WO2022099518A1 - 分离装置和分离方法 - Google Patents

分离装置和分离方法 Download PDF

Info

Publication number
WO2022099518A1
WO2022099518A1 PCT/CN2020/128177 CN2020128177W WO2022099518A1 WO 2022099518 A1 WO2022099518 A1 WO 2022099518A1 CN 2020128177 W CN2020128177 W CN 2020128177W WO 2022099518 A1 WO2022099518 A1 WO 2022099518A1
Authority
WO
WIPO (PCT)
Prior art keywords
chamber
filter membrane
vibrating member
vibration
vibration wave
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/128177
Other languages
English (en)
French (fr)
Inventor
陈欲超
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Huixin Life Technologies Co Ltd
Original Assignee
Shenzhen Huixin Life Technologies Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Huixin Life Technologies Co Ltd filed Critical Shenzhen Huixin Life Technologies Co Ltd
Priority to PCT/CN2020/128177 priority Critical patent/WO2022099518A1/zh
Priority to EP20954324.8A priority patent/EP4246118A4/en
Priority to JP2022522396A priority patent/JP7397529B2/ja
Priority to KR1020237000065A priority patent/KR102878476B1/ko
Priority to CN202080003487.2A priority patent/CN113195080A/zh
Priority to US17/369,836 priority patent/US12196657B2/en
Publication of WO2022099518A1 publication Critical patent/WO2022099518A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/16—Rotary, reciprocated or vibrated modules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
    • B01D65/02—Membrane cleaning or sterilisation ; Membrane regeneration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
    • B01D65/08—Prevention of membrane fouling or of concentration polarisation
    • 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
    • G01N1/34—Purifying; Cleaning
    • 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
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • 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
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/04—Backflushing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/20—By influencing the flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/20—By influencing the flow
    • B01D2321/2033—By influencing the flow dynamically
    • B01D2321/2058—By influencing the flow dynamically by vibration of the membrane, e.g. with an actuator
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/20—By influencing the flow
    • B01D2321/2066—Pulsated flow
    • B01D2321/2075—Ultrasonic treatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/20—By influencing the flow
    • B01D2321/2083—By reversing the flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/26—By suction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/30—Mechanical cleaning, e.g. with brushes or scrapers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/40—Automatic control of cleaning processes
    • 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
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis
    • 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
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

