KR20180039005A - Apparatus for detecting nano particle, and method for detecting nano particle using the same - Google Patents

Abstract

Provided is an apparatus for detecting nanoparticles which can be used more easily by integrating an entire process for detecting nanoparticles from a sample, wherein the apparatus comprises: a disk on which a fluid is transferred by centrifugal force; a sample accommodating part formed on the disk to accommodate the sample; a filter part connected to the sample accommodating part and having a microfiltration membrane for separating nanoparticles by filtering the transferred sample; a supply part connected to the filter part to supply a detection solution for detecting separated nanoparticles into a filtration membrane; a waste solution accommodating part connected to the filter part outlet side to receive a solution filtered through the filtration membrane; a flow path formed on the disk and through which the fluid is transferred; and a valve for selectively opening and closing the flow path.

Description

TECHNICAL FIELD The present invention relates to a nanoparticle detection apparatus and a nanoparticle detection method using the same. BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a nanoparticle detection apparatus,

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

For example, nanosomes are small vesicles of 40-120 nm in size that are generated by cell activity. Nano-vesicles are distinguished from other vesicles by their origin and size. At the time of its discovery, the nanosphere was thought to be a cell by-product, but it was found to be of great importance as it contributes to cellular activities such as tumor progression and metastasis and cell signaling. The nanosphere is present in almost all body fluids of the body and contains the genetic information of the derived cells. For this reason, nanospores are attracting attention as new drug delivery systems as well as new markers for various diseases including cancer.

In recent years, researches on methods for separating and detecting nanospores are continuously increasing. Methods for separating nanospores are broadly classified into those using density, size, and affinity.

As a method for detecting a specific protein or nucleic acid of the nanospores after separating and capturing the nanospores, there is protein analysis for comparing the expression amount of a specific antigen and nucleic acid analysis for analyzing the genetic information of the generated cells. An enzyme-linked immunosorbent assay (ELISA) using a conventional 96-well plate assay method is a method of attaching a specific antibody or protein-specific nano-envelope to a plate and then detecting the antibody using another antibody. However, in the case of the conventional method, there is a limit in the surface area of a 96-well plate to which a nano-vesicle is attached, and there is a limit to the difficulty in analyzing an antibody adjacent to the nano-

A nanoparticle detecting apparatus and a nanoparticle detecting method using the nanoparticle detecting apparatus are provided which are integrated with a life cycle for detecting nanoparticles from a sample so that they can be used more easily.

A nanoparticle detecting apparatus capable of detecting a plurality of antigens and a method of detecting nanoparticles using the same are provided.

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

The detection device of this embodiment includes a disk on which a fluid is transported by centrifugal force, a sample accommodating portion formed on the disk to accommodate the sample, a fine filter connected to the sample accommodating portion and filtering the transferred sample to separate nanoparticles A supply part connected to the filter part to supply a detection solution for detecting nanoparticles separated into a filtration membrane, a waste solution storage part connected to the filter part outlet to receive a solution through the filtration membrane, And a valve for selectively opening and closing the flow path.

The sample accommodating portion includes a sample chamber formed in the disc to accommodate the sample, a first flow path for connecting the sample chamber and the filter portion, a first flow path for transporting the sample according to centrifugal force of the disc, and a first valve for opening and closing the first flow path .

Wherein the sample chamber is formed by centrifugally separating the sample according to the centrifugal force of the disk and extending along the direction of the centrifugal force toward the outer side of the disk so that the sediment part accommodating the centrifuged sample is elongated, The centrifuged supernatant may be connected to the boundary of the settling portion of the sample chamber and transferred to the filter unit.

The settling portion may be inclined with respect to the radial direction of the disc.

The bottom of the settling portion may be inclined upward gradually toward the end along the centrifugal force direction at the boundary point.

And a groove portion formed at an end of the sediment portion and containing impurities centrifuged in the sample.

And a discharge valve connected to the sedimentation outlet side of the sample chamber and the waste solution reservoir for transferring the sedimented solution, and a discharge valve for opening and closing the discharge passage.

The filter unit includes a filter chamber having an inlet space and an outlet space along the fluid moving direction and having the filtration membrane between the inlet space and the outlet space. The inlet space is connected to the sample storage unit, The particles are accommodated, and the outlet space can be connected to the waste liquid receiving portion.

The filtration membrane may have pores of 1 nm to 1000 nm.

The filtration membrane may be made of thermosetting plastic, anodic aluminum oxide, nickel, or silicon material including polycarbonate, polystyrene, polymethyl methacrylate, cyclic olefin copolymer.

At least two or more filtration membranes are stacked between the inlet space and the outlet space, and the pores of the respective filtration membranes may be gradually reduced along the fluid transport direction.

The filter unit may be sequentially arranged along at least two fluid transfer directions, and the filtration membrane provided in each filter unit may have a structure in which nanoparticles of different size ranges are separated by different sizes of pores.

At least two or more of the filter units may be sequentially disposed along the fluid transport direction, and the filtration membrane provided in each filter unit may gradually have pores along the fluid transport direction.

The filtration membrane may be removably installed in the disk.

A lid removably installed on the disc to open and close the filter chamber, and a fastening portion for fastening the lid to the disc.

The supply unit includes an antibody chamber formed on the disk to receive an antibody provided for nanoparticle detection, a second flow path for connecting the antibody chamber to the filter unit and transferring the antibody to the filter unit according to the centrifugal force of the disk, And a second valve that opens and closes.

The supplying unit may include a reagent chamber formed on the disc and containing a reagent provided for labeling an antibody for detecting nanoparticles, a third flow path connecting the reagent chamber and the filter unit and transferring the reagent to the filter unit according to the centrifugal force of the disc, And a third valve for opening and closing the third flow path.

The supply unit includes a substrate liquid chamber formed on the disk to receive a substrate liquid for nanoparticle detection reaction, a fourth flow path for connecting the substrate liquid chamber and the filter unit, and transferring the substrate liquid to the filter unit according to the centrifugal force of the disk, And a fourth valve for opening / closing the fourth flow path.

The supply part includes a stop solution chamber formed in the disc and adapted to stop the nanoparticle detection reaction, a stop solution chamber for stopping the nanoparticle detection reaction, a connection channel for connecting the stop solution chamber and the substrate solution chamber, The stop solution can be supplied to the filter chamber through the substrate liquid chamber, including a connecting valve that opens and closes.

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

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

The cleaning liquid chambers are divided into a plurality of chambers, and the cleaning liquid is contained in each of the cleaning liquid chambers. The outflow channel of each of the cleaning liquid chambers may be provided with an outflow valve for discharging the cleaning liquid.

The waste liquid receiving portion may include a waste liquid chamber formed in the disk to receive the waste liquid, and a sixth flow path that connects the waste liquid chamber and the filter portion and transfers the waste liquid to the waste liquid chamber according to the centrifugal force of the disk.

A recovery chamber formed on the disk and connected to the filter unit to recover nanoparticles separated by a filtration membrane, a seventh flow path connecting the recovery chamber and the filter unit to transfer the nanoparticles to the recovery chamber, The seventh valve may be provided.

The sample may be a blood sample containing a biological particle, a biological sample selected from blood, lymph, tissue fluid, urine, saliva, cerebrospinal fluid and sputum or an aqueous solution in which nanoparticles are dispersed or a combination thereof.

The sample accommodating portion, the filter portion, the supply portion, and the waste liquid accommodating portion constitute one unit, and the units may be arranged with a plurality of intervals along the circumferential direction of the disk.

The flow path connecting the adjacent two chambers is located at the center of the disk than the outlet, and the inlet can be connected to the outlet of the one chamber along the centrifugal force direction of the disk.

The disc may be surface-modified with a protein, a polymer, or an organic molecule so as to prevent nonspecific antibody attachment.

The nanoparticle detection method of this embodiment comprises the steps of feeding a sample to a disk, transferring the sample to a filter section by centrifugal force on the disk, filtering the nanoparticles through a filtration membrane, separating the nanoparticles, And supplying the detection liquid to the filter section to detect nanoparticles on the filtration membrane.

The step of detecting the nanoparticles may include a step of applying a centrifugal force to the disk to supply the antibody to the filter section, detecting the nanoparticles, and supplying a reagent to the filter section to label the antibody attached to the nanoparticles.

And separating the sample by centrifugal force to centrifuge the sample before transferring the sample to the filter section.

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

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

Centrifugal force is applied to the disk, and the washing liquid is supplied to the filter unit to clean the disk.

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

And labeling the nanoparticles and then supplying a substrate solution.

And recovering nanoparticles separated from the filtration membrane.

The step of detecting the nanoparticles may further include supplying nucleic acid extraction reagents to the separated nanoparticles to extract the nucleic acid.

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

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

As the detection is performed by feeding the antibody into the separated space of the nanoparticles, all the antigens on the surface of the nanoparticles can be effectively detected. Therefore, nanoparticles can be detected within a short time, and the efficiency of the antibody for detection can be increased.

Therefore, it becomes possible to effectively diagnose diseases such as cancer and the research using the nano-vesicles.

