CN115990355A - Affinity monolithic column with orderly functionalized aptamer and preparation method thereof - Google Patents
Affinity monolithic column with orderly functionalized aptamer and preparation method thereof Download PDFInfo
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Images
Abstract
The invention discloses an affinity monolithic column with orderly functionalized nucleic acid aptamer and a preparation method thereof, wherein double-block DNA containing poly-A and the nucleic acid aptamer Apt is modified on the surface of nano gold with carboxyl negative charge on the surface to form AuNPs@poly-A-Apt composite nano particles with a core-shell structure, and then the composite nano particles are used as functional probes and directly adsorbed on the surface of a stationary phase matrix formed by a strong cationic polymer through electrostatic action, so that the nucleic acid aptamer can be vertically and orderly arranged on the surface of the nano gold, thereby preparing the affinity monolithic column. According to the invention, through directional assembly of the double-block DNA, the ordering and affinity recognition effects of the aptamer DNA sequence on the surface of the gold nanomaterial are improved, the problems of low utilization efficiency and the like caused by the disorder of aptamer dispersion on the surface of the affinity monolithic column are avoided, and the effective regulation and control and the efficient utilization of the aptamer in the column conformational state are realized.
Description
Technical Field
The invention belongs to the field of material chemistry, and particularly relates to an affinity monolithic column based on ordered functionalization of a nucleic acid aptamer and a preparation method thereof.
Background
The interaction between the aptamer base and the interface material often does not maintain the conformation on the nanointerface well, which can greatly affect the function of the aptamer. Biological binding methods such as streptavidin-biotin, covalent binding methods and nano gold (AuNPs) methods are utilized as effective introduction modes of nucleic acid aptamer, so that good development is achieved, but the nucleic acid aptamer often presents disordered distribution on the surface of a material due to random adsorptivity of the nucleic acid sequence. The interaction between aptamer and AuNPs particles has been widely used for affinity recognition of trace species in complex samples. The ordered distribution of the nucleic acid aptamer on the surface of the AuNPs is realized, and the method has important significance for improving the affinity recognition performance of the aptamer.
The gold nanoparticle loaded aptamer monolithic column is prepared by a salt aging method at present, and is generally prepared by firstly preparing a aptamer-gold nanoparticle complex and then fixing the complex on the monolithic column. Chen et al immobilized three mono-functionalized (amine, thiol and carboxyl) and two di-functionalized (amine and thiol and amine and carboxyl) hybrid monolithic columns using Gold Nanoparticles (GNP) as a vehicle to construct universal substrate modified GNPs with a loading density of the prepared aptamer affinity monolithic column as high as 2205.8 pmol. Mu.L -1 . Yu et al immobilized the Apt@AuNPs complex on a (POSS-PEI) hybrid whole column with rich groups. Lin et al uses nano gold particles as a medium to prepare a series of aptamer affinity monolithic columns, realizes the specific recognition and detection of different toxins such as ochratoxin A, zearalenone, bisphenol A and other small molecular targets, and has a coverage density as high as 3388-3636 pmol.mu.L -1 . The aptamer loading density of the prepared affinity monolithic column reaches a higher level, but the aptamer has a certain adsorption capacity on the Au surface of the aptamer, and nonspecific adhesion cannot be avoided even in a lodging disordered state on the AuNPs surface, so that the effective utilization of the biological functions of DNA is affected. Therefore, achieving effective regulation and efficient utilization of the aptamer in the conformational state of the column is critical to current research.
It was found that DNA charge adsorption is a simple, straightforward and cost-effective model. DNA with negative charges (based on phosphate groups) can be adsorbed on positively charged polymers or support carriers, and strong adhesion of DNA molecules on the surface of AuNPs can be realized by utilizing strong adhesion force between poly adenine (polyA) and the surface of nano gold, and nonspecific adsorption between other base sequences of the DNA and the nano gold can be shielded, so that effective combination and self-assembly ordering of the DNA on the surface of the nano gold can be realized. At present, how to effectively develop double-embedded DNA modification and ordered structure construction of nano gold in the field of affinity monolithic columns is not reported, so that the ordered control of the direction and conformation of a DNA sequence in a monolithic column microscale space is still challenging.
