WO2016078340A1 - 微量液体分配/混合装置、系统及方法 - Google Patents

微量液体分配/混合装置、系统及方法 Download PDF

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Publication number
WO2016078340A1
WO2016078340A1 PCT/CN2015/077630 CN2015077630W WO2016078340A1 WO 2016078340 A1 WO2016078340 A1 WO 2016078340A1 CN 2015077630 W CN2015077630 W CN 2015077630W WO 2016078340 A1 WO2016078340 A1 WO 2016078340A1
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Prior art keywords
liquid
micro
signal
container
fluid
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PCT/CN2015/077630
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English (en)
French (fr)
Inventor
杜文斌
徐鹏
贠娟莉
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中国科学院微生物研究所
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Priority claimed from CN201410655191.5A external-priority patent/CN104324769B/zh
Priority claimed from CN201410655309.4A external-priority patent/CN104450891B/zh
Application filed by 中国科学院微生物研究所 filed Critical 中国科学院微生物研究所
Publication of WO2016078340A1 publication Critical patent/WO2016078340A1/zh
Priority to US15/598,186 priority Critical patent/US20170253914A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
    • B01J2/06—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops in a liquid medium
    • 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
    • B01L3/02—Burettes; Pipettes
    • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
    • 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
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B01L3/0268—Drop counters; Drop formers using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
    • 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
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1016—Control of the volume dispensed or introduced
    • 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
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • 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
    • B01L2400/02—Drop detachment mechanisms of single droplets from nozzles or pins
    • 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
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • 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
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • 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
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid

Definitions

  • the invention relates to the technical field of biochemical detection and screening, in particular to a biochemical reaction detecting device, system and method based on micro-liquid dispensing and/or mixing.
  • the field of life science involves various studies such as genetic engineering, protein engineering, and drug screening. These studies require the transfer and distribution of a large number of liquids containing nucleic acids, proteins, and other organic and inorganic substances, and the scientific development has made people more and more The more prone to high-throughput distribution of trace amounts of liquid.
  • the common micro-liquid distribution technology has contact type (Jia Zhenzhong, et al; research progress of contact type spotting robot and related technology, robot, 2007, 29(2): 179-185) and non-contact type (Liu Yaxin, et al; based on compound Intelligently controlled non-contact liquid quantitative distribution system, Journal of Mechanical Engineering, 2010, 46(20): 175-181).
  • Microfluidic generation technology based on microfluidics has been rapidly developed in recent years (Teh SY, et al., 2008. Droplet microfluidics. Labon a Chip, 8(2): 198-220), the generation of droplets is The interface is unstable based on the intersection of the dispersed phase and the continuous phase in the microchannel. Through the different microfluidic channel chip design, it is possible to generate droplets of uniform size and perform operations such as fusion, reaction and sorting.
  • the most common single-cell analysis methods include the following: (1) micromanipulation techniques; (2) laser capture microdissection; (3) laser-induced fluorescence detection for flow cytometry; (4) micro Flow control flow sorting.
  • Micromanipulation techniques and laser capture microdissection techniques are used to manually separate cells or particles with different optical characteristics under a microscope, so the flux is low, and these operations may destroy the integrity of the sample, not optimal.
  • Single cell isolation method (Espina, V., et al., 2006. Lasercapture microdissection. Nat. Protoc. 1, 586-603).
  • Flow cytometric sorting is a high-throughput single-cell sorting method based on fluorescent labeling.
  • the general analysis speed is 5000-10000 per second. It is generally required that cells need to be fluorescently labeled to be sorted (Rinke, C, et al., 2014. "Obtaining genomes from uncultivated environmental microorganisms using FACS-based Single-cell genomics, "Nat Protocols, 9 [5]: 1038-45.), some cells can be sorted by their scattered light signals. Cells passed through a flow cytometer are dispersed in nanoliter to picoliter volumes. The droplets can be dispensed one by one into the multi-well plate for subsequent reaction and analysis of the single cells.
  • the position of the dropped droplets is usually uncertain, and the microdroplet array is It is easy to occur that the droplets are adsorbed on the sidewalls and the subsequent sample mixing operation cannot be performed.
  • the existing multi-well plate loading operation volume is usually in a microliter volume, and the measured single-cell bacteria scale is only ⁇ 1 micron, which is It brings great uncertainty to the subsequent cultivation, sequencing and other processes. How to reduce the volume of reagents and improve the reliability of analysis is of great significance for improving the sensitivity of analysis and reducing the pollution risk of analysis.
  • Microfluidics is an emerging single-cell, single-molecule separation technology that uses differently designed chips to separate, analyze, and detect single cells, individual DNA molecules, or individual protein molecules by controlling microfluidics.
  • Microfluidic chips can be used for subsequent single cell culture, lysis and sequencing, and are therefore currently recognized as ideal single cell separation assays (Cho, BS, et al., 2003. Passively driven integrated microfluidic system for separation of motile sperm.Anal.Chem. 75, 1671-1675).
  • There are many methods for separating single cells or single molecules using microfluidic chips to generate droplets Marcy Y, et al., 2007. Nanoliter reactors improve multiple displacement amplification of genomes from single cells.
  • PLoS Genet3 (9 ):e155.).
  • the design, processing and operation of microfluidic chips are complex and costly, and many factors need to be considered, such as specific flow rate, oil-water interfacial tension, channel configuration and channel surface modification, etc., to generate droplet volume by various fluid mechanics.
  • the influence of the channel structure cannot be accurately quantified, and the range of adjustment is also restricted by the above factors.
  • specific steps and devices are required to be transferred to the storage container, and it is difficult to customize the conditions of the individual droplets, and the operations of positioning, extracting and analyzing the droplets are inconvenient.
  • Chinese patent application CN103954786A discloses a semi-contact continuous spotting and liquid addition method for oil droplets.
  • a certain amount of oil phase is covered on the chip, and the capillary tip is finely adjusted to maintain a certain distance from the lower surface of the micropore of the chip, and the distance is just enough to make the droplet to be dispensed contact the droplet at the bottom of the container, and simultaneously After the capillary tip pushes a certain amount of liquid to form a small droplet, the droplet contacts the bottom of the container, and since the lower surface of the micropore is previously treated to be hydrophilic, the tip of the droplet is detached from the tip of the capillary and attached to the chip.
  • the difficulty of this method is that the distance between the capillary tip and the lower surface of the micropore of the chip needs to be precisely controlled for each spotting, and in the case of continuous spotting, it is necessary to adjust the lower surface of all the micropores at the same level.
  • the droplet volume is small, the gap between the capillary tip and the small surface of the micropore is very small, so this adjustment requires a microscope, and once adjusted, the entire device including the chip that receives the droplet can no longer move.
  • the current micro-liquid distribution technology has the problems of complicated equipment, high cost, inconvenient operation, low versatility, and a narrow liquid volume adjustment range, which limits the popularization and wide application of the technology.
  • micro-liquid dispensing and/or mixing device that is simple in operation, low in cost, and capable of precise control and adjustment in volume.
  • the device utilizes micro-pipeline-based micro-fluiding technology in droplets. Biological/chemical samples are included and further reactions and/or analyses can be performed for each droplet.
  • micro-liquid dispensing and/or mixing device as well as a handling method for micro-liquid dispensing and/or mixing, for various applications based on biological and/or chemical reactions, for example Single cell culture condition screening, single cell genomic and transcriptome amplification, single nucleic acid molecule amplification, enzyme kinetics studies, protein crystallization conditions screening, drug activity screening, etc., providing microsystems, high throughput, low cost, easy operation A small amount of liquid distribution program.
  • the invention provides a micro-liquid dispensing/mixing device.
  • the device includes a fluid drive unit and a mechanical moving unit, wherein
  • the fluid drive unit includes a fluid drive device for driving a first liquid in a microchannel connected to the fluid drive device to advance toward an outlet end of the microchannel at a flow rate; and
  • the mechanical moving unit is configured to control the micro-pipe and/or the container containing the second liquid to move in at least one-dimensional direction to cooperate with the start and stop of the fluid-driven device to make the outlet end of the micro-pipe Inserting and leaving the second liquid to add a given volume of the first liquid to the container,
  • first liquid and the second liquid are not miscible.
  • the fluid drive unit further comprises a conduit member having one or more fluid passages for sealing connection to the at least one of the microchannels in fluid communication with the fluid drive device.
  • the conduit member has an array of fluid passages corresponding to the arrangement of the containers, wherein the conduit members are coupled to one or more fluid drive devices, and each fluid passageway is associated with a microchannel connection.
  • the conduit member has an array of fluid passages corresponding to standard multi-well plates (e.g., 24, 96, 384 or 1536 orifice plates).
  • the fluid drive unit may comprise two or more fluid drive devices, each fluid drive device being connected to at least one microchannel. In this way, different liquids can be dispensed at the same time, and different volumes of liquid can be dispensed at the same time, thereby improving liquid distribution efficiency and device utilization efficiency.
  • each or several (e.g., one row or column) of fluid passages of the above described conduit components having arrayed fluid passages may be driven by a fluid driven device.
  • the mechanical moving unit of the micro-liquid dispensing/mixing device of the present invention comprises a machine for controlling the movement of the micro-pipe A robotic hand, and/or a translation stage for controlling movement of the container.
  • the robot and the translation stage are each movable in at least one dimension.
  • the movement of the mechanical moving unit is smoothly performed, preferably at a uniform speed, to avoid the shock caused by the movement to the liquid distribution system, and affect the liquid dispensing accuracy.
  • the micro-liquid dispensing/mixing device of the present invention further includes a control and feedback unit, the control and feedback unit comprising: a signal receiving module, a computing module, and a signal output module.
  • the signal receiving module is configured to receive a signal and transmit the signal to the computing module; the computing module receives the signal and performs calculation and/or comparison with a set value, and then calculates and/or The comparison result is transmitted to the signal output module; and the signal output module receives the calculation and/or comparison result and converts the result into an instruction signal output.
  • the signal is a liquid volume signal to be dispensed.
  • the calculation module receives the liquid volume signal to be distributed transmitted by the signal receiving module, selects a suitable fluid driving speed according to the signal, and calculates a running time of the fluid driving device in the fluid driving unit, or according to the a signal and a preset fluid drive speed, calculating a running time of the fluid driving device in the fluid driving unit, and transmitting a calculation result to the signal output module, the signal output module receiving the calculation result and The calculation result is converted into a command signal, which is respectively transmitted to the fluid drive unit and the mechanical movement unit to control the mechanical movement unit to cooperate with the fluid drive unit to start, stop moving the micro-pipe and/or the container to complete the distribution of the first liquid.
  • control and feedback unit further includes a distance detecting module capable of acquiring a relative positional relationship signal between the outlet end of the micro-pipe and the target container, and an exit end and a target of the micro-pipe a distance signal between the liquid levels of the second liquid in the container, and capable of transmitting the acquired relative positional relationship signals and/or the distance signals to the signal receiving module.
  • a distance detecting module capable of acquiring a relative positional relationship signal between the outlet end of the micro-pipe and the target container, and an exit end and a target of the micro-pipe a distance signal between the liquid levels of the second liquid in the container, and capable of transmitting the acquired relative positional relationship signals and/or the distance signals to the signal receiving module.
  • the signal receiving module receives the signal and transmits the signal to the computing module; the computing module receives the signal and compares it with a preset liquid level distance value of the micropipe outlet end and the second liquid, and compares The result is transmitted to the signal output module; the signal output module receives the comparison result and converts it to a command signal for transmission to the mechanical movement unit to adjust the movement of the mechanical movement unit until the microchannel is positioned at a predetermined distance above the second liquid level.
  • the distance detecting module is further capable of acquiring a depth signal of the second liquid and transmitting it to the signal receiving module.
  • the signal receiving module receives the depth signal of the second liquid and transmits the depth signal to the calculation module; the calculation module receives the depth signal and compares it with the set minimum depth value, and transmits the comparison result to the signal output module; when the measured depth value is greater than When the minimum depth value is set, the output module sends a signal for liquid distribution to the fluid drive unit and the mechanical movement unit, and when the measured depth value is less than the set minimum depth value, the output module sends out to the fluid drive unit and the mechanical movement unit.
  • the signal for liquid dispensing is stopped, and more preferably the alarm signal is simultaneously issued.
  • the control and feedback unit is used to precisely control the operation of the fluid drive unit and the mechanical moving unit to increase the liquid Accuracy and precision of distribution.
  • the control and feedback unit can control the micro-liquid dispensing/mixing device of the present invention to automate complex multiple liquid dispensing schemes, thereby Further improve liquid distribution efficiency and device use efficiency.
  • the micro-liquid dispensing/mixing device of the present invention may further comprise a sterilizing/dusting and gas replacing unit as needed.
  • the sterilization/dust and gas displacement unit may be a filtration device that may form an enclosed space, may be disposed, for example, on the outer circumference of the micro-liquid distribution/mixing device, or on the outer circumference of the micro-tube and the container.
  • the sterilization/dust and gas displacement unit may also be an ultraviolet sterilization lamp disposed adjacent to the microchannel and the container. It is also possible to combine the air filtering device and the ultraviolet sterilization lamp to form the sterilization/dust and gas replacement unit. It may also include means for replacing the gas within the apparatus to achieve inert gas protection, anaerobic operation, and a gas atmosphere required to supplement cell culture or biochemical reactions such as carbon dioxide, oxygen, and hydrogen.
  • the micro-liquid dispensing/mixing device of the present invention further comprises a temperature control and feedback unit.
  • the temperature control and feedback unit may include a heating and/or refrigeration device, and a temperature control device.
  • the temperature control and feedback unit directly heats and/or cools the platform for placing the container.
  • the temperature control and feedback unit can also be a separately provided incubator. This provides the desired temperature to the system during the liquid dispensing process for reaction or analysis.
  • the micro-liquid dispensing/mixing device of the invention consists of or consists essentially of the fluid drive unit, the mechanical movement unit and the control and feedback unit.
  • the micro-liquid dispensing/mixing device of the present invention is distinguished from the existing micro-liquid dispensing/mixing device mainly in its structure and operation. In conventional micro-liquid dispensing/mixing devices, it is also common to include equipment that drives a given volume of droplets out of the microchannel, such as piezoelectric devices, heating devices, spraying devices, and the like.
  • the micro-liquid dispensing/mixing device of the present invention consists of or consists essentially of the fluid drive unit and the mechanical moving unit, as well as an optional control and feedback unit.
  • the fluid drive unit is for generating a droplet of a given volume of the first liquid
  • the mechanical movement unit is for generating the droplet by passing the outlet end of the microchannel through the liquid level of the second liquid Assigned to the second liquid.
  • the micro liquid distribution/mixing device of the invention has a simple structure and convenient operation, and the liquid amount can be adjusted within a wide range of the flying up to the nanoliter level. Moreover, the amount of liquid dispensed is accurate and can meet the needs of experiments and tests.
  • the micro-liquid dispensing/mixing device of the present invention can directly use a conventional standard multi-well plate as a container, and can not only perform high-throughput experiments, but also directly utilize existing standard experiments and / or testing equipment, without the need for additional equipment, greatly facilitating the user and greatly reducing the cost of the experiment.
  • a micro-liquid dispensing system comprises the above-described micro-liquid dispensing/mixing device, micro-tubes and containers. Wherein the micro-pipe and the micro-liquid distribution The fluid drive unit of the mixing device is coupled for dispensing a first liquid contained therein; the container containing a second liquid for receiving droplets of the dispensed first liquid.
  • microchannels used in the micro-liquid dispensing system of the present invention have a cylindrical opening or a tapered opening, preferably a tapered opening.
  • the microchannel may be a single single core capillary, a single multi-core capillary, a bundled capillary, an array capillary, a capillary with a cannula, or a microfluidic chip.
  • the microchannel has an enlarged upper end formed as a reservoir.
  • a single single-core capillary such as a glass or quartz capillary, is preferred.
  • Such capillaries can further form a capillary bundle or array.
  • a capillary tube which is further tapered at the outlet end to form a tapered opening can obtain a satisfactory micro-liquid distribution result.
