WO2017069573A1 - Centrifugal force-based nanoparticle separation apparatus and method for separating nanoparticles using the same - Google Patents

Abstract

The present invention relates to a centrifugal force-based nanoparticle separation apparatus and method. Specifically, the present invention is based on having a low centrifugal force and a small size, and can thus separate nanovesicles unrelated to antibody specificity in a short time and without using an ultracentrifuge. Further, the present invention requires no additional professional personnel and enables accurate fluid measurement by integrating and automating all processes following sample injection, and can thus reduce the loss of nanovesicles.

Description

Centrifugal force-based nanoparticle separation device and nanoparticle separation method using the same

The present invention relates to a centrifugal force-based nanoparticle separation device and a nanoparticle separation method using the same.

Nanovesicles are small vesicles of 40-120 nm in size that arise from cellular activity and are distinguished from other vesicles by origin and size. It was considered a cell byproduct at the time of discovery, but its importance was found to contribute to cellular activities such as tumor progression and metastasis, and cellular signal transduction. Nanovesicles are present in almost all body fluids in the body and contain genetic information of derived cells, attracting attention as new drug delivery systems as well as new markers for various diseases including cancer.

Recently, researches for separating nano vesicles are continuously increasing, and methods are classified into using density, size, and affinity. The separation method using density is the most commonly used method to obtain a concentrated nano vesicle with or without antibodies. However, this method requires a lot of time and many processes for sample processing through ultracentrifuges. Affinity method can separate high purity nano vesicles in a short time, but it is expensive and only small amount of nano vesicles can be separated and nano vesicles that do not contain specific antibodies are difficult to separate. Therefore, there is a need for a system for separating nano vesicles irrelevant to antibody specificity in a short time.

Conventional nano vesicle separation using filters has been used to filter out impurities prior to ultracentrifugation. In order to filter the nano-vesicles within a certain range, two types of filtration membranes including small diameter pores and relatively large pores are required. However, the polycarbonate filtration membrane made of the conventional technology is not suitable for separation in the case of small pores (1 nm ~ 100 nm), the size of the pores is not uniform and the porosity is low. The filtration membrane made of anodized aluminum was relatively uniform in size and had high porosity, but it was difficult to apply due to its weak durability and destruction. However, other methods based on size other than the use of a filtration membrane require a sample pretreatment process, which requires a complicated process and requires specialized personnel.

Therefore, the development of a device and method that integrates the whole process of urine treatment and nano-vesicle extraction using disc-shaped chips based on centrifugal force has been the main subject, and research on this has been made (Korea Patent Publication) 10-2016-0017374), which is still inadequate.

The present invention has been made to solve the above problems, the present inventors using a plurality of filters of different sizes, by performing a plurality of particles filtration from the sample using a centrifugal force, the centrifugal force lower than the conventional method for separating the endoplasmic reticulum In the simple method was confirmed the effect of separating the endoplasmic reticulum, based on which the present invention was completed.

Thus, the object of the present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 providing a space supported by injecting a fluid sample including nanoparticles;

A filtration chamber unit 300 including a filtration membrane having pores of 1 nm to 1 μm capable of filtering nanoparticles from the fluid sample;

Waste solution holding unit 400 for storing the filtered sample solution; And

It is to provide a nanoparticle separation device comprising a micro-channel 500 for providing a passage for the flow of the fluid sample.

Another object of the present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

A filtration chamber unit 300 including two or more filtration membranes capable of filtering a sample;

Waste solution holding unit 400 for storing the filtered sample solution; And

It is to provide a nanoparticle separation device comprising a micro-channel 500 for providing a passage for the flow of the fluid sample.

Another object of the present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

A filtration chamber unit 300 for receiving one or more filtration membranes capable of filtering a sample;

Waste solution holding unit 400 for storing the filtered sample solution;

A micro flow path part 500 for providing a passage for the flow of the fluid sample; And

It provides a nanoparticle separation device, characterized in that for filtering the nanoparticles from the sample, including a valve 700 that can selectively control the flow of the fluid in the micro-channel.

Another object of the present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

A filtration chamber unit 300 for receiving one or more filtration membranes capable of filtering a sample;

Waste solution holding unit 400 for storing the filtered sample solution;

A micro flow path part 500 for providing a passage for the flow of the fluid sample;

A valve 700 capable of selectively controlling the flow of fluid in the microchannel; And

It is to provide a nanoparticle separation device, characterized in that it can filter and recover a specific size range of nanoparticles from the sample, including a particle collecting unit 800 that can recover the filtered specific size range of nanoparticles.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 providing a space supported by injecting a fluid sample including nanoparticles;

A filtration chamber unit 300 including a filtration membrane having pores of 1 nm to 1 μm capable of filtering nanoparticles from the fluid sample;

Waste solution holding unit 400 for storing the filtered sample solution; And

It provides a nano-particle separator comprising a micro-channel 500 for providing a passage for the flow of the fluid sample.

Preferably, the fluid sample may be a biological sample selected from the group consisting of urine, blood, saliva, sputum, and the like, including aqueous solutions and cell bodies in which various nanoparticles are dispersed, rare biological particles, and the like.

Preferably, the cleaning chamber unit 600 may further include a space for supporting the cleaning solution.

Preferably, the filtration membrane may be made of a material selected from the group consisting of polycarbonate, polystyrene, polymethyl methacrylate, cyclic olefin copolymer, anodized aluminum, nickel and silicon.

Preferably, the nanoparticle separation device may further include one or more fastening portions for attaching and detaching the filtration chamber 300.

More preferably, the fastening part may be an elastic material selected from the group consisting of polydimethylsiloxane, silicone, latex, rubber, and the like.

Preferably, the micro channel 500 may be connected to a channel through the device to change the channel of the fluid sample.

The present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

A filtration chamber unit 300 including two or more filtration membranes capable of filtering a sample;

Waste solution holding unit 400 for storing the filtered sample solution; And

It provides a nano-particle separator comprising a micro-channel 500 for providing a passage for the flow of the fluid sample.

Preferably, the filtration membrane may be made of a material selected from the group consisting of polycarbonate, polystyrene, polymethyl methacrylate, cyclic olefin copolymer, anodized aluminum, nickel and silicon.