1 is a schematic plan view showing a nanoparticle detecting apparatus according to an embodiment of the present invention.
2 is a schematic plan view showing a nanoparticle detection apparatus according to another embodiment.
3 is a schematic cross-sectional view showing a sample receiving portion of the nanoparticle detecting apparatus according to the present embodiment.
4 is a schematic cross-sectional view showing a filter unit of the nanoparticle detection apparatus according to the present embodiment.
5 is a schematic plan view showing a nanoparticle detection apparatus according to another embodiment.
6 is a schematic view for explaining the principle of nanoparticle detection in the nanoparticle detection apparatus according to the present embodiment.
7 is a graph showing the results of protein detection of nanoparticles according to this embodiment.
Fig. 8 is a graph showing the protein expression level of the urine-derived nano-particles according to the present embodiment in comparison with the conventional one.
FIG. 9 is a diagram showing the result of nucleic acid detection of nanoparticles according to the present embodiment in comparison with the conventional method.
FIG. 10 is a graph showing the results of detecting and separating nanoparticles from a sample spiked with plasma of a cancerous cell-derived nanosporter according to the present embodiment, in comparison with the conventional method.
FIG. 11 is a graph showing the result of detection and separation of nanoparticles according to spin conditions from a sample spiked with plasma from a cancer cell-derived nanospores according to this embodiment.
FIG. 12 is a graph showing the result of detecting and separating nanoparticles according to the volume of a sample spiked with cancerous cell-derived nanospores in plasma according to the present embodiment, in comparison with the conventional method.
FIG. 13 is a diagram showing the results of separation and protein detection of cancer patient plasma and normal plasma-derived nanoparticles according to the present embodiment in comparison with conventional ones.
FIG. 14 is a view showing separation of normal plasma-derived nanoparticles and nucleic acid detection results according to the present embodiment in comparison with conventional ones
FIG. 15 is a view showing the separation of plasma and the plasma-derived nanoparticles that have undergone the proteolytic enzyme treatment step according to the present embodiment and the result of nucleic acid detection.
FIG. 16 is a graph showing nanoparticle protein detection results of the nanoparticle detection apparatus according to the embodiment of FIG.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Wherever possible, the same or similar parts are denoted using the same reference numerals in the drawings.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms as used herein include plural forms as long as the phrases do not expressly express the opposite meaning thereto. Means that a particular feature, region, integer, step, operation, element and / or component is specified, and that other specific features, regions, integers, steps, operations, elements, components, and / And the like.

All terms including technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Predefined terms are further interpreted as having a meaning consistent with the relevant technical literature and the present disclosure, and are not to be construed as ideal or very formal meanings unless defined otherwise.

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

The detection device of the present embodiment is provided with a disk for transferring fluid by centrifugal force, a sample accommodating portion formed on the disk for accommodating the sample, a filtration membrane connected to the sample accommodating portion and for filtering the transferred sample to separate the nanoparticles A filter unit, a supply unit connected to the filter unit to supply a detection solution for detecting the nanoparticles separated in the filter membrane, a waste solution storage unit connected to the filter unit for storing the solution filtered through the filter membrane, And a valve that selectively opens and closes the flow path, the flow path, and the flow path.

Here, detection may mean all the detection and analysis of a specific protein or nucleic acid of a nanoparticle, including detecting and identifying the presence or absence of a specific antigen of the nanoparticle and extracting the nucleic acid of the nanoparticle.

Discs form the body of the device. The disk has its constituent parts inside.

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

The disk may be formed by joining two plate structures of an upper plate and a lower plate in the form of a disk. The disk can be formed by joining a plurality of plates in addition to the two plate-joining structures of the upper plate and the lower plate. For example, the disc may further include a plate structure having a valve formed between the upper plate and the lower plate.

A cavity of a predetermined shape may be formed on the inner surface of the upper plate and the lower plate so as to form the constituent parts and the flow path. A space formed between the upper plate and the lower plate forms a flow path and a chamber for receiving or transferring the fluid. The top and bottom plates making up the disc can be made of a biologically inert material whose surface is in contact with the fluid. Further, the upper plate and the lower plate may be made of a material having optical transparency. For example, the top and bottom plates may be made of polysrene (PS), polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyacrylate, polycarbonate, , Polyaclic olefins, polyimide polyurethanes, and the like.

In addition, the upper and lower plates constituting the disc may be surface-modified with a protein, a polymer, or an organic molecule to prevent nonspecific antibody attachment.

Therefore, when a sample is supplied to the disk and moved along the flow path, the sample does not react with the disk and does not adhere to the surface, thereby securing the biological stability and increasing the reactivity with the antibody. In addition, it is possible to detect and transmit the separated nanoparticles through the upper and lower plates through the optical detector without discharging the nanoparticles to the outside of the disk.

The present embodiment may be a structure for detecting a plurality of different nanoparticles using one disk. To this end, the sample accommodating portion, the filter portion, the supply portion, and the waste liquid accommodating portion, which are necessary for nanoparticle detection, constitute one unit, and a plurality of the units may be arranged along the circumferential direction of the disk. 1 illustrates, for example, a structure in which two units are arranged. A variety of units may be provided on one disk depending on the size of the disk, the size of each unit, and the like.

Therefore, it is possible to simultaneously detect a plurality of nanoparticles with a single disk by preparing samples or detection solutions that are different from each other in a plurality of units provided in the disk.

The sample accommodating portion, the supply portion, the filter portion, and the waste liquid accommodating portion are sequentially positioned along the centrifugal force direction from the rotation center of the disk. Thus, the fluid flows from the sample accommodating portion and the supply portion to the filter portion and from the filter portion to the waste liquid accommodating portion. Therefore, by applying the centrifugal force by rotating the disk, the fluid can be sequentially moved to perform a desired process.

A flow path is formed between the respective constituent parts of the disk. The flow path connects the chambers of neighboring components. The flow path has an inlet through which the fluid flows and an outlet through which the fluid exits, and the inlet may be located closer to the center of the disk than the outlet. Thus, when the centrifugal force of the disk is applied, the fluid can move more smoothly along the outlet at the entrance located relatively to the center of the disk.

The flow path may be, for example, a micro flow path in which impurities are difficult to flow backward. The microfluidic channel may mean a channel formed in a size such that the channel has a resistance capable of preventing backflow of the fluid. Thus, for example, it becomes possible to prevent impurities contained in the fluid discharged to the waste liquid containing portion through the filter portion from flowing back to the filter portion along the flow path.

Further, the flow path may be connected to the outlet of each component along the centrifugal force direction of the disk. The term " output of the component " means the portion of the component to which the fluid flows out, which means the end near the outer end of the disk along the centrifugal force direction.

Accordingly, when centrifugal force is applied to the disk, the fluid contained in each component is collected by the centrifugal force toward the inlet of the flow path, and can be moved along the flow path. Therefore, since the fluid does not remain in the chambers of the respective components, the loss of the fluid can be minimized.

For example, as the flow path is connected to the outflow side of the sample accommodating portion, all of the samples accommodated in the sample accommodating portion can be moved to the filter portion through the flow path. Therefore, the 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 have a variety of configurations that can block or open the flow path. The valve can open / close the flow path by applying power to itself, or can open and close the flow path by receiving driving force from the outside of the disk.

When the valve is driven and the flow path is closed, the fluid transfer between the constituent parts connected by the flow path is blocked. If necessary, the valve is driven to open the flow path, and fluid is transferred between the components to start the necessary process.

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

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

In the following description, each of the flow paths, valves, and chambers formed on the disk will be referred to as being separately classified according to their constituent parts for convenience of explanation. In the case of describing a flow path, a valve, and a chamber, the flow path, the valve, and the chamber formed on the disk may be referred to.

In this embodiment, the disk 10 can be formed by joining two plate structures of an upper plate (see 12 in Fig. 4) and a lower plate (see 14 in Fig. 4) in the form of a disk.

The sample accommodating portion 20 of the present embodiment is formed on the disk 10 to connect the sample chamber 21 accommodating the sample and the sample chamber 21 and the filter portion 30, And a first valve 23 that opens and closes the first flow path 22. The first valve 22 is provided with a first flow path 22,

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 path 22. The sample chamber 21 may have a hole for injecting the sample on one side thereof. The sample accommodated in the sample chamber 21 is moved through the first flow path 22 by the centrifugal force of the disk 10. A first valve (23) for opening and closing the first flow path (22) is installed at one side of the first flow path (22). When the first valve 23 is operated to open the first flow path 22, the sample of the sample chamber 21 is moved to the filter portion 30 along the first flow path 22.

The filter unit 30 includes a filter chamber 31 having the inlet space 32 and the outlet space 33 along the fluid moving direction and having the filtration membrane 34 disposed between the inlet space 32 and the outlet space 33, . ≪ / RTI > The filter chamber 31 is an empty space formed in the disk 10 and can be divided into an inlet space 32 and an outlet space 33 with the filtration membrane 34 as a boundary. The inlet space 32 of the filter chamber 31 is a space into which a fluid such as a sample is introduced and is a space for accommodating the filtered nanoparticles. The detection of the nanoparticles in the inlet space 32 is performed.

A ventilation hole may be formed at one side of the filter chamber 31 so that filtration can be smoothly performed. The vent can be formed, for example, on the top plate. Accordingly, when the sample is filtered, air or the like existing in the filter chamber 31 is discharged to the outside of the disk 10, so that filtration can be smoothly performed.

