KR102425055B1 - Nanoparticle separation device and method of separation of nanoparticles using it - Google Patents

Abstract

The present invention is a nanoparticle separation device including a microfluidic channel, comprising a single injection hole, at least two outlets, an injection channel and a plurality of parallel channel portions, wherein the aspect ratio of the height to the channel width of the parallel channel portion is greater than 1 5 It relates to a nanoparticle separation device and a nanoparticle separation method using the same, characterized in that below.

Description

Nanoparticle separation device and method of separation of nanoparticles using it

The present invention relates to a separation technology of viscoelastic non-Newtonian fluid-based nanoparticles, and more particularly, a device for separating cell-derived nanoparticles from a blood sample using the non-Newtonian fluid flow characteristics in a microfluidic channel, and the same it's about how

Cell-derived nanoparticles are a type of extracellular vesicle and are known as biomarkers that play an important role in physiological processes such as communication between cells, blood clotting, inflammation, and metastasis of cancer cells. These cell-derived nanoparticles have a size of about 100-1000 nm, and are generated from various cells such as red blood cells, white blood cells, platelets, and endothelial cells due to causes such as cell activation, apoptosis, or external stimulation. Separation of cell-derived nanoparticles contained in blood is important for biological research or clinical analysis, so technology development for this is required. In particular, when the amount of cell-derived nanoparticles in the blood is increased, it can attract attention as a useful biomarker that can predict cardiovascular disease, inflammatory disease, or cancer.

The main methods used for isolation of cell-derived nanoparticles are ultracentrifugation and immunoaffinity capture. However, in the ultracentrifugation method, artificial cell-derived nanoparticles can be generated due to the high shear force applied to the cells as a stimulus, the protocol has not yet been established, and the size distribution of the cell-derived nanoparticles during the centrifugation process may be biased. There is a problem that there is. On the other hand, the immunoaffinity capture method has a disadvantage that the nanoparticles may lose their original function or cause a change in the particle state. Therefore, in order to overcome this limitation, it is necessary to develop a technology that does not require separate labeling, does not use a centrifuge, and can separate cell-derived nanoparticles in an intact state.

Technology to separate nano-sized cell-derived nanoparticles such as filter method, immunoaffinity capture method in microfluidic device, inertial hydrodynamic technology, and deterministic lateral displacement according to the development of microfluidic technology These are being developed in various ways. Recently, microfluidic technology based on viscoelastic, non-Newtonian fluids has been receiving great attention due to its characteristic nonlinear elastic force gradient inherent in fluids. The non-uniform distribution of the first normal stress difference (N1) can move the particles and cells suspended in the fluid laterally under the influence of the elastic force (Fe) proportional to the size.

In the above formula, a is the cell/particle diameter, N1 is the primary normal stress difference, λ is the relaxation time, W is the width of the microfluidic channel, and Q is the flow rate. This technique has recently been widely used for focusing and size-based separation of particles/cells within microfluidic channels.

Since the viscoelastic force flowing inside the channel has a small influence when targeting the nanoscale in proportion to the size, the nanoscale extracellular vesicle region has not been dealt with much in the field of viscoelastic fluid-based microfluidics. A study was conducted to isolate exosomes from other extracellular vesicles. As an exosome separated from the cell culture medium, the size cutoff was set to 200 nm. However, blood is a biological fluid in which various cells such as red blood cells, white blood cells, and platelets are suspended. In order to isolate nanoparticles, a technology capable of removing large numbers of blood cells is required. For this purpose, conventionally, centrifugation has been most widely used, but physical stimulation is applied to cells by high shear force generated during the centrifugation process, and by activating platelets that are weak to stimulation, artificial platelet-derived microparticles (PDMP) ) will occur. Therefore, in order to replace the centrifugation method, a technology for extracting 99.99% pure plasma by removing cells such as red blood cells, white blood cells, and platelets from blood has been developed, but the technology has a low yield for plasma extraction of 50 μl/min. It is only a level, and there is a limitation in that nano-sized particles present in plasma are excluded from analysis.

Therefore, the study of isolating cell-derived nanoparticles from high concentration blood containing a large amount of red blood cells without a helper fluid remains a challenge to be solved.

