CN106512958B - A kind of preparation method and application of aptamer modified chitosan nano fiber - Google Patents
A kind of preparation method and application of aptamer modified chitosan nano fiber Download PDFInfo
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- CN106512958B CN106512958B CN201611050215.XA CN201611050215A CN106512958B CN 106512958 B CN106512958 B CN 106512958B CN 201611050215 A CN201611050215 A CN 201611050215A CN 106512958 B CN106512958 B CN 106512958B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28023—Fibres or filaments
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
Abstract
The invention discloses a kind of preparation methods of aptamer modified chitosan nano fiber, and the preparation method comprises the following steps: (1) preparing chitosan solution；(2) chitosan nano fiber is prepared；(3) chemistry is immobilized: taking aptamer solution, after the activation of EDC/NHS solution is added, adds step (2) resulting chitosan nano fiber, concussion reaction under room temperature；(4) it post-processes: step (3) resulting reaction mixture is separated by solid-liquid separation, obtain the aptamer modified chitosan nano fiber.Aptamer modified chitosan nano fiber of the present invention is suitable for the highly selective separation and enrichment of food, Environmental Trace or ultra trace mycotoxin substance or other substances, has the advantages that selectivity is high, loading capacity is big, stability is high and prepares simple.
Description
Technical field
The invention belongs to chemical analysis test technical fields, are related to dispersed solid phase micro-extraction technique, and in particular to a kind of
The preparation method of aptamer modified chitosan nano fiber adsorbent, the nanofiber suitable for complex sample trace or
The highly selective separation and concentration of the components such as ultra trace mycotoxin.
Background technique
Due to by component state to be measured, Coexisting component interference or measuring method sensitivity it is low the problems such as limited, absolutely mostly
Number chemical detection and analysis method require the pretreatment to sample progress effectively and reasonably in advance, to reach the effect of enrichment, purification
Fruit.But relative to the fast development of instrument analysis technology, the progress of Sample Pretreatment Technique is more slow.Currently used sample
Generally existing time-consuming, the inefficient, consumption of organic solvent such as pretreatment technology such as liquid-liquid extraction, column chromatography is greatly, operation is cumbersome, selects
Property it is poor the problems such as, cause sample pre-treatments to become most time-consuming and laborious link in entire analytic process, while also in sample analysis
The error of at least one third is introduced in the process.For this purpose, simple, quick, efficient, green, highly selective sample pre-treatments
Technology is to realize that complex sample such as blood plasma, urine, medicine, environment, trace or ultra trace component separation to be measured are rich in food samples
Collection is badly in need of the key technology of development.
Solid phase extraction techniques are combined by liquid-solid extraction and liquid chromatography technology and are developed, and are had become at present using the most
One of extensive novel sample-pretreating method.Solid phase extraction techniques achieve significant progress in recent years, have derived numerous
Novel solid phase extraction method for example solid phase dynamic extraction, Dispersive solid phase extraction, Stir Bar Sorptive Extraction, solid phase microextraction, from
Dynamic head space dynamic Solid Phase Extraction etc., and core of the adsorbent as these technologies, type also increasingly tend to diversification, it is some
Novel material such as magnetic nano-particle, Nano particles of silicon dioxide, carbon nanomaterial, nanofiber etc. are used as solid phase extraction
Take adsorbent or adsorbent ground.Nanofiber has compared with nanoparticle adsorbent because having continuous, whole structure
Background pressure is low, various informative, extraction is simple, many advantages, such as can be easily separated, and during the experiment only need to by being vortexed or
Concussion can obtain good sorption extraction separating effect, attract extensive attention in Sample Pretreatment Technique field.
Nanofiber can be prepared using a variety of high molecular materials, as polyvinyl alcohol, polyacrylonitrile, polyethylene glycol and
Chitosan etc..Chitosan is always novel within a very long time because it has many advantages, such as that bio-compatible is good, raw material are easy to get
Very powerful and exceedingly arrogant research object in Material Field.There are many kinds of the technologies that nanofiber is prepared using chitosan, such as electrostatic spinning
Technology, ultrasonic grinding method, freeze-drying etc..Wherein, electrostatic spinning technique is the basic skills for preparing nanofiber, but is being tried
It needs to use organic solvent during testing, to influence the biocompatibility of chitosan nano fiber.And the maximum of ultrasonic grinding method
Advantage is easy to operate, but chitosan nano fiber obtained has homogeneity and less reproducible.Freeze-drying
It is the emerging technology of chitosan nano fiber preparation, has the characteristics that simple, convenient and environmentally friendly, this method preparation Nanowire
The principle of dimension is to be removed hydrone in chitosan solution by the method for distillation, in the process due between chitosan molecule
With very strong electrostatic adsorption, head and the tail are connected, and nanofiber, nanofiber obtained are uniform one by one for microcosmic upper formation
Property, good biocompatibility, surface have hydroxyl abundant and amino group, be easy to chemical modification, have a wide range of applications potentiality.