The inlet space 32 is connected to the sample chamber 21 of the sample accommodating portion 20 through the first flow path 22. The outflow space 33 is connected to the waste liquid receiving portion 70. Thus, the sample introduced into the inlet chamber is separated from the nanoparticles via the filtration membrane (34). The separated nanoparticles are accommodated in the inlet space 32 and the filtrate having passed through the filtration membrane 34 is discharged to the waste fluid receiver 70 connected to the outlet space 33.

In this embodiment, the permeate can be received in advance in the outflow space 33 of the filter chamber 31.

The permeate may be the same solution as that in which the nanoparticles are separated from the sample, that is, the same solution as the filtrate filtered out through the filtration membrane (34). Or the permeate can be any solution that does not have particles to be separated and does not affect the sample or the filtrate and that can reduce the capillary pressure of the pores of the filtration membrane 34 at the outlet side of the filtration membrane 34.

Thus, the sample introduced into the inlet space 32 of the filtration membrane 34 can easily pass through 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 permeated liquid can be previously accommodated in the outflow space 33 of the filter chamber 31, for example, when the apparatus is manufactured. Alternatively, a separate inlet port connected to the outlet space 33 of the lower plate of the disk 10 may be formed, and the permeate may be injected into the outlet space 33 through the inlet after the device is manufactured.

The filtration membrane 34 is a membrane structure in which numerous pores are formed on the surface. In this embodiment, the filtration membrane 34 may have pores of 1 nm to 1000 nm for separating the nanoparticles contained in the sample. The filtration membrane 34 may be selected to have an appropriate pore size within the above range depending on the type and 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 lowered.

The filtration membrane 34 may be formed of various materials so as 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, a thermosetting plastic including a cyclic olefin copolymer, anodized aluminum, nickel, silicon, or the like. For example, a filtration membrane formed from an anodized aluminum material has higher porosity and comparatively uniform diameter pores than other materials.

The filtration membrane 34 of the present embodiment may be formed of a biologically inert material so as to be applicable to a biological sample. Further, the filtration film 34 may be formed of a material having optical transparency at the same time. Thus, the nanoparticles can be detected using an optical detector without separating the filtration film 34 from the disk 10.

In addition to the structure for detecting nanoparticles on the disk 10, the present apparatus can perform the detection process by separating the filtration film 34 from the disk 10. The separation structure of the filtration membrane 34 will be described in detail later.

The filter portion may have a single filtration membrane or may have a structure having two or more filtration membranes in one filter chamber. In the case of a structure having two or more filter membranes in the filter chamber, a plurality of filter membranes may be stacked and disposed between the inlet space and the outlet space. Each of the filtration membranes may have a structure in which the pore size gradually decreases from the inlet space toward the outlet space along the fluid transport direction. For example, the filtration membrane disposed on the inlet space side may have a pore size of 50 nm, and the filtration membrane stacked below the outlet space may have a pore size of 5 nm.

The sample containing nanoparticles first separates the primary particles while passing through the large filtration membrane arranged toward the inlet space, and separates the nanoparticles as the next pores pass through the small filtration membrane. Therefore, only the nanoparticles of a specific size range can be selectively collected and collected.

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

In this embodiment, the supply unit 40 is for detecting the separated nanoparticles in the filtration film 34, and may be a structure for supplying the detection liquid to the filter unit 30. [ The detection liquid may be a substance for detecting nanoparticles, for example, an antibody for an antigen, a reagent for labeling an antibody, a reaction substrate, a nucleic acid extraction solution, a washing solution, or the like. The detection liquid is applicable to any substance that can be used for detecting nanoparticles separated from the filter unit.

To this end, the supply unit 40 includes an antibody chamber 41 formed in the disk 10 for receiving the antibody provided for nanoparticle detection, a disk 10 connected to the antibody chamber 41 and the filter unit 30, A second flow path 42 for transferring the antibody to the filter section 30 according to the centrifugal force of the first flow path 42, and a second valve 43 for opening and closing the second flow path 42.

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 path 42. The antibody chamber 41 may have a hole for injecting the antibody on one side thereof. The antibody contained in the antibody chamber 41 is moved through the second flow path 42 by the centrifugal force of the disk 10. A second valve (43) for opening and closing the second flow path (42) is installed at one side of the second flow path (42). Thus, when the second valve 43 is operated to open the second flow path 42, the antibody in the antibody chamber 41 is moved to the filter chamber 31 along the second flow path 42.

In this embodiment, the antibody may be a primary detection antibody that binds to the biomarker of the nanoparticle to capture and fix the biomarker, and may be a detection antibody with biotin attached thereto.

The supply section 40 includes a reagent chamber 44 formed in the disk 10 for accommodating the reagent provided for the labeling of the antibody that detects nanoparticles, a reagent chamber 44 and a filter section 30, A third flow path 45 for transferring the reagent to the filter unit 30 according to the centrifugal force of the disk 10 and a third valve 46 for opening and closing the third flow path 45.

The reagent chamber 44 can be understood as an empty space formed inside the disk 10. [ The reagent chamber 44 is connected to the filter chamber 31 through the third flow path 45. The reagent chamber 44 may have a hole for injecting the reagent on one side thereof. The reagent contained in the reagent chamber 44 is moved through the third flow path 45 by the centrifugal force of the disk 10. A third valve (46) for opening and closing the third flow path (45) is installed at one side of the third flow path (45). When the third valve 46 is operated to open the third flow path 45, the reagent in the reagent chamber 44 is moved to the filter chamber 31 along the third flow path 45.

In this embodiment, the reagent may be a fluorescent secondary attached antibody that binds to the antibody. The reagent can be applied to any substance that can analyze and quantify the biomarker using fluorescent light or the like optical signal. For example, the reagent may be streptavidin-HRP to amplify the antibody signal. The reagent amplifies the signal of the detection antibody and is advantageous for measurement.

The supply unit 40 includes a substrate liquid chamber 47 formed in the disk 10 to receive a substrate liquid for nanoparticle detection reaction and a substrate liquid chamber 47 connected to the substrate liquid chamber 47 and the filter unit 30, A fourth flow path 48 for conveying the substrate liquid to the filter section 30 according to the centrifugal force of the first flow path 10 and a fourth valve 49 for opening and closing the fourth flow path 48.

The substrate liquid chamber 47 can be understood as an empty space formed inside the disk 10. The substrate liquid chamber 47 is connected to the filter chamber 31 through the fourth flow path 48. The substrate liquid chamber 47 may have a hole for injecting the substrate liquid on one side thereof. 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. A fourth valve (49) for opening and closing the fourth flow path (48) is installed at one side of the fourth flow path (48). Thus, when the fourth valve 49 is operated to open the fourth flow path 48, the substrate liquid of the substrate liquid chamber 47 is moved to the filter chamber 31 along the fourth flow path 48.

In this embodiment, the substrate solution may be TMB (Tetramethylbenzidine) solution. TMB is a substrate of HRP, and its color changes as the structure is changed by HRP. TMB (3,3 ', 5,5'-tetramethylbenzidine) is blue when oxidized to hydrogen peroxide by peroxidase catalysis and has the maximum optical density (OD) value at 370 nm and 652 nm. When stopped, it turns yellow and shows the maximum OD value at 450 nm.

In addition, the supply unit 40 may be a structure that provides a stop solution provided to stop the nanoparticle detection reaction. The stopping solution chamber 50 is formed in the disc 10 to receive the stopping solution and the stopping solution chamber 50 is connected to the substrate liquid chamber 47 And the stop solution may be supplied to the filter chamber 31 through the substrate liquid chamber 47 in accordance with the driving of the connection valve 52 provided at one side of the connection channel 51.

The stop solution may be a solution containing a strong acid. The stop solution stops the reaction in which the substrate liquid changes color by the enzyme.

The stop solution chamber 50 connected to the antibody chamber 41, the reagent chamber 44 and the substrate solution chamber 47 and the substrate solution chamber 47 is moved along the centrifugal direction of the disk 10 And is located at the center of the disk 10. When the centrifugal force is applied, the fluid contained in the antibody chamber 41, the reagent chamber 44, the substrate liquid chamber 47 and the stationary solution chamber 50 is transferred to the filter chamber 31 along the flow path by the centrifugal force do. The valves installed on the respective flow paths open or close the corresponding flow paths in accordance with the process order to sequentially supply necessary fluids to the filter chamber 31.

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

The cleaning unit 60 includes a cleaning liquid chamber 61 formed in the disk 10 to receive the cleaning liquid, a cleaning liquid chamber 61 connected to the cleaning liquid chamber 61 and the filter unit 30, A fifth flow path 62 for transferring the cleaning solution to the filter unit 30, and a fifth valve 63 for opening and closing the fifth flow path 62.

The cleaning liquid chamber 61 can 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 cleaning liquid chamber 61 may have a hole for injecting the cleaning liquid on one side thereof. 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. A fifth valve (63) for opening and closing the fifth flow path (62) is installed at one side of the fifth flow path (62). Thus, when the fifth valve (63) is operated to open the fifth flow path (62), the cleaning liquid in the cleaning liquid chamber (61) is moved to the filter chamber (31) along the fifth flow path (62).

