WO2018066979A1

WO2018066979A1 - Apparatus for nanoparticle detection and method for nanoparticle detection using same

Info

Publication number: WO2018066979A1
Application number: PCT/KR2017/011136
Authority: WO
Inventors: 조윤경; 우현경; 순카라비자야
Original assignee: 울산과학기술원
Priority date: 2016-10-07
Filing date: 2017-10-10
Publication date: 2018-04-12
Also published as: KR20180039005A; KR102013819B1

Abstract

The present invention provides an apparatus for nanoparticle detection, which integrates the complete process of nanoparticle detection from a sample so as to be more convenient to use, the apparatus comprising: a disk for transferring a fluid by a centrifugal force; a sample accommodating part, formed on the disk, for accommodating a sample; a filter part connected to the sample accommodating part and having a microfiltration membrane for filtering the transferred sample to separate the nanoparticles; a supply part, connected to the filter part, for supplying a detection liquid for detecting the nanoparticles separated by the filtration membrane; a waste liquid receiving part, connected to an output side of the filter part, for receiving a solution that has passed through the filtration membrane; a flow path, formed on the disk, for transferring the fluid; and a valve for selectively opening or closing the flow path.

Description

【Specification】

[Name of invention]

Nanoparticle Detection Device and Nanoparticle Detection Method Using the Same

Technical Field

The present invention relates to a nanoparticle detection apparatus and a nanoparticle detection method using the same.

[Technique to become background of invention]

For example, nano endoplasmic reticulum is a small 40-120 nm endoplasmic reticulum generated from cellular activity. Nanovesicles are distinguished from other vesicles by their source and size. Nanovesicles were considered to be cellular byproducts at the time of discovery, but their importance was found to contribute to cellular activities such as tumor progression and metastasis and cellular signal transduction. Nanovesicles are present in almost every body fluid in the body and contain the genetic information of the cells from which they are derived. For this reason, nano vesicles are attracting attention as new drug delivery systems as well as new markers for various diseases including cancer.

Recently, research on the method of separating and detecting nano vesicles has been continuously increasing. Methods for separating nano vesicles are classified into density, size, and af f ini ty.

Methods of detecting specific proteins or nucleic acids of nano vesicles after separating and capturing nano vesicles include protein analysis comparing expression levels of specific antigens and nucleic acid analysis analyzing genetic information of generated cells. ELISA (Enzyme-1 inked i 隱 unosorbent assay) using a 96 wel l plate, which is a conventional protein analysis method, is a method of detecting a specific antibody or protein specific nano vesicles on a plate and then detecting them using other antibodies. . However, in the conventional method, there is a limitation in the surface area of the 96 wel l plate attaching the nano endoplasmic reticulum, and it is difficult to analyze the antibody adjacent to the specific area of the nano vesicle after it is attached to the plate.

[Contents of the Invention]

Problem to be solved

The whole process of detecting nanoparticles from a sample is integrated The present invention provides a nanoparticle detection device and a nanoparticle detection method using the same.

Provided are a nanoparticle detection device capable of performing detection of a plurality of antigens and a nanoparticle detection method using the same.

Provided are a nanoparticle detection device capable of more accurate detection and analysis, and a nanoparticle detection method using the same.

[Measures of problem]

The detection device of the present embodiment is a disk for transferring fluid by centrifugal force, a sample accommodating part formed on the disk to accommodate a sample, and a fine particle for separating nanoparticles by filtering a sample transported and conveyed to the sample accommodating part. A filter unit having a filtration membrane, a supply unit connected to the filter unit for supplying a detection liquid for detecting nanoparticles separated from the filtration membrane, a waste solution receiving unit connected to the filter unit outlet side and accommodating a solution having passed through the filtration membrane, to the disk It may include a flow path is formed and the fluid is transferred, and a valve for selectively opening and closing the flow path.

The sample accommodating part may include a sample chamber formed on the disk to accommodate a sample, connecting the sample chamber to the filter part, and having a flow path for transferring the sample according to the disc centrifugal force, and opening and closing the first flow path. Can be. ·

The sample chamber has a centrifugal separation of the sample according to the disc centrifugal force, and a settling portion for accommodating the centrifuged sample is elongated at the distal end of the disc along the centrifugal force direction, and the first flow path is directed toward the center of rotation of the disc. The supernatant centrifuged by being connected to the settling boundary of the sample chamber may be transferred to the filter unit.

The settled portion may be formed to be inclined with respect to the radial direction of the disk.

The settling portion may gradually incline the bottom surface toward the end in the centrifugal force direction at the boundary point.

It may further include a groove portion formed at the end of the settling portion to accommodate the impurities centrifuged from the sample. The discharge chamber may further include a discharge passage configured to be connected to the settling portion exit side of the sample chamber and the waste liquid receiving portion to transfer the settled solution, and a discharge valve to open and close the discharge passage.

The filter part includes a filter chamber having an entrance space and an exit space along a fluid movement direction, and a filter chamber in which the filtration membrane is installed between the entrance space and the exit space, and the entrance space is connected to the sample accommodating part, and the sample is introduced and filtered. Particles are accommodated, and the exit space may be connected to the waste liquid receiving portion. The filtration membrane may be formed with pores of lnm to 100m.

The filtration membrane may be made of polycarbonate, polystyrene ^' , polymethyl methacrylate, a thermosetting plastic including cyclic olefin polymers, anodized aluminum nickel, or silicon. The filter unit may have at least two filtration membranes stacked between the entry space and the exit space, and the pores may gradually decrease in the fluid transport direction.

The filter unit may be sequentially disposed along at least two fluid transfer directions, and the filtration membranes provided in each filter unit may have different pore sizes from each other to separate nanoparticles having different size ranges.

At least two or more filter units may be sequentially disposed along the fluid transfer direction, and the filtration membranes provided in each filter unit may gradually decrease pores along the fluid transfer direction.

The filtration membrane may be installed detachably from the disk.

A cover detachably installed on the disk may further include a cover configured to open and close the filter chamber, and a fastening part configured to fix the cover to the disk.

The supply unit is formed in the disk and the antibody chamber for receiving the antibody provided for nanoparticle detection, the second flow path for connecting the antibody chamber and the filter unit and transfer the antibody to the filter unit in accordance with the centrifugal force of the disk, the second flow path It may include a second valve for opening and closing.

The supply unit includes a reagent chamber containing a reagent formed on the disk for labeling an antibody for detecting nanoparticles, the reagent chamber and a filter unit. It may include three valves for connecting and transferring the reagent to the filter unit according to the centrifugal force of the disk, and three valves for opening and closing the third channel.

The supply part is formed in the disk and the substrate liquid chamber for receiving the substrate liquid provided for the detection of nanoparticles, the fourth flow path for connecting the substrate liquid chamber and the filter unit and transfer the substrate liquid to the filter unit in accordance with the centrifugal force of the disk, The crab may further include four valves for opening and closing the four euros.

The supply part is a supply part is formed in the disc is a stop solution chamber for receiving a stop solution (stop solut ion) provided to stop the nanoparticle detection reaction, a connecting flow path for connecting the stop solution chamber and the substrate liquid chamber, and the connection Including a connecting valve for opening and closing the flow path, the stop solution can be supplied to the filter chamber via the substrate liquid chamber.

The supply unit may further include a washing unit for cleaning the filter unit by transferring the washing liquid to the filter unit.

The washing unit includes a washing liquid chamber formed in the disk to receive the washing liquid, a fifth flow passage connecting the washing liquid chamber to the filter unit and transferring the washing liquid to the filter unit according to the centrifugal force of the disk, and opening and closing the fifth passage. can do.

The washing liquid chamber may be divided into a plurality of washing liquids, and the washing liquid is separately received in each washing liquid chamber, and an outlet valve for discharging the washing liquid may be installed in the outlet flow path of each washing liquid chamber.

The waste liquid receiving part may include a waste fluid chamber formed on the disk to connect the waste liquid chamber and the filter unit to accommodate the waste liquid, and transfer the waste liquid to the waste liquid chamber according to the centrifugal force of the disk.

A recovery chamber formed in the disk and connected to the filter part to recover the nanoparticles separated by the filtration membrane, a seven-channel flow path connecting the recovery chamber and the filter part to transfer the nanoparticles to the recovery chamber, and opening and closing the seven-channel flow path A seventh valve may be further included.

The sample may be an aqueous solution containing a biological sample or nanoparticles selected from blood, lymph, tissue fluid, urine, saliva cerebrospinal fluid, and sputum including bioparticles, or a combination thereof. The sample accommodating part, the filter part, the supply part, and the waste liquid receiving part constitute one unit, and the unit may be arranged at a plurality of intervals along the circumferential direction of the disc.

The flow path connecting two neighboring rambers is located in the disk center side rather than the outlet, the inlet may be connected to the outlet side of the one chamber along the disc centrifugal force direction.

The disc may be surface modified with proteins, polymers, or organic molecules to prevent nonspecific antibody attachment.

Nanoparticle detection method of the present embodiment, the step of supplying a sample to the disk, centrifugal force applied to the disk to transfer the sample to the filter portion and filtering through a filtration membrane to separate the nanoparticles, applying a centrifugal force to the disk for nanoparticle detection Supplying a detection liquid to the filter unit may include detecting the nanoparticles on the filtration membrane.

Detecting the nanoparticles, applying a centrifugal force to the disk to the antibody. The nanoparticles may be supplied to the filter unit to detect the nanoparticles, and the reagents may be supplied to the filter unit to label the antibodies attached to the nanoparticles.

Before the sample is transferred to the filter unit, a separation step of centrifuging the sample by applying a centrifugal force to the disk may be further included.

The separating of the nanoparticles may separate nanoparticles of different size ranges through a plurality of different filtration membranes having different pore sizes.

The separating of the nanoparticles may sequentially separate nanoparticles having different size ranges through a plurality of filtration membranes in which pores gradually decrease.

Applying a centrifugal force to the disk may further comprise the step of supplying the washing liquid to the filter unit to wash.

The washing may include washing and removing excess antibody after detection of nanoparticles, and removing excess reagent after labeling nanoparticles.

The method may further include supplying a substrate solution after labeling the nanoparticles. The method may further include recovering the nanoparticles separated from the filtration membrane.

In the detecting of the nanoparticles, the method may further include extracting nucleic acids by supplying a nucleic acid extracting reagent to the separated nanoparticles.

In the detecting of the nanoparticles, including separating the filtration membrane from the disk, nanoparticle detection may be performed outside the disk.

【Effects of the Invention】

As described above, according to the present exemplary embodiment, the whole process of detecting nanoparticles from a sample may be integrated and simply performed in a single disk, thereby minimizing time and effort required for detecting nanoparticles. Accordingly, analysis and diagnosis of nanoparticles can be performed more easily while minimizing the input of skilled workers.

