KR102577259B1 - A size-selective filtration of extracellular vesicles with a movable-layer system - Google Patents

Abstract

The present invention relates to a fluid control system capable of filtering extracellular vesicles by size, comprising: a rotatable housing; a plurality of chambers disposed in the housing and open at the top; a nanopore filter disposed in the chamber and composed of a filter net having nanopores; a flow pipe connecting the chamber and its neighboring chambers to provide a passage for fluid samples sequentially flowing through the chambers; and a plunger disposed spaced apart from the housing so as to be aligned with the chamber and pumping the fluid sample contained in the chamber by flowing in and out of the chamber.

Description

A fluid control system capable of filtering extracellular vesicles by size {A SIZE-SELECTIVE FILTRATION OF EXTRACELLULAR VESICLES WITH A MOVABLE-LAYER SYSTEM}

The present invention relates to a fluid control system capable of filtering extracellular vesicles by size, and more specifically, to filter extracellular vesicles (EV) from biological fluid samples at various size intervals in a short period of time through an automated and integrated process. The present invention relates to a fluid control system capable of filtering extracellular vesicles by size, which facilitates the analysis of extracellular vesicles and the discovery of extracellular vesicle-based biomarkers.

Extracellular vesicles (EVs) have received considerable attention as biomarkers for various diseases such as cancer, kidney disease, and diabetes.

(Hereinafter, intracellular endoplasmic reticulum is referred to as EV.)

EVs are vesicles ranging in size from 30 to 1000 nm, secreted by most cell types in response to physicochemical and pathological stimuli. In particular, EVs detected in body fluids such as urine, saliva, and blood are attractive for clinical analysis. It becomes a means.

Additionally, pathological conditions can be detected through membrane proteins on the EV surface and nucleic acids, including DNA and miRNA, inside EVs.

However, isolating EVs is not a simple task due to their submicron size and heterogeneous content in body fluids.

Therefore, EV isolation methods for more widespread use of EVs must meet several requirements:

It must be fast and simple, provide high purity and yield, and have size-based isolation capabilities with high separation resolution.

Additionally, separation of EVs by size is necessary because EVs of different sizes may have substantially different molecular contents, and each cell type secretes EVs of different sizes.

Although various EV separation methods have been studied so far, a separation method that satisfies all of the above requirements has not been implemented.

Ultracentrifugation is the most widely used EV separation method, but has the problems of low purity and yield and long process time.

Additionally, precipitation-based methods using precipitation of EVs by reagents such as polyethylene glycol require long incubation times and have low purity, while immunomagnetic bead methods can capture EVs with target surface proteins but are expensive and require downstream analysis. There are many restrictions.

Additionally, the above-described methods are not capable of high-resolution size-based separation of EVs.

Alternatively, micro- and nanofluidic technologies have been applied to EV separation, but although these methods have relatively high purity and separation resolution, they require complex manufacturing processes to make the devices and can only process relatively small quantities, resulting in a variety of sub-scales. Analysis is difficult.

Meanwhile, there is a method of separating EVs with a filter membrane.

The use of filter membranes simplifies the isolation process and enables size-based isolation of EVs at various size intervals.

Recently, a microfluidic centrifugal disk, which has been applied to separate EVs by filter membranes, contains two filters composed of nanometer-sized pores, which can effectively separate EVs in a relatively short period of time. Integrating filters and valves complicates manufacturing and operation, making it difficult to access EVs after filtration.

In addition, although the filter membrane layers are connected in series to achieve classification by size, the serial and modular connection not only results in large dead volume and significant pressure drop, but also has the disadvantage of not automating the operation.

Therefore, there is a need for the development of a fluid control system in which the separation of EVs by various sizes is implemented, the separation process and device are simple and effective, and in particular, the size-specific filtering of extracellular vesicles is possible, allowing sequential filtering in an automated manner. .

[Patent Document] KR 10-2020-0000792 (Published on January 3, 2020)

In order to solve the above problems, the present invention includes a rotatable housing, a plurality of chambers disposed in the housing and open at the top, a nanopore filter disposed in the chamber and composed of a filter net having nanopores, the chamber and its neighbors. By including a flow tube that connects the chambers and provides a passage for the flow of the fluid sample, and a plunger that is spaced apart from the housing to be aligned with the chamber and flows in and out of the chamber to pump the fluid sample contained in the chamber. , Extracellular vesicles, which are biomarkers for various diseases, can be separated into various size intervals in a short period of time with an automated and integrated system. Not only is the separation process and device simple, but in particular, sequential filtering is possible in an automated manner, making it effective. The purpose is to provide a fluid control system capable of filtering by size of extracellular vesicles, which implements filtering by size of extracellular vesicles.

