KR20160015079A - Target sensing method and apparatus based on DNA barcode - Google Patents

Abstract

Provided is a DNA barcode-based target material detection method which comprises the following steps: mixing a magnetic particle bonded with a first probe material, and a nano/microparticle bonded with both a second probe material and DNA along with a target material; forming a composite of the magnetic particle-target material-nano/microparticle based on a first bond between the first probe material and the target material and a second bond between the second probe material and the target material; and determining a kind of the target material depending on a base pair (bp) of DNA bonded onto the nano/microparticle in the formed composite of the magnetic particle-target material-nano/microparticle. The first probe material and the second probe material can bind to the same kind of the target material. According to the present invention, the method enables the detection of various kinds of target materials simultaneously.

Description

[0001] The present invention relates to a DNA barcode-based target substance detection method and apparatus,

The present invention relates to a DNA barcode-based target substance detection method and apparatus, and more particularly, to a DNA barcode-based target substance detection method and apparatus capable of rapidly discriminating and diagnosing a plurality of kinds of target substances at high speed and high sensitivity on the spot by controlling DNA length and base sequence. And more particularly, to a method and apparatus for detecting a barcode-based target material.

The DNA bar code technology is a technique in which gold nanoparticles composed of magnetic particles coated with a monoclonal probe material, a polyclonal probe material and bar coding DNA react with a specific target material protein to form magnetic particles, To form a complex composed of nanoparticles. For example, when blood is introduced into this device, only the target target substance protein forms a complex, and unnecessary proteins and the like are removed while the complex is magnetically fixed.

However, the conventional DNA barcode apparatus and method disclosed in Korean Patent Laid-Open No. 10-2009-0123720 are all for detecting a specific target substance, and there is a problem that it is difficult to detect various kinds of target substances, There is a problem that it is difficult to immediately apply the above-described DNA barcode technology of the prior art to an on-site integrated biosensor system to identify a rapid and accurate pathogen.

Accordingly, the present invention provides a new method of DNA barcode method and apparatus capable of simultaneously detecting various kinds of target substances.

According to an aspect of the present invention, there is provided a method for manufacturing a magnetic sensor, comprising: mixing magnetic particles having a first probe material bonded thereto and nano / microparticles having a second probe material and DNA bonded at the same time together with a target material; Forming a magnetic particle-target material-nano / microparticle complex according to a first bond between the first probe material and the target material and a second bond between the second probe material and the target material; Determining the type of the target material according to bp of the DNA bound to the nano / microparticle of the formed magnetic particle-target material-nano / microparticle complex, wherein the first probe material and the second probe The present invention provides a method for detecting a DNA barcode-based target substance, characterized in that the substance can be bound to the same kind of target substance.

In one embodiment of the present invention, the DNA is double-stranded DNA, wherein one strand is biotin and the other strand is a fluorescent marker.

In one embodiment of the present invention, the first probe material and the second probe material are of a plurality of types, and the plurality of types of the first probe material and the second probe material are mixed together with the target material.

In one embodiment of the present invention, the DNA barcode-based target material detection method further comprises separating the magnetic particle-target material-nanoparticle composite using a magnetic force, The step of separating the target material-nanoparticle complex proceeds in such a manner that the magnetic particle-target material-nanoparticle complex is fixed using a magnetic force.

In one embodiment of the present invention, at least one of the probe substance and the target substance is an antibody and the other is an antigen.

In one embodiment of the present invention, the bp of the DNA is determined from an optical signal from a fluorescent marker bound to the DNA.

In one embodiment of the present invention, the step of determining the type of the target substance comprises the steps of: detecting a first fluorescent signal from a fluorescent marker bound to a first reference DNA; Detecting a fluorescent signal from the fluorescent marker bound to the target DNA bound to the nano / microparticle; Detecting a second fluorescent signal from a fluorescent marker coupled to a second reference DNA, wherein, for a time difference between the first reference fluorescent signal and the second fluorescent signal, the first reference fluorescent signal and the fluorescent marker Lt; RTI ID = 0.0 > DNA < / RTI >

In one embodiment of the present invention, the first reference DNA has a bp shorter than the bp of the target DNA.

In one embodiment of the present invention, the second reference DNA has a bp longer than the bp of the target DNA.

In one embodiment of the present invention, the number of bp of the DNA varies depending on the type of the target substance, and the detection time of the fluorescence signal varies depending on the bp of the DNA.