Preferably, the filtration membrane may be selectively detachable from the housing part 100 by physical force.

Preferably, the filtration membrane, the filtration chamber 300, two or more stacked in the same chamber may collect the nanoparticles through the filtration membrane having two or more sizes during fluid transfer in a single chamber.

Preferably, the filtration membrane comprises a filtration membrane in a single chamber, wherein the plurality of chambers are arranged at different radial coordinates such that the fluid sample passes through the plurality of filtration membranes to perform plural particle filtration to a specific size range. Nanoparticles can be captured.

Preferably, the filtration membrane,

A first filtration membrane having at least one or more pores of 100 nm to 1 μm diameter; And

It may include a second filtration membrane having at least one or more pores of 1 nm to 100 nm in diameter.

Preferably, the micro channel 500 is disposed above or below the filtration chamber 300, and the chambers may be disposed at a distance from the center of the device to minimize loss of solution.

The present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

A filtration chamber unit 300 for receiving one or more filtration membranes capable of filtering a sample;

Waste solution holding unit 400 for storing the filtered sample solution;

A micro flow path part 500 for providing a passage for the flow of the fluid sample; And

It provides a nanoparticle separation device, characterized in that for filtering the nanoparticles from the sample including a valve 700 that can selectively control the flow of fluid in the micro-channel.

Preferably, the sample accommodating part 200 may perform sample purification to purify impurities of a sample.

Preferably, the sample accommodating part 200 may include impurity separation including a space formed at an angle at which a lower portion thereof is distorted than a radial direction.

Preferably, the sample accommodating part 200 may include a groove for preventing separated impurity backflow.

Preferably, the sample accommodating part 200 may be formed of an inclined surface and a curve to minimize the loss and damage of the sample during sample transfer.

Preferably, the valve 700 may be opened and closed according to an external signal.

Preferably, the valve 700, which is external to the rotatable device for the whole process automation, may include a system capable of controlling the opening and closing of the valve and the rotational speed and direction of the body.

Preferably, the waste liquid containing part 400 may be capable of separating high purity nanoparticles without a separate impurity treatment.

Preferably, the filtration chamber part 300 may be connected to one or more of the waste liquid receiving parts 400 to prevent impurity diffusion after purification.

Preferably, the nanoparticle separation device may further include one or more fastening portions for attaching and detaching the filtration chamber 300.

Preferably, the nanoparticle separation device, by injecting a BSA (bovine serum albumin) protein or Pluronic (PEO-PPO-PEO) polymer material can minimize non-specific binding to the surface.

More preferably, the filtration chamber 300 and the waste liquid receiving unit 400 may include a vent for performing smooth filtration.

The present invention

In the nano particle separator,

Rotatable disk-shaped housing portion 100;

A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

A filtration chamber unit 300 for receiving one or more filtration membranes capable of filtering a sample;

Waste solution holding unit 400 for storing the filtered sample solution;

A micro flow path part 500 for providing a passage for the flow of the fluid sample;

A valve 700 capable of selectively controlling the flow of fluid in the microchannel; And

Including a particle collecting unit 800 that can recover the filtered nanoparticles of a specific size range, it provides a nanoparticle separation device, characterized in that to filter and recover a specific size range of nanoparticles from the sample.

Preferably, the recovery of the nanoparticles is a maximum of 3000 rpm or less than the capillary pressure inside the pores present in the filter membrane when the solution to be recovered containing the nanoparticles and the lower surface adjacent to the waste liquid on the upper surface of the filter membrane After the waste liquid adjacent to the lower surface is discarded to the waste liquid receiving unit 400 using a rotation speed, a solution including nanoparticles located on the upper surface of the filtration membrane may be selectively recovered.

Preferably, the particle collecting unit 800 may be connected to the upper surface of the filtration chamber 300 and a micro channel, and the lower surface of the filtration chamber 300 may be connected to the waste liquid receiving unit 400.

The present invention relates to a nanoparticle separation device and method. Specifically, since it is based on low centrifugal force and size, nanovesicles irrelevant to antibody specificity can be separated in a short time without a conventional ultracentrifuge, and by integrating and automating the entire process after sample injection, no additional expert personnel are required. Accurate metering of fluids reduces nanovesicle loss.

1 is a perspective view of a nanoparticle separation device in which a nano vesicle separation process is integrated.

Figure 2 is a result of observing the nanoparticle separation process using the nanoparticle separation device of the present invention over time.

Figure 3 shows a front view of the nanoparticle separation device according to the present invention, (a) perspective view of the microfluidic device, (b) microfluidic device configuration, (c) particle separation process according to the filter and (d) filter I And SEM images of II.

Figure 4 is a process for separating nano vesicles using a nanoparticle separation device according to an embodiment of the present invention, (a) impurity precipitation, (b) nano vesicle concentration, (c) washing, (d) in filter II The remaining solution is removed and (e) nano vesicles collection process is shown.

Figure 5 shows the actual structure of the nanoparticle separation device according to an embodiment of the present invention, (a) separation of the nanoparticle separation device, (b) filter structure of the nanoparticle separation device and (c) nanoparticle separation Side view of the device and filter surface are shown in SEM image.

Figure 6 shows the result of measuring the degree of recovery of the vesicles by coating the nanoparticle separation device according to an embodiment of the present invention with a pluronic solution (pluronic) solution

Figure 7 shows the results confirming the performance of the filter of the nanoparticle separation device according to an embodiment of the present invention, (a) 100 nm particle filter capacity in the combination of AAO filter 200 nm and 100 nm, (b) TEPC 100 nm particle filter capability in filter 600 nm and AAO filter 20 nm combination and (c) filter capacity in 800 nm and 100 nm particle mixture liquid in TEPC filter 600 nm and AAO filter 20 nm filter were confirmed.

8 is a result of analyzing the size and concentration of the nanoparticles using a nanoparticle tracking analysis (NTA) in a solution mixed with nano beads of 100 nm and 800 nm.

9 is a result of analyzing the nano-vesicle concentration before / after performing the disk through NTA using the nanoparticle separation device according to the present invention, (a) results of nano-vesicle concentration separated from the supernatant cultured LNCaP cells, ( c) Powder concentration results from urine of bladder cancer patients, (b) SEM images confirming that vesicles isolated from urine of bladder cancer patients were filtered by filter II and (d) / (e) TEM images of vesicles recovered from filter II. It is shown.

