KR101273963B1 - Aptamer immobilized Nanofibers composite and use of separation and purification of protein using the same - Google Patents

Abstract

The present invention relates to a nanofiber complex having an aptamer immobilized and a protein separation / purification using the same, and more particularly, to a polystyrene-poly (styrene-co-maleic hydride) nanofiber complex having an aptamer immobilized therein The present invention relates to a protein separation / purification method.

Description

Aptamer immobilized Nanofibers composite and use of separation and purification of protein using the same}

The present invention relates to a nanofiber complex having an aptamer immobilized and a protein separation / purification using the same, and more particularly, to a polystyrene-poly (styrene-co-maleic hydride) nanofiber complex having an aptamer immobilized therein The present invention relates to a protein separation / purification method.

BACKGROUND OF THE INVENTION Electrospun functional nanofibers have been widely applied in various fields such as chemical sensors, enzyme immobilization, tissue engineering, drug delivery membrane filters, and fibers. (HS Yoo, TG Kim and TG Park, Adv. Drug Deliv. Rev., 61 (2009) 1033-1042; J. Yoon, SK Chae and J.-M. Kim, J. Am. Chem. Soc., 129 (2007) 3038-3039; A. Greiner and J. Wendorff, Angew.Chem.-Int.Edit., 46 (2007) 5670-5703; JY Lee, CA Bashur, AS Goldstein and CE Schmidt, Biomaterials, 30 (2009) 4325-4335; A. Camposeo, FD Benedetto, R. Stabile, AAR Neves, R. Cingolani and D. Pisignano, Small, 5 (2009), 562-566; G. Ren, X. Xu, Q. Liu, J. Cheng, X. Yuan, L. Wu and Y. Wan, React.Funct.Polym., 66 (2006), 1559-1564; L. Wannatong and A. Sirivat, React.Funct.Polym., 68 (2008), 1646-1651; A. Senecal, J. Magnone, P. Marek and K. Senecal, React. Funct. Polym., 68 (2008), 1492-1434).

These functional nanofibers have a very large surface area with nano-scale sizes, and because they have an unhydride group, ligands such as aptamers can be easily immobilized in high proportions, which can be used for various applications using aptamers. have.

Antibodies have been mainly used as ligands of affinity columns used for protein separation / purification. (L. Hsiung, AN Barclay, NR Brandon, Biochem. J. 1982, 203, 293-298; R. ScoTT Hansen & JA Beavo, Proc. Natd Acad. Sci. USA, 1982, 79, 2788-2792; Lars S Nielsen, Jan G. Hansen, Lars Skriver, s Elaine L. Wilson, Keld Kaltoft, Jesper Zeuthen, and Keld Dana, Biochemistry , 1982, 21, 6410-6415). However, due to the high production cost and the chemical instability of the antibody, the affinity for the target is often decreased during immobilization or reuse, and the amount of immobilization per unit area is limited due to the large molecular weight.

Aptamers, on the other hand, are oligonucleotides obtained through a series of selection processes called selexes and have affinity for specific target materials. They can be applied to medicines, treatments, diagnostics, biosensors (Dzau, VJ, Mann, MJ, Morishita, R. & Kandea, Y. Proc . Natl Acad . Sci . USA 1996, 93 , 11421.11425; Brockstedt, U., Uzaroeska, A., Montpetit, A., Pfau, W. & Labuda, D. Biochem . Biophys . Res . Commun. 2003, 313 , 1004.1008; Liu, J. & Lu, Y. Fast. Angew . Chem . Int . Ed . Engl . 2006, 45 , 90.94), is also applied to the separation / purification of proteins. (Romig, TS, Bell, C. & Drolet, DW J. Chromat . 1999, 731 , 275.284; AC Connor, LB Mc Gown, J. Chromatogr. A 2006, 1111, 215; S. Cho, S. Lee, W. Chung, Y. Kim, Y. Lee , B. Kim, Electrophoresis, 2004, 25, 3730;.. Qiang Zhao, Xing-Fang Li, Yuanhua Shao & X. Chris Le, Anal Chem 2008, 80, 7586- 7593)

In general, microbead or tubular columns, which are used as aptamer immobilization materials for protein separation / purification, are complicated or expensive to produce or are difficult to aptamer immobilization. When the microbead type is used, the aptamer is immobilized only on the bead surface, so the capacity is not high and the void volume is also large and inefficient.