By detecting the antibody by supplying the antibody in the space where the nanoparticles are separated, it is possible to effectively detect all antigens on the surface of the nanoparticles. Accordingly, the nanoparticles can be detected within a short time, and the efficiency of the antibody for detection can be increased.

Therefore, it is possible to effectively diagnose diseases including the research of nano vesicles and cancer using the same.

[Brief Description of Drawings]

1 is a schematic plan view of a nanoparticle detection apparatus according to the present embodiment.

2 is a schematic plan view of a nanoparticle detection apparatus according to another embodiment.

3 is a schematic cross-sectional view showing a sample accommodating part of the nanoparticle detection device according to the present embodiment.

4 is a schematic cross-sectional view showing a filter part of the nanoparticle detection device according to the present embodiment.

5 is a schematic plan view of a nanoparticle detection device according to another embodiment. 6 is a schematic view for explaining the principle of nanoparticle detection of the nanoparticle detection apparatus according to the present embodiment.

7 shows protein detection results of nanoparticles according to the present embodiment.

8 is a view showing the protein expression amount of the urine-derived nanoparticles according to the present embodiment in comparison with the prior art.

9 is a view showing the nucleic acid detection results of the nanoparticles according to the present embodiment compared with the conventional.

10 is a view showing a result of detecting the nanoparticles separated from the sample spiked into the plasma of cancer cell-derived nano endoplasmic reticulum according to the present embodiment compared with the conventional.

FIG. 11 is a diagram illustrating a result of detection after separation of nanoparticles according to spin conditions from a sample spiked with plasma of cancer cell-derived nano endoplasmic reticulum according to the present embodiment.

12 is a view showing a result of detecting the nanoparticles after separation according to the volume of the sample spiked into the plasma of the cancer cell-derived nano endoplasmic reticulum according to this embodiment compared with the conventional.

FIG. 13 is a diagram illustrating the results of separation and protein detection of plasma from cancer patients and normal plasma-derived nanoparticles according to the present embodiment. 14 is a diagram showing the results of separation and nucleic acid detection of normal plasma-derived nanoparticles according to the present embodiment compared with the conventional

FIG. 15 is a diagram illustrating separation and nucleic acid detection results of plasma-derived nanoparticles undergoing proteolytic enzyme treatment and plasma-free nanoparticles according to the present embodiment.

16 is a view showing a nanoparticle protein detection results of the nanoparticle detection device according to the embodiment of FIG.

[Specific contents to carry out invention]

Hereinafter, with reference to the accompanying drawings, it will be described an embodiment of the present invention to be easily implemented by those skilled in the art. Those skilled in the art to which the present invention belongs As can be easily understood, the embodiments described below can be modified in various forms without departing from the spirit and scope of the invention. Where possible, the same or similar parts are represented using the same reference numerals in the drawings.

The terminology used below is merely to refer to specific embodiments, and is not intended to limit the present invention. As used herein, the singular forms “a,” “an,” and “the” include plural forms as well, unless the phrases clearly indicate the opposite. As used herein, the meaning of “comprising” embodies a particular characteristic, domain, integer, step, operation, element and / or component, and other specific characteristic, domain, integer, step, operation, component, component and / or It does not exclude the presence or addition of groups.

All terms including technical terms and scientific terms used below have the same meaning as those commonly understood by those skilled in the art. Terms defined in advance are additionally interpreted to have a meaning consistent with the related technical literature and the presently disclosed contents, and are not interpreted in an ideal or very formal sense unless defined.

In the following description, this embodiment describes a structure for detecting nano vesicles from a sample as an example. Nanovesicles can be captured from a variety of samples, including blood, lymph, tissue fluids, urine, saliva, cerebrospinal fluid, and biological samples containing sputum or aqueous solutions in which nanoparticles are dispersed. This embodiment is not limited to the detection of nano vesicles, it is applicable to the detection of various nanoparticles.

The detection apparatus of the present embodiment includes a disk for transferring fluid by centrifugal force, a sample accommodating portion formed on the disk for accommodating a sample, and a filtration membrane for separating nano particles by filtering the transferred sample. A filter part, a supply part connected to the filter part to supply a detection liquid for detecting nanoparticles separated from the filtration membrane, a waste fluid receiving part connected to the filter part exit side to receive a solution passed through the filtration membrane, and formed on a disk to convey fluid It may include a flow path, and a valve for selectively opening and closing the flow path. Here, the detection may mean all of detecting and confirming the presence or absence of a specific antigen of the nanoparticle, and detecting and analyzing a specific protein or nucleic acid of the nanoparticle, including extracting a nucleic acid of the nanoparticle. The disc (di sc) forms the body of the device. The disc has respective components therein.

The shape of the disc can be variously modified. In this embodiment, the disk may be of circular plate structure. The center of the disk forms the axis of rotation. The disk is rotated about the rotation axis by the driving force provided from the outside. As the disk rotates, centrifugal force is generated and centrifugal force is applied to the fluid to transfer the fluid.

The disk may be formed by joining two plate structures of the upper plate and the lower plate in the form of a disk. The disk may be formed by joining various numbers of plates in addition to the upper plate and the lower plate joining structure. For example, the disk may be further provided with a plate structure formed with a valve between the upper plate and the lower plate.

The inner surface of the upper plate and the lower plate can be formed with a cavity of the type set to form each component and the flow path. The space formed between the upper plate and the lower plate forms a flow path with the chamber for receiving or transporting the fluid. The upper and lower plates constituting the disk may be made of a material whose surface in contact with the fluid is biologically inert. In addition, the upper plate and the lower plate may be made of a material having optical transmission. For example, the top and bottom plates may be made of polystyrene (PS), poly dimethyl siloxane (PDMS), poly methyl methacrylate (PMMA), polyacrylate, polycarbonate, and polycarbonate. ), Polysilic olefins (polyccl ic olef ins), polyimide (polyimide) polyurethane (polyurethanes) and the like.

In addition, the top and bottom plate constituting the disk may be a structure that is surface-modified with proteins, polymers, organic molecules and the like to prevent non-specific antibody adhesion.

Therefore, when the sample is fed into the disk and moved along the flow path, the sample does not react with the disk and does not adhere to the surface, thereby improving biological safety. It can be secured and the reaction with an antibody can be improved. In addition, the separated nanoparticles can be detected through the upper plate and the lower plate through the optical detector without discharging the outside of the disk.

This embodiment may be a structure for detecting a plurality of different nanoparticles using one disk. To this end, the sample accommodating part, the filter part, the supplying part, and the waste liquid receiving part which are necessary for detecting the nanoparticles constitute one unit, and a plurality of units may be arranged at intervals along the circumferential direction of the disk. 1 illustrates, for example, a structure in which two units are arranged. According to the size of the disk, the size of each unit, etc., various numbers of units may be provided in one disk. ^'

Accordingly, different samples or detection liquids may be prepared in a plurality of units provided on the disks, and a plurality of nanoparticles may be detected simultaneously with one disk.

At the center of rotation of the disk, the sample receiving part, the supplying part, the filter part, and the waste liquid receiving part are sequentially positioned along the centrifugal force direction. Accordingly, the fluid flows from the sample receiving part and the supply part to the filter part and from the filter part to the waste liquid receiving part. Thus, by applying the centrifugal force by rotating the disk, it is possible to sequentially move the fluid to perform the desired process.

A flow path is formed between each component of the disk. The flow path connects between the chambers of neighboring components. The flow path has an inlet through which the fluid enters and an outlet through which the fluid exits, and the inlet may be located toward the center of the disk rather than the outlet. Thus, when centrifugal force of the disk is applied, the fluid can move more smoothly along the outlet from the inlet located relatively to the center of the disc.

The flow path may be, for example, a micro flow path in which impurities hardly flow back. The fine flow path may mean a flow path formed in a size having a resistance capable of preventing a back flow of the fluid. Thus, for example, it is possible to prevent the impurities contained in the fluid discharged through the filter portion to the waste liquid receiving portion from flowing back to the filter portion along the flow path.

In addition, the flow path has an inlet in each component part along the disc centrifugal force direction. Can be connected to the exit. The exit of a component is the portion of the fluid outflow from the component and means the side near the outer leading edge of the disk along the direction of centrifugal force.

Thus, when centrifugal force is applied to the disk, the fluid contained in each component portion is able to move toward the inlet of the flow path by the centrifugal force and move all along the flow path. Therefore, no fluid remains in the chamber of each component, thereby minimizing the loss of the fluid. .

For example, as the sample receiving unit is connected to the flow path at the outlet side, all the samples accommodated in the sample receiving unit can move to the filter unit through the channel. Therefore, nanoparticle detection can be effectively performed using fewer samples or antibodies.

The valve is located at one side of the flow path to selectively open and close the flow path. The valve may be of various structures that can block or open the flow path. The valve may open and close the flow path by applying power to itself, or may open and close the flow path by a driving force from outside the disc.

When the valve is driven and the flow path is closed, fluid transfer between components connected to the flow path is interrupted. If necessary, the valve is driven to open the flow path and fluid is transferred between the components to initiate the required process.

As described above, the apparatus can perform a series of processes of separating and detecting nanoparticles from a sample through a sample accommodating part, a supply part, a filter part, and a waste liquid accommodating part provided in the disk.

Hereinafter, each component of the nanoparticle detection apparatus according to an embodiment will be described in detail with reference to FIG. 1.

In the following description, each of the flow paths, valves, and chambers formed in the disk will be referred to separately according to their configuration for convenience of description. In addition, when describing as a flow path, a valve, and a chamber, it may refer to the flow path, a valve, and the whole chamber formed in the disc.

In the present embodiment, the disk ₁₀ may be formed by joining two plate structures of a disk-shaped upper plate (see 12 in FIG. 4) and a lower plate (see 14 in FIG. 4).

The sample accommodating _{portion 20 of the} present embodiment is formed on the disk 10 to provide a sample. It connects the sample chamber 21, the sample chamber 21 and the filter part 30 to receive, and opens and closes the 1st flow path 22 and the 1st flow path 22 which a sample is conveyed according to the centrifugal force of the disk 10. FIG. It may comprise a first valve (23).

The sample chamber 21 can be understood as an empty space formed inside the disk 10. The sample chamber 21 is connected to the filter unit 30 through the first flow passage 22. The sample chamber 21 may be formed with a hole for injecting a sample on one side. The sample contained in the sample chamber 21 is moved through the one flow path 22 by the centrifugal force of the disk 10. One side of the first flow passage 22 is provided with a first valve 23 for opening and closing the first flow passage 22. Accordingly, when the first flow passage 22 is opened by operating the crab valve 23, the sample in the sample chamber 21 is moved to the filter portion 30 along the first flow passage 22.

