KR101941040B1 - Strip and fabricating method for the same - Google Patents

Abstract

In the strip of the present invention, an introduction part into which a result containing DNA is introduced; A guide connected to the one end by a flow of fluid by the capillary phenomenon and guiding the resultant; An absorption part connected to the other end of the guide part by a fluid flow caused by a capillary phenomenon and absorbing the resultant product; A fixing unit connected to the guide unit to allow the resultant product to pass through and to provide graphene oxide for adsorbing and fixing the first DNA in the DNA; And a detecting unit for detecting another DNA passing through the fixing unit. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily and quickly confirm DNA at a lower cost and without any risk.

Description

[0001] Strip and fabricating method for the same [

The present invention relates to a strip and a method for producing the strip, and more particularly, to a strip and a strip for detecting a DNA of a target for detection such as microorganisms, harmful cells, bacteria, and the like which are harmful to the environment such as human body, And a method for producing the same.

Recently, human beings are being attacked by new new species such as Zika virus. These organisms have various expression forms such as viruses and bacteria. Since these organisms have a specific DNA, it is possible to judge whether or not an organism, which is exemplified as a harmful microorganism, is infected by judging whether or not the organism has the DNA of the organism in the human body and the environment.

Real-time PCR, PCR (Polymerase Chain Reaction, hereinafter referred to as PCR), and microarray technology are known as techniques for identifying harmful microorganisms and harmful cells (cancer cells) (Microarray).

These techniques are known to quantitatively amplify DNA and detect it using electrophoresis, high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and fluorescence detection equipment. As a general methodology, a sample suspected of harmful microorganisms in the human body and environment is put into a PCR reagent, a pair of primers capable of inducing specific amplification to a DNA sequence to be detected is put in, and then a PCR reaction is carried out do. When the PCR reaction is performed, double-stranded DNA is quantitatively amplified when harmful microorganisms and harmful cells to be detected are present, and when there is no DNA, double-stranded DNA quantitative amplification does not occur. For example, in the case of specific quantitative amplification, in electrophoresis, which is a technique for identifying double-stranded DNA, quantitative amplification can be confirmed by a fluorescence signal at a portion corresponding to a specific molecular weight. However, when specific quantitative amplification does not occur The fluorescence signal can not be confirmed on the portion corresponding to the specific molecular weight on the electrophoresis. However, in the case of the specific primer used for the PCR reaction, due to the nature of a single strand, fluorescence characteristics may or may not appear depending on the fluorescent substance bound to the drug or the primer on the electrophoresis.

The electrophoresis method is a method of physically separating the amplified DNA according to the size of the resultant using charge characteristics, and it is advantageous in that the product can be relatively easily identified using agarose gel.

However, the electrophoresis method requires an apparatus such as an electrophoresis apparatus and a UV illuminator. Therefore, there is a problem that is difficult for non-experts to use. In addition, it is dangerous when UV rays are irradiated to the eyes or skin, and there is a disadvantage of observing the gel using a separate UV protector. In addition, there is a high possibility that pathogens may leak out when handling is inadvertent. Further, since it takes much time to use, there is a problem that it is difficult to apply it in a real environment requiring promptness.

In order to find ways to improve various problems of the electrophoresis method, the inventor who sold out came to a paper. Non-Patent Document 1 discloses that graphene oxide binds well to single-stranded DNA, but its binding force does not reach the binding force between double-stranded DNA.

The present invention has led to the present invention by examining whether or not DNA of an organism can be confirmed easily and quickly at a lower price, without any risk, using the characteristics of graphene oxide.

1. Lu, Chun-Hua, et al. "A graphene platform for sensing biomolecules." 1 of Angewandte Chemie 121.26 (2009): 4879-4881.

The present invention is proposed under the background described above, and proposes a device capable of confirming the DNA of an organism at a simpler and lower cost, and a manufacturing method of the device.

The present invention proposes a device capable of identifying the DNA of an organism without exposure to ultraviolet rays and exposure to pathogens and a method of manufacturing the device.

The present invention proposes a device capable of easily identifying the DNA of an organism with the naked eye and a manufacturing method of the device.

The present invention proposes a device capable of confirming the DNA of an organism to be identified promptly without having to wait for a long period of time for infecting the patient, and a manufacturing method of the device.

