WO2021007825A1 - 分离装置及分离液体样本中目标颗粒的方法 - Google Patents

分离装置及分离液体样本中目标颗粒的方法 Download PDF

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Publication number
WO2021007825A1
WO2021007825A1 PCT/CN2019/096425 CN2019096425W WO2021007825A1 WO 2021007825 A1 WO2021007825 A1 WO 2021007825A1 CN 2019096425 W CN2019096425 W CN 2019096425W WO 2021007825 A1 WO2021007825 A1 WO 2021007825A1
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Prior art keywords
separation
opening
sample
vacuum pump
chamber
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PCT/CN2019/096425
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English (en)
French (fr)
Inventor
陈欲超
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深圳汇芯生物医疗科技有限公司
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Application filed by 深圳汇芯生物医疗科技有限公司 filed Critical 深圳汇芯生物医疗科技有限公司
Priority to EP19937743.3A priority Critical patent/EP4001914A4/en
Priority to CN201980002362.5A priority patent/CN112601959A/zh
Priority to PCT/CN2019/096425 priority patent/WO2021007825A1/zh
Priority to JP2022503474A priority patent/JP7312507B2/ja
Priority to KR1020227005184A priority patent/KR20220027262A/ko
Publication of WO2021007825A1 publication Critical patent/WO2021007825A1/zh
Priority to US17/576,654 priority patent/US11874209B2/en
Priority to US18/530,661 priority patent/US20240102900A1/en

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • 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
    • 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
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

  • the present invention relates to the field of biotechnology, in particular to a separation device and a method for separating target particles in a liquid sample.
  • Exosomes are small vesicles with a double phospholipid membrane structure of 30 to 150 nm that are continuously secreted by living cells. They are used as a carrier for communication between cells and carry proteins, nucleic acids, and metabolic small molecules derived from mother cells. Components. A large number of studies have shown that exosomes are involved in a variety of events in tumor development, including immune escape, angiogenesis, tumor metastasis, and tumor drug resistance. Exosomes can be released earlier and continuously by cancer cells and enter the patient's blood circulatory system. Its lipid bilayer membrane structure can effectively protect the proteins and nucleic acids that it carries.
  • Exosomes are widely and stably present in a variety of clinical samples, including blood, urine, ascites, tissue fluid, tears, saliva, and cerebrospinal fluid. Among them, the number of exosomes in blood and urine is large, and clinical sampling is easy. Therefore, exosomes are considered to be the key research objects in the field of in vitro diagnostic research and tumor clinical detection. They are expected to be used for early tumor diagnosis, tumor metastasis and recurrence evaluation, tumor heterogeneity evaluation, dynamic detection of tumor development and efficacy, and drug resistance mutations. It has great clinical value in testing and personalized medicine.
  • exosome purification technologies including ultracentrifugation, size exclusion chromatography, magnetic bead-based immunoaffinity capture, polyethylene glycol-based precipitation, ultrafiltration, and microfluidics.
  • the above purification methods have disadvantages: 1) low recovery rate, 2) low purity, 3) poor integrity of the isolated exosomes, 4) poor reproducibility, 5) may introduce unwanted impurities, 6) require biomarkers, 7) Time-consuming and labor-intensive and 8) High cost.
  • Ultracentrifugation is currently the most commonly used method for purification of exosomes, but it also has some limitations, such as low yield (recovery rate: 5% to 25%), cumbersome operation process, long time (>4 hours), and relying on expensive equipment .
  • isolation methods based on immunocapture can collect exosomes with medium to high purity, but these methods are limited by the specificity of antibodies and cumbersome operating steps, which are difficult to standardize and are not suitable for processing large numbers and large volumes of clinical samples.
  • exosomes separation methods based on microfluidic technology have also been reported many times, including hydrodynamic or acoustic separation, immunocapture and dielectrophoresis. However, it is still unable to solve the problems of low throughput, complex operation process and poor repeatability, and it is difficult to achieve the consistency of results between different laboratories.
  • the present invention provides a separation device capable of extracting high-purity exosomes quickly, stably and efficiently.
  • the present invention provides a separation device for separating and purifying target particles from multiple liquid samples.
  • the separation device includes:
  • each separation chip includes:
  • a sample pool for accommodating the liquid sample for accommodating the liquid sample
  • the first opening and the second opening are located on both sides of the sample pool
  • Vacuum system including:
  • a first vacuum pump connected to the first opening of each separation chip, the first vacuum pump generates a negative pressure in the separation chip through the first opening to separate target particles in the liquid sample in the sample pool;
  • a second vacuum pump is connected to the second opening of each separation chip, and the second vacuum pump generates a negative pressure in the separation chip through the second opening to separate target particles in the liquid sample in the sample pool.
  • the present invention also provides a method for separating target particles in a liquid sample by applying the separation device, and the method includes:
  • the separation device has the characteristic of efficiently purifying exosomes from large-volume biological samples.
  • the advantages of the separation device include: 1) high throughput (parallel processing of multiple liquid samples is achieved by setting the separation chip in parallel); 2) automated processing; 3) simple and standardized operation; 4) high yield and purity; 5) Label-free; 6) cost-effective; 7) high stability and reproducibility; 8) can process a variety of different biological samples, including plasma, urine, brain spinal fluid, saliva, tears, emulsion and cell culture fluid.
  • FIG. 1 is a schematic diagram of the structure of a separation chip provided by an embodiment of the present invention.
  • FIG. 2 is a schematic diagram of disassembly of the structure of the separation chip shown in FIG. 1.
  • Fig. 3 is a schematic diagram of the filtration rate of exosomes separation using the separation chip shown in Fig. 1 and the traditional ultrafiltration method respectively.
  • Fig. 4 is a schematic diagram of functional modules of a separation device provided by an embodiment of the present invention.
  • Fig. 5A is a schematic diagram of the liquid path of the separation device shown in Fig. 4.
  • Fig. 5B is a program block diagram of the separation control system of the separation device shown in Fig. 4.
  • Fig. 6 is a schematic diagram of the negative pressure applied to the separation chip in an embodiment of the present invention.
  • Fig. 7 is a working principle diagram of the sampling module of the separation device shown in Fig. 4.
  • First support 1 Second support 2 First shaft 3 Second shaft 4 First preset position 5 Second preset position 6 Third shaft 7 Sample pool 11 Cover sheet on the first side 111 First cover 1111 Second cover 1112 Second side cover 112 The third cover part 1121 The fourth cover part 1122 Sample cell opening 113 First bump 12 First card slot 121 Second bump 13 Third card slot 131 Chip substrate 14 First filter membrane 15 First chamber 16
  • an element when an element is referred to as being “fixed to” another element, it may be directly on the other element or a central element may also exist.
  • an element When an element is considered to be “connected” to another element, it can be directly connected to the other element or an intermediate element may be present at the same time.
  • an element When an element is considered to be “disposed on” another element, it can be directly disposed on the other element or a centered element may exist at the same time.
  • FIG. 1 is a schematic structural diagram of a separation chip 10 provided by an embodiment of the present invention. As shown in FIG. 1 and referring to FIG. 2 at the same time, the separation chip 10 includes a sample pool 11, a first chamber 16 and a second chamber 18. The first chamber 16 and the second chamber 18 are located on opposite sides of the sample cell 11 respectively.
  • the sample pool 11 includes a first side cover piece 111 and a second side cover piece 112, and the first side cover piece 111 and the second side cover piece 112 are disposed on opposite sides of the sample pool 11.
  • the first side cover piece 111 is provided with a first bump 12, and the first bump 12 divides the first side cover piece 111 into a first cover portion 1111 located on one side of the first bump 12 and The second cover portion 1112 on the other side of the first bump 12.
  • the second side cover 112 is provided with a second bump 13 opposite to the first bump 12, and the second bump 13 divides the second side cover 112 into one side of the second bump 13
  • the third cover portion 1121 and the fourth cover portion 1122 on the other side of the second bump 13.
  • the first cover portion 1111, the third cover portion 1121, the first bump 12 and the second bump 13 jointly surround the sample pool 11.
  • the top of the sample pool 11 includes a sample pool opening 113 for adding and/or removing liquid samples.
  • a chip substrate 14 is provided on the bottom of the first side cover 111 opposite to the first bump 12.
  • a first filter film 15 is provided between the first bump 12 and the chip base 14, and the first filter film 15 is opposite to the second cover part 1112.
  • the second cover part 1112 and the first filter The film 15 and the chip substrate 14 jointly surround the first cavity 16.
  • the first chamber 16 is communicated with the sample pool 11 through the first filter membrane 15.
