KR101329300B1 - Method for Detecting Nucleic Acids and Biomolecules Based on Colorimetric Reaction Induced by Peroxidase Activity of Magnetic Nanoparticles - Google Patents

Abstract

The present invention relates to a method for detecting a nucleic acid by using a phenomenon of color reaction by magnetic nanoparticles having peroxidase activity, and more particularly, when a nucleic acid amplification product is present, the nucleic acid is added to the magnetic nanoparticles. The present invention relates to a method for detecting a target nucleic acid by using a phenomenon of inhibiting the catalytic action of magnetic nanoparticles by adsorbing and interacting with a color reaction substrate, thereby reducing the color reaction.
The method for detecting a nucleic acid using magnetic nanoparticles according to the present invention can easily identify the target nucleic acid with the naked eye in a short time, and provide a device and system for on-site diagnosis by providing a magnetic nanoparticle-based color sensor using the nucleic acid detection method. It can be used for diagnosis of various causative infections, genetically modified organisms (GMO) testing, and forensic investigation. In addition, the detection method using the magnetic nanoparticles according to the present invention can be applied not only to the detection of nucleic acids but also to the detection of other biological materials and chemicals.

Description

Method for Detecting Nucleic Acids and Biomolecules Based on Colorimetric Reaction Induced by Peroxidase Activity of Magnetic Nanoparticles}

The present invention relates to a method for detecting a nucleic acid by using a phenomenon of color reaction by magnetic nanoparticles having peroxidase activity, and more particularly, when a nucleic acid amplification product is present, the nucleic acid is added to the magnetic nanoparticles. The present invention relates to a method for detecting a target nucleic acid by using a phenomenon of inhibiting the catalytic action of magnetic nanoparticles by adsorbing and interacting with a color reaction substrate, thereby reducing the color reaction.

Most gene diagnosis is performed by amplifying a target gene by a polymerase chain reaction (PCR) technique and confirming the presence of a gel band through electrophoresis. Since the diagnosis method based on the existing electrophoresis requires a long diagnosis time and skillful technology, it is difficult to carry out directly in a general hospital or a site requiring diagnosis.

In order to solve this problem, color development diagnostic technology has been proposed to quickly and easily diagnose nucleic acids in the field. For this purpose, conjugated polymer (Jung et al . Adv . Funct . Mater . , 18; 701-708, 2008) or metal nanoparticles have been used. Among them, a technique for detecting a nucleic acid including a PCR amplification product by using a gold nanoparticle colloid solution has been attracting attention. For this purpose, gold nanoparticles functionalized with probe oligonucleotides are used. In the presence of target DNA, cross-linking is formed by hybridization with probe oligonucleotides, which causes the colloidal solution to exhibit color transition (Elghanian et. al ., Science , 277 (5329): 1078-1081, 1997). In addition, methods for detecting amplified nucleic acids by color transfer using different electrostatic properties of single- and double-stranded DNA on gold nanoparticles have been reported (Li, HX et. al ., J. Am . Chem . Soc . 126: 10958-10961, 2004). However, the target nucleic acid diagnostic method using the color transfer of gold nanoparticles has a number of problems. Firstly, prior to diagnosis of the target nucleic acid, the process of attaching the probe DNA to the surface of the gold nanoparticles is time consuming and requires timely and skilled techniques. Secondly, after the reaction of the target nucleic acid with the gold nanoparticles, salts must be added into the solution to cause color transfer. However, in this process, the color transition phenomenon is very sensitive to the salt concentration, which may lead to a false diagnosis result, which requires a long time optimization process.

On the other hand, it is known that magnetic nanoparticles have peroxidase activity (Gao et. Al ., Nat . Nanotechnol . 2; 577-583, 2007). On the basis of this fact, the amplified nucleic acid product to identify the principle of inhibiting the peroxidase activity of the magnetic nanoparticles and to provide a nucleic acid detection method using the same.