  • Suitable micro-pipes including various capillary or micro-pipe chips, can be used in the present invention, and the present invention is not particularly limited in form, structure, material, and the like of the micro-pipe. Those skilled in the art will be able to make appropriate selections as needed.
  • the opening of the microchannel is treated with a low surface energy. More preferably, the low surface energy treatment is a silylation treatment. This allows the dispensed droplets to detach more smoothly from the microchannel.
  • the container for the micro-liquid dispensing system of the present invention can be any suitable container.
  • the container is an array of containers that can be arranged in a single container, one or two dimensions.
  • the container is a two-dimensional array of containers, particularly preferably standard 24, 96, 384 or 1536 well plates.
  • the system of the present invention can interface directly with conventional standardized experiments and/or testing equipment without the need for additional dedicated experimental and/or testing equipment.
  • the micro-pipes can be simultaneously arranged into a corresponding two-dimensional array of micro-pipes. This can greatly increase the flux of liquid distribution and improve the efficiency of liquid distribution.
  • the bottom of the container is a pointed bottom, a conical bottom, a rounded bottom or a flat bottom.
  • the sharp bottom, the round bottom or the like facilitates the concentration of the dispensed droplets to the bottom, which is beneficial to the fusion and reaction between the multiple droplets, and also facilitates the rapid and accurate capture of the target to be tested by the detecting device.
  • the first liquid has a specific gravity greater than a specific gravity of the second liquid.
  • the droplets of the first liquid thus dispensed can sink to the bottom of the container.
  • a method of dispensing a small amount of liquid includes:
  • first liquid and the second liquid are not miscible.
  • the first liquid droplets are detached from the micro-fluid under the fluid shear force of the second liquid and the liquid surface tension at the gas-liquid or liquid-liquid interface of the second liquid.
  • the outlet end of the conduit is dispensed into the second liquid.
  • the volume of the droplets of the first liquid to be dispensed is in the range of 100 fL to 100 uL, preferably 100 pL to 1 uL, more preferably 200 pL to 200 nL.
  • the container is a two-dimensional array of containers.
  • the micro-pipes are corresponding two-dimensional arrays of micro-pipes; preferably, the container is a standard 24, 96, 384 or 1536-well plate; more preferably, the bottom of the container is a pointed bottom, a conical bottom, a circular bottom or flat.
  • the first liquid has a specific gravity greater than the second liquid.
  • the container further contains a third liquid covering the second liquid, which is immiscible with the first liquid and the second liquid; or A third liquid is located below the second liquid and is immiscible with the second liquid.
  • the technical solution of the present invention utilizes the interfacial energy and fluid shear force when the gas-liquid or liquid-liquid interface is changed, so that the trace liquid attached to the outside of the micro-pipe outlet overcomes the adhesion with the microchannel outlet. And with the surface tension of the plurality of internal liquids, the droplets flowing out of the micro-pipe nozzle can be smoothly separated from the micro-pipe.
  • the size of the droplets is regulated by the fluid drive unit, the accuracy of which is related to the size of the opening of the microchannel and the precision of the fluid drive device.
  • the present invention distributes liquid that is flying up to the nanoliter level by simply inserting the outlet end of the microchannel into a second liquid that is incompatible with the liquid to be dispensed.
  • the liquid dispensing amount of the present invention can be directly controlled by the liquid flow rate and the driving time in the microchannel, and therefore, the volume of the liquid to be dispensed can be flexibly adjusted as needed.
  • micro-pipe arrays eg, using pipe components with array-like multiple flow channels
  • standardized containers such as porous enzyme plates, etc.
  • It can also realize multi-step ultra-micro standardized biochemical reaction and detection, and has broad application prospects.
  • lysis of a single cell can be carried out by adding microdroplets of the lysate to fuse with single cell microdroplets to prepare a single cell nucleic acid, protein or the like.
  • the prepared single-cell nucleic acid or protein can be taken up by diluting into large droplets, or left in the second liquid to continue the subsequent reaction, and DNA analysis, RNA analysis, protein analysis, etc. can be performed on single cells, according to experiments.
  • the purpose is different, followed by the addition of matching reaction liquid droplets. Because it is wrapped in the oil phase, the droplets will not evaporate, which is more conducive to the extraction and storage of droplets or microspheres.
  • each reaction can be done independently in different wells of the multiwell plate, avoiding cross-contamination; each reaction is completed in the nano-drops, which greatly reduces the cost.
  • FIG. 1 shows a schematic structural view of a micro-liquid dispensing/mixing device in accordance with an embodiment of the present invention.
  • FIG. 2 is a schematic diagram of a method of dispensing a small amount of liquid according to an embodiment of the present invention.
  • FIG. 3 is a schematic diagram showing the structure and micro-filling mode of a two-dimensional array container, micro-pipe and pipe components according to an embodiment of the present invention.
  • FIG. 4 is a schematic illustration of a dual core single capillary dispensing liquid in accordance with the present invention.
  • FIG. 5 is a schematic illustration of a capillary bundle dispensing liquid consisting of three single core single capillaries in accordance with the present invention.
  • Figure 6 is a schematic illustration of a capillary dispensing liquid with a cannula in accordance with the present invention.
  • Figure 7 is a schematic illustration of a four-channel, in-line hybrid chip-type microchannel dispensing liquid in accordance with the present invention.
  • Figure 8 is a schematic illustration of a chip-type microchannel dispensing liquid that can be prepared in accordance with the present invention.
  • Figure 9 is a schematic illustration of the sequential distribution of two solutions into a container and fused to each other in accordance with one embodiment of the present invention.
  • Figure 10 is a photomicrograph of the distribution of two 5 nL droplets of different colors as a 10 nL droplet according to the method of Figure 9.
  • Figure 11 is a bar graph of droplet diameter variations of two approximately 5 nL droplets dispensed into one 10 nL droplet according to the method of Figure 9.
  • Figure 12 is a schematic illustration of a method of dispensing microdroplets into a relatively large amount of system in accordance with one embodiment of the present invention.
  • Figure 13 is a schematic illustration of a method of dispensing a small amount of droplets of two first liquids into a second liquid under a third liquid blanket in accordance with one embodiment of the present invention.
  • Figure 14 is a schematic view showing the connection of a micro liquid distribution/mixing device control module according to an embodiment of the present invention.
  • 15 is a flow chart showing the operation of a control module of a micro-liquid dispensing/mixing device in accordance with an embodiment of the present invention.
  • the first liquid referred to herein is a general term for the liquid to be dispensed, and is not limited to a liquid or a solution.
  • the first liquid may be a plurality of different liquids, such as a solution prepared by separately preparing a plurality of samples; or a mixed solution in which a plurality of liquids are mixed in-line before dispensing.
  • the first liquid is an aqueous solution or an aqueous solution, but is not limited thereto, and may be, for example, a hydrophobic liquid that is insoluble in the second liquid.
  • the second liquid referred to herein is a liquid used to "shear" the first liquid droplets away from the microchannel, and also to receive the first liquid droplets; in some cases, The environment required for the reaction, such as oxygen barrier, light protection, heat preservation, and the like.
  • the second liquid is a liquid or solution that is immiscible with the first liquid and is inert. It is preferably a non-volatile oily liquid.
  • the third liquid may be a liquid or a solution associated with the first liquid, such as a diluent for diluting the first liquid, a large volume reaction solution for reacting with the first liquid, or the like; or an auxiliary liquid of the second liquid, for example, A liquid-sealed liquid or the like is supplied to the second liquid.
  • liquid refers to a liquid which is liquid in a desired environment, such as a solid at normal temperature and a liquid at a higher temperature, or a gas at normal temperature and a liquid at a lower temperature.
  • Trace The trace amount mentioned in this paper, such as the distribution of trace liquid or trace liquid, refers to the amount of liquid that cannot be accurately and repeatedly removed by conventional pipettes, pipettes/guns, etc., usually below the microliter level, especially It is the amount of liquid that rises to the nanoliter level. It also includes the case where the so-called “trace” liquid is mixed with the "constant” liquid and then redistributed.
  • a microchannel as referred to herein is used to dispense a first liquid, which may be any form of conduit containing at least one liquid flow path. The most common and lowest cost is the capillary. It can also be a microchannel of a chip type multi-channel, or a tube with a casing containing at least one micro-pipe.
  • a container as referred to herein is a container that holds a second liquid for receiving a first liquid.
  • the container may be in any form, but a versatile standard multiwell plate is preferred. In particular, containers with narrowed bottoms, such as pointed bottoms, rounded or elliptical bottoms.
  • the present invention is directed to an apparatus capable of accurately performing a small amount of liquid distribution capable of accurately performing the dispensing of a liquid from a flying level to a nanoliter level.
  • the device of the present invention is simple in operation, low in cost, and easy to adjust the volume of the liquid, and can be widely applied to biological and chemical reactions of various microsystems, and high-throughput screening experiments. .
  • microliter liquid dispensing when small volumes, such as nanoliters, picoliters, or even ascending volumes of liquid, are required, tiny droplets exiting the orifice when using conventional capillary tubes Its own gravity is far from enough to overcome its adhesion to the nozzle, surface tension and surface tension with the liquid in the tube, so it cannot be directly obtained. This is also the reason why the current method of dispensing micro-liquids is another way.
  • the micro-liquid dispensing/mixing device of the present invention still utilizes micro-pipes such as capillaries for liquid dispensing.
  • the dress The apparatus includes: a fluid drive unit including a fluid drive device for driving a first liquid in a microchannel connected to the fluid drive device to advance toward an outlet end of the microchannel at a flow rate; and a mechanical movement unit for Controlling the microchannel and/or the container containing the second liquid to move in at least one dimension in cooperation with activation and deactivation of the fluid drive to add a given volume of the first liquid to the vessel Wherein the first liquid and the second liquid are immiscible.
  • Figure 1 shows a schematic representation of one embodiment of a micro-liquid dispensing/mixing device in accordance with the present invention.
  • the fluid drive unit includes a syringe pump 5 as a fluid drive device and a Teflon capillary tube 4 as a pipe member for connecting the syringe pump 5 and the capillary tube 1 as a microchannel.
  • the first liquid is filled with air bubbles and filled with the lumen of the syringe pump 5, the Teflon tubule 4, and the capillary 1.
  • the mechanical moving unit includes a robot 6 that controls the capillary movement up and down along the z-axis in the vertical direction, and a container array 2 for placing the container 3 having a plurality of apertures 3 and drives the container array 2 to move in the horizontal x-axis and/or y-axis directions.
  • Translation stage 7 The mechanical moving unit includes a robot 6 that controls the capillary movement up and down along the z-axis in the vertical direction, and a container array 2 for placing the container 3 having a plurality of apertures 3 and drives the container array 2 to move in the horizontal x-axis and/or y-axis directions.
  • Translation stage 7 7.
  • the first liquid that is, the liquid to be dispensed
  • the first liquid is previously loaded into the microchannel and filled with the microchannel, at least filling the downstream end portion of the microchannel, for example, the first liquid can be driven to the microchannel by the fluid drive device
  • the first liquid can also be drawn into the microchannel by running the fluid drive device in the reverse direction.
  • the second liquid which is an inert liquid that is immiscible with the first liquid, is previously loaded into the container.
  • the container is used to carry out the reaction or to carry out further experiments.
  • Figure 2 shows a schematic representation of liquid dispensing in accordance with the method of the present invention.
  • the microchannel 1 and/or the vessel 3 are moved such that the outlet end of the microchannel extends below the level of the second liquid 9.
  • the flow rate of the fluid driving device 5 is set, and the fluid driving device 5 is turned on for a certain time, so that under the wrapping of the second liquid 9, a drop is formed outside the outlet end of the micro-pipe.
  • the microchannel 1 and/or the container 3 are moved again such that the outlet end of the microchannel 1 is drawn out of the liquid level of the second liquid 9.
  • the droplets 10 at the outlet end are "cut” by the interfacial energy and fluid shear force when the gas-liquid phase interface is changed, so that The droplets are smoothly separated from the nozzle to form a liquid into the second liquid 9.
  • the first liquid 8 has a larger specific gravity than the second liquid 9, so that the droplets 10 formed by the first liquid converge to the bottom of the container 3. This approach is preferred for systems that require further reaction with other droplets, such as the embodiment shown in Figure 9 which will be detailed below.
  • the first liquid has a specific gravity greater than the second liquid, and the tip bottom or the round bottom container can be directly used.
  • the droplets are accurately observed at the fixed position of the bottom center; or the flat bottom container is centrifuged by a centrifuge to realize a plurality of droplets pooled and fused under the action of centrifugal force to perform quantitative reaction.
  • micro-liquid dispensing/mixing device of the present invention The various portions of the micro-liquid dispensing/mixing device of the present invention are described in detail below with reference to FIG.
  • the fluid drive unit of the present invention primarily comprises a fluid drive device.
  • the fluid drive device is in addition to the syringe pump shown, 5 It may be any fluid-driven device capable of continuously driving the liquid flow, and may be, for example, a peristaltic pump, a pressure-driven pump, a pneumatically driven pump, or an electroosmotic drive pump, but is not limited thereto.
  • a microinjection pump is preferred.
  • droplets that are flying up to nano-upgraded can be obtained using a syringe pump of higher precision.
  • the fluid drive speed of the fluid-driven device is typically in the range of 0.5 pL/min to 10 mL/min, preferably 10 nL/s to 100 nL/min. Since the amount of liquid to be dispensed is very small, in order to obtain a high-precision volume of liquid, the accuracy error of the liquid flow rate needs to be less than ⁇ 1%, and the preferred error is less than or equal to ⁇ 0.5%.
  • the fluid drive unit may further include a pipe member 4 having a fluid passage connected to the fluid supply device 5 at one end and a fluid passage at the other end.
  • the pipe component may be a single channel pipe such as the illustrated Teflon tubule 4, or a collective or integral pipe component having multiple fluid passages. In practical applications, the latter can be connected to a plurality of micro-pipes or to a micro-pipe with multiple channels, so that liquid distribution to multiple containers can be performed simultaneously, or multiple liquids can be dispensed at the same time, thereby being more efficient.
  • the multi-channel pipe components can be designed according to the arrangement of the array of containers to be liquid-distributed. For example, for multi-well plates, it is possible to design pipe components with corresponding flow paths to increase liquid distribution efficiency.
  • Figure 3 shows a pipe part 4 having an array-like multiple flow path corresponding to the array of containers 2.
  • the pipe member 4 has a tubular inlet 4-1, a rectangular communication cavity 4-2, and a plurality of array-shaped tubular outlets 4-3.
  • the spacing between the tubular outlets 4-3 corresponds to the arrangement spacing of the respective arrayed containers 2, one for each tubular outlet.
  • the flow path arrangement of the pipe parts can correspond to existing multi-hole plates such as 96-hole, 384-hole or even 1536-hole, so that the user can directly use the experimental equipment provided in the conventional laboratory without being separately purchased.
  • the apparatus of the present invention can be used in combination with an existing detecting device, a drug screening device, a microplate reader, and the like.
  • One end of the duct member 4 may be fixedly or detachably coupled to the fluid drive device 5. Since the apparatus of the present invention is used for the reaction and experiment of a small amount of sample, avoiding contamination is a very critical operation. A detachable connection is preferred if the sample solution is to be contacted in the liquid flow path of the pipe member. However, if the sample solution is not required to be contacted (as one embodiment will be discussed in detail below), the fixed connection is more advantageous because it generally ensures airtightness between the pipe member and the fluid-driven device. Air tightness is a condition necessary to ensure accurate micro-liquid distribution.
  • the other end of the pipe member may be provided with an interface suitable for connecting the micro pipe to be conveniently connected to the micro pipe in a gastight manner.
  • the manner in which the interface is connected to the micropipe is not particularly limited, and any suitable method is possible.
  • it may be a screw interface, a bayonet, a bulge plug, a plug interface, etc., but is not limited thereto.
  • a sealant can be further applied at the interface to ensure The air tightness and tightness of the connection. 1 Since the micro-pipes are usually disposable, the interfaces are preferably those that are easily connected quickly, for example, the interfaces and the corresponding interfaces of the micro-pipes have complementary structures, respectively.
