WO2011145908A2

WO2011145908A2 - Quantitative analysis method using a nanotube having integrated enzymes

Info

Publication number: WO2011145908A2
Application number: PCT/KR2011/003730
Authority: WO
Inventors: 김중배; 오민규; 이혜진; 김병찬
Original assignee: 고려대학교 산학협력단
Priority date: 2010-05-20
Filing date: 2011-05-20
Publication date: 2011-11-24
Also published as: KR20110130384A; WO2011145908A9; WO2011145908A3; KR101273453B1

Abstract

The present invention relates to a quantitative analysis method which involves analyzing an object using an enzyme, aptamer, antibody, nanotube, and a magnetic bead or a substrate. More particularly, a binding aptamer or antibody to be bonded to an analysis object is bonded to a magnetic bead or a substrate, and the resultant structure is placed into a solution to be analyzed, bonded to the analysis object, and separated from the analysis object by magnetic force; another aptamer or antibody to be selectively bonded to the analysis object is bonded to the surface of the nanotube, on which enzymes are integrated, so as to prepare for a quantitative analysis of the analysis object; and the quantitative analysis is performed, through an enzyme reaction, on a composite body which is prepared by binding the resultant structure to the magnetic bead or substrate to which the aptamer or antibody is bonded. Thus, a low concentration analysis object can be diagnosed, and difficulties in quantitative analysis according to conventional detection methods can be overcome using the signal-amplification effects of a nanotube having integrated enzymes.

Description

Quantitative Analysis Using Enzyme-integrated Microtubes

The present invention relates to quantitative assays using enzymes, aptamers or antibodies, magnetic beads or substrates, and microtubes. More specifically, binding the analyte binding aptamer or antibody to a magnetic bead or a substrate, put it in a test solution to be analyzed, bind it to an analyte, and then isolate it, for quantitative analysis of the analyte. A complex prepared by binding another aptamer or antibody that selectively binds to the analyte on the surface of the enzyme-encapsulated microtube and binding it to a magnetic bead or a substrate to which the aptamer or antibody is bound is subjected to an enzyme reaction. Quantitative analysis. Through this, diagnosis can be made even when the concentration of the analyte is low, and it is possible to overcome the disadvantage of the conventional detection method, which is difficult to quantify by using the signal amplification effect of the microtubes in which the enzyme is integrated.

ELISA (Enzyme Linked Immunosoebent Assay) is a method of detecting and quantifying proteins using two kinds of antibodies that bind to the protein to be detected. This method is the most widely used antigen-antibody assay because it is simple, inexpensive, and can be analyzed in large quantities. In particular, this method has the advantage of using a very sensitive reaction such as RIA (Radio Immuno-Assay) but not using radioactivity as in RIA.

However, the conventional ELISA is measured by the antibody to which the enzyme is bound, wherein the enzyme bound to the antibody corresponds to one molecule. Therefore, when the reaction by the enzyme is not large and the activity of the enzyme is inhibited, it is difficult to obtain accurate diagnosis and high signal.

In addition, both the enzyme and the antibody used for ELISA are sensitive to the surrounding environment such as temperature and are easily left at room temperature or modified by other external factors. As a result, the activity is lowered or severely lost. By the way, the diagnosis made by the detection and quantification step generally takes a long time, such safety is a big problem. This is because even though the protein is correctly diagnosed, if the enzyme bound to the antibody loses its activity, it does not show an accurate signal amplification and thus it is difficult to accurately diagnose the protein. As a result, diagnostic materials and enzymes that are stable to temperature, long-term storage at room temperature, and capable of continuous use are urgently needed in biotechnology, medical diagnostics, the environment, and various other fields.

The present invention has been made to solve the above problems, by binding the aptamer 1 or antibody 1 to the magnetic bead or the substrate to capture the analyte, and then isolate the another aptamer 2 or antibody 2 enzyme Aptamer / antibody-enzyme-integrated microtubes were prepared by binding to the integrated microtubes, and then mixed with the separated analyte-aptamer / antibody1-magnetic beads / substrate to convert the analytes into media. Enzyme-fixed microtube-aptamers / antibodies2-analyte-aptamers / antibodies1-magnetic beads / substrate complexes were prepared and the complexes were separated on a solution, and the amount of the complexes was determined by enzymatic reaction of the separated complexes. The object of the present invention is to provide a quantitative analysis method to be measured.

Another object of the present invention is to provide an analyte analysis sensor, which is manufactured according to the method of the analyte quantitative analysis and used in the quantitative analysis.

In order to achieve the object as described above, the analyte quantitative analysis method of the present invention is characterized by including the following steps:

(A) a magnetic bead or a substrate mixed with a first analyte binding aptamer or an antibody, wherein the first analyte binding aptamer or antibody is bound to the surface of the magnetic bead or substrate Preparing a 1-magnetic bead / substrate;

(B) adding the aptamer / antibody-magnetic bead / substrate to the test solution containing the analyte so that the analyte is the first analyte binding app of the aptamer / antibody-magnetic bead / substrate Preparing an analyte-aptamer / antibody1-magnet bead / substrate bound to a tammer or an antibody;

(C) separating the analyte-aptamer / antibody 1-magnetic beads / substrate from the test solution;

(D) mixing an enzyme and a microtube to prepare an enzyme-microtube in which the enzyme is integrated on the surface of the microtube;

(E) mixing the second analyte binding aptamer / antibody binding to the analyte and the enzyme-microtube, so that the second analyte binding aptamer / antibody is bound to the enzyme of the enzyme-microtube. Preparing an aptamer / antibody2-enzyme-microtube;

(F) the second analyte of the aptamer / antibody-enzyme-microtube by mixing the analyte-aptamer / antibody1-magnetic bead / substrate with the aptamer / antibody2-enzyme-microtube Magnetic beads / substrate-aptamers / antibodies1-analytes-aptamers / antibodies-enzymes-microparticles having binding aptamers or antibodies bound to the analytes of the analyte-aptamer / antibody 1-magnetic beads / substrate Manufacturing a tube;

(G) separating the magnetic bead / substrate-aptamer / antibody 1-analyte-aptamer / antibody 2-enzyme-microtube; And

(H) quantitatively analyzing the enzyme of the separated magnetic beads / substrate-aptamers / antibodies-analyte-aptamers / antibodies-enzymes-microtubes.