The strip separating the DNA according to the present invention includes an introduction part into which a result containing DNA is introduced; A guide connected to the one end by a flow of fluid by the capillary phenomenon and guiding the resultant; An absorption part connected to the other end of the guide part by a fluid flow caused by a capillary phenomenon and absorbing the resultant product; A fixing unit connected to the guide unit to allow the resultant product to pass through and to provide graphene oxide for adsorbing and fixing the first DNA in the DNA; And a detecting unit for detecting a substance passing through the fixing unit. According to the present invention, DNA of an organism can be identified easily and quickly at a lower cost, without any risk.

A method of producing a strip for separating DNA according to the present invention includes the steps of providing a guide part for guiding a result including DNA into a capillary phenomenon and providing a detection part to the guide part; Providing a fixing unit for fixing a single strand DNA in the DNA by fixing graphene oxide to a predetermined pad separately from providing the guide unit; And bringing the fixed part into contact with a place where the capillary phenomenon is cut off inside the guide part, so that the flow of the resultant product passes through the fixed part. According to the present invention, a strip more suitable for the detection of DNA can be easily produced.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily and quickly confirm the DNA of an organism (DNA of a causative organism causing a disease) at a lower cost and without any risk.

1 is a structural view of a strip for separating DNA according to an embodiment;
Figure 2 is an enlarged view of the openings disclosed.
3 is a view for explaining the operation of the embodiment;
4 is a flow chart illustrating a method of manufacturing a strip for detecting DNA;
5 is a graph showing the results of adsorption experiments of graphene oxide.
6 is a view showing the results of adsorption test of zirconium dioxide.
7 is a graph showing the results of single-stranded DNA adsorption test according to the concentration of graphene oxide.
8 is a graph showing the results of adsorption tests according to the DNA concentration and the material of the fixing portion.

Hereinafter, specific embodiments of the present invention will be described in detail with reference to the drawings. However, it will be understood by those skilled in the art that the present invention is not limited to the following embodiments, and that those skilled in the art, upon reading the present disclosure, It will be understood that they are also encompassed within the scope of the present invention.

Before describing the embodiments, various basic matters necessary for understanding the embodiments will be described.

The nucleotide bases of single-stranded DNA and graphene oxide can be adsorbed to the graphene oxide by a pi-pi laminate bond, which is a non-covalent interaction.

The strips of the Examples may be formed by a method in which graphene oxide-fixed pads, graphene oxide pad-coupled strips, and multi-stranded DNAs comprising double strands (hereinafter referred to as double strands, A double stranded DNA binding protein, an interfering drug, and the like) that binds to a target nucleic acid molecule (e.g. When single-stranded DNA and multi-stranded DNA are flowed together into this strip, only single-stranded DNA is adsorbed on the grafted oxide-immobilized pad, and the multi-stranded DNA passes through the graphene oxide pad and the zirconium dioxide nanoparticle or antibody, Double-stranded DNA binding proteins, interfering drugs, and the like. By measuring fluorescence, colorimetry, and color development intensity on the pad, it is possible to determine whether the DNA of an organism is in a sample.

Therefore, the present invention can be applied not only to an apparatus for electrophoresis but also to confirm the amplification of PCR products. Further, by simply burning and controlling the used strip, it is possible to prevent the propagation of pathogens that may occur only in one.

The embodiment amplifies the DAN of an organism by using PCR, and at this time, it performs a role of filtering an artificially added primer as ssDNA. The DNA of an organism amplified by the primer can be used as double-stranded DNA to determine whether DNA of an organism corresponding to the specimen exists. For example, if there is a double-stranded DNA in which a single strand of A and a single strand of B bind to each other in the body of the specimen, a double-stranded DNA in which A and C bind to each other using a primer C capable of amplifying A Amplified. Thereafter, the single strand of the primer and B is immobilized on the pad on which graphene oxide is immobilized, and the double stranded DNA of the amplified A and C is detected through the pad on which the graphene oxide is immobilized have.

In addition, since graphene oxide is well dispersed in water and can easily be surface-functionalized, the strip can be produced at a low cost.

1 is a structural view of a strip for separating DNA according to an embodiment.

Referring to FIG. 1, there are shown an introducing part 1 for introducing a result obtained from a specimen, a first guiding part 2 for guiding the introduced result, a fixing part 3 for fixing a single strand DNA by fixing graphene oxide, A detection unit 5 provided in the second guide unit 4 to detect double stranded DNA and a second guide unit 4 for detecting the double stranded DNA, And an absorption part 6 connected with the introduction part 1 to generate a suction force by capillary phenomenon. The resultant may be amplified after being obtained in a sample, and single-stranded DNA, particles of double-stranded DNA may be contained in the liquid

The interval of the guide portions 2 and 4 in which the fixing portion 3 is placed may be opened to provide the opening portion 31. [ Therefore, it is preferable that all of the products guided along the guide portions 2, 4 pass through the fixing portion 3. The opening 31 may be provided not only in a cut-off form but also in the form of a shielding film capable of blocking fluid flow. So that the flow of the fluid passes through the fixing part 3 rather than the guide part.