A fluid control system capable of filtering extracellular vesicles by size according to an embodiment of the present invention to achieve the above object includes a rotatable housing; a plurality of chambers disposed in the housing and open at the top; a nanopore filter disposed in the chamber and composed of a filter net having nanopores; a flow pipe connecting the chamber and its neighboring chambers to provide a passage for fluid samples sequentially flowing through the chambers; and a plunger disposed spaced apart from the housing so as to be aligned with the chamber and pumping the fluid sample contained in the chamber by flowing in and out of the chamber.

In addition, the flow pipe according to the present invention is characterized in that it is connected from the lower part of the nanopore filter of one chamber to the upper part of the nanopore filter of the other chamber adjacent to the one chamber.

In addition, the flow pipe according to the present invention is characterized by including a check valve on one side that controls the unidirectional flow of the fluid sample.

In addition, in the fluid control system capable of filtering extracellular vesicles according to size according to the present invention, when the plunger is introduced into any chamber, the check valve disposed on the flow pipe through which the fluid sample flows into the chamber is closed, and the check valve is closed in the chamber. It is characterized in that the check valve disposed in the flow pipe through which the fluid sample flows is opened.

In addition, the nanopore filter according to the present invention is characterized in that it is selectively attached and detached from the chamber.

In addition, the chamber according to the present invention is characterized by including a waste chamber for storing the filtered fluid sample.

In addition, the nanopore filter according to the present invention is composed of a filtration network having pores with a diameter of 200 nm to 30 nm, and the chamber is equipped with nanopore filters having different pore diameters, sequentially at each stage of flow of the fluid sample. It is characterized by being equipped with a nano-pore filter with small pores.

In addition, the housing according to the present invention includes a rotation motor on one side that controls rotation of the housing, and the plunger includes a vertical motor that controls the lifting and lowering movement of the plunger.

In addition, the rotation motor according to the present invention rotates the housing at a preset angle so that when the plunger moves upward after pumping any first chamber, the second chamber adjacent to the first chamber is aligned with the plunger. When the second chamber is aligned with the plunger, the vertical motor moves the plunger downward to the second chamber to pump the fluid sample and then moves the plunger upward.

In addition, the check valve according to the present invention includes a pair of elastic membranes attached to the inner peripheral surface of the flow pipe to have a fluid sample flow port at the center; And a valve seat disposed adjacent to the flow port to open and close the flow port according to the flow direction of the fluid sample.

In addition, the valve seat according to the present invention is characterized in that it is an elastic material selected from the group consisting of polydimethylsiloxane (PDMS), silicone, latex, and rubber.

According to the fluid control system capable of filtering extracellular vesicles according to size according to the present invention as described above, it is composed of a rotary housing in which a plunger that moves vertically and a chamber that opens through a check valve are sequentially connected, thereby preventing various diseases. Extracellular vesicles as biomarkers can be separated into various size intervals in a short period of time with an automated and integrated system. Not only is the separation process and device simple, but in particular, sequential filtering is possible in an automated manner, making it effective for extracellular vesicles. This has the effect of implementing filtering by size.

In addition, a nanopore filter consisting of a porous membrane filtration network with nanopores is detachably mounted on the chamber to have different pore diameters to separate EVs at various size intervals of 30, 50, 80, 100, and 200 nm. There is an effect.

In addition, the nanopore filter is configured to be attachable and detachable within the chamber, allowing filtering by selectively applying filters with various pore diameters.

In addition, it has the effect of realizing a high separation resolution containing less than 7% of particles larger than the pore size of the nanopore filter and a purity of 8.1 × 10 ¹⁰ particles/mg.

In addition, the use of Pluronic coating on the device surface has the effect of obtaining a high EV recovery rate of 89%.

Figure 1(a) is a diagram showing the overall configuration of a fluid control system capable of filtering extracellular vesicles according to size according to the present invention.
Figure 1(b) is a cross-sectional view showing the detailed configuration of a fluid control system capable of filtering extracellular vesicles according to size according to the present invention.
Figure 2(a) is a diagram showing the configuration of a housing and chamber according to the present invention.
Figure 2(b) is an exploded perspective view showing the detailed configuration of the housing according to the present invention.
Figure 3(a) is a state diagram showing the open state of the check valve according to the present invention.
Figure 3(b) is a state diagram showing the closed state of the check valve according to the present invention.
Figure 3(c) is a diagram showing a valve seat according to the present invention.
Figure 3(d) is a plan view and side view of the valve seat according to the present invention.
Figure 4 is a photograph of a fluid control system capable of filtering extracellular vesicles according to size according to the present invention.
Figure 5 is a block diagram showing the control relationship between the controller, solenoid valve, vertical motor, and rotation motor according to the present invention.
Figure 6 is a graph showing the selective separation performance of EV using plasma.
Figure 7 is a diagram evaluating the performance of selective separation of EVs by size from cell culture supernatant.