On the other hand, as a related prior patent, Korean Patent Publication No. 10-2007-0066902 relates to a dendritic polymer with enhanced amplification and internal functionalities. In the dendritic polymer having a specific structure, (C) denotes a core. (FF) represents the focal functional component of the core; x is independently 0 or an integer from 1 to Nc-1; (BR) represents a branch cell, and if p is greater than 1 (BR) May be the same or different residues; it is described that an aptamer may be selected as a component of FF,

Another related prior patent, Republic of Korea Patent Publication No. 10-2008-003618, relates to an ionic liquid reconstituted cellulose composite as a solid support matrix, the main content of which is a regenerated cellulose matrix, substantially within the regenerated cellulose matrix A cellulose / active substrate composite comprising a primary active material, a linker, and a secondary active material uniformly dispersed in the present invention, wherein the linker is combined with the primary and secondary active materials. An aptamer is described by way of example.

The present invention solves the above problems and the object of the present invention as devised by the necessity of the above is to provide a new complex.

Another object of the present invention is to provide a method for producing the composite.

Still another object of the present invention is to provide an efficient protein separation / purification complex.

In order to achieve the above object, the present invention provides an aptamer-nanofiber complex to which an aptamer is immobilized.

The aptamer-nanofiber composite described in the present invention refers to nanofiber (nanofiber) to which oligonucleotides (aptamers) having affinity for a specific target are bound.

In one embodiment of the invention, the nanofibers are preferably polystyrene (PS) -poly (styrene-co-maleic hydride) nanofibers, but is not limited thereto.

In another embodiment of the present invention, the aptamers are preferably two kinds of aptamers, and the two kinds of aptamers preferably have the nucleotide sequences set forth in SEQ ID NOs: 1 and 2, but are not limited thereto.

The present invention also provides a method for purifying or isolating one or more proteins from a sample, the method comprising contacting the sample with the aptamer-nanofiber complex of the invention for a time sufficient to purify or separate the protein. to provide.

In another aspect, the present invention provides a kit for protein purification comprising the aptamer-nanofiber complex of the present invention.

Kits of the present invention may include, but are not limited to, columns, buffers, instructions for use, etc. for protein purification.

In one embodiment of the present invention, the protein is preferably thrombin, but is not limited thereto.

In another aspect, the present invention includes the step of immobilizing the biotinylated aptamer on the polystyrene (PS) -poly (styrene-co-maleic hydride) nanofiber coated with streptavidin aptamer-nano of the present invention It provides a method for producing a fiber composite.

In one embodiment of the present invention, polystyrene (PS) and poly (styrene-co-maleic anhydride) are mixed in a container, and in this container dimethylformamide (DMF), tetrahydrofuran or trahydrofuran Adding a mixed solvent of acetone and preparing a polystyrene (PS) -poly (styrene-co-maleic hydride) nanofiber composite,

In a preferred embodiment of the present invention, the mixing ratio of the polystyrene (PS) and poly (styrene-co-maleic anhydride) may vary depending on the amount of the electrospinning and the amount of the solvent, various combinations are possible, In the embodiment of the present invention, but adopts a 2: 1 weight ratio is not limited thereto.

In the present invention, a 2: 1 ratio of polystyrene (PS) and polystyrene-co-maleic anhydride (PSMA) was successfully prepared without visual phase bead formation in the fiber and the resulting nanofibers were characterized by scanning electron microscopy (SEM) characterization. .

Eptamer-nanofiber composites are prepared by covalent bonds between unhydride groups and amine groups of nanofibers on the surface of nanofibers. The present inventors studied the most efficient method for protein separation / purification among various methods of immobilizing aptamer on the surface of nanofiber. As a result, the method of immobilizing aptamer with biotin on nanofiber coated with streptavidin is the most effective method. It showed high efficiency. In addition, it was found that the strategy used for protein isolation and purification was more efficient by immobilizing two aptamers simultaneously than using one aptamer. The aptamer-nanofiber complexes developed in the present invention could be successfully applied for protein separation / purification, and nanofibers provide high capacity compared to conventional microbeads or tubular columns to provide aptamer-based protein / separation purification. It seems to be an attractive carrier for.