Accordingly, the present inventors have made diligent efforts to solve the problems of the above-mentioned prior arts and develop a method that can detect the presence or absence of a target substance easily and easily with the naked eye. As a result, when the nucleic acid amplified from the target DNA is present in the substrate mixture solution, In addition, the nucleic acid inhibits the peroxidase activity of the magnetic particles, and compared with the absence of the amplified nucleic acid, it is confirmed that the color difference, this method is the process of labeling the DNA on the magnetic nanoparticles or addition of additional salts By confirming that the nucleic acid can be diagnosed quickly and simply without a process, a magnetic nanoparticle-based color sensor capable of universally diagnosing the presence or absence of a nucleic acid was completed.

An object of the present invention is that when a nucleic acid molecule is present in a magnetic nanoparticle solution, a negatively charged nucleic acid is adsorbed to the magnetic nanoparticle by ionic bonding, and when a color reaction substrate solution is added, the nucleic acid is positively charged. Because of the miracle interaction, the contact between the magnetic nanoparticles and the color reaction substrate is restricted, thereby inhibiting the peroxidase activity of the magnetic nanoparticles. Therefore, the substrate is added to the mixture of the nucleic acid molecule and the magnetic nanoparticle solution to induce color reaction and change color. The present invention provides a method for detecting a nucleic acid.

In order to achieve the above object,

(a) preparing an amplified product of the target nucleic acid;

(b) preparing a mixed solution of the amplification product and the magnetic nanoparticle solution;

(c) inducing a color reaction by adding a color reaction substrate to the mixed solution; And

(D) provides a method for detecting a nucleic acid using the color reaction of the magnetic nanoparticles comprising the step of observing the color change according to the color reaction.

The present invention also relates to

(a) preparing an amplified product of the target nucleic acid;

(b) preparing a mixed solution of the amplification product and the magnetic nanoparticle solution; And

It provides a method for producing a composition for detecting a nucleic acid using the color reaction of the magnetic nanoparticles comprising the step of (c) adding a color reaction substrate to the mixed solution to obtain a composition for detecting the nucleic acid.

The present invention also provides a composition for detecting a nucleic acid using a color reaction of a magnetic nanoparticle containing an amplification product of a target nucleic acid, a magnetic nanoparticle solution and a color reaction substrate.

The method for detecting a nucleic acid using magnetic nanoparticles according to the present invention can easily identify the target nucleic acid with the naked eye in a short time, and provide a device and system for on-site diagnosis by providing a magnetic nanoparticle-based color sensor using the nucleic acid detection method. It can be used for diagnosis of various causative infections, genetically modified organisms (GMO) testing, and forensic investigation. In addition, the detection method using the magnetic nanoparticles according to the present invention can be applied not only to the detection of nucleic acids but also to the detection of other biological materials and chemicals.

Figure 1 is a schematic diagram showing the principle of detecting the target nucleic acid using the color reaction phenomenon by the magnetic nanoparticles having peroxidase activity.
FIG. 2 is a graph showing the results of reduced color reaction due to inhibition of peroxidase activity of magnetic nanoparticles when DNA is added to magnetic nanoparticles having different functional groups, and a graph of the results of scanning with ultraviolet and visible light spectroscopy. (a: magnetic nanoparticles of hydroxyl group, b: magnetic nanoparticles of amine group, c: magnetic nanoparticles of dendrimer).
FIG. 3 is a photograph showing the reduced color reaction phenomenon and the result of scanning by ultraviolet and visible light spectroscopy when the target nanoparticles of different concentrations are added to the magnetic nanoparticles of the hydroxyl group, thereby inhibiting the peroxidase activity of the magnetic nanoparticles. It is a graph (Figure 3 A). Calibration curve for confirming the detectable nucleic acid concentration using the magnetic nanoparticle sensor (Fig. 3B).
FIG. 4 is a graph showing the results of scanning by UV and visible light spectroscopy when the amplification products of target nucleic acids are 200bp, 600bp and 1200bp, respectively, when the peroxidase activity of the magnetic nanoparticles is inhibited and the reduced color reaction phenomenon is reduced. .
5 is a graph of the results of scanning by ultraviolet and visible light spectroscopy to determine whether the magnetic nanoparticles can be efficiently separated and reused using external magnetic force, and the numbers indicate the number of times they have been reused (C: control, T: experimental group).
6 is a table showing the amount of target nucleic acid adsorbed on the upper layer and the magnetic nanoparticles measured by nanodrop spectroscopy to understand the principle of inhibition of peroxidase activity of magnetic nanoparticles by target nucleic acids (FIG. 6A 6, B is a control sample (Fig. 6B (1)) in which there is no amplification product of the target nucleic acid, a sample to which the amplification product of the target nucleic acid is added (Fig. 6B (3)) and the amplification product of the target nucleic acid. After the addition, the magnetic nanoparticles were separated from the supernatant, the supernatant was removed, and the color difference shown by the color reaction of the sample used again (FIG. 6B (2)) is a graph of the result of scanning with ultraviolet and visible spectroscopy.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, based on the known fact that the magnetic nanoparticles have peroxidase activity (Gao et . Al ., Nat . Nanotechnol . 2; 577-583, 2007), the nucleic acid product amplified by PCR is obtained from the magnetic nanoparticles. The principle of inhibiting peroxidase activity was identified, and it was confirmed that the presence or absence of the target nucleic acid could be detected using this.