Definitions

  • the present application relates to the field of biotechnology, and in particular, to a separation device and a separation method for separating target particles in a liquid sample.
  • Exosomes are small vesicles with double phospholipid membrane structure with a diameter of 30-150nm that are continuously secreted by living cells. Molecular and other specific components. Numerous studies have shown that exosomes are involved in various events in tumor development, including immune escape, angiogenesis, tumor metastasis, and tumor resistance. Exosomes can be released by cancer cells earlier and continuously and enter the patient's blood circulation system, and their lipid bilayer membrane structure can effectively protect the carried proteins and encapsulated nucleic acids. Exosomes are widely and stably present in a variety of clinical samples, including blood, urine, ascites, tissue fluid, tears, saliva, and cerebrospinal fluid.
  • exosomes are considered as key research objects in the field of in vitro diagnostic research and tumor clinical detection, and are expected to be used in early tumor diagnosis, tumor metastasis and recurrence assessment, tumor heterogeneity assessment, dynamic detection of tumor development and efficacy, and drug resistance mutations. It has great clinical value in detection and personalized medicine.
  • the present application provides a separation device for separating and purifying target particles from a liquid sample.
  • the separation device includes:
  • a separation chip assembly comprising a separation chip, a first vibrating member and a second vibrating member
  • the separation chip includes a sample pool and a first filter membrane and a second filter membrane located on opposite sides of the sample pool, the first filter membrane
  • the pore size of the filter membrane and the second filter membrane are both smaller than the particle size of the target particles
  • the separation chip further includes a first chamber and a second chamber, and the first chamber passes through the first filter membrane communicates with the sample pool, the second chamber communicates with the sample pool through the second filter membrane, the first vibrating member is fixed on the first filter membrane and the second filter membrane, and the second vibration member a piece is fixed on the outer surface of the first chamber and the outer surface of the second chamber;
  • a frequency conversion module respectively connected with the first chamber and the second chamber through the vacuum system, and the frequency conversion module is used to control the vacuum system to alternate between the first chamber and the second chamber negative pressure is created in the chamber;
  • a controller for controlling the vibration of the first vibrating member and the second vibrating member when the vacuum system stops generating negative pressure in the first chamber; and is also used for controlling the vibration of the first vibrating member and the second vibrating member when the vacuum system stops at When a negative pressure is generated in the second chamber, the first vibrating member and the second vibrating member are controlled to vibrate, wherein the first vibrating member and the second vibrating member respectively generate a first vibrating member when vibrating.
  • a vibration wave and a second vibration wave the frequency of the first vibration wave is greater than the frequency of the second vibration wave, and the amplitude of the first vibration wave is smaller than the amplitude of the second vibration wave.
  • the vibration frequency of the first vibration wave is 5000 Hz-8000 Hz
  • the vibration frequency of the second vibration wave is 100-500 Hz.
  • the vibration frequency of the first vibration wave is equal to the resonance frequency of the first filter membrane or the second filter membrane.
  • the first vibrating member and the second vibrating member are located on the same horizontal plane.
  • the first vibration member is a harmonic oscillator
  • the second vibration member is a vibration motor
  • the present application also provides a separation method for separating and purifying target particles from a liquid sample.
  • the method includes:
  • a negative pressure is generated in the first chamber, so that the components in the liquid sample in the sample cell whose particle size is smaller than the pore size of the first filter membrane enter the first chamber under the action of negative pressure;
  • Stop generating negative pressure in the first chamber, and control the vibration of the first vibrating member and the second vibrating member;
  • Stop generating negative pressure in the second chamber, and control the first vibrating member and the second vibrating member to vibrate, wherein the first vibrating member and the second vibrating member are respectively generated when vibrating A first vibration wave and a second vibration wave, the frequency of the first vibration wave is greater than the frequency of the second vibration wave, and the amplitude of the first vibration wave is smaller than the amplitude of the second vibration wave.
  • the vibration frequency of the first vibration wave is 5000 Hz-8000 Hz
  • the vibration frequency of the second vibration wave is 100-500 Hz.
  • the vibration frequency of the first vibration wave is equal to the resonance frequency of the first filter membrane or the second filter membrane.
  • the first vibrating member and the second vibrating member are located on the same horizontal plane.
  • the first vibration member is a harmonic oscillator
  • the second vibration member is a vibration motor
  • the present application controls the vibration of the first vibrating member and the second vibrating member when the vacuum system stops generating negative pressure in the first chamber/second chamber, and the first vibrating member moves toward the first filter membrane.
  • the first vibration wave is transmitted with the second filter membrane, so that the target particles adsorbed in the membrane pores of the filter membrane can be quickly separated from the membrane pores of the filter membrane and resuspended in the returning liquid sample;
  • the vibration wave and the first vibration wave together perturb the liquid sample and the filter membrane and produce an acoustic effect, preventing the target particles from clogging the filter holes or agglomerating together, and achieving high-efficiency separation.
  • FIG. 1 is a schematic structural diagram of a split chip assembly provided by an embodiment of the present application.
  • FIG. 2 is a schematic view of applying negative pressure to the split chip assembly shown in FIG. 1 .
  • FIG. 3 is a schematic diagram of a first vibration wave and a second vibration wave applied to the split chip assembly shown in FIG. 1 .
  • FIG. 4 is a schematic diagram of functional modules of a separation device provided by an embodiment of the present application.
  • FIG. 5 is a graph showing the variation trend of the content of exosomes obtained in the examples of the present application and comparative examples 1-4 over time.
  • FIG. 7 is a test diagram of the content of exosomes obtained after the application of the present application separates and purifies urine samples of different volumes and concentrations of exosomes.
  • FIG. 8 is a test diagram of the content of exosomes obtained after the application of the present application to separate and purify different types of liquid samples.
  • FIG. 9 is a map obtained by performing western blot analysis on the exosomes obtained in Example 5 of the present application and Comparative Example 5.
  • FIG. 9 is a map obtained by performing western blot analysis on the exosomes obtained in Example 5 of the present application and Comparative Example 5.
  • Figure 10 is a schematic diagram comparing the three dimensions of purification time, exosome yield and exosome concentration between Example 5 of the present application and Comparative Example 5.
  • an embodiment of the present application provides a separation chip assembly 1 for separating and purifying particles of different sizes in a liquid sample to obtain target particles of a specific size.
  • the liquid sample can be human plasma, serum, cerebrospinal fluid, saliva, urine, gastric juice and the like.
  • the target particles can be exosomes or circulating tumor cells, etc.
  • the split chip assembly 1 includes a split chip 10 , a first vibration member 20 and a second vibration member 30 .
  • the separation chip 10 includes a sample cell 13 and a first filter membrane 14 and a second filter membrane 16 located on opposite sides of the sample cell 13 .
  • the sample cell 13 is used to hold a liquid sample.
  • the pore sizes of the first filter membrane 14 and the second filter membrane 16 are both smaller than the particle size of the target particles.
  • the separation chip 10 may further include a first chamber 15 and a second chamber 17 , the first chamber 15 is communicated with the sample cell 13 through the first filter membrane 14 , and the first chamber 15 is provided with a first opening 152 , the first opening 152 communicates with the first chamber 15 and the outside.
  • the second chamber 17 is communicated with the sample cell 13 through the second filter membrane 16 , the second chamber 17 is provided with a second opening 172 , and the second opening 172 communicates with the second chamber 17 and the outside world.
  • the first chamber 15 and the second chamber 17 are respectively located on opposite sides of the sample pool 13 .
  • the liquid sample is added to the sample cell 13, and the first opening 152 and the second opening 172 are respectively connected to the vacuum system 50 (refer to FIG. 4).
  • the vacuum system 50 draws the first chamber 15 through the first opening 152 , a negative pressure is generated in the first chamber 15 .
  • the components in the liquid sample in the sample cell 13 whose size is smaller than the filtration pore size of the first filter membrane 14 (including small particles and liquid components) move toward the first filter membrane 14, It flows into the first chamber 15 via the first filter membrane 14 .
  • the vacuum system 50 draws the second chamber 17 through the second opening 172 , a negative pressure is generated in the second chamber 17 .
  • the components in the liquid sample in the sample cell 13 whose size is smaller than the filtration pore size of the second filter membrane 16 move toward the second filter membrane 16 and flow into the second filter membrane 16 through the second filter membrane 16 .
  • the liquid sample in the sample pool 13 will generate a backflow phenomenon at the first filter membrane 14, thereby reducing or removing the components adhering to the first filter membrane 14, avoiding filtration during the filtration separation process. A situation where the membrane is blocked occurs. Repeated alternation to generate negative pressure in the first chamber 15 and the second chamber 17 can effectively make the liquid sample flow through the first filter membrane 14 and the second filter membrane 16 alternately and repeatedly, so that the size of the liquid sample is larger than that of the first filter membrane 16.
  • the structural design of the separation chip 10 makes the components adsorbed on the surfaces of the first filter membrane 14 and the second filter membrane 16 easy to fall off the surface of the filter membranes during repeated and alternate negative pressure changes, which can effectively prevent the membrane pores of the filter membrane from being blocked. .
  • the negative pressure (NP) generated by the vacuum system 50 in the first chamber 15 and the second chamber 17 alternately forms a periodic trapezoidal pulse signal .
  • the amplitude of the rectangular pulse signal is 10V pp and the frequency is 5000Hz to 7000Hz.
  • the trapezoidal pulse signal can prevent the damage to the first filter membrane 14 and the second filter membrane 16 caused by a sudden change of the negative pressure direction.
  • a negative pressure can be generated in one of the chambers and a positive pressure (AP) can be generated in the other chamber to enhance the Backflow phenomenon at the filter membrane.
  • the number of the first vibrating members 20 is two.
  • One of the first vibrating members 20 is fixed on the surface of the first filter membrane 14 away from the second filter membrane 16 .
  • Another first vibrating member 20 is fixed to the surface of the second filter membrane 16 away from the first filter membrane 14.
  • the first vibrating member 20 is used to generate a transverse first vibration wave when vibrating, and then transmit the first vibration wave to the first filter membrane 14 and the second filter membrane 16, thereby driving the first filter membrane 14 and the second filter membrane 16. high frequency vibration. Therefore, the target particles adsorbed in the membrane pores of the filter membrane can be quickly separated from the membrane pores of the filter membrane and resuspended in the returning liquid sample, thereby further preventing the membrane pores of the filter membrane from being blocked and achieving efficient separation.
  • the number of the second vibrating members 30 is two. One of the second vibrating members 30 is fixed on the outer surface of the first chamber 15 . Another second vibrating member 30 is fixed on the outer surface of the second chamber 17 .
  • the second vibrating member 30 is used to generate a second lateral vibration wave when vibrating. Please also refer to FIG. 3 , wherein the frequency of the first vibrational wave is greater than the frequency of the second vibrational wave, and the amplitude of the first vibrational wave is smaller than that of the second vibrational wave.
  • the second vibration wave is transmitted to the entire separation chip 10 through the first chamber 15 and the second chamber 17 , so that the separation chip 10 vibrates at a low frequency.
  • the second vibration wave can disturb the liquid sample and the filter membrane together with the first vibration wave and produce an acoustic streaming effect, preventing the target particles from clogging the filter holes or gathering together, and improving the separation and purification efficiency.
  • the first vibration member 20 may be a harmonic oscillator
  • the second vibration member 30 may be a vibration motor.
  • the vibration frequency of the first vibration wave is 5000 Hz-8000 Hz
  • the vibration frequency of the second vibration wave is 100-500 Hz. Neither the first vibration wave nor the second vibration wave at the vibration frequency will cause damage to the target particles.