Hereinafter, the present invention will be described in detail.

Aptamer Characteristics of Nanofiber Composites

The images of a and b of FIG. 6 show SEM images of PS-PSMA nanofibers uniformly synthesized at a thickness of 400 nm ± 100 nm. The surface morphology of the nanofibers and aptamer-nanofiber composites is similar, and on the nanofibers Covalently bonded aptamers are small, thin forms.

The degree of immobilization of the aptamers on the nanofibers is shown in FIGS. 6 c and d. CLSM images of aptamer-nanofiber complexes were obtained using TAMRA labeled aptamers. As clearly shown in FIGS. 6 c and d, red fluorescence was observed on all nanofibers or on nanofibers from TAMRA on aptamers, suggesting that aptamers were immobilized in their entire region, not in any part of the nanofibers. This high coupling efficiency and well-distributed phenomena between the two complexes were obtained through simple alcohol pretreatment. The immobilized aptamers were 0.5-0.6 nmole / 0.5 mg of nanofibers in undispersed PS-PSMA nanofibers, while 0.9-1.1 nmoles / 0.5 mg of nanofibers in alcohol-dispersed PS-PSMA nanofibers. The aptamer ability on this high nanofiber is due to the high surface area attributable to the nano-sized material, and other supporting materials previously reported were sepharose, magnetic bead and bead packed columns with 0.068 nmoles, 0.077 nmoles and 0.238 nmoles, respectively. Having a value of support of tamers / mg (TS Romig, C. Bell and DW Drolet, Journal of Chromatography B , 1999, 731 , 275-284; MY Arica, HA Oktem, G. Bayramoglu and VC Ozalp, Biotechnol Progr , 2007, 23 , 146-154; V. Pichon, B. Madru, F. Chapuis-Hugon and E. Peyrin, Anal Chem , 2009, 81 , 7081-7086.

Aptamer On nanofiber composites Thrombin  Specific tablets

The biotinylated aptamer for thrombin, TBA29 (5′-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 ′), was used to determine the type of aptamer (TBA15, TBA29) and the coupling method between aptamers and nanofibers. Selection was made after investigating the effect (FIG. 5). Interesting findings have been made by comparing the purification efficiency of biotinylated aptamers with amine-modified aptamers on nanofibers. The recovery yield of thrombin derived from TBA15 immobilized on the nanofibers through biotin-streptavidin interaction, although TBA15 has a lower affinity for thrombin (K _d ~ 100 nM) than TBA29 (K _d ~ 0.5 nM) It was higher than TBA29 immobilized on nanofibers through amine-maleic anhydride interaction. The amino group of the inner base of the oligonucleotide appears to interact with the maleic anhydride group to some extent on the nanofibers, resulting in an incomplete tertiary structure, reducing the binding affinity for thrombin.

To demonstrate the specific interaction between thrombin and TBA29 on nanofibers, serum-rich proteins such as human serum albumin (HSA), IgG and fibrinogen were tested with thrombin. As shown in FIG. 1, HSA, IgG and fibrinogen were virtually absent after two washes with binding buffer, but at least 80% of thrombin remained strongly on the aptamer-nanofiber complex, indicating that the aptamer-nanofiber complex was specific for thrombin only. It suggests to purify by an enemy. The possibility of nonspecific interactions between thrombin and streptavidin coated PS-PSMA nanofibers was evaluated by simultaneously performing the same experiments on streptavidin coated PS-PSMA nanofibers prepared with the same protocol without aptamer alone. In addition, in order to check the influence of oligonucleotides on thrombin purification, nanofiber complexes having nonspecific oligonucleotides were synthesized. The comparison results of the tablet profiles between the control and aptamer-nanofiber complexes are summarized in FIG. 2. Up to 10% of thrombin was recovered in the elution fraction derived from the control nanofibers, while at least 85% of the thrombin was purified from the aptamer-nanofiber complex. This comparison shows a very specific purification of thrombin for the aptamer-nanofiber complex. Repeatability of the optimized purification protocol was also performed and shown in FIG. 2. The standard deviation value of the recovery yield of the aptamer-nanofiber composite was about 6%, while about 3% with the aptamer-free nanofibers.