Poor selectivity is main problem existing for current commercialization Solid Phase Extraction, in order to realize that specific adsorption, correlation are ground
The person's of studying carefully selection is in sorbent material modification Selective recognition functional group such as crown ether, calixarenes, molecularly imprinted polymer, antibody etc..
Wherein, molecularly imprinted polymer is because having the characteristics that selectivity is high, stability is good, prepares simple grinding as solid absorbent materials
Study carefully hot spot.But molecularly imprinted polymer is inevitably generated nonspecific binding site in the synthesis process, at the same time its
Rigid recognition site is easily destroyed in extraction process, therefore is subject to certain restrictions in analysis applications.Based on antibody-antigene phase
The bio-identification system of interaction also obtains certain application in solid phase extraction techniques.But from nineteen ninety Tuerk and
Ellington is respectively from containing about 1015Since filtering out RNA type aptamer in the library of kind oligonucleotide molecules, antibody skill
Art is by huge challenge.Aptamer is through in-vitro screening technology-index concentration aglucon phyletic evolution technology from random list
The single strand oligonucleotide acid sequence (DNA or RNA) of energy specific bond target ligand obtained in chain oligonucleotide library, usually
It is made of tens nucleotide, with high specificity, high-affinity, repeatable synthesis, stability is good, is easy the spy of modification
Point.When target ligand occurs, aptamers form stable tertiary structure and are formed with target ligand by changing itself secondary structure
New compound, for the dissociation constant of new compound usually within the scope of n mol and μm ol, some is even up to p mol, therefore nucleic acid
Aptamers have high specific recognition capability for target ligand molecule, and can be modified by chemical modification technique in solid
Adsorbent surface has great application potential in solid phase extraction techniques.
Currently, the carrier of aptamer has the materials such as hydrogel, liposome, micelle, metal, silica gel, glass, quantum dot
Material, solid support method have polymer embedding, physical absorption, direct self assembly, indirect self assembly etc., but generally existing immobilized rate compared with
The problems such as low, immobilized insecure, aptamers are easy to run off, material cracky.
Summary of the invention
For existing solid phase extraction techniques poor selectivity, the immobilized rate of aptamer is low the problems such as, the present invention fits nucleic acid
Characteristics and the chitosan nano fiber large specific surface areas, being easy to such as ligand affinity height, high specificity, biological sample compatibility be good
It learns modified advantage to combine, develops a kind of aptamer modified chitosan nano fiber, for trace in complex sample
Or the separation and enrichment of ultra trace material composition, in conjunction with high performance liquid chromatography, it can be achieved that highly selective, high-sensitivity analysis inspection
It surveys.
The invention is realized by the following technical scheme:
A kind of preparation method of aptamer modified chitosan nano fiber, comprising the following steps:
(1) it prepares chitosan solution: weighing Chitosan powder, acetic acid is added, adds distilled water and is made into chitosan
Solution；
(2) it prepares chitosan nano fiber: step (1) resulting chitosan solution being placed in liquid nitrogen and is freezed, then will
It is refrigerated to solid chitosan solution and is placed in the environment of low-temp low-pressure and be freeze-dried, obtain chitosan nano fiber；
(3) chemistry is immobilized: taking aptamer solution, after the activation of EDC/NHS solution is added, adds obtained by step (2)
Chitosan nano fiber, concussion reaction under room temperature；
(4) it post-processes: step (3) resulting reaction mixture is separated by solid-liquid separation, obtain the aptamer
Modify chitosan nano fiber.