The guide portions 2 and 4 may be NC (nitrocellulose) pads. Glass fiber may be used as the material of the fixing portion 3. [ Zirconium dioxide nanoparticles can be used as the detecting portion 5. The detection unit may be, but is not limited to, an antibody or a double-stranded DNA binding protein or an interfering drug.

The function of the strip separating the DNA will be briefly described.

A single strand DNA and a double strand (wherein the double strand may include the concept of multiple strands) DNA is introduced into the introducing section 1 from the sample containing the DNA. The introduced product is moved from the introduction section 1 to the absorption section 6 by the capillary phenomenon occurring between the introduction section 1 and the absorption section 6 in a state in which particles are contained in the liquid. During the movement of the liquid, the single-stranded DNA is adsorbed and fixed on the fixed portion 3, and particles excluding the single-stranded DNA can be continuously moved and detected by the detecting portion 5.

The presence or absence of a substance to be detected can be confirmed in the detecting section 5 by the action described above. Here, the substance detected by the detection unit is preferably a substance amplified by the primer.

2, the liquid reaching the first guide portion 2 does not move directly to the second guide portion 4, and the liquid that reaches the fixed portion 3 Be sure to go. According to this, all of the single-stranded DNA can be fixed by the fixing portion 3.

Fig. 3 shows that, when an indicator exemplified by a fluorescent substance is further provided in the primer, it is confirmed whether DNA of an organism to be visually recognized is present in the specimen.

Fig. 3 (a) shows only single stranded DNA, so that all the particles in the liquid are adsorbed to the fixed portion 3. Fig. In this case, the fluorescent material attached to the primer shows no fluorescence signal due to interaction with graphene oxide present in the fixed portion. Also, in this case, there is no DNA of an organism to be found in the specimen, indicating that amplification using the primer can not be performed.

3 (b) shows that the single-stranded DNA is fixed to the fixed portion 3, but the double-stranded DNA passes through the fixed portion 3 and is detected by the detecting portion 5. [ In this case, the fluorescent substance attached to the primer indicates that the detection unit 5 emits light. Further, in this case, there is DNA of the organism to be found in the specimen, indicating that amplification with the primer has been performed.

4 is a flow chart illustrating a method of producing a strip for detecting DNA.

Referring to FIG. 4, first, the first guide part 2 and the second guide part 4 are provided by cutting the guide part to a predetermined length to form the opening part (S1), and the second guide part (4) is provided with a detection unit (S3). Separately, a pad is provided on which graphene oxide is fixed (S2).

Here, the width of the guide portions 2 and 4 or the length of the open portion 3 may vary depending on various external conditions such as the amount of graphene oxide introduced into the sample. This can be varied depending on various conditions such as the material / density of the open part, the fixing part, the introduction part, and the pad constituting the absorption part.

In the step (S2) of fixing the graphene oxide, a method of dropping 10 μl of the graphene oxide solution on the pad, followed by drying and adsorption, and a method of immersing the pad itself in the graphene oxide solution, followed by drying and adsorption But is not limited thereto. Preferably, 10 μl of the graphene oxide solution is dropped on the pad and dried, followed by adsorption. In one embodiment, pad fabrication to fix graphene oxide can be accomplished by cutting glass fiber 8964 to a size of 3.5 mm x 4 mm, dropping 10 uL of graphene oxide, and drying in a thermostatic chamber at 37 for 15 minutes.

In the step S3 of providing the detection unit, the zirconium dioxide may be fixed to the NC pad. Specifically, the substance that binds to the double-stranded DNA may be an antibody, a double-stranded DNA binding protein, an intervening drug, etc., as well as zirconium dioxide nanoparticles, and preferably zirconium dioxide nanoparticles. no. For example, in the case of various substances other than double-stranded DNA, it is of course possible to use various other substances depending on the substance to be detected.

Specifically, the immobilization of the zirconium dioxide nanoparticles is fixed using a hydrophobic interaction with the NC pad and an electrostatic coupling reaction. In one embodiment, immobilization of the zirconium dioxide nanoparticles may be performed at a temperature of 37 for 10 minutes.