In addition, the binding of the first analyte binding aptamer or antibody and the magnetic bead or the substrate of step (A) may include streptavidin-biotin binding, avidin-biotin binding, EDC [ N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] coupling, sulfhydrylamine coupling, or Ni-NTA (nitrilotriacetic acid) -histidine linkage.

In addition, the binding of the enzyme and the microtube of the step (D) is streptavidin (streptavidin)-biotin (biotin) bond, avidin (avidin)-biotin bond, EDC [N-ethyl-N'- (dimethlaminopropyl) carbodiimide hydrochloride] coupling, sulfhydrylamine coupling, or nitrilotriacetic acid (Ni-NTA) -histidine linkage.

In addition, when the analyte-aptamer / antibody 1-magnetic beads / substrate of step (C) is the analyte-aptamer / antibody 1-magnetic beads, it is preferable to magnetically separate from the test solution.

In addition, the magnetic bead / substrate-aptamer / antibody 1-analyte-aptamer / antibody 2-enzyme-microtubes of the step (G) is a magnetic bead-aptamer / antibody 1-analyte-aptamer / antibody 2 In the case of enzyme-microtubes, it is preferable to separate them by magnetic force.

In addition, the analyte quantitative analysis method of the present invention is to protect the unreacted binding site of the magnetic beads or the substrate surface of the aptamer / antibody 1-magnetic beads / substrate before step (A) and after step (B) It may further comprise.

In addition, the analyte quantitative analysis method of the present invention protects the unreacted binding sites of the magnetic beads or the substrate surface of the analyte-aptamer / antibody 1-magnetic beads / substrate after step (B) and before step (C). It may further comprise the step.

In addition, the analyte quantitative analysis of the present invention further comprises the step of protecting the unreacted binding site of the microtube or enzyme in the aptamer / antibody-enzyme-microtube after the step (E) and before the step (F). It can be included as.

Further, the magnetic beads or the substrate is a neutral carboxyl group (-COOH) on the surface, its ion (-COO ^-), a neutral amine group (-NH _2), its ion (-NH ^- or -NH ₃ ^+), neutral thiol (-SH), and its ion (-S ^-) preferably has a functional group selected from the group consisting of.

Also, the fine tube is neutral carboxyl group (-COOH) on the surface, its ion (-COO ^-), a neutral amine group (-NH _2), its ion ^(-NH-or -NH ₃ ^+), neutral thiol group (- SH), and its ion (-S ^- preferably includes a functional group selected from the group consisting of a).

In addition, the enzyme of the present invention is preferably integrated by crosslinking on the surface of the microtube.

In addition, crosslinking between enzymes integrated on the surface of the microtubes of the present invention can be accomplished by salts, amine bond bifunctional compounds, or salt and amine bond bifunctional compounds.

In addition, before the step (A), the analytical target quantitative analysis of the present invention reacts with a mixture of magnetic beads or a substrate and an EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] solution, followed by an unreacted EDC solution. It may further comprise the step of removing.

In addition, before the step (D), the analyte for quantitative analysis of the present invention reacts by mixing a microtube and an EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] solution, and then removing the unreacted EDC solution. It may further comprise the step.

In addition, the quantitative analysis method of the present invention may remove the unreacted EDC solution by centrifugation after the reaction by mixing the microtube and the EDC solution.

In addition, the first analyte binding aptamer or the second analyte binding aptamer is preferably 10 to 250 of the base constituting the sequence.

In addition, the microtube of the step (D) is preferably treated with an acid (acid).

In addition, the analyte quantitative analysis method of the present invention may further comprise the step of washing after the step (A).

In addition, the analyte quantitative analysis method of the present invention may further comprise the step of washing after the step (C).

In addition, the analyte quantitative analysis method of the present invention may further comprise the step of washing after the step (D).

In addition, the analyte quantitative analysis method of the present invention may further comprise the step of washing after the step (G).

In addition, the analyte quantitative analysis method of the present invention may further comprise the step of washing after the step of protecting the unreacted binding site of the magnetic beads or the substrate surface of the aptamer / antibody 1-magnetic beads / substrate.

In addition, the analyte quantitative analysis method of the present invention may further comprise a step of washing after protecting the unreacted binding site of the surface of the microtube in the enzyme-microtube.

In addition, the analyte may be thrombin.

In addition, the first analyte-binding aptamer is preferably a thrombin specific DNA aptamer of SEQ ID NO: 5'-GGT TGG TGT GGT TGG-3 '.

In addition, the second analysis target binding aptamer is preferably a thrombin specific DNA aptamer of SEQ ID NO: 5'- AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 '.

In addition, the enzyme is preferably glucose oxidase.