Aptamer Determination of Nanofiber Composite Capacity

The ability of the complex was examined with several samples containing increasing amounts of thrombin simultaneously compared to the blank support. The absolute amount of thrombin derived from the aptamer-nanofiber complex and the blank after eluting with 2M NaClO ₄ is reported in FIG. 3. The results obtained from the blank support show a gentle slope of around 7%, indicating that there is little nonspecific binding properties of the nanofibers to thrombin. On the other hand, the amount of thrombin immobilized in the aptamer-nanofiber complex increases substantially in a straight line behavior, suggesting that thrombin can be purified through this complex regardless of its amount. When the thrombin administered was 20 μg or more, the curve obtained from the complex reached a plateau. The ability of the aptamer-nanofiber complex was 20 μg, which corresponds to 0.547 nmoles of thrombin, ie 1.1 μmoles / gram of nanofibers. Decided to be near. An average of 1 nmoles of aptamer could be immobilized on 0.5 mg of nanofibers so that 0.55 molecules of throbin were captured by one aptamer and at this saturation the thrombin / aptamer ratio was higher than that obtained from the SPR base.

Aptamer -Stability and Reusability of Nanofiber Composites ( Reusability )

The storage stability and reusability of the aptamer-nanofiber complex against thrombin tablets were investigated for 7 months. When not using the aptamer-nanofiber complex, it was stored at pH 7.4, 4 ° C. in TE0.1 with 10 mM NaN ₃ and fully conditioned with 4 ml of binding buffer before use. The initial recovery yield of thrombin by aptamers immobilized on the nanofibers was 100% when the relative recovery yield of thrombin was defined as the ratio of yield to initial yield. 4 shows the storage stability and reusability of the aptamer-nanofiber composite. The immobilized aptamer showed high stable stability for seven reuses; They also maintained a preserved initial recovery yield of thrombin for more than 7 months with a standard deviation of 4%. There was no significant decrease in recovery yield of thrombin and the decrease in maximum yield observed was less than 9% of initial yield during six reuses of the aptamer-nanofiber composite at room temperature for 7 months. The addition of TE0.1, including NaN ₃ , inhibits nuclease and bacterial growth allowing for increased storage stability of aptamers.

We used a new aptamer-nanofiber complex for thrombin tablets. The superior ability of affinity ligands has been successfully achieved through aptamers immobilized on electrospun polymer PS-PSMA nanofibers. The aptamer-nanofiber composites in the present invention showed very effective purification of thrombin with a recovery yield of 85%. The system also showed very stable long term storage and repeated use. These results confirmed that the use of nanofibers as a support material for aptamers contributes to overcoming the problem as a support material for protein purification. With regard to the possibility of using nanofibers as packing material in columns, this system can be widely applied to protein purification.

As can be seen from the invention, the protein separation / purification method using PS-PSMA nanofibers with aptamer immobilized in the present invention has a higher capacity than the conventional microbead or tubular column type in terms of efficiency great. In addition, it is easy to mass-produce nanofibers through regular spinning method, and aptamers can be easily immobilized through hydride groups in nanofibers. Separation and purification methods may be universally applied.

Figure 1 shows the purification profile of the aptamer-nanofiber complex and the control. U: 10 μg thrombin was added to each sample in 1 ml of binding butter. W: Washed twice with 1 ml buffer to remove nonspecific binding. E: Thrombin bound with 2M NaClO ₄ was eluted. All experiments were performed at room temperature.
Figure 2 shows the specificity of the aptamer-nanofiber complex for thrombin. Elution profiles show little HSA, IgG and fibrinogen retention for the aptamer-nanofiber complex. 10 μg of each protein was spiked in 1 mL of binding buffer.
Figure 3 shows the capacity curve of the aptamer-nanofiber composite and the blank support.
Figure 4 shows the reusability and storage stability of the aptamer-nanofiber composite. (a) Six reusability of aptamer-nanofiber composites. Relative recovery yields were calculated using residual yields at each time point for those found early on day 0. (b) Represent the storage stability of the aptamer-nanofiber composite for 7 months.
5 shows tablet profiles obtained from Blank and each aptamer-nanofiber composite. 'Amine' represents direct immobilization of amine-modified aptamers to bare PS-PSMA nanofiber amine-anhydride coupling and 'Biotin' represents immobilization of biotinylated aptamers to PS-PSMA nanofibers after streptavidin coating.
U: 10 μg thrombin was added to each sample in 1 ml of binding butter. W: Washed twice with 1 ml buffer to remove nonspecific binding. E: Thrombin bound with 2M NaClO ₄ was eluted. All experiments were performed at room temperature.
Figure 6 is a figure showing the characteristics of the aptamer-nanofiber composite in the present invention. SEM images of (a) and after (b) PS-PSMA nanofibers before aptamer fixation. CLSM images of TAMRA-labeled aptamers for (c) entangled nanofibers and (d) single nanofibers.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