The filter unit 30 has an entrance space 32 and an exit space 33 along the fluid movement direction, and the filter chamber 31 is provided with the filtration membrane 34 between the entrance space 32 and the exit space 33. It may include. The filter chamber 31 is an empty space formed in the disk 10 and may be divided into an entry space 32 and an exit space 33 with a boundary of the filtration membrane 34. The entrance space 32 of the filter chamber 31 is a space into which fluid, such as a sample, flows in, and is a space for receiving filtered nanoparticles. The nanoparticles are detected in the entrance space 32.

One side of the filter chamber 31 may be provided with a vent so that smooth filtration can be made. The vent may be formed in the top plate, for example. As a result, air that is present in the filter chamber 31 when the sample is filtered is discharged to the outside of the disk 10, so that the filtration is more smoothly performed.

The entrance space 32 is connected to the sample chamber 21 of the sample accommodating portion 20 through the first flow passage 22. The exit space 33 is connected to the waste liquid receiving part 70. As a result, the nanoparticles are separated from the sample introduced into the entrance chamber via the filtration membrane 34. The separated nanoparticles are accommodated in the entrance space 32, and the filtrate passing through the filtration membrane 34 is discharged to the waste solution receiving portion 70 connected to the exit space 33.

In this embodiment, the permeate may be previously contained in the exit space 33 of the filter chamber 31.

The permeate is the same solution as the nanoparticles separated from the sample, It may be the same solution as the filtrate which has been filtered through the membrane 34. Alternatively, the permeate is a solution that does not have particles to be separated and does not affect the sample or the filtrate, and any solution can be applied as long as it can reduce the capillary pressure of the pores of the filter membrane 34 at the exit side of the filter membrane 34.

Accordingly, the sample introduced into the entrance space 32 of the filtration membrane 34 can easily pass the fine pores of the filtration membrane 34 even under a smaller pressure. Therefore, the sample can be filtered more quickly to separate the nanoparticles.

The permeate can be accommodated in the filter chamber 31 exit space 33, for example, during device manufacture. Alternatively, by forming a separate injection port connected to the exit space 33 of the lower plate of the disk 10, after the manufacture of the device can be injected into the exit space 33 through the injection hole.

The filtration membrane 34 is a membrane structure in which numerous pores are formed on the surface. In the present embodiment, the filtration membrane 34 may be formed with pores of lnm to 100nm for separation of nanoparticles contained in the sample. The filtration membrane 34 may be selected to an appropriate pore size within the above range according to the type or size of the nanoparticles to be separated. If the pore size of the filtration membrane 34 is out of the above range, the nanoparticles may not be separated or the separation efficiency may be reduced.

The filtration membrane 34 may be formed of various materials to filter living cells, inorganic material particles, organic material particles, and the like. The filtration membrane 34 may be formed of a material such as polycarbonate, polystyrene, polymethyl methacrylate, thermosetting plastic including cyclic olefin copolymer, anodized aluminum, nickel, silicon, or the like. For example, the filtration membrane formed of anodized aluminum has higher porosity and relatively uniform diameter pores than other materials.

The filtration membrane 34 of the present embodiment may be formed of a biologically inert material so that it can be applied to a biological sample. In addition, the filtration membrane 34 may be formed of a material having optical transparency at the same time. Through this, nanoparticles can be detected using an optical detector without separating the filtration membrane 34 from the disk 10. In addition to the structure for detecting nanoparticles on the disk 10, the apparatus may perform the detection process by separating the filtration membrane 34 from the disk 10. The separation structure of the filtration membrane 34 will be described later in detail.

The filter unit may be provided with one filtration membrane or may have a structure including two or more filtration membranes in one filter chamber. In the case where the filter chamber is provided with two or more filtration membranes, a plurality of filtration membranes may be stacked and disposed between the entrance space and the exit space. Each filtration membrane may have a structure in which the pore size gradually decreases along the fluid transfer direction, that is, from the entrance space to the exit space. For example, the filtration membrane disposed in the entrance space may have a pore size of 50 nm, and the filtration membrane stacked below the exit space may have a pore size of 5 nm.

Accordingly, in the sample including nanoparticles, primary particles are separated while the pores disposed toward the entrance space pass through the large filtration membrane, and nanoparticles are separated while the pores pass through the small filtration membrane. Therefore, only nanoparticles of a specific size range can be screened and separated.

In addition to the above-described structure, at least two or more filter units may be sequentially disposed along the fluid transfer direction, and the filtration membranes 34 provided in each filter unit may have a structure in which pores gradually decrease along the fluid transfer direction. This structure is shown in FIG. 2 and will be described later. Also in this structure, the sample containing the nanoparticles is separated from the primary particles while passing through the filter membrane with large pores, and the nanoparticles are separated while passing through the filter membrane with small pores in the next filter portion. Therefore, only the nanoparticles of a specific size range can be separated and collected.

In the present embodiment, the supply unit 40 is for detecting nanoparticles separated from the filtration membrane 34, and may have a structure for supplying the detection liquid to the filter unit 30. The detection solution is a material for detecting nanoparticles, and may be, for example, an antibody attached to an antigen, a reagent for labeling an antibody, a reaction substrate solution, a nucleic acid extracting solution, a washing solution, or the like. The detection liquid is applicable to any material that can be used to detect nanoparticles separated from the filter unit.

To this end, the supply 40 is formed in the disk 10 to detect nanoparticles. A second flow passage 42 which connects the antibody chamber 41, the antibody chamber 41, and the filter portion 30 containing the antibodies provided for the purpose and transfers the antibody to the filter portion 30 according to the centrifugal force of the disk 10. ), Opening and closing the second flow path 42 may include two valves 43.

The antibody chamber 41 can be understood as an empty space formed inside the disk 10. The antibody chamber 41 is connected to the filter chamber 31 through the second flow passage 42. The antibody chamber 41 may be formed with a hole for injecting the antibody on one side. The antibody contained in the antibody chamber 41 is moved through the two flow paths 42 by the centrifugal force of the disk 10. One side of the crab 2 flow passage 42 is provided with a crab 2 valve 43 for opening and closing the second flow passage 42. Accordingly, when the second flow passage 42 is opened by operating the second valve 43, the antibody of the antibody chamber 41 is moved to the filter chamber 31 along the second flow passage 42.

In the present embodiment, the antibody is a primary detection antibody that binds to the biomarker of the nanoparticles and captures and fixes the biomarker, and may be a detection ion ant ibody having a biot in.

The supply section 40 is formed on the disk 10 for reagent supply, and includes a reagent chamber 44, a reagent chamber 44, and a filter section 30 containing a reagent provided for labeling an antibody for detecting nanoparticles. And a third valve 45 for opening and closing the third flow path 45 and the third flow path 45 for transferring the reagent to the filter unit 30 according to the centrifugal force of the disk 10.

The reagent chamber 44 may be understood as an empty space formed inside the disk 10. The reagent chamber 44 ^'is connected to the filter chamber 31 through the three flow paths 45. The reagent chamber 44 may be formed with a hole for injecting a reagent on one side. The reagent contained in the reagent chamber 44 is moved through the third flow path 45 by the centrifugal force of the disk 10. One side of the third flow passage 45 is provided with a crab three valve 46 for opening and closing the crab three flow passage 45. Accordingly, when the third flow passage 45 is opened by operating the crab valve 46, the reagent of the reagent chamber 44 is moved to the filter chamber 31 along the third flow passage 45.

In this embodiment, the reagent may be a fluorescent secondary detection antibody that binds to the antibody. The reagent uses a biosignal such as fluorescent light to biomarker Any material that can be analyzed and quantified is applicable. For example, the reagent may be streptavidin-H P to amplify the antibody signal. The reagent amplifies the signal of the detection antibody, which is advantageous for measurement.

The supply unit 40 is formed in the disk 10 and connects the substrate liquid chamber 47 containing the substrate liquid provided for the nanoparticle detection reaction, the substrate liquid chamber 47 and the filter unit 30. According to the centrifugal force of (10), it may further include a fourth flow passage 48 for transferring the substrate liquid to the filter portion 30 and a fourth valve 49 for opening and closing the fourth flow passage 48.

Substrate liquid chamber 47 can be understood as an internal space formed in the disk (10). The substrate liquid chamber 47 is connected to the filter chamber 31 through the fourth flow path 48. Substrate liquid chamber 47 may be formed with a hole for injecting the substrate liquid on one side. The substrate liquid contained in the substrate liquid chamber 47 is moved through the fourth flow path 48 by the centrifugal force of the disk 10. One side of the fourth flow passage 48 is provided with a fourth valve 49 for opening and closing the fourth flow passage 48. Accordingly, when the fourth valve 49 is operated to open the fourth flow passage 48, the substrate liquid of the substrate liquid chamber 47 is moved to the filter chamber 31 along the fourth flow passage 48.

In this example, the substrate solution may be TMB (Tetramethylbenzidine) solution. TMB is a substrate of HRP and changes color by HRP. ΤΜΒ (3,3 ', 5,5'-tetramethylbenzidine) is blue when oxidized to hydrogen peroxide by Peroxidase catalysis and has maximum absorbance (0D; 0pt i cal densi ty) at 370nm and 652nm. ^{'When the} reaction is stopped, it turns yellow and shows the maximum 0D value at 450nm. ^"

In addition, the supply unit 40 may be a structure that provides a stop solut ion provided to stop the nanoparticle detection reaction. To this end, the supply unit 40 is further provided with a stop solution chamber 50 formed in the disk 10 to receive the stop solution, the stop solution chamber 50 is the substrate liquid chamber 47 through the connecting passage 51 ) And the stop solution may be supplied to the filter chamber 31 through the substrate liquid chamber 47 according to the driving of the connection valve 52 installed at one side of the connection flow path 51.

The stop solution may be a solution containing a strong acid. The stop solution is a substrate solution Stop the reaction of changing the color by the enzyme.

The antibody chamber 41, the reagent chamber 44, and the stationary solution chamber 50 connected to the substrate liquid chamber 47 and the substrate liquid chamber 47 are more than the filter chamber 31 along the centrifugal force direction of the disc 10. Located at the center of the disk (10). Accordingly, when centrifugal force is applied, the fluid contained in the antibody chamber 41, the reagent chamber 44, the substrate liquid chamber 47, and the stop solution chamber 50 is transferred to the filter chamber 31 along the flow path by the centrifugal force. do. Valves provided on each flow path open or close the flow path according to the process sequence to sequentially supply the required fluid to the filter chamber 31.

In addition, the supply unit 40 may further include a washing unit 60 for washing the filter unit 30 for more efficient and accurate detection.