  • the vibration frequency of the first vibration wave may be approximately the same as the resonance frequency of the first filter membrane 14 or the second filter membrane 16, and at this time, the first filter membrane 14 or the second filter membrane 16 may vibrate with a larger amplitude, It is convenient for the target particles adsorbed on the filter membrane to be separated more quickly.
  • the first vibrating member 20 and the second vibrating member 30 are located on the same horizontal plane. That is, the first vibration wave and the second vibration wave face the same direction, so that the first vibration wave and the second vibration wave can be superimposed on each other to form a coordinated vibration.
  • the split chip 10 includes a first side cover sheet 11 and a second side cover sheet 12 .
  • the first side cover sheet 11 includes a first cover sheet body 110 and a first blocking sheet 111 and a second blocking sheet 112 located on opposite sides of the first cover sheet body 110 .
  • the first filter membrane 14 is fixed on the first blocking sheet 111 and the second blocking sheet 112 .
  • the first cover sheet body 110 , the first blocking sheet 111 , the second blocking sheet 112 and the first filter membrane 14 together define the first chamber 15 .
  • the second side cover piece 12 includes a second cover piece body 120 and a third cover piece 121 and a fourth stop piece 122 located on opposite sides of the second cover piece body 120.
  • the third cover piece 121 is opposite to the first cover piece 111.
  • the fourth blocking piece 122 is opposite to the second blocking piece 112, and the second filter membrane 16 is fixed between the third blocking piece 121 and the fourth blocking piece 122 and is opposite to the second cover piece body 120.
  • the second cover piece body 120, The third blocking piece 121 , the fourth blocking piece 122 and the second filter membrane 16 together define the second chamber 17 .
  • the sample cell 13 is located between the first filter membrane 14 and the second filter membrane 16 .
  • the second vibrating member 30 is fixed on the outer surface of the first cover sheet body 110 or the second cover sheet body 120 .
  • the first blocking piece 111 and the third blocking piece 121 are disposed at a distance to define a sample addition port 131 that communicates with the sample cell 13 .
  • the separation chip 10 further includes a sample adding chamber 18 , and the sample adding chamber 18 communicates with the sample pool 13 through a sample adding port 131 .
  • the liquid sample is added into the sample addition chamber 18 , and the sample addition port 131 is used to enable the liquid sample in the sample addition chamber 18 to flow out of the sample addition chamber 18 and into the sample pool 13 .
  • an embodiment of the present application further provides a separation device 100 , including the separation chip assembly 1 , a vacuum system 50 , a frequency conversion module 40 and a controller 60 as described above.
  • the vacuum system 50 is used to generate negative pressures in the first chamber 15 and the second chamber 17 of the separation chip assembly 1 , respectively.
  • the vacuum system 50 can be two separate vacuum systems, or it can be one vacuum system by design.
  • the vacuum system 50 may also include devices such as a micro vacuum pump or a micro air pump. It can be understood that, the vacuum system 50 and the separation chip 10 may be connected by pipes with better air tightness.
  • the vacuum system 50 includes a first vacuum pump 510 and a second vacuum pump 520 , the first vacuum pump 510 is connected to the first opening 152 of the separation chip 10 , and the second vacuum pump 520 is connected to the second opening 172 of the separation chip 10 . connect.
  • the frequency conversion module 40 is electrically connected to the vacuum system 50 , and the frequency conversion module 40 can control the power supply voltage provided to the vacuum system 50 , so as to generate negative pressure alternately in the first chamber 15 and the second chamber 17 .
  • the frequency conversion module 40 includes a frequency converter 410 and a control valve 420 connected to the frequency converter 410 .
  • the control valve 420 may be a fluid circuit converter, including but not limited to a solenoid valve and a rotary valve.
  • the control valve 420 is communicated with one of the first vacuum pump 510 and the second vacuum pump 520 respectively, so that the first vacuum pump 510 and the second vacuum pump 520 work alternately and repeatedly.
  • control valve 420 is connected to the first vacuum pump 510, so that the frequency converter 410 controls the operation of the first vacuum pump 510, and the air is pumped through the first opening 152 to generate a negative pressure in the first chamber 15, and the liquid sample in the sample cell 13 has a negative pressure.
  • the liquid and components smaller than the pore size of the first filter membrane 14 pass through the first filter membrane 14 under the action of negative pressure and enter the first chamber 15; then, the frequency converter 410 controls the first vacuum pump 510 to stop running; after that, the control valve 420 is switched to communicate with the second vacuum pump 520, so that the frequency converter 410 controls the operation of the second vacuum pump 520, and the air is pumped through the second opening 172 to generate a negative pressure in the second chamber 17, so that the liquid in the liquid sample in the sample cell 13 is
  • the components whose size is smaller than the aperture of the second filter membrane 16 pass through the second filter membrane 16 under the action of negative pressure and enter the second chamber 17; after that, the frequency converter 410 controls the second vacuum pump 520 to stop running; repeat the above steps for many times .
  • the controller 60 is configured to control the vibration of the first vibration member 20 and the second vibration member 30 when the suction of the first chamber 15 is stopped, so as to generate the first vibration wave and the second vibration wave respectively.
  • the controller 60 is further configured to control the vibration of the first vibration member 20 and the second vibration member 30 when the suction of the second chamber 17 is stopped, so as to generate the first vibration wave and the second vibration wave respectively.
  • the controller 60 can be electrically connected to the first vacuum pump 510 and the second vacuum pump 520. When the first vacuum pump 510 or the second vacuum pump 520 stops running, the controller 60 determines that the first vacuum pump 510 stops in the first chamber 15. Suction, or judging that the second vacuum pump 520 stops pumping in the second chamber 17 , at this time, the corresponding first vibrating member 20 and the second vibrating member 30 can be notified to start vibrating.
  • the embodiments of the present application further provide a method for separating target particles in a liquid sample applied to the above-mentioned separation chip assembly 1, which includes the following steps:
  • step S1 the separation chip assembly 1 of the present application is provided, and a liquid sample is provided into the sample pool 13 of the separation chip assembly 1 .
  • step S2 the first chamber 15 is sucked through the first opening 152 to generate a negative pressure in the first chamber 15 .
  • the first opening 152 and the second opening 172 are respectively connected to the vacuum system 50 of the separation device 100 .
  • the vacuum system 50 sucks the first chamber 15 through the first opening 152 to generate negative pressure in the first chamber 15 .
  • the liquid in the liquid sample in the sample cell 13 and the components whose size is smaller than the pore size of the first filter membrane 14 move toward the first filter membrane 14 under the action of negative pressure, and enter the first chamber 15 through the first filter membrane 14 .
  • step S3 the pumping of the first chamber 15 is stopped, and the first vibration member 20 and the second vibration member 30 are controlled to vibrate, thereby generating the first vibration wave and the second vibration wave respectively.
  • the second chamber 17 is sucked through the second opening 172 to generate a negative pressure in the second chamber 17 .
  • the first vibration wave drives the first filter membrane 14 to vibrate at a high frequency, so that the target particles adsorbed in the membrane pores of the filter membrane can be quickly separated from the membrane pores of the filter membrane and resuspended in the returning liquid sample.
  • the second vibration wave can prevent the target particles from clumping together.
  • the vacuum system 50 sucks the second chamber 17 through the second opening 172 to generate negative pressure in the second chamber 17 .
  • the components adhering to the surface of the first filter membrane 14 may follow the airflow and/or liquid flow in the sample cell 13, and the liquid in the liquid sample in the sample cell 13 and the components whose size is smaller than the pore size of the second filter membrane 16 are in the negative. Under the action of pressure, it moves toward the second filter membrane 16 and enters the second chamber 17 through the second filter membrane 16 .
  • step S4 the suction of the second chamber 17 is stopped, and the first vibrating member 20 and the second vibrating member 30 are controlled to vibrate.
  • steps S2 to S4 can be cycled for many times, so that the components in the liquid sample smaller than the pore size of the filter membrane are removed, and the components larger than the pore size of the filter membrane are trapped in the sample cell 13 to achieve better separation and purification effect.
  • the frequency of the first vibrating member is 6250 Hz (about the same as the resonance frequency of the filter membrane), and the frequency of the second vibrating member is 200 Hz.
  • a separation chip assembly was used to separate and purify exosomes in a 2 mL urine sample.
  • the difference from the separation chip assembly of the embodiment was that the separation chip of Comparative Example 1 did not contain the first vibrating member and the second vibrating member.
  • the separation chip was used to separate and purify 2 mL of urine samples.
  • the difference from the separation chip assembly of the embodiment is that the separation chip of Comparative Example 2 only contains the second vibrating member (frequency is 200 Hz), and does not contain the first vibration element. Vibration piece.
  • the separation chip was used to separate and purify 2 mL of urine samples.
  • the difference from the separation chip assembly of the embodiment is that the separation chip of Comparative Example 3 only contains the first vibrating member (frequency is 6250 Hz), and does not contain the second vibration Vibration piece.
  • Dead-end filtration uses the same filter membrane as the separation chip of the embodiment, except that the liquid sample is placed in the filter. Upstream of the membrane, driven by the pressure difference, the liquid components and particles smaller than the membrane pores pass through the filter membrane.
  • exosomes obtained in Examples and Comparative Examples 1-4 over time were measured, and the results were recorded in FIG. 5 .
  • the example can separate exosomes of nearly 30 ⁇ m within 10 minutes, and the separation efficiency is much higher than that of the comparative examples 1-4.
  • the exosomes obtained in the examples were further tested by transmission electron microscopy (the scale of the scale was 250 nm), and the test results were shown in Figure 6.
  • the particle size of the isolated exosomes is 50nm to 200nm, which is consistent with the theoretical particle size of exosomes, and the shape is round or cup-shaped, with high integrity.
  • the same separation chip assembly was used to repeat the separation and purification of four urine samples with different exosome concentrations by the same method, and the volume of each urine sample varied from 1 mL to 20 mL. Then, the protein content of the exosomes obtained by each separation was tested by a micro UV-Vis spectrophotometer, and the results were recorded in Figure 7. As shown in Figure 7, at each exosome concentration, the purified exosome content showed a linear increase trend with the increase of the urine sample volume, which indicated that when testing various volumes of urine samples or urine samples Under the condition that the liquid sample has various contents of exosomes, the separation and purification using the separation chip assembly of the embodiment of the present application all have high structural stability.
  • the separation chip assembly provided in this application is also used to separate and purify exosomes from other liquid samples, including plasma, cell culture medium, tear, saliva and cerebrospinal fluid (CSF). )Wait. As shown in Fig. 8, high concentrations of exosomes were isolated from these liquid samples, indicating that the separation chip assembly provided in this application is suitable for the separation and purification of various liquid samples. Moreover, the particle size of the isolated exosomes was also in the range of 50 nm to 200 nm.
  • UC ultracentrifugation
  • PEG polyethylene glycol
  • PS phosphatidylserine
  • SEC size exclusion chromatography
  • MA membrane affinity
  • uromodulin is a protein with high content in urine samples, which can be used to characterize the purity of exosomes after purification.
  • the present application uses the separation chip assembly (denoted as EXODUS in Figure 9) to separate and purify the exosomes obtained from the urine sample, which can simultaneously detect higher concentrations of exosomes
  • the four protein markers indicated that the purification yield was high; and the exosomes did not adsorb a large amount of uromodulin, indicating that the purification accuracy was high.
  • the time required for purification, exosome yield and exosome purity are the three-dimensional parameters used to characterize different isolation and purification methods.
  • the separation chip assembly of the embodiment (denoted as EXODUS in FIG. 10 ) requires a shorter time for separation and purification (reduced by 95%), The yield of exosomes was higher (526% higher) and the purity of exosomes was higher (259% higher), indicating that the separation chip assembly is more competitive than various commercially available exosome separation methods.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Water Supply & Treatment (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Separation Of Solids By Using Liquids Or Pneumatic Power (AREA)
  • Filtration Of Liquid (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