900,000, Pressure Chemicals), Poly (styrene-co-maleic anhydride) (PSMA) (Mw

240,000, Aldrich), N, N-dimethylformamide (DMF) (99.8%, Sigma-Aldrich), Streptavidin (Streptomyces avidinii 유래, Sigma-Aldirich), Fibrinogen(인간 혈장 유래, Sigma-Aldrich), IgG (인간 혈청 유래, Sigma-Aldrich)은 구입하였고 추가적인 정제없이 사용하였다. MicroBCA protein assay kit (Pierce)를 정량화에 사용하였다.Polystyrene (PS) (Mw) used in the present invention

900,000, Pressure Chemicals), Poly (styrene-co-maleic anhydride) (PSMA) (Mw

240,000, Aldrich), N, N-dimethylformamide (DMF) (99.8%, Sigma-Aldrich), Streptavidin (from Streptomyces avidinii, Sigma-Aldirich), Fibrinogen (from human plasma, Sigma-Aldrich), IgG (from human serum, Sigma-Aldrich) was purchased and used without further purification. MicroBCA protein assay kit (Pierce) was used for quantification.

All biotinylated, amine-modified and fluorescent aptamers for thrombin were synthesized and purified by GenoTech (GenoTech Co., Korea). The 29- and 15-mer DNA aptamer targeting thrombin (5′-AGT CCG TGG TAG GGC AGG TTG GGGTGA CT-3 ′ (SEQ ID NO: 1), designated TBA29; 5′-GGT TGG TGT GGT TGG-3 ′ ( SEQ ID NO: 2), designated TBA15), was biotinylated or amine modified in a 5 'modified dTT aptamer via a 15-deoxythymine (dT) spacer arm (LC Bock, LC Griffin, JA Latham, EH Vermaas and JJ Toole, Nature , 1992, 355 , 564-566). Fluorophore labeled 29 aptamers for thrombin were achieved by attaching TAMRA at the 5 ′ end of Apt 29 (named TAMRA-TBA29). Biotinylated aptamers for control experiments had an open sequence: 5′CCA GCG CTG GGG TGT GTG TAG ATA GCA ATC T-3 ′ (SEQ ID NO: 3). They also had a modification of oligo (dT) with spacer arms. All aptamers were PAGE purified by GenoTech. Aptamers were dissolved in deionized water purified with Milli-Q (Millipore).

Example  One: PS - PSMA  Preparation of Nanofibers and Alcohol Dispersion

PS-PSMA nanofibers were prepared by modifying some of the articles of MB Gu, ET Hwang, R. Tatavarty, H. Lee and J. Kim, J Mater Chem , 2011, 21 , 5215-5218. In summary, 120 mg PS and 60 mg PSMA were mixed in 1.5 mL of DMF and completely dissolved at room temperature; The melted solution was sonicated at 25 ° C. for 1 hour. Aliquots obtained from the prepared polymer were each loaded into 10 mL syringes. Electrospinning was performed on a 10 kV Gamma high voltage Research (ES series) with a 10 cm distance from the tip to the collector, and the 23 gauge syringe needle was fixed vertically at a flow rate of 2.5 mL / h. The resulting electrospun PS-PSMA nanofibers were collected on aluminum foil. To disperse the nanofibers completely in water, the electrospun nanofibers were placed in a water soluble alcohol solution (50%, v / v) and stirred at 250 rpm for at least 30 minutes. Then they were washed with lx PBS buffer (pH 7.4) and stored in the same buffer solution until used for aptamer immobilization.