  • the first chamber 16 is provided with a first opening 161 for communicating the first chamber 16 with the outside.
  • Another chip substrate 14 is provided at the bottom of the second side cover 112 and opposite to the second bump 13.
  • a second filter film 17 is provided between the second bump 13 and the chip substrate 14, and the second filter film 17 is opposite to the fourth cover sheet portion 1122.
  • the fourth cover portion 1122, the second filter membrane 17 and the chip substrate 14 jointly surround the second cavity 18.
  • the second chamber 18 communicates with the sample cell 11 through the second filter membrane 17, and the second chamber 18 is provided with a second opening 181 for allowing the second chamber 18 to communicate with the outside world. Connected.
  • a gap (not shown in the figure) is provided between the first bump 12 and the second bump 13, and the gap is used to allow the liquid sample in the sample cell 11 to flow out of the sample cell 11, and Enter the first chamber 16 or the second chamber 18 through the first filter membrane 15 or the second filter membrane 17 respectively.
  • the first bump 12 defines a first slot 121 at a side facing the chip substrate 14, a second slot 121 (not shown in the figure) is formed at a corresponding position of the chip substrate, and the first filter membrane 15 is clamped and fixed between the first slot 121 and the second slot.
  • the second bump 13 defines a third slot 131 on the side facing the chip substrate 14, a fourth slot (not shown in the figure) is opened at the corresponding position of the chip substrate, and the second filter film 17 It is clamped and fixed between the third slot 131 and the fourth slot.
  • the separation chip 10 has a symmetrical structure. It should be noted that the separation chip 10 may also be an asymmetric structure or any other structure that can realize the concept of the present invention.
  • a liquid sample is added to the sample cell 11, and the first opening 161 and the second opening 181 are respectively connected to the vacuum system 30 (refer to FIG. 4).
  • the vacuum system 30 causes the first chamber 16 to be sucked through the first opening 161, a negative pressure is generated in the first chamber 16.
  • components of the liquid sample in the sample pool 11 whose size is smaller than the filter pore size of the first filter membrane 15 flow into the first chamber 16 through the first filter membrane 15.
  • the second chamber 18 is sucked by the vacuum system 30 through the second opening 181, a negative pressure is generated in the second chamber 18.
  • the vacuum system 30 may be two negative pressure pumps, one of which is connected to the first opening 161, the other is connected to the second opening 181, and two negative pressure pumps Alternately provide negative pressure. Repeatedly alternating to generate negative pressure in the first chamber 16 and the second chamber 18 can effectively cause the liquid sample to alternately flow through the first filter membrane 15 and the second filter membrane 17, so that the size of the liquid sample is larger than The components of the pore size of the first filter membrane 15 and the second filter membrane 17 remain in the sample pool 11.
  • the structure design of the separation chip 10 makes the components adsorbed on the surface of the first filter membrane 15 and the second filter membrane 17 easily fall off from the surface of the filter membrane during the repeated alternating negative pressure changes, which can effectively prevent the membrane pores of the filter membrane from being Blocked.
  • the separation chip 10 can process 20 mL of urine and cell culture fluid, far exceeding the traditional ultrafiltration method (3 mL).
  • the main body parts of the sample pool 11, the first chamber 16, and the second chamber 18 of the separation chip 10 may be made of plastic, glass, metal or composite materials.
  • the main parts of the sample pool 11, the first chamber 16, and the second chamber 18 of the separation chip 10 are made of transparent materials such as polyethyleneimine (PEI) or polymethylmethacrylate (PMMA). production.
  • the processing method of the separated chip 10 includes but is not limited to processing molding and injection molding.
  • the first filter membrane 15 and the second filter membrane 17 can be made of the same membrane material, or can be made of different membrane materials.
  • the first filtration membrane 15 and the second filtration membrane 17 may have the same average filtration membrane pore size and/or pore size distribution, or may have different average filtration membrane pore size and/or pore size distribution.
  • the first filter membrane 15 (or the second filter membrane 17) can be made of one kind of membrane material, or can be made of multiple membrane materials.
  • the first filter membrane 15 and the second filter membrane 17 may be porous materials, including but not limited to porous ceramic materials, porous plastic materials, and porous metal materials.
  • the first filter membrane 15 and the second filter membrane 17 can be selected from anodized aluminum membrane (AAO), polycarbonate membrane, cellulose acetate membrane, polyethylene membrane, polypropylene membrane, and polystyrene membrane, respectively. One or more of them. More specifically, in view of the higher porosity and uniform pore size of the anodized aluminum oxide membrane, the first filter membrane 15 and the second filter membrane 17 adopt anodized aluminum oxide membranes.
  • the pore size of the first filter membrane 15 and the second filter membrane 17 can be designed according to the type of the liquid sample and the target particles.
  • the pore size of the first filter membrane 15 and the second filter membrane 17 is 20 nm, which is slightly smaller than the size of exosomes (30-150 nm), and can be used to separate and purify cells that have passed through the 200 nm filter membrane. Exosomes in a medium sample.
  • FIG. 4 shows a schematic diagram of program modules of the separation device 100.
  • the separation device 100 includes a main module 101, an auxiliary module 102, and a human-computer interaction module 103.
  • the main module 101 is used to separate and purify multiple liquid samples at the same time, so as to improve the efficiency of purification.
  • the main module 101 includes a first support table 1, a second support table 2, a liquid supply unit 20, a vacuum system 30, a frequency conversion module 40 and a liquid collection unit 50.
  • FIG. 7 shows the working principle of the sampling module of the separation device 100, and the first support 1 includes a plurality of separation chips 10 as described above.
  • the number of the separation chips 10 in the first support platform 1 is twelve.
  • the first supporting table 1 can rotate around a first rotating shaft 3 under the action of a driving member (such as a motor, not shown), for example, can include clockwise rotation or counterclockwise rotation, thereby driving the first supporting table 1
  • a driving member such as a motor, not shown
  • Each of the separated chips 10 is rotated to move each of the separated chips 10 to the first preset position 5 respectively.
  • the first preset position 5 is the sampling position.
  • a plurality of the separated chips 10 are arranged around the first supporting table 1 with the first rotating shaft 3 of the first supporting table 1 as a center. It can be understood that the number of the separated chips 10 in the first support platform 1 can be increased or decreased accordingly as needed.
  • the second supporting table 2 includes a plurality of containers 21.
  • the number of the containers 21 is the same as the number of the separation chips 10, which is also twelve.
  • the second supporting table 2 can rotate around a second rotating shaft 4 under the action of a driving member (such as a motor, not shown), for example, it can include clockwise rotation or counterclockwise rotation, thereby driving the second supporting table 2
  • a driving member such as a motor, not shown
  • Each container 21 is rotated to move each container 21 to the second preset position 6 respectively.
  • a plurality of the containers 21 are arranged on the second supporting table 2 around the second rotating shaft 4 of the second supporting table 2 as a center.
  • the liquid supply unit 20 includes a sample chamber 210 to be tested, a first control valve 220 and a cleaning liquid chamber 230.
  • the sample chamber 210 to be tested is used for storing the liquid sample, and the liquid sample may be the same sample or multiple different samples.
  • the cleaning liquid chamber 230 is used for storing cleaning liquid.
  • the first control valve 220 is used to control the liquid sample in the sample chamber 210 to be tested and the cleaning liquid in the cleaning liquid chamber 230 to be injected into the container 21.
  • the liquid collection unit 50 includes at least one sampling member 510, which can rotate around a third rotating shaft 7 to form a sampling track T, and the first preset position 5 and the second preset position 6 are located The cleaning liquid sampling trace T.
  • the sampling member 510 is used to collect a liquid sample or cleaning solution in the container 21 at the second preset position 6, and inject the collected liquid sample or cleaning solution into the separation chip 10 at the first preset position 5. So that the separation chip 10 separates and cleans the target particles.
  • the sampling member 510 is a sampling needle, and the number of sampling needles is two.
  • One sampling piece 510 is located on one side of the connecting line between the first supporting table 1 and the second supporting table 2, and the other sampling piece 510 is located between the first supporting table 1 and the second supporting table 2 The other side of the connection.
  • Each sampling piece 510 corresponds to one of the sampling trajectories T, and the number of the first preset position 5 and the second preset position 6 are two respectively.
  • One of the first preset positions 5 and the corresponding second preset position 6 are located on the sampling track T of one of the sampling elements 510, and the other first preset position 5 and the corresponding second preset position 6 are located on the other
  • a sampling piece 510 is on the sampling track T.
  • the two sampling pieces 510 can work simultaneously or independently.