10 is a result of analyzing the size and concentration of nano vesicles between 30 nm and 600 nm using NTA (nanoparticle tracking analysis) in 1 mL of urine.

11 is a result of comparing the nano-vesicle acquisition efficiency according to three methods of nanoparticle separation device according to an embodiment of the present invention and a commercialization kit (Exospin) using the conventional ultracentrifugation (UC) and precipitation reagents, NTA The analysis results are shown.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present invention. Shapes, sizes, ratios, angles, numbers, and the like disclosed in the drawings for describing the embodiments of the present invention are exemplary, and the present invention is not limited to the illustrated items. However, in describing the preferred embodiment of the present invention in detail, if it is determined that the detailed description of the related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. In addition, the same reference numerals are used throughout the drawings for parts having similar functions and functions.

In addition, in the specification, when a part is 'connected' to another part, it is not only 'directly connected' but also 'indirectly connected' with another element in between. Include. In addition, the term 'comprising' of an element means that the element may further include other elements, not to exclude other elements unless specifically stated otherwise.

1A and 3A are perspective views of a nanoparticle separator 10 according to an embodiment of the present invention, and FIGS. 1B and 3B are enlarged views of the nanoparticle separator 10 according to an embodiment of the present invention. . As shown in Figure 1a and 3a, the nanoparticle separation apparatus 10 according to an embodiment of the present invention is a housing portion 100, a sample accommodating portion 200, filtration chamber portion 300, waste liquid receiving portion 400 and the microchannel 500, and as shown in Figure 1b and / or 3b, the cleaning chamber 600, the valve 700 and / or the particle collecting portion 800 is further It can be configured to include.

In the nanoparticle separation device 10 according to an embodiment of the present invention, a fluid sample may be introduced, and centrifugal force by rotation of the device may be used to separate nanovesicles of a desired range from the fluid sample. It is also possible to separate several fluid samples simultaneously. By adopting such a configuration, it is expected that only nano vesicles can be separated at low centrifugal force irrespective of antibody specificity, so that the whole process can be integrated and used as a high recovery rate of automated nano vesicles.

Hereinafter, each component constituting the nanoparticle separation device 10 according to an embodiment of the present invention will be described in detail.

The housing part 100 is a structure capable of self-rotation in order to provide centrifugal force for separating the nanovesicles from the fluid sample while providing a space in which the components to be described later are built. In this case, the housing part 100 is It may be made of polycarbonate (PC) material, but is not limited thereto.

The sample accommodating part 200 is configured to provide a space in which a fluid sample to be separated is carried. The sample accommodating part 200 is installed in the housing part 100 and is applied at the same time while the sample is applied thereto while applying centrifugal force. Has an inclined surface inclined at an angle different from the radial direction, and further includes a groove (not shown) in order to prevent backflow of the separated impurities, and the sample accommodating part 200 is formed of an inclined surface and curved to convey the sample. Loss and damage of the sample can be minimized. In this case, the fluid sample may be a biological sample such as urine, blood, salivary fluid, sputum, and the like, including an aqueous solution in which nanoparticles are dispersed, a cell body, and rare biological particles, and preferably, urine, but is not limited thereto. .

The filtration chamber 300 is configured to collect desired nanoparticles including one or more filtration membranes, and the filtration chamber 300 may be detachable from the housing 100 by applying a physical force as necessary. In this case, in order to smoothly attach and detach, a fastening part (not shown) may be further included. Meanwhile, as illustrated in FIGS. 1B and 3B, the filtration chamber part 300 may include a first filtration part 310 and a second filtration part 320.

Nanoparticle separation device 10 according to the present invention, by using a plurality of filter membranes having different pore sizes, is a principle to obtain a desired range of nano-vesicles from the sample, for example, to filter out impurities having a large particle size By combining the filter with a filter membrane of a size that can pass impurities of particles smaller than the size of the desired particle, the desired particle size nanoparticles collected between the filter membrane and the filter membrane can be filtered out.

Therefore, as described above, the present invention may include a first filtration unit 310 and a second filtration unit 320 having a plurality of different pore sizes, depending on the nanoparticle size required, Or an additional filter for collecting.

At this time, the filtration membrane may be made of a laminated or separated structure according to the user's implementation, this laminated or separated structure may affect the collection of the separated nano-vesicles during the automated process. For example, until the nano-vesicles are collected, the stacked structure can collect nano-vesicles by physically separating and eluting the filters, whereas the separated structures can be integrated into the entire process without the need for separation of the filters, thereby providing more convenient Collection may be possible.

More specifically, the first filtration unit 310 may be connected to the sample accommodating part 200 as shown in FIGS. 1B and 3B to filter out primary impurities in the fluid sample. In this case, the first filtration unit 310 may have a plurality of pores having a diameter of 100 nm to 1 μm, preferably, to filter out impurities having a large particle size, and more preferably, have a 600 nm diameter. Can be.

The second filtration unit 320 is configured to collect only the desired nanoparticles while removing secondary impurities. For example, the second filtration unit 320 allows particles of a size smaller than a desired range to pass therethrough, Only nanovesicles in the range can be captured.

Meanwhile, as shown in FIGS. 1B and 3B, the second filtration unit 320 may be connected to the first filtration unit 310 and the washing chamber unit 600 to be described later. As described above, When the filtration membrane is formed in a stacked structure between the filtration membranes (see FIG. 1B), the first filtration unit 310 and the second filtration unit 320 are filtered in the same chamber. On the other hand, in the case where the structure between the filtration membranes is formed to be separated from each other (see FIG. 3B), the first filtration unit 310 and the second filtration unit 320 are each formed as chambers independent of different radial coordinates, thereby forming the fluid. The sample may be capable of performing a plurality of particle filtration through the plurality of filtration membranes. In addition, the filtration chamber 300 may be connected to one or more washing chamber 600 as necessary.

At this time, in order to filter out impurities having a small particle size except for nano-vesicles in a desired range, the second filtration unit 320 may preferably have a plurality of pores having a diameter of 1 nm to 100 nm, more preferably. May have a 20 nm diameter. At this time, the secondary impurities, which are impurities having a small particle size, may be non-vascular proteins.