NPG Asia Materials 2017, 9, e434, Rapid purification of sub-micrometer particles for enhanced drug release and microvesicles isolation Lab on a Chip 2012, 12, 5202, Microfluidic filtration system to isolate extracellular vesicles from blood Analyst 2019, 144, 5785, Multiplex isolation and profiling of extracellular vesicles using a microfluidic DICE device Biomedical microdevices 2014, 16, 869, Microfluidic isolation of cancer cell -derived microvesicles from hetergeneous extracellular shed vesicle populations ACS nano 2015, 9, 2321, Acoustic Purification of Extracellular Microvesicles Proceedings of the National Academy of Sciences 2017, 114, 10584, Isolation of exosomes from whole blood by integrating acoustics and microfluidics Lab on a Chip 2016, 16, 3919, Continuous plasma extraction under viscoelastic fluid in a straight channel with asymmetrical expansion-contraction cavity arrays ACS nano 2017, 11, 6968, Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows

An object of the present invention is to provide a technology capable of separating cell-derived particles present in blood using a high concentration of blood in a microfluidic channel without a complicated channel design. In this process, there was no physical stimulation applied to the cells, so it was confirmed that there was no artificial generation of nanoparticles due to platelet activation.

An object of the present invention is a nanoparticle separation device including a microfluidic channel, comprising a single injection port, at least two outlets, an injection channel and a plurality of parallel channel portions, wherein the aspect ratio of the height to the channel width of the channel portion is greater than 1 and less than 5. It is to provide a nanoparticle separation device, characterized in that.

Another object of the present invention comprises the steps of injecting a sample through a single inlet, passing the injected sample through a channel, and extracting nanoparticles from a separation device, wherein the aspect ratio of the channel height to the channel width is 1 It is more than 5, and to provide a nanoparticle separation method, characterized in that the nanoparticles in the sample are separated due to the pressure gradient inside the channel having the aspect ratio.

According to an embodiment of the present invention, a nanoparticle separation device including a microfluidic channel, comprising a single injection hole, at least two outlets, an injection channel, and a plurality of parallel channel portions, wherein the aspect ratio of the height of the channel portion to the channel width is It is characterized in that more than 1 and less than 5, a nanoparticle separation device is provided.

According to one side, the separation element is characterized in that it removes blood cells in high concentration blood among the injected samples due to the internal pressure gradient of the parallel channel part having the aspect ratio, and in particular, platelet activation by physical stimulation applied to the cells It can be characterized by removing even platelets without.

According to one side, the separation device may be characterized in that it separates nanoparticles from the injected sample due to a pressure gradient inside the channel having the aspect ratio.

According to one side, the separation device may position the particles having an occlusion factor of 0.1 or more in the channel of the parallel channel portion in the center of the channel.

According to one side, the outlet may include a closed circulation system.

According to one side, the closed circulation system may recirculate the cells concentrated in the center of the parallel channel part to the inlet through the tubing pump.

According to one side, the nanoparticles may be cells-derived nanoparticles having a diameter of 1 μm or less.

According to one side, the nanoparticles may be platelet-derived microparticles (PDMP).

According to another embodiment of the present invention, it comprises the steps of injecting a sample through a single inlet, passing the injected sample through a channel, and extracting nanoparticles from the separation device, the height of the channel versus the channel width An aspect ratio of is greater than 1 and less than 5, and a method for separating nanoparticles is provided, characterized in that the nanoparticles in the sample are separated due to a pressure gradient inside the channel having the aspect ratio.

According to one side, the separation method may include a closed circulation system.

According to one side, the closed circulation system may recirculate the cells concentrated in the center of the parallel channel part through a tubing pump connected to an inlet and at least one outlet.

Using the present invention, cell-derived nanoparticles can be separated from a small amount of blood sample. Specifically, it is possible to remove blood cells, such as red blood cells, white blood cells, and platelets, without activation by physical stimulation without generating artificial nanoparticles due to activation, and to extract cell-derived nanoparticles contained in blood. In addition, since it is possible to separate nano-sized fine particles using a channel having a simple rectangular cross section by using the flow characteristics of the viscoelastic fluid, cumbersome processes such as complicated design and manufacturing can be omitted. In particular, it is possible to extract high-purity nanoparticles using a single inlet and channel internal pressure gradient induced due to a channel structure with a high aspect ratio cross-sectional area.

On the other hand, since separation is possible without using centrifugation technology, physical damage to particles or cells can be minimized, and nanoparticles can be separated within a short time with only a small amount of sample without separate labeling or help fluid.