The washing liquid is removed by washing the excess antibody or reagent remaining in the filter chamber 31. The washing liquid is moved to the filter chamber 31 and then the inlet space 32 of the filter chamber 31. The washing liquid then flows through the filtration membrane 34 to the outlet space 33, And is discharged to the waste liquid containing portion 70 through the waste liquid receiving portion 70.

In this process, an excessive amount of the reagent not bound to the nanoparticles, which is not bound to the antibody or antibody, is washed in the inlet space 32 of the filter chamber 31 and removed together with the washing liquid into the outlet space 33.

In the present embodiment, a plurality of cleaning liquid chambers 61 are provided so that the cleaning liquid can be separately accommodated in each of the cleaning liquid chambers 61. Thus, the inside of the filter chamber 31 can be cleaned a plurality of times, if necessary. With this structure, the outflow channel of each of the washer fluid chambers 61 can be connected to the fifth flow path 62 to supply the washer fluid to the filter chamber 31 through the fifth flow path 62. Also, as shown in FIG. 1, the outlets of the respective washing liquid chambers 61 are connected to each other, and an outflow valve 64 for discharging the washing liquid may be installed in the connecting portion. That is, each of the washer fluid chambers 61 are disposed side by side and the outer ends of the adjacent washer fluid chambers 61 are connected to each other. If necessary, the fifth valve 63 and the outflow valve 64 provided at the connection portion of the respective washing liquid chambers 61 can be sequentially opened to supply the washing liquid in the respective washing liquid chambers 61 to the filter chamber 31 in turn .

The washing liquid chamber 61 is located closer to the center of the disk 10 than the filter chamber 31 along the centrifugal force direction of the disk 10 as in the other chambers of the supplying section 40. Thus, when centrifugal force is applied, the washing liquid, which is in demand water, is sent to the filter chamber 31 along the flow path by centrifugal force in the washing liquid chamber 61.

The waste liquid storage portion 70 is formed in the disk 10 to connect the waste liquid chamber 71 in which the waste liquid is received, the waste liquid chamber 71 and the filter portion 30, And a sixth flow path 72 for transferring the gas to the first flow path 71. A sixth valve (see 73 in FIG. 2) for opening and closing the sixth flow path 72 may be further provided at one side of the sixth flow path 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 output 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. [

Vents may be formed on one side of the waste liquid chamber 71 so that the waste liquid can be smoothly moved to the output space 33 through the filtration film 34 of the filter chamber 31. The vent can be formed, for example, on the top plate. Accordingly, during the sample filtration, the air existing in the outflow space 33 of the filter chamber 31 and the waste fluid chamber 71 is discharged to the outside of the disk 10, and the filtration is more smoothly performed.

With the above-described structure, the present apparatus performs collective processing through the single disk 10 during the extraction and detection of the nanofibers after sample injection. Accordingly, it becomes possible to perform the nanoparticle detection operation more easily and quickly and effectively.

Fig. 2 shows another embodiment of the nanoparticle detection device.

The embodiment shown in FIG. 2 also has a basic configuration of the disk 10, the sample accommodating portion 20, the supply portion 40, the filter portion 30, and the waste liquid accommodating portion 70 provided in the disk 10 Is the same as the embodiment shown in Fig. Therefore, the same reference numerals are used for the same components in the following description, and a detailed description thereof will be omitted.

As shown in FIG. 2, the sample accommodating portion 20 of the present embodiment may be a structure for purifying a sample to remove non-target substances including impurities.

The sample accommodating portion 20 is formed in the disk 10 so that the sample chamber 21 housing the sample centrifips the sample according to the centrifugal force of the disk 10, And the first flow path 22 is extended toward the rotation center of the disk 10 in the sedimented portion of the sample chamber 21 24 to the filter unit 30, and centrifugally transport the supernatant to the filter unit 30. The non-target substance may mean an impurity other than the solution containing the nanoparticles to be purified.

The sample injected into the sample chamber 21 is centrifuged and purified by a centrifugal force according to the rotation of the disk 10. The sample is separated into a solution containing the nanoparticles and impurities outside the nanoparticles. The impurities of the solid phase along the direction of centrifugal force are pushed toward the outer tip of the disk 10 and the solution separated from the impurities is located toward the center of the disk 10. [

In this embodiment, the sedimentation section 24 is elongated along the direction of centrifugal force so that the separation between the solution containing the nanoparticles and the impurity is clearly shown in the space in the sample chamber 21. The settling portion 24 can be formed into a hopper shape whose width becomes narrower toward the outlets so that the impurities can be settled more easily. Thus, separation of impurities and solution precipitated by centrifugation can be surely performed, so that non-target substances other than the solution can be minimally introduced into the filter chamber 31.

The inlet of the first flow path 22 may be connected to a portion of the sample chamber 21 facing the rotation center of the disk 10 at the boundary between the non-target material and the solution. Thus, only the nanoparticle-containing solution centrifuged by the centrifugal force applied in accordance with the rotation of the disk 10 can be transferred to the filter chamber 31 through the first flow path 22. The impurities precipitated in the sedimentation section 24 are located outside the inlet of the first flow path 22 along the direction of centrifugal force so that the impurities can not be transported through the first flow path 22.

As described above, the sample is centrifuged and purified and then supplied to the filter chamber 31, whereby the separation and detection effect of the nanomaterial through the filtration membrane 34 can be further enhanced.

The outlet side of the sedimentation section 24 of the sample chamber 21 is connected to the waste liquid chamber 71 so that purified impurities in the sample chamber 21 can be treated by spouting the waste liquid chamber 71. A discharge passage 26 for transferring impurities is formed between the sedimentation portion 24 of the sample chamber 21 and the waste liquid chamber 71. A discharge valve 27 for opening and closing the discharge passage is formed at one side of the discharge passage 26, Respectively.

After the sample is centrifuged, the target solution is transferred to the filter chamber 31 and then the discharge channel 26 is opened to discharge the non-target material remaining in the sample chamber 21 to the waste liquid chamber 71 Can be removed.

The sample accommodating portion 20 of the present embodiment may be formed so that the settling portion 24 is inclined with respect to the radial direction of the disc 10 in order to improve the sample refining efficiency. 2, the settling portion 24 is formed by being inclined at a predetermined angle A with respect to the radial direction of the disk 10. [ Accordingly, in the centrifugal separation process, solid impurities easily flow down along inclined slopes of the sedimentary portion 24 with respect to the disk radial direction, so that sedimentation can be performed more easily, thereby enhancing the separation effect.

As shown in FIG. 3, the bottom surface 28 of the settling portion 24 is gradually inclined upward from the boundary point toward the end along the centrifugal force direction. At the tip of the settling portion 24, And a groove 25 in which centrifuged impurities are accommodated. 3 shows a cross sectional structure of the disk 10 in the width direction. 3, the upper side is the upper side and the lower side is the lower side along the y-axis direction. The sedimented 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 portion. The outgoing side of the settling portion 24 forms a deep groove (bolt 91) deeply in the vertical direction. Accordingly, the impurities of the solid phase precipitated on the exit side of the sedimentation section 24 due to the centrifugal force move along the inclined bottom surface of the sedimentation section 24 and are prevented from flowing back to the recess section 25.

That is, the groove portion 25 has a hole with a depth, and the inner circumferential surface of the groove portion is blocked. When impurities in the solid phase fall into the groove portion 25, the impurity can not escape from the groove portion 25 even in a state where centrifugal force is not applied. Accordingly, it is possible to minimize the backflow of the impurities to the boundary point of the sample chamber 21, and to supply only the purified sample to the filter chamber 31. The discharge flow path is connected to the groove portion 25 and the impurities which have fallen into the groove portion 25 are discharged to the waste liquid chamber 71 through the discharge flow path 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 transport 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 explanation, the filter chamber disposed in front of the filter chamber 31 in which detection of nanoparticles is finally performed in the two filter chambers is referred to as a preliminary chamber 35. The preliminary chamber 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 path 22 and the filter chamber 31 is connected to the waste liquid chamber 71 through the sixth flow path 72. A transfer passage 36 is formed between the preparatory chamber 35 and the filter chamber 31. On one side of the transfer path 36, there is provided a transfer valve 37 which opens and closes the transfer path. Thus, when the transfer valve 37 is opened, the filtrate filtered in the preliminary chamber 35 is transferred to the filter chamber 31 through the transfer passage 36.

Since the filter unit 30 has two chambers, ie, the preliminary chamber and the filter chamber, the sample containing nanoparticles transferred from the sample chamber 21 passes through the filtration membrane 34 having a large pore size in the preliminary chamber 35 Firstly, particles with a large size of nanoparticles are separated. Finally, the nanoparticles are separated while passing through the filtration membrane 34 having a small pore size in the next filter chamber 31. Therefore, only the nanoparticles of a specific size range can be selectively collected and collected.

The supply section 40 is connected to the filter chamber 31 and supplies the fluid necessary for nanoparticle detection to the inlet space 32 of the filter chamber 31. Thus, the detection of the nanoparticles can finally be performed in the filter chamber 31. Fig.

Also, the present embodiment may be a structure for recovering nanoparticles separated from the filter unit 30 to perform a necessary detection operation. The apparatus includes a recovery chamber 80 formed in the disk 10 and connected to the filter unit 30 to recover nanoparticles separated by the filtration membrane 34, a recovery chamber 80 and a filter unit 30 And a seventh valve 82 for opening and closing the seventh flow path 81. The seventh valve 81 may be formed of a metal or a metal.