Disclosure of Invention
The invention aims to provide an affinity monolithic column with orderly functionalized aptamer and a preparation method thereof, which are based on photoinitiated polymerization reaction, wherein alkenyl free radicals are polymerized in situ to form a monolithic column matrix with the surface rich in strong positive charges, and then nano gold@diblock DNA composite nano particles with negative charges are directly absorbed under the electrostatic action to form the nano gold@diblock DNA self-assembled functionalized affinity monolithic column.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a nucleic acid aptamer orderly functionalized affinity monolithic column is characterized in that double-block DNA containing poly-A and a nucleic acid aptamer (Apt) is modified on the surface of nano-gold (AuNPs) with carboxyl negative charges on the surface to form a nuclear shell structure AuNPs@poly-A-Apt composite nanoparticle with adjustable covering density, the composite nanoparticle is used as a functional probe and is directly adsorbed on the surface of a stationary phase matrix formed by a strong cationic polymer through electrostatic action, and the vertical orderly arrangement of the nucleic acid aptamer on the surface of the nano-gold is realized by utilizing the preferential binding action of poly-A and the nano-gold and the steric hindrance effect between DNA chains, so that the affinity monolithic column is prepared.
Further, the double-block DNA has a poly (adenine) sequence at one end and a nucleic acid aptamer (Apt) having a recognition function at the other end, and its specific base sequence is (A … A) n TTTTTGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA, wherein n=5 to 30.
Further, the strong cationic polymer is formed by photoinitiated in-situ polymerization with the aid of a pore-forming agent and an initiator by taking a quaternary ammonium monomer as an ionization reagent, polysilsesquioxane as a crosslinking agent and a 2-oxazolinone compound as a functional monomer.
Further, the quaternary ammonium monomer is specifically methacryloxyethyl trimethyl ammonium chloride (DMC), the 2-oxazolinone compound is specifically 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone (VDMA), and the polysilsesquioxane is specifically methacrylate branched polyhedral oligomeric silsesquioxane; the pore-foaming agent consists of N, N-Dimethylformamide (DMF) and polyethylene glycol (PEG-10000) with molecular weight of 10000 according to the mass ratio of 19:1; the initiator is benzoin dimethyl ether.
The preparation method of the affinity monolithic column comprises the following steps:
(1) Preparation of AuNPs@polyA-Apt composite nanoparticles:
adding the double-block DNA into a 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution with the pH value of 7.60 and the concentration of 5mM to prepare a HEPES buffer solution containing the double-block DNA 100 nM; taking 1.0 mu L of HEPES buffer solution containing double-block DNA, mixing and incubating with 200 mu L of gold nanoparticle solution of 10 nM at room temperature for 10 min, and then adding 500mM citrate buffer solution with pH value of 3.00 until the final concentration of citrate in the mixed solution is 10 mM; then reacting for 3min at room temperature, adding 500mM HEPES buffer to adjust pH to 7.60, continuing to incubate for 5-10 min at room temperature, centrifuging and washing for three times, removing free nucleic acid aptamer to obtain AuNPs@polyA-Apt composite nano particles, dispersing the AuNPs@polyA-Apt composite nano particles in 5mM HEPES buffer, and preserving the mixture in a refrigerator at 4 ℃ for later use;
(2) Preparation of monolithic column matrix:
weighing quaternary ammonium monomer, 2-oxazolinone compound and polysilsesquioxane according to a proportion, mixing, adding a pore-forming agent and an initiator, and carrying out ultrasonic degassing for 10 min after vortex oscillation for 3min at room temperature to form a uniform solution; then the obtained solution is injected into a quartz capillary tube which is subjected to the olefination pretreatment, and the two ends of the quartz capillary tube are sealed and then are subjected to ultraviolet light irradiation for 5min for polymerization at 365 nm; taking out the prepared monolithic column after the reaction is finished, connecting the monolithic column to a liquid chromatograph, pumping methanol into the liquid chromatograph by using a high-pressure solvent pump to flush and remove residues, and obtaining the cross-linked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges;
(3) Preparation of affinity monolithic column:
filling Tris-HCl buffer solution into the monolithic column prepared in the step (2), then injecting the AuNPs@polyA-Apt composite nanoparticle solution obtained in the step (1) through a liquid phase pump until the column turns from white to reddish brown, stopping injection when the liquid flowing out from the tail end turns into pink, respectively washing the monolithic column with secondary water and a binding buffer solution to remove unbound complexes, and obtaining the AuNPs@diblock DNA modified high-affinity monolithic column, and storing at 4 ℃.