The present invention uses aptamer modified solid phase extraction adsorbents, and aptamer can be high with corresponding target ligand
Effect exclusively combines, therefore the extraction selectivity of adsorbent can be improved, compared with the antibody for being likewise supplied with high binding specificity,
Aptamer also has target ligand range is wide, affinity is high, screens preparation facilitate, stability height, be easily introduced modification group
And the advantage that molecular mass is small, therefore can assign adsorbent wider array of application range, improve stability, the practicability of adsorbent
With immobilized rate.Secondly, the present invention is using the chitosan nano fiber of freeze-drying preparation as solid phase extraction adsorbents, the shell
The homogeneity of glycan nanofiber, good biocompatibility, large specific surface area are easy to chemical modification, have relative to conventional adsorbent
The advantages that pressure that has powerful connections is low, contact area is big, mass transfer rate is fast.Furthermore the present invention is small by molecular mass, it is small to take up space
Aptamer can get very high cover in conjunction with the chitosan nano fiber of large specific surface area, on chitosan nano fiber surface
Lid density, can improve the immobilized rate of aptamer to a greater degree, to further increase the loading capacity of adsorbent.This
Outside, aptamer is fixed on chitosan nano fiber surface by the immobilized mode of chemistry by the present invention, immobilized effect stability,
Solve immobilized insecure, aptamer caused by the modes such as physical absorption and self assembly be easy to run off, material cracky etc.
Problem.
Further, the aptamer solution is matched with the aptamer that carboxyl marks with PBS buffer solution by end
It makes, the concentration of aptamer is 4.5 μ of μ g/mL~7.0 g/mL；The pH value of the PBS buffer solution is 4.5~6.5.
The target ligand that those skilled in the art can detect as needed introduces carboxyl, end in corresponding aptamer end
Amide reaction occurs for the chitosan nano fiber of aptamer and surface amido-containing group with carboxyl label, is adapted to nucleic acid
Body is covalently bonded to the surface of chitosan nano fiber.Relative to the specific phase interaction by Streptavidin and biotin
With, i.e., by with biotin labeling aptamer be fixed on Streptavidin modification adsorbent on non-covalent bonding side
Method, the present invention need to only introduce carboxyl in aptamer, be not required to carry out the amino-containing chitosan nano fiber in surface itself
Modification, operating procedure is easy easily to be realized, preparation cost is low, and is avoided the molecular weight of aptamer after modifying and taken up space
It is excessive, prevent steric restriction from immobilized rate being caused to reduce.
Further, the aptamer solution is zearalenone aptamer solution.
Further, in step (3), under room temperature concussion reaction be no more than 12h, both guaranteed amide reaction can sufficiently into
Row, and avoid material contaminated or side reaction generation.
Further, step (3) are as follows: taking concentration is the aptamer solution of 4.5 μ of μ g/mL~7.0 g/mL, and addition rubs
You than for 1: 1~6 EDC/NHS solution activation after, add step (2) resulting chitosan nano fiber, stir, then plus
Enter the PBS buffer solution that pH value is 4.5~6.5, concussion reaction 6h~12h under room temperature was both obtained.By designing aptamer solution
Concentration, the pH value of PBS buffer solution and reaction time, and add the EDC/NHS solution of suitable proportion, promote aptamer
Carboxyl carries out sufficient amide with the amino of chitosan nano fiber and reacts, and then reaches higher immobilized rate and biggish extraction
Capacity, while avoiding the denaturation and degradation of aptamer.
Further, step (2) are as follows: step (1) resulting chitosan solution is placed in freezing 1h~5h in liquid nitrogen, then
The freeze drier for solid chitosan solution will be frozen into being placed in -60 DEG C~-20 DEG C of temperature, pressure 0.02kPa~0.06kPa
Interior freeze-drying for 24 hours~72h, obtains the chitosan nano fiber being freeze-dried, then clean to it.Utilize freeze-drying
Method prepares chitosan nano fiber, has the advantages that environmentally friendly, easy to operate, equipment is simple, yield is high, chitosan nano obtained
Fiber homogeneity, good biocompatibility, large specific surface area are easy to chemical modification.
Further, the removal of impurities are as follows: with dehydrated alcohol by the chitosan nano fiber being freeze-dried impregnate 10min~
Then chitosan nano fiber containing dehydrated alcohol is dried in vacuo 1h~5h by 60min, it is fine to obtain pure chitosan nano
Dimension.By the immersion of dehydrated alcohol, remaining acetic acid in chitosan nano fiber is removed, then by being dried in vacuo dehydrated alcohol
It removes.