An object of the present invention is to provide a DNA barcode-based target material detection device, which comprises a magnetic particle to which a first probe material is bound, a nano / microparticle to which DNA is bound simultaneously with a second probe material, A passive mixer in which magnetic particles, nano / microparticles and target material injected from the pump layer flow, thereby forming a complex of a first probe material-target material-second probe material; A magnetic field separation chamber capable of separating the composite discharged from the passive mixer and separating the composite; A capillary electrophoresis channel connected to the magnetic field separation chamber; And an electrode unit provided at both ends of the capillary electrophoresis channel to perform electrophoresis in the capillary channel.

In an embodiment of the present invention, the passive mixer and the magnetic field separation chamber are provided in a layer layer provided below the pump layer.

In one embodiment of the present invention, the apparatus further comprises detecting means provided below the capillary channel for detecting DNA of the capillary channel, wherein the detecting means comprises means for detecting a fluorescent marker Optical means.

The present invention provides an integrated DNA barcode-based target material detection device including the aforementioned DNA barcode-based target material detection device.

In one embodiment of the present invention, the integrated device is comprised of a plurality of layers layers joined together.

The present invention uses a magnetic particle to which a first probe substance is bound and nano / microparticles to which a second probe substance and DNA are bound at the same time, and controls a plurality of kinds of target substances at high speed and high sensitivity It is possible to quickly distinguish and diagnose in the field.

1 is a diagram illustrating a method of detecting a DNA barcode-based target material according to an embodiment of the present invention.
FIGS. 2 and 3 are views for explaining the principle of fabricating nano / microparticles in which a first probe material is bonded to a magnetic particle and a second probe material is bonded according to an embodiment of the present invention.
4 is a diagram illustrating the principle that a complex is formed according to a specific binding of a target substance (antigen, 210)) and an antibody bound to magnetic particles and nano / microparticles according to an embodiment of the present invention.
5 is a diagram illustrating a DNA barcode-based target substance detection method for various kinds of target substances.
6 is a schematic diagram showing a target substance detection method according to an embodiment of the present invention.
7 is a view for explaining a process of separating barcode DNA according to capillary electrophoresis (CE) in an apparatus for multi-bio-bar code according to an embodiment of the present invention.
FIG. 8 is a block diagram of a multi-bio-bar code method according to an embodiment of the present invention.
FIG. 9 is a diagram for explaining a method for detecting multiple types of pathogens in accordance with an embodiment of the present invention.
10 is a result of a DNA complex separation test according to an embodiment of the present invention.
11 to 15 are schematic diagrams of a DNA barcode-based target material detection device, according to an embodiment of the present invention.

For the embodiments of the present invention disclosed herein, specific structural and functional descriptions are merely illustrative for purposes of illustrating embodiments of the present invention, and embodiments of the present invention may be embodied in various forms, The present invention should not be construed as limited to the embodiments described in Figs.

The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the text. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed, but on the contrary, is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

When an element is referred to as being "connected" or "coupled" to another element, it may be directly connected or connected to the other element, but other elements may be present in the middle It should be understood. Other expressions that describe the relationship between components, such as "between" and "between" or "neighboring to" and "directly adjacent to" should be interpreted as well.

The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprise", "having", and the like are intended to specify the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, , Steps, operations, components, parts, or combinations thereof, as a matter of principle.

Also, the terms " part, "" module, "" module "," block ", and the like described in the specification mean units for processing at least one function or operation, Lt; / RTI >

In order to solve the above-mentioned problem, the present invention uses magnetic particles having a first probe material bonded thereto and nanoparticles / microparticles having a second probe material and DNA bonded at the same time, Type target substances can be quickly discriminated and diagnosed on the spot at high speed and high sensitivity.

1 is a diagram illustrating a method of detecting a DNA barcode-based target material according to an embodiment of the present invention.

Referring to FIG. 1, a method for detecting a DNA barcode-based target material according to an embodiment of the present invention includes detecting a magnetic particle to which a first probe material is bound and a nano / Mixing with the target material; Forming a magnetic particle-target material-nano / microparticle complex according to a first bond between the first probe material and the target material and a second bond between the second probe material and the target material; And determining the type of the target material according to bp of the DNA bound to the nano / microparticle of the formed magnetic particle-target material-nano / microparticle composite. In the present invention, the nano / microparticle refers to nanometer or micrometer-unit particles in which a second probe material and DNA are simultaneously bonded.

In one embodiment of the present invention, the first probe material and the second probe material may be bonded to the same kind of target material. When the target material binds to the first probe material and the second probe material, the magnetic particles and the nano / The microparticles form a complex by using the target substance as a linking agent.

FIGS. 2 and 3 are views for explaining the principle of fabricating nano / microparticles in which a first probe material is bonded to a magnetic particle and a second probe material is bonded according to an embodiment of the present invention.

