CN110215737B - Affinity monolithic column based on graphene-nanogold composite interface ultrahigh-load aptamer and preparation method thereof - Google Patents
Affinity monolithic column based on graphene-nanogold composite interface ultrahigh-load aptamer and preparation method thereof Download PDFInfo
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- CN110215737B CN110215737B CN201910512438.0A CN201910512438A CN110215737B CN 110215737 B CN110215737 B CN 110215737B CN 201910512438 A CN201910512438 A CN 201910512438A CN 110215737 B CN110215737 B CN 110215737B
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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
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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
- B01D15/3804—Affinity chromatography
- B01D15/3819—Affinity chromatography of the nucleic acid-nucleic acid binding protein type
Abstract
The invention belongs to the field of preparation of monolithic column polymeric materials, and particularly relates to an affinity monolithic column based on a graphene-nanogold composite interface and an ultra-high nucleic acid aptamer and a preparation method thereof. The method comprises the steps of preparing a graphene functionalized hybrid silica gel polymeric monolithic column by a one-pot method by using surface-modified graphene oxide as a catalyst and a modification material, modifying a nanogold column to the surface of a graphene material of the modified silica gel porous monolithic column, and realizing ultrahigh-density loading of an aptamer on the surface of the modified silica gel porous monolithic column based on the bridging action of nanogold. The modified silica gel porous monolithic column matrix has a high specific surface area, the modified graphene oxide on the surface of the modified silica gel porous monolithic column matrix can stably and efficiently load nano gold particles, meanwhile, the agglomeration phenomenon of the graphene and the nano gold particles in the preparation process is avoided, the aptamer can be modified on the monolithic column in a high density manner, and the silica gel hybrid affinity monolithic column can be used for efficient specific recognition and separation of ochratoxin A.
Description
Technical Field
The invention belongs to the field of preparation of monolithic column polymeric materials, and particularly relates to an affinity monolithic column based on a graphene-nanogold composite interface and an ultra-high nucleic acid aptamer and a preparation method thereof.
Background
The aptamer is a short-chain DNA or RNA sequence screened from a large-capacity random oligonucleotide library by an in vitro screening technology (SELEX), can be combined with a corresponding target ligand in a high-affinity and high-specificity manner, and has the advantages of wide applicable target range, easiness in synthesis and modification, stable chemical properties, high specificity and the like. The development of the aptamer provides a new direction for the research of a rapid and efficient recognition technology, the research work of taking the aptamer as an affinity ligand for enrichment, separation, detection and the like is highly concerned, and the aptamer shows a good application prospect.
In recent years, in order to enhance the selective retention capacity of a capillary monolithic column for a specific detection object and reduce the interference of other chemical components, a preparation technology for immobilizing a nucleic acid aptamer on the monolithic column has attracted attention and developed, and the technology plays an important role in selectively identifying, enriching and purifying substances such as small analytical compounds, proteins and cells by utilizing the high specificity between an affinity ligand and a target analyte and combining the characteristics of the capillary monolithic column and the nucleic acid aptamer. The aptamer-modified affinity monolithic column mainly comprises a carrier, a ligand and a spacer arm, wherein the porous structure of the monolithic column and the nucleic acid modification technology are key factors influencing the efficiency of the aptamer-modified affinity monolithic column, and directly influence the specific recognition performance of the affinity monolithic column. At present, the covalent bonding and immobilization of the aptamer on the monolithic column has been reported more, but the coverage density of the aptamer on the affinity monolithic column prepared by most chemical bonding is between 169-568 pmol/μ L, which needs to be improved, needs a long reaction time and harsh conditions, and affects the activity of the aptamer to a certain extent. The aptamer is used as an affinity ligand on an affinity monolithic column, the coverage density of the aptamer on the monolithic column plays a key role in the analysis and detection of trace target substances in the environment, in addition, the non-specific adsorption of the matrix of the monolithic column can cause certain interference on the specific recognition of the analyte, and the quantitative and qualitative analysis of the target substances is influenced. The preferred aptamer affinity monolith materials have high affinity, selectivity, and stability, while avoiding possible non-specific adsorption. Increasing the density of coverage of available aptamers on the surface of capillary monoliths is undoubtedly an effective means to reduce the nonspecific effects of matrix columns. Therefore, a new stationary phase and an immobilization mode are sought, a new technology for preparing the affinity monolithic column is researched and developed, the novel aptamer affinity monolithic column with high aptamer coverage density and high specificity is prepared, and the method has important scientific significance for enriching and perfecting the analysis technology of the affinity monolithic column.