Further, step (4) are as follows: step (3) resulting reaction mixture is put into baking oven and is heated, solid phase is recycled
Extraction equipment is separated by solid-liquid separation the reaction mixture, then rinses solid phase with PBS buffer solution, recycles solid-phase extraction device
It being separated by solid-liquid separation to gained mixed liquor is rinsed, gained solid phase is the aptamer modified chitosan nano fiber,
After being dried in vacuo, it is placed in refrigerator and saves.Unreacted aptamer and chitosan nano fiber are released by heating
Physisorption, then by PBS buffer solution rinse and be separated by solid-liquid separation, thoroughly remove unreacted aptamer, avoid
Adverse effect is caused to the accuracy of subsequent Solid Phase Extraction detection.
Further, in step (4), after product is dried in vacuo 2h~4h under the conditions of 25 DEG C~40 DEG C, 2 DEG C are placed in
It is saved in~6 DEG C of refrigerators, aptamer is avoided to be denaturalized at high temperature.
Aptamer modified chitosan nano fiber of the present invention can apply dispersed solid phase micro-extraction technique, be used for
The separation and enrichment of trace or ultra trace material composition in complex sample.
It should be noted that PBS buffer solution described in text is the abbreviation of phosphate buffered saline solution；The EDC is 1- (3- bis-
Methylaminopropyl) -3- ethyl-carbodiimide hydrochloride abbreviation；The NHS is the abbreviation of n-hydroxysuccinimide.
In order to better understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Detailed description of the invention
Fig. 1 is the preparation of aptamer modified chitosan nano fiber and the process signal of adsorbed target ligand molecular
Figure.
Fig. 2 is aptamer modified chitosan nano fiber membrane electron scanning micrograph (300X).
Fig. 3 is aptamer modified chitosan nano fiber membrane electron scanning micrograph (1150X).
Fig. 4 is the structural formula of zearalenone and its analogue.
Fig. 5 is that the aptamer modified chitosan nano fiber of zearalenone (Apt-CNF), out-of-order single stranded DNA are repaired
Decorations chitosan nano fiber (Scr-CNF), chitosan nano fiber (CNF) extract 50 μ g/L zearalenones, α-jade respectively
The red mould enol of rice, β-zearalenol, α-zearalanol, β-zearalanol standard solution extraction quantity comparison diagram.
Specific embodiment
The present invention provides a kind of preparation methods of aptamer modified chitosan nano fiber, in order to make this field
Technical staff is better understood when technical solution of the present invention, makees with reference to the accompanying drawing with preferred embodiment to the present invention further
Detailed description.
The present embodiment is using zearalenone as target analyte, and with the aptamer modified shell of zearalenone
For the preparation of glycan nanofiber, but do not limited the scope of protection of the present invention with this.
As shown in Figure 1, the preparation method of the aptamer modified chitosan nano fiber of zearalenone includes following
Step:
(1) it prepares chitosan solution: weighing the Chitosan powder of 0.1g, 10mL distilled water is added, adds 60 μ L acetic acid,
Chitosan solution is obtained in 500mL volumetric flask with distilled water constant volume after stirring and dissolving；
(2) it prepares chitosan nano fiber: taking 50mL step (1) resulting chitosan solution respectively with two centrifuge tubes,
Two centrifuge tubes are placed in the 25L container equipped with liquid nitrogen again and freeze 2h, solid chitosan solution then will be frozen into and set
In -42 DEG C of temperature, pressure 0.05kPa freeze drier in be freeze-dried 48h, obtain the chitosan nano being freeze-dried fibre
Dimension, then the chitosan nano fiber being freeze-dried is impregnated into 30min with dehydrated alcohol, remaining acetic acid is removed, nothing then will be contained
The chitosan nano fiber of water-ethanol is placed in a vacuum drying oven, and obtains pure chitosan nano fiber after being dried in vacuo 3h；
(3) chemistry is immobilized: taking 500 μ L concentration is the zearalenone aptamer solution of 6.5 μ g/mL, and 1mL is added
After EDC/NHS solution activates 30min, chitosan nano fiber pure obtained by 5mg step (2) is added, magnetic stirring apparatus is used
2min is stirred, 1mL PBS buffer solution, the concussion reaction 8h at 25 DEG C is then added；
(4) it post-processes: step (3) resulting reaction mixture being put into 90 DEG C of baking ovens and heats 10min, it is then that this is anti-
It answers mixed liquor to pour into the Solid Phase Extraction blank column of 5mL, is separated by solid-liquid separation using solid-phase extraction device, remove solid phase surface and inhale
Then attached liquid phase rinses solid phase three times with PBS buffer solution, solid-phase extraction device is recycled to consolidate to gained mixed liquor is rinsed
Liquid separation, gained solid phase is the aptamer modified chitosan nano fiber of product zearalenone, by product in 30 DEG C
Under the conditions of be dried in vacuo 3h after, be placed in 4 DEG C of refrigerators and save.