As shown in FIG. 2, the recovery chamber 80 can be understood as an empty space formed inside the disk 10. The recovery chamber 80 is connected to the filter chamber 31 through a seventh passage 81. In addition, a hole for injecting a reagent for nucleic acid extraction into the inside of the collection chamber 80 may be formed at one side. Thus, by injecting a nucleic acid extraction reagent containing a phenol component through a hole formed in the recovery chamber, the nucleic acid can be extracted from the sample through the apparatus.

4, the seventh flow path 81 connects between the inlet space 32 of the filter chamber 31 and the collection chamber 80. As shown in Fig. Thus, the nanoparticle-containing fluid contained in the inlet space 32 of the filter chamber 31 is moved through the seventh flow path 81 by centrifugal force. And a seventh valve 82 for opening and closing the seventh flow path 81 is installed at one side of the seventh flow path 81. When the seventh valve 82 is operated to open the seventh flow path 81, the nanoparticle-containing fluid staying in the inlet space 32 in the filter chamber 31 is recovered along the seventh flow path 81 And is then transferred to the chamber 80.

Thus, the nucleic acid extraction operation can be performed by injecting a reagent for extracting nucleic acid including the component of the nano-particle into the nanoparticle-containing fluid transferred to the recovery chamber 80.

The nucleic acid extraction operation can be performed separately from the outside of the apparatus. 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. By separating the filtration membrane 34 from the filter chamber 31 as described above, the nucleic acid extraction operation for the nanoparticles filtered by the filtration membrane 34 outside the disk 10 can be performed separately.

The apparatus may further include a lid 90 detachably attached to the disk 10 for opening and closing the filter chamber 31 and a fastening portion for fastening the lid 90 to the disk 10.

4, the lid 90 is detachably mounted on the upper plate 12 of the disk 10. As shown in Fig. The upper plate 12 is open to the outside on the space for forming the filter chamber 31. The lid 90 is installed on the top plate 12 to form a part of the top plate. In this embodiment, the fastening portion may be a fastening structure using a bolt 91. A variety of structures other than the bolt 91 can be applied to the fastening portion. A fastening hole 92 for fastening the bolt 91 may be formed on the cover 90 and the upper plate corresponding thereto. Thus, by fastening the bolt 91 to the fastening hole 92, the lid 90 can be detachably coupled to the upper plate 12. [

Fig. 5 shows another embodiment of the nanoparticle detecting device.

5 also shows the basic configuration of the disk 10 or the sample accommodating portion 20, the filter portion 30 and the waste liquid accommodating portion 70 provided in the disk 10, Which is the same as the embodiment. Also, although not shown, a configuration for the supply unit (see 40 in FIG. 1) may be equally provided. Therefore, the same reference numerals are used for the same components in the following description, and a detailed description thereof will be omitted.

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

The three filter chambers are sequentially disposed along the fluid transport 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 third 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 path 22. The inlet space of the second filter chamber 312 is connected to the outlet space of the first filter chamber 311 through the flow passage 315. The outflow space of the second filter chamber 312 is connected to the inlet space of the third filter chamber 313 through a separate flow path 317. The fluid is filtered in the sample chamber 21 through the first filter chamber 311, the second filter chamber 312, and the third filter chamber 131 in order to separate the nanoparticles. In this embodiment, a separate opening / closing valve (not shown) is provided at one side of each of the flow paths 315 and 317 to selectively open and close the respective flow paths, if necessary.

The filtration membranes 34 provided in the filter chambers 31 may have different pore sizes to separate nanoparticles of different size ranges. Thus, nanoparticles of different sizes can be separated and recovered through one device.

In this embodiment, the filtration membrane provided in each filter chamber 31 may have a structure in which pores gradually decrease along the fluid transport direction. In this embodiment, the three filter chambers 31 are provided with filtration membranes 34 each having a different pore size from each other to separate nanoparticles of different sizes from each other. The combination of 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 second filter chamber 312 is provided with a filtration membrane having a pore size of 50 nm, and the third filter chamber 313 A filtration membrane having a pore size of 2 nm may be provided and combined in one apparatus. Therefore, nanoparticles of different sizes can be separated and collected in each filter chamber depending on the combination of filtration membranes in one apparatus.

According to this embodiment, a sample containing nanoparticles transferred from the sample chamber 21 is passed through the filtration membrane 34 having a large pore size in the first filter chamber 311, and nanoparticles having a predetermined size are separated. Then, the sample transferred to the second filter chamber 312 is passed through the filtration membrane 34 provided in the second filter chamber to separate nanoparticles having a second predetermined size. Then, the sample transferred to the third filter chamber 313 passes through the filtration membrane provided in the third filter chamber, and the nanoparticles finally set in size are separated. Thus, only the nanoparticles of a specific size range can be sorted and collected in each filter chamber. Therefore, nanoparticles having different size distributions from each other can be separated and recovered in a single disk.

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

First, an antibody, a reagent, a substrate solution, a washing solution, a filtration membrane 34, etc. for detecting nanoparticles are mounted on the disk 10. The valves provided in the respective flow paths of the disk 10 in the ready state can be closed to keep the respective flow paths closed.

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 the centrifugal force is centrifuged and purified first. 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 unit 30 passes through the filtration membrane 34 of the preliminary chamber 35 by the centrifugal force resulting from the rotation of the disk 10, and the particles having a large size outside the nanoparticles are separated.

The solution through the filtration membrane 34 of the preliminary chamber 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 inlet space 32 of the filter chamber 31. The filtrate having passed through the filtration membrane 34 of the filter chamber 31 is discharged to the waste fluid chamber 71.

In this way, the nanoparticles are detected by sequentially supplying the antibody, the reagent, the substrate liquid, and the like to the inlet space 32 of the filter chamber 31 while the nanoparticles are separated and captured through the filtration membrane 34.

6 shows a process of detecting nanoparticles by supplying an antibody, a reagent, and a substrate solution to nanoparticles separated and captured on a space 32 on the upper side of the filtration film 34 in the filter chamber 31 of the filter chamber 31 have.

The antibody supplied to the filter chamber 31 adheres to the nanoparticles in the inlet space 32 on the filtration membrane 34. Since the antibody is attached to the surface of the nanoparticles in an unfixed state by being trapped in space, the antibody may be attached to the entire surface of the nanoparticles within a short period of time. In contrast, when the nanoparticles are immobilized on one surface, the antibody can attach only to a part of the surface of the nanoparticle, and the antibody can not be attached to the entire surface. Thus, in this embodiment, all the nanoparticles can be detected on the entire surface within a short period of time.

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

After washing the inlet space 32 with the washing solution, the reagent is supplied to label the antibody. The reagent binds to the antibody attached to the nanoparticle. The reagent is a secondary detection antibody with fluorescence, which ultimately amplifies the antibody signal on the nanoparticle.

After the reagent supply, the excess reagent not attached to the antibody is removed through the washing solution. When the washing liquid is supplied to the filter chamber 31 and centrifugal force is applied to the disk 10, excess reagent attached to the antibody is removed through the filtration membrane 34 together with the washing liquid.

After washing the inlet space 32 with the washing liquid, the substrate liquid can be supplied into the inlet space 32. After the substrate solution for reaction is supplied into the inlet space 32, the stop solution is supplied.

Then, the nanoparticle-containing 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 collection chamber 80 can be detected and analyzed in a fluorescence-labeled state, for example, using a fluorescence signal-based measurement method.

In this embodiment, unlike the conventional method in which nanoparticles are attached to the surface of a 96-well plate, the separated nanoparticles are detected in a specific chamber, so that all the antigens can be analyzed in a short time. As described above, the detection method of the present embodiment uses streptavidin-HRP to amplify biotin-labeled antibody and signal, and OD (optical density) can be measured by injecting TMB after labeling.

In addition, the nucleic acid extraction operation of the nanoparticles is performed by injecting a nucleic acid extracting reagent containing waste water into the collection chamber 80, or the filter chamber 31 of the disk 10 is opened to separate the filtration membrane 34, The nucleic acid extraction process can be performed.

As described above, the separation and detection of the nanoparticles of the sample in the single disk 10 or the nucleic acid extraction operation can be collectively performed more simply and easily.

[Experimental Example]

FIG. 7 shows the results of detecting and separating nano-vesicles from LNCaP (prostate cancer cell line) cell culture medium and urine sample using the apparatus according to the embodiment of FIG. 2. FIG.

A graph of FIG. 7 shows the detection results of the nanospores according to the combination of the filtration membranes provided in the two filter chambers. The graphs show the results of nanoparticle collection within the range of 100-600 nm, 20-200 nm, and 20-600 nm, respectively. Filter I in the graph refers to the preliminary chamber in Figure 2 and Filter II refers to the filter chamber. Experimental results show that nanofibers are the most abundant when the filtration membranes are combined in the range of 20-600 nm.

The graph B in FIG. 7 shows that as the volume of the cell culture fluid is increased, the amount of CD9 antigen expressed in the nanoembryonic cells increases.