The current research on improving the coverage density of the aptamer on the surface of the capillary affinity monolithic column mainly comprises the following aspects:
1. the multiple derivation method mainly comprises a non-chemical bonding method and a chemical bonding method, and the coverage density of the aptamer on the whole column is further improved by deriving the aptamer after repeated column for multiple times through the affinity action or the chemical coordination action of the aptamer and the whole surface, wherein the non-chemical bonding method mainly comprises a biotin and streptavidin method, and the chemical bonding method mainly comprises a thiol-ene click and glutaraldehyde method, but the coverage density of the aptamer is difficult to be greatly improved by adopting the method due to limited binding sites.
2. The nano gold method, gold nanoparticles have better biocompatibility, easy modification and easy preparation, and have been applied to a plurality of different fields. The gold nanoparticles are modified to the surface of the monolithic column through sulfydryl or amino, and then sulfydryl aptamers are modified to the surface of the gold nanoparticles through sulfydryl or amino, so that high-density functional modification of the surface of the monolithic column can be finally realized. At present, nanogold is used as a connecting medium to fix the aptamer of which the tail end is modified with sulfydryl to a matrix monolithic column of which the surface is provided with sulfydryl groups, and the prepared aptamer-modified monolithic column can enrich, separate and detect thrombin. The covering density of the aptamer in the hybrid silica-based affinity monolithic column prepared by the method is at the level of 277-1314 pmol/muL, and compared with other preparation methods, the covering density of the aptamer on the monolithic column is improved to a certain degree.
After the Graphene Oxide (GO) is subjected to oxidation treatment, the graphite oxide still keeps the layered structure of graphite, the surface of the graphite oxide contains oxygen-containing functional groups such as hydroxyl and epoxy, and the edge of the graphite oxide has carboxyl. Its important application focuses on biosensor, environmental purification, catalysis, RAMAN spectrum base and separation science, GO has good surface-to-volume ratio and higher stability, the surface chemistry of the material can be changed, Graphene Oxide (GO) is introduced into the capillary monolithic column, and the binding sites of the monolithic material can be greatly improved.
Disclosure of Invention
The invention aims to provide an affinity monolithic column based on a graphene-nanogold composite interface and ultrahigh-load nucleic acid aptamer. The graphene-nanogold composite layer is formed by taking graphene oxide with functionalized alkenyl siloxane reagent surface as a catalyst and a modification material, polymerizing the graphene oxide with a mercapto siloxane reagent and an auxiliary siloxane reagent in a one-pot method and constructing the post-column modification nanogold, the graphene-nanogold composite layer can provide a large number of binding sites, and a mercapto aptamer can be combined with nanogold in the graphene-nanogold composite layer and also can be combined with an olefin unsaturated bond on the graphene surface to form an ultrahigh-density modification aptamer affinity interaction interface. The graphene modified silica gel porous monolithic column substrate prepared by the invention has a higher specific surface area, the modified graphene oxide on the surface of the monolithic column can stably and efficiently load nano gold particles, and meanwhile, the agglomeration phenomenon of the graphene and the nano gold particles in the preparation process is avoided, so that aptamer can be modified on the modified silica gel porous monolithic column in a high density manner, and the silica gel hybrid affinity monolithic column can be used for efficient and specific identification and separation of ochratoxin A.
In order to achieve the purpose, the invention adopts the following technical scheme:
an affinity monolithic column based on a graphene-nanogold composite interface and ultra-high loading of a nucleic acid aptamer is characterized in that graphene oxide with surface functionalization of an alkenyl-containing siloxane reagent is used as a catalyst and a modification material, and is polymerized with a mercapto siloxane reagent and an auxiliary siloxane reagent in a one-pot method manner, and the post-column modification nanogold is constructed to form a graphene-nanogold composite layer, so that the ultra-high density loading of the nucleic acid aptamer on the surface of a modified silica gel porous monolithic column is realized based on the bridging effect of the nanogold.
Wherein, in the one-pot polymerization reaction of the graphene oxide with the surface functionalized by the alkenyl siloxane, the mercaptosiloxane reagent and the auxiliary siloxane reagent, no acid catalyst is needed to be added.
Wherein the graphene oxide and the mercaptosiloxane monomer are immobilized through a mercapto-alkene click reaction.
