CN110215737A - A kind of affine integral post and preparation method thereof loading aptamer based on graphene-nanogold compound interface superelevation - Google Patents
A kind of affine integral post and preparation method thereof loading aptamer based on graphene-nanogold compound interface superelevation Download PDFInfo
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- CN110215737A CN110215737A CN201910512438.0A CN201910512438A CN110215737A CN 110215737 A CN110215737 A CN 110215737A CN 201910512438 A CN201910512438 A CN 201910512438A CN 110215737 A CN110215737 A CN 110215737A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
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- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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Abstract
The invention belongs to integral post polymeric material preparation fields, and in particular to a kind of based on graphene-nanogold compound interface superelevation load aptamer affine integral post and preparation method thereof.Using the modified graphene oxide in surface as catalyst and decorative material, graphene functionalized hybridisation silica gel polymerization integer column is prepared by " one pot " method, and modification after nanometer principal column is arrived to the grapheme material surface of the porous integral post of modified silica-gel, bridge linking effect based on nanogold realizes that ultra high density of the aptamer on the porous integral post surface of modified silica-gel loads.The porous integral post matrix of the modified silica-gel has high-specific surface area, the modified graphene oxide on its surface can stablize, efficiently supported nano-gold particle, it avoids in preparation process simultaneously and graphene, nano Au particle agglomeration occurs, it can enable in the modification to integral post of aptamer high density, realizing the affine integral post of the silica gel hybridization can be used for the efficient specific recognition and separation of ochratoxin A.
Description
Technical field
The invention belongs to integral post polymeric material preparation fields, and in particular to one kind is based on the compound boundary of graphene-nanogold
The affine integral post and preparation method thereof of face superelevation load aptamer.
Background technique
Aptamer is screened from the random oligonucleotide library of large capacity by in-vitro screening technology (SELEX)
A kind of short chain DNA or RNA sequence arrived can carry out high affine, high specific bond with corresponding target ligand, have applicable mesh
The advantages that mark range is wide, be easily-synthesized, easily modification, chemical property be stable and high specific.The development of aptamer is quick, high
The research of effect identification technology provides a new direction, using aptamer as affinity ligand for being enriched with, separating and detecting
Etc. research work receive the concern of height, illustrate good application prospect.
In recent years, in order to enhance capillary monolithic column to the selective retention ability of particular detection object, other chemistry are reduced
The immobilized technology of preparing in integral post of aptamer is paid close attention to and is developed by the interference of ingredient, affine by utilizing
High specific between ligand and target analytes, in conjunction with the characteristics of capillary monolithic column and aptamer, so that the technology exists
Important function has been played in the substances such as Selective recognition, enrichment and the small analysis of compounds of purifying, protein and cell.Nucleic acid is suitable
Ligand modified affine integral post is mainly made of carrier, aglucon and spacerarm three parts, wherein integral post porous structure and core
Sour modification technique is the key element for influencing affine integral post bonding aptamers efficiency, directly affects affine integral post
Specific recognition performance.Currently, the immobilized mode in integral post of aptamer covalent bonding has more report, but it is big
The coverage density of aptamer needs between 169-568 pmol/ μ L in the affine integral post of majority chemical bonding preparation
It improves and much needs longer reaction time and exacting terms, will affect the work of aptamer to a certain extent
Property.Aptamer is as affinity ligand in affine integral post, and the coverage density in integral post is for Environmental Trace mesh
The analysis detection of mark object plays key effect, in addition, the matrix non-specific adsorption of integral post can know the specificity of analyte
Certain interference is not caused, and influences the qualitatively and quantitatively analysis of object.Integral post that ideal aptamer is affine
Material be there is high compatibility, selective and high stability, meanwhile, should also avoid as far as possible that may be present non-specific
Property suction-operated.Coverage density by improving capillary monolithic column surface effective nucleic acid aptamers is undoubtedly also a kind of reduction base
The effective means of matter column nonspecific action.Therefore seek new stationary phase and immobilized mode, it is new to research and develop affine integral post preparation
Technology prepares the affine integral post of novel nucleic acids aptamers with high aptamer coverage density and high specific, to abundant
There is important scientific meaning with affine integral post analytical technology is improved.