In the present embodiment, the washing unit ₆₀ is formed in the disk 10 to connect the washing liquid chamber 61, the washing liquid chamber 61 and the filter unit 30 to receive the washing liquid, and to the centrifugal force of the disk 10. Accordingly, the chaff 15 may include the five valves 63 to open and close the fifteen flow passages 62 and the fifth flow passage 62 to transfer the washing liquid to the filter unit 30.

The washing liquid chamber 61 may be understood as an empty space formed inside the disk 10. The washing liquid chamber 61 is connected to the filter chamber 31 through the fifth flow path 62. The washing liquid chamber 61 may be formed with a hole for injecting the washing liquid on one side. The washing liquid contained in the washing liquid chamber 61 is moved through the fifth flow path 62 by the centrifugal force of the disk 10. One side of the fifth flow passage 62 is provided with a five-valve 63 for opening and closing the fifth flow passage 62. Accordingly, when the fifth flow passage 62 is opened by operating the crab 5 valve 63, the washing liquid of the washing liquid chamber 61 is moved to the filter chamber 31 along the fifth flow passage 62.

The washing liquid is removed by washing out any excess antibody or reagent remaining in the filter chamber 31. After being moved to the filter chamber 31 to wash the entrance space 32 of the filter chamber 31, the washing liquid passes through the filtration membrane 34 to the exit space 33, and then passes through a flow path connected to the exit space 33. Through the waste liquid receiving portion 70 is discharged. ·

In this process, the excess antibody that is not bound to the nanoparticles or the excess antibody that is not bound to the antibody is washed in the entry space 32 of the filter chamber 31 and removed together with the washing liquid into the exit space 33. In this embodiment, the washing liquid chamber 61 is provided in plurality, so that the washing liquid can be separately accommodated in each washing liquid chamber 61. Accordingly, the filter chamber 31 may be washed in a plurality of times as necessary. With this structure, the outlet flow passage of each washing liquid chamber 61 is connected to the fifth flow passage 62 so that the washing liquid can be supplied to the filter chamber 31 through the cradle 5 passage 62. In addition, as shown in Figure 1, the outlet side of each washing liquid chamber 61 is connected to each other, the outlet valve 64 for discharging the washing liquid may be provided in this connection portion. That is, each washing liquid chamber 61 is arranged side by side and the outer end is connected to each other between neighboring washing liquid chamber 61. Therefore, if necessary, the outlet valve 64 provided at the connecting portion of the fifth valve 63 and each washing liquid chamber 61 may be sequentially opened to supply the washing liquid of each washing liquid chamber 61 to the filter chamber 31 in turn. .

The washing liquid chamber 61 is located at the center of the disk 10 rather than the filter chamber 31 along the centrifugal force direction of the disk 10 like the other chambers of the supply _{part 40} . Accordingly, when centrifugal force is applied, the washing liquid demanded in the washing liquid chamber 61 is transferred to the filter chamber 31 along the flow path by the centrifugal force.

The waste liquid receiving portion 70 is formed in the disk 10 to connect the waste liquid chamber 71, the waste liquid chamber 71, and the filter portion ₃₀ , in which the waste liquid is received, and the waste liquid chamber according to the centrifugal force of the disk 10. The system may include a sixth flow path 72 that is transferred to the 71. One side of the sixth flow passage 72 may further include a six valve (see 73 of FIG. 2) to open and close the sixth flow passage 72.

The waste liquid chamber 71 can be understood as an empty space formed inside the disk 10. The waste liquid chamber 71 is connected to the filter chamber 31 through the sixth flow path 72. The waste liquid moved to the outlet space 33 of the filter chamber 31 is moved to the waste liquid chamber 71 through the sixth flow path 72 by the centrifugal force of the disk 10.

The waste liquid chamber 71 may have a vent hole at one side thereof so that the waste liquid may be smoothly moved to the exit space 33 through the filtration membrane 34 of the filter chamber 31. The vent may be formed in the top plate, for example. As a result, the air, which is present in the filter chamber 31 exiting space 33 and the waste liquid chamber 71 when the sample is filtered, is discharged to the outside of the disk 10, so that the filtration is more smoothly performed. With the structure described above, the device extracts nano vesicles after sample injection and The whole process of detection is carried out collectively via one disc 10. Accordingly, the nanoparticle detection operation can be performed more simply and quickly and effectively.

2 shows another embodiment of a nanoparticle detection device. In the embodiment shown in FIG. 2, the basic configuration of the sample receiving portion 20, the supply portion 40, the filter portion 30 and the waste liquid containing portion 70 provided in the form of the disc 10 or in the disc 10 is Same as the embodiment shown in FIG. 1. Therefore, in the following description, the same reference numerals are used for the same components, and detailed description thereof will be omitted.

As shown in FIG. 2, the sample accommodating part 20 of the present exemplary embodiment may have a structure for removing a non-target material including impurities by purifying a sample.

. To this end, the sample accommodating part 20 is formed in the disk 10 so that the sample chamber 21 for accommodating the sample centrifugally separates the sample according to the disc 10 centrifugal force, and the disc 10 along the direction of the centrifugal force. The settling part 24 which accommodates the centrifuged sample is extended in the front-end | tip toward the outer side, The crab 1 flow path 22 is the settling part of the said sample chamber 21 toward the rotation center of the disk 10 ( Connected to the boundary point 24 may be a structure for transferring the centrifuged supernatant to the filter unit (30). The non-target material may mean impurities other than a solution containing nano particles to be purified.

The sample injected into the sample chamber 21 is centrifuged and purified by the centrifugal force according to the rotation of the disk 10. Thus, the sample is separated into a solution containing nanoparticles and impurities other than the nanoparticles. Solid impurities along the centrifugal force direction are pushed toward the outer leading end of the disk 10 and a solution separated from the impurities is located toward the center of the disk 10.

In the present embodiment, the sample chamber ₂₁ is formed by extending the settling portion 24 along the centrifugal force direction so that the separation between the impurities and the solution containing the nanoparticles in the space is clearly shown. The settling portion 24 may have a hopper shape that becomes narrower toward the exit side so that impurities can be more easily settled. Therefore, it is possible to ensure separation of the precipitated impurities and the solution by centrifugal separation, so that non-target materials other than the solution flow into the filter chamber 31. It can be minimized.

The inlet of the crab 1 flow passage 22 may be connected to the portion facing the center of rotation of the disk 10 at the boundary point between the non-targeted coating of the sample chamber 21 and the solution. Thus, only the nanoparticle-containing solution centrifuged by the centrifugal force applied as the disk 10 rotates may be transferred to the filter chamber 31 through the one flow path 22. Since the impurities precipitated in the settling portion 24 are located outside the inlet of the first flow passage 22 along the centrifugal force direction, the impurities cannot be transferred through the crab 1 flow passage 22.

As described above, the sample is centrifuged and purified, and then provided to the filter chamber 31, so that the effect of separating and detecting nanomaterials through the filtration membrane 34 can be further enhanced.

The settling part 24 exiting side of the sample chamber 21 is connected to the waste liquid chamber 71, and it can process and process the impurity refine | purified in the sample chamber 21 to the waste liquid chamber 71. FIG. A discharge flow path 26 through which impurities are transferred is formed between the exit of the settling portion 24 and the waste liquid chamber 71 of the sample chamber 21, and a discharge valve 27 that opens and closes the discharge flow path on one side of the discharge flow path 26. Is installed.

Accordingly, after centrifuging the sample, the target material is transferred to the filter chamber 31, and then the discharge passage 26 is opened to discharge the non-target substance remaining in the sample chamber 21 to the waste liquid chamber 71. Can be removed.

In the sample accommodating part _{20 of the} present embodiment, the settling part 24 may be formed to be inclined with respect to the radial direction of the disc 10 so as to increase the sample purification efficiency. 2, the depression 24 is ^formed, it turned to one side inclined at a predetermined angle (A) for the radial direction of the disk 10. Accordingly, the solid phase impurities in the centrifugal separation process are easily pulled down along the inclined surface of the settling portion 24 with respect to the radial direction of the disk, so that the settling is better and the separation effect can be enhanced.

In addition, as shown in Figure 3, the settling portion 24 is formed in the bottom surface 28 is gradually inclined upward toward the end along the direction of the centrifugal force at the boundary point, the end of the settling portion 24 in the sample It may further include a groove portion 25 for receiving the centrifuged impurities. 3 shows a cross-sectional structure in the width direction of the disk 10. In FIG. 3, the upper part is upper part and the lower part is located along the y axis direction. Lower part. The settling portion 24 of the sample chamber 21 is formed to be inclined upward from the lower bottom surface 28 toward the exit side of the end. Then, the exit side of the settling portion 24 forms a deep groove portion (bolt 91) deeply dug in the vertical direction. As a result, the solid impurities settled toward the settling portion 24 exited by the centrifugal force move along the inclined bottom surface of the settling portion 24 and fall back to the groove portion 25 to prevent flow back.

That is, the groove portion 25 is blocked by the inner circumferential surface with a hole having a depth, and when the solid impurities fall into the groove portion 25, the impurities cannot escape from the groove portion 25 even when no centrifugal force is applied. Therefore, it is possible to supply only the purified sample to the filter chamber 31 by minimizing the backflow of impurities to the boundary point of the sample chamber 21. The discharge flow path is connected to the groove portion 25, and the pure water dropped to the groove portion 25 is discharged to the waste liquid chamber 71 through the discharge passage 26.

As shown in FIG. 2, the filter unit of the present embodiment may include two filter chambers.

The two filter chambers are arranged sequentially along the fluid conveying direction. The filtration membrane provided in each filter chamber may have a structure in which pores gradually decrease along the fluid transport direction. For convenience of description below, the filter chamber disposed before the filter chamber 31 in which the detection of the nanoparticles is finally made out of the two filter chambers is referred to as a prechamber 35. The prechamber is connected between the sample chamber 21 and the filter chamber 31.

The preliminary chamber 35 is connected to the sample chamber 21 through the first flow passage 22, and the filter chamber 31 is connected to the waste liquid chamber 71 through the crab 6 flow passage 72. A conveyance passage 36 is formed between the preliminary chamber 35 and the filter chamber 31. One side of the conveyance passage 36 is provided with a conveyance valve 37 for opening and closing the conveying flow passage. Accordingly, when the transfer valve 37 is opened, the filtrate filtered in the prechamber 35 is moved to the filter chamber 31 through the transfer passage 36.

As the filter unit 30 includes the prechamber, the filter chamber, and the two chambers, the sample including the nanoparticles transferred from the sample chamber 21 passes through the filtration membrane 34 having large pores in the prechamber 35. Firstly, large particles other than nanoparticles are separated. In the next filter chamber 31, a small pore filtration membrane 34 is removed. As it passes, the nanoparticles finally separate. Therefore, only the nanoparticles of a specific size range can be separated and collected.

