KR101347722B1 - Detection method of target material using aggregation of aptamer-conjugated gold nanoparticles on fabric - Google Patents

Abstract

The present invention relates to a method for detecting a target substance using a fibrous aptamer-gold nanoparticle complex, which immobilizes a sample to be detected on a support of a fabric material, and sprays an aptamer-gold nanoparticle complex solution. The present invention relates to a method for detecting a target material comprising visually determining a color of a surface of a support.
In particular, the detection method of the target material according to the present invention by controlling the mixing ratio of the aptamer and gold nanoparticles and the concentration of the aptamer-gold nanoparticle complex, a small amount of the target material on the fiber support without special amplification process and the use of the device It can be determined with the naked eye, and it can be determined quickly and in a very simple manner for various target materials in any place.

Description

Detection method of target material using aggregation of aptamer-conjugated gold nanoparticles on fabric}

The present invention relates to a method for detecting a target substance using a fibrous aptamer-gold nanoparticle complex.

Point-of-care (POC) diagnostics are diagnostic tests that are performed at the same location as the patient, which allows the patient to be aware of the results while at the test site, thereby facilitating diagnosis and treatment. This advantage, coupled with the growing demand for easy diagnostics at home prior to the hospital visit, is expected to lead to a significant growth in the POC diagnostics market. This is particularly true in many areas, including blood sugar testing, coagulation testing, diabetes-related testing, pregnancy diagnosis, and drug abuse diagnosis. In fact, it is being applied in practice, and recently, it has been extended to simple cancer diagnosis based on biomarkers.

Bioreceptors have been used as a bioreceptor for diagnosing POC, but antibodies have problems such as chemical instability, high cost, and affinity for a target substance upon immobilization. In order to overcome these shortcomings, studies on the diagnosis using aptamer, a third generation molecular recognition material, have been actively conducted.

Aptamers are oligonucleotides that consist of single-stranded DNA or RNA, and have specificity with affinity with various targets ranging from cells to proteins, peptides, nucleic acids, lipids, inorganic compounds, organic molecules, metal ions, antibiotics, and cells. Through a process called SELEX in It can be easily and cheaply screened in vitro . In addition, because of the chemical synthesis and comparatively flexible to heat and pH changes, it has been spotlighted as a molecular recognition material to replace the antibody.

Representative sensors for diagnosing POC using aptamers include strip-based colorimetric analysis using gold nanoparticles. Gold nanoparticles appear inherent red due to surface plasmon resonance in aqueous solutions. This color changes with the distance between the gold nanoparticles. As the distance between the gold nanoparticles gets closer, the surface plasmons of each gold nanoparticle bind to each other, causing a color change from red to blue. As an application tool in the field of biosensors, gold nanoparticles have a high surface area as well as inherent physical and chemical properties, and thus can be surface modified with various materials. Therefore, studies are being actively conducted to diagnose / detect target materials by immobilizing a sensing material on the surface of gold nanoparticles. However, this color change is in aqueous solution, which requires a large amount of sample and gold nanoparticles, and additional equipment such as UV-vis spectrophotometer is required for high sensitivity detection. Therefore, the research to develop a sensor using the above gold nanoparticle colorimetric analysis is in progress.

Sensors manufactured in the form of conventional strips are similar to sandwich assays using antibodies, and DNA sequences complementary to aptamer sequences immobilized on gold nanoparticles in the control region of the strip surface and target materials in the test region. Another aptamer sequence that specifically binds is immobilized to serve as a probe. Therefore, when the test sample is injected into the strip, the target material binds to the immobilized aptamer sequence in the test region and stays in that region, so that the target material and the secondary DNA aptamer immobilized on the gold nanoparticle bind to the gold nanostructure in the test region. Particle red color is expressed. Without the target material, the gold nanoparticles could not bind to the test region, resulting in no red color. Only in the control region, the aptamer of the gold nanoparticles combined with the complementary DNA sequence would produce red color. Such a strip sensor has a high sensitivity to target material diagnosis, but has a disadvantage in that the design of the strip is not only complicated but also the production cost is high.

Thrombin is a protein that plays an important role in converting fibrinogen to fibrin in the course of aggregation reactions, as well as promoting reactions involved in blood aggregation. Disorders of blood coagulation can cause serious diseases such as heart attacks and heart attacks in the long run. And thrombin is a potential major cause of vascular spasm leading to vascular bleeding. Blood from the bleeding cerebral aneurysm clots near the cerebral artery, and secreted thrombin can cause severe blood vessel blockage and hardening in the long run, resulting in cerebral infarction. Thus, thrombin is a major biomarker capable of diagnosing a variety of diseases associated with abnormal blood aggregation reactions that can occur in the human body.