Hereinafter, embodiments of the present invention will be described with reference to the attached drawings. First of all, it should be noted that the same components or parts in the drawings are given the same reference numerals as much as possible. In describing the present invention, detailed descriptions of related known functions or configurations are omitted so as not to obscure the gist of the present invention.

Figure 1(a) is a diagram showing the overall configuration of a fluid control system capable of filtering by size of extracellular vesicles according to the present invention, and Figure 1(b) is a diagram showing the fluid control capable of filtering by size of extracellular vesicles according to the present invention. It is a cross-sectional view showing the detailed configuration of the system, and Figure 4 is a photograph of the fluid control system capable of filtering extracellular vesicles according to size according to the present invention.

The fluid control system 1 capable of filtering extracellular vesicles according to size according to the present invention includes a housing 100, a chamber 200, a nanopore filter 300, and It may include a pipe 400 and a plunger 500.

Figure 2(a) is a configuration diagram showing the configuration of the housing and chamber according to the present invention, and Figure 2(b) is an exploded perspective view showing the detailed configuration of the housing according to the present invention.

The housing 100 according to the present invention is composed of a rotatable disk shape as shown in FIGS. 2(a) and 2(b), and has a plurality of chambers 200 into which the fluid sample 10 is injected. Includes a space where dogs can be placed.

In addition, the housing 100 may be composed of multiple layers, for example, a first layer 110, a second layer 120, and a third layer 130 as shown in FIG. 2(b). ), the fourth layer 140, and the fifth layer 150.

At this time, the third layer 130 includes an elastic membrane 413 and a silicon gasket of a check valve 410, which will be described later, and the nanopores are formed between the third layer 130 and the fourth layer 140. A filter 300 is disposed.

Additionally, the second layer 120 may have a chamber 200 disposed therein.

Additionally, the housing 100 may include a rotation motor 160 on one side that controls the rotational movement of the housing 100.

The rotation motor 160 moves the plunger 500 upward after pumping any first chamber 210, and the second chamber 220 adjacent to the first chamber 210 moves the plunger 500. It may be a motor that rotates the housing 100 at a preset angle so that it is aligned.

Additionally, the rotation motor 160 is operated under the control of a controller (not shown) for automated operation of the fluid control system 1 according to the present invention.

The controller (not shown) can control the rotation motor 160 together with the vertical motor 510, which will be described later, which controls the lifting and lowering movement of the plunger 500.

Therefore, when the housing 100 moves upward after the plunger 500 descends into the chamber 200 aligned at the bottom and pumps the fluid sample 10 contained in the chamber 200, the rotation motor ( 160 is operated to rotate the housing 100 at a preset angle so that the chamber 200 and the neighboring chamber 200 can be placed at the bottom of the plunger 500.

Additionally, the rotation motor 160 may be a stepper motor that moves one step at a time using a toothed wheel and an electromagnet.

The chamber 200 is disposed in the housing 100 and may be composed of a plurality of chambers 200 with an open top.

Specifically, the chamber 200 is composed of a plurality of chambers 200 open at the top to provide a space in which the fluid sample 10 is injected and supported, and each chamber 200 is connected to the neighboring chamber 200 and the flow pipe ( It is arranged to be connected through 400).

Accordingly, the chamber 200 may be sequentially connected through a flow pipe 400 that allows unidirectional flow movement of the injected fluid sample 10 and a check valve 410 disposed in the flow pipe 400.

Additionally, the chamber 200 may be composed of a filtration chamber equipped with a nanopore filter 300 therein and a waste chamber 260 that stores the filtered fluid sample 10.

The chamber 200 according to one embodiment includes a first chamber 210, a second chamber 220, a third chamber 230, a fourth chamber 240, and a fifth chamber ( The filtration chamber consisting of 250) and the last waste chamber 260 are sequentially connected and arranged.

In addition, the chamber 200 is equipped with a selectively attachable and detachable nanopore filter 300 inside, and the nanopore filter 300 is equipped with filters with different pore diameters in each chamber 200. .

In addition, the chamber 200 is configured to be sequentially equipped with nanopore filters 300 with small pores at each flow stage of the fluid sample 10.

For example, the chamber 200 includes a nanopore filter 300 having a pore diameter of 200 nm in the first chamber 210, and a nanopore filter having a pore diameter of 100 nm in the second chamber 220. (300), a nanopore filter 300 having a pore diameter of 80 nm in the third chamber 230, a nanopore filter 300 having a pore diameter of 50 nm in the fourth chamber 240, the first 5 A nanopore filter 300 having a pore diameter of 30 nm may be installed in chamber 250.