The supply unit 40 is connected to the filter chamber 31 to supply the fluid required for nanoparticle detection to the entrance space 32 of the filter chamber 31. Thus, the detection for the nanoparticles can be finally made in the filter chamber 31.

In addition, the present embodiment may have a structure for recovering the nanoparticles separated from the filter unit 30 to perform a necessary detection operation. To this end, the apparatus is formed in the disk 10 and connected to the filter section 30, the recovery chamber 80, the recovery chamber 80 and the filter section 30 is recovered, the nanoparticles separated by the filtration membrane 34 is recovered. (7) may further include a seventh flow path 81 to transfer the nanoparticles to the recovery chamber 80 and a seventh valve 82 to open and close the seventh flow path 81.

As shown in FIG. 2, the recovery chamber 80 may be understood as an empty space formed inside the disc 10. The recovery chamber 80 is connected to the filter chamber 31 through the crab 7 flow path 81. In addition, the recovery chamber 80 may be formed with a hole for injecting a reagent for extracting nucleic acid into one side. Thus, by injecting a nucleic acid extraction reagent including a phenol component through the hole formed in the recovery chamber, it is possible to extract from the sample to the nucleic acid through the apparatus.

As shown in FIG. 4, the crab 7 flow path 81 connects the entry space 32 of the filter chamber 31 and the recovery chamber 80. Accordingly, the fluid containing nanoparticles contained in the entry space 32 of the filter chamber 31 is moved through the seventh flow path 81 by centrifugal force. A seventh valve 82 is provided on one side of the seventh flow path 81 to open and close the seven flow path 81. Accordingly, when the seventh valve 82 is operated to open the seventh flow path 81, the nanoparticle-containing fluid remaining in the entrance space 32 in the filter chamber 31 is recovered along the seven flow path 81. Is moved to chamber 80. Thus, a nucleic acid extraction operation may be performed by injecting a nucleic acid extraction reagent including a spentol component into the fluid containing nanoparticles transferred to the recovery chamber 80. Nucleic acid extraction can be performed separately from the outside of the device. To this end, the filter unit 30 of the present embodiment may have a structure in which the filtration membrane 34 provided in the filter chamber 31 is detached from the disk 10. In this way, by separating the filtration membrane 34 from the filter chamber 31, the filtration membrane 34 filtered out from the disk 10 outside. Nucleic acid extraction can be performed separately for the nanoparticles.

To this end, the apparatus may further include a cover 90 detachably installed on the disk 10 to open and close the filter chamber 31, and a fastening part fixing the cover 90 to the disk 10.

As shown in FIG. 4, the cover 90 is detachably installed on the top plate 12 of the disk 10. The upper plate 12 is formed to be opened outward on the filter chamber 31 forming space. The cover 90 is installed on the top plate 12 to form part of the top plate. In the present embodiment, the fastening part may be a fastening structure using the bolt 91. The fastening part may be applied to various structures in addition to the bolt 91. A fastening hole 92 for fastening the bolt 91 may be formed on the cover 90 and the upper plate that is opposed thereto. Accordingly, by fastening the bolt 91 to the fastening hole 92, the cover 90 can be detachably coupled to the upper plate 12.

5 shows another embodiment of a nanoparticle detection device. 5 is also in the form of a disk 10 or the basic configuration of the sample receiving portion 20, the filter portion 30 and the waste liquid receiving portion 70 provided in the disk 10 is shown in FIG. Same as the embodiment. In addition, although not shown, the configuration for the supply unit (see 40 of FIG. 1) may also be provided in the same manner. Therefore, in the following description, the same reference numerals are used for the same components, and detailed description thereof will be omitted.

As shown in FIG. 5, the filter unit 30 of the present embodiment may include three filter chambers. The filter unit may be provided in plurality of four or more besides three filter chambers.

The three filter chambers are arranged sequentially along the fluid conveying direction. For convenience of explanation, the three filter chambers are referred to as a first filter chamber 311, a second filter chamber 312 and a crab three filter chamber 313 along the fluid flow direction. The filter chamber 31 may refer to all three filter chambers. Each filter chamber 31 has the same structure except for the pore size of the filtration membrane 34 provided therein. The first filter chamber 311 is connected to the sample chamber 21 through the first flow passage 22. The entrance space of the second filter chamber 312 is connected to the exit space of the first filter chamber 311 through the flow path 315. Of the second filter chamber 312 The exit space is connected to the entrance space of the third filter chamber 313 through a separate flow path 317. Accordingly, the fluid is sequentially filtered while passing through the first filter chamber 311, the second filter chamber 312, and the crab 3 filter chamber 131 in the sample chamber 21 to separate the nanoparticles. In this embodiment, a separate on-off valve (not shown) is installed at one side of each of the flow paths 315 and 317 to selectively open and close each flow path if necessary.

The filter membrane 34 provided in each filter chamber 31 may have a structure in which pores are different in size from each other and separate nanoparticles having different size ranges from each other. Thus, it is possible to separate and recover nanoparticles of different sizes through one device.

In the present embodiment, the filtration membrane provided in each filter chamber 31 may have a structure in which pores gradually decrease along the fluid conveying direction. In the present embodiment, the three filter chambers 31 may be provided with filtration membranes 34 each having a different pore size to separate nanoparticles of different sizes from each other. The combination of the filtration membranes provided in each filter chamber can be variously modified. For example, the first filter chamber 311 is provided with a filtration membrane having a pore size of 100 nm, the crab 2 filter chamber 312 is provided with a filtration membrane having a pore size of 50 mm 3, and the third filter chamber 313. It is equipped with a filtration membrane having a pore size of 2 nm can be combined in one device. Therefore, nanoparticles of different sizes can be separated and recovered in each filter chamber according to the combination of the filtration membranes in one device. ^{According to the} present embodiment, the sample including the nanoparticles transferred from the feed chamber 21 is separated from the nanoparticles having the first set size while passing through the large filtration membrane 34 having the pores of the crab 1 filter chamber 311. . Then, the sample transferred to the second filter chamber 312 passes through the filtration membrane 34 provided in the crab 2 filter chamber, and nanoparticles having a secondary size are separated. Then, the sample transferred to the third filter chamber 313 passes through the filtration membrane provided in the third filter chamber, and finally, nanoparticles having a predetermined size are separated. Thus, only nanoparticles of a specific size range can be separated and collected in each filter chamber. Thus, nanos with different size distributions in one disk Particles can be separated and recovered.

Hereinafter, a nanoparticle detection process using the nanoparticle detection apparatus according to the present embodiment will be described. The following description is made with reference to the apparatus of the embodiment shown in FIG.

First, the disk 10 is prepared by mounting an antibody, a reagent, a substrate liquid, a washing liquid, a filtration membrane 34, and the like for detecting nanoparticles. In the ready state, the valves installed in the respective flow paths of the disc 10 may be closed to maintain the closed state.

When the preparation is completed, the sample is supplied to the sample chamber 21 of the disk 10. Then, the disk 10 is rotated to apply centrifugal force.

The sample supplied to the sample chamber 21 by centrifugal force is centrifuged and purified primarily. The sample is centrifuged to remove impurities and only the solution containing the nanoparticles is transferred to the filter unit 30. The solution transferred to the filter part 30 passes through the filtration membrane 34 of the prechamber 35 by centrifugal force due to the rotation of the disk 10, and the large particles other than the nanoparticles are separated.

The solution which has passed through the filtration membrane 34 of the prechamber 35 is transferred to the filter chamber 31. The nanoparticles are separated through the filtration membrane 34 of the filter chamber 31 and remain on the filtration membrane 34 in the entrance space 32 of the filter chamber 31. The filtrate that has passed through the filtration membrane 34 of the filter chamber 31 is discharged to the waste liquid chamber 71.

As described above, the nanoparticles are detected by sequentially supplying the antibody, the reagent, the substrate liquid, and the like into the entrance space 32 of the filter chamber 31 while the nanoparticles are separated and captured through the filtration membrane 34.

FIG. 6 illustrates a process of detecting the nanoparticles by supplying an antibody, a reagent, and a substrate liquid to the nanoparticles separated and captured on the side space 32 above the filtration membrane 34 of the filter chamber 31 according to the present embodiment. have.

The antibody supplied to the filter chamber 31 adheres to the nanoparticles in the side space 32 on the filtration membrane 34. Since the antibody is confined in space and adheres to the surface of an unfixed nanoparticle, the antibody can be attached to the entire surface of the nanoparticle in a short time. On the other hand, In this case, the antibody may be attached only to some surfaces of the nanoparticles, and the antibody may not be attached to the entire surface. Thus, in the present embodiment, it is possible to detect the antigen on the entire surface of all the nanoparticles within a short time.

After antibody supply, excess antibody not attached to the nanoparticles is removed through the wash solution. When the washing liquid is supplied to the filter chamber 31 and the disk 10 is rotated, the excess antibody remaining after the nanoparticles are attached is removed by passing through the filtration membrane ₃₄ together with the washing liquid by centrifugal force.

After washing the entry space 32 with the washing solution, the reagent is supplied to label the antibody. The reagent binds to the antibody attached to the nanoparticles. Reagents are fluorescent

As a secondary detection antibody, the antibody signal finally attached to the nanoparticles is amplified. After reagent supply, excess reagents not attached to the antibody are removed through the wash solution. When the washing solution is supplied to the filter chamber 31 and centrifugal force is applied to the disk 10, the excess reagent is attached to the antibody and the remaining reagent is removed through the filtration membrane 34 together with the washing solution.

After washing the entry space 32 with the washing liquid, the substrate liquid may be supplied to the entry space 32. After the substrate solution for reaction is supplied in the entrance space 32, the stop solution is supplied.

In addition, the nanoparticle-labeled solution including the stop solution may be transferred to the recovery chamber 80 to measure optical density and the like. The nanoparticles of the solution transferred to the recovery chamber 80 may be fluorescently labeled, for example, to detect and analyze the nanoparticles using a fluorescence signal based measurement method.

In the present embodiment, unlike the conventional method for detecting after attaching the nano vesicles to the surface of 96 wel l plate, all antigens can be analyzed in a short time because the nano vesicles are separated in a specific chamber. As described above, the detection method of the present embodiment uses streptavidin-HRP for amplifying the antibody with the biot in and a signal and can measure ODCopt i cal densi ty) by injecting TMB after lbeling.

In addition, a nucleic acid extraction reagent including spent phenol is injected into the recovery chamber 80 to perform nucleic acid extraction of nanoparticles, or the filter chamber 31 of the disk 10 is removed. After opening to separate the filtration membrane 34, the nucleic acid extraction process can be performed from the outside.