  • the size of the pipe member is not particularly limited, and is usually adapted to the flow rate range of the fluid drive device and the range of liquid volume to be dispensed.
  • the simplest is a flexible Teflon tubule with an inner diameter of 300 ⁇ m, an outer diameter of 600 ⁇ m and a length of 15 cm for easy direct connection and for capillary microchannels with an outer diameter of more than 300 ⁇ m and less than 400 ⁇ m. It does not require the use of an adhesive and can be inserted directly into the end of the Teflon tubing to ensure a tight seal.
  • the fluid drive unit may further comprise a reservoir/storage.
  • the reservoir/storage is in fluid communication with the fluid drive device for continuous supply of the first liquid to the fluid drive device.
  • the injection lumen of the syringe pump can also be considered a reservoir.
  • the reservoir/storage can be one or more. When the reservoir/storage is multiple, it may separately accommodate different first liquids, for example, liquids containing different samples.
  • the number of reservoirs/bins may correspond to the number of fluid passages of the conduit components comprising a plurality of fluid passages.
  • the fluid drive unit does not contain a reservoir/storage, nor does it load any fluid or only an inert fluid rather than a reaction fluid.
  • the first liquid is only loaded into the microchannel, for example, it can be sucked into the microchannel by sucking.
  • the upper portion or other portion of the microchannel may be slightly expanded to form a reservoir to accommodate a sufficient amount of the first liquid.
  • the fluid drive unit indirectly drives the first liquid flow by generating air pressure or by driving an inert fluid. The advantage of this embodiment is that the fluid drive unit does not contact the sample solution and thus does not cause contamination, and it is not necessary to clean/replace contaminated fittings each time, and only need to replace the lower cost micro-pipes.
  • the volume of the reservoir/storage is not particularly limited, but it is usually not very large because the volume of liquid dispensed is very small.
  • it may be in the order of microliters, such as 5 to 500 microliters or may be smaller or larger, and commonly used may be 5, 10, 20, 50, 100, 150, 200, 250, 300, 400, 500 micro. l, etc., but not limited to this.
  • the mechanical moving unit comprises a robot 6 for controlling the microchannels and a translation stage 7 for controlling the movement of the container array 2.
  • the robot 6 may specifically be a vertical lifting translation stage driven by a stepping motor that controls the micro-duct 1 to move along the z-axis in the vertical direction.
  • the microchannel 1 is first moved down along the z-axis, and the outlet end of the microchannel is inserted into the second liquid in the target container 3 (e.g., a perforated container shown in Figure 1).
  • the actuating fluid drive device 5 drives the first liquid 8 to form a given volume of droplets 10 at the outlet end of the microchannel, then stops the drive, and then moves the microchannel 1 upwards, leaving its outlet end away from the second liquid 9, the droplets 10 formed by the first liquid are left in the container 3.
  • the translation stage 7 can be a two-dimensional XY translation stage driven by a stepping motor or a servo motor, or a mechanical arm with a three-dimensional positioning function, and the array of containers 2 to be determined along the x-axis And / or y-axis
  • the next perforated container 3 is moved below the microchannel, and then the robot 6 and the fluid drive assembly 5 are repeated to add a given volume of the first liquid to the perforated container. This repetition may sequentially add a given volume of the first liquid to each container of the container array 2.
  • the robot can move in a vertical mode (z-axis) and a horizontal direction (x-axis or y-axis), while the translation stage can move in only the other horizontal direction.
  • the mechanical movement unit may comprise only a robot or only a translation stage.
  • the mechanical moving unit can be a robot or translation stage that can move in three dimensions.
  • a robot or a translation stage capable of moving only in the vertical direction is sufficient.
  • the micro-liquid dispensing/mixing device of the present invention can be further simplified.
  • the driving device that drives the components of the mechanical moving unit to move is preferably those capable of smooth driving to improve the accuracy of liquid dispensing.
  • the micro-liquid dispensing/mixing device further comprises a control and feedback unit.
  • the control and feedback unit includes a signal receiving module, a computing module, and a signal output module.
  • the signal receiving module 011 of the control and feedback unit 010 receives a signal such as an artificially set liquid volume to be dispensed and transmits the received signal to the calculation module 012 (S1); 012 calculates the running time of the fluid driving device in the fluid driving unit 020 according to a predetermined fluid driving speed or the like, or selects a suitable fluid driving speed according to the volume to be dispensed, and transmits the calculation result to the signal output module 013 (S2)
  • the signal output module 013 controls the mechanical movement unit 030 to cooperate with the start of the fluid drive unit 020 to start and stop moving the micro-pipe and/or the container according to the running time of the fluid drive unit 020 calculated by the calculation module 012 to complete the first liquid. Distribution (S3).
  • the signal output module 013 specifically controls the mechanical moving unit 030 to move the micro-pipe and/or the container, and positions the micro-pipe to the upper part of the target container and extends the outlet end of the micro-pipe into the liquid level of the second fluid in the container. Then, after the control fluid drive unit 020 starts and runs for the calculated length of time, it stops, and then controls the mechanical moving unit 030 to move the outlet end of the micro-pipe to the outside of the liquid surface of the second fluid, thereby completing a reserved volume. The distribution of the first liquid.
  • control and feedback unit further comprises a distance detection module (not shown) to enable detection of the relative positional relationship of the outlet end of the microchannel with the target container, and also to the outlet end of the microchannel The distance between the liquid levels of the second liquid in the target container is detected.
  • a distance detection module (not shown) to enable detection of the relative positional relationship of the outlet end of the microchannel with the target container, and also to the outlet end of the microchannel The distance between the liquid levels of the second liquid in the target container is detected.
  • the liquid level detecting module can acquire the distance information between the outlet end of the micro pipe and the liquid level in the container or the container in real time, and then transmit the information to the signal receiving module 011; the signal receiving module 011 receives the distance signal and transmits the distance signal to the calculating module 012; Module 012 receives the distance signal and pre-
  • the first set microchannel outlet end is compared with the liquid level distance value of the second liquid, and the comparison result is transmitted to the signal output module 013; the signal output module 013 receives the comparison result, and converts it into a command signal and transmits it to the mechanical moving unit.
  • the mechanical moving unit drives the micro-pipe to move to the liquid surface until the micro-pipe is positioned to a predetermined distance above the second liquid level.
  • control and feedback unit then controls the apparatus of the present invention to complete the dispensing of the first liquid in the target container in accordance with the methods described above.
  • the control and feedback unit can also detect whether the amount of the second liquid (or liquid depth) in the target container reaches a desired minimum value (for example, using the distance detecting module to detect the liquid level of the second liquid to the bottom of the container) The distance is) and the signal output module alarms and automatically stops the liquid dispensing of the container if the amount of the second liquid is below the minimum value. For example, when the control and feedback unit detects that there is no second liquid in a target container, it suspends the operation and reminds the target container that it is not suitable for the drop addition experiment.
  • the manner of performing liquid level detection on the liquid level of the second liquid may include an infrared laser ranging sensor, an ultrasonic distance measuring sensor, etc., which are familiar to those skilled in the art and will not be described herein.
  • the control module can repeat the operation according to the calculation result of the calculation module until it is added to each hole Book a volume of liquid. It is of course also possible to use a plurality of micro-pipes arranged in the same manner as the container, for example 96 micro-pipes arranged in a 96-well plate arrangement, the control and feedback unit being capable of controlling the start and stop of the fluid drive unit and controlling the movement of the mechanical moving unit, The dispensing of the first liquid is accomplished in a single container at the same time.
  • the control module 010 can also simultaneously control the plurality of fluid drive units 020 and/or the plurality of mechanical movement units 030 to cooperate to perform different volumes of liquid dispensing simultaneously.
  • the micro-filling device of the present invention further comprises a sterilization/dust unit.
  • the sterilizing/dusting unit may comprise an air filtering device, which may be arranged outside the entire micro-liquid dispensing/mixing device to form a sealable space; or may be arranged only outside the liquid dispensing region, forming a micro-pipe and a container Sealed structure.
  • a closable space is formed on the outer circumference of the micro-pipe 1 and the container 5, a sterilizing/dust filtering member is disposed at an opening allowing gas to pass therethrough, a negative pressure is generated by a vacuum pump, and the micro-filling space is sterilized and dust-removed before the experiment.
  • It may also include means for replacing the gas within the equipment to achieve inert gas protection, anaerobic operation, and a gas atmosphere required to supplement cell culture or biochemical reactions such as carbon dioxide, oxygen, hydrogen, and the like.
  • the sterilizing/dust and gas displacement unit may also or further include an ultraviolet sterilization lamp or other sterilizing/dusting device mounted adjacent to the liquid dispensing region.
  • the micro-liquid dispensing/mixing device of the present invention further includes a temperature control and feedback unit for providing the desired temperature to the liquid system in the container.
  • the temperature control and feedback unit is disposed under the platform for placing the container and may include heating and/or cooling devices, as well as temperature control devices.
  • the temperature control and feedback unit can be used to provide the necessary temperature to the system in the vessel in situ.
  • the temperature control and feedback unit directly heats and/or cools the platform for placing the container to heat or cool the liquid system in the container; or the platform for placing the container is formed into a temperature Part of the control and feedback unit.
  • the temperature control and feedback unit can be arranged to directly heat and/or cool the mesa of the translation stage.
  • the temperature control and feedback unit can be arranged separately, for example as a sealable tank, such as an incubator, a container for liquid dispensing, and/or a container for liquid dispensing can be placed in the incubator. Get the desired temperature or carry out further reactions.
  • a micro-liquid dispensing system comprises the above-mentioned micro-liquid dispensing/mixing device; connected to the fluid driving unit of the micro-liquid dispensing/mixing device, for distributing the micro-channel of the first liquid contained therein; A liquid, a container for receiving droplets of the dispensed first liquid.
  • micro-liquid dispensing system of the present invention can include micro-tubing.
  • commercially available tubes suitable for producing minute droplets such as capillaries, etc., may be employed.
  • the micro-pipe 1 shown in FIG. 1 is a capillary tube which is open at both ends, and one end thereof is fluidly connected to the fluid-driven device 5 through the Teflon tube 4 and is hermetically connected for receiving the first liquid, and the end may be referred to as an inlet. The other end is used to shed the liquid and can be referred to as an outlet. However, in some cases, loading of the liquid can be accomplished by inverting the fluid-driven device to draw the first liquid from the outlet of the micro-duct into the micro-duct.
  • Micropipes are usually disposable because they are difficult to clean inside the microchannel. Therefore, the material of the micro-pipes is preferably those which are lower in cost.
  • the material forming the microchannels may be metal (eg stainless steel), quartz, glass, polymeric materials, and the like. Under certain conditions, capillaries, such as stainless steel capillaries, can be cleaned, dried, etc. for reuse.
  • the microchannel 1 may be any tube suitable for generating a trace amount of liquid, and may be, for example, a single single-core capillary, a single multi-core capillary, an array capillary, a microfluidic channel (chip), or the like, but is not limited thereto.
  • Fig. 3 shows a microchannel 1 in which the upper end is expanded to be airtightly combined with the outer circumference of the tubular outlet 4-3 of the pipe member 4.
  • the enlarged upper end can simultaneously serve as a reservoir for containing the first liquid to be dispensed.
  • FIG. 4 shows a schematic of a single two-core capillary.
  • the capillary 1 has a partition inside to divide it into two mutually non-intersecting passages, I being the first passage and II being the second passage.
  • a capillary having a smaller inner diameter is inserted into the semicircular passage, and the gap between the inserted capillary and the semicircular passage is filled with an adhesive to ensure the tightness at the time of injection.
  • the two thin capillaries are respectively connected to the syringe pump through a Teflon connecting tube, and two different solutions are respectively loaded into the two channels, so that the two-component mixed droplets 10-1 can be obtained.
  • mixed droplets 10-1 of a certain composition having different concentrations can be obtained by setting different flow rates.
  • the size of the dual core capillary can be, for example, 300 micrometers in outer diameter, 200 micrometers in inner diameter, and 50 micrometers in thickness of the intermediate separator.
  • Figure 4 shows only one case of a multi-core capillary, which may also have more "cores" in the multi-core capillary, for example 3, 4 or 5 cores.
  • Figure 5 shows a capillary bundle 1 consisting of three single-channel capillaries, wherein I, II and III are three separate capillary tubes that are closely packed together. The outlet ends are bonded together by an adhesive. The upper ends of the three capillaries are respectively connected with three Teflon connecting tubes, and are connected with three independently controlled syringe pumps, and different solutions are respectively injected into the three capillaries (for example, respectively containing components which cannot be premixed), and the trace amount of the present embodiment is used.
  • the liquid dispensing/mixing device obtains a mixed droplet 10-2 mixed by three solutions.
  • Such capillary bundles can be 2, 3, 4, 5 or more.
  • Figure 6 shows a microchannel 1 with a sleeve.
  • 01 is a hose
  • a capillary 02 is inserted into the side of the hose 01
  • a pointed tube 03 is connected to the lower portion of the hose 01, and the lower end opening of the pointed tube 03 meets the outlet end of the capillary;
  • the solution 8-1 is passed through the capillary 02
  • the solution 8-2 is passed through the hose 01.
  • the mixed droplets 10-3 mixed with the solutions 8-1 and 8-2 can be obtained under the driving of the fluid drive apparatus.
  • This cannulated microchannel can be used to simultaneously dispense large volumes of liquid 8-2 and traces of liquid 8-1 in the same system.
  • a microdroplet containing a cell suspension and a cell lysate is generated, and under a certain reaction condition, for example, incubation at 65 ° C for 10 minutes, a microdroplet coated with a nucleic acid substance released by cell lysis is obtained.
  • the hose 01 is made of a rubber material such as rubber, latex, silicone, or the like, and other materials may be used.
  • the hose 01 has a length of 2 cm and an inner diameter of 0.6 mm.
  • the rubber hose 01 is terminated with a tube 02 with a tip end, such as a Teflon tube having an inner diameter of 300 ⁇ m and an outer diameter of 600 ⁇ m. It can also be made of glass, quartz or the like.
  • a capillary 02 is inserted into the side of the hose 01, for example, a glass or quartz tube having an inner diameter of 50 ⁇ m, an outer diameter of 100 ⁇ m, and a length of 10 cm, having a tipped outlet.
  • Figure 7 shows a chip-type micro-pipeline - a cross-sectional schematic of a four-channel bus bar 1 It is a polydimethoxysilane (PDMS) having a size of, for example, 1.5 cm wide, 2.5 cm high, and 0.5 cm thick, and the end of the chip is cut to obtain a pointed outlet.
  • PDMS polydimethoxysilane
  • four of the four-channel buss are finally combined into one straight channel, where I is the first channel, II is the second channel, III is the third channel, and IV is the fourth channel; 10-4 is The resulting four component mixed droplets.
  • the four channels have a circular opening in the upper part of the chip, which is a sample introduction interface, and the interface is a circular hole perpendicular to the surface of the chip.
  • the four interfaces are connected to four fluid-driven devices carrying different solutions through the pipe parts, and the formation of four-component droplets can be realized.
  • Fig. 8 shows another chip type microchannel, the droplet generating stream chip 1.
  • the droplet generation flow chip 1 has upper and lower inlets.
  • the reaction solution is injected at the lower inlet (for example, may be an aqueous solution containing a sample).
  • An inert liquid for example, a mineral oil to which a surfactant is added
  • the reaction liquid is injected at the above inlet, and is divided into two into two channels.
  • the first liquid "clamps" the reaction liquid into a submerged channel to become a water-in-oil type liquid, and further in the second liquid (which may be water that is immiscible with mineral oil) A water-in-oil-in-water emulsion emulsion 10-5 is formed.
  • the water-in-oil type liquid formed in the merged passage of the chip is the first liquid
  • the second liquid is the aqueous liquid that is immiscible with the oil phase of the first liquid.
  • the oil phase can enclose any number of aqueous phase droplets.
  • chip type microchannels 7 and 8 are only two specific examples of chip type microchannels, and chip type microchannels having various channels can be used in the present invention. This can be specifically selected by those skilled in the art as needed, and will not be described herein.