In addition, the chamber 200 is sequentially connected to the first chamber 210, the third chamber 230, the fifth chamber 250, and the waste chamber 260 to separate EVs of different size intervals, or Various chamber connection combinations can be used, such as a sequential connection from the first chamber 210 to the fourth chamber 240 to the waste chamber 260.

In addition, the chamber 200 includes a first chamber 210 - a second chamber 220 - a third chamber 230 - a waste chamber to implement various chamber connection combinations for separating EVs 20 with different size intervals. In the arrangement leading to 260, a 200 nm nanopore filter 300 is placed in the first chamber 210, an 80 nm nanopore filter 300 is placed in the second chamber 220, and the third chamber 230. Separation of EVs 20 having different size intervals may be implemented by installing a 30 nm nanopore filter 300.

In addition, through the installation of various nanopore filters 300, separation of EVs of different size intervals can be realized by combining the above-described size intervals and connection of different nanopore filters.

The nanopore filter 300 is disposed in the chamber 200 and may be composed of a filter network having nanopores.

Specifically, the nanopore filter 300 is a filter having pores of 200 nm to 30 nm that can filter nanoparticles from the fluid sample 10 flowing into the chamber 200, and selectively removes nanoparticles from the chamber 200. It is detachable, and nanopore filters 300 having different pore diameters are mounted on the chamber 200.

At this time, the nanopore filter 300 having small diameter pores is installed in the sequentially connected chambers 200 at each flow stage of the fluid sample 10.

Therefore, when the pore diameter of the nanopore filter 300 of the second chamber 220 is 100 nm and the pore diameter of the nanopore filter 300 of the third chamber 230 is 80 nm, the second chamber ( After the fluid sample 10 flows into 220), the plunger 500 descends to pump the second chamber 220, and the EV is greater than 100 nm due to the pore size of the nanopore filter 300 of the second chamber 220. (20) remains at the top of the filter of the second chamber (220), and EV (20) of less than 100 nm moves from the bottom of the filter of the second chamber (220) to the third chamber (230) through the flow pipe 400. .

In addition, when the fluid sample 10 moves to the third chamber 230, the housing 100 rotates so that the plunger 500 and the third chamber 230 are aligned, and then the plunger 500 descends. When the fluid sample 10 in the third chamber 230 is pumped, EVs 20 larger than 80 nm remain on the upper part of the filter 300 of the third chamber 230, and EVs 20 less than 80 nm remain in the third chamber 230. It moves from the bottom of the filter 300 of the chamber 230 to the fourth chamber 240 through the flow pipe 400.

The flow pipe 400 connects the chamber 200 and the chamber 200 adjacent to it and provides a passage for the fluid sample 10 to sequentially flow through the chamber 200.

Additionally, the flow pipe 400 may be connected from the lower part of the nanopore filter 300 of one chamber 200 to the upper part of the nanopore filter 300 of the other chamber 200 adjacent to the one chamber 200.

Specifically, the flow pipe 400 is a connection pipe that sequentially connects the chambers 200, and is in communication with each chamber 200 and provides unidirectional flow of the fluid sample 10 flowing in the flow pipe 400. A check valve 410 may be placed on one side for control.

In addition, the flow tube 400 has one end connected to the lower side of the nanopore filter 300 of the previous stage chamber in the flow stage of the fluid sample 10, and the nanopore filter 300 of the next stage chamber 200. The other end is connected to the upper side, allowing the fluid sample 10 that has passed through the nanopore filter 300 to be moved to the next stage chamber 200.

In addition, the flow pipe 400 is provided with a check valve 410 on the center side to control unidirectional flow.

Figure 3(a) is a state diagram showing the open state of the check valve according to the present invention, Figure 3(b) is a state diagram showing the closed state of the check valve according to the present invention, and Figure 3(c) is a state diagram showing the open state of the check valve according to the present invention. This is a diagram showing a valve seat, and Figure 3(d) is a diagram showing a top view and a side view of the belt seat according to the present invention.

The check valve 410 according to the present invention is disposed on one side of the flow pipe 400 as shown in FIGS. 3(a) to 3(d), and the plunger 500 is installed in any chamber 200. When the fluid sample 10 flows into the chamber 200, the check valve 410 disposed on the flow pipe 400 is closed and the fluid sample 10 flows into the next stage chamber 200. The check valve 410 disposed at 400 is opened to control the fluid sample 10 to sequentially flow in one direction.

For example, the plunger 500 descends into the second chamber 220 and pumps the fluid sample 10 held in the second chamber 220 to remove the nanopore filter 300 disposed in the second chamber 220. ) When the fluid sample 10 that has passed through moves to the third chamber 230, the first check valve 411 disposed in the flow pipe 400 that introduces fluid into the second chamber 220 is closed. It prevents the fluid sample 10 from leaking from the second chamber 220 to the first chamber 210.