The graphene oxide physically adsorbs nanogold to form a graphene-nanogold composite layer; the graphene-nanogold composite layer can provide a large number of binding sites, and the sulfhydryl aptamer can be combined with nanogold in the graphene-nanogold composite layer and also can be combined with an olefin unsaturated bond on the surface of graphene to form an ultrahigh-density modified aptamer affinity interaction interface.
Wherein the alkenyl-containing siloxane reagent is methacryloxypropyl tris (trimethylsiloxy) silane.
Wherein the auxiliary siloxane reagent is a silanization reagent and an initiator, wherein the silanization reagent is tetramethoxysilane and 3-mercaptopropyltrimethoxysilane, and the initiator is Azobisisobutyronitrile (AIBN).
Wherein the mercaptosiloxane reagent is mercaptopropyltrimethoxysilane.
The preparation method of the affinity monolithic column based on the graphene-nanogold composite interface ultrahigh-load aptamer comprises the following steps:
(1) preparation of methacryloxypropyl tris (trimethylsiloxy) silane modified graphene oxide solution:
weighing 15 mg of GO solid powder, adding 15 mL of ultrapure water to remove, carrying out ultrasonic treatment for 3h to uniformly disperse the GO solid powder in water to form 1 mg/mL of GO aqueous solution, adding 30 uL of methacryloxypropyl tris (trimethylsiloxy) silane (gamma-MAPS) at room temperature (25 ℃) and stirring for 24 h; centrifuging for 10 min at the rotating speed of 10000 r/min to remove unreacted substances, washing the precipitate for 3 times by using secondary water, and dispersing in ultrapure water again to obtain the methacryloxypropyl tris (trimethylsiloxy) silane modified graphene oxide solution.
(2) preparation of poly (TMOS-co-MPTMS-co-GO) silica gel hybrid monolithic column:
weighing urea and polyethylene glycol as pore-forming agents, weighing a certain amount of methacryloxypropyl tris (trimethylsiloxy) silane modified graphene oxide solution in a round-bottom flask, stirring until the solution is completely dissolved, and continuously stirring at the temperature of 0 ℃ for 600 r/min in an ice bath; weighing a silanization reagent and an initiator in a centrifuge tube according to a certain proportion, carrying out vortex oscillation until the silanization reagent and the initiator are completely dissolved, dropwise adding the mixed reagent into a round-bottom flask at the adding speed of 1 drop/second, carrying out ice bath at 0 ℃ for 45 min, and carrying out ultrasonic degassing; injecting the mixture into a pretreated quartz capillary tube, sealing two ends of the quartz capillary tube, and placing the quartz capillary tube in a 47 ℃ water bath kettle for constant-temperature reaction for 24 hours; and taking out the prepared monolithic column, heating at 120 ℃ for 3h, and removing unreacted residues by using a high-pressure solvent pump to obtain the surface graphene modified porous silica gel hybrid monolithic column.
(3) Preparing a nanogold-modified graphene functional silica gel hybrid monolithic column:
and (3) washing the prepared graphene modified silica gel column (2 cm) with secondary water, and introducing the nano gold particles with the particle size of 15 nm into the monolithic column until the column body becomes dark reddish brown and the liquid flowing out from the tail end is pink to prepare the nano gold-graphene modified silica gel column.
(4) Preparing an aptamer-bonded silica gel hybrid affinity monolithic column:
centrifuging the sulfhydryl modified aptamer for 5 min at 10000 r/min, adding a proper amount of buffer solution A with the pH of 8.0, shaking and dissolving to prepare a aptamer solution with the solubility of 250 mu mol/L, placing at 90 ℃, heating for 3 min, and cooling to room temperature to obtain an activated sulfhydryl aptamer solution; 20 mu L of 250 mu mol/L activated sulfhydryl aptamer is taken, 30 mu L of 5 mmol/L tris (2-carboxyethyl) phosphine (TCEP) is added into the activated sulfhydryl aptamer, and the mixture is incubated for 1 h at room temperature in a shaking table; and (3) injecting the incubated nucleic acid aptamer solution into the nanogold-graphene modified silica gel porous monolithic column obtained in the step (3) at room temperature, washing with water for 1 h for the second time to prepare the silica gel hybrid affinity monolithic column with the nucleic acid aptamer bonded on the surface, introducing a buffer solution B with the pH of 8.0, and storing at the temperature of 4 ℃ for later use.