FIG. 7C shows optical density (OD) results obtained by injecting 400 μl of a urine sample of a normal human and a bladder cancer patient into a disc and driving the disc to finally detect the nanospores. In the C photograph, N represents the results for normal persons, and P represents the results for bladder cancer patients. As a result of the detection, it can be confirmed that the nanoparticle can be sufficiently detected from the urine sample of the bladder cancer patient through the device.

FIG. 8 shows a comparison between the detection result of the nano-vesicle by the device according to the present embodiment and the conventional device.

The results of the example in FIG. 8 are shown as Exodisc, and the results of the detection and detection of the nano-vesicles in urine samples of normal and bladder cancer patients using the apparatus according to the embodiment of FIG. 2 are shown.

In FIG. 8, the comparative examples are denoted by UC and Exospin, and show detection results according to the conventional technique compared with the embodiment. The graph labeled UC is the result of detection of nano-vesicles by ultracentrifugation (UC), which is a widely used method, by ELISA detection method on a 96-well plate. In the exospin graph, the commercial kit was obtained by detecting the exospin-separated nanospores by ELISA detection method on a 96-well plate. As in the case of the comparative examples, the nano-vesicles were separated and detected in urine samples of normal and bladder cancer patients. The isolated nanospores were detected by CD9 and CD81, the protein markers expressed in the nanospores.

As a result of the experiment, it can be confirmed that the nanoparticles separated from the urine sample of the bladder cancer patient (n = 5) are higher than the normal (n = 5). Thus, it can be confirmed that the device can sufficiently detect the nano-vesicles from the urine sample of the bladder cancer patient.

Also, as compared with the comparative examples according to the prior art, it can be confirmed that the detection value of the nanospores for bladder cancer patients is higher in the case of the examples. Thus, it can be seen that the nanoparticle detection effect of the present embodiment is remarkably improved as compared with the conventional art.

FIG. 9 shows the nucleic acid detection results of the nanofibers through a device according to the present embodiment and a conventional device.

9, the results are shown in Exodisc, and the apparatus according to the embodiment of FIG. 2 is used to separate the nanospheres from the LNCaP (prostate cancer cell line) cell culture medium. Then, the phenol component reagent is injected to extract the nucleic acid And the results are shown.

In FIG. 9, comparative examples are indicated by UC and Exospin, and the results of detection according to the conventional technique are shown in comparison with the embodiment. UC and Exospin are the same as described above. Comparative Examples In the same manner as in the Examples, the nanospheres were separated from the LNCaP (prostate cancer cell line) cell culture medium, and the nucleic acid was extracted and detected by injecting a reagent of the worm component.

A graph in FIG. 9 shows the result of RNA electrophoresis using bioanalyzer. The size and concentration of RNA from the isolated nanosomes were analyzed by the experimental and comparative examples. As a result, both the examples and the comparative examples showed the same size pattern and a large amount of RNA of 250 nt or less associated with the nanospores was extracted. In addition, in the case of the RNA concentration detection result, in the case of the nanofibers isolated using the detection device of the present embodiment, the nucleic acid extraction result shows higher RNA concentration than the comparative examples, and the detection effect can be increased.

The graph B in FIG. 9 shows the detected C _T values of GAPDH, CD9, PSA and PSMA by RT-PCR with the extracted nucleic acid.

In FIG. 9B, GAPDH is a house keeping gene, CD9 is a nanoparticle detection marker, PSA (prostate-specific antigen) is a marker used for detecting prostate cancer, and prostate-specific membrane antigen (PSMA) , And C _T means a higher concentration as the value is lower. For example, if the C _T value is 7, it means 2 ⁷ to 128 times.

As shown in the graph B of FIG. 9, when the detection device according to the present embodiment was used as a result of real time-PCR, the concentration of GAPDH, CD9, PSA, PSMA of the nano-vesicles was 100 times higher than that of the conventional UC Can be confirmed.

That is, it can be seen that the difference of GAPDH: 169 times, CD9: 158 times, PSA: 111 times, and PSMA: 208 times are different from the ultracentrifugation (UC) of the comparative example in this embodiment. Thus, it can be seen that the detection effect of the present embodiment can be further enhanced as compared with the conventional and comparative examples.

The graph C in FIG. 9 shows the relative amount of expression relative to UC, which is an ultracentrifugation method, based on the CT value. As a result of the experiment, it can be confirmed that a higher detection amount is obtained than the comparative example when all four genes use the detection apparatus of this embodiment. Thus, it can be seen that the nucleic acid detection effect of the present embodiment is significantly improved as compared with the conventional method.

The graph D in FIG. 9 shows an image obtained by electrophoresis of PCR products. The graph D in FIG. 9 is an image obtained by electrophoresis of PCR products such as the C graph.

As a result of the experiment, it is confirmed that the detection amounts of all four genes are higher than those of the comparative examples using the detection apparatus of this embodiment.

FIG. 10 shows the result of detection and detection of the nanospores using the apparatus and the conventional apparatus according to the present embodiment.

This experiment was performed by spiking LNCaP cell-derived nanospores at 100 microliter of plasma. The initial number of nanoparticles after spike was 3.98 (± ± 0.16) E10 / mL and the total protein was 66.45 (± ± 0.095) mg / mL.

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

The graph A in FIG. 10 shows the results of detection of the nanospores according to the combination of the pore size of the filtration membrane and the rotation speed of the disc 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 nanospores using a plasma not spiked with LNCaP cell culture solution-derived nanospores as a control. As a result of the experiment, when nanoparticles were separated under conditions of 20 nm 3000 rpm (364 G), which is a condition for separating the nanofibers from a conventional urine sample, nanoparticles were detected the most. However, in the case of human plasma, lipid and protein nanoparticles other than pure nanoparticles are present in the blood in a large amount as impurities. Therefore, when 20 nm pore is used, most of the detected nanoparticles are composed of lipid and protein particles.

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

According to the results of the experiment, the purity of the nanospores was lower than that of the case of 100 nm at 1000 rpm (40 G) under the condition of 20 nm 3000 rpm (364 G).

The graph C in FIG. 10 shows the purity of the separated nanofibrous sample according to the combination of the filtration membrane pore size and disc rotation speed of the present embodiment. As a result of the experiment, it was confirmed that the conditions of 100 nm and 1000 rpm (40 G) separated the nanoparticles with the highest purity.

The graph D in FIG. 10 shows the detection results of the marker (EpCAM) and the nanosporter detection marker (CD81) used for detecting the cancer, in accordance with the combination of the pore size of the filtration membrane and the disc rotation speed according to this embodiment. As a result, it was found that the nanoparticles of total nanoparticles and cancerous cells were most detected at 100 nm at 1000 rpm (40 G), and the nanoparticles having high purity can be recovered without losing much nanoparticles .

The graph E in FIG. 10 shows the recovery rate of the nanocomposite of this embodiment and the comparative example.

In the graph E in FIG. 10, the results of the examples are indicated by DISC, and the detection results are shown by the pore size of the filtration membrane and the disk rotation speed, respectively. The comparative example is indicated by UC (ultracentrifugation), and shows the detection result according to the conventional technique compared with the embodiment.

The initial LNCaP cell-derived nanoembryonic cells exhibited an OD value of 1.34 upon detection of CD9 / CD81 (CD9 capture / CD81 detection). As a result of experiments with conventional apparatus spiking nano-vesicles on plasma, OD of 1.18 at 20 nm 3000 rpm (364 G) and OD of 1.12 at 100 nm 1000 rpm (40 G) were obtained. That is, as shown in the graph E of FIG. 10, when the disc according to the present embodiment is irradiated with light of 20 nm at 3000 rpm (364 G), as compared with the conventional method of separating into the ultracentrifuge (UC) And 100 nm at 1000 rpm (40G), respectively.

FIG. 11 shows the results of separation and detection of nanospores using a filtration membrane having a pore size of 100 nm in the apparatus according to the present embodiment.

The experiment was performed by spiking LNCaP cell-derived nanospores at 100 microliter of plasma. The initial number of nanoparticles after spike was 6.34 (± A 0.12) E10 / mL.

The graph of FIG. 11 shows the nano-scale characteristics of the disk according to the disk rotation speed of 600 rpm (15 G), 900 rpm (33 G), 1200 rpm (58 G), 1800 rpm (131 G) and 2400 rpm The results of ER detection are shown.

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

The graph B in FIG. 11 shows the total protein amount according to the disk rotation speed. Experimental results show that the total protein content decreases as the rotation speed is increased from 900 rpm (33G).

The graph C in FIG. 11 shows the time (left side, open circles) and purity (right side, closed squares) of the nanofibers according to the disk rotation speed. As a result, the separation time of nanofibers decreased with increasing the rotation speed from 600 rpm (15 G), and showed high purity in the range of 600 rpm (15 G) to 1200 rpm (58 G) (131 G), the purity gradually decreases.

The graph D in FIG. 11 shows the amount of CD81 antigen expressed on the surface of the nanosome according to the rotation speed of the disk. Experimental results show that CD81 antigen expression is relatively high in the range of 600 rpm (15 G) to 1200 rpm (58 G), and rapidly decreases from 1800 rpm (131 G).

11, it can be confirmed that a good recovery rate, purity and short separation time can be secured at a rotation speed in the range of 900 rpm (33 G) to 1200 rpm (58 G).

