WO2017069573A1 - Appareil de séparation de nanoparticules à force centrifuge, et procédé de séparation de nanoparticules l'utilisant - Google Patents

Appareil de séparation de nanoparticules à force centrifuge, et procédé de séparation de nanoparticules l'utilisant Download PDF

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WO2017069573A1
WO2017069573A1 PCT/KR2016/011917 KR2016011917W WO2017069573A1 WO 2017069573 A1 WO2017069573 A1 WO 2017069573A1 KR 2016011917 W KR2016011917 W KR 2016011917W WO 2017069573 A1 WO2017069573 A1 WO 2017069573A1
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sample
filtration
nanoparticle
nanoparticles
separation device
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PCT/KR2016/011917
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English (en)
Korean (ko)
Inventor
조윤경
우현경
한자령
김태형
김윤근
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울산과학기술원
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Priority to US15/770,454 priority Critical patent/US11154860B2/en
Priority claimed from KR1020160137581A external-priority patent/KR101891890B1/ko
Publication of WO2017069573A1 publication Critical patent/WO2017069573A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B5/00—Other centrifuges
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

Definitions

  • 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.
  • 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.
  • 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 ⁇ m capable of filtering nanoparticles from the fluid sample;
  • 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 is to provide a third object of the present invention.
  • 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
  • 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 is to provide a third object of the present invention.
  • a sample accommodating part 200 which provides a space supported by injecting a fluid sample
  • 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 is to provide a third object of the present invention.
  • a sample accommodating part 200 which provides a space supported by injecting a fluid sample
  • a valve 700 capable of selectively controlling the flow of fluid in the microchannel
  • 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.
  • 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 ⁇ m capable of filtering nanoparticles from the fluid sample;
  • nano-particle separator comprising a micro-channel 500 for providing a passage for the flow of the fluid sample.
  • 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.
  • the cleaning chamber unit 600 may further include a space for supporting the cleaning solution.
  • 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.
  • the nanoparticle separation device may further include one or more fastening portions for attaching and detaching the filtration chamber 300.
  • the fastening part may be an elastic material selected from the group consisting of polydimethylsiloxane, silicone, latex, rubber, and the like.
  • the micro channel 500 may be connected to a channel through the device to change the channel of the fluid sample.
  • the present invention is a.
  • 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
  • nano-particle separator comprising a micro-channel 500 for providing a passage for the flow of the fluid sample.
  • 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.
  • the filtration membrane may be selectively detachable from the housing part 100 by physical force.
  • 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.
  • 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.
  • the filtration membrane Preferably, the filtration membrane,
  • a first filtration membrane having at least one or more pores of 100 nm to 1 ⁇ m diameter
  • It may include a second filtration membrane having at least one or more pores of 1 nm to 100 nm in diameter.
  • 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 is a.
  • a sample accommodating part 200 which provides a space supported by injecting a fluid sample
  • 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.
  • the sample accommodating part 200 may perform sample purification to purify impurities of a sample.
  • 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.
  • the sample accommodating part 200 may include a groove for preventing separated impurity backflow.
  • 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.
  • the valve 700 may be opened and closed according to an external signal.
  • 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.
  • the waste liquid containing part 400 may be capable of separating high purity nanoparticles without a separate impurity treatment.
  • 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.
  • the nanoparticle separation device may further include one or more fastening portions for attaching and detaching the filtration chamber 300.
  • 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.
  • BSA bovine serum albumin
  • Pluronic Pluronic
  • the filtration chamber 300 and the waste liquid receiving unit 400 may include a vent for performing smooth filtration.
  • the present invention is a.
  • a sample accommodating part 200 which provides a space supported by injecting a fluid sample
  • a valve 700 capable of selectively controlling the flow of fluid in the microchannel
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • NTA nanoparticle tracking analysis
  • FIG. 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.
  • NTA nanoparticle tracking analysis
  • FIG. 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.
  • FIGS. 1B and 3B are enlarged views of the nanoparticle separator 10 according to an embodiment of the present invention.
  • the nanoparticle separation apparatus 10 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.
  • 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.
  • 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.
  • 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.
  • 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 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
  • the filter 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.
  • 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.
  • 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.
  • 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.
  • the first filtration unit 310 may have a plurality of pores having a diameter of 100 nm to 1 ⁇ m, 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.
  • 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.
  • the second filtration unit 320 may be connected to the first filtration unit 310 and the washing chamber unit 600 to be described later.
  • the first filtration unit 310 and the second filtration unit 320 are filtered in the same chamber.
  • 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.
  • the filtration chamber 300 may be connected to one or more washing chamber 600 as necessary.
  • 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.