Example  2: on nanofiber Aptamer  Immobilization

To prepare streptavidin attached on nanofibers, streptavidin solution was prepared at 50 μg / mL in 1 × PBS buffer (pH 7.4). For incubation, 0.5 mg of PS-PSMA nanofibers were cut and mixed in 1 mL of streptavidin solution in EP tubes, stirred for 30 minutes at room temperature and stirred (30 rpm) overnight at 4 ° C. for maximum coupling. The nanofibers were transferred to a new EP tube and washed with lx PBS + 0.05% tween 20 (pH 7.4).

The unreacted maleic anhydride groups were quenched by incubating streptavidin coated nanofibers in 100 mM ethanolamine (pH 8.0) for 2 hours. Washed with Streptavidin coated nanofibers 1 × PBS + 0.05% tween 20 (pH 7.4) and stored in TE0.1 (TE buffer with 0.1M NaCl) containing 10 mM NaN ₃ (pH7.4) until further use.

Example  3: Aptamer  Immobilized PS - PSMA  Characterization of Nanofibers

A Zeiss Axiovert 200 microscope equipped with a confocal laser scanning microscope (CLSM) and a Zeiss LSM 510 scanning device (Carl Zeiss Co. Ltd., Germany) was used to examine the aptamer nanofiber composition by fluorescence microscopy. In order to excite the TAMRA-labeled aptamers present on the nanofibers, 543 nm UV diode lasers were used. The morphology of the aptamer-immobilized nanofibers was examined by scanning electron microscopy (SEM; S-2360N, Hitachi Co.).

Example  4: protein purification process

Protein purification was performed at room temperature using EP tubes. The aptamer-nanofiber complex was conditioned with 1 mL of Binding Buffer (140 mM NaCl, 20 mM Tris-HCl, 5 mM KCl, 2 mM MgCl ₂ , 1 mM CaCl ₂ , 0.02% Tween 20, pH 7.4). The aptamer-nanofiber complex was incubated with varying amounts of thrombin for 30 minutes and then washed twice with 1 mL of binding buffer. Thrombin was then eluted with 2M NaClO ₄ . To check purification efficiency, the thrombin concentration in each fraction was measured using the MicroBCA protein assay kit (Pierce). For experiments to determine stability and reusability, the aptamer-nanofiber complexes were washed three times with TE0.1 containing 10 mM NaN ₃ (pH 7.4) and stored at 4 ° C. for subsequent use. . The same experimental protocol was involved to check the storage stability and reusability of the aptamer-nanofiber composites.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Aptamer immobilized Nanofibers composite and use of separation          and purification of protein using the same <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Aptamer <400> 1 agtccgtggt agggcaggtt ggggtgact 29 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> aptamer <400> 2 ggttggtgtg gttgg 15 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> control aptamer <400> 3 ccagcgctgg ggtgtgtgta gatagcaatc t 31

Claims (10)

An aptamer-nanofiber composite in which an aptamer is immobilized to polystyrene-polystyrene-co-maleinc anhydride (PS-PSMA). The aptamer-nanofiber composite according to claim 1, wherein the aptamer is two kinds of aptamers. The aptamer-nanofiber complex according to claim 2, wherein the two aptamers have the nucleotide sequences shown in SEQ ID NOs: 1 and 2. A method for purifying or isolating one or more proteins from a sample, the method comprising contacting the sample with the aptamer-nanofiber complex of claim 1 for a time sufficient to purify or separate the protein. How to include. 3. A protein purification kit comprising the aptamer-nanofiber complex of claim 1. 6. The protein purification kit of claim 5, wherein the protein is thrombin. 6. The protein purification kit according to claim 5, wherein the aptamer has the nucleotide sequences set forth in SEQ ID NOs: 1 and 2. The aptamer-nanofiber of claim 1 or 2, comprising immobilizing the biotinylated aptamer on the streptavidin-coated polystyrene (PS) -poly (styrene-co-maleic anhydride) nanofiber. Method for preparing a composite. 9. The method of claim 8, wherein the aptamer is two kinds of aptamers. 10. The method of claim 9, wherein the two aptamers have a nucleotide sequence as set forth in SEQ ID NOs: 1 and 2.

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