Thereafter, the fixing portion 3 is placed on the opening 31 (S4). More specifically, the fixing portion 3 is placed on the upper side of the opening portion 31 in a state in which both end portions touch the first guide portion 2 and the second guide portion 4, respectively. Thus, the capillary force acts on the contact surface between the fixing portion 3 and the guide portions 2, 4 as it is. Adjustment of the area of the contacting surface and its interface can be changed by various external factors such as the size of the capillary force and the density / size of the pad.

The strip prepared by the above-described method can proceed to DNA detection of an organism by the following procedure.

First, a liquid containing single-stranded DNA is introduced into the lead-in portion of the strip, and is moved using a capillary phenomenon. And the single strand DNA is adsorbed to the fixing portion while the liquid is moving.

The liquid can be amplified with primers added by PCR, and when the single-stranded DNA is present, a large amount of double-stranded DNA can be present in the liquid.

Thereafter, in the state where the single-stranded DNA is removed, the liquid containing the double-stranded DNA further migrates. Zirconium dioxide nanoparticles. The combined double-stranded DNA is exemplified by a fluorescent substance, so that the double-stranded DNA can be precisely detected.

- Experimental Example -

The strips according to the examples were produced in the following manner.

& Lt ; Production of Fixing Member Fixed with Graphene Oxide & gt;

Cut the glass fiber conjugate pad (Grade 8964;) to 3.5mm × 4mm size. The graphene oxide stock solution (763705-100ML; Sigma Aldich, USA) was diluted with DW at 1 mg / ml and sonicated for 30 minutes. Then, 10 ul of 1 mg / ml graphene oxide aqueous solution was dropped on the above-mentioned aliquotted glass fiber conjugate pad, followed by drying in a thermostat for 15 minutes.

≪ Zirconium Dioxide  Immobilization of nanoparticles>

After cutting the 180s NC pad (HF180MC100; Millipore;) to a size of 25mm x 3.5mm, the predetermined interval of the NC pad is removed so as to provide the opening 31 with a size of 1.5mm at the center of the pad. 1ul of zirconium dioxide nanoparticles (US3659; US Research Nanomaterials; US), which was dispersed in DW at a concentration of 2 mg / ml, was injected onto the top of the manufactured second guide portion 4 using a pipette, And dried in a humidifier for 10 minutes.

≪ Fixing part to guide part >

The fixing portion having the graphene oxide fixed to the opening portion was mounted and fixed with a tape. Both the sample pad and the absorbent pad are prepared by being cut into a size of 4 mm × 20 mm.

A strip for DNA detection can be prepared through the above-described process.

Hereinafter, experiments using various methods using strips for DNA detection according to the examples were performed to confirm the effects thereof, and the results are described below.

- Test Example 1-

Images were obtained by using Chemidoc ™ XRS + imaging system (Bio-rad) to confirm single-stranded DNA adsorption and fluorescence signal differences according to the concentration of double-stranded DNA in each of the fixed portion and the detection portion. Fluorescence was then measured using Image lab TM 4.0 software (Bio-rad). Specific experimental methods for each configuration are as follows.

Graphene oxide was prepared by spotting 200 μg / ml graphene oxide on the NC pad 1 μl, 1ul of 2 mg / ml zirconium dioxide nanoparticles was spotted on the upper part of the predetermined distance and dried in a thermostat for 10 minutes. Then, single strand DNA was prepared in DNA hybridization buffer (20 mM Tris-HCl pH 7.4 + 100 mM NaCl + 10 mM KCl + 10 mM MgCl 2) at a concentration of 100 μl per well on a 96-well plate and graphene oxide was spotted on the membrane .

As a result, as shown in FIG. 5, it was shown that single-stranded DNA having a concentration of 2 nM or less was adsorbed on graphene oxide and no fluorescence signal was seen.

For zirconium dioxide nanoparticles, 1 ul of 2 mg / ml zirconium dioxide nanoparticles was spotted onto the membrane and then dried in a constant-temperature air humidifier for 10 minutes. The single strand DNA probe (primer) and the target single strand DNA (target organism DNA) were mixed in a DNA hybridization buffer at a ratio of 1: 1 for each concentration and reacted at room temperature for 30 minutes. The reacted solution was loaded on a 96-well plate in an amount of 100 μl, and a membrane was spotted with zirconium dioxide nanoparticles spotted thereon.

 As a result, as shown in Fig. 6, it was confirmed that fluorescence signals of double-stranded DNA of 0 nM at 100 nM could be distinguished.

