solution to be detected on the graphene aerogel.
The mode of the solution to be detected passing through the graphene aerogel is not particularly limited, and in a specific embodiment of the invention, the graphene aerogel is preferably filled at the bottom of an injector, and the solution to be detected is injected into the injector, so that the purpose that the solution to be detected passes through the graphene aerogel is achieved.
The invention also provides an elution method of food-borne pathogenic microorganisms on the graphene aerogel, which comprises the following steps:
placing the graphene aerogel adsorbed with the food-borne pathogenic microorganisms at the bottom of an injector, eluting by using an eluent, and collecting an elution solution;
mixing the elution solution with a treatment reagent to obtain a solution to be treated;
and standing and centrifuging the solution to be treated, fixing the volume of the obtained centrifugal precipitate by using a PBS buffer solution, and taking supernatant to perform bacterial culture or polymerase chain reaction.
According to the invention, the graphene aerogel adsorbed with food-borne pathogenic microorganisms is placed at the bottom of an injector, elution is carried out by adopting eluent, and an elution solution is collected.
In the invention, the graphene aerogel adsorbing the food-borne pathogenic microorganisms is preferably obtained by the technical scheme.
In the present invention, the pH of the eluent is 9. In the present invention, the eluent is an aqueous solution comprising the following components in mass concentration: 15g/L of sodium chloride, 20g/L of sodium hydroxide, 38.5g/L of glycine, 15g/L of beef powder, 30g/L of peptone, 34g/L of Tris-Base and 58.7g/L of MOPS.
In the present invention, the dosage ratio of the graphene aerogel adsorbing the food-borne pathogenic microorganisms to the eluent is preferably 30 mg: 30 mL.
In the present invention, the elution is preferably performed in a dynamic manner; the dynamic elution preferably comprises the steps of: and adding the eluent into an injector, dripping or injecting the eluent by gravity, and collecting an elution solution below the injector.
After the elution solution is obtained, the elution solution is mixed with a treatment reagent to obtain a solution to be treated.
In the invention, the treatment reagent is preferably PEG-6000 or PEG-8000, and the dosage ratio of the elution solution to the treatment reagent is 10 mL: 1g of the total weight of the composition.
After the liquid to be treated is obtained, the liquid to be treated is stood and centrifuged, the obtained centrifugal precipitate is subjected to constant volume by using PBS buffer solution, and the supernatant is taken for bacterial culture or polymerase chain reaction.
In the invention, the temperature of the standing is preferably 4 ℃, and the time of the standing is preferably 16 h; the rotation speed of the centrifugation is preferably 10000r/min, and the time is preferably 30 min.
The parameters of the bacterial culture and Polymerase Chain Reaction (PCR) are not specifically limited, and the conventional selection can be carried out according to different food-borne pathogenic microorganisms.
The following will describe the graphene aerogel provided by the present invention, the preparation method and the application thereof, and the method for eluting food-borne pathogenic microorganisms on the graphene aerogel in detail by referring to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparing the graphene aerogel:
mixing 30mg of graphene oxide with 12mL of deionized water, and carrying out ultrasonic treatment for 1h to obtain 2.5mg/mL of graphene oxide dispersion liquid;
adding 120 mu L of ammonia water (with the mass concentration of 25-28%), 48 mu L of triethylene tetramine and 108 mu L of boric acid (with the concentration of 2mg/mL) into the graphene oxide dispersion liquid, fully mixing, putting into a polytetrafluoroethylene lining of a 50mL reaction kettle, and reacting for 6 hours at 120 ℃ to obtain graphene hydrogel, wherein the picture of the obtained graphene hydrogel is shown in figure 1;
washing the obtained graphene hydrogel with 300mL of pure water, soaking the graphene hydrogel in 100mL of an ethanol aqueous solution with the volume concentration of 10% for 8 hours, pre-freezing the graphene hydrogel at-80 ℃ for 8 hours, and performing vacuum freeze drying for 24 hours to obtain the graphene aerogel, wherein a picture of the obtained graphene aerogel is shown in figure 2.