Commonly used techniques for diagnosing proteins such as thrombin include radio-immunoassay, enzyme-immunoassay (ELISA), fluorescence, and surface enhanced Raman spectroscopy (SERS). Although accurate and sensitive protein diagnosis is possible, it still requires a lot of time and money to purify, incubate or separate samples. In addition, there is a limitation that expensive equipment and specialized personnel to handle and analyze the equipment are required to be performed under laboratory conditions.

Looking at the previous study applying the colorimetric analysis method using the gold nanoparticles to the sensor, by coating the gold nanoparticles on the cellulose paper makes it possible to easily detect the target material with a small sample that is portable [Non-Patent Document 1]. However, there is a disadvantage that the enzyme must be processed and the sensitivity is low.

Strip biosensor [Non-Patent Document 2], it is also possible to detect the protein with high sensitivity, but has a limitation that the process of manufacturing the strip is complicated and a strip reader is required separately.

W. Zhao, M. Monsur All, S. D. Aguirre, M. A. Brook, and Y. Li, Anal. Chem. 80 (2008) 8431-8437 H. Xu, X. Mao, Q. Zeng, S. Wang, A.-N. Kawde, and G. Liu, Anal. Chem. 81 (2009) 669-675

Accordingly, the present inventors have developed a detection method capable of visually identifying a target material in a short time by using a common fiber (or disposable medical band) as a support and using an aptamer-gold nanoparticle complex without a complicated design and fabrication process as in the conventional technology. By this, the present invention was completed.

Accordingly, an object of the present invention is to detect a variety of target materials with high sensitivity using aptamer-immobilized gold nanoparticles more stably than other sensing materials, while reacting quickly within a few minutes on a simple and inexpensive fiber surface, resulting in a separate measurement. The present invention provides a method for detecting a target substance that can observe color changes with the naked eye without an instrument.

As means for solving the above problems,

Placing the sample to be detected on the support of the fabric material, and spraying the aptamer-gold nanoparticle composite solution to visually determine the color of the surface of the support

It provides a method for detecting a target material comprising a.

As another means for solving the above problems,

Provided are a kit or a sensor for detecting a target material comprising a support of a fiber material and an aptamer-gold nanoparticle complex.

The present invention relates to a method for detecting a target material by using two different aptamers immobilized on the gold nanoparticles together with a target material by using a color change that appears as the distance between the gold nanoparticles becomes closer.

In particular, the present invention is capable of chromaticity analysis having a higher sensitivity under stable conditions than color analysis using the adsorption method between the conventional aptamer and gold nanoparticles by immobilizing the aptamer on the gold nanoparticles. In addition, by controlling the mixing ratio of the aptamer and gold nanoparticles and the concentration of the aptamer-gold nanoparticle complex, it is possible to visually discriminate a small amount of the target material on the fibrous support without special amplification process and the use of a device. The shortcomings of the complicated manufacturing process were solved. In addition, it is possible to determine the presence or absence of a variety of target materials in any place in a quick and very easy way.

Figure 1 is an enlarged schematic diagram showing the detection process of the fiber-based simple target material according to an embodiment of the present invention and agglomerated gold nanoparticles.
Figure 2 shows a complex in which aptamer is immobilized on the gold nanoparticles according to Example 1 [left: TBA15 immobilized gold nanoparticle complex, right: TBA29 immobilized gold nanoparticle complex]
Figure 3 shows the stabilized state as aptamer-gold nanoparticle complexes before binding to the target material.
Figure 4 is a result of confirming the generated gold nanoparticles by Transmission Electron Microscopy (TEM).
5 is a result of observing the degree of color change of the gold nanoparticles according to the aptamer-gold nanoparticle composite having various mixing ratios on the disposable medical band.
6 is a result of observing the degree of color change of the gold nanoparticles according to the aptamer-gold nanoparticle complex of various concentrations on the disposable medical band.
7 is a result of observing the degree of color change of the gold nanoparticles according to the target material of various concentrations on the fiber support and disposable medical band.
FIG. 8 is a comparison of color changes for target proteins and other proteins to show the specificity of gold nanoparticles with aptamer immobilized on a fibrous support and / or disposable medical band.
9 is a result showing the degree of color change of the gold nanoparticles according to the concentration of the target material in the urine test (urine test) on the fiber support and disposable medical band.