In contrast, the second check valve 412 disposed in the flow pipe 400 that flows out the fluid sample 10 from the second chamber 220 to the third chamber 230 is opened, thereby causing the second chamber ( Fluid flow from 220) to the third chamber 230 is allowed.

In addition, the check valve 410 includes a pair of elastic membranes 413 attached to the inner circumferential surface of the flow pipe 400 so as to have a flow port for the fluid sample 10 at the center, and is disposed adjacent to the flow port so that the fluid sample 10 flows. ) may include a valve seat 414 that selectively opens and closes the flow port according to the flow direction.

Additionally, the valve seat 414 may be made of polydimethylsiloxane (PDMS).

Additionally, the valve seat 414 may be made of an elastic material selected from the group consisting of silicone, latex, and rubber.

Therefore, when the fluid sample 10 shows a forward flow in the check valve 414, the elastic membrane 413 is separated from the valve seat 414 by receiving an upward force, allowing the fluid sample 10 to flow. A flow port is formed to enable forward flow of the advancing fluid sample 10.

In addition, when the fluid sample 10 shows a backward flow, the check valve 410 is attached to the valve seat 414 so that the elastic membrane 413 receives downward force and forms a flow port for the flow of the fluid sample 10. is closed to block the reverse flow of the fluid sample 10 moving backward.

Therefore, the check valve 410 controls the forward flow of the fluid sample 10.

Figure 5 is a block diagram showing the control relationship between the controller, solenoid valve, vertical motor, and rotation motor according to the present invention.

The plunger 500 is arranged to be spaced apart from the housing 100 so as to be aligned with the chamber 200, and flows into and out of the chamber 200 to pump the fluid sample 10 contained in the chamber 200. It is provided.

Specifically, the plunger 500 is disposed on the upper side of the housing 100 and flows into the chamber 200 aligned at the bottom, pumps the fluid sample 10 in the chamber 200, and then moves upward, and the housing 100 When the next chamber 200 is aligned at the bottom by the rotational movement of ), the fluid sample 10 flows into the next chamber 200 and is pumped. This process is performed sequentially to move the first chamber 210 to the waste chamber ( Pumping is performed up to 260).

Accordingly, the plunger 500 may be configured to include a vertical motor 510 and a plunger arm 520 that control the lifting and lowering movement of the plunger 500.

As shown in FIG. 5, the vertical motor 510 lowers and then moves the plunger 500 upward to pump an arbitrary first chamber 210, and when pumping is completed, the housing ( When 100) rotates and aligns the plunger 500 with the second chamber 220 adjacent to the first chamber 210, the plunger 500 moves downward to collect the fluid sample 10 in the second chamber 220. Allows pumping.

Additionally, the above-described process is repeatedly performed from the first chamber 210 to the waste chamber 260, so that the fluid sample 10 can be sequentially pumped.

Additionally, the vertical motor 510 may be a stepper motor that moves one step at a time using a toothed wheel and an electromagnet.

Meanwhile, when the filtering of the fluid sample 10 is completed, a cleaning process of the chamber 200 is performed to increase the EV purity in the chamber 200. During this process, the pumping process of the plunger 500 is performed in the same manner.

Specifically, the cleaning process of the chamber 200 is facilitated by injecting a cleaning solution into the filtered chamber 200 and having the plunger 500 pump the cleaning solution to increase EV purity.

In addition, the injection volume of the fluid sample 10 and the cleaning solution buffer into the first chamber 210 is each 800 μL, and the insertion depth of the plunger 500 in the chamber 200 is controlled so that a volume of 100 μL remains after filtering.

Additionally, to prevent contact between the plunger 500 and the solution, the initial gap between the plunger 500 and the fluid sample 10 is set to 3.5 mm.

Additionally, the inflow and outflow velocities of the plunger 500 for each chamber 200 are 5 μm/s and 0.5 mm/s, respectively, and cleaning can be performed three times.

Hereinafter, an example will be described in which the separation performance of the fluid control system (1) capable of filtering extracellular vesicles according to size according to the present invention is evaluated.

Figure 6 is a graph showing the selective separation performance of EV using plasma.

Figure 6 evaluates EV selective separation performance using plasma.

Figure 6(a) shows the size distribution of EV 20 in plasma, and Figure 6(b) shows the distribution of the first chamber 210 - second chamber 220 - third chamber 230 - fifth chamber ( When the EVs 250 are sequentially connected, EVs 20 of different sizes are separated by size in each chamber 200.

Figure 6(c) shows changing parameters including Pluronic coating time, flanger speed, and EV concentration on the surface of the chamber 200 and analyzing their effects on EV filtration. First, EVs 20 on the surface of the chamber 200 are changed. To prevent non-specific adsorption, Pluronic was used and the coating time was changed.