FIGS. 3 to 6 are scanning electron micrographs of the used graphene oxide at different magnifications, which can be seen from FIGS. 3 to 6: the graphene oxide is of a lamellar structure, and the surface of the graphene oxide is provided with folds, the reason is that concentrated sulfuric acid oxidizes the surface of the graphene oxide during production and preparation, the graphene oxide is represented as a surface defect that the folds curl and is rich in various oxygen-containing functional groups, and more reaction sites are provided for subsequent hydrothermal reaction.
Fig. 7 to 10 are scanning electron micrographs of the graphene aerogel obtained in the present embodiment under different magnifications, and it can be seen from fig. 7 to 10 that: the graphene aerogel has an extremely abundant pore structure and is represented as a honeycomb-like structure with regular arrangement. The structure is derived from a self-assembly process that graphene oxide lamella is stable, ordered and controllable during preparation. In the vacuum freeze drying link, the space left after ice crystal sublimation forms abundant pore structures, the diameter is several microns to dozens of microns, and the pore structures with micropores, mesopores and macropores which coexist provide a larger specific surface area for the graphene aerogel, so that the adsorption performance of the graphene aerogel is obviously enhanced.
FIGS. 11 to 12 are transmission electron micrographs of graphene oxide at different magnifications, as can be seen from FIGS. 11 to 12: the graphene oxide is transparent and flexible and has a small amount of wrinkles.
FIGS. 13 to 14 are transmission electron micrographs of graphene aerogel under different magnifications, and it can be seen from FIGS. 13 to 14 that: the graphene aerogel has a large number of folds, and the graphene oxide is folded, curled and self-assembled in the hydrothermal reaction process to generate a large number of folds, so that the graphene aerogel can have a stable three-dimensional network structure and an active site.
Fig. 15 is a graph of energy spectrum analysis of graphene aerogel, and it can be seen from fig. 15 that: carbon atoms, oxygen atoms, nitrogen atoms and boron atoms are uniformly distributed on the graphene plane.
Fig. 16 is a C1s spectrogram, fig. 17 is a C1s spectrogram, fig. 18 is an O1s spectrogram, fig. 19 is an O1s spectrogram, and fig. 20 is an N1s spectrogram of graphene aerogel. As can be seen from FIGS. 16-20: the graphene oxide has a complexAnd the hybrid C1s spectrum comprises components such as sp2-C, sp3-C, C-O, C ═ O and O ═ C-O, the binding energies of the components are respectively 284.0eV, 284.7eV, 286.6eV, 287.8eV and 288.8eV, and the graphene aerogel also comprises extra C-N, and the binding energy is 285.5 eV. Compared with graphene oxide, the C-O content of the graphene aerogel is greatly reduced, which indicates that most oxygen-containing functional groups of the graphene oxide are reduced in the synthesis process. The O1s peaks for graphene oxide and graphene aerogel have the same three components, C O, C-O and OH, respectively, with binding energies of 530.9eV, 532.2eV, and 533.2eV, respectively. The N1s in the graphene aerogel has three peaks, which show that nitrogen atoms added during synthesis are successfully connected to the graphene plane, namely N-H, C-N and RON3The binding energies were 398.8eV, 400.5eV and 406.0eV, respectively.
Fig. 21 is an infrared spectrum of graphene oxide, fig. 22 is an infrared spectrum of graphene aerogel, and a comparison between fig. 21 and fig. 22 shows that: the absorption peak of chemical bonds such as-COOH and-COO-in the graphene oxide after treatment is obviously reduced, and the absorption peak of C ═ C appears, so that the chemical bonds such as-COOH and-COO-can be effectively reduced through treatment, the relative content of functional groups such as-COOH and-COO-is reduced, and the relative content of C ═ C is increased.