  • the opening (including the inlet and the outlet) of the micro-pipe 1 of the present invention may be cylindrical, and the outlet may also be tapered.
  • the outer diameter of the outlet of the micro-pipe is 0.05 to 1000 ⁇ m, preferably 5 ⁇ 400 microns; the microchannel has at least one fluid channel having an inner diameter of from 0.025 to 500 microns, preferably from 2.5 to 200 microns, more preferably from 10 to 150 microns.
  • the outlet configuration of the microchannel be a tapered opening having an inner diameter of 10 to 100 microns and an outer diameter of 20 to 200 microns.
  • the present invention may employ a micro-pipe outlet having a small outer diameter, preferably having an initial inner diameter of 100 microns and an outer diameter of 200 microns.
  • the capillary exit tip of the glass or quartz has a tapered configuration with an inner diameter of 30 microns and an outer diameter of 50 microns.
  • micro-pipe can be selected according to specific needs, and the present invention is not particularly limited.
  • the microchannel is preferably a microchannel that is treated with a low surface energy at the opening.
  • the low surface energy treatment can be a low surface energy coating treatment, or Silylation treatment
  • the present invention preferably employs perfluorosilane (e.g., 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane, Fluorochem Ltd., Derbyshire, UK) for the art of microchannels, such as the outer walls of capillaries, in the art.
  • Silanization treatment well known to the person.
  • the micro-liquid dispensing system of the present invention also includes a container.
  • the container can be any suitable container.
  • commercially available containers suitable for holding a small amount of droplets can be used.
  • the container may be a standard multiwell plate, such as a 24-well plate, a 96-well plate, a 384-well plate, a 1536-well plate, and the like.
  • the number of holes of the perforated plate can be arranged as needed, and is not limited to those enumerated above.
  • standard multiwell plates after liquid dispensing can be used directly for further standardized experiments and/or assays.
  • This is in contrast to many micro-liquid dispensing/mixing devices, where dedicated reaction, detection, and the like are typically provided, thereby greatly increasing the cost of experiments requiring micro-liquid dispensing.
  • the laboratory can be directly used for further experiments/tests. Convenient for the user, while greatly reducing the cost of the experiment. Conducive to the popular use of experiments / tests related to the distribution of trace amounts of liquid.
  • the corresponding microchannels can also be arranged in the same array to achieve high throughput liquid distribution.
  • the container 2 is for loading a second liquid and containing droplets of the first liquid from the microchannel 1. Further, the container 2 can also store and transfer droplets of the first liquid, as will be detailed below, or to dilute the droplets of the first liquid, or to add two or more firsts in the same container. Liquid droplets are fused together to form larger droplets for reaction.
  • the container 2 is any container capable of storing microliters to a volume of milliliters of liquid, which may also be referred to as a well; each well may store more than one droplet of the first liquid.
  • the container is preferably a single-hole, one-dimensional or two-dimensional array of pores, more preferably a standard well-labeled plate or a polymerase chain reaction (PCR) plate, a small flat bottom or a cone. Bottom glass containers, small test tubes, and PCR tubes.
  • the bottom of the container 2 may be a flat bottom, a plurality of droplets may be fused by low-speed centrifugation, and observed at a fixed corner of the bottom of the perforated plate.
  • the container 2 may also be a tapered or nearly conical bottom.
  • a pointed bottom, a rounded bottom or an elliptical bottom so that the added droplets converge to the center of the bottom of the container.
  • the detection device can quickly capture the target to be tested, and avoid the current micro-dispensing method, the micro-droplet sticks to the side wall of the container, resulting in the detection device. The target is not detected.
  • the size and number of the containers are not particularly limited, and those skilled in the art can select according to specific needs.
  • a first liquid refers to a liquid that is loaded into a microchannel and subsequently subjected to microdispensing.
  • Type of first liquid There are no restrictions and can be selected and prepared according to specific needs. For example, it may be a solution containing a sample, a solution containing any reactant, or a solution or solvent which functions as a dilution, buffering or solubilization.
  • the first liquid may also be a mixed solution of a plurality of solutions (see the case of the examples shown in Figs. 3 to 6), and may even be an emulsion (see the case of the example shown in Fig. 8).
  • the second liquid is a liquid that is immiscible with the first liquid.
  • the second liquid is typically present as an inert liquid whose gas-liquid interface provides shear forces for the resulting microdroplets to exit the micropipe opening.
  • the second liquid is an oil phase.
  • the kind of the second liquid is not particularly limited, but it is chemically stable which does not react with the substance in the first liquid, and has a liquid which is not volatile and does not interfere with characteristics such as subsequent detection.
  • mineral oil including n-tetradecane, etc.
  • vegetable oil including n-tetradecane, etc.
  • silicone oil such as silicone oils.
  • perfluoroalkane oil such as n-tetradecane, etc.
  • the first liquid is an oil phase
  • the second liquid may be water or an aqueous solution such as deionized water, sterile water, or the like, or other liquid that is immiscible with the first liquid, such as an ionic liquid or a magnetic liquid.
  • the second liquid not only provides the necessary shear force for the first liquid to form droplets, but also provides a stable environment for the liquid droplets, and also provides a reaction space for the liquid droplets to be isolated from the external environment, avoiding the risk of contamination of the liquid droplets, and avoiding the liquid. Volatilization of the liquid in the drop brings inaccuracies to the experimental results.
  • the specific gravity of the first liquid is greater than that of the second liquid, so that the generated liquid droplets will sink to the bottom of the container, facilitating the easy capture of the position of the liquid droplets during the detection, and also facilitating the convergence of the plurality of liquid droplets, thereby The substances in each droplet mix and react (see the case shown in Figure 9).
  • a third liquid may also be involved in the micro-liquid dispensing method performed by the micro-liquid dispensing/mixing device of the present invention.
  • a third liquid may also be involved in the micro-liquid dispensing method performed by the micro-liquid dispensing/mixing device of the present invention.
  • a third liquid may also be involved in the micro-liquid dispensing method performed by the micro-liquid dispensing/mixing device of the present invention.
  • a third liquid may also be involved in the micro-liquid dispensing method performed by the micro-liquid dispensing/mixing device of the present invention.
  • Fig. 12 a case where the second liquid 9 and the third liquid 11 are accommodated in the container 3 is shown in Fig. 12.
  • the droplet 10 of the first liquid 8 in the microchannel 1 enters the second liquid 9 as the outlet end of the microchannel moves under the liquid surface of the second liquid, and falls to the second liquid 9 and the third under the action of gravity.
  • the interface of the liquid 11 At the interface of the liquid 11.
  • the third liquid 11 is miscible with the first liquid 8, and under the action of the interfacial tension, the droplets 10 of the first liquid are fused with the third liquid 11, thereby bringing the substances in the first liquid into the first A solution 11' is formed in the three liquids.
  • the first liquid may be a sample solution or a reactant solution (aqueous solution, specific gravity is about 1)
  • the second liquid is silicon.
  • the oil (specific gravity is about 0.9) and the third liquid is mineral oil (specific gravity is 0.76).
  • a method of dispensing a micro-liquid comprises the steps of: extending an outlet end of a microchannel having at least a lower end filled with a first liquid below a level of a second liquid contained in the container; driving the chamber in the micro-channel Moving the first liquid toward the outlet end, forming a droplet having a given volume of the first liquid outside the outlet end of the microchannel; and withdrawing the open end of the microchannel from the second liquid Thereby, droplets having a given volume of the first liquid are dispensed from the outlet end of the microchannel into the second liquid, wherein the first liquid is immiscible with the second liquid.
  • micro-liquid distribution is carried out using the micro-liquid dispensing/mixing device of the present invention, and the accuracy of the liquid volume is affected by the accuracy of the fluid-driven device and the size of the micro-pipe outlet. Therefore, a highly accurate micro liquid can be obtained.
  • commercially available microinjection pumps such as the PHD Ultra syringe pump (Harvard Apparatus, USA), have a minimum flow rate of 25 fL/s and an accuracy of ⁇ 0.25%.
  • the outlet end of the commercial micro-pipe can reach an inner diameter of 2 microns.
  • the distribution of a small amount of liquid can be theoretically obtained with a liquid volume as low as 50 fL and an absolute error of less than ⁇ 5 fL. And as the level of mechanical manufacturing technology continues to increase, this precision will be further improved.
  • the amount of liquid to be dispensed can be adjusted by controlling the fluid driving speed of the fluid driving device and the starting time.
  • the adjustment method is simple and easy, and the adjustable range can reach the level of flying to nanoliter. It is usually adjustable from 100 fL to 100 uL.
  • the volume of the liquid to be dispensed is preferably from 100 pL to 1 uL, more preferably from 200 pL to 200 nL.
  • the volume of droplets that can be ejected by the nozzle can be as small as 5 pL, but the volume of the droplets is set in advance during the manufacturing process of the device and cannot be adjusted as needed.
  • the volume of the droplet amount can be controlled in the range of 20 pL to 1 nL, but is used. The volume needs to be calibrated, the control of the system is complicated, and the cost is high.
  • FIG. 2 there is shown a schematic diagram of the most typical micro-liquid dispensing method in accordance with the present invention.
  • the micro liquid dispensing method of the present invention will be exemplified with reference to Fig. 2 in accordance with the apparatus shown in Fig. 1.
  • the fluid driving speed of the syringe pump 5 is set to 4 nL/s, and the volume of liquid to be dispensed is 20 nL.
  • the microchannel 1 is located above the level of the second liquid 9 in the vessel 3, ensuring that the microchannel 1 is filled with the first liquid 8 and its liquid level is flush with the nozzle.
  • the microchannel 1 moves perpendicularly to the liquid surface of the second liquid, and is inserted below the second liquid level at the first moment and remains stationary; the syringe pump 5 is started, the flow rate is 4 nL/s, and the mode is volume mode, after 5 In seconds, the syringe pump 5 is turned off.
  • a droplet 10 having a diameter of 336.8 ⁇ m and a volume of 20 nL was formed at the outlet of the microtube. Then, the microchannel 1 is moved vertically upwards and lifted to 5 mm above the liquid level of the second liquid. At the third moment, when the microchannel is separated from the liquid level of the second liquid, the droplet 10 at the outlet of the microchannel is separated from the capillary opening due to surface tension or the like, remains in the second liquid, and sinks into the bottom of the container 3. Since the container 3 has a rounded bottom, the droplets 10 can be brought by gravity and eventually collected to the center of the bottom. When the microchannel 1 was moved above the liquid level of the second liquid of the next container 3, the above operation was repeated, and 20 nL of the first liquid was also added to the container.
  • the microchannel 1 is stably moved up and down at a uniform speed to avoid disturbance of the droplet volume caused by the movement of the microchannel.
  • the prepared solution can be added to the container for reaction/detection.
  • two or more solutions may be gradually added to the container, and the reaction may be reacted and/or inspected after the added solutions are fused.
  • droplets 10 of one solution and droplets 10' of another solution are sequentially added to the second solution 9 of the container 3, and the droplets 10 and 10' are concentrated at the bottom of the container 3, and Droplets 10-6 are obtained by gravity or centrifugal force fusion.
  • Figure 10 is a graph showing the change in diameter before and after fusion of two droplets of different colors.
  • the container used therein is a 96-well plate with a tapered bottom.
  • the upper left and upper right corners of the figure are single red and green droplets, respectively, and the volume is about 5nL.
  • the lower left corner and the lower right corner of Fig. 10 are photographs before and after the fusion of a red droplet and a green droplet of a volume of about 5 nL, respectively. Due to the tapered bottom, the droplets naturally collect into the center of the cone due to gravity. The droplets spontaneously fuse. The time required for fusion is approximately 1 minute to 1 hour.
  • a 96-well plate In order to accelerate the fusion of the two droplets, a 96-well plate can be placed on a low-speed centrifuge and rotated at 2000 rpm for 30 seconds to achieve efficient droplet fusion.
  • Figure 11 shows the diameters of red, green and brown (red + green) droplets before and after fusion. All diameters are the average of 5 parallel experiments. It can be seen that the diameter of the droplets has good reproducibility.
  • Yet another embodiment, as shown in Figure 12, is to add one or more traces of liquid to a larger amount of system for mixing/reaction/detection.
  • the round bottom vessel was loaded with the aqueous phase liquid 11 having a volume of 100 ⁇ l, and the aqueous phase liquid 11 was covered with the oil phase liquid 9.
  • a 5 nanoliter drop 10 is added to the container; the droplet sinks due to gravity and is incorporated into the aqueous phase liquid 11.
  • the solute in the droplet was diluted about 2000 times. Therefore, with the method of the present invention, high-fold dilution can be obtained quickly without the need for a step-by-step multiple dilution method.
  • micro-pipes are only in contact with the oil, and are not in contact with the aqueous phase liquid 11 at the bottom, thereby avoiding contamination and improving the reliability of the method.
  • the application manner of the method of the present invention is not limited thereto, and those skilled in the art can select according to actual needs.
  • FIG. 13 Another embodiment, as shown in Fig. 13, is a case where a third liquid is present in addition to the first liquid 1 and the second liquid to protect the liquid filling operation of the liquid droplets.
  • the conical bottom container 3 is loaded with the oil phase liquid 9 as a second liquid having a volume of 100 ⁇ l, and the oil phase liquid 9 is covered with the oil phase liquid 11 as a third liquid.
  • the oil phase liquid 9 and the oil phase liquid 11 do not fuse, and the oil phase liquid 11 has a specific gravity lower than that of the oil phase liquid 9.
  • the microchannel is always under the liquid surface of the oil phase liquid 11, and the oil phase liquid 9 is inserted from the top to the bottom of the interface of the oil phase liquid 11 and the oil phase liquid 9, and the microvolume is injected.
  • Drop 10-2 (as the second first liquid) and withdraw the oil phase liquid 9.
  • the aqueous phase droplets 10-2 are retained in the oil phase liquid 9 and sink to the bottom of the vessel due to gravity, merging with the aqueous phase droplets 10-1 into a large droplet 10-3.
  • the oil phase liquid 11 acts as a gas barrier and also serves as a protective phase for the oil phase liquid 9. The probability of contamination of the reaction is further reduced, which is beneficial to improve the reliability of single-cell single molecule analysis.
  • the apparatus for dispensing a small amount of liquid according to the present invention is simple in equipment, low in cost, simple in operation, high-throughput liquid distribution, and capable of easily adjusting a liquid dispensing volume with high precision, it has a wide application space in drug screening. , cytotoxicity research, protein crystallization conditions screening, single cell enzyme activity analysis, single cell whole genome sequencing and transcriptome sequencing sample preparation, digital PCR quantitative nucleic acid amplification analysis, cell-cell interaction research, etc. Application prospects.