In addition, the method for separating nano vesicles using the filtration membrane according to the present invention requires two or more kinds of filtration membranes including small pores and large pores, as described above, to filter nano vesicles within a certain range, and conventionally, polycarbonate Filter membrane was prepared by using the material, and when the filtration membrane of small diameter pores (1 nm ~ 100 nm) using the polycarbonate material, the size of the pores is not uniform, the porosity is low, uniform vesicle separation Was not suitable. In addition, when manufacturing the filter membrane using aluminum anodized, it may have a relatively uniform size and high porosity, but there is a problem that the durability is weak and well broken. Thus, the nanoparticle separation device 10 according to an embodiment of the present invention uses a low centrifugal force to separate the nano vesicles, thereby preventing degradation of durability due to the use of the anodized aluminum. For this reason, it is possible to use a filtration membrane of a material having a high porosity while having a uniform pore size.

Therefore, preferred materials of the filtration membrane forming the first filtration unit 310 and the second filtration unit 320 are polycarbonate, polystyrene, polymethyl methacrylate, cyclic olefin polymer, anodized aluminum, nickel, silicon Etc., but most preferably, anodized aluminum.

The waste solution accommodating part 400 is configured to provide a space for accommodating the sample solution filtered from the first filtration part 310 and the second filtration part 320, as shown in FIGS. 1B and 3B. In order to accommodate the filtered sample, it may be connected to the first filtration unit 310 and the second filtration unit 320. At this time, the waste liquid receiving unit 400, may be composed of a single or a plurality depending on the position formed between the first filtration unit 310 and the second filtration unit 320. For example, when the position structure between the filtration chambers 300 is a stack type, the first filtration unit 310 and the second filtration unit 320 are directly connected in one chamber, so that only a single waste liquid receiving unit 400 is provided. The filtered sample may be accommodated, and when the filtration chamber 300 is formed as a separate chamber, due to centrifugal force, it prevents the diffusion of impurities after purification due to the reverse flow of the previously separated filtrate, and For accurate metering, the waste liquid receiving unit 400 is preferably formed of one or more, but is not limited thereto.

The micro channel 500 is a configuration for providing a space for the flow of the sample between the above-described configuration, as shown in Figure 1b and 3b, respectively disposed between the chambers, the filtration chamber 300 ) May be disposed above or below the chambers, and the chambers may be disposed at a distance from the center of the apparatus to minimize loss of solution. In addition, the micro channel 500 may be connected to a channel through the device to change the channel of the fluid sample. On the other hand, the micro-channel unit 500 may be composed of first to third and / or seventh micro-channel unit 500, the micro-channel unit 500, as described above, the user's embodiment According to the arrangement between the filters according to, the arrangement of the micro channel 500 may vary.

For example, when the position structure between the filtration chambers 300 is a stacked type, the first micro channel 510 connects the sample receiving part 200 and the waste liquid receiving part 400 to the second micro channel 520. ) Connects the washing chamber 600 and the first filtration unit 310 to the second filtration unit 320 to be described later, and the third micro channel 530 is the first micro channel 510. And a first filtration unit 310-a second filtration unit 320.

On the other hand, when the position structure between the filtration chambers 300 is formed as an independent chamber, the first microchannel 510 connects the sample accommodating part 200 and the waste liquid accommodating part 400-1. The second microchannel unit 520 connects the sample receiving unit 200 and the first filtration unit 310, and the third microchannel unit 530 includes the first filtration unit 310 and the second filtration unit ( 320 is connected, and the fourth microchannel 540 connects the cleaning chamber 600 and the second filtration unit 320, and the fifth microchannel 550 is the third microchannel. 530 and the waste liquid accommodating part 400-1, and the sixth microfluidic part 560 connects the second filtration part 320 and the waste liquid accommodating part 400-2, and the seventh microfluidic path. The unit 570 is connected to the second filtration unit 320 and the particle collecting unit 800 to be described later.

The cleaning chamber part 600 is configured to provide a space in which a cleaning solution for cleaning the filtration membrane of the filtration chamber part 300 is supported. As shown in FIGS. 1B and 3B, the filtration chamber part 300 is provided. It can be connected with. At this time, the preferred washing solution may be phosphate buffer saline (PBS).

The valve 700 is a configuration for opening and closing the flow path between the components in order to prevent the flow in the undesired direction due to the centrifugal force in the flow of the sample between the above-described components, on the fine flow path 500 connected to each component Can be placed in. At this time, the valve 700 may be automatically opened and closed through an external signal. Meanwhile, as described above, the arrangement of the valve 700 may vary according to the arrangement between the filtration membranes according to the embodiment of the user.

For example, when the position structure between the filtration membranes is stacked, the first valve 710 is disposed on the first microchannel 510, and the second valve 720 is the second microchannel 520. The third valve 730 is disposed on the third microchannel 530.

On the other hand, when the filtration membrane position structure is formed as an independent chamber, the first valve 710 is disposed on the first microchannel 510 and the second valve 720 is the second microchannel. 520, the third valve 730 is disposed on the third microchannel 530, the fourth valve 740 is disposed on the fourth microchannel 540, and The fifth valve 750 is disposed on the sixth microchannel portion 560, and the sixth valve 760 is disposed on the seventh microchannel portion 570.

On the other hand, as described above, the nanoparticle separation device 10 according to an embodiment of the present invention, according to the arrangement of the filtration membrane of the filtration chamber 300, may affect the collection of nano-vesicles. For example, in the case of the structure in which the filtration membranes are stacked in a stack, after the sample injection and separation of the nano vesicles are separated, the second filtration unit 320 is separated from the nanoparticle separation device 10 to obtain the vesicles on the filter. You may have to go through the process. Therefore, in order to separate the vesicles more efficiently, when the filtration chamber 300 is formed as a structure independent from the filtration chamber 300, the particle collecting part 800 collecting the vesicles from the second filtration part 320 having the independent chamber is further added. It can be configured to include.

More specifically, the particle collecting unit 800 is configured to provide a space for collecting the obtained nano vesicles, as shown in Figure 3b, it may be connected to the second filter unit 320, thereby, After filtration in the second filter unit 320, the remaining nano vesicles may be accommodated in the particle collecting unit 800 due to centrifugal force. More specifically, the waste liquid is discarded to the waste liquid receiving portion 400 using a rotation speed of less than 3000 rpm or less than the capillary pressure inside the pores present in the filtration membrane, and then, on the upper surface of the filtration membrane of the second filter portion 320. Only located nanoparticles can be selectively recovered.