  • Each liquid supply unit 20 may also include a power component, such as a power pump or a suction pump, to provide power for the liquid flow.
  • the vacuum system 30 is used to generate negative pressure in the first chamber 16 and the second chamber 18 of each separate chip 10 on the first support table 1.
  • the vacuum system 30 can be two independent vacuum systems or a designed vacuum system.
  • the vacuum system 30 may also include equipment such as a micro vacuum pump or a micro air pump. It can be understood that the vacuum system 30 and each separation chip 10 can be connected by a pipeline with better airtightness.
  • the vacuum system 30 includes a first vacuum pump 310 and a second vacuum pump 320, and a plurality of the separation chips 10 are connected in parallel between the first vacuum pump 310 and the second vacuum pump 320, that is, the first vacuum pump 310 and the second vacuum pump 320.
  • a vacuum pump 310 is connected to the first opening 161 of each separate chip 10, and the second vacuum pump 320 is connected to the second opening 181 of each separate chip 10.
  • the frequency conversion module 40 is electrically connected to the vacuum system 30.
  • the frequency conversion module 40 can control the power supply voltage provided to the vacuum system 30, so that negative pressure is alternately generated in each first chamber 16 and each second chamber 18 . Since a plurality of the separation chips 10 are connected in parallel, the alternating negative pressure of the same intensity can be generated to act on all the separation chips 10 at the same time. In other embodiments, when the number (greater than 6) of the separation chips 10 on the first support table 1 working at the same time is large, the plurality of separation chips 10 can be divided into two or more groups and connected in parallel respectively. Multiple vacuum systems 30 can ensure stable operation of negative pressure without attenuation.
  • the frequency conversion module 40 includes a frequency converter 410 and a second control valve 420 connected to the frequency converter 410.
  • the second control valve 420 may be a liquid circuit converter, including but not limited to solenoid valves and rotary valves.
  • the second control valve 420 is respectively communicated with one of the first vacuum pump 310 and the second vacuum pump 320, so that the first vacuum pump 310 and the second vacuum pump 320 alternately work repeatedly.
  • the second control valve 420 is connected to the first vacuum pump 310, so that the frequency converter 410 controls the operation of the first vacuum pump 310, and draws air through each first opening 161 to generate negative pressure in each first chamber 16 ,
  • the liquid in the liquid sample in each sample pool 11 and the components whose size is smaller than the pore size of the respective first filter membrane 15 pass through the respective first filter membrane 15 under negative pressure and enter the respective first chamber 16 at the same time ,
  • the liquid sample in each sample pool 11 will have a backflow phenomenon at the respective second filter membrane 17, thereby reducing or removing the components adhering to the respective second filter membrane 17, avoiding the filtration and separation process
  • the filter membrane is blocked; then, the frequency converter 410 controls the first vacuum pump 310 to stop running; afterwards, the second control valve 420 is switched to communicate with the second vacuum pump 320, so that the frequency converter 410 controls the first vacuum pump 320.
  • the two vacuum pumps 320 operate to pump air through each second opening 181 to generate a negative pressure in the respective second chamber 18, so that the liquid in the liquid sample in the respective sample pool 11 and the group whose size is smaller than the pore size of the respective second filter membrane 17 Under the action of negative pressure, the components pass through the respective second filter membranes 17 and enter the respective second chambers 18. At the same time, the liquid samples in each sample pool 11 will have backflow phenomenon at the respective first filter membranes 15, thereby reducing Or remove the components adhering to the respective first filter membrane 15 to avoid clogging of the filter membrane during the filtration and separation process; then, the frequency converter 410 controls the second vacuum pump 320 to stop running; repeat the above steps several times . Please refer to FIG. 6.
  • the negative pressure generated in each first chamber 16 and each second chamber 18 alternately by the vacuum system 30 forms a periodic trapezoidal pulse signal.
  • the intensity of the trapezoidal pulse signal is -10 to -80 kpa.
  • the rectangular pulse signal can also be replaced with a periodic sinusoidal signal or rectangular signal.
  • negative pressure in view of the high protein content in the plasma sample, in order to further avoid clogging of the filter membrane, negative pressure can be generated in one chamber while positive pressure is generated in the other chamber to enhance the backflow phenomenon at the filter membrane. .
  • the intensity, alternating time, cycle, and total operating time of the alternating negative pressure can be adjusted according to the type of liquid sample, so as to optimize the return effect at the filter membrane.
  • RNA, DNA nucleic acid molecules
  • lipoproteins lipids, proteins, and peptide chains
  • RNA nucleic acid molecules
  • lipoproteins lipoproteins
  • lipids lipoproteins
  • proteins lipids
  • peptide chains peptide chains
  • the main body module 101 may further include a plurality of first liquid storage chambers 330 and a plurality of second liquid storage chambers 340.
  • the first liquid storage chamber 330 is disposed between the first vacuum pump 310 and the first opening 161 of each separation chip 10, and the first liquid storage chamber 330 is connected to the first vacuum pump 310 and the first opening 161 of each separation chip 10, respectively.
  • a chamber 16 is connected.
  • the second liquid storage chamber 340 is disposed between the second vacuum pump 320 and the second opening 181 of each separation chip 10, and the second liquid storage chamber 340 is connected to the second vacuum pump 320 and the second opening 181 of each separation chip 10, respectively.
  • the two chambers 18 are connected.
  • Each first liquid storage chamber 330 and each second liquid storage chamber 340 can be used as a safety bottle to prevent the liquid in each separation chip 10 from entering the vacuum pump, or can be used as a waste liquid bottle to collect the residue in each separation after each separation.
  • the auxiliary module 102 is used to ensure the stable operation of the separation device 100 and improve the separation and purification effect.
  • the auxiliary module 102 includes a detector 60 and a controller 70.
  • the detector 60 is used to detect the liquid level in the sample pool 11 of each separation chip 10.
  • the controller 70 is electrically connected to the detector 60 and the frequency conversion module 40.
  • the controller 70 is used to obtain the height of the liquid level detected by the detector 60, and combine the height of the liquid level and the preset added amount of the liquid sample to determine whether it is necessary to continue adding the liquid sample and the cleaning solution, or the separation and purification has been completed.
  • the controller 70 controls the frequency conversion module 40 to stop the operation, thereby stopping the generation of negative pressure in the first chamber 16 and the second chamber 18 of the separation chip 10.
  • the controller 70 may be a set of logical relationships embedded in hardware or firmware, or may be a series of programs written in a programming language and stored in memory or other firmware.
  • the controller 70 is also used to control the frequency conversion module 40 to alternately generate corresponding negative pressures in the first chamber 16 and the second chamber 18 according to preset negative pressure parameters.
  • the human-computer interaction module 103 is used to meet the use requirements in the actual separation and purification process, and make the separation device 100 operability.
  • the human-computer interaction module 103 includes a human-computer interaction interface 80.
  • the human-computer interaction interface 80 is used for the operator to input separation and purification parameters through the input unit (such as touch screen, keyboard, mouse, etc.) of the separation device 100, that is, the operator can preset the separation and purification parameters through the human-machine interface 80
  • the separation and purification parameters include a preset amount of liquid sample, a preset amount of cleaning solution, and a negative pressure parameter.
  • the negative pressure parameter includes at least one of the intensity of the negative pressure, alternating time, period, and total operating time.
  • the controller 70 is also electrically connected with the man-machine interface 80. Therefore, the controller 70 can obtain the separation and purification parameters input through the human-machine interface 80, and control the corresponding operations of the frequency conversion module 40 or the liquid supply unit 20 according to the separation and purification parameters.
  • the human-computer interaction module 103 may further include a transmission interface 81, which is used to connect an external device (for example, an operator's USB flash drive, mobile phone, etc.) to transmit the data to the external device.
  • an external device for example, an operator's USB flash drive, mobile phone, etc.
  • the separation and purification parameters allow the operator to review the relevant separation and purification data after the separation and purification of each liquid sample is completed.
  • the transmission interface can be a USB interface or a wireless interface.
  • the separation device provided by the present invention can automatically separate the target particles in the liquid sample, separate the components in the sample pool that cannot pass the filter membrane, and change the sample through the negative pressure changes in the cavities on both sides of the sample pool.
  • the gas-liquid flow direction in the pool reduces the components adhering to the surface of the filter membrane and avoids the blocking of the filter membrane during the filtration and separation process.
  • the separation device is low in cost and convenient to use, which greatly reduces the workload of experimenters.
  • the embodiment of the present invention further provides a separation control system 200 applied to the separation device 100.
  • the auxiliary module 102 of the separation device 100 may further include a memory 71, and the separation control system 200 is stored in the memory 71.