The dosage of each component in the preparation steps is calculated according to the sum of the mass percentages of 100 percent: 19.98-22.19% of tetramethoxysilane, 6.95-8.80% of 3-mercaptopropyltrimethoxysilane, 53.06-59.29% of methacryloxypropyltris (trimethylsiloxy) silane modified graphene oxide solution, 7.97-8.93% of urea, 5.81-6.89% of polyethylene glycol and 0.46-0.51% of azobisisobutyronitrile.
Wherein the polyethylene glycol has a molecular weight of 10000.
Wherein the aptamer is anti-ochratoxin A, and the base sequence is 5'-SH-C6-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3'.
Wherein the buffer solution A consists of 10 mmol/L Tris-HCl, 120 mmol/L NaCl and 5 mmol/L KCl; the buffer solution B consists of 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl and 20mmol/L CaCl2And (4) forming.
The invention has the following remarkable advantages:
according to the method, graphene oxide surface modification containing alkenyl siloxane surface functionalization is adopted as a catalyst and a modification material, a graphene functionalized hybrid silica gel polymerization monolithic column is prepared through a one-pot method, a nanogold column is modified on the surface of a graphene material of a modified silica gel porous monolithic column, and the ultrahigh-density loading of a nucleic acid aptamer on the surface of the modified silica gel porous monolithic column is realized based on the bridging effect of nanogold. The modified silica gel porous monolithic column matrix prepared by the method has a high specific surface area, the modified graphene oxide on the surface of the monolithic column can stably and efficiently load nano-gold particles, and simultaneously, the phenomenon of agglomeration of graphene and nano-gold particles in the preparation process is avoided, so that a large number of binding site graphene-nano-gold composite layers can be provided for the aptamer; in addition, the sulfhydryl aptamer can be combined with nanogold in the graphene-nanogold composite layer and also can be combined with an olefin unsaturated bond on the surface of the graphene to form an ultrahigh-density modified aptamer affinity interaction interface. The graphene modified porous silica gel hybrid affinity monolithic column can be used for efficient and specific identification and separation of ochratoxin A, and the surface coverage density of the aptamer is as high as 5327 pmol/muL, which is far greater than 113-1413 pmol/muL reported in the literature.
Drawings
Fig. 1 is a structural schematic diagram of an affinity monolithic column based on a graphene-nanogold composite interface ultrahigh-load aptamer.
Fig. 2 is an electron microscope morphology image of a nanogold-graphene porous silica gel hybrid capillary monolithic column before and after being modified by an aptamer, wherein a is a cross-sectional scanning electron microscope image of a graphene-nanogold composite interface monolithic column in which gold nanoparticles are modified but the aptamer is not modified, and a B is an interface scanning electron microscope image of a graphene-nanogold composite interface affinity monolithic column after the aptamer is modified.
FIG. 3 is a comparison graph of the recognition of ochratoxin A by different monolithic columns, wherein A1-A3 are graphene-nanogold composite interface monolithic columns which modify gold nanoparticles but do not modify aptamer and serve as control columns, and A1-A3 respectively represent detection maps of enrichment liquid, cleaning liquid and eluent; B1-B3 are graphene-nanogold composite interface affinity monolithic columns for modifying nucleic acid aptamers, and B1-B3 respectively represent detection maps of the enriched liquid, the cleaning liquid and the eluent. Peak position 1 represents ochratoxin a.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
An affinity monolithic column based on a graphene-nanogold composite interface ultrahigh-load aptamer and a preparation method thereof comprise the following specific steps:
(1) capillary pretreatment:
the silicon hydroxyl is the base of the prepolymerization of the inner wall of the capillary, and the larger the number of the exposed silicon hydroxyl on the tube wall is, the more favorable the prepolymerization is. However, the number of silicon hydroxyl groups of the common fused silica capillary is small, which is not beneficial to prepolymerization. It is therefore necessary to pre-treat the capillary tube,
the pretreatment process is as follows:
washing a capillary empty column with 1.0 mol/L HCl solution for 30 min, introducing secondary water to be neutral, washing with 1.0 mol/L NaOH solution for 30 min, heating at 100 ℃ for 3h, introducing secondary water to be neutral, washing with 0.1M hydrochloric acid for 30 min, introducing secondary water to be neutral, washing with methanol for 30 min, and blowing with nitrogen at 180 ℃ and 0.4 MPa for 3h to obtain a pretreated capillary column;