Fig. 23 is a raman spectrum of graphene oxide and graphene aerogel, wherein the black curve is graphene oxide and the red curve is graphene aerogel. As can be seen from fig. 23: both the D peak and the G peak are Raman characteristic peaks of the carbon atom crystal, and the D peak (1331 cm)-1) Represented by a defect in the lattice of carbon atoms, the G peak (1593 cm)-1) Representative is the in-plane stretching vibration of carbon atom sp2 hybridization. The I (D)/I (G) values are generally used to characterize the degree of defect in the carbon atom crystal. The value of I (D)/I (G) of the graphene aerogel is higher than that of graphene oxide, which shows that the graphene aerogel has higher carbon atom crystal defect degree, more active sites when adsorbing viruses and better electrochemical performance.
Fig. 24 is an XRD spectrum of graphene oxide and graphene aerogel, and it can be seen from fig. 24 that: the graphene oxide curve has a diffraction peak around 2 theta 12 degrees, which is a characteristic peak of a relatively typical graphene oxide. The graphene aerogel synthesized by the hydrothermal method does not have a strong diffraction peak near 2 theta 12 degrees, but has a new diffraction peak at 2 theta 22.4 degrees. Due to the fact that the graphene oxide is rich in functional groups, steric hindrance effect of the graphene oxide on space is obvious, and interlayer spacing is large; a large number of functional groups in the graphene aerogel are reduced, the steric effect is obviously weakened, and therefore the interlayer spacing is reduced.
Fig. 25 is a schematic contact angle diagram of a graphene aerogel. Contact angle is a measure of how well a liquid wets a solid. When the liquid is water, theta is less than 90 degrees, which means that the solid is hydrophilic and is easy to wet; when θ >90 °, it means that the solid material is hydrophobic and not easily wetted. The graphene aerogel theta synthesized by the traditional method is more than 90 degrees, is a typical hydrophobic material, does not absorb water, and can be used for adsorbing organic pollutants in water. If the material is very hydrophobic, it cannot be enriched with microorganisms. In the graphene aerogel provided by the invention, nitrogen is considered to be introduced to improve the hydrophilicity at the beginning of synthesis. As shown in fig. 25, the average contact angle of the graphene aerogel provided by the invention is about 34 °, which indicates that the effect of introducing nitrogen to improve hydrophilicity is obvious, and the better hydrophilicity can greatly improve the speed of treating a water sample, and provide a good mass transfer channel for virus adsorption and elution.
Elemental analysis was performed on graphene oxide and graphene aerogel, and the results are shown in table 1.
Table 1 elemental composition analysis results of graphene oxide and graphene aerogel
As can be seen from table 1: the content of oxygen element is reduced from 39.67% in the graphene oxide to 16.03% in the graphene aerogel, which shows that a large amount of oxygen-containing groups are reduced and removed, and the content of carbon element is increased. The nitrogen element is successfully doped into the graphene aerogel, and the content of the nitrogen element is 14.82%, so that the material is endowed with better hydrophilicity. Boron is used as a cross-linking agent and is introduced into the graphene aerogel, and the content of the boron is 0.15%.
Example 2
Preparing the graphene aerogel:
mixing 30mg of graphene oxide with 10mL of water, and carrying out ultrasonic treatment for 1h to obtain 3mg/mL of graphene oxide dispersion liquid; adding 90 mu L of ammonia water (with the mass concentration of 25-28%), 60 mu L of triethylene tetramine and 120 mu L of boric acid (with the concentration of 2mg/mL) into the graphene oxide dispersion liquid, fully mixing, putting into a polytetrafluoroethylene lining of a 50mL reaction kettle, and reacting for 6 hours at 120 ℃ to obtain graphene hydrogel; and (3) washing the obtained graphene hydrogel with 200mL of pure water, soaking the graphene hydrogel in 100mL of 20% ethanol aqueous solution in volume concentration for 8h, pre-freezing the graphene hydrogel at-80 ℃ for 8h, and performing vacuum freeze drying for 24h to obtain the graphene aerogel.