  • the separation control system 200 includes one or more program modules composed of program codes.
  • the controller 70 is used to load and execute each program module of the separation control system 200, so as to realize the separation and purification function of the separation device 100.
  • the separation control system 200 includes a liquid circuit and mechanical module 202 and a main control module 203.
  • the liquid path and mechanical module 202 is used to control each sampling piece 510 to inject a liquid sample and a cleaning solution into the sample pool 11 of the corresponding separation chip 10.
  • the main control module 203 is used for controlling the vacuum system 30 to alternately generate negative pressure in the first chamber 16 and the second chamber 18 of each separation chip 10 through the frequency conversion module 40.
  • the vacuum system 30 includes a first vacuum pump 310 and a second vacuum pump 320, the first vacuum pump 310 is connected to the first opening 161 of each separate chip 10, and the second vacuum pump 320 is connected to each separate chip 10.
  • the second opening 181 of 10 is connected.
  • the frequency conversion module 40 includes a frequency converter 410 and a second control valve 420 connected to the frequency converter 410.
  • the main control module 203 is used to control the second control valve 420 to communicate with the first vacuum pump 310, so that the frequency converter 410 controls the operation of the first vacuum pump 310, and draws air through each first opening 161 to make the respective first chamber 16 Negative pressure is generated inside.
  • the main control module 203 is also used to control the second control valve 420 to switch to communicate with the second vacuum pump 320, so that the frequency converter 410 controls the operation of the second vacuum pump 320, and pumps air through each second opening 181 to make the respective first Negative pressure is generated in the second chamber 18.
  • the separation device 100 further includes a detector 60.
  • the detector 60 is used to detect the liquid level in the sample pool 11 of each separation chip 10.
  • the separation control system 200 further includes a drive control module 201, which is used to obtain the liquid level detected by the detector 60, and combine the liquid level and the preset added amount of the liquid sample to determine whether the liquid sample is Separation and purification have been completed.
  • the drive control module 201 sends a stop instruction to the main control module 203.
  • the main control module 203 responds to the stop instruction and controls the frequency conversion module 40 to stop operation, thereby stopping the generation of negative pressure in the first chamber 16 and the second chamber 18 of the corresponding separation chip 10.
  • the drive control module 201 is further configured to obtain preset negative pressure parameters, and send a first control instruction including the preset negative pressure parameters to the main control module 203.
  • the main control module 203 is used to respond to the first control instruction, and according to the preset negative pressure parameters, control the frequency conversion module 40 to alternately generate corresponding signals in each first chamber 16 and each second chamber 18 Negative pressure.
  • the drive control module 201 is also used to obtain the preset adding amount of each liquid sample, and send a second control instruction to the liquid path and mechanical module 202.
  • the liquid path and mechanical module 202 is used to respond to the second control instruction, and control the working time of each sample 510 according to the preset amount of each liquid sample, so that the liquid sample that meets the preset amount of liquid flows into the Sample pool 11.
  • the drive control module 201 is also used to obtain the preset added amount of cleaning fluid, and send a third control command to the fluid path and mechanical module 202.
  • the fluid path and mechanical module 202 is also used to respond to the third control instruction, and control the working time of each sample 510 according to the preset amount of cleaning fluid, so that the cleaning fluid that meets the preset amount of cleaning fluid flows into Each corresponding sample pool 11.
  • the embodiment of the present invention further provides a method for separating target particles in a liquid sample, which includes the following steps:
  • Step one place the plurality of separation chips 10 on the first support table 1, and place the plurality of liquid samples on the second support table 2.
  • Step 2 Control the rotation of the second supporting table 2 so that the multiple liquid samples can move to the second preset positions 6 respectively.
  • Step three controlling the rotation of the first support platform 1 so that the plurality of separation chips 10 can move to the first preset position 5 respectively.
  • Step 4 Control the rotation of the sampling piece 510 to form the sampling trajectory T, and the first preset position 5 and the second preset position 6 are located on the sampling trajectory T, so that the sampling piece 510 collects The liquid sample located at the second preset position 6 and the collected liquid sample are added to the separation chip 10 located at the first preset position 5.
  • Step 5 Suction the corresponding first chamber 16 through the first opening 161 of each separation chip 10 to generate a negative pressure in the corresponding first chamber 16.
  • the first opening 161 and the second opening 181 of each separation chip 10 are respectively connected to the vacuum system 30 of the separation device 100.
  • the vacuum system 30 sucks the corresponding first chamber 16 through each first opening 161 to generate a negative pressure in the corresponding first chamber 16.
  • the liquid in the liquid sample in each sample pool 11 and the components whose size is smaller than the pore size of the respective first filter membrane 15 pass through the corresponding first filter membrane 15 under the action of negative pressure and enter the corresponding first chamber 16.
  • the liquid and the components whose size is smaller than the pore size of the first filter membrane 15 may also be It further flows out through the first opening 161 and enters the first liquid storage chamber 330.
  • the sample pool opening 113 can be further closed.
  • the sample cell opening 113 is closed, during the suction process, the liquid flow velocity between the corresponding first filter membrane 15 and the corresponding second filter membrane 17 in each sample cell 11 increases, thereby enhancing the corresponding
  • the backflow phenomenon of the first filter membrane 15 or the corresponding second filter membrane 17 reduces the components adhering to the surface of the filter membrane and prevents the filter membrane from being blocked during the filtration and separation process.
  • step 5 may further include generating a positive pressure in each second chamber 18 to enhance the backflow phenomenon at the filter membrane.
  • Step six stop sucking each first chamber 16.
  • Step 7 sucking the corresponding second chamber 18 through the second opening 181 of each separation chip 10 to generate a negative pressure in the corresponding second chamber 18.
  • the vacuum system 30 sucks the corresponding second chamber 18 through each second opening 181 to generate a negative pressure in the corresponding second chamber 18.
  • the components adhering to the surface of the corresponding first filter membrane 15 can flow back into the corresponding sample cell 11 along with the air flow and/or liquid flow.
  • the liquid and size of the liquid sample in each sample cell 11 are smaller than the corresponding first filter membrane.
  • the pore components of the second filter membrane 17 pass through the corresponding second filter membrane 17 under the action of negative pressure and enter the corresponding second chamber 18.
  • step four and step five can be executed sequentially one after another, or simultaneously.
  • step 5 may further include generating a positive pressure in each first chamber 16 to enhance the backflow phenomenon at the filter membrane.
  • Step eight stop sucking each second chamber 18.
  • steps 5 to 8 can be cycled multiple times to remove the components smaller than the corresponding filter membrane pore size in each liquid sample, and the components larger than the corresponding filter membrane pore size are trapped in the corresponding sample pool 11. Achieve better separation and purification effect.
  • the alternating changes of the negative pressure in each first chamber 16 and each second chamber 18 can improve the permeability of the filter membrane during the filtration process, reduce the blocking of the filter membrane, and improve the filtration effect.
  • the method may further include the following steps:
  • Step 7 Provide a cleaning solution to the sample pool 11 of each separation chip 10. Then, each separation chip 10 is cleaned by repeatedly performing steps five to eight.
  • a higher yield of exosomes can be separated within 30 minutes.
  • the separation device 100 has the characteristic of efficiently purifying exosomes from large-volume biological samples.
  • the innovations of the separation device 100 include: 1) high throughput (realizing parallel processing of multiple liquid samples: 2-100); 2) automated processing; 3) simple and standardized operation; 4) high yield and purity; 5) Label-free; 6) cost-effective; 7) high stability and reproducibility; 8) can process a variety of different biological samples, including plasma, urine, brain spinal fluid, saliva, tears, emulsion and cell culture fluid.

