CN108435107B - Preparation and application of DNA affinity nano-microspheres - Google Patents
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Abstract
The invention discloses preparation and application of DNA affinity nano-microspheres, and belongs to the technical field of biological detection. The invention provides a DNA affinity nano-microsphere, establishes a novel DNA extraction technology, comprises a DNA affinity nano-microsphere and various buffers, is of a double-layer structure, has a lipophilic material-formed lipophilic layer on the surface layer, is internally provided with a plurality of internal cavities and capillary micro-channels connected with the internal cavities, establishes an operation method and experimental steps for extracting DNA from various animal and vegetable oils, has high purity of the obtained DNA and stable and reliable quality, and solves the problems of 'no extraction' and 'impurity extraction' of the traditional reagent.
Description
Technical Field
The invention relates to preparation and application of DNA affinity nano-microspheres, in particular to application in DNA extraction, and belongs to the technical field of biological detection.
Background
Biological species all contain DNA sequences that are capable of calibrating their individual identities. The molecular detection technology can be used for carrying out the most specific species identification on the species, and can identify various animal and plant impurities in a certain biological product; the DNA with high purity and good integrity is obtained by separating and purifying biological materials containing various interfering impurities, which is the premise of success of molecular detection. At present, the traditional DNA extraction methods include: (1) using organic solvents, including: the CTAB method, the SDS method and the alcohol precipitation method (2) adsorption method, which comprises using a material having a siliceous surface, such as a microfiltration column, a magnetic nanomaterial or a glass filter; (3) ion exchange resin method.
However, the above techniques have disadvantages in the case of oils and fats, and cannot effectively and sufficiently extract and purify DNA in oils (fats), such as various edible vegetable oils, animal oils, fish oils, and the like. In particular, the first-stage refined (manufactured) edible oil is subjected to the process flows of drying, degumming, impurity removal, decoloration, deodorization, degreasing, dewaxing and the like, the DNA content is very rare, and valuable DNA fragments are difficult to separate from the edible oil by a common method. Various DNA extraction reagents sold in the market at present can not meet the requirements of conventional experiments for extracting DNA of edible oil; the extracted DNA is of low quality or can not be extracted.
The main components of the edible oil are fat, lipoid and other various kinds of grease. For example: vegetable oils, including rapeseed oil, soybean oil, peanut oil, sesame oil, corn germ oil, etc.; the animal fat refers to fat extracted from animal tissues qualified in quarantine, such as: edible sheep oil, edible goat oil, edible lard, edible chicken oil, edible duck oil, chafing dish sheep oil, baking lard, refined sheep oil, refined chicken oil, special sheep oil for seasonings, etc. The most sensitive and specific identification can be carried out on the authenticity of the mark of the grease product by applying a molecular detection technology, so that the inferior oil is blocked from flowing into the market, and the method comprises the following steps: (1) the dining table reclaimed oil (illegal cooking oil), namely 'active carbon or active clay instant clear oil'; (2) the label is used as fake oil, such as: no transgene or false identity; (3) mixing false oil, i.e. mixing low-value oil such as "pseudo sesame oil" mixed from chemical raw materials into high-value oil; (4) no harmful components are identified, such as high erucic acid, gossypol, toxins, etc., in the oil.
The separation of DNA with high purity and good integrity from the grease material is an important prerequisite for the success of molecular traceability detection of grease.
Disclosure of Invention
The invention aims to overcome the defects and provide preparation and application of DNA affinity nano microspheres.
The purpose of nucleic acid extraction is to provide DNA molecules suitable for PCR or gene sequencing analysis. The quality of the DNA extract includes the average length of the DNA molecules, chemical purity and integrity of the DNA sequence. The basic principles of DNA extraction are: the DNA in the sample is first released, followed by purification of the DNA and removal of various impurities. Quantification of the extracted DNA is critical for the success of the subsequent PCR and can generally be done by physical methods (measuring light absorption at a specific wavelength), chemical-physical methods (binding substances that can fluoresce).