Example 3
Preparing the graphene aerogel:
mixing 30mg of graphene oxide with 10mL of water, and carrying out ultrasonic treatment for 1h to obtain 3mg/mL of graphene oxide dispersion liquid; adding 120 mu L of ammonia water (with the mass concentration of 25-28%), 90 mu L of triethylene tetramine and 150 mu L of boric acid (with the concentration of 2mg/mL) into the graphene oxide dispersion liquid, fully mixing, putting into a polytetrafluoroethylene lining of a 50mL reaction kettle, and reacting for 6.5 hours at 125 ℃ to obtain graphene hydrogel; and (3) washing the obtained graphene hydrogel with 200mL of pure water, soaking the graphene hydrogel in 100mL of 20% ethanol aqueous solution in volume concentration for 8h, pre-freezing the graphene hydrogel at-80 ℃ for 8h, and performing vacuum freeze drying for 24h to obtain the graphene aerogel.
Example 4
The graphene aerogel obtained in example 1 is used for enriching hepatitis a virus in an aqueous solution:
place about 30mg of graphene aerogel (about 2cm in diameter, about 1.5cm in height) into the bottom of a 20mL syringe;
200mL of pure water is taken, autoclaved at 121 ℃, and added with the water with the order of magnitude of 106Copying hepatitis A virus to obtain an aqueous solution containing hepatitis A virus;
pouring the aqueous solution containing the hepatitis A virus into a syringe in batches or placing the syringe in a container to be dripped into the syringe dropwise; a beaker was placed below the syringe to collect the filtrate, and 50. mu.L of hepatitis A Virus RNA was obtained from 200. mu.L of the filtrate using an RNA extraction kit (EZ-10 column type Total RNA extraction kit, Shanghai's Co., Ltd.).
mu.L of hepatitis A Virus RNA was collected and used with a cDNA Synthesis kit (PrimeScript)TM1st Strand cDNA Synthesis Kit, Dalianbao Bio Inc.) to obtain 20. mu.L of hepatitis A Virus cDNA. Taking 2 mu L of hepatitis A virus cDNA as a template, and carrying out virus quantitative detection by using an Applied Biosystems Viia 7 real-time fluorescent quantitative PCR instrument, wherein the upstream primers are as follows: 5'-GGT AGG CTA CGG GTGAAA C-3' (nucleotide sequence is shown as SEQ ID NO. 1), 5'-AAC AAC TCACCAATATCC GC-3' as downstream primer (nucleotide sequence is shown as SEQ ID NO. 2), 5'-CTTAGG CTAATACTT CTATGAAGA GAT GC-3' as probe (nucleotide sequence is shown as SEQ ID NO. 3), FAM fluorescent group modified at 5 'end of probe, and TAMRA quenching group modified at 3'. The reaction system is as follows:
the total reaction system is 20 mu L, and after uniform mixing and centrifugation, the reaction is carried out according to the following conditions: 50-2 min; 95-10 min; 95-15 s, 60-1 min and 40 times of circulation. Each cDNA template was set with 3 replicates and negative controls (enzyme-free water as template) and an applied biosystems via 7 real-time fluorescent quantitative PCR instrument generated the average of the CT values of 3 replicates, substituted into the standard curve equation: the concentration of hepatitis A Virus was found to be-3.24 x +39.199 (y in the equation is the average value of CT, x is found, and the number of copies of hepatitis A Virus per microliter is 10xThe unit of virus concentration is: copy/. mu.L). Calculating the hepatitis A virus concentration as C copy/. mu.L, and multiplying by the total volume 2 × 105μ L, the virus copy number C in the filtrate can be obtainedGeneral assembly。
And another same amount of hepatitis A virus stock solution added with the standard volume is taken to carry out the same operations of RNA extraction, reverse transcription and fluorescence quantitative PCR so as to confirm the total copy number of the added standard hepatitis A virus.
And taking pure water with the same volume without hepatitis A virus to perform the same operations of RNA extraction, reverse transcription and fluorescence quantitative PCR so as to confirm that the background is free of hepatitis A virus.
And (4) calculating the enrichment rate of the hepatitis A virus, wherein the enrichment rate (%) (the copy number of the added standard total virus-the copy number of the virus in the filtrate)/the copy number of the added standard total virus.
According to the method of example 1, 6 graphene aerogels were prepared in the same batch, and the normalized total virus copy number is 4543227, and the virus enrichment rate is shown in table 2.