As such, even nanoparticle separation and detection or nucleic acid extraction of a sample in a single disc 10 can be performed more simply and easily.

Experimental Example

FIG. 7 shows the results obtained after separation of the nano endoplasmic reticulum from the LNCaP (prostate cancer cell line) cell culture medium and the urine sample using the apparatus according to the example of FIG. 2.

A graph of FIG. 7 shows the results of nano-vesicle detection according to the combination of the filtration membranes provided in the two filter chambers. The graph shows nano vesicle recovery results within the range of 100-600 nm, 20-200 nm, and 20-600 nm, respectively. In the graph, filter I refers to the prechamber in FIG. 2 and filter π refers to the filter chamber. As a result of the experiment, when the filter membranes were combined in the range of 2600 nm, the nano vesicles were detected the most. 'B graph of Figure 7 shows that the increase in the infusion volume of the cell culture _. The expression level of CD9 also increases.

FIG. 7C shows the results of 0D (opt i cal densi ty) in which the nano vesicles were finally detected by injecting 400 ^ urine samples of normal people and bladder cancer patients into the discs. . In the C picture, N represents the result for a normal person, and P represents the result for a bladder cancer patient. As a result of the detection, it can be confirmed that the nanovesicles can be sufficiently detected from the urine sample of the bladder cancer patient through the device.

Figure 8 shows the comparison results of the nano-vesicle detection results by the device according to the present embodiment and the conventional device.

In FIG. 8, the results of the examples are indicated by Exodi sc, and the results obtained after separation of the nano-vesicles from urine samples of normal people and bladder cancer patients using the apparatus according to the example of FIG. 2 are shown.

In addition, in FIG. 8, comparative examples are represented by UC and Exospin, and show detection results according to the related art compared with the embodiment. To UC The displayed graph is the result of detecting the nano vesicles through ultra-ultracentrifugation (UC; ul tracentr i fugat ion), which is a widely used method, and detected by ELISA detection method using 96 wel l piate. The graph labeled Exospin shows the results of commercialization kit detecting nano vesicles separated by Exospin by ELISA detection method through 96wel l plate. Comparative Examples were also detected by separating nano vesicles from urine samples of normal people and bladder cancer patients as in Example. The separated nano vesicles were detected through CD9 and CD81, protein markers expressed in nano vesicles.

Experimental results, in the example in the urine samples of bladder cancer patients (n = 5) _. It can be seen that the isolated nano vesicles appear higher than normal (n = 5). Thus, it can be confirmed that the present invention can sufficiently detect the nano-vesicles from the urine samples of bladder cancer patients.

In addition, compared with the comparative examples according to the prior art, it can be seen that in the case of the nano-vesicle detection value for the bladder cancer patient in the case of the embodiment is higher. Thus, it can be seen that the nanoparticle detection effect of the present embodiment is significantly improved compared to the conventional.

9 shows a comparison of nucleic acid detection results of nano-vesicles by the device according to the present embodiment and the conventional device.

In FIG. 9, the results of the examples are represented by Exodi sc. After the separation of the nano endoplasmic reticulum from the LNCaP (prostate cancer cell line) cell culture medium using the apparatus according to the example of FIG. The result of extraction and detection is shown.

In addition, in FIG. 9, comparative examples are represented by UC and Exospin, and show detection results according to the related art compared with the embodiment. UC and Exospin are the same as described above. Comparative Examples were also isolated from the endoplasmic reticulum in the LNCaP (prostate cancer cell line) cell culture medium, and the nucleic acid was extracted and detected by injecting the reagents of the phenol.

A graph of FIG. 9 shows RNA electrophoresis results using a bioanalyzer. RNA size and concentration were analyzed from the nano vesicles separated by the experimental and comparative examples. Experimental results, the same H group in both Examples and Comparative Examples Many RNAs of 250 nt or less associated with the endoplasmic reticulum were extracted. In addition, it can be seen that the nano-vesicles separated by the detection apparatus of this embodiment in the concentration detection result showed higher RNA concentration than the nucleic acid extraction results and the comparative examples, thereby increasing the detection effect.

9B shows extracted C _T values of GAPDH, CD9, PSA, and PSMA through RT-PCR.

In the B graph of Figure 9, GAPDH is a house keeping gene, CD9 nano ER detection mageo, PSA (prostate-speci fic ant igen) is a marker, PSMA (prostate-speci fic membrane ant igen) used in the ^"prostate cancer detection It represents a marker used for detecting prostate cancer, and C _T means a high concentration as the value is lower. For example, if the value of C _T is seven orders of magnitude, 2 ⁷ means 128 times. -As shown in the graph B of Figure 9, Real time-PCR results, when using the detection apparatus according to this embodiment, the mRNA of the GAPDH, CD9, PSA, PSMA of the nano endoplasmic reticulum is more than 100 times higher than the conventional UC of the comparative example It can be seen that the concentration.

That is, in the case of the present embodiment, it can be seen that there is a difference of GAPDH: 169 times, CD9: 158 times, PSA: 111 times, and PSMA: 208 times compared to ultracentrifugal separation (UC) which is a comparative example. Thus, it can be seen that the present embodiment can further increase the detection effect compared with the conventional and comparative examples.

9C shows the relative expression amount compared to UC, which is an ultracentrifugal separation technique, based on CT values. As a result of the experiment, it can be seen that when all four genes were used in the detection apparatus of this example, a higher detection amount was shown than in the comparative example. Thus, in the case of the present embodiment it can be seen that the nucleic acid detection effect is significantly improved compared to the conventional.

9D shows an image obtained by electrophoresis of the PCR product. 9 is an image of electrophoresis of a PCR product such as a C graph. As a result of the experiment, it can be seen that all four genes showed higher detection amounts than the comparative examples when the detection device of the present example was used.

Figure 10 shows the results of the detection after separation of the nano-vesicles using the apparatus according to the present embodiment and the conventional apparatus. This experiment was accomplished by spiked LNCaP cell-derived nano endoplasmic reticulum into plasma 100 mi crol iter. The initial number of nanoparticles after s ike is 3.98 (±± 0.16) E10 / mL and total protein is 66.45 (±± 0.095) mg / mL.

First, the optimization values for the filtration membrane, pore size and disk rotation speed of the apparatus according to the present embodiment were obtained through experiments.

A graph of FIG. 10 shows the results of nanovesicle detection according to the pore size of the filtration membrane and the rotational speed of the disc (di sc) according to the present embodiment. The graph shows the results of nanoparticle recovery by NTA at 20 nm 3000 rpm (364 G) and 100 nm, 1000 rpm (40 G) combination conditions, respectively. The blue line in the graph shows the result of detecting the nano endoplasmic reticulum using plasma which did not spike the nano endoplasmic reticulum derived from the LNCaP cell culture medium as a control. As a result of the experiment, when the nanoparticles were separated under the condition of 20 nm 3000 rpm (364 G), which is a condition when the nanovesicles were separated from the conventional urine sample, the nanoparticles were most detected. However, in the case of human pl asma, since lipid and protein nanoparticles other than pure nano vesicles are present in the blood as impurities, most of the nanoparticles detected using 20 nm pores seem to be composed of lipid and protein particles.

10B shows the total protein detection result according to the pore size of the filtration membrane and the rotation speed of the disc (di sc) according to the present embodiment. The graph shows the results of total protein detection by BCA at 20 nm 3000 rpm (364 G), 100 nm 3000 rpm (364 G) and 100 nm 1000 rpm (40G) combination conditions, respectively. As a result of the experiment, when the nanoparticles were separated under the condition of 20 nm 3000 rpm (364 G), the total protein was detected the most. That is, in the case of the pore size of 20 nm and the rotational speed of 300 rpm, the number of nanoparticles appears to be large when measured using NTA. However, the purity of Pur i ty decreases due to the large amount of protein as measured by BCA. Able to know.

According to the experimental results, it can be seen that in the case of 20 nm 3000 rpm (364 G) condition, the purity of the nano-vesicle separation purity is lower than in the case of 100 nm 1000 rpm (40 G) conditions.

C graph of Figure 10 shows the filter membrane pore size and disk (di sc) of this embodiment The purity of the separated nano vesicle samples according to the rotational speed combination is shown. Experimental results, it can be seen that the conditions of 100 nm 1000 rpm (40 G), the nano-vesicles are separated in the highest purity.

FIG. 10D shows the detection results of the marker (EpCAM) and the nano vesicle detection marker (CD81) used for cancer detection, according to the pore size of the filtration membrane and the rotation speed of the disc (di sc) according to the present embodiment. Experimental results show that the total number of nano vesicles and cancer cell-derived endoplasmic reticulum was detected at 100 nm and 1000 rpm (40 G), and high-purity nanoparticles can be recovered without losing many nanoparticles. .

The E graph of FIG. 10 shows the recovery rate of the nano vesicles of the present example and the comparative example.

In the E graph of FIG. 10, the results of the examples are represented by DISC, and the detection results are divided according to the pore size and the disk rotation speed of the filtration membrane. The comparative example is represented by UC (ul tracentr i fugat ion) and shows the detection result by the conventional technique compared with an Example.

Early LNCaP cell-derived nano vesicles exhibited a 0D value of 1.34 upon detection of CD9 / CD81 (CD9 capture / CD81 detect ion). Nano-vesicles spiked into the plasma and tested by a conventional apparatus showed 0D of 1.18 at 20 nm 3000 rpm (364 G) and 0D value of 1.12 at 100 nm 1000 rpm (40 G). Indicated. That is, as shown in the E graph of Fig. 10, 20 nm 3000 rpm in the disc (di sc) according to the present embodiment than when separated by the conventional nano-vesicle standard separation method Ul tracentr i fuge (UC) When separated under (364 G) and 100 nm 1000 rpm (40G) conditions it was confirmed that the excellent recovery of more than 80%.

FIG. 11 shows the detection result after the nano vesicles were separated using a filtration membrane having a pore size of 100 nm in the apparatus according to the present embodiment. In the experiment, LNCaP cell-derived nano endoplasmic reticulum was spiked into 100 microliters of plasma. The initial number of nanoparticles after the spike is 6.34 (±± 0.12) ElO / mL.

The graphs in FIG. 11 are 600 rpm (15 G), 900 rpm (33 G) and 1200 rpm (58, respectively). G), nano vesicle detection results according to the rotational speed of the disc (di sc) at 1800 rpm (131 G) and 2400 rpm (233 G) are shown.

A graph of FIG. 11 shows the number of nanoparticles according to the disk rotation speed. Experimental results show that the number of nanoparticles decreases as the rotation speed increases from 900 rpm (33 G).