(2) preparation of poly (TMOS-co-MPTMS-co-GO) silica gel hybrid monolithic column:
weighing urea and polyethylene glycol as pore-forming agents according to the proportion of the formula in the table 1, weighing a certain amount of methacryloxypropyl tris (trimethylsiloxy) silane modified graphene oxide solution in a round-bottom flask, stirring until the solution is completely dissolved, and continuously stirring at the temperature of 0 ℃ in an ice bath of 600 r/min; weighing tetramethoxysilane and 3-mercaptopropyltrimethoxysilane (MPTMS) in proportion in a centrifuge tube, carrying out vortex oscillation until the mixture is completely dissolved, adding a mixed reagent into a round-bottom flask at the adding speed of 1 drop/second, carrying out ice bath at 0 ℃ for 45 min, and carrying out ultrasonic degassing; injecting the mixture into a pretreated quartz capillary tube, sealing two ends of the quartz capillary tube, and placing the quartz capillary tube in a 47 ℃ water bath kettle for constant-temperature reaction for 24 hours; taking out the prepared monolithic column, heating at 120 ℃ for 3h, and removing unreacted residues by using a high-pressure solvent pump to obtain a surface graphene modified porous silica gel hybrid monolithic column;
(3) preparing a nanogold-modified graphene functional silica gel hybrid monolithic column:
and (3) washing the prepared graphene silica gel column (2 cm) with secondary water, introducing the nano gold particles with the particle size of 15 nm into the treated monolithic column until the column body becomes dark reddish brown and the liquid flowing out from the tail end is pink, and thus obtaining the nano gold-graphene modified silica gel column.
(4) Preparing an aptamer-bonded silica gel hybrid affinity monolithic column:
centrifuging the sulfhydryl modified aptamer for 5 min at 10000 r/min, adding a proper amount of buffer solution A with the pH of 8.0, shaking and dissolving to prepare a aptamer solution with the solubility of 250 mu mol/L, placing at 90 ℃, heating for 3 min, and cooling to room temperature to obtain an activated sulfhydryl aptamer solution; 20 mu L of 250 mu mol/L activated sulfhydryl aptamer is taken, 30 mu L of 5 mmol/L tris (2-carboxyethyl) phosphine (TCEP) is added into the activated sulfhydryl aptamer, and the mixture is incubated for 1 h at room temperature in a shaking table; and (3) injecting the incubated nucleic acid aptamer solution into the nanogold-graphene modified silica gel porous monolithic column obtained in the step (3) at room temperature, washing with water for 1 h for the second time to prepare the silica gel hybrid affinity monolithic column with the nucleic acid aptamer bonded on the surface, introducing a buffer solution B with the pH of 8.0, and storing at the temperature of 4 ℃ for later use.
Table 1 component content table of graphene modified porous silica gel hybrid monolithic column
a, PEG is MWPolyethylene glycol of = 10000;
TMSPMA-modified GO is a modified graphene aqueous solution with the concentration of 1 mg/mL;
FIG. 2 is an electron microscope morphology of a nanogold-graphene porous silica gel hybrid capillary monolithic column before and after modification with an aptamer, wherein a is a cross-sectional scanning electron microscope of a graphene-nanogold composite interface monolithic column with gold nanoparticles modified but without the aptamer, and a B is an interface scanning electron microscope of a graphene-nanogold composite interface affinity monolithic column with the aptamer modified.
Example 2
The preparation method comprises the following steps of respectively preparing aptamers for modifying gold nanoparticles without modifying ochratoxin A from a formula B as a control column and an affinity monolithic column for modifying ochratoxin A aptamers, and respectively carrying out balancing, enrichment, cleaning and elution, wherein the preparation method comprises the following specific steps:
(1) balancing: control and modified anti-ochratoxin a aptamer affinity columns were equilibrated with binding buffer. Binding buffer: 10 mM Tris-HCl (pH 8.0), 120 mM NaCl, 5 mM KCl, 20mM CaCl2A 500 psi back pressure valve with flow rate of 0.05 mL/min, balance for 0.5 h;
(2) enrichment: 20 μ L of 5 ng/mL ochratoxin A (OTA) solution was injected and enriched for 0.5 h on the control column and the modified OTA aptamer affinity column, respectively. And (5) a 500 psi back pressure valve with the flow rate of 0.05 mL/min, collecting the enrichment solution and detecting.