Table 2 virus enrichment ratio of graphene aerogel
As can be seen from table 2: the graphene aerogel synthesized by the method can enrich more than 99% of hepatitis A virus in water, and has the advantages of strong adsorption capacity, high enrichment efficiency, small material volume, light weight and convenient operation.
Fig. 26 is a negative staining electron micrograph of the graphene aerogel enriched hepatitis a virus, from which hepatitis a virus particles enriched by the graphene aerogel can be observed.
Example 5
The elution method of the food-borne pathogenic microorganisms on the graphene aerogel comprises the following steps:
the pH value of the eluent is 9;
the eluent is an aqueous solution comprising the following components in mass concentration: 15g/L of sodium chloride, 20g/L of sodium hydroxide, 38.5g/L of glycine, 15g/L of beef powder, 30g/L of peptone, 34g/L of Tris-Base and 58.7g/L of MOPS.
The graphene aerogel adsorbing the hepatitis A virus in example 4 is continuously placed in an injector, 30mL of eluent is added, and the elution solution is collected below the injector. Adding 3g of PEG-8000 into the elution solution, and fully shaking to dissolve the PEG-8000 to obtain the solution to be treated. Standing the obtained solution to be treated at 4 ℃ for 16h, centrifuging at 10000r/min for 30min, carefully removing the supernatant to obtain a precipitate attached to the wall of the centrifugal tube, and adding 1ml PBS solution to fix the volume.
200. mu.L of the above solution was taken, and 50. mu.L of hepatitis A Virus RNA was obtained using an RNA extraction kit (EZ-10 column type Total RNA extraction kit, Shanghai Biotech Co., Ltd.).
mu.L of hepatitis A Virus RNA was collected and used with a cDNA Synthesis kit (PrimeScript)TM1st Strand cDNA Synthesis Kit, Dalianbao bio) to obtain 20. mu.L of hepatitis A Virus cDNA.
Taking 2 mu L of hepatitis A virus cDNA as a template, and carrying out virus quantitative detection by using an Applied Biosystems Viia 7 real-time fluorescent quantitative PCR instrument, wherein the upstream primers are as follows: 5'-GGTAGG CTA CGG GTGAAA C-3' (nucleotide sequence shown as SEQ ID NO. 1), 5'-AACAAC TCA CCAATATCC GC-3' as downstream primer (nucleotide sequence shown as SEQ ID NO. 2), 5'-CTT AGG CTAATA CTT CTATGAAGA GAT GC-3' as probe (nucleotide sequence shown as SEQ ID NO. 3), FAM fluorescent group modified at 5 'end of probe, and TAMRA quenching group modified at 3'; the reaction system is as follows:
the total reaction system is 20 mu L, and after uniform mixing and centrifugation, the reaction is carried out according to the following conditions: 50-2 min; 95-10 min; 95-15 s, 60-1 min and 40 times of circulation. Each cDNA template was set with 3 replicates and negative controls (enzyme-free water as template), and an Applied Biosystems via 7 real-time fluorescent quantitative PCR instrument generated the average of the CT values of 3 replicates, substituted into the standard curve equation: the concentration of hepatitis A Virus was found to be-3.24 x +39.199 (the average value of CT was y in the equation, the value of x was found, and the number of copies of hepatitis A Virus per microliter was 10xThe unit of virus concentration is: copy/. mu.L). Calculating the hepatitis A virus concentration as Mcopy/. mu.L, and multiplying by the total volume of 103mu.L, the number of copies M of the virus eluted in PBS solution can be obtainedGeneral assembly。
And another same amount of hepatitis A virus stock solution added with the standard volume is taken to carry out the same operations of RNA extraction, reverse transcription and fluorescence quantitative PCR so as to confirm the total copy number of the added standard hepatitis A virus.
The hepatitis a virus elution rate, which (%) ═ virus copy number in PBS solution/spiked total virus copy number, was calculated.
The total virus copy number was 4543227, and the rate of virus elution is shown in Table 3.
Table 3 elution rate of graphene aerogel
As can be seen from table 3: the elution method provided by the invention can enable the elution rate of the hepatitis A virus to reach more than 90%, has high elution efficiency, and provides convenience for subsequent qualitative and quantitative detection.