On the other hand, the analytical sensor used in the analytical target quantitative analysis method of the present invention

By mixing a magnetic bead or a substrate (magnetic bead) and the first analyte binding aptamer or antibody, the aptamer / antibody 1-magnet wherein the first analyte binding aptamer or antibody is bound to the surface of the magnetic bead or substrate Beads / substrates, and

By mixing an enzyme and a microtube to prepare an enzyme-microtubes in which the enzyme is integrated on the surface of the microtube, and mixing the enzyme-microtubes with a second analyte binding aptamer or an antibody which binds to a target analyte, The aptamer / antibody2-enzyme-microtube wherein the second analyte binding aptamer or antibody is bound to the enzyme of the enzyme-microtube

Characterized in that it comprises a.

The analyte quantitative assay of the present invention is based on the immunogenic capacity and stability between the analyte and a particular aptamer or antibody. Detection of the analyte consists of specific binding by magnetic beads or substrates to which aptamers or antibodies are bound. The use of the aptamer or antibody binding magnetic beads or substrates is useful to isolate a particular analyte in an environment containing various components. And aptamers or antibodies have the advantage of having a strong affinity for the analyte. In addition, since the microtubes in which the enzyme is integrated can maintain activity for a long time, when the enzyme is left at room temperature, it can overcome the conventional disadvantage that the activity is not caused to cause an enzyme reaction. In addition, one enzyme molecule does not cause an enzymatic reaction, but a number of enzymes accumulated in the microtubes perform enzymatic reactions and thus show a higher signal than the existing enzymatic reactions. Based on this concept, the quantitative analysis using the signal amplification effect by immunomagnetism separation and enzymatic integration of the present invention improves the specificity of binding and the speed, convenience and stability of the separation in detection. It has the effect of improving sensitivity and accuracy.

Figure 1 is a schematic diagram showing the process of one embodiment of the analyte quantitative analysis method of the present invention for the analysis of thrombin.

2 is a graph measuring the concentration of optimal aptamer / antibody 2-enzyme-microtubes.

3 is a photograph of the separation result by magnetic force when the target analyte exists and when it is not.

4 is a SEM photograph of a magnetic bead and its composite after separation by magnetic force when the target analyte is present and when it is not.

5 is a graph showing the results of thrombin analysis using the present invention quantitative analysis.

6 and 7 are graphs showing the results of electrochemical analysis of thrombin using a voltage current meter.

8 is a graph showing the results of measuring the thermal stability of the analyte analysis sensor of the present invention.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. In addition, it will be apparent to those skilled in the art that the following description is provided only to provide a more general understanding of the present invention, and the present invention may be practiced without these features. In the following description of the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

The inventors have discovered that an immunomagnetic approach using analyte detection aptamer or antibody bound to a magnetic bead or substrate can detect and isolate an objective analyte with high efficiency. Furthermore, it was found that the signal amplification effect of the enzyme-integrated microtube can be used to diagnose and quantify the analyte detected and separated.

In the present invention, the analyte to be detected and quantified is for all particles that are worth analyzing, such as bioparticles such as proteins, antigens, cells, or inorganic particles.

In addition, aptamers used in the present invention are single-stranded nucleic acid ligands selected for specific target molecules for desired functions such as peptides, small molecules, binding to nucleic acids and catalysis of various chemical reactions. Aptamers achieve specific binding to target molecules with a high affinity comparable to antigen-antibody conjugates. Aptamers have several advantages over antibodies, making them an attractive alternative to antibodies in affinity analysis of analytes. That is, aptamers are smaller, more stable, easier to modify chemically, can be screened through systematic evolution of ligands by exponential augmentation (SELEX), and can be synthesized in large quantities in vitro.

Conventional ELISA is measured by the antibody to which the enzyme is bound, wherein the enzyme bound to the antibody corresponds to one molecule. Therefore, if the reaction by the enzyme is not large and the activity of the enzyme is inhibited, it is difficult to obtain accurate diagnosis and high signal. However, when the enzyme is immobilized in the microtube, the aptamer or the antibody that recognizes the target analyte can be combined and used in the reaction to give a higher signal than the conventional ELISA, and the stabilization of the enzyme is also enhanced.

Accordingly, the present inventors have developed a method for diagnosing and quantifying a target analyte bound to a magnetic bead or a substrate by binding an aptamer or an antibody capable of recognizing the target analyte to an enzyme-immobilized microtube. This confirmed that the signal amplification of the immobilized enzyme can detect and quantitate up to 1 ng of analyte per ml that could not be detected by the conventional ELISA method.

The analyte quantitative assay of the present invention first begins by binding an aptamer or an antibody that binds the analyte to a magnetic bead or substrate for capture of the analyte of interest for which presence and amount thereof are to be analyzed. Herein, the binding of the magnetic beads or the substrate and the analyte binding aptamer or the antibody may be used without limitation as long as it is a method known in the biosciences for the binding. First, a neutral carboxyl group (-COOH), its ion (-COO ^-), a neutral amine group (-NH _2), its ion (-NH ^- or -NH ₃ ^+), neutral thiol group (-SH), and its ion (-S ^-) from the group consisting of It is desirable to have selected functional groups. For example, by mixing and reacting a magnet bead or a substrate with an EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] solution, the surface of the magnet bead or substrate is provided with a carboxyl group as a functional group, and the magnet bead or substrate After magnetically fixing, the unreacted EDC solution is removed.

As a specific method of binding the magnetic bead or substrate having a functional group to the surface and the aptamer or the antibody, streptavidin-biotin bond, avidin-biotin bond, EDC [N-ethyl- N '-(dimethlaminopropyl) carbodiimide hydrochloride] coupling, sulfhydrylamine coupling, or nitrilotriacetic acid-histidine coupling, and the like, and amide binding is particularly preferred. . The amide binding is a reaction formed by binding of a magnetic bead or a carboxyl group (-COOH) of a substrate with an aptamer 5 'end or an amine group (-NH ₂ ) of an antibody.