Further, the mass percentages of the components used for preparing the monolithic column matrix in the step (2) are as follows: 7.84% of quaternary ammonium salt monomer, 6.86% of polysilsesquioxane, 4.90% of 2-oxazolinone compound, 78.4% of pore-forming agent and 2.00% of initiator.
Further, the pH of the binding buffer in step (2) was 8.00, which contained 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl and 20mmol/L CaCl 2 。
The invention takes the nano gold@double-block DNA composite nano particle containing a poly-A block and a nucleic acid aptamer (Apt) as a functional probe, and directly adsorbs the nano gold@double-block DNA composite nano particle based on strong cations on the surface of a polymer to form the affinity monolithic column, meanwhile, as the poly-A and the nano gold have preferential combination and the steric hindrance effect between DNA chains, the nucleic acid aptamer can be vertically and orderly arranged on the surface of the nano gold, and the coverage density of the nano gold@double-block DNA composite nano particle on the monolithic column can be regulated and controlled by regulating the number of A bases of a poly A block part (the modification density of the nano gold@double-block DNA composite nano particle is reduced along with the increase of the number of A bases), thereby realizing high-specificity identification of a target object.
The invention has the remarkable advantages that:
aiming at the limitation that the non-specific adhesion and even lodging disorder state of a single-stranded linear nucleic acid aptamer can be generated on the Au surface, so that the conformation state of the nucleic acid aptamer in a whole column limited space is difficult to effectively regulate and control and effectively utilize, the invention provides a novel technology for orderly self-assembling nano gold@double-block DNA composite nano particles on the micro-size surface of a whole column. Based on the strong electrostatic combination effect of quaternary ammonium salt cations on the surface of the matrix of the whole column, the high-efficiency self-assembly of the nano gold@double-block DNA composite nano particles is realized; based on the preferential action of the poly adenine and the nano gold surface and the steric hindrance effect between DNA chains, the nucleic acid aptamer on the nano gold@double-block DNA composite nanoparticle is vertically and orderly arranged on the nano gold surface, so that the bottleneck problems of conformational regulation and efficient utilization of the nucleic acid aptamer in a limited space are solved.
Compared with the traditional covalent bond bonding method, the method is based on direct charge adsorption of the aptamer, the preparation process is direct and rapid, chemical modification is not needed, the synthesis cost of the aptamer is reduced, good economic benefits are achieved, the activation step required by disulfide bond formation is avoided, and the method has the advantages of simplicity in preparation, high immobilization efficiency of the aptamer and the like; the poly-A is utilized to carry out saturated coverage on the surface of the nano gold, so that the nonspecific adsorption of DNA on the surface of AuNPs can be effectively inhibited; and the assembly density of the poly-A block can be controlled by simply adjusting the length of the poly-A block, thereby providing a new way for regulating and preparing the high-performance aptamer functionalized affinity monolithic column and application.
Drawings
FIG. 1 is an electron microscope topography of the cross-linked polymeric monolith (A) and the nanogold@diblock DNA modified affinity monolith (B) prepared in example 1.
FIG. 2 is a dynamic light scattering plot of hydrodynamic radius analysis of example 2.
FIG. 3 is a graph comparing the effect of the nanogold@thiol aptamer modified affinity monolith (A) and the nanogold@double-block DNA modified affinity monolith (B) on a target at similar coverage densities in example 3.