Referring to FIG. 2, in one embodiment of the present invention, magnetic particles 110 made of a magnetic material and having streptavidin bonded to its surface are shown. (First probe substance) 120 is bound to streptavidin on the surface of the magnetic particle, and a magnetic particle capable of specifically binding to the target substance can be bound to the target substance Is completed.

Referring to FIG. 3, nano / microparticles 130 with streptavidin bound to the surface are shown. In one embodiment of the present invention, the nano / microparticle 130 is a polystyrene particle, and the biotin-linked antibody (second probe material 140) is connected to the nano / microparticle surface streptavidin. The second probe material 140 and the barcode DNA 150 are bonded to the surface of the nano / microparticle 130 at the same time on the surface of the nano / .

In one embodiment of the present invention, it is possible to determine whether the target substance is bound through the bp length of the barcode DNA. To this end, the DNA in the embodiment of the present invention is a double stranded DNA, Biotin is attached to one strand and fluorescent marker is attached to the other strand.

4 is a diagram illustrating the principle that a complex is formed according to a specific binding of a target substance (antigen, 210)) and an antibody bound to magnetic particles and nano / microparticles according to an embodiment of the present invention.

4, the target substance 210, which is an antigen, is bound to an antibody (a first probe substance 120 and another antibody (second probe substance 140) bound to nano / In this embodiment, the bp of the DNA (bar code DNA) bound to the surface of the nano / microparticle is determined according to the target substance The number of barcode DNA complexes is different when the bar code DNA complex is separated by the capillary electrophoresis (CE) process.

5 is a diagram illustrating a DNA barcode-based target substance detection method for various kinds of target substances.

Referring to FIG. 5, there are shown five types of pathogens (target material) and magnetic and nano / micro particles to which probes corresponding to the pathogens are combined. When mixing the target substance with magnetic and nano / microparticles, magnetic particles, nano / microparticles having an antibody (probe) specifically binding to the pathogen are bound to pathogenic bacteria to form a complex. At this time, the barcode DNAs bound to the nano / microparticles have different lengths, so that the detection sequence is changed by a capillary electrophoresis process, and thus multiple detection of a plurality of target substances becomes possible.

6 is a view for explaining a composite separation and a barcode DNA dissociation method according to an embodiment of the present invention.

Referring to FIG. 6, nano / microparticles bonded with barcode DNA determined by DNA length are combined with a magnetic particle-target material to form a complex. The complex can then be fixed and purified by an externally applied magnetic field. At this time, the single-stranded DNA labeled with the fluorescent marker is dissociated and released by applying heat, and analysis of the single-stranded DNA enables determination of whether or not the target substance is present.

In particular, instead of detecting the pathogen itself, the present invention can detect a pathogen by using a plurality of barcode DNAs, which can be specifically bound to the pathogen, have a greater number of pathogens and are detected with a high probability, The detection probability of the substance is remarkably increased, and the sensing sensitivity can be greatly improved.

In addition, the present invention can simultaneously detect a plurality of types of target materials. In this case, the first probe material and the second probe material may be of a plurality of types depending on the type of the target material.

 FIG. 7 is a view for explaining a process of separating bar code DNA dissociated according to capillary electrophoresis (CE) in an apparatus for multi-bio-bar code according to an embodiment of the present invention.

Referring to FIG. 7, a composite is fixed to a channel by a magnetic force (for example, a magnetic force generated by a magnet provided under the channel), and heat is applied to allow only the bar code DNA to be separated from the composite. This is based on the principle of separating single-stranded DNA by applying heat to the double-stranded DNA. Then, the separated bar-code DNA is subjected to capillary electrophoresis.

Referring to FIG. 7, a cathode 320 and an anode 330 formed on a single plate 310 and a capillary 340 provided between the cathode 320 and the anode 330 are provided. The composite sample flows into the capillary tube 340 and the barcode DNA flows into the capillary tube by an electrical force applied between the cathode 320 and the anode 330. At this time, the length of the bar code DNA is determined from the detection time of the optical signal from the fluorescent marker bonded to the bar code DNA, and the type of the target substance is determined accordingly.

Thus, under the single plate according to an embodiment of the present invention, detection means 350 capable of optically detecting fluorescent markers is provided.

That is, according to the present invention, the short-length bar-code DNA flows more rapidly than the long-length bar-code DNA, so that the plural kinds of complexes have a detection order that is distinguished from each other according to the DNA length.

In order to more clearly identify the detection peak, in one embodiment of the present invention, the DNA of the bp (first reference DNA) shorter than the bp of the DNA corresponding to the actual target substance (the first reference DNA) bp DNA (second reference DNA) is used.