一种分离装置和分离方法，包括分离芯片组件(1)、真空系统(50)、变频模块(40)和控制器(60)。分离芯片组件(1)包括分离芯片(10)、第一振动件(20)和第二振动件(30)，分离芯片(10)包括样本池(13)、位于样本池(13)两侧的第一过滤膜(14)和第二过滤膜(16)、第一腔室(15)和第二腔室(17)。第一振动件(20)固定于第一过滤膜(14)和第二过滤膜(16)，第二振动件(30)固定于第一腔室(15)及第二腔室(17)的外表面。变频模块(40)用于控制真空系统(50)交替地在第一腔室(15)和第二腔室(17)中产生负压。控制器(60)用于在真空系统(50)停止在第一腔室(15)中产生负压时，控制第一振动件(20)和第二振动件(30)振动；还用于在真空系统(50)停止在第二腔室(17)中产生负压时，控制第一振动件(20)和第二振动件(30)振动。

Description

分离装置和分离方法 技术领域
本申请涉及生物技术领域，具体地，涉及一种用于分离液体样本中目标颗粒的分离装置和分离方法。
背景技术
外泌体(exosome)是由活细胞持续大量分泌的一种直径为30-150nm的双磷脂膜结构小囊泡，其作为细胞间通信交流的载体携带来源于母细胞的蛋白、核酸、代谢小分子等特异性组分。大量的研究表明，外泌体参与了肿瘤发展的多种事件，包括免疫逃逸、血管生成、肿瘤转移、肿瘤耐药等。外泌体能更早、持续地被癌细胞释放并进入患者血液循环系统，其脂质双层膜结构能够有效保护所携带的蛋白质和包裹的核酸类物质。外泌体广泛且稳定地存在于多种临床样本中，包括血液、尿液、腹水、组织液、眼泪、唾液和脑脊液等。其中血液和尿液中的外泌体数量多、临床取样容易。因此，外泌体被认为是体外诊断研究和肿瘤临床检测领域中的重点研究对象，有望在肿瘤早期诊断、肿瘤转移复发评估、肿瘤异质性评估、动态检测肿瘤发生发展和疗效、耐药突变检测、个性化用药等方面发挥巨大的临床价值。
目前，实现外泌体临床应用的主要障碍是分离过程中发生过滤膜堵孔现象，通量低、纯度低。
发明内容
有鉴于此，有必要提供一种能够解决上述问题的分离装置和分离方法。
本申请提供一种分离装置，用于从液体样本中分离提纯出目标颗粒。所述分离装置包括：
分离芯片组件，包括分离芯片、第一振动件和第二振动件，所述分离芯片包括样本池和位于所述样本池相对的两侧的第一过滤膜和第二过滤膜，所述第一过滤膜和所述第二过滤膜的孔径均小于所述目标颗粒的粒径，所述分离芯片还包括第一腔室和第二腔室，所述第一腔室通过所述第一过滤膜与所述样本池连通，所述第二腔室通过所述第二过滤膜与所述样本池连通，所述第一振动件固定于第一过滤膜和第二过滤膜，所述第二振动件固定于所述第一腔室的外表面以及所述第二腔室的外表面；
真空系统；
变频模块，通过所述真空系统分别与所述第一腔室和所述第二腔室连接，所述变频模块用于控制所述真空系统交替地在所述第一腔室和所述第二腔室中产生负压；以及
控制器，用于在所述真空系统停止在所述第一腔室中产生负压时，控制所述第一振动 件和所述第二振动件振动；还用于在所述真空系统停止在所述第二腔室中产生负压时，控制所述第一振动件和所述第二振动件振动，其中，所述第一振动件和所述第二振动件在振动时分别产生第一振动波和第二振动波，所述第一振动波的频率大于所述第二振动波的频率，所述第一振动波的振幅小于所述第二振动波的振幅。
在一些可能的实现方式中，所述第一振动波的振动频率为5000Hz-8000Hz，所述第二振动波的振动频率为100-500Hz。
在一些可能的实现方式中，所述第一振动波的振动频率等于所述第一过滤膜或所述第二过滤膜的共振频率。
在一些可能的实现方式中，所述第一振动件和所述第二振动件位于同一水平面上。
在一些可能的实现方式中，所述第一振动件为谐波振荡器，所述第二振动件为振动马达。
本申请还提供一种分离方法，用于从液体样本中分离提纯出目标颗粒。所述方法包括：
提供上述分离装置，并向所述样本池提供液体样本；
在所述第一腔室产生负压，从而使所述样本池内的液体样本中粒径小于所述第一过滤膜的孔径的组分在负压作用下进入所述第一腔室；
停止在所述第一腔室中产生负压，并控制所述第一振动件和所述第二振动件振动；
在所述第二腔室中产生负压，从而使所述样本池内的液体样本中粒径小于所述第二过滤膜的孔径的组分在负压作用下进入所述第二腔室；以及
停止在所述第二腔室中产生负压，并控制所述第一振动件和所述第二振动件振动，其中，所述第一振动件和所述第二振动件在振动时分别产生第一振动波和第二振动波，所述第一振动波的频率大于所述第二振动波的频率，所述第一振动波的振幅小于所述第二振动波的振幅。
在一些可能的实现方式中，所述第一振动波的振动频率为5000Hz-8000Hz，所述第二振动波的振动频率为100-500Hz。
在一些可能的实现方式中，所述第一振动波的振动频率等于所述第一过滤膜或所述第二过滤膜的共振频率。
在一些可能的实现方式中，所述第一振动件和所述第二振动件位于同一水平面上。
在一些可能的实现方式中，所述第一振动件为谐波振荡器，所述第二振动件为振动马达。
相较于现有技术，本申请在真空系统停止在第一腔室/第二腔室中产生负压时，控制第一振动件和第二振动件振动，第一振动件向第一过滤膜和第二过滤膜传递第一振动波，使吸附于滤膜的膜孔中的目标颗粒能够快速与过滤膜的膜孔分离并重新悬浮于回流的液体样本中；第二振动件产生的第二振动波和第一振动波共同扰动液体样本和过滤膜并产生声学效应，防止目标颗粒堵塞滤孔或者聚集在一起，实现高效分离。
附图说明
图1为本申请实施例所提供的分离芯片组件的结构示意图。
图2为向图1所示的分离芯片组件上所施加负压的示意图。
图3为向图1所示的分离芯片组件上施加的第一振动波和第二振动波的示意图。
图4为本申请实施例所提供的分离装置的功能模块示意图。
图5为对本申请实施例与对比例1-4得到的外泌体的含量随时间的变化趋势图。
图6为本申请实施例得到的外泌体的扫描电镜图。
图7为本申请对不同体积和外泌体浓度的尿液样本进行分离提纯后得到的外泌体的含量测试图。
图8为本申请对不同类型的液体样本进行分离提纯后得到的外泌体的含量测试图。
图9为对本申请实施例和对比例5得到的外泌体进行蛋白质印迹分析得到的图谱。
图10为本申请实施例和对比例5在提纯时间、外泌体产量和外泌体浓度三个维度的比较示意图。
主要元件符号说明
分离芯片组件 1
分离芯片 10
第一侧盖片 11
第二侧盖片 12
样本池 13
第一过滤膜 14
第一腔室 15
第二过滤膜 16
第二腔室 17
加样室 18
第一振动件 20
第二振动件 30
变频模块 40
真空系统 50
控制器 60
分离装置 100
第一盖片本体 110
第一挡片 111
第二挡片 112
第二盖片本体 120
第三挡片 121
第四挡片 122
加样口 131
第一开口 152
第二开口 172
变频器 410
控制阀 420
第一真空泵 510
第二真空泵 520
如下具体实施方式将结合上述附图进一步说明本申请。
具体实施方式
下面将结合本申请的优选实施方式及实施例对本申请的技术方案进行描述。需要说明的是，当一个单元被描述为“连接”于另一个单元，它可以是直接连接到另一个单元或者可能同时存在居中单元。当一个单元被描述为“设置于”另一个单元，它可以是直接设置在另一个单元上或者可能同时存在居中单元。除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。在本申请的说明书中所 使用的元件或设备的名称只是为了描述具体的实施例的目的，不是旨在于限制本申请。