On the other hand, the nanoparticle separation device 10 according to an embodiment of the present invention, as described above, depending on the position of the plurality of filtration membranes according to the user's embodiment, the configuration can be changed. Such a difference in configuration may bring a difference in the integration up to the separation process of the nano-vesicles.

For example, as illustrated in FIG. 1B, when the filtration membrane position structure of the filtration chamber part 300 is a stacked type, each configuration is as follows.

1) Put the urine sample, the sample receiving unit 200 to separate the impurities

2) cleaning chamber part 600 containing a cleaning solution to increase the purity of the nano-vesicles

3) 600 nm first filter 310 for separating other vesicles

4) 30 nm second filter 320 for separating the nano-vesicles

5) Waste solution containing part 400 for accommodating small impurities other than nano vesicles

6) connecting the respective components to the flow of the sample, micro-channel 500

7) Valve 700 to regulate the flow of the selective fluid between the configurations

Through this configuration, as shown in Figure 1c, it is possible to filter the desired range of nanoparticles from the sample using two different sizes of filtration membrane.

In addition, as shown in Figure 1a, the nanoparticle separation device 10 may be composed of four identical units at the same time it is possible to separate the four types of samples, after the assembly of the adhesive layer 30 nm of the second filtration unit 320 Can be fixed and detachable smoothly. In this case, in order to attach and detach the filtration unit, it may be fixed by a fastening unit having elasticity, and the preferred gasket material may be polydimethylsiloxane, silicone, latex or rubber, but is not limited thereto.

Next, in the case where the filtration membrane arrangement of the filtration chamber part 300 is a lamination type, the vesicle separation process is performed after the injection of the sample (red water) and the washing liquid (yellow water), as shown in FIG. 2. The process is automated and the process is as follows.

1) The disk is composed of three valves and four chambers (sample receiving part 200, filtration chamber part 300, waste liquid receiving part 400 and washing chamber part 600), the red circle is a closed valve Blue circle means open valve (see FIG. 2A).

2) Like the red arrow, the impurity treatment chamber extracts impurities from the sample using centrifugal force, and the blue arrow shows the filter washing (see FIG. 2B).

3) After the other vesicles have been filtered out, they are transferred to a chamber containing a 600 nm filter through an open valve 1 to filter out the nano vesicles and other small impurities are moved centrifugally to the discard chamber. At this time, the valve 2 may be blocked to prevent backflow of the solution (see FIG. 2C).

4) After all sample transfer, nano vesicles are filtered over a 30 nm filter (see FIG. 2D).

5) The cleaning solution is transferred to a chamber with a filter to remove impurities other than nanovesicles. At this time, the valve 3 may be blocked to prevent backflow of the solution (see FIG. 2E).

6) The nano vesicles from which impurities are removed after washing remain on the 30 nm filter, after which the nano vesicles can be obtained by separating and eluting the filter (see FIG. 2F).

As shown in 6) above, when the position structure of the filtration membrane of the filtration chamber part 300 is a lamination type, the filtration membrane is separated to separate nano vesicles.

On the other hand, when formed as a structure as an independent chamber between the filtration chamber 300, the entire process of separation of the vesicles are integrated so that each configuration is as shown in Figure 3b, as follows.

1) Put the urine sample, the sample receiving unit 200 to separate the impurities

2) cleaning chamber part 600 containing a cleaning solution to increase the purity of the nano-vesicles

3) first filtration unit 310 for separating other vesicles

4) second filter 320 for separating nano-vesicles

5) Waste solution containing part 400 for accommodating small impurities other than nano vesicles

6) connecting the respective components to the flow of the sample, micro-channel 500

7) Valve 700 to regulate the flow of the selective fluid between the configurations

8) Particle collecting unit 800 for collecting nano-vesicles

Through such a configuration, as shown in FIG. 3C, the nanoparticle separator 10 further includes a particle collecting part 800 capable of collecting nano vesicles. Unlike the case where the structure is stacked, the vesicles can be obtained without a separate filter after injection of the sample, and the entire process up to the separation process of the nano vesicles is integrated.

The filtration membrane of the nanoparticle separation device 10 may include a filtration membrane made of anodized aluminum, and the filtration membrane made of anodized aluminum is made of pores having a higher porosity and a relatively uniform diameter compared to other materials. Have

On the other hand, the process for separating the nano vesicles can be automated. At this time, in order to automate the whole process, the control system for controlling the opening and closing of the valve 700, the rotational speed and the direction of the housing portion 100 may be further included.

More specifically, Figure 4 shows a state using the nanoparticle separation device 10 according to an embodiment of the present invention. As shown in Figure 4, in order to use the nanoparticle separation device 10 according to an embodiment of the present invention, a sample (maximum 1 ml) and a buffer solution (600 µl) are first included in the sample accommodating part 200 and the washing chamber. The unit 600 is loaded. Thereafter, when the housing part 100 is rotated at a rotation speed of 3000 rpm, impurities of the sample are precipitated in the inclined chamber (FIG. 4A), and then the second valve 720 is opened to clear the supernatant. It is filtered through the filtration unit 310 and the second filtration unit 320 is moved to the waste liquid receiving unit (400-1) (Fig. 4b). Subsequently, during the filtration process, large particles are filtered out of the first filtration unit 310 having a diameter of 600 nm, and non-vascular proteins are passed through the second filtration unit 320 having a diameter of 20 nm to be removed. Only the nano vesicles are concentrated on the second filter unit 320. Thereafter, the third valve 730 is closed and the fourth valve 740 is opened so that the washing buffer flows from the washing chamber 600 to the second filtration unit 320 to perform washing (FIG. 4C). When the fifth valve 750 is opened and the housing part 100 is rotated at a rotational speed of 1500 rpm, the solution remaining under the second filtration part 320 completely moves to the waste liquid receiving part 400-2. (Figure 4d). Finally, when the sixth valve 760 is opened and the housing part 100 is rotated at a rotation speed of 1500 rpm, the concentrated vesicles filtered by the second filtration part 320 are moved to the particle collecting part 800. (FIG. 4E). Because of this, nano vesicles can be separated in a short time using a low centrifugal force.