The present invention provides a method for detecting a target material comprising placing a sample to be detected on a support of a fabric material, and visually determining the color of the surface of the support by spraying an aptamer-gold nanoparticle complex solution. It is about.

In the present invention, the fabric (fabric) is cotton, wool, linen, asbestos, glass fibers, synthetic fibers (nylon, acrylic, polyester, spandex, etc.), carbon fibers, cellulose-based pulp, polymer nanofibers or nonwoven fabrics, It is not limited. In particular, the present invention can use a medical gauze, disposable pad, disposable medical band, sanitary napkin, diaper, etc. can be easily purchased as the fiber has the advantage that the target material can be detected anywhere.

The aptamer is for detecting a target substance, and in the present invention, two aptamers should be used. The two aptamers are “first aptamer” and “second aptamer,” meaning different aptamers.

Thus, the aptamer-gold nanoparticle complex includes a first aptamer-gold nanoparticle complex and a second aptamer-gold nanoparticle complex.

The aptamer-gold nanoparticle composite is preferably prepared by mixing the gold nanoparticles and aptamer in a molar ratio of 1: 8 to 22 to prepare the aptamer-gold nanoparticle composite. If the aptamer is used too small, there is a problem in that the effect of stabilizing the gold nanoparticles due to the aptamer falls, and if the aptamer is used too much, there is a problem in terms of efficiency in terms of economy.

When the concentration of the aptamer-gold nanoparticle complex, that is, the sum of the concentrations of the first aptamer-gold nanoparticle complex and the second aptamer-gold nanoparticle complex is 6 to 16 nM, and the complex concentration is low, Colors are too light on the fiber, making it difficult to analyze the chromaticity. Higher complex concentrations are less economically efficient.

In the present invention, the target material may be a protein, a cell, a peptide, a nucleic acid, a lipid, an inorganic compound, a low molecular organic material, a metal ion, a hormone, or an antibiotic, and the protein has two aptamers developed for one target material, such as thrombin, IgG or platelet-derived growth factor-BB (PDGF-BB) and the like, but is not limited thereto.

The detection method of the target material according to the present invention has the advantage that even a small amount of the target material can be detected. Since the amount of the target substance that can be detected is obvious that the larger the target substance, the easier it is to detect, preferably 0.01 pmol or more, more preferably 0.015 pmol or more, and still more preferably 0.02 pmol or more.

For example, detection of the target substance may be used for diagnosis of a disease or diagnosis of a physical condition, and for such diagnosis, the target substance may be detected from the sample substance by the method according to the present invention. , Lymph, tissue fluids and the like, urine, runny nose, tears, saliva, sweat, sebum, pus and the like.

In the method for detecting a target material according to the present invention, the surface of the support is changed to blue when the target material is present, and when the target material is not present, the surface of the support is kept red.

Hereinafter, as an example of a method for detecting a target substance according to the present invention, a process of detecting thrombin will be described in detail with reference to FIG. 1.

The aptamer-immobilized gold nanoparticle complex has a form close to a sphere, and is a form in which an aptamer for detecting a target substance having a tertiary structure is immobilized on a surface of gold nanoparticles (see FIG. 2).

The aptamer-gold nanoparticle complex detection sample prepared at an appropriate concentration using a buffer solution is reddish by gold plasmon resonance due to its unique surface plasmon resonance because each gold nanoparticle is stabilized by an aptamer immobilized in an aqueous solution. [See FIG. 3]. When the aptamer-gold nanoparticle complex detection sample is processed on a fabric containing a thrombin, a target material to be detected, or a gauze attached to a disposable medical band, two aptamers immobilized on different gold nanoparticles are used as one target material. Because they can bind to different binding sites, they react like aggregates between the target material and the aptamer-gold nanoparticle complex. Due to the affinity between the target material and the aptamer, the distance between the gold nanoparticles is close, so that the surface plasmons of the gold nanoparticles are combined to change the color of the detection sample to blue.

The present invention also relates to a kit for detecting a target substance comprising a support of a fiber material and an aptamer-gold nanoparticle complex.

The present invention also relates to a sensor for detecting a target substance comprising a support of fibrous material and an aptamer-gold nanoparticle composite.

In the present invention, the shape of the kit or sensor for detecting the target material is not particularly limited, and may be any type as long as it includes a support of the fiber material and an aptamer-gold nanoparticle composite according to the present invention. In addition, the aptamer-gold nanoparticle complex may include a first aptamer-gold nanoparticle complex and a second aptamer-gold nanoparticle complex.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention and Comparative Examples which are not based on the present invention, but the scope of the present invention is not limited by the following Examples.