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Abstract

本发明提出一种分离装置，用于同时从多个液体样本中分离提纯出目标颗粒，所述分离装置包括：多个分离芯片，每一分离芯片包括：样本池，用于容置所述液体样本；以及第一开口和第二开口，位于所述样本池的两侧；真空系统，包括：第一真空泵，连接每一分离芯片的第一开口，所述第一真空泵通过所述第一开口在所述分离芯片内产生负压以分离所述样本池内所述液体样本中的目标颗粒；以及第二真空泵，连接每一分离芯片的第二开口，所述第二真空泵通过所述第二开口在所述分离芯片内产生负压以分离所述样本池内所述液体样本中的目标颗粒。本发明还提供一种应用所述的分离装置分离液体样本中目标颗粒的方法。

Description

分离装置及分离液体样本中目标颗粒的方法 技术领域
本发明涉及生物技术领域，尤其涉及一种分离装置及分离液体样本中目标颗粒的方法。
背景技术
外泌体是由活的细胞持续大量分泌的一种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、肿瘤转移以及肿瘤耐药等。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。外泌体广泛且稳定的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、耐药突变检测以及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缺乏快速、稳定、高效提取高纯度外泌体的标准方法。市场上存在多种外泌体纯化技术，包括超速离心、尺寸排阻色谱、基于磁珠的免疫亲和捕获、基于聚乙二醇的沉淀法、超滤和微流体等方法。然而，上述纯化方法存在缺点：1)回收率低，2)纯度低，3)分离的外泌体完整性差，4)重现性差，5)可能引入不需要的杂质，6)需要生物标记，7)耗时耗力和8)成本高。超速离心目前是最常用的外泌体纯化方法， 但它也存在一些限制，例如产量低(回收率：5％～25％)，操作过程繁琐，耗时长(>4小时)，依赖昂贵的设备。另外基于免疫捕获的分离方法可以收集具有中等至高纯度的外泌体，但这些方法受限于抗体的特异性和繁琐操作步骤，难以标准化，不适合处理大数量及大体积的临床样本。最近，基于微流控技术的外泌体分离方法也被多次报道，包括流体动力或声力分离，免疫捕获和介电电泳。然而，依然无法解决通量低、操作过程复杂和重复性差的问题，难以实现不同实验室间结果的一致性。
发明内容
有鉴于此，本发明提供一种能够快速、稳定、高效提取高纯度外泌体的分离装置。
另，还有必要提供一种利用上述分离装置分离液体样本中目标颗粒的方法。
本发明提供一种分离装置，用于从多个液体样本中分离提纯出目标颗粒，所述分离装置包括：
多个分离芯片，每一分离芯片包括：
样本池，用于容置所述液体样本；以及
第一开口和第二开口，位于所述样本池的两侧；
真空系统，包括：
第一真空泵，连接每一分离芯片的第一开口，所述第一真空泵通过所述第一开口在所述分离芯片内产生负压以分离所述样本池内所述液体样本中的目标颗粒；以及
第二真空泵，连接每一分离芯片的第二开口，所述第二真空泵通过所述第二开口在所述分离芯片内产生负压以分离所述样本池内所述液体样本中的目标颗粒。
本发明还提供一种应用所述的分离装置分离液体样本中目标颗粒的方 法，所述方法包括：
向所述分离芯片的样本池提供液体样本；
运行所述第一真空泵并通过所述第一开口在所述分离芯片内产生负压；
停止运行所述第一真空泵；
运行所述第二真空泵并通过所述第二开口在所述分离芯片内产生负压；
停止运行所述第二真空泵，重新运行所述第一真空泵；以及
重复循环以上过程，让所述第一真空泵与所述第二真空泵交替运行，实现负压在所述第一开口与所述第二开口交替切换，从而分离所述样本池内所述液体样本中的目标颗粒。
所述分离装置具有从大体积生物样本中高效纯化外泌体的特点。所述分离装置的优点包括：1)高通量(通过分离芯片并联设置，实现多个液体样本并行处理)；2)自动化处理；3)简单和标准化操作；4)高产率和纯度；5)免标记；6)具有成本效益；7)高稳定性和可重复性；8)可处理多种不同的生物样本，包括血浆、尿液、脑髓液、唾液、泪液、乳液和细胞培养液等。
附图说明
图1是本发明实施例所提供的分离芯片的结构示意图。
图2是图1所示的分离芯片的结构拆解示意图。
图3是分别使用图1所示的分离芯片和传统超滤法进行外泌体分离的过滤速度示意图。
图4是本发明实施例所提供的分离装置的功能模块示意图。
图5A是图4所示的分离装置的液路示意图。
图5B是图4所示的分离装置的分离控制系统的程序模块图。
图6是本发明一实施例中施加于分离芯片的负压的示意图。
图7是图4所示的分离装置的采样模块工作原理图。
符号说明
分离芯片 10
第一支撑台 1
第二支撑台 2
第一转轴 3
第二转轴 4
第一预设位置 5
第二预设位置 6
第三转轴 7
样本池 11
第一侧盖片 111
第一盖片部 1111
第二盖片部 1112
第二侧盖片 112
第三盖片部 1121
第四盖片部 1122
样本池开口 113
第一凸块 12
第一卡槽 121
第二凸块 13
第三卡槽 131
芯片基底 14
第一过滤膜 15
第一腔室 16
第一开口 161
第二过滤膜 17
第二腔室 18
第二开口 181
液体供应单元 20
容器 21
真空系统 30
变频模块 40
液体采集单元 50
检测器 60
控制器 70
存储器 71
人机交互界面 80
传输接口 81
分离装置 100
主体模块 101
辅助模块 102
人机交互模块 103
分离控制系统 200
驱动控制模块 201
液路与机械模块 202
主控模块 203
待测样本室 210
第一控制阀 220
清洗液室 230
第一真空泵 310
第二真空泵 320
第一液体存储室 330
第二液体存储室 340
变频器 410
第二控制阀 420
采样件 510
如下具体实施方式将结合上述附图进一步说明本发明。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，当元件被称为“固定于”另一个元件，它可以直接在另一个元件上或者也可以存在居中的元件。当一个元件被认为是“连接”另一个元件，它可以是直接连接到另一个元件或者可能同时存在居中元件。当一个元件被认为是“设置于”另一个元件，它可以是直接设置在另一个元件上或者可能同时存在居中元件。
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发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本发明实施例提供一种分离芯片，该分离芯片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、泪液、乳液、胃液以及细胞培养液等。图1是本发明一实施例所提供的分离芯片10的结构示意图。如图1所示，同时请参阅图2，分离芯片10包括样本池11、第一腔室16和第二腔室18。该 第一腔室16以及该第二腔室18分别位于该样本池11相对的两侧。
该样本池11包括第一侧盖片111和第二侧盖片112，该第一侧盖片111与该第二侧盖片112设置于该样本池11相对的两侧。该第一侧盖片111上设有第一凸块12，该第一凸块12将该第一侧盖片111划分为位于该第一凸块12一侧的第一盖片部1111以及位于该第一凸块12另一侧的第二盖片部1112。该第二侧盖片112上设有与该第一凸块12相对的第二凸块13，该第二凸块13将该第二侧盖片112划分为位于该第二凸块13一侧的第三盖片部1121以及位于该第二凸块13另一侧的第四盖片部1122。该第一盖片部1111、该第三盖片部1121、该第一凸块12以及该第二凸块13共同围设形成该样本池11。该样本池11的顶端包括样本池开口113，用于加入和/或取出液体样本。
该第一侧盖片111的底部与该第一凸块12相对的位置设有一芯片基底14。该第一凸块12与该芯片基底14之间设置有第一过滤膜15，且该第一过滤膜15与该第二盖片部1112相对，该第二盖片部1112、该第一过滤膜15以及该芯片基底14共同围设形成该第一腔室16。该第一腔室16与该样本池11通过该第一过滤膜15相连通。该第一腔室16设置有第一开口161，该第一开口161用于使该第一腔室16与外界连通。
该第二侧盖片112的底部且与该第二凸块13相对的位置设有另一芯片基底14。该第二凸块13与该芯片基底14之间设置有第二过滤膜17，且该第二过滤膜17与该第四盖片部1122相对。该第四盖片部1122、该第二过滤膜17以及芯片基底14共同围设形成该第二腔室18。该第二腔室18与该样本池11通过该第二过滤膜17相连通，该第二腔室18设置有第二开口181，该第二开口181用于使该第二腔室18与外界连通。