请参阅图1，本申请实施例提供一种分离芯片组件1，用于对液体样本中不同尺寸的颗粒进行分离提纯，以得到特定尺寸的目标颗粒。该液体样本可为人体血浆、血清、脑髓液、唾液、尿液以及胃液等。目标颗粒可为外泌体或循环肿瘤细胞等。分离芯片组件1包括分离芯片10、第一振动件20和第二振动件30。
分离芯片10包括样本池13以及位于样本池13相对的两侧的第一过滤膜14和第二过滤膜16。样本池13用于容置液体样本。第一过滤膜14和第二过滤膜16的孔径均小于目标颗粒的粒径。
进一步地，分离芯片10还可包括第一腔室15和第二腔室17，第一腔室15与样本池13通过第一过滤膜14相连通，第一腔室15设置有第一开口152，第一开口152连通第一腔室15与外界。第二腔室17与样本池13通过第二过滤膜16相连通，第二腔室17设置有第二开口172，第二开口172连通第二腔室17与外界。其中，第一腔室15与第二腔室17分别位于该样本池13相对的两侧。
使用分离芯片组件1时，将液体样本加入样本池13，将第一开口152和第二开口172分别与真空系统50(参图4)相连接。当真空系统50通过第一开口152使第一腔室15受到抽吸时，第一腔室15中产生负压。在第一腔室15的负压作用下，样本池13中的液体样本中尺寸小于第一过滤膜14的过滤孔径的组分(包括小颗粒和液体组分)朝向第一过滤膜14移动，经由第一过滤膜14流入第一腔室15。当真空系统50通过第二开口172使第二腔室17受到抽吸时，第二腔室17中产生负压。在第二腔室17的负压作用下，样本池13中的液体样本中尺寸小于第二过滤膜16的过滤孔径的组分朝向第二过滤膜16移动，经由第二过滤膜16流入第二腔室17，与此同时，样本池13中的液体样本在第一过滤膜14处会产生回流现象，从而减少或移除粘附于第一过滤膜14的组分，避免过滤分离过程中过滤膜被堵塞的情况发生。反复交替使第一腔室15和第二腔室17内产生负压，可以有效地使液体样本反复交替地流过第一过滤膜14和第二过滤膜16，使液体样本中尺寸大于第一过滤膜14和第二过滤膜16孔径的组分(即目标颗粒)留在样本池13中。分离芯片10的结构设计使吸附于第一过滤膜14和第二过滤膜16表面的组分在反复交替的负压变化中易于从滤膜表面脱落，可以有效地防止滤膜的膜孔被堵塞。
请一并参照图2，在一实施例中，该真空系统50交替地在该第一腔室15以及该第二腔室17内产生的负压(Negative pressure,NP)形成周期的梯形脉冲信号。矩形脉冲信号的幅度为10V pp，频率为5000Hz至7000Hz。其中，梯形脉冲信号可防止负压方向突然改变对第一过滤膜14以及第二过滤膜16的损坏。在一实施例中，鉴于血浆样本中蛋白含量较多，为了进一步避免过滤膜堵塞现象，可以在其中一腔室内产生负压的同时在另一腔室内产生正压(Air pressure,AP)，加强过滤膜处的回流现象。
如图1所示，第一振动件20的数量为两个。其中一第一振动件20固定于第一过滤膜14远离第二过滤膜16的表面。另一第一振动件20固定于第二过滤膜16远离第一过滤膜14的表 面。第一振动件20用于在振动时产生横向的第一振动波，然后向第一过滤膜14和第二过滤膜16传递第一振动波，从而带动第一过滤膜14和第二过滤膜16高频振动。因此，吸附于滤膜的膜孔中的目标颗粒能够快速与过滤膜的膜孔分离并重新悬浮于回流的液体样本中，从而进一步防止滤膜的膜孔被堵塞，实现高效分离。
第二振动件30的数量为两个。其中一第二振动件30固定于第一腔室15的外表面。另一第二振动件30固定于第二腔室17的外表面。第二振动件30用于在在振动时产生横向的第二振动波。请一并参阅图3，其中，第一振动波的频率大于第二振动波的频率，第一振动波的振幅小于第二振动波的振幅。第二振动波通过第一腔室15和第二腔室17向整个分离芯片10传递，使分离芯片10低频振动。第二振动波能够与第一振动波共同扰动液体样本和过滤膜并产生声流效应(acoustic streaming)，防止目标颗粒堵塞滤孔或者聚集在一起，提高分离提纯效率。在一实施方式中，第一振动件20可为谐波振荡器，第二振动件30可为振动马达。
在一实施方式中，第一振动波的振动频率为5000Hz-8000Hz，第二振动波的振动频率为100-500Hz。该振动频率下的第一振动波和第二振动波均不会对目标颗粒造成损坏。优选的，第一振动波的振动频率可与第一过滤膜14或第二过滤膜16的共振频率大致相同，此时，第一过滤膜14或第二过滤膜16可以更大的振幅振动，便于吸附于过滤膜上的目标颗粒更快速地分离下来。
在一实施方式中，第一振动件20和第二振动件30位于同一水平面上。即，第一振动波与第二振动波朝向同一方向，使得第一振动波和第二振动波可以相互叠加，形成协同振动。
在一实施方式中，分离芯片10包括第一侧盖片11和第二侧盖片12。第一侧盖片11包括第一盖片本体110以及位于第一盖片本体110相对两侧的第一挡片111和第二挡片112，第一过滤膜14固定于第一挡片111和第二挡片112之间且与第一盖片本体110相对。第一盖片本体110、第一挡片111、第二挡片112和第一过滤膜14共同围设出第一腔室15。第二侧盖片12包括第二盖片本体120以及位于第二盖片本体120相对两侧的第三挡片121和第四挡片122，第三挡片121与第一挡片111相对，第四挡片122与第二挡片112相对，第二过滤膜16固定于第三挡片121和第四挡片122之间且与第二盖片本体120相对，第二盖片本体120、第三挡片121、第四挡片122和第二过滤膜16共同围设出第二腔室17。样本池13位于第一过滤膜14和第二过滤膜16之间。其中，第二振动件30固定于第一盖片本体110或第二盖片本体120的外表面。
进一步地，第一挡片111和第三挡片121相距设置以限定出与样本池13连通的加样口131。分离芯片10还包括加样室18，加样室18与样本池13通过加样口131连通。操作时，将液体样本加入加样室18中，加样口131用于使得加样室18内的液体样本能够流出加样室18并进入样本池13。
请参阅图4，本申请实施方式进一步提供了一种分离装置100，包括如上的分离芯片组件1、真空系统50和变频模块40和控制器60。
真空系统50用于分别使分离芯片组件1的第一腔室15和第二腔室17产生负压。真空系 统50可以是两个独立的真空系统，也可以是经过设计的一个真空系统。真空系统50也可以包括微型真空泵或微型抽气泵等设备。可以理解的是，真空系统50与分离芯片10之间可以通过气密性较佳的管道连接。在一实施方式中，真空系统50包括第一真空泵510和第二真空泵520，第一真空泵510与分离芯片10的第一开口152相连接，第二真空泵520与分离芯片10的第二开口172相连接。