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Abstract

一种微量液体分配/混合装置、系统及方法。其中，微量液体分配/混合装置包括：具有流体驱动设备（5）的流体驱动单元，用于以一定流速驱动与所述流体驱动设备（5）连接的微管道（1）中的第一液体（8）向所述微管道（1）的出口端推进；和机械移动单元（6、7），用于控制所述微管道（1）和/或容纳有第二液体（9）的容器（2）在至少一维方向上移动，以配合所述流体驱动设备（5）的启动和停止，使所述微管道（1）的出口端插入和离开所述第二液体（9），从而向所述容器（2）中加入给定体积的所述第一液体（8），其中，所述第一液体（8）与所述第二液体（9）不互溶。上述装置、系统及方法利用微量液体在气液或液液界面变换时的界面能和流体剪切力，克服液体在微管道（1）出口的表面张力和附着力，使流出微管道（1）管口的液滴能顺利地脱离微管道（1）。上述装置结构简单、成本低廉、易于操控，液滴的大小可以调节，且能够方便地进行标准化的反应、筛选及检测。

Description

微量液体分配/混合装置、系统及方法 技术领域
本发明涉及生化检测和筛选技术领域，具体涉及一种基于微量液体分配和/或混合的生化反应检测装置、系统及方法。
背景技术
生命科学领域涉及基因工程、蛋白质工程、药物筛选等各种研究，这些研究需要对大量的包含有核酸、蛋白以及其它有机和无机物质的液体进行转移、分配等操作，而科学的发展使得人们越来越倾向于对微量液体进行高通量的分配。目前常见的微量液体分配技术有接触式(贾振中，等；接触式点样机器人及相关技术研究进展，机器人，2007，29(2)：179-185)和非接触式(刘亚欣，等；基于复合智能控制的非接触式液体定量分配系统，机械工程学报，2010，46(20)：175-181)。基于微流控的微液滴生成技术在最近几年得到快速发展(Teh SY,et al.,2008.Droplet microfluidics.Labon a Chip,8(2):198-220)，其液滴的生成是基于分散相和连续相在微通道中交汇时的界面失稳。通过不同的微流控通道芯片设计，能够生成大小均一的液滴，并进行融合、反应和分选等操作。
随着对细胞生理研究的逐渐深入，科学家们开始从单细胞、单分子水平上对生命现象进行分析。如利用单分子技术研究DNA与蛋白质之间的相互作用(Jessen,W.J.,et al.,2006.Active PHO5 chromatin encompasses variable numbers of nucleosomes at individual promoters.Nat.Struct.Mol.Biol.13,256-263.)。利用光镊捕获间单个DNA分子并研究其结构的变化等(James T.Inman,et al.,2014.DNA Y Structure:A Versatile,Multidimensional Single Molecule Assay.Nano Lett.14,6475-6480)。
迄今为止，最常见的单细胞分析方法包括以下几种：(1)显微操作技术；(2)激光捕获显微切割技术；(3)激光诱导荧光检测流式细胞分选；(4)微流控流式分选。
显微操作技术和激光捕获显微切割技术是在显微镜下人工地将具有不同光学特征的细胞或颗粒分离出来，因此通量低，且这些操作有可能会破坏样品的完整性，并不是最优的单细胞分离方法(Espina,V.,et al.,2006.Lasercapture microdissection.Nat.Protoc.1,586-603)。
激光诱导荧光检测流式细胞分选是基于荧光标记的高通量单细胞分选方法，一般分析速度为5000～10000个每秒。一般需要细胞需要带有荧光标记才能被分选(Rinke,C,et al.,2014.“Obtaining genomes from uncultivated environmental microorganisms using FACS-based  single-cell genomics,”Nat Protocols,9[5]:1038-45.)，部分细胞可以通过其散射光信号进行分选。通过流式细胞分选仪的细胞被分散在纳升至皮升体积的液滴中，可以被逐一分配到多孔板里面，以便对单细胞进行后续的反应和分析。但是目前的多孔板由于体积较大，落入的液滴的位置通常不确定，微液滴阵列容易出现液滴吸附在侧壁而无法进行后续试样混合操作的情况。现有的多孔板加样操作体积通常在微升体积，而被测的单细胞细菌尺度仅为<1微米大小，这给后续的培养、测序等过程带来和很大的不确定性，如何将试剂的体积减小，提高分析的可靠性，对于提高分析的灵敏度，降低分析的污染风险具有非常重要的意义。
微流控(Microfluidics)是新兴的单细胞、单分子分离技术，它应用不同设计的芯片，通过对微量流体的控制，对单细胞、单个DNA分子或单个蛋白分子等进行分离、分析和检测。微流控芯片可以用于后续单细胞培养、裂解和测序，因此，是目前被公认的理想的单细胞分离分析手段(Cho,B.S.,et al.,2003.Passively driven integrated microfluidic system for separation of motile sperm.Anal.Chem.75,1671-1675)。目前已经有许多关于使用微流控芯片生成液滴的方法对单细胞或单分子进行分离分析(Marcy Y,et al.,2007.Nanoliter reactors improve multiple displacement amplification of genomes from single cells.PLoS Genet3(9):e155.)。但是，微流控芯片的设计、加工及操作复杂，成本高昂，且需要考虑许多因素，如特定的流速、油水界面张力以及通道构型和通道表面修饰等，生成液滴体积受各种流体力学和通道结构的影响，无法精确定量，调节的范围也受到以上因素的制约。另外，液滴在微流控芯片通道内生成后，需要特定的步骤和装置转移到储存容器中，难以对单个液滴的条件进行定制，液滴的定位、提取和分析等操作较为不便。
中国专利申请CN103954786A中公开了一种半接触式的油下液滴连续点样和加液方法。该方法中在芯片上覆盖一定量的油相，精细调节毛细管尖端与芯片微孔下表面之间保持一定的距离，且该距离刚好能够使待分配的液滴接触到容器底部液滴，同时向毛细管尖端推出一定量的液体形成小液滴后，液滴接触容器底部，并由于微孔下表面事先处理为亲水性，因而微滴脱离毛细管的尖端附着到芯片上。该方法的难点在于每次点样都需精确控制毛细管尖端与芯片微孔下表面的距离，而且对于连续点样的情况，还需调整所有微孔的下表面在同一水平面上。此外，由于液滴体积微小，毛细管尖端与微孔小表面之前的间隙十分微小，因此这种调节需要借助显微镜，而且一旦调整好，整个设备包括承接液滴的芯片就不能再移动。
可见，目前的微量液体分配技术存在设备复杂、成本高昂、操作不便、通用性不高等问题，而且液体量调节范围窄，限制了该项技术的普及和广泛应用。
发明内容
有鉴于此，本发明的目的在于提供一种操作简便、成本低、体积可精确控制和调节的微量液体分配和/或混合装置，该装置利用基于微管道的微加液技术，在微滴中包含生物/化学样品，并针对每个微滴可进行进一步的反应和/或分析。此外，本发明的目的还在于提供一种基于该微量液体分配和/或混合装置的系统，以及微量液体分配和/或混合的操控方法，为基于生物和/或化学反应的各种应用，例如单细胞培养条件筛选、单细胞基因组和转录组扩增、单个核酸分子扩增、酶动力学研究、蛋白质结晶条件筛选、药物活性筛选等，提供微体系、高通量，且成本低、操作简便的微量液体分配方案。
本发明一方面提供一种微量液体分配/混合装置。该装置包括流体驱动单元和机械移动单元，其中,
所述流体驱动单元包括流体驱动设备，用于以一定流速驱动与所述流体驱动设备连接的微管道中的第一液体向所述微管道的出口端推进；和
所述机械移动单元用于控制所述微管道和/或容纳有第二液体的容器在至少一维方向上移动，以配合所述流体驱动设备的启动和停止，使所述微管道的出口端插入和离开所述第二液体，从而向所述容器中加入给定体积的所述第一液体，
其中，所述第一液体与所述第二液体不互溶。
根据一种实施方式，所述流体驱动单元进一步包括与所述流体驱动设备密闭连接的、用于连接至少一个所述微管道的具有一个或多个流体通路的管道部件。优选，所述管道部件具有阵列状流体通路，所述阵列状流体通路与容器的排列方式相对应，其中所述管道部件与一个或多个流体驱动设备连接，且每个流体通路与一个微管道连接。更优选，所述管道部件具有与标准多孔板(如24、96、384或1536孔板)对应的流体通路阵列。这样用本发明的装置分配液体后，可以直接采用常规的设备、仪器进行进一步的反应和/或检测，而不需额外特殊的配套装置或检测设备，从而大大降低了实验/检测成本。而且能够进行大通量实验，例如药物筛选、实验条件筛选等。
根据一种实施方式，所述流体驱动单元可包括两个或更多个流体驱动设备，每个流体驱动设备连接至少一个微管道。这样可同时进行不同液体的分配，也可以同时分配不同体积的液体，从而提高液体分配效率和装置利用效率。
在这种实施方式下，上述具有阵列状流体通路的管道部件的每个或若干个(如一行或一列)流体通路可由一个流体驱动设备驱动。
本发明的微量液体分配/混合装置的机械移动单元包括用于控制所述微管道移动的机 械手，和/或用于控制所述容器移动的平移台。优选，所述机械手和平移台分别能够在至少一维方向上移动。优选机械移动单元的移动平稳进行，优选匀速运动，以避免移动对液体分配体系带来震荡，而影响液体分配精度。
本发明的微量液体分配/混合装置进一步包括控制与反馈单元，所述控制与反馈单元包括：信号接收模块、计算模块和信号输出模块。
其中，所述信号接收模块用于接收信号并将所述信号传输给所述计算模块；所述计算模块接收所述信号并进行计算和/或与设定值进行比较，然后将计算和/或比较结果传输给所述信号输出模块；和所述信号输出模块接收所述计算和/或比较结果，并将所述结果转换为指令信号输出。
在本发明的实施方式中，所述信号是要进行分配的液体体积信号。所述计算模块接收由信号接收模块传输的所述要进行分配的液体体积信号，根据该信号选择合适的流体驱动速度，并计算所述流体驱动单元中的流体驱动设备的运行时间，或者根据该信号以及预先设定的流体驱动速度，计算所述流体驱动单元中的流体驱动设备的运行时间，并将计算结果传输给所述信号输出模块，所述信号输出模块接收所述计算结果并将该计算结果转换为指令信号，分别传输给流体驱动单元和机械移动单元，以控制机械移动单元配合流体驱动单元的启动、停止移动微管道和/或容器，以完成对第一液体的分配。
根据优选的实施方式，所述控制与反馈单元进一步包括距离探测模块，所述距离探测模块能够获取所述微管道的出口端与目标容器的相对位置关系信号以及所述微管道的出口端与目标容器中第二液体的液面之间的距离信号，并能够将所获取的所述相对位置关系信号和/或所述距离信号传输给所述信号接收模块。在该实施方式中，信号接收模块接收所述信号并传递给计算模块；计算模块接收所述信号并与预先设定的微管道出口端与第二液体的液面距离值进行比较，并将比较结果传输给信号输出模块；信号输出模块接收比较结果并转换为指令信号传输给机械移动单元，以调整机械移动单元的移动方式直至使微管道定位到第二液体液面上方的预订距离处。
根据更优选的实施方式，所述距离探测模块还能够获取第二液体的深度信号，并传输给信号接收模块。信号接收模块接收第二液体的深度信号并传输给计算模块；计算模块接收所述深度信号并与设定的最小深度值进行比较，并将比较结果传输给信号输出模块；当测定的深度值大于设定的最小深度值时，输出模块向流体驱动单元和机械移动单元发出进行液体分配的信号，当测定的深度值小于设定的最小深度值时，输出模块向流体驱动单元和机械移动单元发出停止进行液体分配的信号，更优选地同时发出报警信号。
所述控制与反馈单元用于精确控制流体驱动单元和机械移动单元的运行，以提高液体 分配的准确性和精密度。特别是对于具有多个流体驱动设备，以及各流体驱动设备用于分配不同液体体积的情况，控制与反馈单元可控制本发明的微量液体分配/混合装置自动完成复杂的多种液体分配方案，从而进一步提高液体分配效率和装置使用效率。
根据需要，本发明的微量液体分配/混合装置还可进一步包括除菌/尘及气体置换单元。所述除菌/尘及气体置换单元可以是可形成封闭空间的过滤装置，可布置在例如微量液体分配/混合装置的外周，或者布置在微管道和容器的外周。所述除菌/尘及气体置换单元还可以是紫外灭菌灯，布置在微管道和容器附近。也可以将空气过滤装置和紫外灭菌灯结合起来形成所述除菌/尘及气体置换单元。还可以包括对装置内的气体进行置换的装置，实现惰性气体保护、厌氧操作、以及补充二氧化碳、氧气、氢气等细胞培养或生化反应所需的气体氛围。
优选地，本发明的微量液体分配/混合装置还进一步包括温度控制与反馈单元。所述温度控制与反馈单元可包括加热和/或制冷装置，以及控温装置。根据一种实施方式，温度控制与反馈单元直接对用于放置所述容器的平台进行加热和/或制冷。温度控制与反馈单元还可以是单独设置的恒温箱。这样在进行液体分配的过程中可为体系提供所需的温度，以利反应或分析。
根据本发明的特定实施方式，本发明的微量液体分配/混合装置由或者主要由所述流体驱动单元、机械移动单元和控制与反馈单元组成。
本发明的微量液体分配/混合装置区别于现有的微量液体分配/混合装置主要在于其结构及操作简单。在常规的微量液体分配/混合装置中，通常还要包括驱动给定体积的液滴脱离微管道的设备，例如压电装置、加热装置、喷射装置等等。而本发明的微量液体分配/混合装置由或者主要由所述流体驱动单元和机械移动单元，以及可选的控制与反馈单元组成。在本发明中，流体驱动单元用于生成给定体积的第一液体的液滴，机械移动单元用于通过使微管道的出口端通过从第二液体的液面抽出，从而将产生的液滴分配到第二液体中。
本发明的微量液体分配/混合装置结构简单，操作方便，液体量可在飞升到纳升量级的宽泛范围内调节。而且，所分配的液体量精度好，能够满足实验、检测需要。
而且根据本发明采用阵列容器的实施方式，本发明的微量液体分配/混合装置能够直接使用常规的标准多孔板作为容器，不但能够进行高通量的实验，还能够直接利用现有的标准实验和/或检测设备，而无需额外配备，大大方便了使用者并大幅降低的了实验成本。
根据本发明的第二方面，提供一种微量液体分配系统。本发明的微量液体分配系统包括上述的微量液体分配/混合装置、微管道和容器。其中，所述微管道与所述微量液体分配 /混合装置的流体驱动单元相连接，用于分配其中容纳的第一液体；所述容器中容纳有第二液体，用于承接所分配的第一液体的液滴。
用于本发明的微量液体分配系统的微管道具有圆柱形开口或锥形开口，优选锥形开口。
所述微管道可以是单根单芯毛细管、单根多芯毛细管、集束毛细管、阵列毛细管、具有套管的毛细管或微流控芯片。优选地，所述微管道具有膨大的上端形成为储液仓。从成本的角度考虑，优选单根单芯毛细管，例如玻璃或石英毛细管。这样的毛细管可进一步组成毛细管集束或阵列。采用这种毛细管，特别是出口端进一步拉细形成为锥形开口的毛细管可以获得令人满意的微量液体分配结果。
合适的微管道，包括各种毛细管或微管道芯片均可用于本发明，本发明对于微管道的形式、结构、材质等没有特别限制。本领域技术人员能够根据需要进行恰当的选择。
优选地，所述微管道的开口处经低表面能处理。更优选地，所述低表面能处理是硅烷化处理。这样可以使被分配的液滴更顺利地脱离微管道。