Example 6
Preparing 5 graphene aerogels in the same batch according to the method of example 2;
enrichment of hepatitis a virus with graphene aerogel was performed according to the method of example 4, except that the spiked total virus copy number was 222604; the virus enrichment ratio of the 5 graphene aerogels obtained is shown in table 4.
The graphene aerogel adsorbing the virus was eluted in the elution manner described in example 5, and the virus elution rate of the 5 graphene aerogels obtained was shown in table 5.
Table 4 virus enrichment ratio of graphene aerogel
Table 5 elution rate of graphene aerogel
As can be seen from tables 4 and 5: after the synthesis method of the graphene aerogel is finely adjusted, the graphene aerogel still can keep strong adsorption performance, hepatitis A virus in water can be efficiently enriched, and the virus can be efficiently eluted after the virus enrichment is finished.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that several modifications and embellishments can be made for enriching other pathogens such as viruses, bacteria, parasites without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention.
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Claims (9)
1. The graphene aerogel is characterized by having a three-dimensional net structure and being loaded with active groups, wherein the active groups comprise carboxyl groups, carbonyl groups, hydroxyl groups, epoxy groups and nitrogen-containing groups.
2. The method for preparing the graphene aerogel according to claim 1, comprising the following steps:
mixing graphene oxide with water, and performing ultrasonic treatment to obtain a graphene oxide dispersion liquid;
mixing the graphene oxide dispersion liquid, ammonia water, triethylene tetramine and boric acid, and carrying out redox reaction to obtain graphene hydrogel;
and washing, freezing and drying the graphene hydrogel to obtain the graphene aerogel.
3. The preparation method according to claim 2, wherein the mass concentration of the ammonia water is 25-28%, and the concentration of the boric acid is 2 mg/mL; the dosage ratio of the graphene oxide, the ammonia water, the triethylene tetramine and the boric acid is 10 mg: 10-40 μ L: 16-40 μ L: 36-160 μ L.
4. The preparation method according to claim 2, wherein the temperature of the redox reaction is 120-125 ℃ and the time is 6-7 h.
5. The method of claim 2, wherein the washing comprises: washing with water and ethanol in sequence; the washing mode is washing; the ethanol washing mode is soaking, and the soaking time is 8-16 h; the reagent for washing with ethanol is an ethanol water solution, and the volume concentration of the ethanol water solution is 5-20%.
6. The method of claim 2, wherein the freeze-drying comprises the steps of: pre-freezing at-80 deg.C for 8 hr, and vacuum drying for 24 hr.
7. Use of the graphene aerogel according to claim 1 or the graphene aerogel prepared by the preparation method according to any one of claims 2 to 6 for enriching food-borne pathogenic microorganisms.
8. The use according to claim 7, wherein the graphene aerogel, when used for enriching food-borne pathogenic microorganisms, comprises the following steps:
and (3) passing the solution to be detected through the graphene aerogel, and adsorbing the food-borne pathogenic microorganisms in the solution to be detected on the graphene aerogel.
9. The elution method of food-borne pathogenic microorganisms on the graphene aerogel is characterized by comprising the following steps of:
placing the graphene aerogel adsorbed with the food-borne pathogenic microorganisms at the bottom of an injector, eluting by using an eluent, and collecting an elution solution;
mixing the elution solution with a treatment reagent to obtain a solution to be treated;
standing and centrifuging the solution to be treated, fixing the volume of the obtained centrifugal precipitate by using a PBS buffer solution, and taking supernatant to perform bacterial culture or polymerase chain reaction;
the pH value of the eluent is 9;
the eluent is an aqueous solution comprising the following components in mass concentration: 15g/L of sodium chloride, 20g/L of sodium hydroxide, 38.5g/L of glycine, 15g/L of beef powder, 30g/L of peptone, 34g/L of Tris-Base and 58.7g/L of MOPSS;
the treatment reagent is PEG-6000 or PEG-8000, and the dosage ratio of the elution solution to the treatment reagent is 10 mL: 1g of the total weight of the composition.
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