The invention establishes a novel DNA extraction technology, the material part comprises DNA affinity nano-microspheres and various buffers, and the action principle is as follows:
most of the surface layer (except the opening) of the DNA affinity nano-microsphere is oleophilic, the nano-microsphere can be uniformly dispersed in the oil phase, and a larger specific surface area participates in the reaction;
2. because of the hydrophilic property of DNA, the DNA in the grease exists in a water-in-oil mode, when DNA micro-droplets enter the internal cavity through the opening of the nano-microsphere and contact the hydrophilic surface, the DNA micro-droplets can be quickly dispersed and wet the surface of the internal cavity, and can be combined with DNA affinity molecules under the conditions of certain salt concentration and proper pH. Also, the DNA affinity molecule can be dissociated under certain salt concentration and pH conditions. The uptake and release of the DNA by the nano-microspheres can be controlled by adjusting the conditions of salt concentration, pH, temperature and the like;
3. the capillary micro-channel of the internal cavity contains inert gas components which contract and expand along with the temperature change to play a pump-like role in spitting or sucking, and the heating or refrigeration can enhance the capacity of the DNA affinity nano-microspheres for containing or discharging micro-droplets.
The invention is realized by the following steps:
the invention provides a DNA affinity nano microsphere, wherein the inside of the DNA affinity nano microsphere is polymethyl methacrylate, a plurality of internal cavities and capillary micro channels connected with the outer surfaces of the internal cavities are distributed in the DNA affinity nano microsphere, inert gas is contained in the capillary micro channels, one part of the capillary micro channels can be mutually wound and extended, and the other part of the capillary micro channels are tortuous and connected with the adjacent internal cavities; the outer surface layer of the DNA affinity nanospheres is composed of a lipophilic layer composed of a lipophilic material including but not limited to polystyrene and a surface layer opening, the surface layer opening is inwardly coupled to the inner cavity and opens the inner cavity, the inner surface of the inner cavity is covered with a hydrophilic material and then grafted with aminated DNA affinity molecules, and the hydrophilic material is a hydrophilic-NH 2 coating.
Optionally, the DNA affinity molecule comprisesBut are not limited to: 5' -aminated Polyinosinic acid (NH)2-[CH2]6-Poly I), fragment length: 20-300 bp.
Optionally, the DNA affinity molecule is a thermostable DNA binding protein.
Furthermore, the diameter control range of the nano-microspheres is 10-200 nm.
Further, the size control range of the opening on the surface layer of the nano microsphere is 1-15 nm.
Furthermore, the number of the inner cavities of the nano-microspheres is 20-2000.
The invention further provides a preparation method of the DNA affinity nano-microsphere, which comprises the following steps:
step one, synthesis of nano microspheres:
i. preparing a surfactant buffer solution: dropwise adding a certain amount of surfactant and surface gap forming molecules into 50ml of distilled water, and stirring for 2 hours at 25 ℃;
add 1.0mL of 1.0g/mL methyl methacrylate solution and sparge well with inert gas for 1 hour, including but not limited to: nitrogen, carbon dioxide, helium, neon; adding a crosslinking initiator, and then dropwise adding the solution, keeping the temperature at 70 ℃, and stirring for 2 hours while adding;
the crosslinking initiators include, but are not limited to: azobisisobutyronitrile (azobisisobutyronitrile) or potassium persulfate (potassium persulfate);
dropwise adding styrene and styrene solution; keeping the temperature at 70 ℃ and stirring for 2 hours;
iv, stopping the reaction, namely adding 100mL of distilled water containing 2% Triton X-100 to stop the reaction, adding 150mL of 95% ethanol, centrifuging 12000g for 30 minutes to recover the precipitate, and drying;
controlling the diameter of the nano-microsphere to be 10-200 nm by adjusting the concentration of the surfactant; the proportion of molecules formed by the surfactant and the surface gaps is adjusted, so that the number of generated cavities in the surface active agent is 20-2000;
step two, erosion of the internal cavity:
putting the dried nano-microspheres into dichloromethane (methylene chloride) at the temperature of 25 ℃, and keeping the ultrasonic output power at 100-500W for 0.5-4 hours; then cleaning with methanol and water, and drying for later use; the diameter of the inner cavity can be controlled to be 1-20 nm by controlling the ultrasonic output power and the reaction time;
step three, carrying out surface modification by using a hexamethylenediamine method modified by alkaline boric acid to provide amino and form a hydrophilic coating on the surface; meanwhile, glutaric acid treatment, EDC and NHS are combined to further graft DNA affinity molecules:
further, in step one, the surfactant buffer solution: is composed of surfactant and surface gap forming molecule; surfactant buffer solutions referred to herein include, but are not limited to: various phospholipids and their derivatives, polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymers, surface void-forming molecules referred to herein include, but are not limited to: glucose pentaacetate (glucose pentaacetate), pentaglucopyranosylphenylacid (glucopyranosyloxy) and glucose pentaacetate (glucopyranosyloxy).