Furthermore, in order to prevent unreacted functional groups present on the surface of the magnet bead or the substrate after the aptamer or antibody binding, it is preferable to block with bosa serum albumin (BSA) or the like to prevent unexpected reactions. .

The aptamer or antibody-bound magnetic beads or substrates obtained through the above process, that is, aptamer / antibody-magnetic beads or substrates, were separated by magnetic force and mixed with the test solution containing the analyte to be analyzed. The analyte prepares an analyte-aptamer / antibody 1-magnetic bead / substrate coupled to the aptamer 1 or antibody 1 of the aptamer / antibody-magnetic bead / substrate.

Likewise, even after capturing the analyte, it is preferable to protect with BSA or the like to prevent unreacted functional groups present on the surface of the magnet bead or the substrate after binding the analyte to cause unexpected results in subsequent reactions.

On the other hand, the enzyme for generating a signal from the analyte is coupled with another aptamer or antibody that interacts with the analyte.

In the quantitative analysis method of the present invention, the analyte to be detected or quantitatively analyzed may be the subject of all analyses without any limitation.

The analyte-aptamer / antibody 1-magnet bead / substrate is another aptamer 2 or antibody 2 that binds to the analyte for binding to the microtubes in which the enzyme, which is the final analytical signal generator of the present invention, is integrated. Is bonded to the integrated microtube. Microtubes here refer to all materials that are smaller than millimeters in size and have a tube shape. In the present invention, carbon nanotubes are particularly preferable, and in particular, it is more preferable to undergo an acid treatment in which an acid solution is immersed in an acid solution before being integrated into an enzyme.

The binding of the microtube and the enzyme is the same as that of the binding of the magnetic bead or the substrate to the aptamer or the antibody in the aptamer / antibody 1-magnetic bead / substrate.

That is, the first neutral carboxyl group (-COOH) on the surface of the fine tube, its ion (-COO ^-), a neutral amine group (-NH _2), its ion (-NH ^- or -NH ₃ ^+), neutral thiol group ( -SH), and its ion (-S ^-) as it is preferable to be provided with a functional group selected from the group consisting of, for example, the fine tube and EDC [N-ethyl-N ' - (dimethlaminopropyl) carbodiimide hydrochloride] and the reaction solution was mixed In this way, the surface of the microtube is provided with a carboxyl group as a functional group.

In addition, as a specific binding method of the enzyme and the microtube having a functional group on the surface, streptavidin (biotin) bond, avidin (avidin) -biotin bond, EDC [N-ethyl-N'- (dimethlaminopropyl) carbodiimide hydrochloride] coupling, sulfhydrylamine coupling, or nitrilotriacetic acid (Ni-NTA) -histidine linking, and the like, and amide binding is particularly preferred.

This enzyme binds an aptamer or antibody specific for the target analyte. However, if the combination of the aptamer / antibody and the enzyme is a monoantibody-monoenzyme and free form as in the conventional ELISA, it is difficult to fully express the effects of the present invention. That is, if one molecule of an enzyme or antibody bound to the target analyte binds to the target analyte, the enzyme cannot be captured even if it binds to the target analyte. In particular, the enzyme in the free form is a high disadvantage of the above-described activity degradation or deactivation acts as a big disadvantage.

Therefore, in the present invention, a large amount of signaling enzymes are first bound to microtubes, and then aptamers or antibodies are bound to the enzymes. These enzymes bound to the microtubes are not free forms, which enhances their stability, and a large amount of enzymes can generate sufficient signals even if some enzymes are inhibited or inactivated. There are possible advantages.

As such, the present invention is particularly characterized by crosslinking between enzymes in order to bind enzymes to microtubes in large quantities.

In general, enzymes exist in water one molecule apart, but when salts such as ammonium sulphate are added, agglomeration or precipitation occurs several times. Examples of salts that cause this phenomenon include sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, and potassium phosphate. have

In addition, the addition of an amine-binding bifunctional compound having two or more functional groups that react with the amine group of the enzyme, such as glutaraldehyde, can form crosslinks by intercalating between the enzymes and binding to both enzymes. Bifunctional compounds that cause this phenomenon include glutaraldehyde, bis (imido esters), bis (succinimidyl esters), bis (succinimidyl esters) and diisocyanate. And diacid chloride.

By adding these compounds, more enzymes can be accumulated in the microtubes, thereby further enhancing the signal amplification effect.

Furthermore, in order to prevent the unreacted functional groups present on the surface of the microtubes after the aptamer or antibody binding to cause unexpected results in subsequent reactions, it is preferable to block with BSA (bovime serum albumin).

The microtubes in which the aptamer 2 or the antibody 2 is coupled to the enzyme-encapsulated microtubes are separated by a known method such as centrifugation, and then mixed with the analyte-aptamer / antibody 1-magnetic beads / substrate prepared above. The analyte is formed as a complex of microtube-enzyme-aptamer / antibody2-analyte-aptamer / antibody 1-magnet bead / substrate and separated using magnetic force.

The separated microtube-enzyme-aptamer / antibody2-analyte-aptamer / antibody 1-magnetic bead / substrate is amplified by the enzyme-specific reaction and thereby rapidly and quantitatively exists and quantifies the target analyte. I can figure it out correctly.

Furthermore, it is preferable to wash with a buffer or the like after each step for accurate analysis of the present invention. In particular, the aptamer / antibody 1-magnet bead / substrate preparation step, enzyme-microtube production step, magnetic separation step, It is more preferable to wash after protecting the magnetic beads or the unreacted bonded portion of the substrate or the microtube surface. It is, of course, possible to perform the washing step in addition to the above steps.