It mainly include at present following several improving the research of the affine integral post surface nucleic acid aptamers coverage density of capillary
A aspect:
1. more than derivatization method mainly includes that non-bonding is legal and chemical bonding, passes through aptamer and overall surface
Affinity interaction or chemical coordination effect, are repeated several times post-column derivation aptamer to further increase aptamer whole
Coverage density on scapus, wherein non-bonding is legal based on biotin and Streptavidin method, and chemical bonding mainly wraps
Mercapto-alkene click and glutaraldehyde method are included, but since binding site is limited, the coverage density using this method aptamer is difficult
To significantly being promoted.
2. nanogold method, gold nanoparticle has preferable biocompatibility, easy modified and easy preparative, has also been employed that
To multiple and different fields.Gold nanoparticle by sulfydryl or it is amido modified arrive integral post surface, subsequent sulfydryl aptamers are logical
It crosses sulfydryl or amido modified to gold nanoparticle surface, can finally realize the highdensity functional modification on integral post surface.
Currently, being to connect medium the end modified aptamer for having sulfydryl is fixed to the base that surface has mercapto groups with nanogold
In matter integral post, the aptamer modified integral post of preparation can be enriched with fibrin ferment, separated and be detected.The above method
The coverage density for integral post amplifying nucleic acid aptamers that the hybrid silicon matrix of preparation is affine all in the level of 277-1314 pmol/μ L,
Compared to other preparation methods, coverage density of the aptamer in integral post has obtained a degree of promotion.
For oxide graphene (GO) after oxidation processes, graphite oxide still keeps the layer structure of graphite, contains on surface
There are the oxygen-containing functional groups such as hydroxyl and epoxy, there is carboxyl on edge.It is net that its important application concentrates on biosensor, environment
Change, be catalyzed, RAMAN spectrum substrate and separation science, GO can be changed with good surface volume ratio and higher stability
Graphene oxide (GO) is introduced capillary monolithic column by the surface chemistry of material, can greatly improve the combination of whole firmly material
Site.
Summary of the invention
The purpose of the present invention is to provide a kind of based on graphene-nanogold compound interface superelevation load aptamer
Affine integral post.The present invention using the surface-functionalized graphene oxide of reagent containing alkenyl siloxane as catalyst and modification material
Material, with sulfydryl silicone agent and assist silicone agent " one kettle way " polymerize and column after modified nano gold construct to form graphite
Alkene-nanogold composite layer, graphene-nanogold composite layer can provide a large amount of binding site, and sulfydryl aptamer is not only
It can also be formed in conjunction with the alkene unsaturated bond of graphene surface in conjunction with nanogold in graphene-nanogold composite layer
Ultra high density modification of nucleic acids aptamers affinity interaction interface.The porous integral post matrix of graphene modified silica-gel prepared by the present invention
Specific surface area with higher, the modified graphene oxide on integral post surface can stablize, efficiently supported nano-gold particle, together
When avoid in preparation process and graphene, nano Au particle agglomeration occur, so as to enable aptamer high density to repair
It adorns in the porous integral post of modified silica-gel, realizing the affine integral post of the silica gel hybridization can be used for the efficient spy of ochratoxin A
Different identification and separation.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of affine integral post loading aptamer based on graphene-nanogold compound interface superelevation is using one kind
The graphene oxide surface-functionalized using reagent containing alkenyl siloxane is as catalyst and decorative material, with sulfydryl silicone agent
It constructs to form graphene-nanogold composite layer, base with modified nano gold after auxiliary silicone agent " one kettle way " polymerization and column
In the bridge linking effect of nanogold, realize that ultra high density of the aptamer on the porous integral post surface of modified silica-gel loads.
Wherein containing the surface-functionalized graphene oxide of alkenyl siloxane and sulfydryl silicone agent and auxiliary siloxanes
In reagent " one kettle way " polymerization reaction, without adding acidic catalyst.
Wherein graphene oxide and sulfydryl siloxanyl monomers pass through mercapto-alkene click-reaction immobilization.
Wherein graphene oxide forms graphene-nanogold composite layer by physical absorption nanogold；The graphene-
Nanogold composite layer can provide a large amount of binding site, and sulfydryl aptamer not only can be compound with graphene-nanogold
Nanogold combines in layer, can also form ultra high density modification of nucleic acids aptamers in conjunction with the alkene unsaturated bond of graphene surface
Affinity interaction interface.