FIG. 8 is a block diagram of a multi-bio-bar code method according to an embodiment of the present invention.

Referring to FIG. 8, a first fluorescent signal is detected from a fluorescent marker coupled to a first reference DNA. In this case, the first reference DNA has a small bp smaller than the bp of the DNA (second probe) bound to the nano / microparticle due to the specific binding with the actual target substance, It is detected the earliest.

Fluorescence signals are then detected from the fluorescent markers bound to the target DNA bound to the nano / microparticles upon binding to the target material. If the target substance is not present, the magnetic particles do not bind to the nano / micro particle, so that the nano / micro particle bound to the target DNA is removed during the separation and purification by the magnetic field.

Thereafter, the second fluorescent signal is detected from the fluorescent marker coupled to the second reference DNA. The second reference DNA has a bp greater than the bp of the DNA bound to the nano / microparticle. The present invention is characterized in that the time difference of the fluorescence signal of the target substance detected between the first reference fluorescence signal and the second fluorescence signal And the corresponding DNA and the target substance are determined.

FIG. 9 is a diagram for explaining a method for detecting multiple types of pathogens in accordance with an embodiment of the present invention.

Referring to FIG. 9, a 15 bp DNA was used as a first reference DNA, a 45 bp DNA was used as a second reference DNA, and a DNA of a probe substance binding to pathogens was inserted between a first reference DNA and a second reference DNA Belongs. That is, the present invention designs barcode DNAs (20, 25, 30, 35, and 40 bp) having different lengths for each pathogen and uses a reference DNA to detect which pathogen the corresponding peak corresponds to do.

10 is a result of bar code DNA separation experiment according to an embodiment of the present invention.

)을 통해 검출된 바코드 DNA를 정확히 규명할 수 있으며, 이로써 해당 피크가 어떤 병원체인지 알 수 있었다. 10, in this embodiment, the bar code DNAs 20, 25, 30, 35, 40, and bp having different lengths are separated and two types of reference DNAs (Bracket ladder (15,45 bp) The ratio of the time difference of peaks (time difference ratio =

) Can be accurately identified, and thus the peak can be identified as a hospital.

Normally, when the peak position is determined based on the absolute elution time, since CE, the state of the gel, the temperature, the heating time, and the experimenter are changed, the absolute elution time is changed. Analytical possibilities exist. However, since the present invention calculates bp of the target substance bar code DNA at a peak time difference ratio using a reference DNA (bracket ladder), it has an advantage that peak position analysis can be accurately performed regardless of the CE environment.

The present invention provides an apparatus for performing the above-described method in one device.

To this end, there is provided an integrated chip device capable of collecting a composite containing magnetic particles by an external magnet, applying heat to dissociate the barcode DNA, and then detecting it by capillary electrophoresis.

11 to 15 are schematic diagrams of a DNA barcode-based target material detection device, according to an embodiment of the present invention.

11, a DNA barcode-based target material detecting device according to an embodiment of the present invention includes a micro pump 410 for automatically injecting a sample and a probe material, a passive mixer 410 for forming a complex of a target material and a probe, (420); A magnetic field separation chamber 430 for separation and purification of the first probe-target material-second probe complex; And a capillary electrophoresis channel 440 for analyzing the dissociated barcode DNA.

12, the chip includes an upper layer (a pump layer) having a pathogen inlet and a probe injection port, and is connected to the target material solution through the probe inlet and the pathogen inlet by an external pump (three-valve pump system) The probe particle solution is injected into the chip.

A PDMS membrane (not shown) capable of flowing up and down by the air applied by the external pump is provided below the pump layer of FIG. 12, and the PDMS membrane flows up and down according to the air pressure by the pump, The target substance solution and the probe particle solution migrate to the layer layer of the target substance solution.

Referring to FIG. 13, the target material solution and the probe particle solution moved to the layer layer below the pump layer through a via hole connected to the pump layer pass through the passive mixer 420 provided in the lower layer layer , And the magnetic particle-target material-nano / micro particle composite is formed in the passive mixer 420. In an embodiment of the present invention, a circular column is provided in the channel of the passive mixer 420 to promote mixing of particles.

Referring to FIG. 14, the composite in the mixed solution passing through the passive mixer 420 is collected in the separation chamber 430 to which magnetic force by the permanent magnet is applied. At this time, permanent magnets are provided above or below the separation chamber 430, so that the magnetic particles of the composite are attracted to the magnet and fixed in the separation chamber 430. As a result, materials other than the complex (noncomplexed pathogens, polystyrene probes, etc.) can not be attached to the magnet and thus are washed out through the outlet.