在本实施方式中，该第一凸块12与该第二凸块13之间设有间隙(图未标)，该间隙用于使样本池11内的液体样本能够流出该样本池11，并经该第一过滤膜15或该第二过滤膜17分别进入该第一腔室16或该第二腔室18。更具体地，该第一凸块12朝向该芯片基底14的一侧开设有一第一卡槽121， 该芯片基底14对应的位置开设有一第二卡槽(图未标)，该第一过滤膜15卡设并固定于该第一卡槽121与该第二卡槽之间。同理，该第二凸块13朝向该芯片基底14的一侧开设有一第三卡槽131，该芯片基底14对应的位置开设有一第四卡槽(图未标)，该第二过滤膜17卡设并固定于该第三卡槽131与该第四卡槽之间。
所述分离芯片10具有对称结构。需要说明的是，分离芯片10也可以是不对称结构或其他任何能够实现本发明构思的结构。
使用该分离芯片10时，将液体样本加入样本池11，将该第一开口161和该第二开口181分别与真空系统30(参图4)相连接。当真空系统30通过该第一开口161使该第一腔室16受到抽吸时，该第一腔室16中产生负压。在该第一腔室16的负压作用下，样本池11中的液体样本中尺寸小于第一过滤膜15的过滤孔径的组分经由第一过滤膜15流入该第一腔室16。当真空系统30通过该第二开口181使该第二腔室18受到抽吸时，该第二腔室18中产生负压。在该第二腔室18的负压作用下，样本池11中的液体样本中尺寸小于第二过滤膜17的过滤孔径的组分经由第二过滤膜17流入该第二腔室18。在一实施例中，所述真空系统30可以为两个负压泵，其中一个负压泵与该第一开口161连接，另一个负压泵与该第二开口181连接，两个负压泵交替提供负压。反复交替使该第一腔室16和该第二腔室18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15和第二过滤膜17，使液体样本中尺寸大于第一过滤膜15和第二过滤膜17孔径的组分留在样本池11中。该分离芯片10的结构设计使吸附于第一过滤膜15和第二过滤膜17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。
在交替负压作用下产生交换式过滤及膜振荡，使得在分离期间外泌体可再悬浮以避免膜堵塞。与传统的膜分离相比，实验结果如图3显示，在30分钟内，该分离芯片10可以处理20mL尿液和细胞培养液，远超过传统超滤法 (3mL)。
该分离芯片10的样本池11、第一腔室16、第二腔室18的主体部分可以由塑料、玻璃、金属或复合材料制成。在一实施例中，该分离芯片10的样本池11、第一腔室16、第二腔室18的主体部分由聚乙烯亚胺(PEI)或聚甲基丙烯酸甲酯(PMMA)等透明材料制成。该分离芯片10的加工方法包括但不仅限于加工成型和注模成型。该第一过滤膜15和该第二过滤膜17可以由相同的膜材料制成，也可以由不同的膜材料制成。该第一过滤膜15和该第二过滤膜17可以具有相同的平均过滤膜孔径和/或孔径分布，也可以具有不同的平均过滤膜孔径和/或孔径分布。该第一过滤膜15(或该第二过滤膜17)可以是由一种膜材料制成的，也可以是由多种膜材料复合而成的。该第一过滤膜15和该第二过滤膜17可以是多孔材料，包括但不仅限于多孔陶瓷材料、多孔塑料材料和多孔金属材料。具体地，该第一过滤膜15和该第二过滤膜17可以分别选自阳极氧化铝膜(AAO)、聚碳酸酯膜、醋酸纤维膜、聚乙烯膜、聚丙烯膜和聚苯乙烯膜中的一种或几种。更具体地，鉴于阳极氧化铝膜具有较高的孔隙率和较均匀的孔径，该第一过滤膜15和该第二过滤膜17采用阳极氧化铝膜。
该第一过滤膜15和该第二过滤膜17的孔径可根据该液体样本以及目标颗粒的类型进行设计。在一实施例中，该第一过滤膜15和该第二过滤膜17的孔径为20纳米，略小于外泌体尺寸(30～150nm)，可以用于分离提纯已通过200纳米过滤膜的细胞培养液样本中的外泌体。
本发明实施例进一步提供了一种分离装置。图4示出了该分离装置100的程序模块示意图。该分离装置100包括主体模块101、辅助模块102以及人机交互模块103。
该主体模块101用于同时对多个液体样本进行分离提纯，以提高提纯的效率。该主体模块101包括第一支撑台1、第二支撑台2、液体供应单元20、真空系统30、变频模块40以及液体采集单元50。
图7所示的是该分离装置100的采样模块的工作原理，该第一支撑台1包括多个如上文所述的分离芯片10。在一实施方式中，该第一支撑台1中的该分离芯片10的数量为12个。该第一支撑台1可以在一驱动件(如马达，图未示)的作用下绕一第一转轴3旋转，例如可以包括顺时针旋转或逆时针旋转，从而带动该第一支撑台1上的每一分离芯片10旋转，以使每一分离芯片10分别移动至第一预设位置5。所述第一预设位置5即为采样位置。其中，多个该分离芯片10以该第一支撑台1的第一转轴3为中心围绕设置在该第一支撑台1上。可以理解，该第一支撑台1中的该分离芯片10的数量可以根据需要进行相应的增加或减少。
该第二支撑台2包括多个容器21。在一实施例中，该容器21的数量与该分离芯片10的数量相同，也为12个。该第二支撑台2可以在一驱动件(如马达，图未示)的作用下绕一第二转轴4旋转，例如可以包括顺时针旋转或逆时针旋转，从而带动该第二支撑台2上的每一容器21旋转，以使每一容器21分别移动至第二预设位置6。其中，多个该容器21以该第二支撑台2的第二转轴4为中心围绕设置在该第二支撑台2上。
该液体供应单元20包括待测样本室210、一第一控制阀220以及清洗液室230。该待测样本室210用于存放该液体样本，该液体样本可以是同一个样本，也可以是多个不同的样本。该清洗液室230用于存放清洗液。该第一控制阀220用于控制该待测样本室210中的液体样本以及该清洗液室230中的清洗液注入到该容器21中。
该液体采集单元50包括至少一采样件510，该采样件510能够绕一第三转轴7转动以形成一采样轨迹T，所述第一预设位置5和所述第二预设位置6位于所述清洗液采样轨迹T上。该采样件510用于采集位于第二预设位置6的容器21中的液体样本或清洗液，并将所采集的液体样本或清洗液注入到位于第一预设位置5的分离芯片10中，以便该分离芯片10分离和清洗目标颗粒。在一实施例中，该采样件510为采样针，该采样针的数量为两个。其中 一采样件510位于所述第一支撑台1与所述第二支撑台2的连线的一侧，另一采样件510位于所述第一支撑台1与所述第二支撑台2的所述连线的另一侧。每一采样件510对应其中一采样轨迹T，所述第一预设位置5和所述第二预设位置6的数量分别为两个。其中一第一预设位置5和对应的一第二预设位置6位于其中一采样件510的采样轨迹T上，另一第一预设位置5和对应的一第二预设位置6位于另一采样件510的采样轨迹T上。两个该采样件510能够同时工作或单独工作。每一液体供应单元20还可以包括一动力部件，如动力泵或抽气泵，为液流提供动力。
该真空系统30用于分别使该第一支撑台1上的每一分离芯片10的第一腔室16和第二腔室18产生负压。该真空系统30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。该真空系统30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，该真空系统30与每一分离芯片10之间可以通过气密性较佳的管道连接。在一实施方式中，该真空系统30包括第一真空泵310和第二真空泵320，多个该分离芯片10并联连接于所述第一真空泵310和所述第二真空泵320之间，即，该第一真空泵310与每一分离芯片10的第一开口161相连接，该第二真空泵320与每一分离芯片10的第二开口181相连接。
该变频模块40与该真空系统30电连接，该变频模块40可以控制提供给该真空系统30的电源电压，从而使每一第一腔室16和每一第二腔室18内交替产生负压。由于多个该分离芯片10并联连接，所以可产生相同强度的交替负压同时作用于所有的该分离芯片10上。在其他实施例中，当该第一支撑台1上的该分离芯片10同时工作的数量(大于6个)较多时，可将多个该分离芯片10分成两组或者更多组，分别并联到多个真空系统30上，这样可保证负压稳定工作，不会衰减。