Further, in step two, the ultrasound conditions are: the temperature is 50 ℃, the output power is 300W, and the duration is 90 minutes.
The invention also provides an application of the DNA affinity nano-microsphere in nucleic acid extraction, because DNA in edible animal and vegetable oil exists in a water-in-oil mode, when the DNA micro-droplet contacts the hydrophilic surface of the inner cavity of the nano-microsphere, the DNA micro-droplet can be rapidly dispersed and wetted on the surface of the inner cavity of the nano-microsphere, and is combined with DNA affinity molecules, and the uptake and release of the nano-microsphere to the micro-droplet are controlled by adjusting the salt concentration, pH, temperature and time conditions.
The invention has the beneficial effects that:
the invention relates to preparation of DNA affinity nano-microspheres and application thereof in nucleic acid extraction, establishes a novel DNA adsorption technology, comprises DNA affinity nano-microspheres and various buffers in material parts, establishes an operation method and experimental steps for extracting DNA from various animal and vegetable oils, obtains DNA with high purity and stable and reliable quality, and solves the problems of 'no extraction' and 'impurity extraction' of the traditional reagent.
Drawings
FIG. 1 is a structural diagram of DNA affinity nanospheres;
wherein, 1 is an internal cavity DNA affinity molecule
2 is micro-pore channel in micro-sphere
3 is a polymethyl methacrylate core layer
4-NH 2 hydrophilic coating on the surface of the internal cavity
5 is a polystyrene microsphere surface layer;
FIGS. 2A-2D are UV spectra of DNA extracts of first-stage refined vegetable oils and animal fats obtained using the DNA affinity liquid phase nano-adsorbent of the present invention,
wherein: FIG. 2A is a UV spectrum of a DNA extract of primary refined corn oil;
FIG. 2B is an ultraviolet spectrum of a DNA extract of primary soybean oil;
FIG. 2C is a UV spectrum of a DNA extract from refined first-grade rapeseed oil;
FIG. 2D is a UV spectrum of DNA extract from sheep fat.