The present invention in one aspect, (a) preparing an amplification product of the target nucleic acid; (b) preparing a mixed solution of the amplification product and the magnetic nanoparticle solution; (c) inducing a color reaction by adding a color reaction substrate to the mixed solution; And (d) relates to a method for detecting a nucleic acid using the color reaction of the magnetic nanoparticles comprising the step of observing the color change according to the color reaction.

In the present invention, any of the color reaction substrates of the magnetic nanoparticles can be used, TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid) and OPD (o-phenylenediamine) may be selected from the group consisting of. Preferably, it may be characterized as a positively charged OPD (o-phenylenediamine) capable of inhibiting access to the magnetic nanoparticles as much as possible through electrical coupling with a negatively charged target nucleic acid.

In the present invention, the gene amplification product and the magnetic nanoparticle solution of 5mg / ml in the step (b) may be characterized in that it is mixed in a volume ratio of 7-9: 1.

In the present invention, the size of the magnetic nanoparticles may be characterized in that 11-15nm.

In the present invention, the solvent of the magnetic nanoparticle solution can be used without limitation as long as it is an inorganic solvent, it is preferable to use water.

In the present invention, the concentration of the magnetic nanoparticle solution may be characterized in that 4-6 mg / ml.

In the present invention, the color change may be characterized in that when the substrate solution is added to the mixed solution of the amplification product of the target nucleic acid and the magnetic nanoparticle solution, the color changes to yellow by the color reaction of the magnetic nanoparticles.

The present invention in another aspect, the method comprising the steps of (a) preparing an amplification product of the target nucleic acid; (b) preparing a mixed solution of the amplification product and the magnetic nanoparticle solution; And (c) adding a color reaction substrate to the mixed solution to obtain a composition for detecting a nucleic acid.

In still another aspect, the present invention relates to a nucleic acid detection composition using a color reaction of a magnetic nanoparticle containing an amplification product of a target nucleic acid, magnetic nanoparticles, and a color reaction substrate.

The nucleic acid detection method according to the present invention utilizes the properties of a negatively charged target nucleic acid due to the deoxyribose-phosphate basic framework. Specifically, the negatively charged target nucleic acid in the test solution electrostatically interacts with the positively charged chromogenic reaction substrate, thereby preventing the chromogenic reaction substrate from accessing the magnetic nanoparticles. By adsorbing on the magnetic nanoparticles, the chromogenic substrate is prevented from coming into direct contact with the magnetic nanoparticles, thereby limiting the formation of the enzyme-substrate complex. As a result, the magnetic nanoparticles have reduced peroxidase activity, and due to the oxidation of the reduced color reaction substrate, the absorbed wavelength is reduced in the absorption wavelength of the color reaction substrate, and the color difference is used to diagnose the presence of the target nucleic acid. can do.