As shown in Table 1 below, as a result of comparing the separation method of the present invention and the conventional ultracentrifugation method and commercialization kit, the conventional method requires a lot of time and various processes for sample processing using an ultracentrifuge or a precipitation reagent. On the other hand, in the case of the nanoparticle separation device 10 having a separated filter unit, the total operating time is within 30 minutes and the G force operating range is also significantly lower than the ultracentrifugation separation and commercialization kit.

[Correction under Rule 91 06.12.2016]

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Experimental Preparation

1-1. Nanoparticle Separator Manufacture according to the Invention

In order to manufacture the microfluidic device according to the present invention, the microfluidic device was designed using a 3D CAD program and manufactured using a numerical milling machine (CNC milling machine). More specifically, according to the design by using a polycarbonate (PC, PC, I-Components Co. Ltd, Korea) was divided into a Top, Body and Base layer (layer) to process the nanoparticle separation device (see Figure 5). When processing was complete, all layers were laminated using two pressure-sensitive papers, double sided adhesive (DFM 200 clear 150 POLY H-9V-95, FLEXcon, USA) and a customized compression device. On the other hand, the valve according to an embodiment of the present invention can be disposed on the top layer, and can be opened and closed automatically by an external signal as needed. According to a general procedure for manufacturing a lab-on-a-disc fused to a conventionally known membrane filter, each layer is processed according to computer numerical control as described above, and the opposite side of the filtration chamber portion according to an embodiment of the present invention. The face is filter I and filter II, respectively, inserting commercially available membranes such as track-etched PC membranes (SPI, 13 mm, pore diameter of 0.6 μm) and aluminum oxide anodes (Whatman, 13 mm, 0.02 μm). Carved for.

On the other hand, all chambers and channels were coated with 1% pluronic solution (PEO-PPO-PEO block copolymer) to obtain nonspecific adsorption and maximum yield of nano vesicles (see FIG. 6). More specifically, the 1% Pluronic solution was allowed to react in all chambers and channels for 1 hour, respectively, after which the Pluronic solution was removed and washed with PBS buffer.

1-2. Cell culture

LNCaP cells, a prostate cancer cell line, were grown in RPMI medium (Gibco, UK) supplemented with 10% exo-free FBS (System Biosciences Inc., CA), and 1% antibiotic / antifungal, and at 5% CO _2, 37 ° C. conditions. Proceed in an incubator. Cell culture supernatants were harvested after 24 hours and extracellular vesicles were collected as described in each protocol.

1-3. Clinical Sample Storage and Processing

In accordance with the guidelines of the Institutional Review Board, urine samples from healthy donors were collected as in bladder cancer patients, and the first urine (15 ml) was collected from bladder cancer patients. Collected urine was stored until use at -80 ℃.

To isolate nanovesicles, the samples were thawed and used at RT, and 5 ml urine each was used to separate extracellular vesicles using ultracentrifugation (UC) and Exospin, and the nanoparticles according to the present invention. For separation of nano vesicles in the separator, 400 μl urine was used.

Example 2. Confirmation of the separation effect of the nano beads using a mixed solution and urine

2-1. Check disk performance effect by filter combination

In order to confirm the effect of the combination of the filter diameter of the nanoparticle separation device according to the present invention, the experiment was carried out according to the combination of the filter size.

More specifically, when the filter I of the 200 nm AAO membrane and the filter II of the 20 nm membrane are combined, as shown in FIG. 7A, most 100 nm PS nanoparticles are sandwiched within a 200 nm diameter and PS nanoparticles on the 20 nm filter. Did not find. Next, when the filters were combined at 600 and 100 nm, the nanoparticles passed through the 600 nm filter, but many particles were present on the 100 nm filter, but the particles did not recover. Finally, when the filters were combined at 600 and 20 nm, high recovery of the PS nanoparticles could be confirmed (see FIG. 7B).

Next, using a solution mixed with 800 nm and 100 nm PS nanoparticles, the size-selective separation performance experiment according to the filter size was conducted.

More specifically, as a result of performing a disk using a solution in which the filters were combined at 600 and 20 nm and mixed with 800 nm and 100 nm PS nanoparticles, as shown in FIG. 7C, 800 nm nanoparticles passed through the filter I. It was filtered without, and only 100 nm nanoparticles were found to be concentrated in the filter II.

In this example, nanoparticle tracking was carried out through nanoparticle tracking analysis (NTA) to analyze the size and concentration.

FIG. 8 shows experimental data showing that 100 nm particles are filtered as a result of performing a disk experiment in a solution mixed with 100 nm and 800 nm nano beads. The mixed solution contains both 100 nm and 800 nm particles and has a low concentration value. On the other hand (see FIG. 8A), disc performance showed that only 100 nm beads were detected and concentrated in the filter (see FIGS. 8B and C).

2-2. Determine ability to carry out extracellular vesicle enrichment

After confirming the separation effect of the disc according to the PS nanoparticles of different sizes according to Example 2-1, the ability to perform extracellular vesicle enrichment from the CCS and urine samples of the disc was evaluated.

More specifically, 1 mL of the urine samples of patients with CCS or bladder cancer by performing a disk experiment to analyze the concentration through NTA, as shown in Figure 9a and b, about 5 times compared to before the disk experiment Showed a high concentration.

FIG. 9C shows that large impurities are actually filtered on filter I and extracellular vesicles of urine of bladder cancer patients are filtered by filter II, and FIGS. 9D and e are results confirming that the nanovesicles are recovered in a rounded form. .

FIG. 10 shows experimental data showing that nano vesicles are filtered between 30 nm and 600 nm as a result of performing disk experiments in 1 mL of urine, while particles of various sizes are observed in urine (see FIG. 10A). In the filter, nano vesicles were detected between 30 nm and 600 nm, showing the concentration of nano vesicles in the range, and the total separation time was within 40 minutes (see FIG. 10B).

Example 3 Parcel Separation and Quantitative Comparative Analysis

In order to confirm the effect of the vesicle separation using the nanoparticle separation device according to the present invention from the conventional vesicle separation method, ultracentrifugation, Exospin and the method using the nanoparticle separation device according to the present invention are as follows. The experiment was conducted.