The B graph of FIG. 11 shows the total protein amount according to the disk rotation speed. Experimental results show that the total protein decreases with increasing rotation speed from 900 rpm (33G). ^'

FIG. 11C shows the nano vesicle separation time (left, open cicles) and purity (right, closed squares) according to disk rotation speed. Experimental results show that the separation time of nano endoplasmic reticulum decreases with increasing rotation speed from 600 rpm (15 G), and shows high purity in the range of 600 rpm (15 G) to 1200 rpm (58 G), 1800 rpm From 131 G, purity gradually decreases.

FIG. 11D shows the amount of CD81 antigen expression on the surface of the nano vesicles according to the disk rotation speed. As a result of the experiment, CD81 antigen expression was relatively high in the range of 600rpm (15G) to 1200rpm (58G), and rapidly decreased from 1800rpm (131G).

Comprehensive confirmation through Figure L1, it can be seen that a good recovery rate, purity, a short separation time can be obtained in the rotational speed range from 900rpm (33G) to 1200rpm (58G).

Figure 12 shows the detection results after the separation of nano-vesicles for this Example and Comparative Example.

The results for the examples in FIG. 12 are labeled Di sc. The example shows the result of detecting the nano vesicles after separating the disks with the filtration membrane having the pore size of 100 nm by operating at 900 rpm (33 G). The comparative example is denoted by UC (ult racentr i ligat ion), and shows the detection result according to the prior art compared with the example.

The experiments were performed by spiked plasma of LNCaP cell-derived nano endoplasmic reticulum in the same manner as in Examples and Comparative Examples. The initial nanoparticle count after the spike 6.34 (±± 0.12) ElO / mL.

A graph of FIG. 12 shows the number of nano-vesicles detected. As shown in the graph A of FIG. 12, in the example, the number of particles separated according to the injection volume of the plasma spiked with the LNCaP cell-derived nano-endoplasmic reticulum, was linear relationship in the range of 5 mi crol i to 200 mi crol i ter (l inear relat ionship). Thus, in the case of the embodiment it can be seen that the nanoparticles can be separated in a larger number compared to the comparative example UC is superior.

FIG. 12B shows the result of quantitative analysis of surface proteins by ELISA of the detected nano vesicles. As shown in the graph B of FIG. 12, in the case of the example, the expression volume of CD9 / CD81 (CD9 capture / CD81 detect ion) on the surface of the nano-endoplasmic reticulum was changed according to the injection volume of plasma spiked with LNCaP cell-derived nano-endoplasmic reticulum. It can be seen that the linear increase in the range ter to 200 microl i ter. Thus, in the case of the example, it can be seen that the higher expression amount, compared to the comparative example UC. Therefore, the quantitative analysis of the blood endoplasmic reticulum is possible by utilizing the disc (di sc) of the present embodiment.

The graph C of FIG. 12 shows the result of separating R A derived from a nano endoplasmic reticulum.

The experiments were performed in the same manner as in Examples and Comparative Examples, spiked the LNCaP cell-derived nano endoplasmic reticulum in plasma 100 microliter, separated the nano vesicles, and injected the reagent of the phenol component to extract the nano-vesicle-derived RNA. Bioanalyzer was used and the extracted RNA was subjected to electrophoresis to analyze the size and concentration of RNA.

As shown in the C graph of FIG. 12, the experimental results showed that both the Examples and Comparative Examples had the same size, and many RNAs of 250 nt or less associated with the nano-vesicles were extracted. In addition, it can be confirmed that the RNA concentration detection results can be increased by the nano-vesicle nucleic acid extraction results separated using the detection device of the present embodiment, showing a higher RNA concentration than the comparative examples to increase the detection effect. As described above, RNA was difficult to detect in UC, which is a comparative example, even in the result of separating the nano-vesicle-derived RNA, whereas Exosomal RNA was detected in the sample separated using the disk of the example. Figure 13 shows the detection results of the nano-vesicles separated from the normal plasma and plasma 200 mi Crol iter of the cancer patients according to the conditions of the embodiment compared with the conventional. In Figure 13, H is the normal plasma, L is the plasma of lung cancer patients, S means the plasma of gastric patients.

The results for the examples in FIG. 13 are labeled Di sc. Di sc-20 shows the experimental results under the conditions of rotating and driving a disk having a filtration membrane having a pore size of 20 nm at 3000 rpm (364 G), and Di sc-100 has a disk having a filtration membrane having a pore size of 100 nm. The experimental results are shown under the condition of driving rotationally at 1200 rpm (58 G). The comparative example is represented by UC (ul tracentr i fugat ion) and shows the detection result by the conventional technique compared with an Example.

Graph A of Figure 13 shows the total protein amount according to the nano-vesicle separation method. As a result, the total protein content was the highest in Di sc-20 at 20 nm 3000 rpm (364 G) and similar for UC and Di sc-100 (100 nm 1200 rpm (58 G)). Total protein was low.

Graph B of FIG. 13 shows a result of recovering nanoparticles according to a method for separating nanovesicles. As a result, the largest amount of nanoparticles was recovered in the Di sc-20 example at 20 nm 3000 rpm (364 G), but there was no difference in the number of nanoparticles between the normal sample and the cancer patient sample. In the di sc-100 example, which was carried out at 100 nm 1200 rpm (58 G), the amount of ^. The nanoparticles were recovered and there was a difference in the number of nanoparticles between normal and cancer patients.

Graph C of Figure 13 shows the purity of the nanoparticles according to the nano-vesicle separation method. The experimental results, in all of the sample groups, exhibited the highest purity, if the exemplary embodiment as a di sc-100 100 nm 1200 rpm (58 G) conditions coming from ^"example. Graph D of FIG. 13 shows the expression level of a marker (EpCAM) used for cancer detection of the surface of the nano endoplasmic reticulum according to the method for separating nano vesicles. As a result of the experiment, when compared to the UC isolation method of the comparative example, both Di sc-20 and Di sc-100 showed high antigen expression overall, and showed a large difference in the expression level of EpCAM antigen between normal patients and cancer patients. As shown in the graphs A, B, C, and D of FIG. 13, the patient's blood-derived nano endoplasmic reticulum (BCA), total number of nanoparticles (NTA), and purity (NTA measured by NTA) were measured through four experiments. The number divided by the total protein mass measured by BCA), the nano-vesicle surface markers analyzed by ELISA results, it can be seen that excellent results in the Di sc-100 Example.

FIG. 14 compares and shows detection CT values of nano-vesicle detection results through the device according to the present embodiment and the conventional device. The experiment was performed by extracting the nucleic acid of blood-derived nano endoplasmic reticulum isolated from the normal plasma of 200 microliters and detecting it by real-time PCR.

In FIG. 14, the results of the examples are denoted by Exodi sc, and ᅳ Comparative Examples are denoted by UC and Exospin, and show detection results according to the prior art compared with the examples.

As a result of the experiment, the expression level and detection rate of the nano vesicle surface antigens CD81, CD63, and CD9 were significantly higher than those of the UC and Exospin of the comparative example.

Figure 15 shows the extraction of nucleic acid of blood-derived nano-endoplasmic reticulum from the plasma subjected to the sample pretreatment step and the plasma pretreatment step using the proteinase K solution using the apparatus according to the present embodiment and real-t ime PCR (RT- PCR shows a result of detection.

The experiment was performed by extracting nucleic acids from nano vesicles separated from the normal plasma of 200 microliters using the device according to the present example. The graph of FIG. 15 shows detection CT values of GAPDH (House keeping gene), CD9, CD63, and CD81 (nano vesicle detection marker) through RT-PCR with nucleic acids extracted according to the present example.

As shown in the experimental results, when sample pretreatment was performed using Proteinase K (enzyme that degrades protein), mRNA expression was about five times higher than that of untreated pl asma sample. That is, it means that other proteins in the plasma, other than the nano-vesicles, may enjoy the nucleic acid detection results, and nucleic acid amplification performance may be improved by sample pretreatment using Proteinase K. FIG. 16 is a graph illustrating separation of nanovesicles from BT474 breast cancer cell line) cell culture medium according to size differences and analysis of surface proteins of nanovesicle fract ions having respective size distributions using the apparatus according to the embodiment of FIG. 5. For example, in a structure using two filtration membranes with pore sizes of 20 nm and 600 nm, a mixture of all nanoparticles ranging in size from 20 nm to 600 nm will be present on the 20 nm AA0 filter. Surface protein and molecular properties are as shown in FIG.

As such, the nanoparticles may be separated by a size difference, and analysis for the nanoparticles having each size distribution may be performed.

In this experiment, the size of the nanoparticles recovered in each filtration membrane was analyzed using a disk having a M0 filtration membrane having a pore size of 200 nm, 100 nm, and 20 nm as in the example of FIG. 5. Graph A of FIG. 16 shows the results of nano-vesicle separation according to the combination of the filtration membranes provided in the three filter chambers. In the A graph, on AA0 200 nm is a nano vesicle detected in the filtration membrane (200 nm) of the first filter chamber, on M0 100 nm is a nano vesicle detected in the filtration membrane (100 nm) of the crab 2 filter chamber, and on AA0 20 nm is The nano vesicles detected in the filtration membrane (20 nm) of the three filter chambers are shown. Post-f l trat ion refers to a solution after passing through a filtration membrane (20 nm) in a low 13 filter chamber.

As shown in the graph A of FIG. 16, the nanoparticles having the average size of 255 nm, 141 nm, 78 nm, and 10 nm, respectively, in the solution (post-f il trat ion) passed through all the filters, AA0 200 nm, AA0 100 nm and AA0 20 nm The particle solution can be recovered.

Therefore, as a result of the experiment, as shown in Graph A, nano-vesicles of a desired size were detected on the filtration membranes of the respective filter chambers. Thus, it can be seen that the present invention can be detected by separating the nano-vesicles of various sizes. The graph B of FIG. 16 shows the detection results of BT474 breast cancer cell-derived nano vesicles separated from the filter membrane of each filter chamber. The graph B of FIG. 16 shows normal i ze based on the amount of surface proteins of EpCAM and Sial i c acid of the nano vesicles.

In the case of 20 nm M0 only, it means a nano-vesicle recovered on the 20 nm filter membrane using a 20 nm M0 filter membrane. That is, in the case of the embodiment having three filter chambers It refers to a nano-vesicle having a size of 20 nm or more detected in the third filter chamber (20 nm filtration membrane) without passing through the first filter chamber (200 nm filtration membrane) and the second filter chamber (100 nm filtration membrane). In the case of exofree medium, it means that the pure culture solution without the nano-vesicles was passed through a 20 nm filtration membrane.