Referring to FIG. 15, a capillary suction channel 440 connected to the separation chamber 430 is opened, and the capillary suction channel 440 is filled with a gel. If the composite is all under the magnet, the cathode and anode are filled with TTE buffer and heat is applied to the composite through a heater. That is, the barcode DNA is dissociated from the complex by keeping the temperature at 70 ° C through the heater provided below the square portion in FIG. 15, and then when the voltage is applied to the cathode and the anode, the barcode DNA is electrophoresed , The target substance can be effectively detected by optically analyzing the electrophoretic barcode DNA.

As described above, the DNA barcode-based target material detecting device including the DNA barcode-based target material detecting device according to the present invention can vertically combine a plurality of layer layers performing independent functions to continuously detect DNA barcode- Based target material detection process can be performed, so that the target material can be detected with high sensitivity, multiple, and high speed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art.

Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (17)

Magnetic particles having the first probe material bonded thereto, and nano / micro particles having the DNA and the second probe material simultaneously bonded together with the target material;
Forming a magnetic particle-target material-nano / microparticle complex according to a first bond between the first probe material and the target material and a second bond between the second probe material and the target material;
Determining the type of the target material according to bp of the DNA bound to the nano / microparticle of the formed magnetic particle-target material-nano / microparticle complex, wherein the first probe material and the second probe Wherein the substance is capable of binding to the same kind of target substance. The method according to claim 1,
Wherein the DNA is double-stranded DNA, wherein one strand is bound to biotin and the other strand is bound to a fluorescent marker. The method according to claim 1,
A method for detecting a target substance based on a DNA barcode, characterized in that a plurality of types of the first probe material and a second probe material are used, and the plurality of types of first probe material and second probe material are mixed together with the target material . 4. The method of claim 3, wherein the DNA barcode-
A method for detecting a target substance based on DNA barcode, characterized by further comprising the step of separating the magnetic particle-target material-nanoparticle complex using a magnetic force. 5. The method of claim 4,
Wherein the step of separating the magnetic particle-target material-nanoparticle composite using the magnetic force is performed by fixing the magnetic particle-target material-nanoparticle composite by using a magnetic force. A method for detecting a target substance. The method according to claim 1,
Wherein at least one of the probe material and the target material is an antibody and the other is an antigen. 3. The method of claim 2,
Wherein the bp of the DNA is determined from an optical signal from a fluorescent marker bound to the DNA. 7. The method of claim 6, wherein the type of the target material is determined by:
Detecting a first fluorescent signal from a fluorescent marker coupled to a first reference DNA
Detecting a fluorescent signal from the fluorescent marker bound to the target DNA bound to the nano / microparticle;
Detecting a second fluorescent signal from a fluorescent marker coupled to a second reference DNA,
Wherein a target material corresponding to a time difference ratio between the first reference fluorescence signal and DNA to which the fluorescence marker is bound is determined with respect to a time difference between the first reference fluorescence signal and the second fluorescence signal. DNA barcode-based target substance detection method. 9. The method of claim 8,
Wherein the first reference DNA has a bp shorter than the bp of the target DNA. 9. The method of claim 8,
Wherein the second reference DNA has a bp longer than the bp of the target DNA. 10. The method of claim 9,
Wherein the number of bp of the DNA varies depending on the type of the target substance and the detection time of the fluorescent signal varies depending on bp of the DNA. A DNA barcode-based target material detection apparatus,
A magnetic particle to which the first probe material is bound, a nano / microparticle to which the second probe material and the DNA are bound at the same time, and a pump layer into which the target material is injected;
A passive mixer in which magnetic particles, nano / microparticles and target material injected from the pump layer flow, thereby forming a complex of a first probe material-target material-second probe material;
A magnetic field separation chamber capable of separating the composite discharged from the passive mixer and separating the composite;
A capillary electrophoresis channel connected to the magnetic field separation chamber; And
And an electrode unit provided at both ends of the capillary electrophoresis channel to perform electrophoresis in the capillary channel. 13. The method of claim 12,
Wherein the passive mixer and the magnetic field separation chamber are provided in a layer layer provided below the pump layer. 14. The method of claim 13,
Further comprising detection means provided below the capillary channel for detecting DNA of the capillary channel. 15. The method of claim 14,
Wherein the detecting means is an optical means for detecting a fluorescent marker coupled to the DNA. An integrated DNA barcode-based target material detection device comprising a DNA barcode-based target material detection device according to any one of claims 12 to 15. 17. The method of claim 16,
Wherein the integrated device comprises a plurality of layers of mutually coupled layers.

Images