Wherein reagent containing alkenyl siloxane is methacryloxypropyl three (trimethylsiloxane group) silane.
Wherein auxiliary silicone agent be silylating reagent and initiator, wherein silylating reagent be tetramethoxy-silicane and
3- mercaptopropyl trimethoxysilane, the initiator are azodiisobutyronitrile (AIBN).
Wherein the sulfydryl silicone agent is mercaptopropyl trimethoxysilane.
It is described a kind of based on the affine integral post preparation side of graphene-nanogold compound interface superelevation load aptamer
Method the following steps are included:
(1) preparation of methacryloxypropyl three (trimethylsiloxane group) silane-modified graphene oxide solution:
15 mg GO solid powders are weighed, 15 mL are added and remove ultrapure water, 3 h of ultrasound make it be uniformly dispersed to form 1 mg/ in water
30 uL methacryloxypropyls three (trimethylsiloxane group) are added in the GO aqueous solution of mL under the conditions of room temperature (25 DEG C)
Silane (γ-MAPS) stirs 24 h；It is centrifuged 10 min under the revolving speed of 10000 r/min and removes unreacting substance, uses secondary water
Washing precipitating 3 times, is scattered in ultrapure water again, and it is silane-modified to obtain methacryloxypropyl three (trimethylsiloxane group)
Graphene oxide solution.
(2) preparation of poly (TMOS-co-MPTMS-co-GO) silica gel hybridization integral post:
Perforating agent urea and polyethylene glycol are weighed, a certain amount of methacryloxypropyl three (trimethylsiloxane group) silicon is weighed
Alkane modified graphene oxide solution in a round bottom flask, is stirred to being completely dissolved, is persistently stirred as 0 DEG C of 600 r/min of ice bath
It mixes；Silylating reagent and initiator are weighed in proportion in centrifuge tube, and concussion of mediating is to being completely dissolved, by the mix reagent with 1
Drop/sec adding speed round-bottomed flask is added dropwise, in 0 DEG C of 45 min of ice bath, ultrasonic degassing；It injects the mixture into and is located in advance
In the quartz capillary of reason, both ends sealing is placed on 24 h of isothermal reaction in 47 DEG C of water-baths；The integral post prepared is taken
Out, 120 DEG C of heating 3h remove unreacted residue with high-pressure solvent pump, and it is miscellaneous to obtain the modified Bio-sil of surface graphene
Change integral post.
(3) preparation of the graphene functionalized silica gel hybridization integral post of decorated by nano-gold:
(2cm) in the modified silicagel column of the graphene prepared is rinsed well with secondary water, is 15 nm nano-scale gold particles by partial size
Son is passed through in the integral post until cylinder becomes the liquid pinkiness of reddish brown and end outflow, obtained nanogold-stone
Black alkene modified silicon rubber column gel column.
(4) preparation of the affine integral post of aptamer bonded silica gel hydridization:
10000 r/min of mercapto-modified aptamer are centrifuged 5 min, the buffer solution A concussion of appropriate pH 8.0 is added
Dissolution, being made into solubility is 250 μm of ol/L aptamer solution, is placed in 90 DEG C of 3 min of heating, after being cooled to room temperature, obtains
To the sulfydryl aptamer solution of activation；30 are added in the sulfydryl aptamer for taking 250 μm of ol/L of 20 μ L to activate
Three (2- carboxyethyl) phosphines (TCEP) of 5 mmol/L of μ L, shaking table is incubated for 1 h at room temperature；The aptamer solution that will hatch
At room temperature in nanogold-porous integral post of graphene modified silica-gel obtained by injection step (3), 1 h is cleaned with secondary water, is made
Surface bond has the affine integral post of the silica gel hybridization of aptamer, after being passed through the buffer solution B of pH 8.0, save at 4 DEG C standby
With.
The dosage of each component in above-mentioned preparation step is counted the sum of by weight percent for 100 %: tetramethoxy-silicane
6.95 ~ 8.80 % of 19.98 ~ 22.19 %, 3- mercaptopropyl trimethoxysilane, three (trimethicone of methacryloxypropyl
Base) silane-modified 53.06 ~ 59.29 % of graphene oxide solution, 7.97 ~ 8.93 % of urea, 5.81 ~ 6.89 % of polyethylene glycol,
Azodiisobutyronitrile 0.46 ~ 0.51%.