用于本发明微量液体分配系统的容器可以是任何适合的容器。所述容器为可单个容器、一维或二维排列的容器阵列。优选地，所述容器为二维排列的容器阵列，特别优选标准24、96、384或1536孔板。这样本发明系统就能够与常规的标准化实验和/或检测设备直接对接，而无需额外配备专用的实验和/或检测设备。与所述容器相对应，所述微管道可同时设置为相应的二维排列的微管道阵列。这样可大大提高液体分配的通量，提高液体分配效率。
根据一种实施方式，所述容器底部为尖底、锥形底、圆形底或平底。其中尖底、圆底或类似的结构便于被分配的液滴汇聚到底部，既有利于多液滴之间融合、反应，也有利于检测设备快速准确地捕捉到待测目标。
优选地，所述第一液体的比重大于所述第二液体的比重。这样被分配的第一液体的液滴可沉到容器底部。
本发明的第三方面，提供一种微量液体分配方法。所述方法包括：
A、使至少下端充满第一液体的微管道的出口端伸入容纳在容器中的第二液体的液面以下；
B、驱动所述微管道中的所述第一液体向所述出口端移动，在所述微管道的出口端外形成具有给定体积的第一液体的微滴；和
C、使所述微管道的开口端从所述第二液体中抽出，从而使具有给定体积的第一液体的微滴脱离所述微管道的出口端而分配到所述第二液体中，
其中，所述第一液体与所述第二液体不互溶。
根据本发明，不希望受任何理论的限制，所述第一液体微滴在第二液体的流体剪切力以及第二液体的气液或液液界面处的液体表面张力作用下脱离所述微管道的出口端而分配到所述第二液体中。
根据本发明的方法，被分配的第一液体的微滴的体积在100fL～100uL范围内，优选为100pL～1uL，更优选为200pL～200nL。
根据优选的方法，所述容器为二维排列的容器阵列。所述微管道为相应二维排列的微管道阵列；优选，所述容器为标准24、96、384或1536孔板；更优选，所述容器底部为尖底、锥形底、圆形底或平底。
根据优选的实施方式，所述第一液体的比重大于所述第二液体。
根据一种实施方式，所述容器中还容纳有第三液体，所述第三液体覆盖在所述第二液体上，与所述第一液体和所述第二液体均不互溶；或者所述第三液体位于所述第二液体之下，与所述第二液体不互溶。
与现有技术相比，本发明技术方案利用气液或液液界面变换时的界面能和流体剪切力，使附着在微管道出口外侧的微量液体克服与微通道出口之间的附着力，以及与为多个内液体的表面张力，使流出微管道管口的液滴能顺利地脱离微管道。液滴的大小受到流体驱动单元的调控，其精确度与微管道的开口大小以及流体驱动装置装置的精密程度影响。因此，本发明通过简单地将微管道的出口端插入与抽出与待分配的液体不互溶的第二液体，便可对飞升到纳升量级的液体进行分配。
另外，本发明的液体分配量，可通过微管道内的液体流速和驱动时间来直接控制，因此，对待分配的液体体积能够根据需要灵活调节。此外，通过布置微管道阵列(例如利用具有阵列状多流道的管道部件)，可以利用标准化的容器，如多孔酶标版等，从而既可以用于实现大批量的微体积高通量筛选，也可以实现多步骤的超微量标准化的生化反应和检测，具有广阔的应用前景。
例如，对单细胞的裂解可以通过加入裂解液的微液滴，使之与单细胞微液滴融合，从而制备单细胞的核酸、蛋白等物质。制备的单细胞核酸或蛋白等物质可以通过稀释至大的液滴中吸出，也可以留在第二液体中继续后面的反应，可以对单细胞进行DNA分析、RNA分析、蛋白质分析等，根据实验的目的不同，后续加入相匹配的反应液液滴。由于包裹在油相中，液滴不会蒸发，更利于液滴或微球的提取以及储存。且每个反应可以在多孔板的不同孔中独立完成，避免了交叉污染；每个反应在纳升级别的液滴中完成，大大降低了成本。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1示出了根据本发明一种实施方式的微量液体分配/混合装置的结构示意图。
图2为根据本发明一种实施实施方式微量液体分配方法的示意图。
图3为根据本发明一种实施方式的二维阵列容器、微管道和管道部件的结构及微加液方式的示意图。
图4为根据本发明的双芯单根毛细管分配液体的示意图。
图5为根据本发明的由三根单芯单根毛细管组成的毛细管集束分配液体的示意图。
图6为根据本发明的具有套管的毛细管分配液体的示意图。
图7为根据本发明的具有四通道的可在线混合的芯片型微管道分配液体的示意图。
图8为根据本发明的可制备复乳液滴的芯片型微管道分配液体的示意图。
图9为根据本发明的一种实施方式将两种溶液依次分配到容器中并相互融合的示意图。
图10为根据图9所示方法，所分配的两个5nL不同颜色的液滴融合为一个10nL液滴的显微成像照片。
图11为根据图9所示方法，所分配的两个约5nL液滴融合为一个10nL液滴的液滴直径变化的柱形图。
图12为根据本发明一种实施方式将微量液滴分配到一个较大量体系中的方法示意图。
图13为根据本发明一种实施方式在第三液体覆盖下将两种第一液体的微量液滴分配到第二液体中的方法示意图。
图14为根据本发明一种实施方式的微量液体分配/混合装置控制模块的连接示意图。
图15为根据本发明一种实施方式的微量液体分配/混合装置的控制模块的工作流程示意图。
具体实施方式
下面结合附图及实施例对本发明作进一步描述。显然，所描述的实施例仅用于解释本发明的基本原理，而不是对本发明进行限制。基于本发明中的实施例，本领域普通技术人员在没有做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。
定义：
第一液体：本文所称第一液体是待进行分配的液体的总称，而不限于一种液体或溶液。例如第一液体可以是多种不同的液体，如多个样品分别配制而成的溶液；也可以是多种液体在分配之前在线混合而成的混合溶液。多数情况下，第一液体是水溶液或水性溶液，但不限于此，例如可以是不溶于第二液体的疏水性液体。
第二液体：本文所称第二液体是用于“剪切”第一液体的微滴，使其脱离微管道的液体，也用于承接第一液体微滴；在某些情况下，还提供反应所需的环境，如隔氧、避光、保温等。第二液体是与第一液体不互溶，且惰性的液体或溶液。最好是不易挥发的油性液体。
第三或其他液体：除以上第一和第二液体之外，在本发明的系统或方法中用到的液体统称为第三液体，或者成为其他液体。第三液体可以是与第一液体相关的液体或溶液，例如用于稀释第一液体的稀释液、与第一液体反应的大体积反物溶液等；也可以是第二液体的辅助液，例如，为第二液体提供液封的液体等。
在此所说的液体，是指在所需环境中为液体的情况，例如常温下为固体而在较高温度下为液体，或者常温下为气体而在较低温度下为液体。
微量：本文所说的微量，如微量液体或微量液体分配，是指用常规的移液管、移液器/枪等无法准确、重复移取的液体量，通常指微升量级以下，特别是飞升到纳升量级的液体量。其中也包括在线将所称的“微量”液体与“常量”液体混合后再分配的情况。
微管道：本文所称的微管道用于对第一液体进行分配，可以是任何形式的管道，其中包含至少一个液体流道。最常见、同时成本最低的是毛细管。也可以是芯片型多流道的微管道，还可以是包含至少一个微管道的、具有套管的管道。
容器：本文所称容器是容纳第二液体，用于承接第一液体的容器。容器可为任何形式，但是优选通用的标准多孔板。特别是底部变窄的容器，如尖底、圆或椭圆底等。
如前所述，本发明旨在提供一种能够精确进行微量液体分配的装置，能够精确地进行从飞升量级到纳升量级液体的分配。与现有的微量液体分配/混合装置不同，本发明的装置操控简单、成本低廉、且液体的体积易于调节，可广泛应用于各种微体系的生物、化学反应，以及高通量的筛选实验。
与宏观的，例如毫升量级、微升量级的液体分配不同，当需要微小体积，如纳升、皮升、甚至飞升体积的液体时，用常规的毛细管时，流出管道口的微小液滴，其自身重力远远不足以克服其与管口之间的附着力、表面张力以及与管内液体间的表面张力，因而无法直接获取。这也是目前的微量液体分配方法都另辟它途的原因。
本发明的微量液体分配/混合装置仍然利用诸如毛细管的微管道来进行液体分配。该装 置包括：流体驱动单元，其包括流体驱动装置，用于以一定流速驱动与所述流体驱动设备连接的微管道中第一液体向所述微管道的出口端推进；和机械移动单元，用于配合所述流体驱动装置的启动和停止，控制所述微管道和/或容纳有第二液体的容器在至少一维方向上移动，以向所述容器中加入给定体积的所述第一液体，其中，所述第一液体与所述第二液体不互溶。
图1示出了根据本发明的微量液体分配/混合装置的一种具体实施方式的示意图。图1所示的装置中，流体驱动单元包括作为流体驱动设备的注射泵5和作为管道部件的特氟龙(Teflon)细管4，用于连接注射泵5和作为微管道的毛细管1。第一液体被排除气泡后充满注射泵5的管腔、特氟龙细管4及毛细管1。机械移动单元包括控制毛细管沿垂直方向的z轴上下移动的机械手6和用于放置具有多个孔型容器3的容器阵列2并带动容器阵列2在水平的x轴和/或y轴方向移动的平移台7。
在本发明中，第一液体，即待分配的液体，事先加载到微管道中，并充满微管道，至少充满微管道的下游端部分，例如可通过流体驱动设备将第一液体驱动到微管道中，也可以通过反向运行流体驱动设备，将第一液体吸入微管道中。第二液体，是与第一液体不互溶的一种惰性液体，事先加载到容器中。容器是用于在其中进行反应或进行进一步实验。图2示出了根据本发明的方法进行液体分配的示意图。参见图2，移动微管道1和/或容器3，使微管道的出口端伸到第二液体9的液面以下。根据要加入的第一液体8的体积，设定流体驱动设备5的流速，开启流体驱动设备5一定的时间，这样在第二液体9的包裹下，在微管道的出口端外形成一滴具有给定体积的液滴10。再次移动微管道1和/或容器3，使微管道1的出口端抽出第二液体9的液面之外。当微管道1的出口端移动到第二液体9的液面处时，通过气液相界面变换时的界面能和流体剪切力，将出口端处的微滴10“剪”下来，使其顺利地脱离管口形成液滴进入第二液体9中。在图2所示的实施方式中，第一液体8的比重大于第二液体9，因此由第一液体形成的液滴10汇聚到容器3的底部。这种方式对于需要与其他液滴进行进一步反应的体系(如以下将要详述的图9所示的实施方式)来说是优选的。此外，对于后续需要进行检测的体系来说，由于液滴很微小，为了便于检测仪捕捉到液滴，也优选第一液体的比重大于第二液体，配合尖底或圆底容器，就可以直接在容器的中心位置或通过重力作用，在底部中心固定位置准确观测液滴；或者配合平底容器，通过离心机离心，实现多个液滴在离心力作用下汇集融合，进行定量反应。
以下参考图1，对本发明微量液体分配/混合装置的各个部分进行详细的说明。
流体驱动单元
本发明流体驱动单元主要包括流体驱动设备。流体驱动设备除了图示的注射泵外5， 可以是任何能连续驱动液体流动的流体驱动设备，例如，还可以是蠕动泵、压力驱动泵、气压驱动泵或电渗驱动泵等，但不限于此。优选微量注射泵。特别是，使用精度较高的注射泵，可获得飞升～纳升级的液滴。
流体驱动设备的流体驱动速度通常在0.5pL/min～10mL/min的范围内，优选为10nL/s～100nL/min。因要分配的液体量甚为微小，为了获得高精度体积的液体，液体流速的精度误差需达到±1％以下，优选误差小于等于±0.5％。
流体驱动单元还可以包括一端连接流体驱动设备5，另一端连接微管道1的具有流体通道的管道部件4。管道部件可以是诸如图示的特氟龙(Teflon)细管4一类的单通道管道，也可以是具有多个流体通道的集合式或一体式管道部件。在实际应用中，后者可连接多个微管道或连接具有多通道的微管道，这样可同时完成对多个容器的液体分配，或者同时对多种液体进行分配，因而效率更高。
多流道的管道部件可以根据需进行液体分配的容器阵列的排列进行设计。例如针对多孔板，可设计具有相应流道分布管道部件，以提高液体分配效率。
图3示出了一种具有与容器阵列2相应的阵列状多流道的管道部件4。该管道部件4具有1个管状入口4-1、1个长方形的连通腔体4-2和多个阵列状管状出口4-3。管状出口4-3间的间距与相应的阵列式容器2的排列间距相对应，每个管状出口连接一个微管道1。为了进一步降低成本，管道部件的流道设置可与现有的例如96孔、384孔甚至1536孔等多孔板相对应，因而用户可直接使用常规实验室均配备的实验器材而不需另购。而且由于采用了此类具有标准排列的阵列多流道管道部件，本发明的装置可以与现有的检测装置、药物筛选装置、酶标仪等联合使用。与多通道或多个微管道相匹配的，流体驱动设备也可以为多个，这样针对不同的通道或微管道，可以设定不同的流速和/或启动时长，从而方便同时分配不同体积的液体。
管道部件4的一端可固定地或可拆卸地连接在流体驱动设备5上。由于本发明的装置用于微量样品的反应和实验，因此避免污染是非常关键的操作环节。如果管道部件的液体流道中需要接触样品溶液，那么可拆卸的连接方式是优选的。但是如果不需接触样品溶液(如下面将会详细讨论的一种实施方式)，则固定连接更为有利，因为这样通常能够确保管道部件与流体驱动设备之间的气密性。而气密性是保障进行精确的微量液体分配所必须的条件。
管道部件的另一端可布置有适合连接微管道的接口，以方便地与微管道气密性地连接。接口与微管道的连接方式没有特别限制，任何合适的方式都是可以的。例如，可以是螺旋接口、卡口、胀塞、插接接口等，但不限于此。在接口处可进一步涂覆密封胶以确保 连接的气密性和紧密性。1因为微管道通常是一次性使用的，因此所述接口优选是容易进行快速连接的那些，例如所述接口与微管道相应接口分别具有互补性的结构。
管道部件的尺寸并没有特别的限制，通常是与流体驱动设备的流速范围及被分配的液体体积范围相适应。例如，最简单的，可采用内径为300微米，外径为600微米，长度为15厘米的柔性特氟龙细管，方便直接连接，且对于外径大于300微米，小于400微米的毛细管微管道，不需要使用胶粘剂，可直接插入特氟龙细管的末端并保证密闭。
流体驱动单元还可进一步包括储液池/仓。所述储液池/仓与流体驱动设备密闭连接且流体联通，用于连续向流体驱动设备提供第一液体。在图1中，注射泵的注射管腔也可视作是储液仓。储液池/仓可以是一个也可以是多个。当储液池/仓为多个时，其中可以分别容纳不同的第一液体，例如可为包含不同样品的液体。优选地，所述储液池/仓的数目可与包含多个流体通道的管道部件的流体通道数目相对应。根据另一种实施方式，流体驱动单元并不包含储液池/仓，也不载入任何流体或者仅载入惰性流体而非反应液。第一液体仅加载到微管道中，例如可利用倒吸的方式吸入微管道中。微管道的上部或其他部位可以略膨大从而形成储液仓以容纳足够量的第一液体。而流体驱动单元通过产生气压或通过驱动一种惰性流体从而间接驱动第一液体流动。这种实施方式的优点是流体驱动单元并不接触样品溶液，因而不会造成污染，不需每次都进行清洗/更换被污染的配件，而只需更换成本较低的微管道即可。
储液池/仓的体积没有特别限制，但通常不会很大，因为所分配的液体体积是十分微小的。例如，可以是微升量级的，如5～500微升或者可以更小或更大，常用的可为5、10、20、50、100、150、200、250、300、400、500微升等，但不限于此。
机械移动单元
在图1所示的实施方式中，机械移动单元包括一个用于控制微管道的机械手6和用于控制容器阵列2移动的平移台7。
在该实施方式中，机械手6具体地可为以步进电机驱动的垂直升降平移台，控制微管道1在垂直方向上沿z轴移动。参考图2，在开始进行液体分配时，先沿z轴向下移动微管道1，将微管道的出口端伸入目标容器3(如，图1所示的一个孔型容器)中第二液体9的液面以下，启动流体驱动设备5驱动第一液体8在微管道的出口端形成给定体积的液滴10，然后停止驱动，再向上移动微管道1，使其出口端离开第二液体9，而将由第一液体形成的液滴10留在容器3中。