The nanoparticle separation device of the present invention has the advantage of being easy to use because the nanoparticles can be extracted by pushing by hand without a pump. Additionally, when using a tubing pump, a closed circulation system may be applied.

The nanoparticle separation device of the present invention can be used for liquid biopsy-based in vitro diagnosis, and can be used for sample pretreatment. In addition, since the device can be used as a high-concentration cell concentrator by applying a pulmonary circulation type fluid control device to the device, there is an advantage in that it is possible to concentrate and evaluate the high-concentration concentration and evaluation of cells that are rarely present in biological samples, such as circulating tumor cells, leukocytes, and malaria-infected red blood cells.

1 schematically shows a cell-derived nanoparticle separation device according to an embodiment of the present invention.
Figure 2 shows a conceptual diagram comparing the separation technology of platelet-derived microparticles (PDMP) using the device of the present invention and the prior art.
3 is an evaluation of flow characteristics of a device according to an embodiment of the present invention.
4 is a graph showing the effect of PDMP separation in blood diluted with hematocrit 5% of the device according to an embodiment of the present invention.
5 is an evaluation of the flow characteristics of each nanoparticle size of the device of the present invention.
6 is a comparative evaluation of the PDMP recovery rate using the device of the present invention and the prior art.
7 schematically shows the principle of high concentration concentration in which a loop is additionally applied to the device of the present invention.
Figure 8 confirms the concentration effect of the recirculation loop system using the device of the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all modifications, equivalents and substitutes for the embodiments are included in the scope of the rights.

Terms used in the examples are used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It is to be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

In addition, in the description with reference to the accompanying drawings, the same components are assigned the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In the description of the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

The sample described herein means a biological sample obtained from an animal, preferably a mammal, and most preferably a human. On the other hand, such a biological sample may be an aqueous solution in which nanoparticles are dispersed, urine containing cell bodies and rare biological particles, blood, fetal fluid, sputum, and in particular, blood. The biological sample may be pre-treated before being used for detection, and may include, for example, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. On the other hand, the cell-derived nanoparticles as used herein means nano-sized particles derived from cells in the biological sample, and refers to nanoparticles generated from various cells such as red blood cells, white blood cells, platelets, and endothelial cells. As mentioned above, cell-derived nanoparticles have important significance as useful biomarkers for predicting cardiovascular diseases, inflammatory diseases, and other diseases.

According to an embodiment of the present invention, a nanoparticle separation device including a microfluidic channel, comprising a single injection hole, at least two outlets, an injection channel, and a plurality of parallel channel portions, and an aspect ratio of the height to the channel width of the parallel channel portion is more than 1, characterized in that 5 or less, a nanoparticle separation device is provided. The microfluidic channel is largely divided into an injection channel and a parallel channel part. The injection channel means a channel connected to the injection hole, and the main channel substantially involved in the flow of particles may be some or all of the parallel channel part. Hereinafter, unless otherwise specified, the term 'channel' is preferably understood to mean a part or all of the parallel channel unit.

10 μm의 높이를 갖는 채널의 설계가 필요했으며, 해당 채널에서 백혈구 크기의 세포(10~15마이크론의 원형)들은 사용될 수가 없으므로 고순도의 나노입자를 획득하기 어려운 문제가 있었다. 그러나, 본 발명의 소자는 고종횡비를 이용하여 채널폭보다 높이가 큰 단면형상의 소자를 구현함으로써, 특정 폐색율 이상의 크기를 갖는 입자는 모두 중앙부로 집중시킬 수 있으므로, 혈소판 크기 이상의 입자는 모두 중앙부로 집중시킨 뒤 중앙 채널과 연결되는 출구(출구 A)로 제거할 수 있고, 혈소판 보다 직경이 작은 나노크기의 입자는 측면 채널과 연결된 다른 출구(출구 B)로 추출할 수 있다.When the aspect ratio of the height of the channel to the width of the channel is within the above range (more than 1 and less than or equal to 5), it is advantageous for the separation of cell-derived nanoparticles. That is, when a channel with a low aspect ratio cross section is used, the condition of 0.3 β < 0.7 (β is the occlusion rate, β is the diameter of the cell, and H is the height of the channel) so that platelets are patterned toward the side of the channel. for 2.85 < H

It was necessary to design a channel with a height of 10 μm, and there was a problem in that it was difficult to obtain high-purity nanoparticles because leukocyte-sized cells (10-15 micron round cells) could not be used in the channel. However, since the device of the present invention implements a device having a cross-sectional shape having a height greater than the channel width by using a high aspect ratio, all particles having a size greater than or equal to a specific occlusion rate can be concentrated in the central portion. After concentration, it can be removed through the outlet (exit A) connected to the central channel, and nano-sized particles smaller in diameter than platelets can be extracted through the other outlet (exit B) connected to the side channel.