3-1. Vesicle Separation by Ultracentrifugation

Ultracentrifugation (UC) is centrifuged at 300 x g for 10 minutes to remove cell debris from the sample samples obtained in Examples 1-3. Thereafter, the resulting pellet was centrifuged at 20,000 x g for 30 minutes, and the resulting pellet was discarded. The supernatant was then transferred to an 80 ml polypropylene ultracentrifuge tube and centrifuged at 4 ° C. and 50,000 × g for 1 hour on a Ti45 fixed angle rotor. After the centrifugation, the resulting pellet was discarded, and the supernatant was transferred to a new ultracentrifuge tube, followed by centrifugation at 4 ° C. and 150,000 × g for 2 hours in a Ti45 fixed angle rotor to defoamer pellets. Collected. The supernatant is then discarded, and the pellet is resuspended with 1 ml of 10 nm pre-filtered PBS and transferred to a 1 ml polycarbonate ultracentrifuge tube in a MLA-130 fixed angle rotor. Centrifugation was performed at 4 ° C. and 150,000 × g for 2 hours. The resulting pellet was resuspended in 1 ml of 10 nm pre-filtered PBS and stored at 4 ° C. for immediate use or at −80 ° C. for long term storage.

3-2. Vesicle Separation Using Exospin Exosome Purification Kit

As described above in Example 3-1, in order to confirm the effect of the vesicle separation using the nanoparticle separation apparatus according to the present invention from the conventional vesicle separation method, the vesicles are separated using the Exospin exosome purificaition kit. The experiment was conducted.

More specifically, in order to remove the cell debris of the sample obtained through Example 1-3, centrifugation for 10 minutes at 300 × g, the supernatant centrifuged for 30 minutes at 20,000 × g, the resulting pellet Discarded. Again, the supernatant was mixed gently with half the volume of buffer A, and the resulting pellet was centrifuged at 20,000 × g for 1 hour at 4 ° C., and the resulting pellet was resuspended with 100 μl of PBS provided in the kit. The vesicle pellets were purified using a spin column provided according to the manufacturer's instructions, and 200 분리 of the separated vesicles were stored at 4 ° C. for immediate use / short term storage or at −80 ° C. for long term storage.

3-3. Vesicle separation and quantification according to nanoparticle separation device according to the present invention

An experiment was performed to separate the vesicles by using the nanoparticle separation device according to an embodiment of the present invention. More specifically, large particles or cell debris in a sample sample (urine or complex medium) is precipitated at 300 x g for 2 minutes, and the clear supernatant is transferred to the filtration chamber and filtered through filters I and II for 15 minutes at 500 x g. Let's do it. After the filter II was washed with PBS solution at 500 x g for 10 minutes, the filtered sample solution was discarded into the waste solution holding part 2. At this time, the vesicles filtered in Filter II (˜100 μl) were transferred to the particle collection part, and Filter II was washed with 100 μl PBS. The solution containing the vesicles moved to the particle collection was used for further analysis.

Example 4 Enzyme linked immunosorbent assay (ELISA)

In order to compare the separation effect according to each vesicle separation method, the vesicle solution according to the separation method of Example 3 was analyzed using an enzyme immunoassay (ELISA).

Antifoam solution was prepared by maintaining the same input capacity for the three separation methods of Example 3, the plate was coated with an antibody (anti-CD9 antibody, MEM61, abcam, MA, US) overnight at 4 ℃, 1 hour Blocked with 1% BSB-PBS buffer at 37 ° C. Thereafter, the cells were washed with 0.1% BSA-PBS buffer (wash buffer), incubated in PBS buffer (100 µl) with antifoam solution at 37 ° C for 1 hour, and then the solution was removed, and the plate was then washed twice with washing buffer. Washed. At this time, the biotin-conjugate detection antibody solution (anti CD81 antibody, biotin, LifeSpan Biosciences, INC, WA, US), washed three times with washing buffer and diluted with PBS buffer (100 μl, 500 ng / ml) Add and incubate in the room for 1 hour. After washing three times with wash buffer, plates were incubated for 30 min at RT with HRP-conjugate streptavidin solution diluted in PBS buffer (100 μl, 1: 1000 in PBS). Then, add 100 μl of TMB solution and incubate for 15 minutes in the room, finally add 50 μl of termination solution to each well to stop the reaction, and the absorbance of the solution was measured at 450 nm with a plate reader spectrophotometer (TECAN ) Was measured.

Example 5 Confirmation of Results of Separation of Vesicles

The efficiency according to the separation method of each endoplasmic reticulum through the above example was analyzed. More specifically, nano vesicles were isolated by the three methods using 1 ml LNCaP CCS.

As a result, as shown in Figure 11, NTA results show a high concentration yield of nano vesicles separated from the nanoparticle separator according to the present invention.

More specifically, as shown in FIG. 11, the concentrations of the detected extracellular vesicles were 1.33 ± 0.07, 1.32 ± 0.06 and 7.67 ± 1.5 × 10 ⁹ particles / ml, respectively. It confirmed that it was 5.8 times higher than the method.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The present invention relates to a nanoparticle separation device and method using a microfluidic device. Specifically, since it is based on low centrifugal force and size, nano-vesicles irrelevant to antibody specificity can be separated in a short time without a conventional ultracentrifuge, and by integrating and automating the entire process after sample injection, no additional expert personnel are required. Accurate metering of fluids reduces the loss of nano vesicles.