变频模块40与真空系统50电连接，变频模块40可以控制提供给真空系统50的电源电压，从而使第一腔室15和第二腔室17内交替产生负压。在一实施方式中，变频模块40包括变频器410以及与变频器410连接的控制阀420。控制阀420可以是液路转换器，包括但不仅限于电磁阀、旋转阀。控制阀420分别与第一真空泵510以及第二真空泵520中的其中一个连通，从而使第一真空泵510和第二真空泵520反复交替工作。例如，将控制阀420连通第一真空泵510，使得变频器410控制第一真空泵510运行，通过第一开口152抽气使第一腔室15内产生负压，样本池13中的液体样本中的液体和尺寸小于第一过滤膜14孔径的组分在负压作用下通过第一过滤膜14，进入第一腔室15；然后，变频器410控制第一真空泵510停止运行；之后，将控制阀420切换至与第二真空泵520连通，使得变频器410控制第二真空泵520运行，通过第二开口172抽气使第二腔室17内产生负压，使得样本池13中的液体样本中的液体和尺寸小于第二过滤膜16孔径的组分在负压作用下通过第二过滤膜16，进入第二腔室17；再之后，变频器410控制第二真空泵520停止运行；反复上述步骤多次。
控制器60用于在第一腔室15停止抽吸时，控制第一振动件20和第二振动件30振动，从而分别产生第一振动波和第二振动波。控制器60还用于在第二腔室17停止抽吸时，控制第一振动件20和第二振动件30振动，从而分别产生第一振动波和第二振动波。其中，控制器60可与第一真空泵510和第二真空泵520电性连接，当第一真空泵510或第二真空泵520停止运行时，控制器60即判断第一真空泵510在第一腔室15停止抽吸，或判断第二真空泵520在第二腔室17停止抽吸，此时便可通知对应的第一振动件20和第二振动件30开始振动。
本申请实施例进一步提供一种应用于上述分离芯片组件1的分离液体样本中目标颗粒的方法，其包括如下步骤：
步骤S1，提供本申请的分离芯片组件1，向该分离芯片组件1的样本池13中提供液体样本。
步骤S2，通过第一开口152抽吸第一腔室15，使第一腔室15内产生负压。
其中，在进行抽吸之前，将第一开口152、第二开口172分别与分离装置100的真空系统50相连。如此，真空系统50通过第一开口152抽吸第一腔室15，使第一腔室15内产生负压。样本池13中的液体样本中的液体和尺寸小于第一过滤膜14孔径的组分在负压作用下朝向第一过滤膜14移动，经由第一过滤膜14进入第一腔室15。
步骤S3，停止抽吸第一腔室15，控制第一振动件20和第二振动件30振动，从而分别产生第一振动波和第二振动波。同时，通过的第二开口172抽吸第二腔室17使第二腔室17内产生负压。
其中，第一振动波带动第一过滤膜14高频振动，使吸附于滤膜的膜孔中的目标颗粒能够快速与过滤膜的膜孔分离并重新悬浮于回流的液体样本中。第二振动波可以防止目标颗粒聚集在一起。同时，真空系统50通过第二开口172抽吸第二腔室17，使第二腔室17内产生负压。粘附在第一过滤膜14表面的组分可以随着气流和/或液流样本池13中，样本池13中的液体样本中的液体和尺寸小于第二过滤膜16孔径的组分在负压作用下朝向第二过滤膜16移动，经由第二过滤膜16进入第二腔室17。
步骤S4，停止抽吸第二腔室17，控制第一振动件20和第二振动件30振动。
然后，步骤S2至步骤S4可循环多次，使液体样本中小于过滤膜孔径的组分被去除，大于过滤膜孔径的组分被截留在样本池13中，以实现更好的分离提纯效果。
以下将结合实施例和对比例对本申请进行具体描述。
实施例
使用本申请提供的分离芯片组件对2mL的尿液样本进行外泌体分离提纯，第一振动件的频率为6250Hz(与过滤膜的共振频率大致相同)，第二振动件的频率为200Hz。
对比例1
使用分离芯片组件对2mL的尿液样本进行外泌体分离提纯，与实施例的分离芯片组件不同之处在于，对比例1的分离芯片未含有第一振动件和第二振动件。
对比例2
使用分离芯片对2mL的尿液样本进行外泌体分离提纯，与实施例的分离芯片组件不同之处在于，对比例2的分离芯片仅含有第二振动件(频率为200Hz)，未含有第一振动件。
对比例3
使用分离芯片对2mL的尿液样本进行外泌体分离提纯，与实施例的分离芯片组件不同之处在于，对比例3的分离芯片仅含有第一振动件(频率为6250Hz)，未含有第二振动件。
对比例4
使用死端过滤法(dead-end filtration)对2mL的尿液样本进行外泌体分离提纯，死端过滤采用与实施例的分离芯片相同的过滤膜，不同之处在于，将液体样本置于过滤膜的上游，在压力差的推动下，使液体组分和粒径小于膜孔的颗粒透过过滤膜。
对实施例和对比例1-4随时间推进得到的外泌体的含量进行测定，结果记录于图5中。如图5所示，实施例在10min内便可分离出将近30μm的外泌体，分离效率远大于对比例1-4。进一步对实施例得到的外泌体进行透射电镜测试(标尺刻度为250nm)，测试结果如图6所示。其中，分离得到的外泌体粒径在50nm至200nm，与外泌体的理论粒径相符，且形状为圆形或杯形，具有较高的完整性。
进一步地，使用同一个分离芯片组件并以相同的方法重复对四种具有不同外泌体浓度的尿液样本进行分离提纯，每一尿液样本的体积从1mL至20mL不等。然后，采用微量紫外-可见光分光光度计对每次分离得到的外泌体进行蛋白含量测试，结果记录于图7中。如图7所示，每一外泌体浓度下，提纯得到的外泌体含量均随尿液样本体积的增加而呈现线 性增加的趋势，这表明在测试各种不同体积的尿液样本或尿液样本具有各种不同含量的外泌体的情况下，使用本申请实施例的分离芯片组件进行分离提纯均具有较高的结构稳定性。此外，重复使用同一个分离芯片组件对体积为10mL的尿液样本进行分离提纯，重复二十遍之后发现得到的外泌体含量之间变异系数(CV)小于9.9％，表明使用该分离芯片组件进行分离提纯具有较高的可重复性。
进一步地，还采用本申请提供的分离芯片组件对其它液体样本进行外泌体分离提纯，包括血浆(plasma)、细胞培养液(culture medium)、泪液(tear)、唾液(saliva)和脑脊液(CSF)等。如图8所示，从这几种液体样本中均分离出较高浓度的外泌体，表明本申请提供的分离芯片组件适用于多种液体样本的分离提纯。而且，分离得到的外泌体粒径同样在50nm至200nm。
对比例5
使用传统的超速离心(UC)、聚乙二醇(PEG)沉淀、磷脂酰丝氨酸(PS)亲和、排阻色谱(SEC)、膜亲和(MA)等方法分别从同样的尿液样本中分离外泌体。
具体地，使用蛋白质印迹法对实施例和对比例5中各分离提纯方法得到的外泌体中的ALIX、CD63、TSG101、CD81蛋白标志物进行测试。其中，尿调节素(UMOD)是尿液样本中含量较高的蛋白，可用于表征外泌体提纯后的纯度。如图9所示，相对于传统的分离提纯方法，本申请使用分离芯片组件(在图9中记为EXODUS)对尿液样本分离提纯后得到的外泌体中能够同时检测到较高浓度的四种蛋白标志物，表明提纯产量较高；而且外泌体未吸附有大量的尿调节素，表明提纯精度较高。
其中，提纯所需时间、外泌体产量和外泌体纯度是用于表征不同分离提纯方法的三个维度的参数。如图10所示，相较于对比例5中各种传统的分离提纯方法，实施例的分离芯片组件(在图10中记为EXODUS)进行分离提纯所需时间较短(减少95％)、外泌体产量较高(提高526％)、外泌体纯度较高(提高259％)，表明该分离芯片组件相较于市售的各种外泌体分离手段具有较强的竞争力。
上述实施例为本申请较佳的实施方式，但本申请的实施方式并不受上述实施例的限制，以上实施方式仅是用于解释权利要求书。然本申请的保护范围并不局限于说明书。任何熟悉本技术领域的技术人员在本申请披露的技术范围内，可轻易想到的变化或者替换，都包含在本申请的保护范围之内。