Manufacturing example  One

1) Manufacture of Gold Nanoparticles

Citrate stabilized gold nanoparticles were generated for use in making aptamer-gold nanoparticle composites. A large beaker HCl and HNO ₃ to 3: 1 ratio mixed litter placed behind prepared made aqua regia, at least more than 15 minutes to 200 ml flask and the magnet used to make the gold nanoparticles to remove contaminants. 98 ml of tertiary distilled water was added to the flask prepared as described above, and 2 ml of 50 mM HAuCl ₄ solution was added while stirring on a hot plate such that the final concentration of HAuCl ₄ was 1 mM. As soon as this solution began to boil, 10 ml of 38.8 mM sodium citrate solution was added quickly. Then the color of the solution changed from bright yellow to dark red within 1 minute. After boiling for another 20 minutes, let it stir at room temperature until it cools down. The solution was removed using a 0.45 um nitrocellulose filter paper to remove aggregated gold nanoparticles or other impurities.

Gold nanoparticles produced as shown in Figure 4 was confirmed the size and shape by using Transmission Electron Microscopy (TEM). The completed gold nanoparticles were refrigerated at 4 ℃.

2) Aptamer Production

The aptamers used to make the aptamer-gold nanoparticle complexes were synthesized from two previously studied thrombin aptamers 15-mer and 29-mer (hereinafter referred to as TBA 15 and TBA 29, respectively) [GenoTech (GenoTech Co., Korea). , Purified] was synthesized by thiol-modification.

TBA 15

5'-thiol (C6) -TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3 '(SEQ ID NO: 1)

TBA 29

5'-thiol (C6) -TTT TTT TTT TTT TTT AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3 '(SEQ ID NO: 2)

3) Preparation of Aptamer-Gold Nanoparticle Composite

Two vials to store the aptamer-gold nanoparticle complexes were prepared by soaking in DW for 1 hour in 12 M NaOH. 5 μl of 50 μM TBA 15 and TBA 29 were added to two centrifuge tubes, and 0.5 μl of 500 mM acetic acid buffer (pH 5.2) and 1 μl of 10 mM TCEP were added to thiol-modified at room temperature for 1 hour. DNA was activated. In each vial prepared above, 1 ml of gold nanoparticles prepared in advance, and activated TBA15 and TBA29 aptamers were added, respectively, and then shaken by hand. The vials were capped and stored for at least 16 hours in the dark. Thereafter, 10.2 μl of 500 mM free-acetate buffer (pH 8.2) was added slowly to bring the final concentration of this buffer to 5 mM. Thereafter, 102 μl of 1 M NaCl solution was slowly added thereto, mixed, and stored in a dark place for at least one day. Two solutions of the resulting TBA15-AuNP complex and TBA29-AuNP complex [FIG. 2] were respectively transferred to a centrifuge tube and centrifuged at 16,000 g for 25 minutes to remove the supernatant, followed by 3 times with 5.0 mM Tris-HCl buffer. After washing, the aptamer-gold nanoparticle complex was resuspended in 1 ml of the same buffer and then refrigerated at 4 ° C.

Example  1: Detection of target material using a support of fiber material

In the present invention, two different aptamers immobilized on the gold nanoparticles are bonded to the target material together to develop a chromaticity sensor using a color change that appears as the distance between the gold nanoparticles becomes closer.

Two aptamer-gold nanoparticle complexes synthesized in Preparation Example 1 were prepared in a buffer solution (Thrombin binding buffer pH 7.4 contained 140 mM NaCl, 20 mM Tris-HCl, 5 mM KCl, 1 mM MgCl ₂ , 1 mM CaCl _2). , 0.02% Tween 20) was used to prepare detection reagents at appropriate concentrations. This reagent was sprayed onto the target material, the fabric attached to the thrombin, or a gauze attached to a disposable medical band, indicating the presence of color change within 10 to 15 minutes, depending on the presence of thrombin.

For the efficient use of aptamers and gold nanoparticles and the detection of highly sensitive targets, the conditions for the ratio between the gold nanoparticles and the aptamers and the concentration of the aptamer-gold nanoparticle complexes were optimized.

First, the gold nanoparticles and the TBA15 aptamer were mixed at a molar ratio of 1:10, 1:20, and 1:30, respectively. The immobilized sample and the gold nanoparticles and the TBA29 aptamer were mixed at a molar ratio of 1:10, 1:20, and 1:30, respectively. Immobilized samples were generated to find the rate of detection of the target material with the highest sensitivity.