EMBODIMENT OF THE INVENTION Hereinafter, the Example of this invention is described.

실시예EXAMPLE

Example 1 Preparation of Aptamer 1-Magnetic Beads

When the analyte was used as a thrombin, a magnetic bead bound to the first thrombin binding aptamer was produced by EDC coupling method (Mahmoud et al. 2005) as follows. Magnetic beads (10 μl, 7-12 × 10 ⁹ beads / ml) were washed with 0.01 M aqueous NaOH solution and deionized water. After washing, the solution was placed on a magnet for 1 minute to remove the washing solution. EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] solution (500 μl, 20 mg / ml) was then added and incubated at 4 ° C. for 30 minutes with stirring (200 rpm). After incubation, the EDC solution was removed and the magnetic beads were resuspended in 1000 μl of PBS (phosphate buffered saline) solution (10 mM, pH 7.4), and the first thrombin binding aptamer (5'-NH _2- GGT TGG TGT GGT). TGG-3 ′, 20 μl, 100 μM, Geno-Tech Co. Korea) and incubated with stirring (200 rpm) for 1 hour at room temperature. After incubation, the mixed solution of the magnetic beads and the first thrombin-bound aptamer was placed on the magnet for 1 minute to remove unbound aptamers. The amount of bound aptamer was analyzed by measuring the absorbance at 260 nm with a spectrometer. Aptamer bound magnetic beads (Aptamer1-Magnetic beads) were washed three times with 1000 μl of 10 mM PBS (pH 7.4). Subsequently, in order to protect the unreacted carboxyl group on the surface of the magnetic beads, 1000 μl of protective solution (0.1% BSA [bovine serum albumin] and 0.05% Tween 20) was added and incubated with stirring (200 rpm) at room temperature. After incubation, the solution was placed on a magnet for 1 minute to remove the protective solution, washed three times with 1000 μl of 10 mM PBS solution (pH 7.4), followed by aptamer-coupled magnetic beads (aptamer1-magnetic beads) Was resuspended in 10 μl of 10 mM PBS solution, pH 7.4.

Example 2: Preparation of analyte-aptamer1-magnet beads

Thrombin was detected using the aptamer1-magnet beads of Example 1 as follows. The aptamer1-magnet beads (10 μl, 0.7-1.2 × 10 ⁸ beads) of Example 1 were suspended in a PBS solution (thrombin concentration: 1 ng-1 ug / ml) containing thrombin (Abcam. USA). For detection of thrombin analyses, the mixture was incubated with stirring (200 rpm) for 1 hour at room temperature. After incubation, the thrombin-aptamer1-magnet bead complex was separated by magnet, and the supernatant was transferred to a separate tube to measure the amount of captured thrombin by Micro BCA method. The thrombin-aptamer1-magnet bead complex was washed five times with 1000 μl of 10 mM PBS solution (pH 7.4), then 1000 μl of protective solution (0.1% BSA and 0.05% Tween 20) was added and stirred at room temperature. (200 rpm) and incubated. After incubation, the solution was placed on a magnet for 1 minute to remove the protective solution, washed three times with 1000 μl of 10 mM PBS solution (pH 7.4), and the thrombin-aptamer1-magnet bead complex was added to 1000 μl of 10 Resuspend in mM PBS solution (pH 7.4).

Example 3: Preparation of Aptamer2-Enzyme-Microtubes

When carbon nanotubes were used as microtubes and glucose oxidase was used as an enzymatic enzyme, aptamer2-enzyme-microtubes having a second thrombin binding aptamer were prepared as follows. Carbon nanotubes (multi-walled, outer diameter 30 ㅁ 15 nm, length 1-5 μm, purity> 95%) Purchased from Newton, MA, USA, 100 mg of the carbon nanotubes were added to an acid solution containing H ₂ SO ₄ (98%, 7.5 ml) and HNO ₃ (70%, 2.5 ml), followed by room temperature. Treatment was continued overnight with stirring at. The treated carbon nanotubes were washed with deionized water and dried in a vacuum oven at 80 ° C. Subsequently, glucose oxidase (Sigma-Aldrich. USA) was coupled to carboxylated carbon nanotubes by the EDC coupling method. To introduce the functional groups on the carbon nanotube surface, acid treated carbon nanotubes (0.1 mg) were suspended in deionized water, followed by MES (2- [N-morpholino] ethane sulfonic acid) buffer (0.1 M, pH 6.5). 0.4 ml, EDC solution (10 mg / ml) 0.2 ml and NHS (N-hydroxysuccinimde) solution (50 mg / ml) were mixed and incubated with stirring (200 rpm) for 30 minutes at room temperature. After incubation, the EDC solution was removed by centrifugation, and then resuspended in PB (phosphate buffer. 0.1 M, pH 7.4) to adjust the concentration to 2 mg / ml. Then, 0.5 ml of glucose oxidase solution (10 mg / ml) was added to 1 ml of the carbon nanotube mixture and incubated with stirring (100 rpm) for 1 hour at room temperature. After incubation, 0.67 ml of a 65% aqueous solution of ammonium sulfate was added and incubated for 30 minutes. Enzyme-microtubes prepared for crosslinking were treated with 35 μl of glutaraldehyde and incubated with stirring (50 rpm) overnight at 4 ° C. The cultured enzyme-microtubes were washed by centrifugation with PB (0.1 M, pH 7.4). The final enzyme-microtubes were washed three times with PB (0.1 M, pH 7.4) and separated by centrifugation.