Detailed Description
The DNA affinity nano-microsphere is of a double-layer structure, the diameter control range of the DNA affinity nano-microsphere is 10-200 nm, the inner core layer of the DNA affinity nano-microsphere is polymethyl methacrylate, referring to figure 1, a plurality of inner cavities and capillary micro-channels connected with the outer surfaces of the inner cavities are arranged in the polymethyl methacrylate. Generally, the number of the inner cavities is 20-2000. The capillary micro-channel has irregular length and width, and has inert gas therein, the inert gas includes but is not limited to: nitrogen, carbon dioxide, helium, neon. A portion of the capillary microchannels are intertwined and extend with each other and another portion of the capillary microchannels are tortuous and connect to adjacent internal cavities. The surface layer of the nano microsphere is made of oleophylic materials, a surface layer opening is formed on the surface, and the size control range of the surface layer opening is 1-15 nm. The surface layer opening is inwardly coupled to and opens the internal cavity, the inner surface of which is covered with a hydrophilic material to which the aminated DNA affinity molecules are grafted. Wherein, the oleophilic material includes but is not limited to polystyrene, and the hydrophilic material is a hydrophilic-NH 2 coating. The functions are as follows: the surface of the DNA affinity nano microsphere consists of an oleophilic layer which can be uniformly dispersed in liquid grease, and the inner surface of the inner cavity is covered with a hydrophilic coating and DNA affinity molecules which are in a water wetting state and can dissolve biological macromolecules and combine DNA; the inert gas in the capillary micro-channel extending to the deep part of the nano microsphere expands along with the rise of the temperature and contracts along with the fall of the temperature, thereby generating the 'spitting or absorbing' effect on the liquid in the internal cavity.
The preparation method of the DNA affinity nano-microsphere comprises the following steps:
step one, synthesis of nano microspheres: and (3) separately casting a double-layer structure by adopting an emulsion template method.
i. Preparing a surfactant buffer solution: dropwise adding a certain amount of surfactant and surface gap forming molecules into 50ml of distilled water, and stirring for 2 hours at 25 ℃;
surfactant buffer solutions referred to herein include, but are not limited to: various phospholipids and their derivatives, polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymers, such as: p123(EO20PO70EO20, molecular weight 5800), and the amount of the surfactant buffer solution used is 0.5-60 mg. Surface void-forming molecules as referred to herein include, but are not limited to: glucose pentaacetate (glucose pentaacetate), pentaglucopyranosylphenylacid (glucopyranosyloxy) and glucose pentaacetate (glucopyranosyloxy). The using amount is 0.05-50 mu g;
adding 1.0mL of 1.0g/mL methyl methacrylate solution, and fully aerating with inert gas for 1 hour to fully form capillary micro-channels; inert gases include, but are not limited to: nitrogen, carbon dioxide, helium, neon; adding a crosslinking initiator, and then dropwise adding the solution, keeping the temperature at 70 ℃, and stirring for 2 hours while adding;
crosslinking initiators as referred to herein include, but are not limited to: azobisisobutyronitrile (azobisisobutyronitrile) or potassium persulfate (potassium persulfate);
dropwise adding styrene and styrene solution; keeping the temperature at 70 ℃ and stirring for 2 hours; iv, stopping the reaction, namely adding 100mL of distilled water containing 2% Triton X-100 to stop the reaction, adding 150mL of 95% ethanol, centrifuging 12000g for 30 minutes to recover the precipitate, and drying; controlling the diameter of the nano-microsphere to be 10-200 nm by adjusting the concentration of the surfactant; the proportion of the surfactant to the molecules formed by the surface voids is adjusted, so that the number of the generated inner cavities is 20-2000.
Step two, erosion of the internal cavity
Ultrasonic erosion: putting the dried nano-microspheres into dichloromethane (methylene chloride) at the temperature of 25 ℃, and keeping the ultrasonic output power of 100-500W for 0.5-4 hours; washing with methanol and water, and drying; the diameter of the inner cavity can be controlled to be 1-20 nm by controlling the ultrasonic output power and the reaction time; the size control range of the opening on the surface layer of the nano microsphere is 1-15 nm; the number of the inner cavities is 20-2000.
Under the following sonication conditions: the temperature is 50 ℃, the output power is 300W, and the duration is 90 minutes, so that the satisfactory result can be obtained. The internal cavity formed after ultrasonic erosion is shown in figure 1.
And step three, carrying out surface modification by using a hexamethylenediamine method modified by alkaline boric acid to provide amino groups and form a hydrophilic coating on the surface.