Specifically, a single injection port is connected to the injection channel, and the injection channel is connected to a plurality of parallel channel units. The injection channel preferably has a width of 30 μm to 60 μm, but is not limited thereto. On the other hand, the parallel channel portion may have a structure connected to at least two outlets, in this case, the inlet may be an interference-fit connection, and at least one outlet may be a reservoir or an interference-fit connection. The reservoir is one of the components of the pulmonary circulation system, and in the form of a box, it can accommodate a sample containing separated and concentrated cells and a sample containing the removed blood cells, and the liquid inside the reservoir is supplied by a flow pressure from the pump. may be provided to flow along a predetermined path.

For example, the nanoparticle separation device of the present invention may include the configuration of FIG. 1 . 1 schematically shows a cell-derived nanoparticle separation device 100 according to an embodiment of the present invention, in which (a) is a perspective view, (b) is a plan view, (c) is a side view , (d) shows the manufactured prototype. Referring to FIG. 1 , the injection port 110 of the device 100 is a portion into which a sample containing particles to be separated is injected, and may include an interference fit connection part. The injection port 110 is connected to one injection channel 120 , and the injection channel 120 is connected to a parallel channel unit. At this time, the parallel channel part has a shape in which a plurality of channels of a parallel structure are divided from the main channel, and the device 100 of the present invention includes at least one branch point at which the injection channel 120 and the parallel channel part 130 are connected. will do Each channel of the parallel channel part includes one branch point in the outlet area, of which the central channel is connected to the outlet A 140 through the central outlet merged channel part connected to the upper plate of the device 100, and the side channel is the device 100 ) may be merged in the wake of and connected to the outlet B 150 . Particles located in the center (focusing) due to these structural features are discharged to the outlet A 140, and the particles not located in the center may be extracted to the outlet B 150 through the side channels of the parallel channel unit. At this time, the outlet may further include a collecting unit capable of recovering particles of a specific size range. Figure 1 (d) shows the result of a prototype of the separation device of the present invention according to an embodiment. As shown in Figure 1 (d), four outlets A are manufactured and located on the upper plate of the device. It is connected to the reservoir and may be manufactured with one outlet B, but this is only an embodiment and is not limited thereto.

According to one side, the separation device may be characterized in that it separates nanoparticles from the injected sample due to a pressure gradient inside the channel having the aspect ratio. For example, the separation element may be to remove blood cells in high concentration blood and to separate platelet-derived nanoparticles. Here, the high-concentration blood may mean blood having a hematocrit of 25% or less.

That is, it is possible to separate cell-derived nanoparticles due to the pressure gradient inside the channel without destroying the cells without using the ultracentrifugation method or the immunoaffinity capture method.

5 임이 가장 바람직하다.By separating the cell-derived nanoparticles due to the pressure gradient inside the channel, it is possible to extract high-purity nanoparticles without a help fluid or labeling. On the other hand, with the prior art, it was difficult to remove even platelets when the hematocrit is 1% or more. However, even if a blood sample with a hematocrit of 25% or less is injected using the device of the present invention, even platelets are removed without activation by physical stimulation, and high-purity nanoparticles can be extracted. In addition, if the aspect ratio of the channel is increased, the extraction yield of nanoparticles can be increased even under the same flow condition, and the height or width of the channel can be adjusted according to the particle size of the target to be extracted. Specifically, the aspect ratio of the channel cross section is preferably 1 or more, and 1 < aspect ratio

5 is most preferred.