(3) Cleaning: and (4) washing the monolithic column by using a binding buffer solution, and collecting a washing solution to be tested after washing by a certain volume.
(4) And (3) elution: with 30% ACN: OTA was eluted from the monolithic column with 70% TE buffer (10 mM Tris-HCl pH 8.0, 2.5 mM EDTA), 20. mu.L of the eluate was collected under conditions of 500 psi back pressure, flow rate 0.1 mL/min, and 20. mu.L of the eluate was collected.
(5) And (3) detection: respectively injecting the collected enrichment liquid, cleaning liquid and eluent into HPLC-RF-20A for detection, and detecting OTA conditions: mobile phase: 2% water acetate acetonitrile =38:62, Ex=333 nm，Em=460 nm, 1 mL/min, the detection results are shown in fig. 3.
As can be seen from fig. 3, ochratoxin a is detected in both the enriched liquid and the cleaning liquid of the aptamer control column which only modifies gold nanoparticles but does not modify anti-ochratoxin a, ochratoxin a is not detected in the eluent, the ochratoxin a is not effectively retained by the control column, ochratoxin a is not detected in both the enriched liquid and the cleaning liquid of the affinity monolithic column, and ochratoxin a is detected in the eluent, so that the affinity monolithic column based on the graphene-nanogold composite interface ultra-high load aptamer can realize high specific recognition of ochratoxin a.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> department of building
<120> affinity monolithic column based on graphene-nanogold composite interface and ultrahigh-load aptamer and preparation method thereof
Preparation method
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> Artificial sequence
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gatcgggtgt gggtggcgta aagggagcat cggaca 36
Claims (8)
1. An affinity monolithic column based on a graphene-nanogold composite interface ultrahigh-load aptamer is characterized in that: the affinity monolithic column is an organic-inorganic hybrid silica gel aptamer affinity monolithic column which takes graphene oxide with surface functionalization of an alkenyl siloxane reagent as a catalyst and a modification material, is polymerized with a mercapto siloxane reagent and an auxiliary siloxane reagent by a one-pot method and is constructed by post-column modification of nanogold to form a graphene-nanogold composite layer, and based on the bridging action of nanogold, the graphene-nanogold composite layer and a mercapto aptamer are bonded to form an ultra-high density modification aptamer affinity action interface; the graphene oxide in the affinity monolithic column and a mercaptosiloxane monomer of a mercaptosiloxane reagent are immobilized through a mercapto-alkene click reaction; the graphene forms a graphene-nanogold composite layer through physical adsorption of nanogold; the graphene-nanogold composite layer can provide a large number of binding sites, and the sulfhydryl aptamer can be combined with nanogold in the graphene-nanogold composite layer and also can be combined with an olefin unsaturated bond on the surface of graphene to form an ultrahigh-density modified aptamer affinity interaction interface.
2. The affinity monolithic column based on the ultra-high nucleic acid aptamer load on the graphene-nanogold composite interface as claimed in claim 1, wherein: wherein, in the one-pot polymerization reaction of the graphene oxide with the surface functionalized by the alkenyl siloxane, the mercaptosiloxane reagent and the auxiliary siloxane reagent, no acid catalyst is needed to be added.
3. The affinity monolithic column based on the ultra-high nucleic acid aptamer load on the graphene-nanogold composite interface as claimed in claim 1, wherein: the alkenyl-containing siloxane reagent is methacryloxypropyl tris (trimethylsiloxy) silane; the auxiliary siloxane reagent is a silanization reagent and an initiator, wherein the silanization reagent is tetramethoxysilane and 3-mercaptopropyltrimethoxysilane, and the initiator is azobisisobutyronitrile; the mercaptosiloxane reagent is mercaptopropyltrimethoxysilane.
4. The affinity monolithic column based on the ultra-high nucleic acid aptamer load on the graphene-nanogold composite interface as claimed in claim 1, wherein: the aptamer is anti-ochratoxin A, and the base sequence is 5'-SH-C6-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3'.