In one embodiment of the present invention, a positively charged OPD (o-phenylenediamine) capable of inhibiting access to the magnetic nanoparticles as much as possible through electrical coupling with a negatively charged target nucleic acid as a substrate was used. The negatively charged target nucleic acid electrostatically interacts with the positively charged chromogenic substrate OPD and ionic bonds with the magnetic nanoparticles, which inhibits the chromogenic reaction substrate OPD from contacting the magnetic nanoparticles, thus forming the enzyme-substrate complex. Inhibition of the color reaction substrate resulted in decreased absorbance at 450 nm of the absorption wavelength of the color development substrate OPD, and it was found that the presence of the target nucleic acid can be diagnosed through the color difference.

In the present invention, in order to examine the effect of the ionic bond between the magnetic nanoparticles and the target nucleic acid on the reduced peroxidase activity of the magnetic nanoparticles, the magnetic nanoparticles have various functional groups (hydroxyl group, amine group, dendrimer) It was chemically synthesized, and as a result, no differences were found depending on the functional groups. In the present invention, the magnetic nanoparticles may have a functional group selected from a hydroxyl group, an amine group, and a dendrimer, and may be characterized in that nucleic acid detection is performed using magnetic nanoparticles of the hydroxyl group which is most easily synthesized.

In one embodiment of the present invention, Chlamydia , which is one of the urogenital infections clinically useful as the target nucleic acid of step (a) trachomatis Select Chlamydia using a real clinical sample A portion of the gene expressing the toxic protein of trachomatis was amplified. PCR amplification is carried out using primers designed using the Primer 3 ( http://biotools.umassmed.edu/bioapps/primer3_www.cgi ) program to obtain amplification products of the target nucleic acid. Purified amplified nucleic acid product was obtained by removing primers, salts of buffer and dNTPs using DNA purification kit (QIAGEN). In step (c), 45 μl of the mixed solution was added to 95 μl 0.2 M sodium acetate buffer (pH 4.0), 40 μl 60 mM OPD, and 20 μl H ₂ O _2. After adding (100 mM) in sequence, the color reaction was observed by reacting at 43 ° C for 30 minutes.

According to the present invention, the sample in which the target nucleic acid is present is electrostatically interacted with the negatively charged target nucleic acid in the test solution with the positively charged color reaction substrate OPD, thereby preventing the color reaction substrate from accessing the magnetic nanoparticles. On the other hand, the target nucleic acid is adsorbed on the magnetic nanoparticles by ionic bonding, thereby limiting the direct contact of the color development substrate OPD with the magnetic nanoparticles, which in turn inhibits the formation of the enzyme-substrate complex. As a result, the magnetic nanoparticles have a reduced peroxidase activity, and due to the reduced oxidation of the color reaction substrate, the absorbance at 450 nm of the absorption wavelength of the color development substrate OPD is yellow and yellow. On the other hand, the control sample without the target nucleic acid does not inhibit the peroxidase activity of the magnetic nanoparticles, and thus, the color reaction substrate OPD is sufficiently oxidized to show high absorbance at 450 nm of the absorption wavelength of the color reaction substrate. Will be displayed. This color difference can be used to easily detect the target nucleic acid (FIG. 1).

Nucleic acid detection method using the color reaction phenomenon of the magnetic nanoparticles according to the present invention is made through the addition of a color reaction substrate solution after mixing the target nucleic acid obtained through the amplification process, such as PCR, with the magnetic nanoparticle solution, Inhibition of the peroxidase activity of the target nucleic acid resulted in a reduced color reaction phenomenon, which is characterized by easy detection of the target nucleic acid using this color difference. As a result, when the target nucleic acid is present, primers of a specific gene cause PCR amplification to generate an amplification product of the target nucleic acid, and the amplified target nucleic acid electrostatically interacts with the coloring substrate in the solution. On the other hand, the target nucleic acid is adsorbed on the magnetic nanoparticles by ionic bonding, thereby limiting the direct contact of the chromogenic substrate with the magnetic nanoparticles, resulting in enzyme-substrate complex formation. Will be inhibited. As a result, the magnetic nanoparticles have reduced peroxidase activity, exhibit reduced absorbance at the absorption wavelength of the chromogenic substrate, and exhibit color change. On the other hand, if the target nucleic acid is not present, PCR amplification does not occur, there is no factor that inhibits the peroxidase activity of the magnetic nanoparticles, so it shows a high absorbance at the absorption wavelength of the color reaction substrate.