The operation steps are as follows:
i. surface amination treatment
Washing the nano microspheres with a solution containing 10% of NaOH solution and 50% of ethanol in sequence; then immersing in 1.0g/L polyvinyl alcohol solution for 20 minutes; at room temperature, with 1% NaIO4Soaking for 1 hour and washing with water; immersed in 100mM boric acid, pH 11, containing 10% hexamethylenediamineIn a buffer solution, carrying out amination reaction for 2 hours; washing with boric acid buffer of pH 11 and 8.2 for 15 minutes in sequence; washing with deionized water for 2 times, each time for 10 minutes; drying in a drying oven at 30 ℃ overnight to obtain the product with the surface containing-NH2Hydrophilic coated nanospheres.
Glutaric acid treatment, EDC and NHS in combination, further grafting DNA affinity molecules:
washing the nano microspheres for 2 times by using 10mL of coupling buffer solution; soaking in 5.0mL of coupling buffer solution;
weighing 6.6mg of glutaric acid, dissolving in 5.0mL of activation buffer solution, uniformly mixing, adding 20mg of water-soluble carbodiimide EDC and 10mg of NHS or DMAP, stirring for 15 minutes at room temperature, adjusting the pH value to 7.8, mixing with liquid containing nano microspheres, and reacting for 6 hours at room temperature; after the reaction is finished, washing for 2 times by using an activation buffer solution, and immersing the nano-microspheres in 5.0mL of the activation buffer solution;
20mg of water-soluble 1- (3-dimethylaminopropyl) carbodiimides (EDC) and 10mg of N-hydroxysuccinimide (NHS) are added with stirring; reacting for 15 minutes at room temperature; washing with 5.0mL of coupling buffer solution for 2 times and immersing the nanospheres in 5.0mL of coupling buffer solution;
taking 50uL amino modified DNA affinity molecules (10OD, diluted in 1.0mL PBS solution), uniformly mixing in 5.0mL coupling buffer solution, and mixing the nano microspheres (in the coupling buffer solution) with the DNA affinity molecule solution; shaking for reaction for 6 hours at room temperature; after washing, immersing the washed solution in 5.0mL of storage buffer solution; storing at 4 deg.C for use.
Grafted aminated DNA affinity molecules include, but are not limited to: 5' -aminated Polyinosinic acid (NH)2-[CH2]6-Poly I), fragment length: 20-300 bp, generally 40bp in length can obtain satisfactory results, and the A-type nano microspheres are obtained after grafting; and (3) thermally stabilizing the DNA binding protein, and grafting to obtain the B-type nano microsphere.
Step four, post-treatment: the grafted nanospheres were washed three times with PBS buffer pH 7.2 and stored in the same buffer containing preservative.
Step five, the storage condition of the DNA affinity nano-microspheres is as follows: storing in PBS buffer solution with pH of 7.2 at 4-25 ℃.
Before use, 20-2000 mg (wet weight) of the nano-microspheres are mixed with one of the extracting solution I, the extracting solution II and the extracting solution III according to the condition to form the DNA affinity nano-microsphere adsorption reagent.
Specifically, the extract I is 15mM sodium citrate,150mM NaCl, pH 7.0;
extract II, 125mM NaCl,10mM EDTA, 1% CTAB,50mM Tris-HCl, pH 7.4, and 2% Tween 20, pH 7.6;
and (3) extracting solution III: 120mM NaCl,10mM EDTA,40mM Tris-HCl, 0.25% (w/v) Sodium Didecyl Sulfate (SDS) and l% 2-mercaptoethanol, 1% Tween 20; the pH was 7.6.
The invention will be further described with reference to the following examples.
The DNA affinity nano microsphere adsorption reagent is suitable for animal and vegetable oil sold in the market at present, such as vegetable oil, such as rapeseed oil, soybean oil, corn oil, peanut oil, sunflower seed oil, olive oil, linseed oil, sesame oil, camellia oil and the like, and animal oil, such as sheep oil, goat oil, lard oil and the like.