Wherein the molecular weight of the polyethylene glycol is 10000.
Wherein the aptamer is anti-ochratoxin A, and base sequence is 5'-SH-C6-GAT CGG GTG TGG
GTG GCG TAA AGG GAG CAT CGG ACA-3'。
Wherein the buffer solution A is by 10 mmol/L Tris-HCl, 120 mmol/L NaCl and 5 mmol/L KCl groups
At；The buffer solution B is by 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl and 20mmol/L
CaCl2Composition.
Remarkable advantage of the invention is:
The present invention is to be used as catalyst and modification material using the surface-functionalized surface of graphene oxide containing alkenyl siloxane is modified
Material prepares graphene functionalized hybridisation silica gel polymerization integer column by " one pot " method, and modified silicon is arrived in modification after nanometer principal column
The grapheme material surface of the porous integral post of glue, the bridge linking effect based on nanogold realize that aptamer is more in modified silica-gel
The ultra high density on hole integral post surface loads.The porous integral post matrix of the modified silica-gel prepared in the present invention has high-ratio surface
Product, the modified graphene oxide on integral post surface can stablize, efficiently supported nano-gold particle, while avoid preparation process
In there is graphene, nano Au particle agglomeration, formation can provide a large amount of binding site graphenes-for aptamer and receive
The golden composite layer of rice；In addition, sulfydryl aptamer can also not only may be used in conjunction with nanogold in graphene-nanogold composite layer
In conjunction with the alkene unsaturated bond of graphene surface, ultra high density modification of nucleic acids aptamers affinity interaction interface is formed.It realizes
Integral post that the modified porous silica gel hybridization of the graphene is affine can be used for the efficient specific recognition and separation of ochratoxin A, nucleic acid
Aptamers surface coverage up to 5327 pmol/ μ L, is much larger than 113 ~ 1413 pmol/ μ L reported in the literature.
Detailed description of the invention
Fig. 1 is that the structure for the affine integral post for loading aptamer based on graphene-nanogold compound interface superelevation is shown
It is intended to.
Fig. 2 is nanogold-graphene Bio-sil hybridization capillary tube monolithic column using aptamer modified front and back
Electronic Speculum shape appearance figure, wherein figure A is the graphene-nanogold compound interface for modifying gold nanoparticle but unmodified aptamer
The cross-sectional scans electron microscope of integral post, figure B are graphene-nanogold compound interface the is affine integral post after modification of nucleic acids aptamers
Interface scanning electron microscope.
Fig. 3 is different integral posts to the identification situation comparison diagram of ochratoxin A, and wherein A1-A3 is modification Jenner's grain of rice
The graphene of sub but unmodified aptamer-nanogold compound interface integral post respectively indicates enrichment as control column, A1-A3
The test map of liquid, cleaning solution and eluent；B1-B3 is graphene-nanogold compound interface parent for modification of nucleic acids aptamers
And integral post, B1-B3 respectively indicate the test map of pregnant solution, cleaning solution and eluent.Peak position 1 represents ochratoxin A.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
A kind of affine integral post and preparation method thereof loading aptamer based on graphene-nanogold compound interface superelevation,
Specific steps are as follows:
(1) pretreatment:
Silicone hydroxyl is that capillary tube inner wall carries out prepolymerized basis, and the exposed silicone hydroxyl number of tube wall the more more are conducive to pre-polymerization
It closes.But the silicone hydroxyl quantity of normal melt quartz capillary is seldom, is unfavorable for prepolymerization.Therefore it needs to carry out capillary pre-
Processing,
Preprocessing process is as follows:
30 min of HCl solution flushing of 1.0 mol/L, secondary water are successively used to pass to neutrality, 1.0 mol/L capillary void column
NaOH solution rinse 30 min, then heat 3 h in 100 DEG C of environment, then again successively with secondary water pass to it is neutral, 0.1
M hydrochloric acid rinses 30 min, secondary water passes to neutrality, finally rinses 30 min, nitrogen under the conditions of 180 DEG C, 0.4 MPa with methanol