In another embodiment of the present invention, when the DNA is added to the magnetic nanoparticles having different functional groups, the peroxidase activity of the magnetic nanoparticles is inhibited, resulting in a reduced color reaction phenomenon. It was confirmed that the absorbance at 450 nm of the absorption wavelength of the chromophore substrate OPD was reduced depending on the presence of oligonucleotides in the nanoparticles, but no difference was observed according to the functional group. Thus, further experiments were performed by selecting the magnetic nanoparticles of the hydroxyl group which is the easiest to manufacture (FIG. 2).

In another embodiment of the present invention, in order to confirm the difference in color reaction when different concentrations of target nucleic acids are added to the magnetic nanoparticles of the hydroxyl group, the amplification products were purified and reacted with the magnetic nanoparticles at different concentrations, respectively. Thereafter, the color reaction was added by adding a color reaction solution and reacted at 43 ° C. for 30 minutes using ultraviolet and visible spectroscopy to measure absorbance. In the presence of the amplified nucleic acid, the absorbance of the magnetic nanoparticles inhibited the peroxidase activity, resulting in a reduced absorbance at 450 nm of the absorption wavelength of the chromogenic substrate OPD, and the absorbance was linearly changed according to the concentration of the amplified nucleic acid. 3).

In addition, in order to check whether the color reaction is different depending on the length of the amplified product of the target nucleic acid, the target nucleic acid was changed to 200bp, 600bp and 1200bp, respectively, and reacted with the magnetic nanoparticles, respectively, and then the coloring reaction solution was added. The absorbance was measured by UV and visible spectroscopy for 30 minutes at 43 ° C, but the absorbing wavelength 450nm of the color reaction substrate OPD was inhibited by inhibiting the peroxidase activity of the magnetic nanoparticles regardless of the length of the amplified nucleic acid. It was confirmed that the reduced absorbance at. This indicates that magnetic nanoparticle based sensors can be universally applied regardless of the length of the amplification product (FIG. 4).

In another embodiment of the present invention, in order to confirm that the magnetic nanoparticles can be separated and reused after detecting the nucleic acid using the magnetic nanoparticles, after mixing the nucleic acid material and the magnetic nanoparticles, a color reaction solution is added. After reacting at 43 ° C. for 30 minutes, the magnetic nanoparticles were easily separated using an external magnetic force, and the nucleic acid material adsorbed on the magnetic nanoparticles was removed using 0.1 M PBS (pH 8). This process is based on the ion exchange reaction, which can easily remove the nucleic acid material attached to the magnetic nanoparticles, and confirmed the DNA removal using SYBR green fluorescence dye. The magnetic nanoparticles (C) separated through the above process were able to maintain the peroxidase activity even after several reuses and showed a difference in color from the sample (T) in which DNA is present (FIG. 5).

6 is a table showing the amount of target nucleic acid adsorbed on the upper layer and the magnetic nanoparticles measured by nanodrop spectroscopy for understanding the principle of peroxidase activity inhibition of magnetic nanoparticles by target nucleic acids (FIG. 6A) As shown in the above table, only 40% of the target nucleic acid present in the solution was bound to the magnetic nanoparticles. In addition, the control sample without the amplification product of the target nucleic acid was found to have high absorbance at 450 nm of the absorption wavelength of the color reaction substrate OPD through the color reaction by the magnetic nanoparticles through ultraviolet and visible light spectroscopy (FIG. 6B (1)). On the other hand, the sample to which the amplification product of the target nucleic acid was added showed reduced absorbance through the inhibition of peroxidase activity of the target nucleic acid (Fig. 6B (3)). However, after adding the amplification product of the target nucleic acid, the magnetic nanoparticles were separated from the supernatant, the supernatant was removed, and the used sample was compared with the reduced absorbance of the sample to which the amplification product of the target nucleic acid was added ((3) of FIG. 6B). Only 40% was found to be reduced (Fig. 6B (2)). This result indicates that the peroxidase activity of the magnetic nanoparticles is limited to two different forms by the target nucleic acid present in the solution as well as the target nucleic acid adsorbed on the magnetic nanoparticles.