Example 1: DNA extraction in primary refined soybean oil
The use method of the DNA affinity nano microsphere adsorption reagent comprises the following operation steps:
step one, adding 20mL of extracting solution I or extracting solution II or extracting solution III (containing 50mg of A-type nano microspheres) into a 50mL centrifugal tube, and carrying out water bath at 70 ℃ for 20 minutes; adding 20mL of preheated soybean oil (brand: ninety three, leaching method, primary refining) at 45 deg.C, mixing; centrifuge at 6000g for 15 minutes at room temperature, and add the bottom aqueous phase to a new 50mL centrifuge tube using a syringe. Before the edible oil is used, the edible oil needs to be shaken up; the edible vegetable oil should be uniform liquid, and if the room temperature is low, the edible vegetable oil is placed in a constant temperature box at 37 ℃ for overnight; before use, animal fat is heated in water bath at 45 deg.C.
And step two, adding equal volume of chloroform, mixing uniformly, centrifuging (20 ℃, 6000g, 10min), and taking an upper aqueous phase. Adjusting the pH value to 8.0-8.5; mu.g proteinase K per ml of aqueous phase was added and the mixture was incubated in a water bath at 55 ℃ for 60 minutes.
And step three, dropwise adding 30mL of cleaning solution preheated at 25 ℃, and centrifuging (20 ℃, 6000g, 15min) to take the supernatant. Adding equal volume of chloroform, mixing, centrifuging, and collecting the upper aqueous phase (20 deg.C, 6000g, 10 min). The cleaning solution comprises the following components: 80mM NaCl,10mM EDTA,50mM Tris-HCl, 0.1% (w/v) Sodium Didecyl Sulfate (SDS) and 0.5% Triton X-100; the pH was 7.4.
Step four, adding 30mL of dissociation liquid, and carrying out water bath at 65 ℃ for 20 minutes; centrifuging, taking out the supernatant, cooling to 20 ℃, adding chloroform (10-15 mL), centrifuging (20 ℃, 12000g, 10min), and taking out the supernatant; placing the supernatant in a refrigerator at-20 ℃ for 5 minutes, adding chloroform (10-15 mL), and centrifuging (4 ℃, 12000g, 10min) to obtain the supernatant; the extraction was repeated once with chloroform at 4 ℃. The dissociation liquid comprises the following components: 100mM GuSCN,30mM EDTA pH 8.0,200mM Tris-HCl pH 8.2, 0.5% Triton X-100, 5% Tween-20, 0.5% PVP.
Step five, adding 1/10 volumes of 3M sodium acetate buffer solution (pH 5.2) into the supernatant, adding absolute ethyl alcohol (about 23.3mL) with the total volume of liquid phase being 2.25 times of the total volume of liquid phase, and freezing at-20 ℃ overnight; centrifuging to remove supernatant (4 deg.C, 15000g, 30min), adding 4.0mL of 75% ethanol pre-cooled at-20 deg.C into the precipitate, centrifuging (4 deg.C, 15000g, 30min), washing the precipitate for 2 times, and keeping the precipitate in the middle of the operation. OD was measured after dissolving the precipitate.
Remarking: during centrifugation, the handle end of the pipe is placed into a centrifuge towards the outer side of the rotor; taking out the supernatant as soon as possible after centrifugation; when liquid is added or extracted, the liquid phase is inserted into the gun head close to the wall opposite to the handle end, a 100-200 mu L sample injector is used carefully, no more than 200 mu L of liquid is slowly added or extracted each time, and the operation is completed as soon as possible.