Detailed Description
Preparation of a nucleic acid aptamer affinity monolithic column based on DNA ordered functionalization, comprising the following steps:
(1) Preparation of AuNPs@polyA-Apt composite nanoparticles:
adding the double-block DNA into 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution with the pH of 7.60 and the concentration of 5mM to prepare HEPES buffer solution containing double-block DNA of 100 nM, taking 1.0 mu L of the HEPES buffer solution, mixing and incubating the HEPES buffer solution with 200 mu L of gold nano-solution of 10 nM for 10 minutes at room temperature, and then adding citrate buffer solution with the pH of 3.00 and the pH of 500mM until the final concentration of citrate in the mixed solution is 10 mM; then reacting for 3min at room temperature, adding 500mM HEPES buffer to adjust pH to 7.60, continuing to incubate for 5-10 min at room temperature, centrifuging and washing for three times, removing free nucleic acid aptamer to obtain AuNPs@polyA-Apt composite nano particles, dispersing the AuNPs@polyA-Apt composite nano particles in 5mM HEPES buffer, and preserving the mixture in a refrigerator at 4 ℃ for later use;
(2) Preparation of monolithic column matrix:
weighing methacryloxyethyl trimethyl ammonium chloride (DMC), 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone (VDMA) and polyhedral oligomeric silsesquioxane grafted by methacrylate according to a proportion, mixing, adding N, N-Dimethylformamide (DMF), polyethylene glycol with molecular weight of 10000 (PEG-10000) and benzoin dimethyl ether, and carrying out ultrasonic degassing for 10 min after vortex oscillation for 3min at room temperature to form a uniform solution; then the obtained solution is injected into a quartz capillary tube which is subjected to the olefination pretreatment, and the two ends of the quartz capillary tube are sealed and then are subjected to ultraviolet light irradiation for 5min for polymerization at 365 nm; taking out the prepared monolithic column after the reaction is finished, connecting the monolithic column to a liquid chromatograph, pumping methanol into the liquid chromatograph by using a high-pressure solvent pump to flush and remove residues, and obtaining the cross-linked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges;
(3) Preparation of affinity monolithic column:
filling Tris-HCl buffer solution into the monolithic column prepared in the step (2), then injecting the AuNPs@polyA-Apt composite nanoparticle solution obtained in the step (1) through a liquid phase pump until the column turns from white to reddish brown, stopping injection when the liquid flowing out from the tail end turns into pink, respectively washing the monolithic column with secondary water and a binding buffer solution to remove unbound complexes, and obtaining the AuNPs@diblock DNA modified high-affinity monolithic column, and storing at 4 ℃.
The base sequence of the double-block DNA in step (1) is (A … A) n TTTTTGATCGGGTGTGGGT GGCGTAAAGGGAGCATCGGACA, wherein n=5 to 30.
The mass percentages of the components used for preparing the monolithic column matrix in the step (2) are as follows: 7.84% of methacryloxyethyl trimethyl ammonium chloride, 6.86% of methacrylate branched polyhedral oligomeric silsesquioxane, 4.90% of 2-vinyl-4, 4-dimethyl-2-oxazolin-5-one, 74.48% of N, N-dimethylformamide, 3.92% of polyethylene glycol and 2.00% of benzoin dimethyl ether.
The pH of the binding buffer in step (3) was 8.00, which contained 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl and 20mmol/L CaCl 2 。
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
The preparation method of the affinity monolithic column with orderly functionalized aptamer comprises the following steps:
(1) Preparation of AuNPs@polyA-Apt composite nanoparticles:
the preparation of gold nanoparticles (AuNPs) solutions by sodium citrate in literature (Chemical Communications, 2008, (19): 2242-2244); then adding the double-block DNA into HEPES buffer solution with the pH value of 7.60 and the concentration of 5mM to prepare HEPES buffer solution containing double-block DNA 100 nM; taking 1.0 mu L of HEPES buffer solution containing double-block DNA, mixing with 200 mu L of nano gold solution (10 nM) at room temperature for incubation for 10 min, adding citrate buffer solution (500 mM, pH=3.00) until the final concentration of citrate in the mixed solution is controlled to be 10 mM, reacting at room temperature for 3min, adding HEPES buffer solution (500 mM) to adjust the pH of the solution to 7.60, continuing to incubate at room temperature for 5-10 min, centrifuging and washing for three times, and removing free nucleic acid aptamer; finally, the obtained AuNPs@polyA-Apt composite nanoparticle is dispersed in HEPES buffer solution of 5mM, and is stored at 4 ℃ for standby.