Pretreatment capillary column is made in 3 h of air-blowing；
(2) preparation of poly (TMOS-co-MPTMS-co-GO) silica gel hybridization integral post:
Perforating agent urea and polyethylene glycol are weighed by formula rate in table 1, weighs a certain amount of methacryloxypropyl three (three
Methyl siloxane-based) silane-modified graphene oxide solution in a round bottom flask, stir to being completely dissolved, as 0 DEG C of ice bath
600 r/min are persistently stirred；Tetramethoxy-silicane and 3- mercaptopropyl trimethoxysilane (MPTMS) are weighed in proportion in centrifuge tube
In, to being completely dissolved round-bottomed flask is added with 1 drop/sec of adding speed, in 0 DEG C of ice bath 45 in mix reagent by concussion of mediating
Min, ultrasonic degassing；It injects the mixture into pretreated quartz capillary, both ends sealing is placed in 47 DEG C of water-baths permanent
24 h of temperature reaction；The integral post prepared is taken out, 120 DEG C of heating 3h, removes unreacted residue with high-pressure solvent pump,
Obtain the modified Bio-sil hydridization integral post of surface graphene；
(3) preparation of the graphene functionalized silica gel hybridization integral post of decorated by nano-gold:
Into the graphene silicagel column prepared, (2 cm) is rinsed well with secondary water, is that 15 nm nano Au particles lead to by partial size
Enter to handle well in integral post until cylinder becomes the liquid pinkiness of reddish brown and end outflow, obtained nanogold-graphite
Alkene modified silicon rubber column gel column.
(4) preparation of the affine integral post of aptamer bonded silica gel hydridization:
10000 r/min of mercapto-modified aptamer are centrifuged 5 min, the buffer solution A concussion of appropriate pH 8.0 is added
Dissolution, being made into solubility is 250 μm of ol/L aptamer solution, is placed in 90 DEG C of 3 min of heating, after being cooled to room temperature, obtains
The sulfydryl aptamer solution of activation；30 μ L are added in the sulfydryl aptamer for taking 250 μm of ol/L of 20 μ L to activate
Three (2- carboxyethyl) phosphines (TCEP) of 5 mmol/L, shaking table is incubated for 1 h at room temperature；By the aptamer solution hatched in room
In nanogold-porous integral post of graphene modified silica-gel obtained by temperature lower injection step (3), 1 h is cleaned with secondary water, surface is made
It is bonded with the affine integral post of silica gel hybridization of aptamer, after being passed through the buffer solution B of pH 8.0, is saved backup at 4 DEG C.
The constituent content table of the modified porous silica gel hybridization integral post of 1 graphene of table
A:PEG is MW=10000 polyethylene glycol；
B:TMSPMA-modified GO is that concentration is 1 mg/mL modified graphite aqueous solution；
Fig. 2 is nanogold-graphene Bio-sil hybridization capillary tube monolithic column Electronic Speculum using aptamer modified front and back
Shape appearance figure, wherein figure A is modification gold nanoparticle but the graphene of modification of nucleic acids aptamers-nanogold compound interface is not whole
The cross-sectional scans electron microscope of column, figure B are the boundary of graphene-nanogold compound interface the is affine integral post after modification of nucleic acids aptamers
Surface scan electron microscope.
Embodiment 2
It selects formula B to prepare modification gold nanoparticle respectively but does not modify the aptamer of anti-ochratoxin A as control
Column and the affine integral post for modifying anti-ochratoxin A aptamer, are balanced respectively, are enriched with, clean and elute, and have
Steps are as follows for body:
(1) it balances: control column and the anti-ochratoxin A aptamer affinity column of modification is balanced with combination buffer.In conjunction with
Buffer: 10 mM Tris-HCl (pH 8.0), 120 mM NaCl, 5 mM KCl, 20 mM CaCl2500 psi back-pressures
Valve, 0.05 mL/min of flow velocity balance 0.5 h；
(2) it is enriched with: being injected separately into 20 μ L 5 ng/mL ochratoxin A (OTA) solution, respectively in control column and modification OTA core
Sour aptamers affinity column is enriched with 0.5 h.500 psi backpressure valves, 0.05 mL/min of flow velocity collect pregnant solution, to be measured.
(3) it cleans: cleaning integral post with combination buffer, after certain volume cleaning, collect cleaning solution, it is to be measured.