The EDC activity is rerun to bind another thrombin binding Eptamer 2 to the final enzyme-microtube. The final enzyme-microtubes were resuspended in 1 ml of deionized water. Then 0.4 ml of MES buffer (0.1 M, pH 6.5), 0.2 ml of EDC solution (10 mg / ml) and 0.4 ml of NHS solution (50 mg / ml) were mixed and incubated with stirring (200 rpm) for 30 minutes at room temperature. did. After incubation, the EDC solution was removed and the enzyme-microtubes were then resuspended in 950 μl of PBS solution (10 mM, pH 7.4) and a second thrombin binding aptamer (29 bases, 5′-NH ₂ -AGT CCG TGG TAG 50 μl, 50 μM, Geno-Tech Co. Korea) of GGC AGG TTG GGG TGA CT-3 ') and then incubated with stirring (200 rpm) for 1 hour at room temperature. After incubation, the unbound aptamer was removed by centrifugation of the mixed solution of the enzyme-microtube and the second thrombin-bound aptamer. The amount of bound aptamer was analyzed by measuring the absorbance at 260 nm with a spectrometer. Aptamer bound enzyme-microtubes (Aptamer2-enzyme-microtubes) were washed three times with 1000 μl of 10 mM PBS (pH 7.4). Subsequently, in order to protect the enzyme and the unreacted carboxyl group of the microtube, 1000 μl of protective solution (0.1% BSA and 0.05% Tween 20) was added and incubated with stirring (200 rpm) at room temperature. After incubation, the protective solution was removed by centrifugation, washed three times with 1000 μl of 10 mM PBS solution (pH 7.4), and then washed again with 0.1 M Tris-HCl buffer (0.1 M, pH 7.4), 30 Protected by incubation with stirring (100 rpm) in Tris-HCl buffer (0.1 M, pH 7.4) for minutes. Aptamer bound enzyme-microtubes were resuspended in 1000 μl of 10 mM PBS solution, pH 7.4.

Example 4: Analysis of an Analysis Subject

For detection of captured thrombin, 1 ml of thrombin-aptamer1-magnet bead (0.1 mg / ml) of Example 2 was combined with 1 ml of aptamer-enzyme-microtube (1.5 mg / ml) of Example 3 Incubated with stirring at 200 rpm for 1 hour. After standing for 1 minute on a magnetic device, separated and washed three times with wash buffer (10 mM PBS containing 0.1% BSA and 0.05% Tween 20), the prepared immune complexes were subjected to conventional GO assay (Bergmeyer et al. 1974). Was measured.

Total immunoassays were performed at room temperature, and electrochemical analysis was also performed.

Specifically, to investigate the concentration of aptamer2-enzyme-microtubes required for the detection of 1 μg of thrombin, the maximum amount that can be detected through 0.1 mg of magnetic beads, the aptamer2-enzyme- of Example 3 The experiment was performed while varying the concentration of the microtubes, and the results are shown in FIG. 2. In the absence of thrombin (when other analytes are present), the binding signal aptamer specifically targets the target analyte by confirming that the reaction signal does not increase even when the concentration of the aptamer2-enzyme-microtube increases. It was found that only to combine.

In addition, in order to assess the detection limit and quantification capacity in the present invention, analysis was performed on thrombin samples of various concentrations (1000 ng, 500 ng, 100 ng, 10 ng, 1 ng or 0 ng / ml) diluted with PBS. It became. The thrombin-aptamer1-magnet beads combined with the various concentrations of analytes thus obtained were quantified by the aptamer2-enzyme-microtubes at the optimized concentrations described in FIG. 2 described above.

The complex in the form of the microtube-enzyme-aptamer2-analyte-aptamer1-magnet bead causes an enzymatic reaction according to the addition of glucose by glucose oxidase, an enzyme in the complex (Fig. 1). The signal was amplified, which allowed for a rapid and accurate determination of the presence and quantitation of thrombin.

3 is a photograph taken a state in which the actual sample is separated. The left BSA control group does not bind to the aptamer that selectively binds to the target analyte because there is no target thrombin and another protein, BSA. Therefore, the aptamer2-enzyme-microtubes do not complex with the aptamer1-magnet beads and the aptamer2-enzyme-microtubes. However, when the target analysis target thrombin is present in the right embodiment, the aptamer 1-magnetic beads and the aptamer 2-enzyme-microtubes form a sandwich bond by using thrombin as a medium. Therefore, when the magnetic beads are separated by magnetic force, the complexed magnetic bead-aptamer1-purpose protein (thrombin) -aptamer2-enzyme-microtube complex is separated toward the magnet. Then, the concentration of the target protein can be quantified according to the amount of the aptamer2-enzyme-microtubes thus separated.

4 is a photograph taken with a scanning electron microscope (SEM) by separating only the samples forming a complex through a washing process after the separation step by magnetic force as shown in FIG. 3. The left sample, BSA Control, removes the aptamer2-enzyme-microtubes that were dispersed during the wash. Only Aptamer1-Magnetic Beads can be recovered. Right Example The thrombin sample can remove only the supernatant that is clearly separated and recover the magnetic bead-aptamer1-purpose protein (thrombin) -aptamer2-enzyme-microtube complex. Observation by SEM shows that in the absence of the target analyte, only the microtubule complexes due to very fine and non-specific binding are shown, but mostly the magnetic beads, whereas when thrombin is present on the right, the magnetic beads and the microtubes are entangled with each other. You can see it. The upper row is when viewed at 10000 magnification and the lower row is when viewed at high magnification of 30 million times.