Experimental results show that EPCAM (Epi theel Cel l adhesion molecule, a marker used for cancer detection) is the highest expression level on On 20nm MO, more in relatively small nano-vesicle fract ion with a range between 20-100 nm It can be seen that a lot of detection. In addition, the expression level of Sialic acid (marker used for cancer detection) is the highest in On 100nm AA0, it can be confirmed that more detected in the nano-vesicle fract ion having a range between 100-200 nm.

Thus, it can be seen that the present invention can selectively recover the nano vesicles fract ion having a different size distribution.

Through such experiments, it can be seen that the nanoparticles can be detected more effectively using the detection apparatus according to the present embodiment.

In the above description of the preferred embodiment of the present invention, the present invention is not limited thereto, and various modifications and changes can be made within the scope of the claims and the detailed description of the invention and the accompanying drawings. Naturally, it belongs to the range of.

[Explanation of code]

10 ： Disc 12 ： Top plate

14 ： Bottom plate 20 ： Sample holding part

21: sample chamber 22: first flow path

23 ： U-valve 24 ： Sedimentation part

25 ： Groove 30 ： Filter

31 ： Filter chamber 311: Giant 11 filter chamber

312: 2nd filter chamber 313: 3rd filter chamber

315, 317: Euro 32: Entrance space

33 ： Reduced space 34 ： Filtration membrane

35: preliminary chamber 36: transfer path

37 ： Transfer valve 40 ： Goggles

ᄋ ᄇ 丁 Antibody chamber 42 2nd flow path 2 valve 44 Reagent chamber 3rd flow path 46 3rd valve Substrate fluid chamber 48th flow path 4th valve 50 Stopper chamber cleaning part 61 Washing liquid chamber 5th flow path 63 ^ 15 Valve waste liquid receiving part 71 Waste liquid chamber ger 16 Euro 73 Geo 16 Valve return chamber 81 7 Euro 7 valve 90 Cover bolt

Claims

[Claim]

[Claim 1]

A disk carrying fluid by centrifugal force,

A sample accommodating part formed on the disc to accommodate a sample;

A filter part connected to the sample accommodating part and having a fine filtration membrane for separating the nanoparticles by filtering the transferred sample;

A supply unit connected to the filter unit and supplying a detection liquid for detecting nanoparticles separated from the filtration membrane;

A waste liquid containing part connected to the filter part outlet side to receive the solution passed through the filtration membrane;

A flow path formed in the disk to transfer fluid,

A valve for selectively opening and closing the flow path

Nanoparticle detection device comprising a.

[Claim 2]

The method of claim 1,

The filter unit includes a filter chamber having an entrance space and an exit space along a fluid movement direction, and the filtration membrane is installed between the entrance space and the exit space.

The entrance space is connected to the sample receiving portion and the sample is introduced and the filtered nanoparticles are accommodated, and the exiting space is connected to the waste liquid receiving portion.

[Claim 3]

The method of claim 2,

The filtration membrane is a nanoparticle detection device the pores are formed of lnm to 100m.

[Claim 4]

The method of claim 2,

The filtration membrane is made of a polycarbonate, polystyrene, polymethyl methacrylate, a thermosetting plastic containing a cyclic olefin copolymer, anodized aluminum, nickel, or silicon material Particle detection device.

[Claim 5]

The method of claim 2,

The filter unit is at least two filtration membrane is stacked between the entrance space and the exit space, each of the filtration membrane nanoparticle detection device that the pores gradually decrease along the fluid transport direction.

[Claim 6]

The method of claim ² ,

The filter unit is disposed sequentially along at least two or more fluid transfer direction, the filter membrane provided in each filter unit is different in size of pores, each nanoparticle detection device for separating the nanoparticles of different size range.

[Claim 7]

The method of claim 6,

At least two filter units are disposed sequentially in the fluid transfer direction, and the filtration membranes provided in each filter unit gradually decrease pores along the fluid transfer direction.

[Claim 8]

The method of claim 2,

And the filtration membrane is detachably installed in the filter chamber of the disk.

[Claim 9]

The method of claim 8,

The filter unit further comprises a cover detachably installed on the disk for opening and closing the filter chamber, the fastening unit for fixing the cover to the disk.

[Claim 10]

According to claim 1,

The sample accommodating part is formed in the disc to accommodate a sample chamber for accommodating the sample, and connects the sample chamber and the filter part according to the centrifugal force of the disc. And a first flow path through which a sample is transferred, and a first valve for opening and closing the first flow path.

[Claim 11]

The method of claim 10,

The sample chamber is a centrifugal separation of the sample in accordance with the disc centrifugal force, and the settling portion for receiving the sample centrifuged at the leading end toward the outside of the disc in the direction of the centrifugal force is formed elongated,

The first flow path is nano-particle detection apparatus is towards the center of rotation of the disk connected to the depression border of the sample chamber, transferring the centrifuged supernatant portion ^"filter.

[Claim 12]

The method of claim 11,

The settling portion is nanoparticle detection device formed inclined inclined with respect to the radial direction of the disk.

[Claim 13]

The method of claim 11,

The settling portion further comprises a groove portion in which the bottom surface is gradually inclined upward toward the end along the direction of the centrifugal force at the boundary point, is formed at the end of the settling portion to accommodate the impurities centrifuged from the sample.

[Claim 14]

According to the clause 1 1,

The sample is a nanoparticle detection device that is a biological sample or a nanoparticles dispersed in the blood, lymph, tissue, urine, saliva, cerebrospinal fluid and sputum containing bioparticles or a combination thereof.

[Claim 15]

The method of claim 1,

And the sample accommodating part, the filter part, the supplying part, and the waste liquid receiving part constitute one unit, and the unit is arranged in a plurality spaced apart along the circumferential direction of the disk.

[Claim 16]

The method of claim 1,

The flow path connecting the two adjacent components of the disk inlet is located in the center of the disk than the outlet, the inlet is connected to the outlet side of the chamber along the direction of the centrifugal force of the disk.

[Claim 17]

The method of claim 1,

The disk is a nanoparticle detection device surface-modified with proteins, polymers, or organic molecules to prevent non-specific antibody adhesion.

[Claim 18]

The method according to any one of claims 1 to 17,

The supply unit is formed in the disk and the antibody chamber for receiving the antibody provided for nanoparticle detection, the second flow path for connecting the antibody chamber and the filter unit and transfer the antibody to the filter unit in accordance with the centrifugal force of the disk, and the second flow path Nanoparticle detection device comprising two valves to open and close the.

[Claim 19]

The method of claim 18,

The supply part is formed in the disk, a reagent chamber containing a reagent provided for labeling an antibody for detecting nanoparticles, a third flow path connecting the reagent chamber and the filter part and transferring the reagent to the filter part according to the centrifugal force of the disk, and The nanoparticle detection device further comprises a three-valve valve for opening and closing the three-channel crab. .

[Claim 20]

According to claim 19,

The supply part is formed in the disk and the substrate liquid chamber for receiving the substrate liquid provided for the detection of nanoparticles, the fourth flow path for connecting the substrate liquid chamber and the filter unit and transfer the substrate liquid to the filter unit in accordance with the centrifugal force of the disk, And a fourth valve for opening and closing the fourth flow path.

[Claim 21]

In accordance with paragraph 20, The supply part is a supply part is formed in the disk is a stop solution chamber for receiving a stop solution (stop solut ion) provided to stop the nanoparticle detection reaction, a connection flow path for connecting the stop solution chamber and the substrate liquid chamber, and the connection flow path Nanoparticle detection device further comprising a connection valve for opening and closing the.

[Claim 22]

The method of claim 21,

The supply unit further comprises a washing unit for cleaning the filter unit by transferring the washing liquid to the filter unit.

[Claim 23]

The method of claim 22,

The washing unit includes a washing liquid chamber formed in the disk to receive the washing liquid, a fifth flow passage connecting the washing liquid chamber to the filter unit and transferring the washing liquid to the filter unit according to the centrifugal force of the disk, and a fifth valve opening and closing the fifth flow passage. Nanoparticle detection device.

[Claim 24]

The method of claim 22,

The cleaning liquid chamber is divided into a plurality, the cleaning liquid is contained in each of the cleaning liquid chamber, and the nanoparticle detection device is provided with an outlet valve for discharging the cleaning liquid in the outlet flow path of each washing liquid chamber.

[Claim 25]

The method of claim 22,

To 7 flow path is formed in the disc transport of nanoparticles in recovery chamber to ^open, is determined connection recovery chamber where the number of the nanoparticles separated by a filtration membrane, the recovery chamber and the filter unit to the filter unit, the crab 7 euros Nanoparticle detection device further comprises a seventh valve for opening and closing the.

[Claim 26]

Feeding the sample to the disc,

Applying a centrifugal force to the disk and transferring the sample to the filter filter and separating the nanoparticles by filtration through the filtration membrane; and

The centrifugal force is applied to the disk and the detection liquid is supplied to the filter unit to Detecting nanoparticles

Nanoparticle detection method comprising a.

[Claim 27]

The method of claim 26,

Separating the nanoparticles, nanoparticle detection method for separating the nanoparticles of different size range through a plurality of different membranes with different pore sizes.

[Claim 28]

The method of claim 27,

Separating the nanoparticles, nanoparticle detection method for separating the nanoparticles of different size range sequentially through a plurality of filtration membranes the pores are gradually smaller.

[Claim 29]

The method of claim 26,

And a purification step of centrifuging the sample by applying a centrifugal force to the disk before transferring the sample to the filter portion.

[Claim 30]

30. The method according to any one of claims 26 to 29

The detecting of the nanoparticles includes applying a centrifugal force to the disk to supply the antibody to the filter unit to detect the nanoparticles, and supplying a reagent to the filter unit to label the antibody attached to the nanoparticles.

[Claim 31]

According to clause 1 29

The nanoparticle detection method further comprises the step of applying a centrifugal force to the disk and supplying the washing solution to the filter.

[Claim 32]

The method of claim 31, wherein

The washing step includes detecting the nanoparticles by supplying the antibody to the filter unit and washing and removing the excess antibody, and supplying the reagent to the filter unit to label the antibody and washing and removing the extra reagent. Way .

[Claim 33]

The method of claim 32,

The detecting of the nanoparticles may further include supplying a substrate solution to the nanoparticles by applying a centrifugal force to the disk.

[Claim 34]

The method of claim 33,

The substrate solution was fed after the nanoparticle is detected further includes the step of recovering the nanoparticles detection solution ^"method.

[Claim 35]

The method of claim 26,

In the detecting of the nanoparticles, the nanoparticle detection method further comprises the step of extracting the nucleic acid by supplying a nucleic acid extraction reagent to the separated nanoparticles.

[Claim 36]

The method of claim 26,

In the step of detecting the nanoparticles, comprising the step of separating the filter membrane from the disk, the nanoparticle detection method for detecting nanoparticles from the outside of the disk.