  • the secondary impurities, which are impurities having a small particle size may be non-vascular proteins.
  • 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.
  • 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.
  • the nanoparticle separation device 10 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.
  • 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
  • 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.
  • the micro channel 500 may be connected to a channel through the device to change the channel of the fluid sample.
  • 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.
  • the first micro channel 510 connects the sample receiving part 200 and the waste liquid receiving part 400 to the second micro channel 520.
  • 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).
  • PBS phosphate buffer saline
  • 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.
  • the first valve 710 is disposed on the first microchannel 510
  • the second valve 720 is the second microchannel 520
  • the third valve 730 is disposed on the third microchannel 530.
  • the first valve 710 is disposed on the first microchannel 510 and the second valve 720 is the second microchannel.
  • the third valve 730 is disposed on the third microchannel 530
  • the fourth valve 740 is disposed on the fourth microchannel 540
  • the fifth valve 750 is disposed on the sixth microchannel portion 560
  • the sixth valve 760 is disposed on the seventh microchannel portion 570.
  • the nanoparticle separation device 10 may affect the collection of nano-vesicles.
  • 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.
  • 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.
  • 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.
  • 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.
  • each configuration is as follows.
  • 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.
  • the filtration unit 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.
  • 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.
  • 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).
  • the impurity treatment chamber extracts impurities from the sample using centrifugal force, and the blue arrow shows the filter washing (see FIG. 2B).
  • nano vesicles are filtered over a 30 nm filter (see FIG. 2D).
  • the cleaning solution is transferred to a chamber with a filter to remove impurities other than nanovesicles.
  • the valve 3 may be blocked to prevent backflow of the solution (see FIG. 2E).
  • the filtration membrane of the filtration chamber part 300 is a lamination type, the filtration membrane is separated to separate nano vesicles.
  • first filtration unit 310 for separating other vesicles
  • the nanoparticle separator 10 further includes a particle collecting part 800 capable of collecting nano vesicles.
  • 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.
  • the process for separating the nano vesicles can be automated.
  • 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.
  • Figure 4 shows a state using the nanoparticle separation device 10 according to an embodiment of the present invention.
  • a sample maximal 1 ml
  • a buffer solution 600 ⁇ l
  • 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).
  • the conventional method requires a lot of time and various processes for sample processing using an ultracentrifuge or a precipitation reagent.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m) and aluminum oxide anodes (Whatman, 13 mm, 0.02 ⁇ m). Carved for.
  • LNCaP cells a prostate cancer cell line
  • RPMI medium Gibco, UK
  • FBS System Biosciences Inc., CA
  • antibiotic / antifungal 1% antibiotic / antifungal
  • Proceed in an incubator Cell culture supernatants were harvested after 24 hours and extracellular vesicles were collected as described in each protocol.
  • 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 °C.
  • 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.
  • UC ultracentrifugation
  • Exospin Exospin
  • 400 ⁇ l urine was used for separation of nano vesicles in the separator.
  • nanoparticle tracking was carried out through nanoparticle tracking analysis (NTA) to analyze the size and concentration.
  • NTA nanoparticle tracking analysis
  • 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.
  • disc performance showed that only 100 nm beads were detected and concentrated in the filter (see FIGS. 8B and C).
  • 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
  • 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).
  • 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).
  • Ultracentrifugation 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.
  • 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.
  • Example 1-3 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 ⁇ l of PBS provided in the kit. The vesicle pellets were purified using a spin column provided according to the manufacturer's instructions, and 200 ⁇ of the separated vesicles were stored at 4 ° C. for immediate use / short term storage or at ⁇ 80 ° C. for long term storage.
  • the vesicle solution according to the separation method of Example 3 was analyzed using an enzyme immunoassay (ELISA).
  • ELISA enzyme immunoassay
  • 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 °C, 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 ⁇ l) 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.
  • 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 ⁇ l, 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 ⁇ l, 1: 1000 in PBS).
  • NTA results show a high concentration yield of nano vesicles separated from the nanoparticle separator according to the present invention.
  • 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 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.

Abstract

La présente invention concerne un appareil et un procédé de séparation de nanoparticules à force centrifuge. Spécifiquement, la présente invention est basée sur le fait d'avoir une faible force centrifuge et une petite taille et, de ce fait, peut séparer des nanovésicules non associées à une spécificité d'anticorps en peu de temps et sans utiliser d'ultracentrifugeuse. En outre, la présente invention ne nécessite aucun personnel professionnel supplémentaire, et permet une mesure précise de fluide par intégration et automatisation de tous les processus suivant une injection d'échantillon et, de ce fait, peut réduire la perte de nanovésicules.
PCT/KR2016/011917 2015-10-23 2016-10-21 Appareil de séparation de nanoparticules à force centrifuge, et procédé de séparation de nanoparticules l'utilisant WO2017069573A1 (fr)

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KR10-2015-0147883 2015-10-23
KR1020160137581A KR101891890B1 (ko) 2015-10-23 2016-10-21 원심력 기반 나노 입자 분리장치 및 이를 이용한 나노 입자 분리방법
KR10-2016-0137581 2016-10-21

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CN112940910A (zh) * 2019-12-09 2021-06-11 中国科学院大连化学物理研究所 一种胰岛囊泡富集装置及其制备方法和应用

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