在一实施方式中，该变频模块40包括变频器410以及与该变频器410连接的第二控制阀420。该第二控制阀420可以是液路转换器，包括但不仅限 于电磁阀、旋转阀。该第二控制阀420分别与该第一真空泵310以及该第二真空泵320中的其中一个连通，从而使第一真空泵310和第二真空泵320反复交替工作。例如，将该第二控制阀420连通该第一真空泵310，使得该变频器410控制该第一真空泵310运行，通过每一第一开口161抽气使每一第一腔室16内产生负压，每一样本池11中的液体样本中的液体和尺寸小于各自第一过滤膜15孔径的组分在负压作用下通过各自第一过滤膜15，进入各自第一腔室16，与此同时，每一样本池11中的液体样本在各自第二过滤膜17处会产生回流(back flow)现象，从而减少或移除粘附于各自第二过滤膜17的组分，避免过滤分离过程中过滤膜被堵塞的情况发生；然后，该变频器410控制该第一真空泵310停止运行；之后，将该第二控制阀420切换至与该第二真空泵320连通，使得该变频器410控制该第二真空泵320运行，通过每一第二开口181抽气使各自第二腔室18内产生负压，使得各自样本池11中的液体样本中的液体和尺寸小于各自第二过滤膜17孔径的组分在负压作用下通过各自第二过滤膜17，进入各自第二腔室18，与此同时，每一样本池11中的液体样本在各自第一过滤膜15处会产生回流现象，从而减少或移除粘附于各自第一过滤膜15的组分，避免过滤分离过程中过滤膜被堵塞的情况发生；再之后，该变频器410控制该第二真空泵320停止运行；反复上述步骤多次。请参阅图6，在一实施例中，该真空系统30交替地在每一第一腔室16以及每一第二腔室18内产生的负压形成周期的梯形脉冲信号。所述梯形脉冲信号的强度为-10～-80kpa。在其它实施例中，该矩形脉冲信号还可以替换为周期性的正弦信号或矩形信号。在其它实施例中，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，加强过滤膜处的回流现象。工作中，可根据液体样本的类型相应调整该交替变化的负压的强度、交替时间、周期以及总操作时间等，以使得过滤膜处回流效果最佳。在交替负压作用下，液体样本中的小尺寸杂质，包括但不限于核酸分子(RNA、DNA)、脂蛋白、脂质、 蛋白质以及肽链等，可以经过该各自第一过滤膜15和各自第二过滤膜17由各自第一开口161和各自第二开口181被吸出，而尺寸较大的外泌体则留在各自样本池11中。最终从各自样本池11中得到纯度较高的浓缩的外泌体样品。各自第一过滤膜15和各自第二过滤膜17相互对立。
进一步地，如图5A所示，该主体模块101还可以包括多个第一液体存储室330和多个第二液体存储室340。该第一液体存储室330设置于该第一真空泵310与每一分离芯片10的第一开口161之间，且该第一液体存储室330分别与第一真空泵310和每一分离芯片10的第一腔室16相连通。该第二液体存储室340设置于该第二真空泵320与每一分离芯片10的第二开口181之间，且该第二液体存储室340分别与第二真空泵320和每一分离芯片10的第二腔室18相连通。每一第一液体存储室330和每一第二液体存储室340可以作为安全瓶，避免每一分离芯片10中的液体进入真空泵，也可以作为废液瓶收集每次分离后残余在每一分离芯片10中的液体或清洗液。
该辅助模块102用于确保该分离装置100稳定运行且提高分离提纯效果。该辅助模块102包括一检测器60以及一控制器70。
该检测器60用于检测每一分离芯片10的样本池11中的液面高度。
该控制器70与该检测器60以及该变频模块40电连接。该控制器70用于获取该检测器60检测到的液面高度，并结合该液面高度以及液体样本的预设加入量判断是否需要继续加入液体样本和清洗液，或者已经完成分离提纯。当判断液体样本已经完成分离提纯时，该控制器70控制该变频模块40停止作业，从而停止在该分离芯片10的第一腔室16和第二腔室18内产生负压。其中，该控制器70可以是内嵌在硬件或固件(firmware)上的逻辑关系集合，也可以是用编程语言所编写的一系列存储在存储器或其他固件中的程序。在一实施例中，该控制器70还用于根据预设的负压参数控制该变频模块40在该第一腔室16以及第二腔室18内交替产生相应的负压。
该人机交互模块103用于满足实际分离提纯过程中的使用需求，使该分 离装置100具有可操作性。该人机交互模块103包括一人机交互界面80。该人机交互界面80用于供操作者通过该分离装置100的输入单元(如：触摸屏、键盘、鼠标等)输入分离提纯参数，即，操作者可通过该人机交互界面80预先设置分离提纯过程中所需的分离提纯参数。在一实施例中，该分离提纯参数包括液体样本的预设加入量、清洗液的预设加入量以及负压参数。该负压参数包括该负压的强度、交替时间、周期以及总操作时间等中的至少一种。该控制器70还与该人机交互界面80电连接。从而，该控制器70可获取经该人机交互界面80输入的分离提纯参数，并根据该分离提纯参数控制该变频模块40或该液体供应单元20相应作业。
在一实施例中，该人机交互模块103还可以包括一传输接口81，该传输接口81用于连接一外部设备(如，操作者的U盘、手机等)，从而向该外部设备传输该分离提纯参数，使操作者可在每一液体样本分离提纯完成后回顾相关的分离提纯数据。其中，该传输接口可以是USB接口或无线接口。
使用本发明所提供的分离装置可以自动化地对液体样本中的目标颗粒进行分离，将样本池中无法通过过滤膜的组分分离出来，同时通过样本池两侧空腔内的负压变化改变样本池中的气液流动方向，减少粘附在过滤膜表面的组分，避免过滤分离过程中过滤膜被堵塞的情况发生。该分离装置成本较低、使用方便，极大地降低了实验人员的工作量。
本发明实施例进一步提供一种分离控制系统200，其应用于该分离装置100中。该分离装置100的辅助模块102还可包括一存储器71，该分离控制系统200存储于该存储器71中。该分离控制系统200包括一个或多个由程序代码组成的程序模块。该控制器70用于加载并执行该分离控制系统200的各个程序模块，从而实现该分离装置100的分离提纯功能。其中，如图4和5B所示，该分离控制系统200包括液路与机械模块202以及主控模块203。
该液路与机械模块202用于控制每一采样件510向对应的分离芯片10的样本池11中注入液体样本以及清洗液。
该主控模块203用于通过该变频模块40控制该真空系统30分别在每一分离芯片10的第一腔室16和第二腔室18内交替产生负压。在一实施方式中，该真空系统30包括第一真空泵310和第二真空泵320，该第一真空泵310与每一分离芯片10的第一开口161相连接，该第二真空泵320与每一分离芯片10的第二开口181相连接。该变频模块40包括变频器410以及与该变频器410连接的第二控制阀420。该主控模块203用于控制该第二控制阀420连通该第一真空泵310，使得该变频器410控制该第一真空泵310运行，通过每一第一开口161抽气使各自第一腔室16内产生负压。该主控模块203还用于控制该第二控制阀420切换至与该第二真空泵320连通，使得该变频器410控制该第二真空泵320运行，通过每一第二开口181抽气使各自第二腔室18内产生负压。
在一实施例中，该分离装置100还包括检测器60。该检测器60用于检测每一分离芯片10的样本池11中的液面高度。该分离控制系统200还包括一驱动控制模块201，该驱动控制模块201用于获取该检测器60检测到的液面高度，并结合该液面高度以及液体样本的预设加入量判断液体样本是否已经完成分离提纯。当判断液体样本已经完成分离提纯时，该驱动控制模块201向该主控模块203发送一停止指令。该主控模块203响应该停止指令，并控制该变频模块40停止作业，从而停止在相应的分离芯片10的第一腔室16和第二腔室18内产生负压。
在一实施例中，该驱动控制模块201还用于获取预设的负压参数，并向该主控模块203发送包括所述预设的负压参数在内的一第一控制指令。该主控模块203用于响应该第一控制指令，并根据所述预设的负压参数控制该变频模块40在每一第一腔室16以及每一第二腔室18内交替产生相应的负压。该驱动控制模块201还用于获取每一液体样本的预设加入量，并向该液路与机械模块202发送一第二控制指令。该液路与机械模块202用于响应该第二控制指令，并根据每一液体样本的预设加入量控制每一采样件510的工作时 间，从而使符合该预设加入量的液体样本流入该样本池11。该驱动控制模块201还用于获取清洗液的预设加入量，并向该液路与机械模块202发送一第三控制指令。该液路与机械模块202还用于响应该第三控制指令，并根据每一清洗液的预设加入量控制每一采样件510的工作时间，从而使符合该预设加入量的清洗液流入各自对应的样本池11。
本发明实施例进一步提供一种分离液体样本中目标颗粒的方法，其包括如下步骤：
步骤一，将所述多个分离芯片10放置于所述第一支撑台1上，将所述多个液体样本放置于所述第二支撑台2上。
步骤二，控制所述第二支撑台2转动以使所述多个液体样本能够分别移动至第二预设位置6。
步骤三，控制所述第一支撑台1转动以使所述多个分离芯片10能够分别移动至第一预设位置5。
步骤四，控制所述采样件510转动以形成所述采样轨迹T，所述第一预设位置5和所述第二预设位置6位于所述采样轨迹T上，使得所述采样件510采集位于所述第二预设位置6上的液体样本并将所采集的液体样本加入位于所述第一预设位置5的所述分离芯片10中。
步骤五，通过每一分离芯片10的第一开口161抽吸相应的第一腔室16，使相应的第一腔室16内产生负压。