The base sequence of the double-block DNA is (A … A) nTTTTTGATCGGGTGTGGGT GGCGTAAAGGGAGCATCGGACA, and n=5-30.
(2) Alkenyl formation of capillary column wall surfaces
And (3) washing the capillary empty column for 3 hours by adopting a hydrochloric acid solution with the concentration of 0.1 mol/L and a NaOH solution with the concentration of 1.0 mol/L in sequence, washing with deionized water for 15 minutes, washing with methanol for 15 minutes, then drying with nitrogen at the temperature of 50 ℃, injecting a mixture with the volume ratio of methanol to methacryloxypropyl trimethoxy silane of 1:1, reacting for 12 hours at the temperature of 60 ℃, washing with methanol for 15 minutes, and drying with nitrogen at the temperature of 70 ℃ to obtain the styrenated pretreated quartz capillary.
(3) Preparation of monolithic column matrix
Weighing DMC 7.84%, methacrylate grafted polyhedral oligomeric silsesquioxane 6.86%, VDMA 4.90%, DMF 74.48%, PEG-10000.3.92% and benzoin dimethyl ether 2.00%, vortex oscillating for 3min at room temperature, ultrasonic degassing for 10 min to form a uniform solution, injecting into a quartz capillary tube subjected to olefination pretreatment, sealing two ends, and irradiating for 5min with 365 nm ultraviolet light for polymerization; and after the reaction is finished, the prepared monolithic column is taken out and connected to a liquid chromatograph, methanol is pumped into the monolithic column by a high-pressure solvent pump to flush and remove residues, and the crosslinked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges is obtained.
(4) Preparation of affinity monolithic columns
Continuously introducing AuNPs@polyA-Apt composite particle solution into the whole column prepared in the step (3) by means of a liquid phase pump until the column turns from white to reddish brown, stopping injection when the liquid flowing out from the tail end turns into pink, and then respectively using secondary water and a binding buffer solution (containing 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl and 20mmol/L CaCl) 2 Ph=8) washing the monolith to remove unbound complex, resulting in aunps@diblock DNA modified affinity monolith and preserving at 4 ℃.
FIG. 1 is an electron microscope topography of a cross-linked polymeric monolith (A) and a nanogold@diblock DNA modified affinity monolith (B) prepared in this example. As shown in figure 1, the monolithic column matrix is formed by stacking uniform particles to form a cluster morphology, the hole distribution is obvious, and the composite nano particles on the affinity monolithic column modified by the nano gold@double-embedded DNA are densely attached to the surface of the monolithic column matrix, so that the stability of the affinity monolithic column modified by the double-embedded DNA in the use process is ensured.
For experimental comparison, a corresponding control monolith was prepared using a thiol aptamer with a base sequence of 5' -SH-C6-GAT CGG GTG TGGGTG GCG TAA AGG GAG CAT CGG ACA.
Example 2
To elucidate the ordered assembly of the poly-A-bearing diblock DNA on the surface of the nanogold, a hydrodynamic radius analysis of the nanogold @ diblock DNA block was performed based on the Dynamic Light Scattering (DLS) method, the results of which are shown in FIG. 2.