(4) it elutes: will with 30 % ACN:70 %TE buffers (10 mM Tris-HCl pH, 8.0,2.5 mM EDTA)
OTA is eluted from integral post, and collection 20 μ L of eluent is to be measured, and chromatographic condition is 500 psi backpressure valves, 0.1 mL/ of flow velocity
Min, the eluent for collecting 20 μ L are to be measured.
(5) it detects: the pregnant solution, cleaning solution and eluent of collection being injected separately into HPLC-RF-20A detection, detect OTA
Condition: mobile phase: 2 % acetic acid water: acetonitrile=38:62, Ex=333 nm, Em=460 nm, 1 mL/min, testing result such as Fig. 3.
As seen from Figure 3, the richness of the aptamer control column of gold nanoparticle but unmodified anti-ochratoxin A is only modified
Ochratoxin A is detected in liquid collecting and cleaning solution, and ochratoxin A is not detected in eluent, compares column to reddish brown song
Mould toxin A, which has no, to be effectively retained, and ochratoxin A is not detected in the pregnant solution and cleaning solution of affine integral post, and is washed
Ochratoxin A is detected in de- liquid, it was demonstrated that the present invention is based on graphene-nanogold compound interface superelevation to load aptamer
Affine integral post can be realized to the high specific recognition of ochratoxin A.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Xiamen Xanadu institute
<120>a kind of based on graphene-nanogold compound interface superelevation load aptamer affine integral post and its system
Preparation Method
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213>artificial sequence
<400> 1
gatcgggtgt gggtggcgta aagggagcat cggaca 36
Claims (9)
1. a kind of based on the affine integral post of graphene-nanogold compound interface superelevation load aptamer, it is characterised in that:
The affine integral post is a kind of graphene oxide surface-functionalized using reagent containing alkenyl siloxane as catalyst and modification
Material, with sulfydryl silicone agent and auxiliary silicone agent " one kettle way " polymerize and column after modified nano gold construct to form stone
Black alkene-nanogold composite layer, the bridge linking effect based on nanogold, graphene-nanogold composite layer and sulfydryl aptamer key
Cooperation is affine with the hybrid inorganic-organic silica gel aptamer for forming ultra high density modification of nucleic acids aptamers affinity interaction interface
Integral post.
2. according to claim 1 a kind of based on graphene-nanogold compound interface superelevation load aptamer parent
And integral post, it is characterised in that: wherein containing the surface-functionalized graphene oxide of alkenyl siloxane and sulfydryl silicone agent and
It assists in silicone agent " one kettle way " polymerization reaction, without adding acidic catalyst.
3. according to claim 1 a kind of based on graphene-nanogold compound interface superelevation load aptamer parent
And integral post, it is characterised in that: the sulfydryl siloxanes list of graphene oxide and sulfydryl silicone agent in the affine integral post
Body passes through mercapto-alkene click-reaction immobilization；It is compound to form graphene-nanogold by physical absorption nanogold for the graphene
Layer；The graphene-nanogold composite layer can provide a large amount of binding site, and sulfydryl aptamer not only can be with graphite
Nanogold combines in alkene-nanogold composite layer, can also form ultra high density in conjunction with the alkene unsaturated bond of graphene surface and repair
Adorn aptamer affinity interaction interface.
4. according to claim 1 a kind of based on graphene-nanogold compound interface superelevation load aptamer parent
And integral post, it is characterised in that: the reagent containing alkenyl siloxane is methacryloxypropyl three (trimethylsiloxane group)
Silane；The auxiliary silicone agent is silylating reagent and initiator, and wherein silylating reagent is tetramethoxy-silicane and 3-
Mercaptopropyl trimethoxysilane, the initiator are azodiisobutyronitrile；The sulfydryl silicone agent is mercapto propyl trimethoxy
Base silane.