According to one side, the separation device may position particles having an occlusion factor of 0.1 or more in the channel of the parallel channel portion in the middle of the channel. The blockage ratio (β) is the size of the particle and the dimension ratio of the channel (d/H, where d is the diameter and hydraulic diameter of the particle (aspherical shape), H is the height or By width, the shorter length of the cross-section is H). At this time, the sample may be a viscoelastic fluid, and may be diluted with HA (Hyaluronic acid), but is not limited thereto. While the diluted sample is injected into the separation element and flows through the main channel, the elastic force is affected by the viscoelastic fluid, and the effect of the elastic force is proportional to the size of the cell. For example, when the sample is blood, various cells in the blood, such as red blood cells, white blood cells, and platelets, are focused in the center of the channel with an occlusion rate of 0.1 or more, but PDMP (100~1000 nm), a small-sized nanoparticle, is not focused in the center. , widely distributed throughout the channel.

According to the same principle as described above, it is possible to separate nano-sized particles without using centrifugation. When the particle to be separated is platelet-derived nanoparticles, when centrifugation is used, platelet-derived nanoparticles are additionally generated due to platelet activation, so the accuracy of analysis may be reduced. Since there is little change in the number of platelet-derived nanoparticles without additional generation of platelet-derived nanoparticles due to subsequent platelet activation, there is an advantage in that platelet-derived nanoparticles can be separated with high purity.

According to one side, the outlet may be using a closed circulation system.

When the closed circulation system is applied to the separation device of the present invention, the cells discharged through at least one outlet connected to the parallel channel unit of the separation device may be concentrated and recirculated. Specifically, the outlet may be any one outlet (eg, outlet A) connected to the central channel among the parallel channel units and the other outlet (eg, outlet B) connected to the side channels among the parallel channel units.

The closed circulation system may include a sample reservoir, a tubing pump, a tube, and a waste reservoir, and may further include one or more conduits connecting the reservoir and the pump. The conduit may be a supply conduit or an exhaust conduit. If the closed circulation system is used, a high concentration of desired particles can be achieved by recycling the sample.

According to one side, the closed circulation system may send the cells concentrated in the center of the parallel channel part to the sample reservoir through a tubing pump, and recirculate them to the device inlet. By receiving the flow pressure from the tubing pump and flowing the liquid inside the sample reservoir along a predetermined path, the sample can be recirculated and only particles of a desired size can be highly concentrated.

According to one side, the nanoparticles may be cells-derived nanoparticles having a diameter of 1 μm or less. Specifically, it is preferably about 100 to 1000 nm, but is not limited thereto. For example, when the sample is blood, the cell-derived nanoparticles may be a type of extracellular vesicle, and have important functions in physiological processes such as communication between cells, blood clotting, inflammation, and metastasis of cancer cells. It may be a biomarker that performs

The nanoparticles may be platelet-derived microparticles (PDMPs). PDMPs have an average size distribution of 100 to 1000 nm, and 90% of them have a size of 500 nm or less.

According to another embodiment of the present invention, it comprises the steps of injecting a sample through a single inlet, passing the injected sample through a channel, and extracting nanoparticles from the separation device, the height of the channel versus the channel width An aspect ratio of is greater than 1 and less than 5, and a method for separating nanoparticles is provided, characterized in that the nanoparticles in the sample are separated due to a pressure gradient inside the channel having the aspect ratio. For example, when separating the process of separating PDMP from whole blood, the nanoparticle separation method of the present invention may be separated by the same process as in FIG. same as

2 is a conceptual diagram comparing platelet-derived microparticle (PDMP) separation technology using the device of the present invention and the prior art, and a conventional multiple centrifugation method for separating 100 μl of PDMP from whole blood A separation method using the device of the present invention is compared. Figure 2 (a) uses a multiple centrifugation method, and in order to extract 100 μl of PDMP from whole blood with a high hematocrit, several centrifugation steps are performed, so 5 ml of whole blood is required and a processing time of about 60 minutes. This takes On the other hand, when the device of the present invention is used as shown in FIG. 2(b), 100 μl of PDMP can be extracted with only 0.3 ml of whole blood in about 2 minutes of treatment.

Referring to FIG. 2(b) in more detail, first, whole blood diluted with 0.1% hyaluronic acid (HA) solution is injected into a single inlet. Whole blood contains high concentrations of red blood cells, white blood cells, platelets, and PDMP, and when diluted whole blood is added, all cells are randomly distributed throughout the channel width at the inlet. Thereafter, while flowing through the injection channel and the parallel channel part connected to the injection port, the elastic force is affected by the viscoelastic fluid. In this case, the effect of the elastic force is proportional to the cell size. When the occlusion rate in the channel is 0.1 or more, the cells included in the whole blood except for PDMP, which are nano-sized particles, are focused in the center due to the influence of the elastic force, and the PDMP is widely distributed throughout the channel.