Fig. 12 shows the detection results of the nanofibers after separation according to the present embodiment and the comparative example.

In FIG. 12, the results for the embodiment are indicated by Disc. In this example, a disk having a filtration membrane having a pore size of 100 nm is operated at 900 rpm (33 G) to detect the result of separating the nanospores. The comparative example is indicated by UC (ultracentrifugation), and shows the detection result according to the conventional technique compared with the embodiment.

The experiment was carried out by spiking the plasma from the LNCaP cell-derived nanospores in both of the examples and the comparative examples. The initial number of nanoparticles after spike was 6.34 (± A 0.12) E10 / mL.

A graph in Fig. 12 shows the number of detected nanospores. As shown in the graph A of FIG. 12, in the examples, it was confirmed that the number of particles separated according to the injection volume of plasma spiked with LNCaP cell-derived nanospores increased linearly in a range of 5 microliter to 200 microliter . Therefore, it can be seen that, in the case of the embodiment, more number of nanoparticles can be separated as compared with the UC of the comparative example.

The graph B in FIG. 12 shows the results of quantitative analysis of surface proteins by ELISA of the detected nanosomes. As shown in the graph B of FIG. 12, in the case of the example, the amount of expression of CD9 / CD81 (CD9 capture / CD81 detection) on the surface of the nanospheres varies from 5 microliter to 200 microliter according to the volume of injection of plasma spiked with LNCaP cell- In the linear relationship. Thus, in the case of the example, a higher expression amount is shown as compared with the UC of the comparative example. Therefore, it is possible to quantitatively analyze the nano-vesicles in the blood using the disc of this embodiment.

The graph C in FIG. 12 shows the result of separating the RNA derived from the nanofibers.

In the experiment, the nanoparticles derived from LNCaP cells were spiked with 100 microliter of plasma, and the nanoparticles were separated and the phenol component reagent was injected to extract the RNA from the nanoparticles. The size and concentration of RNA were analyzed by using Bioanalyzer and electrophoresis of extracted RNA.

As shown in the graph C of FIG. 12, the results of the experiment showed that both the examples and the comparative examples showed the same size pattern, and a large amount of RNA of 250 nt or less associated with the nanoembryonic was extracted. In addition, as a result of RNA concentration detection, it was confirmed that the extraction efficiency of the RNA was higher than that of the comparative examples, as a result of the extraction of the nano-deficient nucleic acid separated using the detection device of the present embodiment. As described above, even when the RNA derived from the nanofibers is separated, it is difficult to detect the RNA in the UC of the comparative example, whereas the exosomal RNA is detected in the sample separated using the disk of the embodiment.

FIG. 13 shows the results of detection of nanospores isolated from 200 microliter plasma of normal plasma and cancer patients according to the conditions of the embodiment, in comparison with the conventional method. 13, H means normal plasma, L means plasma of lung cancer patients, and S means plasma of gastric cancer patients.

The results for the embodiment in FIG. 13 are denoted as Disc. Disc-20 represents a test result under the condition that a disk having a filtration membrane having a pore size of 20 nm is rotated at 3000 rpm (364 G). Disc-100 is a disk having a filtration membrane having a pore size of 100 nm, (58 G). The results are shown in Fig. The comparative example is indicated by UC (ultracentrifugation), and shows the detection result according to the conventional technique compared with the embodiment.

Graph A in FIG. 13 shows the total protein amount according to the method of separating nano-cells. Experimental results showed that the total protein content was the highest in the Disc-20 example performed at 20 nm at 3000 rpm (364 G), and similarly for the UC and Disc-100 (100 nm 1200 rpm (58 G) The amount of protein was low.

Graph B in Fig. 13 shows the result of nanoparticle recovery according to the method of separating nanofibers. As a result, the largest amount of nanoparticles was recovered in the Disc-20 example performed at 20 nm at 3000 rpm (364 G), but no difference in the number of nanoparticles between the normal sample and the cancer patient sample was observed. In the disc-100 example conducted at 100 nm at 1200 rpm (58 G), a larger amount of nanoparticles than the comparative UC was recovered and showed a difference in the number of nanoparticles between a normal sample and a cancer patient sample.

Graph C in FIG. 13 shows the purity of the nanoparticles according to the method of separating nanofibers. Experimental results showed the highest purity in the disc-100 example performed at 100 nm at 1200 rpm (58 G) in all the sample groups.

Graph D in FIG. 13 shows the expression amount of a marker (EpCAM) used for cancer detection on the surface of the nano-embryo according to the method of separating nano-cells. As a result of comparison with the comparative UC separation method, both Disc-20 and Disc-100 exhibited a high antigen expression level and showed a large amount of EpCAM antigen expression between normal and cancer patients.

As shown in the graphs of A, B, C and D in FIG. 13, the patient blood-derived nanospores were analyzed by four experiments to measure total protein amount (BCA), total nanoparticle number (NTA), purity Number divided by the total protein amount measured by BCA). As a result of the nano-vesicle surface markers analyzed by ELISA, it can be confirmed that the Disc-100 example shows excellent results.

Fig. 14 shows CT values of nano-vesicle detection results obtained by comparing the apparatus according to the present embodiment with a conventional apparatus. The experiment was performed by extracting nucleic acid from blood-derived nanospores isolated from normal plasma of 200 microliter and detecting by real-time PCR.

In FIG. 14, the results for the embodiment are shown as Exodisc, and the comparative examples are shown as UC and Exospin, which show the detection results according to the conventional technique compared with the embodiment.

As a result of the experiment, when the device according to the present embodiment was used, the expression amount and detection rate of nanosomal surface antigen CD81, CD63, and CD9 were much higher than those of UC and Exospin of the comparative example.

FIG. 15 is a graph showing the results of real-time PCR (RT-PCR) using the apparatus according to the present embodiment, extracting nucleic acid of blood-derived nano-vesicle from plasma that has undergone sample preprocessing step with Proteinase K solution and plasma not subjected to the pre- As shown in FIG.

The experiment was performed by extracting the nucleic acid from the nanoparticles separated from the normal plasma of 200 microliter using the apparatus according to the present embodiment. The graph of FIG. 15 shows the CT values of GAPDH (House keeping gene), CD9, CD63, and CD81 (nanoembradent detection markers) detected by RT-PCR using the nucleic acid extracted according to the present embodiment.

As shown in the experimental results, when the sample was pretreated with Proteinase K (protein degrading enzyme), the amount of mRNA expression was about 5 times higher than that of untreated plasma sample. In other words, it means that proteins other than nanoembryonic proteins present in plasma can affect the nucleic acid detection result, and it can be confirmed that the nucleic acid amplification performance is improved by the sample pretreatment using Proteinase K.

FIG. 16 is a graph showing the analysis of the surface proteins of the nanosuspension fraction having different size distributions by separating the nanospores according to the size difference in the cell culture of BT474 (breast cancer cell line) using the apparatus according to the embodiment of FIG.

For example, for 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 is present on the 20 nm AAO filter, Protein and molecular characteristics are shown in FIG. 7, FIG.

Thus, the nanoparticles can be separated by size difference, and the analysis of nanoparticles having respective size distributions can be performed separately.

In this experiment, the sizes of the nanoparticles collected in the respective filtration membranes were analyzed by using disks having sequentially arranged AAO filtration membranes having pore sizes of 200 nm, 100 nm and 20 nm as in the embodiment of FIG. The graph A in FIG. 16 shows the result of separation of the nanospores according to the combination of the filtration membranes provided in the three filter chambers. On the A graph, on AAO 200 nm is the nanofibers detected in the filtration membrane (200 nm) of the first filter chamber, and on AAO 100 nm is the nanofibers detected in the filtration membrane (100 nm) of the second filter chamber and on AAO 20 nm 3 shows the nanospores detected in the filter membrane (20 nm) of the filter chamber. Post-filtration means the solution after passing through the filtration membrane (20 nm) of the third filter chamber.

As shown in the graph A of FIG. 16, the nanoparticle solution having an average size of 255 nm, 141 nm, 78 nm, and 10 nm in a solution (Post-filtration) passed through all the filters of AAO 200 nm, AAO 100 nm and AAO 20 nm Can be recovered.

Therefore, as a result of the experiment, as shown in the graph A, a desired size of nanofibers was detected on the filter membranes of each filter chamber. Thus, it can be seen that the nanoparticles of various sizes can be separated and detected through the apparatus.

The graph B in FIG. 16 shows the detection results of BT474 breast cancer cell-derived nanospores isolated from the filter membrane of each filter chamber. The graph B in FIG. 16 shows normalization based on the amount of surface protein of EpCAM and sialic acid of the corresponding nanosecomposite.

In the case of 20 nm AAO only, it refers to nanospores recovered on a 20 nm filtration membrane using a 20 nm AAO filtration membrane. That is, in the case of the embodiment having three filter chambers, the 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 ≪ / RTI > In the case of Exofree medium, it means that a pure culture solution having no nanospores is passed through a 20 nm filtration membrane.

As a result, EPCAM (markers used for cancer detection) showed the highest expression level on On 20 nm AAO, and it was detected in the relatively small nanospheres fraction having a range of 20-100 nm . In addition, the expression level of sialic acid (markers used for cancer detection) is highest in On 100 nm AAO, and is detected more in nano-sister fraction having a range of 100-200 nm.