5. according to claim 1 a kind of based on graphene-nanogold compound interface superelevation load aptamer parent
And integral post, it is characterised in that: the aptamer is anti-ochratoxin A, and base sequence is 5'-SH-C6-GAT CGG
GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3'。
6. a kind of one kind prepared as described in claim 1-5 any one is negative based on graphene-nanogold compound interface superelevation
The method for carrying the affine integral post of aptamer, it is characterised in that: the following steps are included:
(1) preparation of methacryloxypropyl three (trimethylsiloxane group) silane-modified graphene oxide solution:
15 mg GO solid powders are weighed, 15 mL are added and remove ultrapure water, 3 h of ultrasound make it be uniformly dispersed to form 1 mg/ in water
30 uL methacryloxypropyl three (trimethylsiloxane group) silicon are added in the GO aqueous solution of mL under the conditions of 25 DEG C of room temperature
Alkane (γ-MAPS) stirs 24 h；It is centrifuged 10 min under the revolving speed of 10000 r/min and removes unreacting substance, with secondary washing
It washs precipitating 3 times, is scattered in ultrapure water again, obtains methacryloxypropyl three (trimethylsiloxane group) silane-modified oxygen
Graphite alkene solution；
(2) preparation of poly (TMOS-co-MPTMS-co-GO) silica gel hybridization integral post:
Perforating agent urea and polyethylene glycol are weighed, a certain amount of methacryloxypropyl three (trimethylsiloxane group) silicon is weighed
Alkane modified graphene oxide solution in a round bottom flask, is stirred to being completely dissolved, is persistently stirred as 0 DEG C of 600 r/min of ice bath
It mixes；Tetramethoxy-silicane, 3- mercaptopropyl trimethoxysilane and azodiisobutyronitrile are weighed in proportion in centrifuge tube, shake of mediating
It swings to being completely dissolved, round-bottomed flask is added dropwise in mixing, in 0 DEG C of 45 min of ice bath, ultrasonic degassing；Inject the mixture into through
In pretreated quartz capillary, both ends sealing is placed on 24 h of isothermal reaction in 47 DEG C of water-baths；The integral post that will be prepared
It takes out, 120 DEG C of heating 3h, removes unreacted residue with high-pressure solvent pump, obtain the modified Bio-sil of surface graphene
Hydridization integral post；
(3) preparation of the graphene functionalized silica gel hybridization integral post of decorated by nano-gold:
It is rinsed well in the silicagel column modified to the 2 cm graphenes prepared with secondary water, is 15 nm nano-scale gold particles by partial size
Son, which is passed through, to be handled well in integral post until cylinder becomes the liquid pinkiness of reddish brown and end outflow, obtained nanogold-
The modified silicagel column of graphene；
(4) preparation of the affine integral post of aptamer bonded silica gel hydridization:
10000 r/min of mercapto-modified aptamer are centrifuged 5 min, the buffer solution A oscillation of appropriate pH 8.0 is added
Dissolution, being made into solubility is 250 μm of ol/L aptamer solution, is placed in 90 DEG C of 3 min of heating, after being cooled to room temperature, takes
Three (2- carboxyethyl) phosphines of 30 μ L, 5 mmol/L are added in the sulfydryl aptamer that 250 μm of ol/L of 20 μ L have been activated
(TCEP), shaking table is incubated for 1 h at room temperature；By nanometer obtained by the aptamer solution hatched at room temperature injection step (3)
In gold-porous integral post of graphene modified silica-gel, 1 h is cleaned with secondary water, obtained surface bond has the silica gel of aptamer
Integral post that hydridization is affine after being passed through the buffer solution B of pH 8.0, saves backup at 4 DEG C.
7. preparation method according to claim 6, it is characterised in that: polymer fluid formula composition described in step (2) is pressed
The sum of mass percent is that 100 % are counted: 19.98 ~ 22.19 % of tetramethoxy-silicane, 3- mercaptopropyl trimethoxysilane 6.95 ~
8.80 %, the silane-modified graphene oxide solution 53.06 ~ 59.29 of methacryloxypropyl three (trimethylsiloxane group)
%, 7.97 ~ 8.93 % of urea, 5.81 ~ 6.89 % of polyethylene glycol, azodiisobutyronitrile 0.46 ~ 0.51%.
8. preparation method according to claim 6, it is characterised in that: the molecular weight of the polyethylene glycol is 10000.
9. preparation method according to claim 6, it is characterised in that: the buffer solution A by 10 mmol/L Tris-HCl,
120 mmol/L NaCl and 5 mmol/L KCl composition；The buffer solution B is by 10 mmol/L Tris-HCl, 120 mmol/L
NaCl, 5 mmol/L KCl and 20mmol/L CaCl2Composition.
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