When the channel of the separation element is designed to have a high aspect ratio, the particle separation time can be further shortened. For example, in order to remove even platelets, it is preferable that the occlusion ratio is 0.1 or more, that is, the channel width is designed to be 30 μm or less at the maximum so that the particles of the platelet size (2 to 3 μm) can be centrally removed. However, when the height of the channel cross-section is designed to be 70 μm or more, a larger amount can be processed under the same flow condition, so the separation yield of PDMP can be improved. In order to separate high-purity PDMP from whole blood, platelet removal is one of the important functions, so it is preferable that the maximum channel width is 30 μm or less and the height is at least 30 μm. However, the above-described separation method is only an embodiment, and the width, height, aspect ratio, and state of the sample, etc. of the channels constituting the device may be appropriately changed according to the particles to be separated.

According to one side, the separation method may use a closed circulation system. The closed circulation system may include a sample reservoir, a tubing pump, a tube, and a waste reservoir, and may further include one or more conduits connecting the reservoir and the pump. The conduit may be a supply conduit or an exhaust conduit. If the closed circulation system is used, a high concentration of desired particles can be achieved by recycling the sample. According to one side, the closed circulation system may recirculate the cells concentrated in the center of the parallel channel part through a tubing pump connected to an inlet and at least one outlet.

After injecting the sample from the sample reservoir to the inlet through the tubing pump, the occlusion rate among the injected samples is 0.1 or more (β is the occlusion rate, β=a/H, where a is the diameter of the cell and H is the channel The particle satisfying the condition of height of Buffer solution (buffer) other than particles discharged through High-concentration separation of particles (ex. cells) is possible through a recirculation loop method that repeats this process.

A more specific method will be described later through examples.

(Example)

Example 1. Evaluation of flow characteristics of nanoparticle separation device

Flow characteristics for each condition of the nanoparticle separation device according to an embodiment of the present invention were evaluated. The flow characteristics for each flow rate, channel length, and red blood cell concentration were evaluated, respectively, as shown in FIG. 3 .

3 (a) shows the evaluation of flow characteristics by flow rate, (b) by channel length, and (c) flow characteristics by red blood cell concentration, respectively. Referring to (a) of FIG. 3 , it can be seen that, first, 2 μm particles suspended in 0.1% hyaluronic acid (HA) solution at a flow condition of 2 μl/min are concentrated in the middle region of the channel, but are focused at a low level. However, as a result of increasing the flow rate to 20 μl/min, 100 μl/min, and 200 μl/min, it was confirmed that the particles were gradually focused in the middle row. That is, it was verified that all particles with a size of 2 μm were focused on the central region of the channel within the flow rate range of 20 to 200 μl/min. 3 (b) shows the evaluation of the flow characteristics of 2 μm particles to determine the minimum channel length that can be removed from blood to platelets. 3 (b), from the left, the flow characteristics were analyzed in channels of lengths of 7.5, 15, 22.5, and 30 mm, respectively, and in the channel length of 15 mm or more, it was confirmed that all 2 μm particles were tightly focused to the center of the channel. . The length of the channel is closely related to the flow resistance, and the longer the channel, the greater the flow resistance, which may hinder the improvement of the device yield. Therefore, in the experiment to be described later, the minimum length of the channel on which the 2 μm particles are focused was applied to the device as 15 mm, and the flow rate at this time was fixed at 200 μl/min.

Example 2. Effect of Nanoparticle Separation Device on PDMP Separation in Blood

PDMP was separated from blood diluted by 5% hematocrit using the nanoparticle separation device of the present invention, and the effect was as shown in FIG. 4 .

4 (a) and (b) show the flow characteristics of fluorescently-stained platelets. In the channel inlet, it seems that fluorescently-stained platelets are randomly distributed throughout the channel, but at the outlet after passing through the device, platelets are It was confirmed that it was focused to the center and exited through the exit A.

4 (c) to (e) are injection samples before and after passing through the device using blood diluted to 5% hematocrit (hematocrit in blood) to evaluate the performance of the device of the present invention, outlets A and B The distribution in the sample was confirmed by flow cytometry. Flow cytometry gates for erythrocytes and PDMPs were set using particles of 10 and 1 μm in size. At this time, the proportion of red blood cells among the total cells included in the blood sample before separation into the device was about 78%. 85.4% was detected at the PDMP exit gate as shown in FIG. 4(e).