Thus, it can be seen that the nanofabric fraction having different size distributions can be selectively recovered through the present embodiment.

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

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course.

10: Disk 12: Top plate
14: lower plate 20: sample receiving portion
21: sample chamber 22: first flow path
23: first valve 24: settling portion
25: groove portion 30: filter portion
31: filter chamber 311: first filter chamber
312: second filter chamber 313: third filter chamber
315,317: Euro 32: Inlet space
33: output space 34: filtration membrane
35: preliminary chamber 36:
37: feed valve 40:
41: antibody chamber 42: second channel
43: second valve 44: reagent chamber
45: third flow path 46: third flow path
47: Substrate chamber 48: Fourth fluid line
49: fourth valve 50: stop chamber
60: cleaning section 61: cleaning liquid chamber
62: fifth valve 63: fifth valve
70: waste liquid storage part 71: waste liquid chamber
72: sixth valve 73: sixth valve
80: Collection chamber 81: Seventh channel
82: seventh valve 90: cover
91: Bolt

Claims (36)

A disk on which fluid is transferred by centrifugal force,
A sample storage portion formed on the disk to receive the sample,
A filter unit connected to the sample storage unit and having a microfiltration membrane for separating the nanoparticles by filtering the transferred sample,
A supply part connected to the filter part to supply a detection solution for detecting nanoparticles separated in the filtration membrane,
A waste solution receiving part connected to the filter part outlet side to receive a solution having passed through the filtration membrane,
A flow path formed on the disk and through which the fluid is transferred, and
A valve for selectively opening and closing the flow path
The nanoparticle detection device comprising: The method according to claim 1,
Wherein the filter unit includes a filter chamber having an inlet space and an outlet space along the fluid movement direction and having the filtration membrane disposed between the inlet space and the outlet space,
Wherein the inlet space is connected to the sample receiving portion to receive the sample and the filtered nanoparticles, and the outlet space is connected to the waste liquid containing portion. 3. The method of claim 2,
Wherein the filtration membrane has pores of 1 nm to 1000 nm. 3. The method of claim 2,
Wherein the filtration membrane is made of thermosetting plastic, anodic aluminum oxide, nickel, or silicon material including polycarbonate, polystyrene, polymethyl methacrylate, cyclic olefin copolymer. 3. The method of claim 2,
Wherein at least two filtration membranes are laminated between the inlet space and the outlet space, and the filter membranes have pores gradually becoming smaller along the fluid transport direction. 3. The method of claim 2,
Wherein the filter unit is sequentially disposed along at least two fluid transfer directions, and the filtration membrane provided in each filter unit has different pore sizes to separate nanoparticles of different size ranges. The method according to claim 6,
Wherein at least two of the filter units are sequentially disposed along the fluid transport direction, and the filtration membrane provided in each filter unit gradually decreases in pore size along the fluid transport direction. 3. The method of claim 2,
Wherein the filtration membrane is detachably installed in a filter chamber of the disk. 9. The method of claim 8,
Wherein the filter unit further comprises a lid detachably installed on the disk and opening / closing the filter chamber, and a coupling part fixing the lid to the disk. The method according to claim 1,
The sample accommodating portion includes a sample chamber formed on the disk and accommodating a sample, a first flow path for connecting the sample chamber and the filter portion, a first flow path for transporting the sample according to the centrifugal force of the disk, and a first valve for opening and closing the first flow path The nanoparticle detection device. 11. The method of claim 10,
The sample chamber centrifuges the sample according to the centrifugal force of the disk,
A sedimentation section in which a centrifuged sample is accommodated at a distal end thereof facing the outer side of the disk along a centrifugal force direction is elongated,
Wherein the first flow path is connected to a boundary point of the sediment portion of the sample chamber toward the rotation center of the disk to transfer the centrifuged supernatant to the filter portion. 12. The method of claim 11,
Wherein the settling portion is inclined with respect to the radial direction of the disk. 12. The method of claim 11,
Wherein the settling portion further comprises a groove portion formed at an end of the settling portion, the bottom surface of which is gradually inclined upwardly from the boundary point along the centrifugal force direction to accommodate impurities centrifugally separated from the sample. The method according to claim 1,
Wherein the sample is a biological sample selected from blood, lymph fluid, tissue fluid, urine, saliva, cerebrospinal fluid and sputum containing bioparticles, or an aqueous solution in which nanoparticles are dispersed, or a combination thereof. The method according to claim 1,
Wherein the sample accommodating portion, the filter portion, the supply portion, and the waste liquid accommodating portion constitute one unit, and the unit is disposed with a plurality of intervals along the circumferential direction of the disk. The method according to claim 1,
Wherein the channel connecting the two adjacent constituent parts of the disk is located at the center of the disk than the outlet and the inlet is connected to the outlet of the one chamber along the centrifugal force direction of the disk. The method according to claim 1,
Wherein the disk is surface-modified with a protein, a polymer, or an organic molecule so as to prevent nonspecific antibody attachment. 18. The method according to any one of claims 1 to 17,
Wherein the supply unit comprises an antibody chamber formed on the disk to receive an antibody provided for nanoparticle detection, a second flow path for connecting the antibody chamber and the filter unit and transferring the antibody to the filter unit according to the centrifugal force of the disk, And a second valve that opens and closes the nanoparticle detection device. 19. The method of claim 18,
The supply unit may include a reagent chamber formed in the disc and accommodated with a reagent provided for labeling an antibody that detects nanoparticles, a third flow path connecting the reagent chamber and the filter unit and transferring the reagent to the filter unit according to the centrifugal force of the disc, And a third valve for opening and closing the third flow path. 20. The method of claim 19,
The supply unit includes a substrate liquid chamber formed on the disk to receive a substrate liquid for nanoparticle detection reaction, a fourth flow path for connecting the substrate liquid chamber and the filter unit, and transferring the substrate liquid to the filter unit according to the centrifugal force of the disk, And a fourth valve for opening and closing the fourth flow path. 20. The method of claim 20,
The supply part includes a stop solution chamber formed in the disc and adapted to stop the nanoparticle detection reaction, a stop solution chamber for stopping the nanoparticle detection reaction, a connection channel for connecting the stop solution chamber and the substrate solution chamber, And a connection valve that opens and closes the nanoparticle detection valve. 22. The method of claim 21,
Wherein the supplying unit further comprises a washing unit for washing the filter unit by transferring the washing liquid to the filter unit. 23. The method of claim 22,
The washing unit may include a washing liquid chamber formed in the disk to receive the washing liquid, a fifth flow path connecting the washing liquid chamber and the filter unit and transferring the washing liquid to the filter unit according to centrifugal force of the disk, and a fifth valve opening / closing the fifth flow path The nanoparticle detection device. 24. The method of claim 23,
Wherein the cleaning liquid chamber is divided into a plurality of chambers, the cleaning liquid is contained in each of the cleaning liquid chambers, and the outflow valve for discharging the cleaning liquid is provided in the outflow channel of each of the cleaning liquid chambers. 23. The method of claim 22,
A recovery chamber formed on the disk and connected to the filter unit to recover nanoparticles separated by a filtration membrane, a seventh flow path connecting the recovery chamber and the filter unit to transfer the nanoparticles to the recovery chamber, And a seventh valve for controlling the flow of the gas. Supplying a sample to the disk,
Applying a centrifugal force to the disk to transfer the sample to the filter section, filtering through the filtration membrane to separate the nanoparticles, and
Applying a centrifugal force to the disk and supplying the detection liquid to the filter section to detect nanoparticles on the filtration membrane
/ RTI > 27. The method of claim 26,
Wherein the separating the nanoparticles comprises separating nanoparticles of different size ranges through a plurality of filtration membranes having different pore sizes. 28. The method of claim 27,
Wherein the separating of the nanoparticles comprises separating the nanoparticles of different size ranges sequentially through a plurality of filtration membranes in which pores gradually decrease. 27. The method of claim 26,
Further comprising a purification step of centrifuging the sample by applying centrifugal force to the disk before transferring the sample to the filter section. 30. The method according to any one of claims 26 to 29,
Detecting the nanoparticles comprises: applying centrifugal force to the disk to supply the antibody to the filter section to detect the nanoparticles, and supplying reagents to the filter section to label the antibody attached to the nanoparticles. 30. The method of claim 29,
Applying a centrifugal force to the disk and supplying a washing solution to the filter unit to clean the disk. 32. The method of claim 31,
Wherein the step of washing comprises washing the excess antibody after supplying the antibody to the filter part and removing the excess antibody, and supplying the reagent to the filter part to label the antibody and then washing and removing the excess reagent. Way. 33. The method of claim 32,
Wherein the step of detecting the nanoparticles further comprises the step of applying a centrifugal force to the disk to supply the substrate liquid to the nanoparticles. 34. The method of claim 33,
And recovering the nanoparticle detection solution after supplying the substrate liquid. 27. The method of claim 26,
Wherein the step of detecting the nanoparticles further comprises the step of extracting the nucleic acid by supplying a reagent for nucleic acid extraction to the separated nanoparticles. 27. The method of claim 26,
A method of detecting nanoparticles in a disk, the method comprising: separating a filter membrane from the disk in the step of detecting the nanoparticles.

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