In the present embodiment, the Gibberella zeae that the zearalenone aptamer solution is marked by end with carboxyl
Ketenes aptamer is formulated with PBS buffer solution, and the concentration of zearalenone aptamer is 6.5 μ g/mL.Institute
The pH value for stating PBS buffer solution is 5.5, wherein the concentration of NaCl is 0.1mol/L, Na2HPO4With NaH2PO4Total concentration be
10mmol/L, MgCl2Concentration be 5mmol/L.In the EDC/NHS solution, the molar ratio of EDC and NHS are 1: 4.
In step (4), adsorbent is not loaded inside the Solid Phase Extraction blank column, sieve plate is only housed, the sieve plate is to anti-
Answer mixed liquor and rinse gained mixed liquor and be filtered, products obtained therefrom be entirely seated in the Solid Phase Extraction blank column to get
Using aptamer modified chitosan nano fiber as the solid-phase extraction column of adsorbent, dispersed solid phase micro-extraction can be directly used for,
It is convenient and efficient.The solid-phase extraction device is general commercial product, can adjust flow rate of liquid, pressure, high-efficient, treating capacity
Greatly, play the role of accelerating to be separated by solid-liquid separation rate in this step.
The present invention selects freeze-drying to prepare chitosan nano fiber, has without using organic solvent, environmentally protective, behaviour
Make the advantages that simple, yield is high, as shown in Fig. 2, chitosan nano fiber homogeneity obtained is good, large specific surface area.
Aptamer has high specific recognition capability for target ligand molecule, and the present embodiment selects chitosan
Nanofiber is sorbent material, and the aptamer and surface that end is marked with carboxyl have the chitosan nano of amino group fine
Amide reaction occurs for dimension, and aptamer is covalently bonded on chitosan nano fiber surface, and immobilized condition is easily realized, operation
Simply, it as shown in figure 3, the supported quantity of aptamer modified chitosan nano fiber obtained is high, can be improved to complex sample
Extraction selectivity, increase loading capacity.
The aptamer modified chitosan nano fiber of zeranol (Apt-CNF) of the present embodiment 5mg, random ordering are single-stranded
DNA modification chitosan nano fiber (Scr-CNF), chitosan nano fiber (CNF) extract respectively 50 μ g/L zearalenones,
α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol standard solution, as a result such as 1 institute of table
Show.
The different adsorbents of table 1 compare the extraction quantity of zearalenone and its analogue
Note: end is 5 '-COOH-TCA TCT ATC TAT with the zearalenone aptamer that carboxyl marks
GGT ACA TTA CTA TCT GTA ATG TGATAT G-3′；The out-of-order single stranded DNA of terminal carboxyl group label is 5 '-COOH-
TTT CGT AATTTC GTA ACG AAT TTC GAA TTT CGA ACG ATT T-3′。
It is analyzed in conjunction with table 1, Fig. 4 and Fig. 5, it may be concluded that under the same conditions, zearalenone aptamer is repaired
Adoring chitosan nano fiber is 45.0ng to the extraction quantity of zearalenone, is 1.6 to other structures analog extraction quantity
~13.4 times, so it has very high Selective Separation and accumulation ability to zearalenone；In contrast, out-of-order single-stranded
DNA modification chitosan nano fiber is poor to the extraction selectivity of zearalenone and other 4 kinds of analogues, without jade
Extraction of the aptamer modified chitosan nano fiber of Zearlenone to zearalenone and other 4 kinds of analogues
Selectivity is also poor.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.