In the present invention, a target nucleic acid is a gene associated with a disease, a gene associated with an infection (bacterial, viral, etc.), a gene associated with genetically modified organisms (GMO), a gene for forensic investigation or a mixture of one or more thereof. Include. Therefore, the present invention not only provides a diagnostic method capable of quickly and simply detecting a nucleic acid universally, but also provides a possibility of being used as a biosensor for on-site diagnosis. In addition, the above phenomenon can be used to detect not only nucleic acids but also biological materials and chemicals.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

In particular, in the following examples, only the detection of nucleic acids was confirmed, but it is possible to detect biological materials or chemicals in addition to nucleic acids by using the present invention for observing color changes caused by inhibition of peroxidase activity of magnetic nanoparticles by target materials. It will be obvious to you.

Nucleic Acid Detection Using Color Reaction Phenomenon of Magnetic Nanoparticles

1-1. Target nucleic acid Amplification  Produce

Chlamydia , the genitourinary tract infection Amplification of the trachomatis gene was performed using the PCR method. The primers selected as above were used to obtain a final amplification product of 600 bp. C. trachomatis gene extracted from a real patient sample, 0.42 μM primer pair (SEQ ID NO: 1 and SEQ ID NO: 2), 1X PCR reaction buffer (30 mM Tris-HCl, 30 mM KCl, 30 mM (NH ₄ ) ₂ SO _{4 ,} 2 mM MgCl ₂ ), 1.07 mM dNTPs and 4 U i-starMAX ^TM II DNA polymerase (Intronbio, Korea) were used to amplify the reaction mixture using a Perkin-Elmer 9200 thermo-cycler (Perkin-Elmer, Norwalk, CT). I was. After denaturation of double-stranded DNA by heating at 94 ° C for 5 minutes, the cycle was repeated 35 times for 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C, followed by the last extension of 7 minutes at 72 ° C. The nucleic acid amplification process was completed. PCR products were agarose gel electrophoresis (agarose gel electrophoresis) to determine the size and PCR. The obtained amplification product was obtained by using a DNA purification kit (QIAGEN) to remove the primer, the salt of the buffer and dNTPs purely purified amplified nucleic acid product.

SEQ ID NO: 5'-CCATCTTCTTTGAAGCGTTGT-3 '(Forward primer)

SEQ ID NO: 2'-ACAGGATGACTCAAGGAATAG-3 '(Reverse primer)

1-2. Preparation of Mixed Solution of Amplified Target Nucleic Acid and Magnetic Nanoparticle Solution

Chlamydia prepared in Example 1-1 trachomatis 40 μl of the gene amplification product (100-600 nM) and 5 μl (5 mg / ml) of the magnetic nanoparticle solution were mixed to prepare a mixed solution. In addition, magnetic nanoparticles were prepared by the following method. 13 nm magnetic nanoparticles are dissolved in 0.25 M Fe ^{2 +} and Fe ^{3 +} ions (Fe ^{3 +} / Fe ^{2 +} = 2) in water to form an aqueous solution, then boiled at 80 ° C, by adding 1 M sodium hydroxide Prepared by reduction (Gao et al . J. Magn . Magn. Mater . , 293,1; 48-54, 2005). The prepared magnetic nanoparticles were sized using Transmission Electron Microscopy (TEM) and analyzed using an element analyzer and a zetasizer. The magnetic nanoparticles used herein had a size of 13 ± 2 nm, and the magnetic nanoparticles were stored dispersed in 0.2 M acetate buffer at a concentration of 5 mg / ml.