As shown in FIG. 2, the nano-gold particles have a hydrodynamic diameter of about 21.9 and nm, and the poly-adenine mediated series AuNPs@polyA-Apt composite nano-particles have a hydrodynamic diameter of 34.2-36.5 nm, which indicates that the polyA block is fully adsorbed on the nano-gold surface, thus keeping the state of the aptamer base sequence chain portion similar; the fluidic diameter of the nano gold compound modified by the sulfhydryl aptamer is about 29.7 and nm, which is obviously smaller than that of the AuNPs@polyA-Apt compound nano particle, and analysis shows that the sulfhydryl aptamer is possibly entangled and lodged on the surface of the nano gold, so that the chain part of the nano gold compound is extended and has low freeness.
Example 3
To further illustrate the performance of the nanogold@diblock DNA self-assembled affinity monolith, a nanogold@diblock DNA modified affinity monolith with a substantially consistent coverage density and a comparative nanogold@thiol aptamer modified affinity monolith (monolith lengths of 5 cm) were prepared. And taking ochratoxin A (OTA) as an application object, and evaluating the specific recognition effect of the prepared affinity column on a target object. The method comprises the following specific steps:
(1) Balance: the binding buffer (containing 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5 mmol/L KCl and 20mmol/L CaCl) was used 2 Its ph=8) equilibrated affinity monolith for 30-40 min (flow rate 0.10mL/min, pressure 500 psi);
(2) Loading: 20. Mu.L of the OTA-containing sample solution was separately injected into the monolith (flow rate 0.05mL/min, pressure 250 psi) and allowed to bind for 0.5 hours;
(3) Cleaning: loading the enrichment column onto a liquid chromatographic pump, and eluting the OTA which remains and is not specifically captured in the control column and the whole column by using 10 mM Tris-HCl buffer solution;
(4) Eluting: eluting the OTA captured by the monolithic column with a Tris-HCl-EDTA buffer solution containing 40% acetonitrile (containing 10 mM Tris-HCl pH 8.00 and 2.5mM EDTA) (250 psi back pressure valve, flow rate 0.05 mL/min), and collecting the eluate to be tested;
(5) And (3) detection: the collected eluent is injected into HPLC-RF-20A for detection under the following conditions: mobile phase: 2% acetic acid water acetonitrile=38:62 (v/v), E x =333 nm，E m Sample injection amount 20 μl sample injection analysis was performed at flow rate of 1.0 mL/min=460 nm, and the results are shown in fig. 3.
As shown in FIG. 3, the peak response of the diblock DNA modified affinity monolith to OTA was significantly higher than that of the AuNPs@SH-Apt affinity monolith. The result of the combination of the example 2 shows that the nano gold@mercapto aptamer cannot effectively regulate the configuration of the aptamer on the nano gold surface, so that the prepared aptamer on the surface of the affinity monolithic column cannot be completely in an ideal state and interact with a target, and the nano gold@double-embedded DNA modified aptamer on the surface of the affinity monolithic column has better activity due to the fact that the nano gold@double-embedded DNA modified aptamer can maintain a good space configuration, and thus has better affinity recognition capability for OTA.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (7)
1. A preparation method of an affinity monolithic column with orderly functionalized aptamer is characterized by comprising the following steps: the double-block DNA containing poly-A and aptamer Apt is modified on the surface of the gold AuNPs with carboxyl negative charge on the surface to form the nuclear shell structure AuNPs@polyA-Apt composite nano particle with adjustable covering density; then the composite nano particles are used as functional probes, and are directly adsorbed on the surface of a stationary phase matrix formed by a strong cationic polymer through electrostatic action, and the upright ordered arrangement of the nucleic acid aptamer on the surface of the nano gold is realized by utilizing the preferential binding action of poly-A and the nano gold and the steric hindrance effect between DNA chains, so that the affinity monolithic column is prepared;
the strong cationic polymer is formed by photoinitiated in-situ polymerization with the aid of a pore-forming agent and an initiator by taking a quaternary ammonium monomer as an ionization reagent, polysilsesquioxane as a crosslinking agent and a 2-oxazolinone compound as a functional monomer.