Claims (10)

  1. 一种分离装置，用于从液体样本中分离提纯出目标颗粒，其特征在于，所述分离装置包括：
    分离芯片组件，包括分离芯片、第一振动件和第二振动件，所述分离芯片包括样本池和位于所述样本池相对的两侧的第一过滤膜和第二过滤膜，所述第一过滤膜和所述第二过滤膜的孔径均小于所述目标颗粒的粒径，所述分离芯片还包括第一腔室和第二腔室，所述第一腔室通过所述第一过滤膜与所述样本池连通，所述第二腔室通过所述第二过滤膜与所述样本池连通，所述第一振动件固定于第一过滤膜和第二过滤膜，所述第二振动件固定于所述第一腔室的外表面以及所述第二腔室的外表面；
    真空系统；
    变频模块，通过所述真空系统分别与所述第一腔室和所述第二腔室连接，所述变频模块用于控制所述真空系统交替地在所述第一腔室和所述第二腔室中产生负压；以及
    控制器，用于在所述真空系统停止在所述第一腔室中产生负压时，控制所述第一振动件和所述第二振动件振动；还用于在所述真空系统停止在所述第二腔室中产生负压时，控制所述第一振动件和所述第二振动件振动，其中，所述第一振动件和所述第二振动件在振动时分别产生第一振动波和第二振动波，所述第一振动波的频率大于所述第二振动波的频率，所述第一振动波的振幅小于所述第二振动波的振幅。
  2. 如权利要求1所述的分离装置，其特征在于，所述第一振动波的振动频率为5000Hz-8000Hz，所述第二振动波的振动频率为100-500Hz。
  3. 如权利要求2所述的分离装置，其特征在于，所述第一振动波的振动频率等于所述第一过滤膜或所述第二过滤膜的共振频率。
  4. 如权利要求1所述的分离装置，其特征在于，所述第一振动件和所述第二振动件位于同一水平面上。
  5. 如权利要求1所述的分离装置，其特征在于，所述第一振动件为谐波振荡器，所述第二振动件为振动马达。
  6. 一种分离方法，用于从液体样本中分离提纯出目标颗粒，其特征在于，所述方法包括：
    提供如权利要求1所述的分离装置，并向所述样本池提供液体样本；
    在所述第一腔室产生负压，从而使所述样本池内的液体样本中粒径小于所述第一过滤膜的孔径的组分在负压作用下进入所述第一腔室；
    停止在所述第一腔室中产生负压，并控制所述第一振动件和所述第二振动件振动；
    在所述第二腔室中产生负压，从而使所述样本池内的液体样本中粒径小于所述第二过滤膜的孔径的组分在负压作用下进入所述第二腔室；以及
    停止在所述第二腔室中产生负压，并控制所述第一振动件和所述第二振动件振动，其 中，所述第一振动件和所述第二振动件在振动时分别产生第一振动波和第二振动波，所述第一振动波的频率大于所述第二振动波的频率，所述第一振动波的振幅小于所述第二振动波的振幅。
  7. 如权利要求6所述的分离方法，其特征在于，所述第一振动波的振动频率为5000Hz-8000Hz，所述第二振动波的振动频率为100-500Hz。
  8. 如权利要求7所述的分离方法，其特征在于，所述第一振动波的振动频率等于所述第一过滤膜或所述第二过滤膜的共振频率。
  9. 如权利要求6所述的分离方法，其特征在于，所述第一振动件和所述第二振动件位于同一水平面上。
  10. 如权利要求6所述的分离方法，其特征在于，所述第一振动件为谐波振荡器，所述第二振动件为振动马达。
PCT/CN2020/128177 2020-11-11 2020-11-11 分离装置和分离方法 Ceased WO2022099518A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
PCT/CN2020/128177 WO2022099518A1 (zh) 2020-11-11 2020-11-11 分离装置和分离方法
EP20954324.8A EP4246118A4 (en) 2020-11-11 2020-11-11 SEPARATION APPARATUS AND METHOD
JP2022522396A JP7397529B2 (ja) 2020-11-11 2020-11-11 分離装置および分離方法
KR1020237000065A KR102878476B1 (ko) 2020-11-11 2020-11-11 분리 장치 및 분리 방법
CN202080003487.2A CN113195080A (zh) 2020-11-11 2020-11-11 分离装置和分离方法
US17/369,836 US12196657B2 (en) 2020-11-11 2021-07-07 Isolation device and isolation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/128177 WO2022099518A1 (zh) 2020-11-11 2020-11-11 分离装置和分离方法