在一个孔型容器3中完成液体分配后，平移台7，可为步进电机或伺服电机驱动的二维X-Y平移台，或者是带有三维定位功能的机械臂，待定容器阵列2沿x轴和/或y轴方 向移动，将下一个孔型容器3移动到微管道的下方，然后机械手6和流体驱动装5置重复上述操作，在该孔型容器中加入给定体积的第一液体。如此重复可依次在容器阵列2的各容器中加入给定体积的第一液体。
根据一种实施方式，机械手可以在垂直方式(z轴)和一个水平方向(x轴或y轴)移动，而平移台则可以仅在另一个水平方向移动。
根据其他实施方式，机械移动单元可以仅包括机械手或者仅包括平移台。例如，在图1所示的这种有一个微管道和多个容器的情况下，机械移动单元可以是一个能够在三个维度上移动的机械手或平移台。当然，如图3所示的这种微管道数目与容器数目相匹配的情况来说，一个能够仅在垂直方向移动的机械手或平移台就已经足够。在这种情况下，本发明的微量液体分配/混合装置可得到进一步的简化。
驱动机械移动单元各部件进行移动的驱动设备优选那些能够进行平稳驱动的驱动设备，以提高液体分配的精度。
控制与反馈单元
根据一种实施方式，微量液体分配/混合装置进一步包括控制与反馈单元。控制与反馈单元包括信号接收模块、计算模块和信号输出模块。参考图12和13，首先，控制与反馈单元010的信号接收模块011接收诸如人为设定的要进行分配的液体体积等信号并将其接收到的信号传递给计算模块012(S1)；计算模块012根据预先设定的流体驱动速度等条件，或者根据要分配的体积选择合适的流体驱动速度，计算流体驱动单元020中的流体驱动设备的运行时间并将计算结果传输给信号输出模块013(S2)；信号输出模块013根据计算模块012所计算出的流体驱动单元020的运行时间，控制机械移动单元030配合流体驱动单元020的启动、停止移动微管道和/或容器，以完成对第一液体的分配(S3)。
其中，信号输出模块013具体地是先控制机械移动单元030移动微管道和/或容器,使微管道定位于目标容器上部并使微管道的出口端伸入所述容器中第二流体的液面下，接着控制流体驱动单元020启动并运行所计算的时长后停止，然后再控制机械移动单元030使所述微管道的出口端移动到第二流体的液面之外，从而完成一次预订体积的第一液体的分配。
根据一种实施方式，控制与反馈单元还进一步包括距离探测模块(未图示)，以便能够对微管道的出口端与目标容器的相对位置关系进行探测，并且还能够对微管道的出口端到目标容器中第二液体的液面之间的距离进行探测。液面探测模块能够实时获取微管道的出口端与容器或容器中液面间的距离信息，然后将该信息传递给信号接收模块011；信号接收模块011接收距离信号并传递给计算模块012；计算模块012接收距离信号，并与预 先设定的微管道出口端与第二液体的液面距离值进行比较，并将比较结果传输给信号输出模块013；信号输出模块013接收比较结果，并转换为指令信号传输给机械移动单元，以调整机械移动单元的移动方式(例如实际距离值大于设定值，则机械移动单元带动微管道向液面移动)，直至使微管道定位到第二液体液面上方的预订距离处。
然后，控制与反馈单元按照上述方法控制本发明的装置在该目标容器中完成对第一液体的分配。
当存在阵列容器时，控制与反馈单元还能探测目标容器中第二液体的量(或者液体深度)是否达到所需的最低值(例如利用距离探测模块探测第二液体的液面到容器底部间的距离)并对第二液体的量低于最低值的情况由信号输出模块进行报警并自动停止对该容器进行液体分配。例如当控制与反馈单元检测到某目标容器中没有第二液体时，会暂停操作并提醒该目标容器不适合进行液滴加入实验。
对第二液体的液面进行液位检测的方式可以包括，红外激光测距传感器，超声波测距传感器等，这些是所属领域技术人员所熟悉的，在此不再赘述。
对于多个容器的情形，例如以96孔板作为容器，要在每个孔中分配同样体积的第一液体时，控制模块根据计算模块的计算结果可以进行重复操作，直至在每个孔中加入预订体积的液体。当然也可以采用与容器同样排列的多个微管道，例如按照96孔板的排列方式排列的96个微管道，控制与反馈单元能够控制流体驱动单元的启动和停止并控制机械移动单元的移动，以同时在多个容器中一次性完成第一液体的分配。
控制模块010还可同时控制多个流体驱动单元020和/或多个机械移动单元030分别配合进行动作，以便同时进行不同体积的液体分配。
除菌/尘单元
由于本发明的装置用于极微量液体的分配，因此即便微管道的尖端出口处仅有极短暂的时间是暴露在空气中的，微管道尖端部分的第一液体仍然有被空气中的微生物/灰尘污染的风险。这些微生物，无论是有生命的还是已经死亡的，以及吸附了各种杂质的灰尘，只要其中包含有可能与第一液体中的反应物发生反应的分子，或者包含任何会产生干扰的物质，都会干扰检测的结果。因此，根据优选的实施方式，本发明的微加液装置进一步包括除菌/尘单元。
所述除菌/尘单元可以包括空气过滤装置，可布置在整个微量液体分配/混合装置外部，形成可密封的空间；也可以仅布置在液体分配区域的外部，形成为能将微管道和容器密封起来的结构。例如在微管道1和容器5的外周形成可封闭的空间，在允许气体通过的开口处设置除菌/尘过滤部件，通过真空泵产生负压，在实验之前对微加液空间进行除菌、除尘。 还可以包括对装备内的气体进行置换的装置，实现惰性气体保护、厌氧操作、以及补充二氧化碳、氧气、氢气等细胞培养或生化反应所需的气体氛围。
所述除菌/尘及气体置换单元还可以是或进一步包括安装在液体分配区域附近的紫外灭菌灯或其他可以除菌/尘的装置。
温度控制与反馈单元
根据一种实施方式，本发明的微量液体分配/混合装置还包括温度控制与反馈单元，用于为容器中的液体系统提供所需的温度。温度控制与反馈单元布置在用于放置容器的平台下，可包括加热和/或冷却装置，以及控温装置。温度控制与反馈单元可以用于原位给容器中的体系提供必要的温度。在这种情况下，温度控制与反馈单元直接对用于放置所述容器的平台进行加热和/或制冷，从而加热或冷却容器中的液体体系；或者用于放置所述容器的平台形成为温度控制与反馈单元的一部分。在有平移台的实施方式中，温度控制与反馈单元可布置为直接加热和/或冷却平移台的台面。另一种情况下，温度控制与反馈单元可单独布置，例如设置为一个可密封的箱体，如恒温箱，待进行液体分配的容器，和/或完成液体分配的容器可放入恒温箱中以获得所需温度，或者进行进一步的反应。
根据本发明的第二方面，提供一种微量液体分配系统。本发明的微量液体分配系统，包括上述微量液体分配/混合装置；与所述微量液体分配/混合装置的流体驱动单元相连接，用于分配其中容纳的第一液体的微管道；和容纳有第二液体，用于承接所分配的第一液体的液滴的容器。
微管道
本发明的微量液体分配系统可以包括微管道。根据本发明的系统，可以采用已商品化的、适于产生微量液滴的管，例如毛细管等。
如图1所示的微管道1是两端开口的毛细管，其中一端与流体驱动设备5通过特氟龙细管4流体联通且密闭相连，用于接收第一液体，该端可以称为入口，另一端用于流出液体，可以称为出口。然而，在某些情况下，可以通过使流体驱动设备反向运行，从而从微管道的出口将第一液体吸入微管道中来完成液体的加载。
由于微管道内部难以清洗，因此微管道通常为一次性使用的。因此，微管道的材质优选为成本较低的那些。对于不同应用的需要，形成微管道的材料可以是金属(例如不锈钢)、石英、玻璃、聚合物材料等。在某些条件下，毛细管，如不锈钢材质的毛细管可以经过清洗、烘干等操作，实现重复使用。
微管道1可以是任何适合产生微量液体的管道，例如可为单根单芯毛细管、单根多芯毛细管、阵列毛细管、微流控通道(芯片)等，但不限于此。
图3示出了一种上端膨大以与管道部件4的管状出口4-3的外周气密性结合的微管道1。该膨大的上端可同时作为储液仓，容纳要进行分配的第一液体。
图4示出了单根双芯毛细管的示意图。毛细管1内部有一隔板而将其分隔成两个互不相通的通道，I为第一通道，II为第二通道。在毛细管的上端，分别在半圆形通道中插入一条内径较小的毛细管，并用胶粘剂填充插入的细毛细管和半圆形通道间的空隙，保证注射时的密闭性。两条细毛细管再分别通过Teflon连接管连接注射泵，向两通道内分别加载两种不同的溶液，从而可获得双组分混合液滴10-1。两个通道如果分别用不同的流体驱动设备驱动，则可通过设定不同的流速获得具有不同浓度(如系列梯度)的某种组分的混合液滴10-1。该双芯毛细管的尺寸可为例如外径300微米、内径200微米、中间隔板厚50微米。
当然，图4仅示出了多芯毛细管的一种情况，多芯毛细管中也可以具有更多的“芯”，例如可以有3、4或5芯。
图5示出了一种由三根单通道毛细管组成的毛细管集束1，其中I、II和III分别为三根紧密排列在一起的独立的毛细管。出口端通过胶粘剂粘结在一起。三根毛细管的上端分别连接三条Teflon连接管，并与三个独立控制的注射泵连接，向三个毛细管中分别注入不同的溶液(如分别含有不能预先混合的组分)，用本实施方式的微量液体分配/混合装置可获得一个由三种溶液混合的混合液滴10-2。
这种毛细管集束可以是2根、3根、4根、5根或更多根。
图6示出了具有套管的微管道1。如图所示，01为一软管，在软管01的侧面插入毛细管02，并在软管01的下部连接一个尖头管03，该尖头管03的下端开口与毛细管的出口端汇合；在毛细管02中通入溶液8-1，在软管01中通入溶液8-2。在流体驱动设备的驱动下可获得混合有溶液8-1和8-2的混合液滴10-3。这种具有套管的微管道可用于同时完成较大体积液体8-2和微量液体8-1在同一系统中的分配。例如，生成包裹有细胞悬液和细胞裂解液的微液滴，在一定反应条件下，如65℃孵育10分钟，即可得到包裹有经细胞裂解释放细胞核酸物质的微液滴。
在该示例中，软管01为橡胶材质，如橡胶、乳胶、硅胶等，也可采用其它材质。软管01的长度为2厘米，内径为0.6毫米。橡胶软管01下端接一根带有尖端的管02，如内径为300微米，外径为600微米的特氟龙管，也可采用玻璃、石英等材质制成。在软管01的侧面插入毛细管02，例如采用具有拉尖的出口的内径为50微米、外径为100微米、长度为10厘米的玻璃或石英管。
图7示出了一个芯片型微管道——具有四通道的汇流芯片1的剖面示意，芯片的材质 为聚二甲氧基硅烷(PDMS)，尺寸例如为1.5厘米宽、2.5厘米高、0.5厘米厚，芯片末端采用裁切的方法得到尖头出口。在该示例中，四通道汇流芯片中有四个通道最终汇合成一个直通道，其中I为第一通道，II为第二通道，III为第三通道，IV为第四通道；10-4为生成的四组分混合液滴。四条通道在芯片的上部具有圆形开口，为样品引入接口，接口为垂直于芯片表面的圆孔。四个接口通过管道部件分别连接四个载有不同溶液的流体驱动设备，可以实现四组分液滴的生成。
图8示出了另一种芯片型微管道——液滴生成流芯片1。该液滴生成流芯片1具有上、下两个入口。在下面的入口注入反应液(例如可以是包含样品的水溶液)。在上面的入口注入与反应液不互溶的惰性液体(例如添加了表面活性剂的矿物油)，分成两股进入两个通道中。在流体驱动设备的驱动下，第一液体将反应液“夹”断，进入下方合并的通道中成为油包水型液体，并进一步在第二液体(可为与矿物油不互溶的水)中形成水包油包水型的复乳液滴10-5。
该示例中通过改变芯片各通道的液体流速，可以获得包裹不同数目反应液液滴的复乳液滴。在该示例中，在芯片的合并通道中形成的油包水型液体为第一液体，而第二液体为与第一液体的油相不互溶的水性液体。形成的复乳液滴中，油相可包裹任意数量的水相液滴。
图7和8所示仅仅是芯片型微管道的两种具体示例，具有各种通道的芯片型微管道均可用于本发明。这是本领域技术人员根据需要可以具体选择的，在此不再赘述。
进一步地，本发明的微管道1的开口(包括入口和出口)都可以是圆柱形，出口还可以是锥形.举例来说，对于微管道的出口外径为0.05～1000微米，优选为5～400微米；所述微管道具有至少一个流体通道，所述流体通道的内径为0.025～500微米，优选2.5～200微米、更优选10～150微米。对于单芯微管道，最优选微管道的出口构型为内径10～100微米，且外径为20微米至200微米的锥形开口。考虑到生成液滴的均一性，本发明可以采用小外径的微管道出口，所述微管道优选初始内径为100微米、外径为200微米。在本发明的一个实施例中，玻璃或石英的毛细管出口拉尖为锥形构型，内径为30微米，外径为50微米。
当然，微管道的具体结构和尺寸可根据具体需要进行选择，对此本发明并没有特别的限制。
为了精确控制加液量以及使生成的液滴更加均一，还可以进一步对微管道1的出口表面进行低表面能处理，使流出的第一液体更容易脱离管口，以便形成液滴。即，所述微管道优选为开口处经低表面能处理的微管道。所述低表面能处理可为低表面能涂层处理，或 硅烷化处理，本发明优选采用全氟硅烷(如1H,1H,2H,2H-全氟辛基三氯硅烷，Fluorochem Ltd.,Derbyshire,英国)对微管道，如毛细管的外壁，进行本领域技术人员熟知的硅烷化处理。
容器
本发明的微量液体分配系统还包括容器。根据本发明的系统，容器可以是任何适宜的容器。例如可以使用已商品化的、适于容纳微量液滴容器。优选，容器可以是标准多孔板，例如24孔板、96孔板、384孔板，1536孔板等。多孔板的孔数可根据需要布置，而不限于以上列举的那些。
对于采用标准多孔板的情况，进行液体分配后的标准多孔板可直接用于进一步的标准化实验和/或检测。这与很多微量液体分配/混合装置不同，在这些装置中通常都配套有专用的反应、检测等设备，因而大大增加了需要进行微量液体分配的实验的成本。而本发明的微量液体分配/混合装置或系统进行液体分配后，可直接利用实验室原有设备进行进一步的实验/检测。方便了使用者，同时大大降低了实验成本。利于与微量液体分配相关实验/检测的普及使用。
在这种情况下，对应的微管道也可以呈相同的阵列排列，从而实现高通量的液体分配。
容器2用于装载第二液体，并容纳来自微管道1的第一液体的液滴。进一步地，容器2也能储存和转移第一液体的液滴，如下面将要详述的，还可以对第一液体的液滴进行稀释，或者在同一容器中加入两种或更多种第一液体的液滴，并相互融合形成更大的液滴而进行反应。
所述容器2为任意能够存储微升至毫升体积液体的容器，也可称为孔；每个孔可储存一个以上第一液体的液滴。在本发明中，所述容器优选为单个孔、一维或二维排列的孔阵列，更优选为标准孔酶标板或聚合酶链反应(PCR，Polymerase Chain Reaction)板，小型平底或锥形底玻璃容器，小试管以及PCR管等。在本发明中，容器2的底部可以为平底，多个液滴可通过低速离心实现融合，并在多孔板底部的固定角落进行观察.容器2也可以是锥形的或近锥形的底部，例如尖底、圆形底或椭圆形底，以便加入的液滴汇聚到容器底部的中央。这样不但能快速实现多个液滴之间融合和进行反应，也有利于检测设备快速捕捉到待测目标，避免了目前一些微量液体分配方法中，微小液滴粘在容器侧壁，导致检测设备检测不到目标的情况。
容器的大小和数量都没有特别的限制，本领域技术人员可根据具体需要选择。
第一液体
在本文中，第一液体是指载入微管道中并随后进行微量分配的液体。第一液体的种类 并无限制，根据具体需要来选择和制备。例如，可以是含有样品的溶液，也可以是含有任何反应物的溶液，还可以是起稀释、缓冲或增溶等作用的溶液或溶剂。如前文所述，第一液体也可以是多种溶液的混合溶液(参见图3～6所示例的情况)，甚至可以是一种乳液(参见图8所示例的情况)。
第二液体
第二液体是与第一液体不互溶的液体。在本发明中，第二液体通常作为惰性液体存在，其气液界面为生成的微量液滴脱离微管道口提供剪切力。
第一液体为水性溶液时，第二液体为油相。第二液体的种类没有特别限制，但是应该与第一液体中的物质不发生反应的化学性质稳定的，具有不易挥发、不干扰后续检测等特性的液体。例如，如矿物油(包括正十四烷等)、植物油、硅油和全氟烷烃油等。