At this time, the third chamber 230 was used for filtration, and EVs were collected in the third chamber 230 and the waste chamber 260. Additionally, unlike polymer particles, which showed a recovery rate of up to 89%, the EV(20) recovery rate was as low as 66% without Pluronic coating. Additionally, as the coating time increases to 2 hours, the recovery rate increases to 88%. Additionally, as the coating time increases from 2 to 4 hours, the recovery rate increases by only 1% to 89%. This suggests that Pluronic was coated on the surface of the chamber 200 to almost the maximum extent to prevent non-specific adsorption of EVs 20 on the surface of the chamber 200 during the coating time of 2 hours. Therefore, a coating time of 2 hours is used for EV(20) isolation.

In addition, in order to study the effect of the insertion speed of the plunger 500 on the separation of the EV 20, sequential connection of the first chamber 210, the third chamber 230, and the waste chamber 260 was used, resulting in the third chamber. (230) and waste chamber (260) contain EVs with diameters (D) of 80 ≤ D ≤ 200 nm and D ≤ 80 nm, respectively, and larger particles, EVs with D > 200 nm, are stored in the third chamber (230 ), particles with D > 80 nm are separated in the waste chamber 260, and the larger particle fraction above is defined as the larger particle concentration divided by the total particle concentration in each chamber 200.

Figure 6(d) shows that as the plunger 500 insertion speed decreases from 50 to 10 μm/s, the proportion of larger particles decreases significantly from 35 to 16% for both chambers 200.

Additionally, the larger particle fractions described above decrease by a further 7% in the third chamber 230 and by 11% in the waste chamber 260 as the plunger 500 insertion speed decreases from 10 to 5 μm/s, which results in Due to changes in chamber 200 pressure, chamber 200 pressure increases according to the plunger 500 insertion speed. (Figure 6(e))

Here, the sequential chamber 200 connection of the first chamber 210 - the third chamber 230 - the fifth chamber 250 - the waste chamber 260 was used, and the EV 20 is elastic and deformable. Therefore, the modified EV 20 can pass through the pores of the nanopore filter 300. Therefore, at high pressure, EV 20 larger than the pore diameter of the nanopore filter 300 can pass through the filter pores.

Next, when analyzing the effect of EV concentration on filtration by EV size, when using the connection of the first chamber 210 - the third chamber 230 - the waste chamber 260 as shown in FIG. 6(d) , as the EV 20 concentration in the third chamber 230 decreases from 109 to 105 particles/mL, the proportion of larger particles in the third chamber 230 decreases from 7% to 3% (FIG. 6 (left panel of (f))

As a result, it can be seen that at a higher concentration of EVs 20, the EVs 20 passed from the first chamber 210 to the third chamber 230 through the nanopore filter 300 at a higher rate.

Additionally, to determine the relative amount of EVs 20 that are smaller than the pores of the nanopore filter 300, the concentration of EVs 20 with D ≤ 80 nm was divided by the total particle concentration obtained in the third chamber 230 to determine the smaller particles. The ratio was defined. The proportion of smaller particles remains almost unchanged despite the decrease in EV (20) concentration in the third chamber (230) (right panel of FIG. 6(f)). This means that EV (20) is smaller than the pore size of the nanopore filter 300. ) This suggests that it passes through the nanopore filter 300 at the same speed regardless of EV concentration.

Additionally, the effect of washing cycle on EV(20) purity is analyzed. At this time, the filtered solution is collected in the first chamber 210 and the third chamber 230, and EV (20) purity is defined as particle concentration per total protein concentration. Therefore, purity increases as total protein concentration decreases. It can be seen in Figure 6(g) that the total protein concentration rapidly decreases by 3-fold after the first wash. Afterwards, as the number of washings increases from 1 to 5, it decreases from 4.7 mg/mL to 3.7 mg/mL. The EV(20) concentration gradually increases with increasing number of washings, which leads to improved purity (Figure 6(h)). When using the inlet velocity (VI) of the plunger 500 = 5 μm/s and performing three washes in one chamber 200, filtration in that chamber 200 takes 1 hour.

In addition, under the above conditions, EV filtration by size takes 3 hours for chambers 200 with three sequential connections, such as the first chamber 210, the third chamber 230, and the fifth chamber 250. However, if the inlet velocity (VI) of the plunger 500 = 10 μm/s is used and each chamber 200 is cleaned once, the time required can be reduced to 45 minutes. Additionally, increasing the VI from 5 to 10 μm/s can slightly increase the proportion of larger particles from 7-11% to 16% (Figure 6(d)), and reducing the number of cleaning cycles from 3 to 1 Purity may slightly decrease from 8.1 × 10 ¹⁰ to 5.8 × 10 ¹⁰ particles/mg (Figure 6(h)).