Claims (29)

1. In the nano particle separator,

   Rotatable disk-shaped housing portion 100;

   A sample accommodating part 200 providing a space supported by injecting a fluid sample including nanoparticles;

   A filtration chamber unit 300 including a filtration membrane having pores of 1 nm to 1 μm capable of filtering nanoparticles from the fluid sample;

   Waste solution holding unit 400 for storing the filtered sample solution; And

   And a micro flow path portion 500 for providing a passage for the flow of the fluid sample.
2. The method of claim 1, wherein the fluid sample is a biological sample selected from the group consisting of urine, blood, saliva, sputum and the like, including aqueous solutions and cell bodies, rare biological particles and the like dispersed in various nanoparticles, Nano particle separator.
3. According to claim 1, The nanoparticle separation device, Nanoparticle separation device, characterized in that further comprising a washing chamber 600 for providing a space in which the cleaning solution is carried.
4. The method of claim 1, wherein the filtration membrane is made of a material selected from the group consisting of polycarbonate, polystyrene, polymethyl methacrylate, cyclic olefin copolymer, anodized aluminum, nickel and silicon. Particle separator.
5. According to claim 1, The nanoparticle separation device, Nanoparticle separation device, characterized in that it further comprises one or more fastening portion for detachable attachment of the filtration chamber (300).
6. The nanoparticle separator of claim 5, wherein the fastening part is an elastic material selected from the group consisting of polydimethylsiloxane, silicone, latex, rubber, and the like.
7. The nanoparticle separation device of claim 1, wherein the microchannel unit 500 is connected to a channel that penetrates the device to change a channel of the fluid sample.
8. In the nano particle separator,

   Rotatable disk-shaped housing portion 100;

   A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

   A filtration chamber unit 300 including two or more filtration membranes capable of filtering a sample;

   Waste solution holding unit 400 for storing the filtered sample solution; And

   And a micro flow path portion 500 for providing a passage for the flow of the fluid sample.
9. The method of claim 8, wherein the filtration membrane is nano, characterized in that composed of a material selected from the group consisting of polycarbonate, polystyrene, polymethyl methacrylate, cyclic olefin copolymer, anodized aluminum, nickel and silicon. Particle separator.
10. According to claim 8, The filtration membrane, Nanoparticle separation device, characterized in that the detachable detachment from the housing portion (100) by a physical force.
11. The method of claim 8, wherein the filtration membrane, the filtration chamber 300, two or more stacked in the same chamber to collect the nanoparticles through the filtration membrane having two or more sizes during fluid transfer in a single chamber A nanoparticle separator.
12. The method of claim 8, wherein the filtration membrane comprises a single filtration membrane in a single chamber, and the plurality of chambers are disposed at different radial coordinates so that the fluid sample passes through the plurality of filtration membranes to perform plural particle filtration to a specific size. Nanoparticle separator, characterized in that to collect a range of nanoparticles.
13. The method of claim 8, wherein the filtration membrane,

    A first filtration membrane having at least one or more pores of 100 nm to 1 μm diameter; And

    And a second filtration membrane having at least one or more pores having a diameter of 1 nm to 100 nm.
14. The method of claim 8, wherein the micro-channel 500 is disposed above or below the filtration chamber 300, the chambers are disposed at a distance away from the center of the device to minimize the loss of solution Nano particle separator.
15. In the nano particle separator,

    Rotatable disk-shaped housing portion 100;

    A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

    A filtration chamber unit 300 for receiving one or more filtration membranes capable of filtering a sample;

    Waste solution holding unit 400 for storing the filtered sample solution;

    A micro flow path part 500 for providing a passage for the flow of the fluid sample; And

    And a valve (700) capable of selectively controlling the flow of fluid within the microchannel, characterized in that the nanoparticles are filtered from the sample.
16. The method of claim 15,

    The sample accommodating part 200, the nanoparticle separation device, characterized in that to perform a sample purification that can purify the impurities of the sample.
17. The nanoparticle separation device of claim 15, wherein the sample accommodating part 200 performs impurity separation by including a space formed at an angle at which a lower portion thereof is distorted than a radial direction.
18. The nanoparticle separator according to claim 15, wherein the sample accommodating part 200 includes a groove for preventing separated impurity backflow.
19. The nanoparticle separation device of claim 15, wherein the sample accommodating part 200 is formed of an inclined surface and a curve to minimize loss and damage of the sample during sample transfer.
20. The nanoparticle separator according to claim 15, wherein the valve 700 can be opened or closed in response to an external signal.
21. The method of claim 15, wherein the valve 700 is located outside the rotatable device for the whole process automation, characterized in that it comprises a system capable of controlling the opening and closing of the valve and the rotational speed and direction of the body, Particle separator.
22. The nanoparticle separator according to claim 15, wherein the waste liquid receiving part 400 is capable of separating nanoparticles of high purity without a separate impurity treatment.
23. The apparatus of claim 15, wherein the filtration chamber part 300 is connected to one or more of the waste liquid receiving parts 400 to prevent impurity diffusion after purification.
24. 16. The nanoparticle separator according to claim 15, wherein the nanoparticle separator further comprises at least one fastening part for detachable attachment of the filtration chamber part (300).
25. The nanoparticle separator of claim 15, wherein the nanoparticle separator minimizes nonspecific binding to a surface by injecting a BSA (bovine serum albumin) protein or a Pluronic (PEO-PPO-PEO) polymer material. .
26. According to claim 1, wherein the filtration chamber 300 and the waste liquid receiving portion 400, characterized in that it comprises a vent (vent) for performing a smooth filtration, nanoparticle separation device.
27. In the nano particle separator,

    Rotatable disk-shaped housing portion 100;

    A sample accommodating part 200 which provides a space supported by injecting a fluid sample;

    A filtration chamber unit 300 for receiving one or more filtration membranes capable of filtering a sample;

    Waste solution holding unit 400 for storing the filtered sample solution;

    A micro flow path part 500 for providing a passage for the flow of the fluid sample;

    A valve 700 capable of selectively controlling the flow of fluid in the microchannel; And

    Nanoparticle separation device, characterized in that it can filter and recover a specific size range of nanoparticles from a sample, including a particle collecting unit (800) capable of recovering the filtered specific size range of nanoparticles.
28. 28. The method of claim 27, wherein the recovery of the nanoparticles, the upper surface of the membrane and the solution to be recovered containing the nanoparticles, when the lower surface adjacent to the waste liquid, the maximum 3000 rpm less than the capillary pressure inside the pores present in the filter membrane After discarding the waste liquid adjacent to the lower surface using the following rotational speed to the waste liquid receiving portion 400, characterized in that for selectively recovering a solution containing the nanoparticles located on the upper surface of the filter membrane, nanoparticle separation device.
29. 28. The method of claim 27, wherein the particle collecting unit 800, the upper surface of the filtration chamber 300 is connected to the micro channel, the lower surface of the filtration chamber 300 is characterized in that it is connected to the waste liquid receiving unit 400 Nanoparticle separation apparatus.

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