As a result, it was confirmed that the target material detection ability of the aptamer-immobilized gold nanoparticles made by mixing the gold nanoparticles and the aptamer in a molar ratio of 1:10 was shown to be the most excellent [FIG. 5]. Was used at optimum conditions. At this time, the amount of the target material is 0.025 pmol.

In addition, the TBA15 aptamer-gold nanoparticle complex and the TBA29 aptamer-gold nanoparticle complex were made with 4, 6, and 8 nM, respectively (concentrations of the two complex sums were made with 8 nM, 12 nM, and 16 nM). The concentration was able to detect the target substance with high sensitivity while being well confirmed.

As a result, when using a complex solution of 12 nM it was able to observe the visible color change with the naked eye, but showed the ability to detect the target material with high sensitivity [Fig. 6]. Therefore, a complex solution of 12 nM was used as the optimum condition when detecting the target substance. At this time, the amount of the target material is 0.2 pmol.

Test Example  One

The experiment was carried out under the optimized conditions in Example 1.

In order to see the color change of the gold nanoparticles according to the concentration of the target material, the color change was observed by treating the gold nanoparticle solution to thrombin from 10 pmol to the detection limit concentration on each fabric or disposable medical band.

As a result, it can be seen that as the concentration of the target material is lowered, the degree of turning the gold nanoparticles to blue becomes weaker [FIG. 7].

Test Example  2

The experiment was carried out under the optimized conditions in Example 1.

To demonstrate that aptamer-immobilized gold nanoparticles specifically detect thrombin, gold nanoparticle solutions were treated with Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), and IgG instead of thrombin as targets. When the color change was observed. The amount of the target material was 20 pmol.

As a result, it can be seen that the gold nanoparticles characterized by the change in blue only in thrombin can be detected by specifically binding to thrombin [FIG. 8].

Test Example  3

The experiment was carried out under the optimized conditions in Example 1.

Even when the sample to be detected is a mixed solution containing various substances in addition to the target substance, the limit of detection by adding thrombin, a target substance, is added to the urine to prove that the aptamer-immobilized gold nanoparticles specifically detect thrombin. Measured.

As a result, it can be seen that in the control region without thrombin, the red color is changed in a dose-dependent blue color in the urine sample to which the thrombin is added (FIG. 9).

Attach an electronic file to a sequence list

Claims (11)

Placing a sample to be detected on a support of a fabric material and spraying an aptamer-gold nanoparticle complex solution having an aptamer immobilized on the gold nanoparticles to visually determine the color of the surface of the support; ,
The aptamer-gold nanoparticle complex may include a complex of first aptamer-gold nanoparticles; And a second aptamer-gold nanoparticle complex, wherein the first aptamer and the second aptamer are different aptamers capable of binding to different binding sites of the target material.
The method of claim 1,
Fiber is a method of detecting a target material is cotton, wool, linen, asbestos, glass fiber, synthetic fiber, carbon fiber, cellulose-based pulp, polymer nanofiber or nonwoven fabric.
delete The method of claim 1,
The aptamer-gold nanoparticle complex is a method for detecting a target material prepared by mixing gold nanoparticles and aptamers in a molar ratio of 1: 8 to 22.
The method of claim 1,
A method for detecting a target material, wherein the concentration of the aptamer-gold nanoparticle complex is 6 to 16 nM.
The method of claim 1,
The target material is a protein, cell, peptide, nucleic acid, lipid, inorganic compound, low molecular weight organic material, metal ion, hormone or antibiotic.
The method according to claim 6,
Method for detecting a target material is a protein thrombin, IgG or PDGF-BB (platelet-derived growth factor-BB).
delete The method of claim 1,
A method for detecting a target material in which the surface of the support turns blue when the target material is present.
Including a support of fiber material and an aptamer-gold nanoparticle composite,
The aptamer-gold nanoparticle complex may include a complex of first aptamer-gold nanoparticles; And a second aptamer-gold nanoparticle complex, wherein the first aptamer and the second aptamer are different aptamers capable of binding to different binding sites of the target material.
Including a support of fiber material and an aptamer-gold nanoparticle composite,
The aptamer-gold nanoparticle complex may include a complex of first aptamer-gold nanoparticles; And a second aptamer-gold nanoparticle complex, wherein the first aptamer and the second aptamer are different aptamers capable of binding to different binding sites of the target material.

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