Example 2: DNA extraction from edible sheep oil
The invention relates to a using method of a DNA affinity nano microsphere adsorption reagent; the operation steps are as follows:
step one, adding 20mL of extracting solution I or extracting solution II or extracting solution III (containing 80mg of B-type nano microspheres) into a 50mL centrifuge tube, and carrying out water bath at 70 ℃ for 20 minutes; adding 20mL of white lard preheated at 65 deg.C (brand: Gong), mixing and mixing; 7000g was centrifuged for 15 minutes at room temperature and the bottom aqueous phase was added to a new 50mL centrifuge tube using a syringe. Before the edible oil is used, the edible oil needs to be shaken up; the edible lard should be white solidified body, and before use, it should be melted by heating in water bath at 65 deg.C.
The rest of the steps andexample 1The same is true.
According to the using method of the DNA affinity nano microsphere adsorption reagent, DNA in animal and vegetable oil is extracted, all endogenous and exogenous DNA can be extracted, and the extract is detected by an ultraviolet spectrophotometer to determine the purity and concentration of the extracted DNA. As shown in table 1.
Table 1 lists the recommended amounts of various animal and vegetable oils and their purities to be obtained for DNA extraction. The purity of the extracted DNA should meet the reference ranges in Table 1. Recommended dosage of the sample to be extracted: 10-20 g of animal oil and 10-20 ml of vegetable oil. As can be seen from Table 1: generally, 28-200 ng of pure DNA can be obtained from 10-20 ml of commercially available animal and vegetable oil;
wherein the DNA concentration is OD260X 50(μ g/mL), total amount of DNA ═ DNA concentration × volume.
Uv absorption data of extracted DNA molecules: 260/280, 260/230, should meet the requirements in the table. They represent the residues of various impurities of protein and polysaccharide in the sample respectively, and the CV of the repeated experiment should be less than 15%.
TABLE 1 reference values of DNA extraction effect and purity of various animal and vegetable oils and fats
The ultraviolet spectra of the DNA extracts of the first-stage refined vegetable oil and animal fat obtained by using the DNA affinity nanoparticle adsorption reagent of the present invention are shown in fig. 2A-2D, wherein fig. 2A is the first-stage refined corn oil, and the ultraviolet absorption data of the extracted DNA molecules thereof: 260/280, 260/230 are 1.39, 1.03, respectively; fig. 2B is a graph of uv absorption data for primary soybean oil, extracted DNA molecules: 260/280, 260/230 are 1.31, 0.98 respectively; fig. 2C shows uv absorption data of DNA molecules extracted from refined first-grade rapeseed oil: 260/280, 260/230 are 1.32, 1.31 respectively; fig. 2D is the uv absorbance data for the extracted DNA molecules of sheep oil: 260/280, 260/230 are 1.84, 2.03 respectively.
Therefore, the DNA affinity nano-microsphere adsorption reagent and the method thereof can be used for extracting DNA of various animal and vegetable oils sold in the market, and can achieve corresponding expected effects; the extract can be used for further analysis. Some samples have certain characteristics, such as hot-pressed vegetable oil, after high-temperature and high-pressure treatment, DNA molecules are damaged to a certain extent, large fragments of the DNA molecules are few, but the further PCR detection of multi-copy genes is generally not influenced, and before carrying out PCR on certain single-copy genes, pre-amplification is possibly needed, namely, the copy number is increased by using a constant-temperature amplification method or a PCR method, and then secondary amplification is carried out.
The DNA affinity nano microsphere adsorption reagent provides an efficient molecular tracing analysis method for safety evaluation of edible oil and provides complete technical support for molecular detection of oil. The technology can play a promoting role in food safety detection, teaching, scientific research and popularization.
Therefore, any modification, equivalent change and modification of the above embodiments according to the technical spirit of the present invention by those skilled in the art are within the scope of the present invention as defined in the claims.