5 is a mixture of 990 μl of the reaction solution (glucose 100 mg / ml, 10 mg / ml of o-Dianisidine and 3.79 mg / ml of Peroxidase [Sigma-Aldrich. USA]) into 10 μl of the complex sample solution in an acrylic cuvette. 6 and 7 show the results of electrochemical analysis using a voltammetry consisting of an Ag / AgCl reference electrode, a glass carbon working electrode, and a platinum counter electrode.

Example 5: Stability Test

The analyte analysis of Example 4 was carried out while storing the aptamer 1-magnet beads and the aptamer 2-enzyme-microtubes prepared in Examples 1 and 3 at a high temperature of 40 ° C. As a result of quantifying the analyte at room temperature again with the materials stored at high temperature, it was confirmed from FIG. 8 that the activity was maintained at 90% or more for 100 hours. In the comparative example, ammonium sulfate and glutaraldehyde were not treated. In this case, only one layer of enzyme was formed on the microtube. As a result, it can be easily seen that the stability is lost.

In the present invention, in order to examine the presence and amount of the target analyte, two kinds of aptamers or antibodies which bind to the analyte were respectively bound to different substances, and a complex was prepared by binding them to the analyte. This complex can be easily and quickly separated by the magnetic bead or the magnetic force of the substrate, there is an advantage that can be obtained a large signal amplification effect in a small amount by the microtubule immobilized enzyme. In addition, there is an advantage in that it can detect and quantify a wide range of analytes from 1 ug to 0.5 ng, and it can detect and quantify low concentrations of analytes by supplementing and improving the shortcomings of the stability of the conventional ELISA. And it was found.

Although the above has been illustrated and described with respect to the preferred embodiment of the present invention, the present invention is not limited to the above-described specific embodiment, those skilled in the art without departing from the gist of the present invention various modifications It will be appreciated that implementation is possible. Therefore, the scope of the present invention should not be construed as being limited to the above embodiments, but should be defined by the claims below and equivalents thereof.

The present invention is a nanobiotechnology (K2060100000209E010000210) for the early diagnosis of acute respiratory tract infection and severe sepsis through the use of Battelle Research Institute of Korea Research Foundation, the development of high activity, high-definition nano-analytical target technology of the Korea Science Foundation and its application in ELISA (task No.:R0911491, Assignment No .: 20090082314) and Korea Research Foundation's study on the stability and activity mechanism of trypsin hydrolysis and nanoenzyme coating for biofuel cells (Task No.:R0904822, Assignment No .: 20100016464) Has been done.

<110> Korea University Industry and Academy Cooperation

<120> Quantitative Analysis Using Minute Tube with Accumulated Enzyme

<130> M09-7373-PCT

<160> 2

<170> KopatentIn 1.71

<210> 1

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> Aptamer 1 for Thrombin

<400> 1

ggttggtgtg gttgg

15

<210> 2

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Aptamer2 for Thrombin

<400> 2

agtccgtggt agggcaggtt ggggtgact

29

Claims

(A) a magnetic bead or a substrate mixed with a first analyte binding aptamer or an antibody, wherein the first analyte binding aptamer or antibody is bound to the surface of the magnetic bead or substrate Preparing a 1-magnetic bead / substrate;

(B) adding the aptamer / antibody-magnetic bead / substrate to the test solution containing the analyte so that the analyte is the first analyte binding app of the aptamer / antibody-magnetic bead / substrate Preparing an analyte-aptamer / antibody1-magnet bead / substrate bound to a tammer or an antibody;

(C) separating the analyte-aptamer / antibody 1-magnetic beads / substrate from the test solution;

(D) mixing an enzyme and a microtube to prepare an enzyme-microtube in which the enzyme is integrated on the surface of the microtube;

(E) the second analyte-binding aptamer or antibody which binds to the analyte and the enzyme-microtube are mixed so that the second analyte-binding aptamer or antibody is bound to the enzyme of the enzyme-microtube. Preparing an aptamer / antibody2-enzyme-microtube;

(F) the second analyte of the aptamer / antibody-enzyme-microtube by mixing the analyte-aptamer / antibody1-magnetic bead / substrate with the aptamer / antibody2-enzyme-microtube Magnetic beads / substrate-aptamers / antibodies1-analytes-aptamers / antibodies-enzymes-microparticles having binding aptamers or antibodies bound to the analytes of the analyte-aptamer / antibody 1-magnetic beads / substrate Manufacturing a tube;

(G) separating the magnetic bead / substrate-aptamer / antibody 1-analyte-aptamer / antibody 2-enzyme-microtube; And

(H) quantitatively analyzing the enzyme of the separated magnetic beads / substrate-aptamers / antibodies-analyte-aptamers / antibodies-enzymes-microtubes

Quantitative analysis method comprising a.
The method according to claim 1,

The binding of the first analyte binding aptamer or antibody to the magnetic beads or the substrate of step (A) may include streptavidin-biotin binding, avidin-biotin binding, EDC [N− Ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] coupling, sulfhydrylamine coupling, or analyte quantitative analysis characterized in that it is made by nitrilotriacetic acid (H-NTA) -histidine bond.
The method according to claim 1,

The binding of the enzyme and the microtube of the step (D) is streptavidin (biotin) bond, avidin (avidin) -biotin bond, EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride Analyte quantitative analysis characterized in that it is by coupling, sulfhydrylamine coupling, or nitrilotriacetic acid (Ni-NTA) -histidine binding.
The method according to claim 1,

After step (A) and before step (B),

Protecting the unreacted binding portion of the magnetic bead or the substrate surface of the aptamer / antibody 1-magnetic bead / substrate