1-3. Induction of color reaction of magnetic nanoparticles by adding color reaction solution to mixed solution

In 45 µl of the mixed solution prepared in Example 1-2, 95 µl 0.2 M sodium acetate buffer (pH 4.0), 40 µl 60 mM OPD and 20 µl H ₂ O ₂ (100 mM) was added sequentially, followed by reaction for 30 minutes at 43 ° C.

1-4. Observation of Color Difference by Color Reaction of Magnetic Nanoparticles

As a result of observing the color difference generated through the oxidation reaction using the magnetic nanoparticles and the color reaction solution in Example 1-3, when the PCR amplification product is not present, the absorption wavelength of the color development substrate OPD is high at 450 nm. It shows absorbance and red color. On the other hand, when PCR was generated due to the presence of the target nucleic acid, it was confirmed that yellow color was obtained by decreasing absorbance at 450 nm of the wavelength of OPD through inhibition of the peroxidase activity of the magnetic nanoparticles of the target nucleic acid.

1-5. Efficient Separation and Reuse of Magnetic Nanoparticles

After confirming the presence or absence of nucleic acid in Example 1-4, magnetic nanoparticles were efficiently separated using magnetic force, washed with 0.1 M PBS buffer (pH 8), and then added by a color reaction solution for reuse. It was confirmed whether or not, and the magnetic nanoparticles isolated through the above process was able to confirm the color difference enough to maintain the peroxidase activity and to detect the presence of DNA even after several reuses.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (15)

Nucleic acid detection method using the color reaction of magnetic nanoparticles comprising the following steps:
(a) preparing an amplified product of the target nucleic acid;
(b) preparing a mixed solution of the 11-15 nm magnetic nanoparticle solution having the amplification product and peroxidase activity;
(c) inducing a color reaction by adding a color reaction substrate to the mixed solution; And
(d) monitoring the color change according to the color reaction.
The method of claim 1, wherein the color development substrate is TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (O-phenylenediamine) A method for detecting a nucleic acid using a color reaction of magnetic nanoparticles, characterized in that selected from the group consisting of.
According to claim 1, wherein in step (b) the nucleic acid using the color reaction of the magnetic nanoparticles, characterized in that the amplification product of the target nucleic acid and 5mg / ml magnetic nanoparticle solution is mixed in a volume ratio of 7-9: 1 Detection method.
delete The method of claim 1, wherein the solvent of the magnetic nanoparticle solution is an inorganic solvent.
The method of claim 1, wherein the concentration of the magnetic nanoparticle solution is 4-6 mg / ml.
The method of claim 1, wherein the color change of the mixed solution of the nucleic acid amplification product and the magnetic nanoparticle solution is yellow by the addition of a substrate.
Method for preparing a nucleic acid detection composition using a color reaction of magnetic nanoparticles comprising the following steps:
(a) preparing an amplified product of the target nucleic acid;
(b) preparing a mixed solution of the 11-15 nm magnetic nanoparticle solution having the amplification product and peroxidase activity; And
(c) adding a color reaction substrate to the mixed solution to obtain a nucleic acid detection composition.
According to claim 8, wherein the color development substrate is TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (o-phenylenediamine) A method for producing a composition for detecting a nucleic acid, characterized in that selected from the group consisting of.
The nucleic acid detection method according to claim 8, wherein the amplification product of the target nucleic acid and the 5 mg / ml magnetic nanoparticle solution are mixed in a volume ratio of 7-9: 1 in step (b). Method for producing a composition for.
delete The method of claim 8, wherein the solvent of the magnetic nanoparticle solution is an inorganic solvent. 9.
The method of claim 8, wherein the concentration of the magnetic nanoparticle solution is 4-6 mg / ml method for producing a composition for detecting a nucleic acid using a color reaction of the magnetic nanoparticles.
A nucleic acid detection composition using the amplification product of the target nucleic acid, a magnetic nanoparticle solution of 11-15 nm having a peroxidase activity and a color reaction of the magnetic nanoparticles containing the color reaction substrate.
The method of claim 14, wherein the color reaction substrate is TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD Nucleic acid detection composition using the color reaction of the magnetic nanoparticles, characterized in that selected from the group consisting of (o-phenylenediamine).

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