第一液体为油相，则第二液体可为水或水性溶液，如去离子水、无菌水等，也可以是其他与第一液体不互溶的液体，例如离子液体或磁性液体。
第二液体不但为第一液体形成液滴提供必须的剪切力以及稳定存在的环境，而且还为液滴提供与外部环境隔离的反应空间，避免了液滴被污染的风险，并避免了液滴中液体挥发给实验结果带来不精确的因素。
本发明中，第一液体的比重大于第二液体，这样所产生的液滴会沉到容器底部，便于检测时易于捕捉到液滴的位置，也有利于多个液滴汇聚到一起，从而使各液滴中的物质混合，并发生反应(参见图9所示情况)。
其他液体
根据本发明的微量液体分配/混合装置进行的微量液体分配方法中还可能涉及其他液体，例如第三液体。参考图12，图12中示出了在容器3中容纳了第二液体9和第三液体11的情形。微管道1中的第一液体8的微滴10随微管道出口端在第二液体的液面上下的移动而进入第二液体9中，并在重力作用下下落到第二液体9和第三液体11的界面处。在该示例中，第三液体11与第一液体8是互溶的，在界面张力的作用下，第一液体的微滴10与第三液体11融合，从而将第一液体中的物质带入第三液体中形成溶液11’。
进一步地，另一种情况是第三液体存在于第二液体之上。第一、第二、第三液体互不相容。微管道的出口端插入第二液体的液面以下，启动流体驱动设备一定时间，产生所需体积的液滴后，提起微管道至第三液体之外。当微管道的出口端通过第二和第三液体的界面处时，由于界面间的表面张力使液滴脱离微管道的出口端而留在第二液体中。这种实施方式可以用于第二液体易挥发的情形，或者需要与外界环境隔绝的情形，如无氧或其他情况。例如，第一液体可以是样品溶液或反应物溶液(水溶液,比重约为1)，第二液体为硅 油(比重约为0.9)，第三液体为矿物油(比重为0.76)。
微量液体分配方法
根据本发明的原理，微量液体分配方法包括以下步骤：使至少下端充满第一液体的微管道的出口端伸入容纳在容器中的第二液体的液面以下；驱动所述微管道中的所述第一液体向所述出口端移动，在所述微管道的出口端外形成具有给定体积的第一液体的微滴；和使所述微管道的开口端从所述第二液体中抽出，从而使具有给定体积的第一液体的微滴脱离所述微管道的出口端而分配到所述第二液体中，其中，所述第一液体与所述第二液体不互溶。
采用本发明的微量液体分配/混合装置进行微量液体分配，液体体积的精度受到流体驱动设备的精度以及微管道出口的大小的影响。因此能够获得较高精度的微量液体。例如，目前已商品化微量注射泵，如PHD Ultra注射泵(Harvard Apparatus公司，美国)最低流速可以达到25fL/s，精度可达到±0.25％。于此相配合的，已商品化的微管道的出口端可达到2微米的内径。因此采用本发明的方法进行微量液体的分配，理论上可以获得的液体体积可低至50fL，绝对误差达可到小于±5fL。而且随着机械制造工艺水平的不断提升，这一精度将更进一步地提高。
在本发明中，通过控制流体驱动设备的流体驱动速度以及启动时长就能够调节欲分配的液体的量。调节方法简单易行，而且可调节的范围可达到飞升～纳升量级。通常可在100fL～100uL范围内调节。被分配液体的体积优选在100pL～1uL，更优选在200pL～200nL。
这与很多现有的微量液体分配方法不同。例如，利用喷墨打印原理进行液体分配时，喷头能喷射的液滴的体积可以小到5pL，但是液滴的体积是设备制造过程中事先设定好的，不能根据需要再调节。再例如，在基于压电驱动液滴喷射方法的Microdrop Microdispenser system(型号：MD-E-3000，microdrop Technologies GmbH，德国)中，液滴量的体积虽然可以在20pL至1nL区间进行控制，但是使用时需要对体积进行校准，系统的控制复杂，成本较高。
再回到图2，其中示出了根据本发明的最典型的微量液体分配方法的示意图。以下根据图1所示的装置，参考图2，举例说明本发明的微量液体分配方法。
例如，设定注射泵5的流体驱动速度为4nL/s，要分配的液体体积为20nL。参考图2，在时间为0时刻时，微管道1位于容器3中第二液体9液面上方，保证微管道1内充满第一液体8且其液面与管口齐平。微管道1垂直于第二液体的液面向下运动，在第1时刻插入第二液体液面以下并保持静止；启动注射泵5，流速为4nL/s，模式为体积模式，经过5 秒钟，关闭注射泵5。此时(在第2时刻)，在微管道出口处形成了一个直径为336.8微米、体积为20nL的液滴10。然后，微管道1垂直向上运动，提起至第二液体液面以上5毫米。在第3时刻，当微管道脱离第二液体的液面时，由于表面张力等作用，微管道出口处的液滴10脱离毛细管口，留在第二液体中，并沉入容器3底部。由于容器3具有圆形的底部，液滴10能够通过重力作用，最终汇集到底部的中心。将微管道1移动到下一个容器3的第二液体的液面以上，重复以上操作，则在该容器中也加入了20nL的第一液体。
根据本发明的方法，优选微管道1以均匀的速度，稳定地上下移动，以避免因微管道移动对微滴体积造成的扰动。
当然，也可以固定微管道，而上下移动容器。
对于容器的移动，也同样优选进行稳定的移动，以避免对容器中的液体体系产生扰动。
本发明的方法可以有多种应用形式。根据一种实施方式，如图2所示，可将已经配制好的溶液一次性加入容器中进行反应/检测。另一种方式如图9所示，可将两种或更多种溶液逐步加入容器中，使这些加入的溶液融合为一体后发生反应和/或进行检查。。例如，图9中所示，依次将一种溶液的液滴10和另一种溶液的液滴10’加入容器3的第二溶液9中，液滴10和10’在容器3底部汇聚，并通过重力或离心力融合获得液滴10-6。
图10为两个不同颜色的液滴融合前和融合后的直径的变化。其中采用的容器为锥形底部的96孔板。图的左上角和右上角分别为单一红色液滴和绿色液滴，体积均为约5nL。图10的左下角和右下角分别为体积约5nL红色液滴与绿色液滴融合前后的照片。由于采用了锥形的底部，液滴由于重力作用自然汇集到锥形的中心。液滴发生自发的融合现象。融合所需的时间大概为1分钟至1小时。为了加速这两个液滴的融合，可以将96孔板放在低速离心机上，以2000rpm的转速转动30秒，即可实现高效的液滴融合。图11为融合前后红色、绿色和棕色(红+绿)液滴的直径。所有直径均为5次平行实验的平均值。可以看到液滴的直径具有较好的重现性。
还有一种实施方式如图12所示，是将一种或多种微量液体加入一个较大量的体系中进行混合/反应/检测。如图12所示，在圆底容器中装载有水相液体11，其体积为100微升，在水相液体11上覆盖有油相液体9。按照上述方法，向容器中加入一个5纳升的液滴10；液滴由于重力作用下沉，并融入水相液体11。融合后，该液滴内的溶质被稀释约2000倍。故而，采用本发明的方法，可以快速的获得高倍数的稀释，而不需要采用逐级多次稀释的方法。此外，微管道只与油相接触，而不与底部的水相液体11接触，从而避免了污染，提高了方法的可靠性。本发明的方法的应用方式不限于此，本领域技术人员能够根据实际需要进行选择。
另一种实施方式如图13所示，是除第一液体1和第二液体之外，存在第三液体，保护液滴的加液操作的情况。如图13所示，在锥形底容器3中装载有油相液体9作为第二液体，其体积为100微升，在油相液体9上覆盖有油相液体11作为第三液体。油相液体9与油相液体11不互融，且油相液体11的比重比油相液体9轻。在油相液体9的底部锥形位置，有预先加入的水相液体微液滴10-1(作为第一种第一液体)。在加液操作时，微管道一直处于油相液体11的液面之下，并从油相液体11和油相液体9的界面处，从上往下插入油相液体9，注射微体积液滴10-2(作为第二种第一液体)，并抽出油相液体9。水相液滴10-2被保留在油相液体9中，并由于重力作用沉入容器底部，与水相液滴10-1融合为一个大液滴10-3。在此操作过程中，油相液体11起到了隔绝气体的作用，同时也可以作为油相液体9的保护相。使反应出现污染的概率进一步下降，有利于提高单细胞单分子分析的可靠性。
由于本发明的微量液体分配方法所用设备简单、价格低廉，操作简便，可进行高通量的液体分配，且能够容易地调节液体的分配体积，精度高，因此具有广阔的应用空间，在药物筛选、细胞毒理性研究、蛋白结晶条件筛选、单细胞酶活性分析、单细胞全基因组测序和转录组测序的样品制备、数字PCR定量核酸扩增分析、细胞-细胞相互作用研究等领域具有广阔的应用前景。

Claims (22)

  1. 一种微量液体分配/混合装置，包括流体驱动单元和机械移动单元，其中：
    所述流体驱动单元包括流体驱动设备，用于以一定流速驱动与所述流体驱动设备连接的微管道中的第一液体向所述微管道的出口端推进；和
    所述机械移动单元用于控制所述微管道和/或容纳有第二液体的容器在至少一维方向上移动，以配合所述流体驱动设备的启动和停止，使所述微管道的出口端插入和离开所述第二液体，从而向所述容器中加入给定体积的所述第一液体，
    其中，所述第一液体与所述第二液体不互溶。
  2. 根据权利要求1所述的微量液体分配/混合装置，其中所述流体驱动单元进一步包括与所述流体驱动设备密闭连接的、用于连接至少一个所述微管道的具有一个或多个流体通路的管道部件。
  3. 根据权利要求2所述的微量液体分配/混合装置，其中所述管道部件具有阵列状流体通路，所述阵列状流体通路与容器的排列方式相对应，其中所述管道部件与一个或多个流体驱动设备连接，且每个流体通路与一个微管道连接；优选，所述管道部件具有与标准多孔板对应的流体通路阵列。
  4. 根据权利要求1所述的微量液体分配/混合装置，其中所述流体驱动单元包括两个或更多个流体驱动设备，每个流体驱动设备连接至少一个微管道。
  5. 根据权利要求1所述的微量液体分配/混合装置，其中所述机械移动单元包括用于控制所述微管道移动的机械手，和/或用于控制所述容器移动的平移台；优选所述机械手和平移台分别能够在至少一维方向上移动。
  6. 根据权利要求1～5中任意一项所述的微量液体分配/混合装置，进一步包括控制与反馈单元，所述控制与反馈单元包括信号接收模块、计算模块和信号输出模块，其中：
    所述信号接收模块用于接收信号并将所述信号传输给所述计算模块；
    所述计算模块接收所述信号并进行计算和/或与设定值进行比较，然后将计算和/或比较结果传输给所述信号输出模块；和
    所述信号输出模块接收所述计算和/或比较结果，并将所述结果转换为指令信号输出。
  7. 根据权利要求6所述的微量液体分配/混合装置，其中所述信号是要进行分配的液体体积信号，所述计算模块接收由信号接收模块传输的所述要进行分配的液体体积信号，根据该信号选择合适的流体驱动速度，并计算所述流体驱动单元中的流体驱动设备的运行时间，或者根据该信号以及预先设定的流体驱动速度，计算所述流体驱动单元中的流体驱动设备的运行时间，并将计算结果传输给所述信号输出模块，所述信号输出模块接收所述 计算结果并将该计算结果转换为指令信号，分别传输给流体驱动单元和机械移动单元，以控制机械移动单元配合流体驱动单元的启动、停止移动微管道和/或容器，以完成对第一液体的分配。
  8. 根据权利要求6所述的微量液体分配/混合装置，其中所述控制与反馈单元进一步包括距离探测模块，所述距离探测模块能够获取所述微管道的出口端与目标容器的相对位置关系信号以及所述微管道的出口端与目标容器中第二液体的液面之间的距离信号，并能够将所获取的所述相对位置关系信号和/或所述距离信号传输给所述信号接收模块；优选地，信号接收模块接收所述信号并传递给计算模块；计算模块接收所述信号并与预先设定的微管道出口端与第二液体的液面距离值进行比较，并将比较结果传输给信号输出模块；信号输出模块接收比较结果并转换为指令信号传输给机械移动单元，以调整机械移动单元的移动方式直至使微管道定位到第二液体液面上方的预订距离处。
  9. 根据权利要求8所述的微量液体分配/混合装置，其中所述距离探测模块还能够获取第二液体的深度信号，并传输给信号接收模块；优选地，信号接收模块接收第二液体的深度信号并传输给计算模块；计算模块接收所述深度信号并与设定的最小深度值进行比较，并将比较结果传输给信号输出模块；当测定的深度值大于设定的最小深度值时，输出模块向流体驱动单元和机械移动单元发出进行液体分配的信号，当测定的深度值小于设定的最小深度值时，输出模块向流体驱动单元和机械移动单元发出停止进行液体分配的信号和可选地发出报警信号。
  10. 根据权利要求1所述的微量液体分配/混合装置，进一步包括除菌/尘及气体置换单元，优选地，更进一步包括温度控制与反馈单元。
  11. 根据权利要求6所述的微量液体分配/混合装置，由所述流体驱动单元、机械移动单元和控制与反馈单元组成。
  12. 一种微量液体分配系统，包括：
    根据权利要求1～11中任意一项所述的微量液体分配/混合装置、微管道和容器，
    其中所述微管道与所述微量液体分配/混合装置的流体驱动单元相连接，用于分配其中容纳的第一液体，所述容器中容纳有第二液体，用于承接所分配的第一液体的液滴。
  13. 根据权利要求12所述的微量液体分配系统，其中所述微管道具有圆柱形开口或锥形开口；优选地，所述微管道是单根单芯毛细管、单根多芯毛细管、集束毛细管、阵列毛细管或微流控芯片；更优选地，所述微管道具有膨大的上端形成为储液仓。
  14. 根据权利要求12所述的微量液体分配系统，其中所述微管道是开口处经低表面能处理的微管道，优选地，所述低表面能处理是硅烷化处理。
  15. 根据权利要求12所述的微量液体分配系统，其中，所述容器为单个容器、一维或二维排列的容器阵列；优选地，所述容器为二维排列的容器阵列，更优选标准24、96、384或1536孔板，且所述微管道为相应的二维排列的微管道阵列；进一步优选，所述容器底部为尖底、锥形底、圆形底或平底。
  16. 根据权利要求15所述的微量液体分配系统，其中所述第一液体的比重大于所述第二液体的比重。
  17. 一种微量液体分配方法，包括：
    A、使至少下端充满第一液体的微管道的出口端伸入容纳在容器中的第二液体的液面以下；
    B、驱动所述微管道中的所述第一液体向所述出口端移动，在所述微管道的出口端外形成具有给定体积的第一液体的微滴；和
    C、使所述微管道的开口端从所述第二液体中抽出，从而使具有给定体积的第一液体的微滴脱离所述微管道的出口端而分配到所述第二液体中，
    其中，所述第一液体与所述第二液体不互溶。
  18. 根据权利要求17所述的微量液体分配方法，其中所述第一液体微滴在第二液体的流体剪切力以及第二液体的气液或液液界面处的液体表面张力作用下脱离所述微管道的出口端而分配到所述第二液体中。
  19. 根据权利要求17所述的微量液体分配方法，其中所述第一液体微滴的体积为100fL～100uL，优选为100pL～1uL，更优选为200pL～200nL。
  20. 根据权利要求17所述的微量液体分配方法，其中所述容器为二维排列的容器阵列，且所述微管道为相应二维排列的微管道阵列；优选，所述容器为标准24、96、384或1536孔板；更优选，所述容器底部为尖底、锥形底、圆形底或平底。
  21. 根据权利要求19所述的微量液体分配方法，其中所述第一液体的比重大于所述第二液体。
  22. 根据权利要求17～21所述的微量液体分配方法，其中所述容器中还容纳有第三液体，所述第三液体覆盖在所述第二液体上，与所述第一液体和所述第二液体均不互溶；或者所述第三液体位于所述第二液体之下，与所述第二液体不互溶。
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