Claims (10)
1. a kind of preparation method of aptamer modified chitosan nano fiber, comprising the following steps:
(1) it prepares chitosan solution: weighing Chitosan powder, acetic acid is added, adds distilled water and is made into chitosan solution；
(2) it prepares chitosan nano fiber: step (1) resulting chitosan solution being placed in liquid nitrogen and is freezed, it then will freezing
It is placed in the environment of low-temp low-pressure and is freeze-dried to solid chitosan solution, obtain chitosan nano fiber；
(3) chemistry is immobilized: taking aptamer solution, after the activation of EDC/NHS solution is added, adds step (2) resulting shell
Glycan nanofiber, concussion reaction under room temperature；The aptamer that the aptamer solution is marked by end with carboxyl with
PBS buffer solution is formulated；
(4) it post-processes: step (3) resulting reaction mixture is separated by solid-liquid separation, obtain described aptamer modified
Chitosan nano fiber.
2. the preparation method of aptamer modified chitosan nano fiber as described in claim 1, it is characterised in that: the core
The concentration of sour aptamers nucleic acid in solution aptamers is 4.5 μ of μ g/mL~7.0 g/mL；The pH value of the PBS buffer solution be 4.5~
6.5。
3. the preparation method of aptamer modified chitosan nano fiber as claimed in claim 2, it is characterised in that: described
Aptamer solution is zearalenone aptamer solution.
4. the preparation method of aptamer modified chitosan nano fiber as claimed in claim 3, it is characterised in that: in step
(3) in, concussion reaction is no more than 12h under room temperature.
5. the preparation method of aptamer modified chitosan nano fiber as claimed in claim 4, it is characterised in that: step
(3) are as follows: taking concentration is the aptamer solution of 4.5 μ of μ g/mL~7.0 g/mL, and the EDC/NHS that molar ratio is 1:1~6 is added
After solution activation, step (2) resulting chitosan nano fiber is added, is stirred, the PBS that pH value is 4.5~6.5 is then added
Buffer, concussion reaction 6h~12h under room temperature, both.
6. the preparation method of aptamer modified chitosan nano fiber as claimed in claim 5, it is characterised in that: step
(2) are as follows: step (1) resulting chitosan solution is placed in freezing 1h~5h in liquid nitrogen, then will be frozen into solid chitosan
Solution is placed in the interior freeze-drying for 24 hours~72h of freeze drier of -60 DEG C~-20 DEG C of temperature, pressure 0.02kPa~0.06kPa,
The chitosan nano fiber being freeze-dried is obtained, then is cleaned to it.
7. the preparation method of aptamer modified chitosan nano fiber as claimed in claim 6, it is characterised in that: described to remove
It is miscellaneous are as follows: the chitosan nano fiber being freeze-dried is impregnated into 10min~60min with dehydrated alcohol, then will contain dehydrated alcohol
Chitosan nano fiber be dried in vacuo 1h~5h, obtain pure chitosan nano fiber.
8. the preparation method of aptamer modified chitosan nano fiber, feature exist as described in claim any one of 1-7
In: step (4) are as follows: step (3) resulting reaction mixture is put into baking oven and is heated, recycles solid-phase extraction device anti-to this
It answers mixed liquor to be separated by solid-liquid separation, then rinses solid phase with PBS buffer solution, solid-phase extraction device is recycled to mix to gained is rinsed
Liquid is separated by solid-liquid separation, and gained solid phase is the aptamer modified chitosan nano fiber, after being dried in vacuo,
It is placed in refrigerator and saves.
9. the preparation method of aptamer modified chitosan nano fiber as claimed in claim 8, it is characterised in that: in step
(4) it in, after product is dried in vacuo 2h~4h under the conditions of 25 DEG C~40 DEG C, is placed in 2 DEG C~6 DEG C refrigerators and saves.
10. aptamer modified chitosan nano fiber made from preparation method described in claim 1 is in the micro- extraction of dispersed solid phase
Application in taking.
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| CN111992186A (en) * | 2020-08-07 | 2020-11-27 | 武汉瑞法医疗器械有限公司 | New corolla pneumovirus receptor ACE2 affinity adsorbent and preparation method and application thereof |
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| CN105056915A (en) * | 2015-08-07 | 2015-11-18 | 兴义民族师范学院 | Preparation and application for magnetic metal organic framework medium modified by nucleic acid aptamer |
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|---|---|---|---|---|
| CN103990443A (en) * | 2014-03-27 | 2014-08-20 | 华南师范大学 | Single-stranded DNA nucleic acid modified chitosan magnetic microsphere preparation method |
| CN105056915A (en) * | 2015-08-07 | 2015-11-18 | 兴义民族师范学院 | Preparation and application for magnetic metal organic framework medium modified by nucleic acid aptamer |
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