其中，在进行抽吸之前，将每一分离芯片10的第一开口161、第二开口181分别与分离装置100的真空系统30相连。如此，真空系统30通过每一第一开口161抽吸相应的第一腔室16，使相应的第一腔室16内产生负压。每一样本池11中的液体样本中的液体和尺寸小于各自第一过滤膜15孔径的组分在负压作用下通过相应的第一过滤膜15，进入相应的第一腔室16。在某些情况下，如第一腔室16的体积相对较小，亦或是，第一腔室16内的负压变化过快，液体和尺寸小于第一过滤膜15孔径的组分也可能进一步通过第一 开口161流出，进入第一液体存储室330。
更具体地，在进行抽吸之前，若液体样本通过该样本池开口113加入该样本池11，可进一步将该样本池开口113封闭。当该样本池开口113被封闭时，在抽吸过程中，每一样本池11中位于对应的第一过滤膜15以及对应的第二过滤膜17之间的液体流动速度增强，从而增强对应的第一过滤膜15或对应的第二过滤膜17的回流现象，减少粘附在过滤膜表面的组分，避免过滤分离过程中过滤膜被堵塞的情况发生。
在其它实施例中，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，步骤五可进一步包括在每一第二腔室18内产生正压，加强过滤膜处的回流现象。
步骤六，停止抽吸每一第一腔室16。
步骤七，通过每一分离芯片10的第二开口181抽吸对应的第二腔室18使对应的第二腔室18内产生负压。
其中，真空系统30通过每一第二开口181抽吸对应的第二腔室18，使对应的第二腔室18内产生负压。粘附在对应额第一过滤膜15表面的组分可以随着气流和/或液流回流到对应的样本池11中，每一样本池11中的液体样本中的液体和尺寸小于对应的第二过滤膜17孔径的组分在负压作用下通过对应的第二过滤膜17，进入对应的第二腔室18。在某些情况下，如第二腔室18的体积相对较小，亦或是，第二腔室18内的负压变化过快，液体和尺寸小于第二过滤膜17孔径的组分也可能进一步通过第二开口181流出，进入第二液体存储室340。可以理解地，步骤四和步骤五可以依次先后执行，也可以同时执行。
在其它实施例中，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，步骤五可进一步包括在每一第一腔室16内产生正压，加强过滤膜处的回流现象。
步骤八，停止抽吸每一第二腔室18。
然后，步骤五至步骤八可循环多次，使每一液体样本中小于相应的过滤膜孔径的组分被去除，大于相应的过滤膜孔径的组分被截留在对应的样本池11中，以实现更好的分离提纯效果。通过每一第一腔室16、每一第二腔室18内负压的交替变化可以改善过滤过程中过滤膜的通透性，降低过滤膜堵孔，提高过滤效果。
在一实施例中，在该步骤八之后，该方法可进一步包括如下步骤：
步骤七：向每一分离芯片10的样本池11中提供清洗液。然后，通过反复执行步骤五至步骤八对每一分离芯片10进行清洗。
使用本发明实施例提供的分离装置100对20mL的细胞培养液样本进行分离提纯，30min内便可分离出较高产量的外泌体。
该分离装置100具有从大体积生物样本中高效纯化外泌体的特点。该分离装置100的创新点包括：1)高通量(实现多个液体样本并行处理：2～100个)；2)自动化处理；3)简单和标准化操作；4)高产率和纯度；5)免标记；6)具有成本效益；7)高稳定性和可重复性；8)可处理多种不同的生物样本，包括血浆、尿液、脑髓液、唾液、泪液、乳液和细胞培养液等。
上述实施例为本发明较佳的实施方式，但本发明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。然本发明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发明披露的技术范围内，可轻易想到的变化或者替换，都包含在本发明的保护范围之内。

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  1. 一种分离装置，用于同时从多个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：
    多个分离芯片，每一分离芯片包括：
    样本池，用于容置所述液体样本；以及
    第一开口和第二开口，位于所述样本池的两侧；
    真空系统，包括：
    第一真空泵，连接每一分离芯片的第一开口，所述第一真空泵通过所述第一开口在所述分离芯片内产生负压以分离所述样本池内所述液体样本中的目标颗粒；以及
    第二真空泵，连接每一分离芯片的第二开口，所述第二真空泵通过所述第二开口在所述分离芯片内产生负压以分离所述样本池内所述液体样本中的目标颗粒。
  2. 如权利要求1所述的分离装置，其特征在于，还包括：
    第一支撑台，能够绕一第一转轴转动，所述多个分离芯片放置于所述第一支撑台，所述分离芯片围绕所述第一转轴设置且在所述第一支撑台转动时分别移动至第一预设位置；
    第二支撑台，能够绕一第二转轴转动且用于放置所述多个液体样本，所述液体样本围绕所述第二转轴设置且在所述第二支撑台转动时分别移动至第二预设位置；以及
    液体采集单元，包括至少一采样件，所述采样件能够绕一第三转轴转动以形成一采样轨迹，所述第一预设位置和所述第二预设位置位于所述采样轨迹上，所述采样件用于采集位于所述第二预设位置上的液体样本并将所采集的液体样本加入位于所述第一预设位置的所述分离芯片中。
  3. 如权利要求2所述的分离装置，其特征在于，所述采样件的数量为两 个，其中一采样件位于所述第一支撑台与所述第二支撑台的连线的一侧，另一采样件位于所述第一支撑台与所述第二支撑台的所述连线的另一侧，每一采样件对应其中一采样轨迹，所述第一预设位置和所述第二预设位置的数量分别为两个，其中一第一预设位置和对应的一第二预设位置位于其中一采样件的采样轨迹上，另一第一预设位置和对应的一第二预设位置位于另一采样件的采样轨迹上。
  4. 如权利要求1所述的分离装置，其特征在于，所述分离芯片还包括：
    第一过滤膜，所述第一过滤膜的孔径小于目标颗粒的粒径；
    第二过滤膜，所述第二过滤膜的孔径小于目标颗粒的粒径；
    第一腔室，所述第一腔室与所述样本池通过所述第一过滤膜相连通，所述第一腔室设置有所述第一开口，所述第一开口用于使所述第一腔室与外界连通；以及
    第二腔室，所述第二腔室与所述样本池通过所述第二过滤膜相连通，所述第二腔室设置有所述第二开口，所述第二开口用于使所述第二腔室与外界连通，其中，所述第一腔室与所述第二腔室分别位于该样本池相对的两侧。
  5. 如权利要求1所述的分离装置，其特征在于，所述分离装置还包括变频模块，所述变频模块通过所述真空系统分别与每一所述第一开口和每一所述第二开口连接。
  6. 如权利要求5所述的分离装置，其特征在于，所述变频模块包括变频器以及与所述变频器连接的控制阀，所述控制阀分别与所述第一真空泵以及所述第二真空泵中的其中一个连通，从而使所述第一真空泵和第二真空泵反复交替工作。
  7. 如权利要求4所述的分离装置，其特征在于，所述真空系统交替地在每一所述第一腔室以及每一所述第二腔室内产生的负压形成周期的矩形脉冲信号、正弦信号或梯形信号。
  8. 一种应用如权利要求1-7中任一项所述的分离装置分离液体样本中目 标颗粒的方法，其特征在于，所述方法包括：
    向所述分离芯片的样本池提供液体样本；
    运行所述第一真空泵并通过所述第一开口在所述分离芯片内产生负压；
    停止运行所述第一真空泵；
    运行所述第二真空泵并通过所述第二开口在所述分离芯片内产生负压；
    停止运行所述第二真空泵，重新运行所述第一真空泵；以及
    重复循环以上过程，让所述第一真空泵与所述第二真空泵交替运行，实现负压在所述第一开口与所述第二开口交替切换，从而分离所述样本池内所述液体样本中的目标颗粒。
  9. 如权利要求8所述的分离装置分离液体样本中目标颗粒的方法，其特征在于，向所述分离芯片的样本池提供液体样本具体包括：
    将所述多个分离芯片放置于所述第一支撑台上，将所述多个液体样本放置于所述第二支撑台上；
    控制所述第二支撑台转动以使所述多个液体样本能够分别移动至第二预设位置；
    控制所述第一支撑台转动以使所述多个分离芯片能够分别移动至第一预设位置；以及
    控制所述采样件转动以形成一采样轨迹，所述第一预设位置和所述第二预设位置位于所述采样轨迹上，使得所述采样件采集位于所述第二预设位置上的液体样本并将所采集的液体样本加入位于所述第一预设位置的所述分离芯片中。
  10. 如权利要求8所述的分离装置分离液体样本中目标颗粒的方法，其特征在于，还包括向所述分离芯片的样本池提供液体样本和清洗液。
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