2. The method for preparing the affinity monolithic column for ordered functionalization of the aptamer according to claim 1, wherein the method comprises the following steps: the double block DNA has one end containing poly-A sequence and the other end being nucleic acid aptamer Apt with recognition function, and its specific base sequence is (A … A) n TTTTTGATCGGGTGTGGGT GGCGTAAAGGGAGCATCGGACA, wherein n=5 to 30.
3. The method for preparing the affinity monolithic column for ordered functionalization of the aptamer according to claim 1, wherein the method comprises the following steps: the quaternary ammonium monomer is specifically methacryloyloxyethyl trimethyl ammonium chloride, the 2-oxazolinone compound is specifically 2-vinyl-4, 4-dimethyl-2-oxazoline-5-ketone, and the polysilsesquioxane is specifically polyhedral oligomeric silsesquioxane grafted by methacrylate; the pore-foaming agent consists of N, N-dimethylformamide and polyethylene glycol with molecular weight of 10000 according to the mass ratio of 19:1; the initiator is benzoin dimethyl ether.
4. The method for preparing the affinity monolithic column for ordered functionalization of the aptamer according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) Preparation of AuNPs@polyA-Apt composite nanoparticles:
adding the double-block DNA into HEPES buffer solution with the pH value of 7.60 and the concentration of 5mM to prepare HEPES buffer solution containing double-block DNA 100 nM; taking 1.0 mu L of HEPES buffer solution containing double-block DNA, mixing and incubating with 200 mu L of gold nanoparticle solution of 10 nM at room temperature for 10 min, and then adding 500mM citrate buffer solution with pH value of 3.00 until the final concentration of citrate in the mixed solution is 10 mM; then reacting for 3min at room temperature, adding 500mM HEPES buffer to adjust the pH to 7.60, continuously incubating for 5-10 min at room temperature, centrifuging and washing for three times to obtain AuNPs@polyA-Apt composite nano particles, dispersing the AuNPs@polyA-Apt composite nano particles in 5mM HEPES buffer, and preserving the AuNPs@polyA-Apt composite nano particles in a refrigerator at 4 ℃ for later use;
(2) Preparation of monolithic column matrix:
weighing quaternary ammonium monomer, 2-oxazolinone compound and polysilsesquioxane according to a proportion, mixing, adding a pore-forming agent and an initiator, and carrying out ultrasonic degassing for 10 min after vortex oscillation for 3min at room temperature to form a uniform solution; then the obtained solution is injected into a quartz capillary tube which is subjected to the olefination pretreatment, and the two ends of the quartz capillary tube are sealed and then are subjected to ultraviolet light irradiation for 5min for polymerization at 365 nm; taking out the prepared monolithic column after the reaction is finished, connecting the monolithic column to a liquid chromatograph, and flushing with methanol to remove residues to obtain a cross-linked polymeric monolithic column with the surface rich in quaternary ammonium salt positive charges;
(3) Preparation of affinity monolithic column:
filling Tris-HCl buffer solution into the monolithic column prepared in the step (2), then injecting the AuNPs@polyA-Apt composite nanoparticle solution obtained in the step (1) until the column body turns from white to reddish brown, stopping injection when the liquid flowing out from the tail end turns into pink, and then respectively washing the monolithic column by using secondary water and a binding buffer solution to obtain the AuNPs@diblock DNA modified high-affinity monolithic column.
5. The method for preparing the aptamer ordered functionalized affinity monolithic column according to claim 4, wherein the method comprises the following steps: the mass percentages of the components used for preparing the monolithic column matrix in the step (2) are as follows: 7.84% of quaternary ammonium salt monomer, 6.86% of polysilsesquioxane, 4.90% of 2-oxazolinone compound, 78.4% of pore-forming agent and 2.00% of initiator.
6. The method for preparing the affinity monolithic column for ordered functionalization of the aptamer according to claim 4, wherein the method comprises the following steps: the pH of the binding buffer was 8.00, which contained 10 mmol/L Tris-HCl, 120 mmol/L NaCl, 5mmmol/L KCl and 20mmol/L CaCl 2 。
7. A nucleic acid aptamer ordered functionalized affinity monolith prepared according to the method of any one of claims 1-6.
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