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/369,836 Continuation-In-Part US12196657B2 (en) 2020-11-11 2021-07-07 Isolation device and isolation method

Publications (1)

Publication Number Publication Date
WO2022099518A1 true WO2022099518A1 (zh) 2022-05-19

Family

ID=76973868

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/128177 Ceased WO2022099518A1 (zh) 2020-11-11 2020-11-11 分离装置和分离方法

Country Status (6)

Country Link
US (1) US12196657B2 (zh)
EP (1) EP4246118A4 (zh)
JP (1) JP7397529B2 (zh)
KR (1) KR102878476B1 (zh)
CN (1) CN113195080A (zh)
WO (1) WO2022099518A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114225698B (zh) * 2021-12-31 2025-01-28 深圳汇芯生物医疗科技有限公司 分离装置和分离方法
CN114377549B (zh) * 2022-01-18 2023-04-11 深圳汇芯生物医疗科技有限公司 自动化分离设备及分离液体样本中目标颗粒的方法
CN114377550A (zh) * 2022-01-18 2022-04-22 深圳汇芯生物医疗科技有限公司 一种过滤设备
US20240151614A1 (en) * 2022-01-28 2024-05-09 Shenzhen Huixin Life Technologies Co., Ltd Isolation device and isolation method
CN116083214A (zh) * 2023-03-21 2023-05-09 深圳汇芯生物医疗科技有限公司 洗脱结构及分离装置

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020183677A1 (en) * 2000-02-02 2002-12-05 Chang Yu-An Apparatus for enhanced plasmapheresis and methods thereof
CN103007759A (zh) * 2012-12-03 2013-04-03 北京清大国华环保科技有限公司 超声震动型卷式超滤膜组器及其超滤装置
CN107576555A (zh) * 2017-06-02 2018-01-12 温州汇芯生物科技有限公司 分离收集液体样本中目标微粒的装置及方法
CN108126522A (zh) * 2017-12-21 2018-06-08 深圳汇芯生物医疗科技有限公司 分离芯片、分离装置及分离液体样本中目标颗粒的方法
CN207478341U (zh) * 2017-10-26 2018-06-12 河南华禹环保科技有限公司 超声波卷式膜组件清洗系统
CN109439533A (zh) * 2017-11-30 2019-03-08 深圳汇芯生物医疗科技有限公司 分离装置、分离控制系统及分离方法
CN211784638U (zh) * 2019-09-16 2020-10-27 深圳汇芯生物医疗科技有限公司 分离芯片组件和分离装置

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU8129798A (en) * 1997-07-11 1999-02-08 Kabushiki Kaisha Yokota Seisakusho Filter
KR100733529B1 (ko) * 2003-06-17 2007-06-29 아사히 가세이 케미칼즈 가부시키가이샤 막 카트리지, 막 분리 장치 및 막 분리 방법
US20120000851A1 (en) * 2010-02-04 2012-01-05 Dxv Water Technologies, Llc Water treatment systems and methods
GR1008540B (el) * 2014-06-27 2015-07-28 Κωνσταντινος Χατζηκωνσταντινου Χρηση υψηλης συχνοτητας μηχανικης δονησης για την βελτιωση λειτουργιας διεργασιων διαχωρισμου ρευστων με μεμβρανες
GB2529179A (en) * 2014-08-12 2016-02-17 Membrane Recovery Ltd Forward osmotic and water hammer method of membrane cleaning
US10758867B2 (en) * 2017-11-30 2020-09-01 Wellsim Biomedical Technologies, Inc Isolation device and isolation method
CN107917835A (zh) * 2017-12-30 2018-04-17 广州阳普医疗科技股份有限公司 一种筛选循环肿瘤细胞的采血装置
KR102101082B1 (ko) * 2018-08-02 2020-04-16 경남대학교 산학협력단 막 파울링 저감 시스템 및 이를 이용한 막 파울링 저감 방법
JP7111355B2 (ja) 2018-10-17 2022-08-02 株式会社アクト 排水処理装置
WO2020202200A1 (en) * 2019-03-29 2020-10-08 S Nikhil Das Apparatus and method for reducing concentration polarization and membrane fouling on membrane surface in a filter unit
CN111870258B (zh) * 2020-07-31 2023-07-21 武汉轻工大学 动物血清高效分离针

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020183677A1 (en) * 2000-02-02 2002-12-05 Chang Yu-An Apparatus for enhanced plasmapheresis and methods thereof
CN103007759A (zh) * 2012-12-03 2013-04-03 北京清大国华环保科技有限公司 超声震动型卷式超滤膜组器及其超滤装置
CN107576555A (zh) * 2017-06-02 2018-01-12 温州汇芯生物科技有限公司 分离收集液体样本中目标微粒的装置及方法
CN207478341U (zh) * 2017-10-26 2018-06-12 河南华禹环保科技有限公司 超声波卷式膜组件清洗系统
CN109439533A (zh) * 2017-11-30 2019-03-08 深圳汇芯生物医疗科技有限公司 分离装置、分离控制系统及分离方法
CN108126522A (zh) * 2017-12-21 2018-06-08 深圳汇芯生物医疗科技有限公司 分离芯片、分离装置及分离液体样本中目标颗粒的方法
CN211784638U (zh) * 2019-09-16 2020-10-27 深圳汇芯生物医疗科技有限公司 分离芯片组件和分离装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4246118A4 *

Also Published As

Publication number Publication date
KR20230096955A (ko) 2023-06-30
CN113195080A (zh) 2021-07-30
EP4246118A4 (en) 2024-07-10
US12196657B2 (en) 2025-01-14
US20220146384A1 (en) 2022-05-12
JP7397529B2 (ja) 2023-12-13
EP4246118A1 (en) 2023-09-20
KR102878476B1 (ko) 2025-10-31
JP2023506110A (ja) 2023-02-15

Similar Documents

Publication Publication Date Title
WO2022099518A1 (zh) 分离装置和分离方法
CN211784638U (zh) 分离芯片组件和分离装置
Aran et al. Microfiltration platform for continuous blood plasma protein extraction from whole blood during cardiac surgery
CN209555218U (zh) 分离芯片
US12253447B2 (en) Isolation method for isolating target particles from liquid samples
US20210353844A1 (en) Acoustophoresis device having improved dimensions
KR20150005624A (ko) 적혈구로부터의 지지 파티클의 음향영동 분리
KR101533230B1 (ko) 다단 미세유체 칩 및 이를 이용한 시료의 선택적 분리방법
JP2020056799A (ja) 生物学的なサンプルを濾過するための濾過セルおよび方法
JP2024507437A (ja) 分離装置及び分離方法
CN114225698B (zh) 分离装置和分离方法
CN108126522B (zh) 分离芯片、分离装置及分离液体样本中目标颗粒的方法
CN214764552U (zh) 分离芯片组件
Chernyshev et al. Filtration-based technologies for isolation, purification and analysis of extracellular vesicles
CN103111337A (zh) 一种用于研究声电场助滤动态过程的微流控实验装置
WO2022099517A1 (zh) 分离芯片组件
CN110669637A (zh) 基于微孔滤膜的真菌孢子分离装置及其系统和分离方法
CN216677745U (zh) 分离装置
CN113913367A (zh) 一种基于外泌体富集装置的外泌体分离富集方法
Faridi et al. Milliliter Scale Acoustophoresis Based Bioparticle Processing Platform
CN118345029A (zh) 一种干细胞外泌体提取方法
US20200340893A1 (en) Hydrogel Particle-Enabled Large Volume Fluid Processing Device and System

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2022522396

Country of ref document: JP

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20954324

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020954324

Country of ref document: EP

Effective date: 20230612