Claims (9)
1. The DNA affinity nano microsphere is characterized in that the inside of the DNA affinity nano microsphere is polymethyl methacrylate, a plurality of internal cavities and capillary micro channels connected with the outer surfaces of the internal cavities are distributed in the DNA affinity nano microsphere, inert gas is contained in the capillary micro channels, one part of the capillary micro channels can be wound and extended mutually, and the other part of the capillary micro channels are tortuous and connected with the adjacent internal cavities; the outer surface layer of the DNA affinity nanospheres is composed of a lipophilic layer composed of a lipophilic material including but not limited to polystyrene and a surface layer opening, the surface layer opening is inwardly coupled to the inner cavity and opens the inner cavity, the inner surface of the inner cavity is covered with a hydrophilic material and then grafted with aminated DNA affinity molecules, and the hydrophilic material is a hydrophilic-NH 2 coating.
2. The DNA affinity nanosphere of claim 1, wherein said DNA affinity molecules include but are not limited to: 5' -aminated Polyinosinic acid (NH)2-[CH2]6-Poly I), fragment length: 20-300 bp.
3. The DNA affinity nanosphere of claim 1 wherein said DNA affinity molecule is a thermostable DNA binding protein.
4. The DNA affinity nanospheres according to any one of claims 1 to 3, wherein the diameter of said nanospheres is controlled in the range of 10 to 200 nm.
5. The DNA affinity nanospheres according to any one of claims 1 to 3, wherein the size control range of the surface layer opening of the nanospheres is 1-15 nm.
6. The DNA affinity nanosphere according to any one of claims 1 to 3, wherein the number of internal cavities of the nanosphere is 20 to 2000.
7. The preparation method of the DNA affinity nano-microsphere is characterized by comprising the following steps:
step one, synthesis of nano microspheres:
i. preparing a surfactant buffer solution: dropwise adding a certain amount of surfactant and surface gap forming molecules into 50ml of distilled water, and stirring for 2 hours at 25 ℃;
add 1.0mL of 1.0g/mL methyl methacrylate solution and sparge well with inert gas for 1 hour, including but not limited to: nitrogen, carbon dioxide, helium, neon; adding a crosslinking initiator, and then dropwise adding the solution, keeping the temperature at 70 ℃, and stirring for 2 hours while adding;
the crosslinking initiators include, but are not limited to: azobisisobutyronitrile (azobisisobutyronitrile) or potassium persulfate (potassium persulfate);
dropwise adding styrene and styrene solution; keeping the temperature at 70 ℃ and stirring for 2 hours;
iv, stopping the reaction, namely adding 100mL of distilled water containing 2% Triton X-100 to stop the reaction, adding 150mL of 95% ethanol, centrifuging 12000g for 30 minutes to recover the precipitate, and drying;
controlling the diameter of the nano-microsphere to be 10-200 nm by adjusting the concentration of the surfactant; the proportion of molecules formed by the surfactant and the surface gaps is adjusted, so that the number of generated cavities in the surface active agent is 20-2000;
step two, erosion of the internal cavity:
putting the dried nano-microspheres into dichloromethane (methylene chloride) at the temperature of 25 ℃, and keeping the ultrasonic output power at 100-500W for 0.5-4 hours; then cleaning with methanol and water, and drying for later use; the diameter of the inner cavity can be controlled to be 1-20 nm by controlling the ultrasonic output power and the reaction time;
step three, carrying out surface modification by using a hexamethylenediamine method modified by alkaline boric acid to provide amino and form a hydrophilic coating on the surface; meanwhile, glutaric acid treatment, EDC and NHS are combined to further graft DNA affinity molecules:
8. the preparation method of the DNA affinity nanospheres of claim 7, wherein in step one, the surfactant buffer solution: is composed of surfactant and surface gap forming molecule; surfactant buffer solutions referred to herein include, but are not limited to: various phospholipids and their derivatives, polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymers, surface void-forming molecules referred to herein include, but are not limited to: glucose pentaacetate (glucopyranose acetate), and glucopyranose benzenepropanoic acid (glucopyranose pentabenzoate).
9. The preparation method of the DNA affinity nanospheres of claim 7, wherein in the second step, the ultrasonic conditions are as follows: the temperature is 50 ℃, the output power is 300W, and the duration is 90 minutes.
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