The quantitative analysis method characterized in that it further comprises.
The method according to claim 1,

After step (B) and before step (C),

Protecting unreacted binding sites of the magnetic beads or the substrate surface of the analyte-aptamer / antibody 1-magnetic beads / substrate;

The quantitative analysis method characterized in that it further comprises.
The method according to claim 1,

After step (E) and before step (F),

Protecting the unreacted binding site of the microtube or enzyme in the aptamer / antibody-enzyme-microtube

The quantitative analysis method characterized in that it further comprises.
The method according to claim 1,

The magnetic beads or the substrate is a neutral carboxyl group (-COOH) on the surface, its ion (-COO -), a neutral amine group (-NH 2), its ion (-NH-or -NH 3 +), neutral thiol group (- SH), and its ion (-S -) in analyte quantitation method comprising the functional group selected from the group consisting of.
The method according to claim 1,

It said fine tube is a neutral carboxyl group (-COOH), its ion (-COO -) on the surface, a neutral amine group (-NH 2), its ion (-NH - or -NH 3 +), neutral thiol (-SH) , and its ion (-S -) in analyte quantitation method comprising the functional group selected from the group consisting of.
The method according to claim 1,

Before step (A),

After reacting the magnetic beads or the substrate and the EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] solution by mixing, magnetically fixing the magnetic beads or the substrate, and removing the unreacted EDC solution

The quantitative analysis method characterized in that it further comprises.
The method according to claim 1,

Prior to step (D),

Reacting the microtubes with an EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] solution and then removing the unreacted EDC solution

The quantitative analysis method characterized in that it further comprises.
The method according to claim 1,

The first analyte binding aptamer or the second analyte binding aptamer is a quantitative analysis method, characterized in that the number of bases constituting the sequence is 10 to 250.
The method according to claim 1,

The microtube of step (D) is quantitative analysis method, characterized in that the treatment with acid (acid).
The method according to claim 1,

The analyte is quantitative analysis method, characterized in that the thrombin.
The method according to claim 13,

The first analyte binding aptamer is a thrombin specific DNA aptamer of SEQ ID NO: 5'- GGT TGG TGT GGT TGG-3 '.
The method according to claim 13,

The second analyte binding aptamer is a thrombin specific DNA aptamer of SEQ ID NO: 5'- AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 '.
The method according to claim 13,

The enzyme is a quantitative analysis method characterized in that the glucose oxidase (glucose oxidase).
By mixing a magnetic bead or a substrate (magnetic bead) and the first analyte binding aptamer or antibody, the aptamer / antibody 1-magnet wherein the first analyte binding aptamer or antibody is bound to the surface of the magnetic bead or substrate Beads / substrates, and

By mixing an enzyme and a microtube to prepare an enzyme-microtubes in which the enzyme is integrated on the surface of the microtube, and mixing the enzyme-microtubes with a second analyte binding aptamer or an antibody which binds to a target analyte, The aptamer / antibody2-enzyme-microtube wherein the second analyte binding aptamer or antibody is bound to the enzyme of the enzyme-microtube

Analysis sensor used in the analytical target quantitative analysis method of any one of claims 1 to 16 comprising a.
The method according to claim 17,

The binding of the first analyte binding aptamer or antibody to the magnetic beads or the substrate may include streptavidin-biotin binding, avidin-biotin binding, EDC [N-ethyl-N '-( dimethlaminopropyl) carbodiimide hydrochloride] coupling, sulfhydrylamine coupling, or Ni-NTA (nitrilotriacetic acid)-histidine coupling.
The method according to claim 17,

The combination of the enzyme and the microtube is streptavidin-biotin bonds, avidin-biotin bonds, EDC [N-ethyl-N '-(dimethlaminopropyl) carbodiimide hydrochloride] coupling, sulfhigh Analytical sensor characterized in that it is made by the sulphhydrylamine coupling, or Ni-NTA (nitrilotriacetic acid) -histidine bond.
The method according to claim 17,

And an unreacted binding portion of the magnetic bead or the substrate surface of the aptamer / antibody 1-magnetic bead / substrate is protected.
The method according to claim 17,

The unreacted binding site of the microtube or enzyme in the aptamer / antibody-enzyme-microtube is protected.
The method according to claim 17,

The magnet bead or substrate has a neutral carboxyl group (-COOH), an ion thereof (-COO-), a neutral amine group (-NH2), an ion thereof (-NH- or -NH3 +), a neutral thiol group (-SH), And a functional group selected from the group consisting of ions (-S-) thereof.
The method according to claim 17,

The microtube has a neutral carboxyl group (-COOH), an ion thereof (-COO-), a neutral amine group (-NH2), an ion thereof (-NH- or -NH3 +), a neutral thiol group (-SH), and Analytical sensor characterized in that it comprises a functional group selected from the group consisting of ions (-S-).
The method according to claim 17,

The first analyte binding aptamer or the second analyte binding aptamer is an analysis sensor, characterized in that the number of bases constituting the sequence is 10 to 250.
The method according to claim 17,

The microtube is an analysis sensor, characterized in that the treatment with acid (acid).
The method according to claim 17,

The analyte sensor, characterized in that the analysis target is thrombin.
The method of claim 26,

The first analyte binding aptamer is a thrombin specific DNA aptamer of SEQ ID NO: 5'- GGT TGG TGT GGT TGG-3 '.
The method of claim 26,

The second analyte binding aptamer is a thrombin specific DNA aptamer of SEQ ID NO: 5'- AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 '.
The method of claim 26,

The enzyme is an analysis sensor, characterized in that glucose oxidase (glucose oxidase).