- Test Example 2-

In order to confirm the range of adsorption concentration of single stranded DNA depending on the concentration of graphene oxide immobilized on the glass fiber, experiments were performed to measure fluorescence by the same method. Specific experimental methods are as follows.

A 180s NC pad was made to have a size of 3.5 mm x 25 mm and an opening 31 having a length of 1.5 mm was provided at an intermediate portion of the guide as shown in Fig. 1 2 of 2 mg / ml zirconium dioxide nanoparticles was spotted on the prepared broken-down guide, and then dried in a thermostat for 10 minutes. Glass fiber 8964 was cut into a size of 3.5 mm x 4 mm and 10ul of graphene oxide having a concentration of 500ug / ml and 10ul of graphene oxide having a concentration of 1mg / ml were loaded on the pad, followed by drying in a thermostat for 15 minutes. 10ul of graphene oxide having a concentration of 1mg / ml was loaded on one side, and graphen oxide having the same concentration and volume was further loaded on the back side of the dried pad and dried.

A sample was prepared in the same manner as in the adsorption experiment (Test Example 1), and 120ul each was loaded on a 96-well plate to bind the pad fixed with graphene oxide. The guide was inserted.

As a result, as shown in FIG. 7, in the case of a pad in which 10 μl of graphene oxide having a concentration of 500 ug / ml was immobilized, a single strand DNA probe (primer) having a concentration of 10 nM or less was adsorbed on graphene oxide, / ml, the single stranded DNA probe (primer) at a concentration of 100 nM or less was adsorbed to the graphene oxide and no fluorescence signal was observed. In addition, a single-stranded DNA probe with a concentration of 200 nM or less was adsorbed on graphene oxide, indicating that no fluorescence signal was observed in the pads fixed with 10 ul of graphene oxide having a concentration of 1 mg / ml on both sides.

- Test Example 3-

Using the strip prepared in Test Example 2, a fluorescence signal corresponding to the concentration of a single strand DNA probe (primer) and a complementary target single strand DNA (DNA of an organism to be detected) was confirmed, and according to the type of graphene oxide fixing pad In order to confirm the detection efficiency, fluorescence was measured by the same method as the above experiment. Specific experimental methods are as follows.

To the solution containing the sample solution, ie, 100 nM single-stranded DNA probe, the target single-stranded DNA complementary to the probe DNA is added at each concentration and hybridized at room temperature for 30 minutes or more. The hybridized sample solution was loaded on a 96-well plate in an amount of 120 l each, and a strip made of a glass fiber conjugate pad (Grade 8964; and Fusion 5) The structure was inserted into a 96-well plate containing the sample solution.

As a result, as shown in FIG. 8A, when the graphene oxide was immobilized on the glass fiber conjugate pad, it was confirmed that the target single strand DNA concentration in the range of 1 nM to 100 nM was detected when compared with the sample without the target single strand DNA . On the other hand, when graphene oxide was immobilized on the Fusion 5 pad, the target single strand DNA concentration of 10 nM or more could be detected as shown in FIG. 8B, and the linearity of the fluorescence signal according to the concentration was not better than that of the glass fiber pad Respectively.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily and quickly confirm the DNA of an organism (in particular, a DNA of a causative organism causing disease) at a lower cost and without any risk.

3:
31:
5:

Claims (10)

An introduction part into which a result containing DNA is introduced;
A guide connected to the one end by a flow of fluid by the capillary phenomenon and guiding the resultant;
An absorption part connected to the other end of the guide part by a fluid flow caused by a capillary phenomenon and absorbing the resultant product;
A fixing unit connected to the guide unit to allow the resultant product to pass through and to provide graphene oxide for adsorbing and fixing the first DNA in the DNA; And
And a detection unit for detecting a substance passing through the fixing unit,
The guide portion includes an opening portion that is opened to guide the resultant to the fixing portion,
The opening portion is provided in a shielding film shape
strip. The method according to claim 1,
The DNA is further provided with an indicator, and the result is a PCR-treated strip. delete delete The method according to claim 1,
Wherein the first DNA is a single stranded DNA and the material that has passed through the anchoring portion comprises a double stranded DNA. 6. The method of claim 5,
Wherein the single stranded DNA is a primer. The method according to claim 1,
The guide portion is a NC pad material,
Wherein the fixing portion is made of a glass fiber material,
Wherein at least one of the zirconium dioxide or antibody, the double stranded DNA binding protein, and the interfering drug is immobilized on the detection portion. delete delete delete

Images