Figure 7 is a diagram evaluating the performance of selective separation of EVs by size from cell culture supernatant.

Meanwhile, Figure 7 evaluates the size-specific selective separation performance of EVs from cell culture supernatants. To characterize EV production in endothelial cells under shear flow, HUVEC cells were cultured with shear flow (SF) or without shear flow (NF). Obtain EVs from cell culture supernatants. At this time, the EV is operated in a selective manner using a housing 100 having a first chamber 210, a second chamber 220, a third chamber 230, a fifth chamber 250, and a waste chamber 260. EVs are separated by size.

Figure 7(a) shows the size distribution of EVs obtained in SF (top panel) and NF (bottom panel) conditions. In addition, Figure 7(b) shows the sum of EV 20 concentrations in each chamber 200 based on size distribution. At this time, under SF conditions, if a smaller group of EVs (20) is selected from the first chamber 210 to the fifth chamber 250, the EV (20) concentration ranges from 7 × 10 ¹⁰ to 5 × 10 ⁹ particles/mL. decreases to This shows that HUVECs secrete more EVs of larger size. Additionally, EV concentration in NF conditions shows a similar trend as in SF conditions, but has values that are at least one order of magnitude lower.

This indicates that HUVECs secreted a higher number of EVs in SF conditions, and the concentration ratio is defined for each chamber 200 to analyze the above-described trends in each size group. This is the EV concentration in SF conditions divided by the concentration in NF conditions.

Figure 7(c) shows that the concentration ratios are 17 and 20, respectively, in the first chamber 210 and the second chamber 220, where a larger size EV group exists. The ratio greatly increases to 139 in the fifth chamber 250, where the smallest size EV group (30 ≤ D ≤ 80 nm) belongs. Therefore, the EV group with the smallest size appears higher in SF. Additionally, Figure 7(d) qualitatively shows the expression levels of EV surface (CD63) and internal (TSG101) proteins in groups with different EV sizes. Under SF conditions, CD63 protein in the first chamber 210 and the second chamber 220 is expressed at similar levels. In contrast, under NF conditions the expression level of the first chamber 210 is significantly lower than that of the second chamber 220. The EV concentration in the first chamber 210 and the NF concentration in the second chamber 220 appear similar (FIG. 7(b)). This suggests that under NF conditions, the expression of CD63 protein per EV is lower in the first chamber (210) than in the second chamber (220). Under SF conditions, the expression of CD63 in the third chamber 230 and the fifth chamber 250 is lower than that in the first chamber 210 and the second chamber 220. This may be because the EV concentration in the third chamber 230 and the fifth chamber 250 is low. This trend is the same in the case of NF. Additionally, the expression level of TSG101 protein appears similar in both NF and SF. This means that a large amount of TSG101 protein was expressed in EVs under NF conditions.

Therefore, the fluid control system (1) capable of filtering extracellular vesicles according to their size according to the present invention is selectively equipped with a nanopore filter (300) having pore diameters of 30, 50, 80, 100, and 200 nm to filter filters of various size intervals. By separating EVs, a high separation resolution containing less than 7% of particles larger than the pore size of the nanopore filter 300 and a purity of 8.1 × 10 ¹⁰ particles/mg are achieved. Additionally, a high EV recovery rate of 89% is obtained using a plunger (500) insertion speed of 5 μm/s and a Pluronic coating on the device surface after three cleaning cycles. This results in a separation time of 1 hour in each chamber 200 for a raw fluid sample volume of 800 μL, which can be significantly increased to 15 minutes in each chamber 200 with a plunger 500 speed of 10 μm/s. It can be reduced, and the separation resolution and purity do not decrease significantly after one wash.

Additionally, we performed size-selective EV isolation on endothelial cells under shear flow and found that cells secreted more EVs of larger sizes and that the expression of CD63 protein was higher in EVs of larger sizes, and its expression level was significantly reduced by shear flow. It appears higher in shear flow than in the case without it.

In contrast, the expression level of TSG101 protein was similar in both flow conditions, indicating that a large amount of TSG101 protein was expressed under NF conditions.

Therefore, the fluid control system (1) capable of size-specific filtering of extracellular vesicles according to the present invention operates in a simple and effective manner and does not require an expensive and complex off-chip controller.

Therefore, it may be suitable for molecular analysis and EV-based biomarker discovery with broad applications in prognosis, diagnosis, and treatment monitoring of patients with cancer and other diseases.

According to the fluid control system capable of filtering extracellular vesicles according to size according to the present invention as described above, it is composed of a rotary housing in which a plunger that moves vertically and a chamber that opens through a check valve are sequentially connected, thereby preventing various diseases. Extracellular vesicles as biomarkers can be separated into various size intervals in a short period of time with an automated and integrated system. Not only is the separation process and device simple, but in particular, sequential filtering is possible in an automated manner, making it effective for extracellular vesicles. This has the effect of implementing filtering by size.