4(f) shows the measurement of the number of red blood cells in the sample at the inlet and outlets A and B. Of the total 10000 events, 7,874±890 at the inlet and 8,688±1,100 and 108±98 at the outlets A and B, respectively. was detected. Through this, it was confirmed that high concentration red blood cells could be effectively separated and removed using the device of the present invention.

4 (g) and (h) were used to evaluate platelet activity using the device of the present invention, CD41-FITC antibody reagent was used for platelet detection, and CD62-PE antibody was used for platelet activity evaluation. . A CD41-positive result indicates the detection of platelets, and a CD62-positive result indicates the detection of activated platelets. At this time, the device had a width of 20 μm, a height of 70 μm, and a length of 15 mm, and the shear force applied under the flow condition of 200 μl/min was calculated to be 209.4 Pa. Such a condition is a condition in which platelets are not activated even during flow inside the device, which can be confirmed by the CD62 reagent-based analysis result. As a result of the analysis, among the total platelets detected as CD41-positive, the number of platelets that appeared to be activated as CD62-positive was 24.73%, which was similar to that before passing through the device. Therefore, it was confirmed that platelets were not activated even when the device of the present invention was used.

Example 3. Evaluation of flow characteristics by nanoparticle size

Using the nanoparticle separation device of the present invention, flow characteristics for each nanoparticle size were evaluated. In order to confirm the separation effect of PDMP having a size distribution of 100-1000 nm, of which about 90% have a size of 500 nm or less, 200 μl/min of fluorescent particles of 300, 500, 700, and 1000 nm, respectively It was separated under flow conditions, and the result was as shown in FIG. 5 .

As shown in (a) of FIG. 5 , it can be seen that the 300 nm particles are less affected by the elastic force due to their small size and exit through the outlet while being distributed throughout the channel. The larger the particle size, the greater the effect of the elastic force, which causes the particle to move to the center of the channel, resulting in a wider 'particle free layer' near the channel wall. Referring to (b) to (d) of FIG. 5, it can be seen that the width of the 'particle-free region' gradually increases as the particle size increases (in the order of b, c, and d, 2.25±0.05 μm and 2.71±0.09 μm, respectively). , and 3.12±0.05 μm).

As shown in (e) of FIG. 5 , the concentration of particles included in each sample was measured through flow cytometry analysis of the samples exiting through outlets A and B. As a result of the measurement, the concentrations of samples coming out of outlets A and B were almost the same for 300 nm particles, but in the case of 500 nm and 700 nm particles, the concentrations of samples coming out of outlet A were 4.8% and 8.6% higher than those of outlet B, respectively. . On the other hand, in the case of 1000 nm particles, the concentration at the outlet A was 42.5% higher than that at the outlet B, which was confirmed because the particles were focused into the center of the channel by the viscoelastic force.

It was confirmed that the difference in concentration for each outlet as described above was due to the fact that the outlet branch of the device used in the experiment was designed with a width ratio of 3:2:3, and by adjusting this, the size and concentration distribution of collectable nanoparticles for each outlet can be controlled. can

Example 4. PDMP recovery rate comparison and effectiveness evaluation

The recovery rate of PDMP using the nanoparticle separation device of the present invention and centrifugation (prior art) was compared and evaluated. At this time, blood (same as Example 2) diluted with 5% hematocrit was used as a sample, and the result was as shown in FIG. 6 . Figure 6 (a) shows the results of analyzing the PDMP separated by the centrifugation method (left) and the PDMP separated through the separation device of the present invention through a flow cytometer (CytoFLEX). Centrifugation conditions were 1500g-15min, 13000g-2min, followed by a continuous process of 25000g-30min. CD41 was used as a means for detecting PDMP in the flow cytometry results, and it is shown in the yellow shaded area in the two graphs of FIG. 6(a), respectively. Comparing the analyzed results, the PDMP separated through centrifugation has a concentration of 5.4x10 ³ /μl, and the PDMP separated through the separation device of the present invention has a concentration of 2.6x10 ⁴ /μl (Fig. b)). That is, when PDMPs were isolated using the same blood sample, it was confirmed that the number of separated PDMPs differed by about 4.8 times because the loss of PDMPs occurred in the multi-processing process when centrifugation was used.