5. A method for preparing the affinity monolithic column based on the ultra-high aptamer loading of the graphene-nanogold composite interface as claimed in any one of claims 1 to 4, wherein the method comprises the following steps: the method comprises the following steps:
(1) preparation of methacryloxypropyl tris (trimethylsiloxy) silane modified graphene oxide solution:
weighing 15 mg of GO solid powder, adding 15 mL of ultrapure water to remove, carrying out ultrasonic treatment for 3h to uniformly disperse the GO solid powder in water to form 1 mg/mL of GO aqueous solution, adding 30 uL of methacryloxypropyl tris (trimethylsiloxy) silane (gamma-MAPS) at room temperature of 25 ℃, and stirring for 24 h; centrifuging at 10000 r/min for 10 min to remove unreacted substances, washing the precipitate with secondary water for 3 times, and dispersing in ultrapure water again to obtain methacryloxypropyl tris (trimethylsiloxy) silane modified graphene oxide solution;
(2) preparation of poly (TMOS-co-MPTMS-co-GO) silica gel hybrid monolithic column:
weighing urea and polyethylene glycol as pore-forming agents, weighing a certain amount of methacryloxypropyl tris (trimethylsiloxy) silane modified graphene oxide solution in a round-bottom flask, stirring until the solution is completely dissolved, and continuously stirring at the temperature of 0 ℃ for 600 r/min in an ice bath; weighing tetramethoxysilane, 3-mercaptopropyltrimethoxysilane and azodiisobutyronitrile in a centrifugal tube according to a certain proportion, carrying out vortex oscillation until the tetramethoxysilane, the 3-mercaptopropyltrimethoxysilane and the azodiisobutyronitrile are completely dissolved, dropwise adding the mixture into a round-bottom flask, carrying out ice bath at 0 ℃ for 45 min, and carrying out ultrasonic degassing; injecting the mixture into a pretreated quartz capillary tube, sealing two ends of the quartz capillary tube, and placing the quartz capillary tube in a 47 ℃ water bath kettle for constant-temperature reaction for 24 hours; taking out the prepared monolithic column, heating at 120 ℃ for 3h, and removing unreacted residues by using a high-pressure solvent pump to obtain a surface graphene modified porous silica gel hybrid monolithic column;
(3) preparing a nanogold-modified graphene functional silica gel hybrid monolithic column:
washing the prepared 2cm graphene modified silica gel column with secondary water, introducing nano gold particles with the particle size of 15 nm into the treated monolithic column until the column body becomes dark reddish brown and the liquid flowing out from the tail end is pink, and preparing the nano gold-graphene modified silica gel column;
(4) preparing an aptamer-bonded silica gel hybrid affinity monolithic column:
centrifuging the sulfhydryl modified aptamer for 5 min at 10000 r/min, adding a proper amount of buffer solution A with pH of 8.0, oscillating and dissolving to prepare a aptamer solution with the solubility of 250 mu mol/L, placing the aptamer solution at 90 ℃ for heating for 3 min, cooling to room temperature, adding 30 mu L of 5 mmol/L tris (2-carboxyethyl) phosphine (TCEP) into 20 mu L of 250 mu mol/L activated sulfhydryl aptamer, and incubating for 1 h in a shaking table at room temperature; and (3) injecting the incubated nucleic acid aptamer solution into the nanogold-graphene modified silica gel porous monolithic column obtained in the step (3) at room temperature, washing with water for 1 h for the second time to prepare the silica gel hybrid affinity monolithic column with the nucleic acid aptamer bonded on the surface, introducing a buffer solution B with the pH of 8.0, and storing at the temperature of 4 ℃ for later use.
6. The method of claim 5, wherein: the polymerization liquid formula in the step (2) comprises the following components in percentage by mass, the sum of the mass percentages is 100%: 19.98-22.19% of tetramethoxysilane, 6.95-8.80% of 3-mercaptopropyltrimethoxysilane, 53.06-59.29% of methacryloxypropyltris (trimethylsiloxy) silane modified graphene oxide solution, 7.97-8.93% of urea, 5.81-6.89% of polyethylene glycol and 0.46-0.51% of azobisisobutyronitrile.
7. The method of claim 5, wherein: the molecular weight of the polyethylene glycol is 10000.
8. The method of claim 5, wherein: the buffer solution A consists of 10 mmol/L Tris-HCl, 120 mmol/L NaCl and 5 mmol/L KCl; the buffer solution B consists of 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl and 20mmol/L CaCl2And (4) forming.
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| CN112316492A (en) * | 2020-10-22 | 2021-02-05 | 厦门华厦学院 | Aptamer affinity monolithic column capable of being simultaneously used for specific recognition of various mycotoxins and preparation method thereof |
| CN112870764B (en) * | 2021-02-25 | 2021-12-21 | 福州大学 | Functional micro-extraction probe with polysiloxane-modified graphene polymeric coating and preparation method thereof |
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