In addition, a nanopore filter consisting of a porous membrane filtration network with nanopores is detachably mounted on the chamber to have different pore diameters to separate EVs at various size intervals of 30, 50, 80, 100, and 200 nm. There is an effect.

In addition, the nanopore filter is configured to be attachable and detachable within the chamber, allowing filtering by selectively applying filters with various pore diameters.

In addition, it has the effect of realizing a high separation resolution containing less than 7% of particles larger than the pore size of the nanopore filter and a purity of 8.1 × 10 ¹⁰ particles/mg.

In addition, the use of Pluronic coating on the device surface has the effect of obtaining a high EV recovery rate of 89%.

The terms and words used in the present specification and claims described herein should not be construed as limited to their usual or dictionary meanings, and the present inventor has appropriately used the concept of terms to explain his/her invention in the best way. It must be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle of definability.

Therefore, the configuration shown in the drawings and examples described in this specification is only one of the most preferred embodiments of the present invention, and does not represent the entire technical idea of the present invention, so they cannot be replaced at the time of filing the present application. It should be understood that various equivalents and variations may exist.

1: Fluid control system capable of filtering extracellular vesicles by size
10: fluid sample 20: extracellular vesicles, EV
100: Housing 110: First layer
120: 2nd layer 130: 3rd layer
140: 4th layer 150: 5th layer
160: rotation motor 200: chamber
210: first chamber 220: second chamber
230: 3rd chamber 240: 4th chamber
250: Fifth chamber 260: Waste chamber
300: Nanopore filter 400: Flow pipe
410: check valve 411: first check valve
412: second check valve 413: elastic membrane
414: valve seat 500: plunger
510: Vertical motor 520: Plunger arm

Claims (11)

Rotatable housing;
a plurality of chambers disposed in the housing and open at the top;
a nanopore filter disposed in the chamber and composed of a filter net having nanopores;
a flow pipe connecting the chamber and its neighboring chambers to provide a passage for fluid samples sequentially flowing through the chambers; and
A plunger is disposed spaced apart from the housing so as to be aligned with the chamber and pumps the fluid sample carried in the chamber by flowing in and out of the chamber.
The flow pipe is,
It includes a check valve on one side that controls the unidirectional flow of the fluid sample,
The check valve is,
A pair of elastic membranes attached to the inner peripheral surface of the flow pipe to have a fluid sample flow port at the center; and
a valve seat disposed adjacent to the flow port and opening and closing the flow port according to the flow direction of the fluid sample;
A fluid control system capable of filtering extracellular vesicles by size, comprising:
According to clause 1,
The flow pipe is,
A fluid control system capable of filtering extracellular vesicles by size, characterized in that the lower part of the nanopore filter of one chamber is connected to the upper part of the nanopore filter of the other chamber adjacent to the one chamber.
delete According to clause 1,
When the plunger is introduced into any chamber, the check valve disposed on the flow pipe through which the fluid sample flows into the chamber is closed, and the check valve disposed on the flow pipe through which the fluid sample flows out of the chamber is opened. A fluid control system capable of filtering extracellular vesicles by size.
According to clause 1,
The nanopore filter is,
A fluid control system capable of filtering extracellular vesicles by size, characterized in that they are selectively attached and detached from the chamber.
According to clause 1,
The chamber is,
a waste chamber for storing filtered fluid samples;
A fluid control system capable of filtering extracellular vesicles by size, comprising:
According to clause 1,
The nanopore filter is,
It consists of a filter network with pores with a diameter of 200 nm to 30 nm,
The chamber is,
A fluid control system capable of filtering by size of extracellular vesicles, wherein nanopore filters having different pore diameters are installed, and nanopore filters with smaller pores are sequentially installed at each flow stage of the fluid sample.
According to clause 1,
The housing is,
Includes a rotation motor on one side that controls rotational movement of the housing,
The flanger,
A vertical motor that controls the lifting and lowering movement of the plunger;
A fluid control system capable of filtering extracellular vesicles by size, comprising:
According to clause 8,
The rotation motor is,
When the plunger moves upward after pumping an arbitrary first chamber, the housing is rotated at a preset angle so that the second chamber adjacent to the first chamber is aligned with the plunger,
The vertical motor is,
When the second chamber is aligned with the plunger, the plunger is moved downward to the second chamber to pump the fluid sample, and then the plunger is moved upward. A fluid control system capable of filtering by size of extracellular vesicles.
delete According to clause 1,
The valve seat is,
A fluid control system capable of filtering extracellular vesicles by size, characterized in that it is an elastic material selected from the group consisting of polydimethylsiloxane (PDMS), silicone, latex, and rubber.