On the other hand, the PDMP was isolated through the separation device of the present invention by artificially activating platelets using a chemical reagent. This is a necessary process for the standardization of the PDMP separation method to secure the validity of the developed device. As a chemical reagent for platelet activation, collagen, thrombin, adenosine, diphosphate (ADP), calcium ionophore, etc. may be used, and in this experiment, a calcium carrier was used. After the chemical reagent is mixed with the blood sample, the number of PDMPs generated increases according to the incubation time, which was confirmed through analysis of the number of PDMPs separated through the device (FIG. 6(c)) . Each time-dependent increase in the number of PDMPs was 3700±959/μl, 4233±1165/μl, 8700±1710/μl, 13166±6109/μl, and 15166±10900 over 1, 2, 3, 4, and 5 hours. /μl appeared like this. The samples not treated with the calcium carrier and the samples cultured for 5 hours after the treatment were analyzed by FE-SEM, respectively, to confirm the actually generated PDMP, as shown in FIGS. 6 (d) and (e), respectively.

Example 5. Separation effect of nanoparticles using closed circulation system

7 schematically shows a case of applying a closed circulation system to the outlet of the nanoparticle separation device of the present invention according to an embodiment. As shown in FIG. 7, when a sample is first injected from the sample reservoir into the inlet of the parallel chip through a tubing pump (①), the occlusion rate is 0.1 or more (βa is the diameter of the cell, W is the width of the channel) Cells that satisfy the condition of It comes out and is removed to the waste reservoir through the tubing pump (③). That is, a recirculation loop system is constructed, and the separation/concentration is repeated so that a high concentration of target particles to be separated is possible.

As a result of observing the fluorescence stream after 1, 5, and 10 minutes of system operation to confirm the concentration effect using the closed circulation system, it was confirmed that the concentration of the fluorescence stream gradually increased over time as shown in FIG. 8(a). . In addition, as a result of checking the concentration of the sample before and after concentration, it was confirmed that the concentration was remarkably concentrated as shown in FIG.

As described above, although the embodiments have been described with reference to the limited drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the described techniques are performed in a different order than the described method, and/or the described components of the system, structure, apparatus, circuit, etc. are combined or combined in a different form than the described method, or other components Or substituted or substituted by equivalents may achieve an appropriate result.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

100: nanoparticle separation device
110: inlet (Inlet)
120: injection channel
130: parallel channel unit
140: exit A (outlet A)
150: exit B (outlet B)

Claims (10)

A nanoparticle separation device including a microfluidic channel,
a single inlet, at least two outlets, an infusion channel and a plurality of parallel channel portions;
The aspect ratio of the height to the channel width of the parallel channel part is characterized in that it is greater than 1 and less than or equal to 5,
The nanoparticle is a cell-derived nanoparticle having a diameter of 1 μm or less, a nanoparticle separation device. According to claim 1,
The separation device is a nanoparticle separation device, characterized in that for separating the nanoparticles from the injected sample due to the pressure gradient inside the parallel channel portion having the aspect ratio. According to claim 1,
The separation device, the nanoparticle separation device for locating particles having an occlusion factor of 0.1 or more in the channel of the parallel channel portion in the center of the channel. According to claim 1,
The outlet, including a closed circulation system, a nanoparticle separation device. 5. The method of claim 4,
The closed circulation system, the nanoparticle separation device that recirculates the cells concentrated in the center of the parallel channel part through a tubing pump. delete The method of claim 1,
The nanoparticles are platelet-derived microparticles (PDMP), which is a nanoparticle separation device. injecting the sample through a single inlet;
passing the injected sample through the channel; and
Including the step of extracting nanoparticles from the separation device,
The aspect ratio of the height of the channel to the width of the channel is greater than 1 and less than 5;
characterized in that the nanoparticles in the sample are separated due to the pressure gradient inside the channel having the aspect ratio,
The nanoparticle is a cell-derived nanoparticle having a diameter of 1 μm or less, a nanoparticle separation method. 9. The method of claim 8,
The separation method, including a closed circulation system, nanoparticle separation method. 10. The method of claim 9,
The closed circulation system recirculates the cells concentrated in the center of the parallel channel part through the tubing pump connected to the inlet and at least one outlet, the nanoparticle separation method.

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