WO2021007710A1 - 基于微流控电喷雾的单细胞全基因组扩增系统和方法 - Google Patents

基于微流控电喷雾的单细胞全基因组扩增系统和方法 Download PDF

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WO2021007710A1
WO2021007710A1 PCT/CN2019/095797 CN2019095797W WO2021007710A1 WO 2021007710 A1 WO2021007710 A1 WO 2021007710A1 CN 2019095797 W CN2019095797 W CN 2019095797W WO 2021007710 A1 WO2021007710 A1 WO 2021007710A1
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ultra
droplets
small droplets
flow channel
sample
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French (fr)
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门涌帆
陈艳
冯鸿涛
敖婷婷
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • 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

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  • the invention relates to the technical field of microfluidics, in particular to a single cell whole genome amplification system and method based on microfluidic electrospray.
  • Water-in-oil emulsion droplet generation technology is an important direction of current life science and industrial production research, and it is also a key link in mainstream biological experiments, institutional testing and industrial production at home and abroad, such as digital PCR, single-cell sequencing, pharmaceuticals and daily Production with chemicals, etc.
  • Microfluidic technology based on droplets has the advantages of reagent consumption far lower than the volume required for traditional experimental operations, and performing a large number of reactions while keeping the device miniaturized, so it has a broader prospect .
  • the microfluidic technology generates and manipulates discrete droplets inside microdevices, and performs independent control of individual droplets, thereby creating a microreactor that can be transported, mixed and analyzed separately.
  • this technology can form multiple identical microreactor units in a short period of time, effectively simplifying subsequent processing and experimental procedures, thereby improving the efficiency of big data collection.
  • This technology provides support for the increase in droplet flux and droplet scalability.
  • EHD electrohydrodynamics
  • the purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide a single-cell whole-genome amplification system and method based on microfluidic electrospray.
  • a single-cell whole-genome amplification system based on microfluidic electrospray includes a microfluidic chip and an AC power module, wherein the microfluidic chip is provided with sample flow channels for injecting discrete and continuous phases, and the AC power module is used to apply AC power to the sample solution in the sample flow channel,
  • the sample solution in the sample flow channel generates droplets under the action of the electric field force and the liquid force, and the droplets are broken into ultra-small droplets by the stretching of the AC electric field within a certain distance.
  • a first electrode and a second electrode are fixed on the microfluidic chip, one end of the first electrode is electrically connected to the continuous phase sample flow channel, and the other end is used to connect to the AC voltage, so One end of the second electrode is electrically connected to the sample flow channel that generates droplets, and the other end is used to connect to an AC voltage.
  • the shape of the sample flow channel is set such that droplet generation occurs at the tip of the Taylor cone, the injected discrete phase carries ions and has a set conductivity, and the injected continuous phase is mixed with surface activity. Agent.
  • the opening angle of the Taylor cone is controlled according to the electric field force applied on the microfluidic chip and the liquid force in the sample flow channel, thereby controlling the particle size and frequency of the generated droplets.
  • the generation of ultra-small droplets is controlled by controlling one or more of the following items: the temperature of the sample solution, the shape of the electrode, the distance between the first electrode and the second electrode, the size of the sample solution The conductivity, the viscosity of the sample solution, the shape of the sample flow channel, the size of the sample flow channel, the flow rate of the continuous phase, the flow rate of the discrete phase, the voltage and frequency of the applied alternating current.
  • the system of the present invention further includes that the generated ultra-small droplets are cured into solid pellets by a UV-triggered curing method, and the surface topography is analyzed by scanning electron microscope or atomic force microscope to obtain ultra-small droplets.
  • the particle size characteristics of the droplet and the constant temperature nucleic acid amplification reaction are used to determine the biological activity inside the ultra-small droplet through the fluorescent signal.
  • the generation of ultra-small droplets is controlled and optimized based on the obtained particle size characteristics of the ultra-small droplets and the influence of the biological activity inside the ultra-small droplets on the amplification reaction.
  • the particle size of the generated ultra-small droplets is less than 5 microns.
  • a single-cell whole-genome amplification method based on microfluidic electrospray includes: generating ultra-small droplets using the single-cell whole-genome amplification system based on microfluidic electrospray provided by an embodiment of the present invention; determining the correlation between the size of the ultra-small droplets, the size of nucleic acid fragments and the amplification efficiency Relationship: Perform gene sequencing on the results of single-cell whole genome amplification, and control and adjust the characteristics of the generated ultra-small droplets according to the sequencing results.
  • the particle size and frequency of ultra-small droplets generated are controlled by controlling the voltage and frequency of the alternating current applied to the microfluidic chip.
  • the present invention has the advantages of: by developing a microfluidic system based on the alternating current electrospray method in the oil phase, it realizes the generation of high-efficiency, high-throughput, and small-volume droplets. Observe and study the cooperative regulation law of multi-parameters on droplet generation, and then master the regulation method to realize ultra-high-throughput micro-droplet generation; and use the monodisperse and high-throughput characteristics of the generated droplets to apply it In terms of single-cell whole-genome amplification and the establishment of sequencing libraries, it helps eliminate amplification bias and achieve more uniform amplification of rare nucleic acids.
  • Fig. 1 is a schematic diagram of a single-cell whole-genome amplification system based on microfluidic electrospray according to an embodiment of the present invention
  • Fig. 2 is a schematic diagram of a process of generating ultra-small droplets according to an embodiment of the present invention
  • Fig. 3 shows a flow chart of a single-cell whole-genome amplification method based on microfluidic electrospray according to an embodiment of the present invention.
  • a single-cell whole-genome amplification system based on microfluidic electrospray is provided as a highly integrated ultra-small droplet generation experiment platform.
  • the system includes microfluidic The chip 110 and the AC power source 120 are electrically connected to the microfluidic chip 110 through electrodes 121 and 121.
  • the system as a whole includes a liquid sample injection process, a process of using a microfluidic chip to generate droplets of a target size, a microdroplet collection process, and a sequencing inspection process.
  • the microfluidic chip 120 integrates multiple functional units, including liquid sample injection, Liquid sheath flow generation, micro-droplet lysis, micro-droplet collection, etc., can complete the function of lysing liquid samples into micro-droplets of target size.
  • the liquid sample a is passed into the microfluidic chip b (also labeled as the microfluidic chip 110) for generating ultra-small droplets in the embodiment of the present invention; in the microfluidic chip Automatically complete the lysis of hundreds of thousands to millions of droplets to obtain ultra-small droplets; collect the lysed ultra-small droplet samples; perform single-cell whole-genome amplification on the collected ultra-small droplet samples; and finally , Send it to sequencer c for sequencing inspection.
  • the microfluidic chip b also labeled as the microfluidic chip 110
  • the microfluidic chip Automatically complete the lysis of hundreds of thousands to millions of droplets to obtain ultra-small droplets; collect the lysed ultra-small droplet samples; perform single-cell whole-genome amplification on the collected ultra-small droplet samples; and finally , Send it to sequencer c for sequencing inspection.
  • a micro-droplet generation chip (or called a microfluidic chip) is used to effectively lyse the sample solution into a target particle size by an electrospray method, for example, into ultra-fine droplets (less than 5 microns).
  • the droplet generation mechanism is: in the droplet generation unit, the flow focusing structure generates the sheath flow of the liquid sample, and the fluid dynamics analysis is used to calculate and simulate the microfluidic chip Flow field distribution, optimized design to generate the shape and characteristics of sheath flow, so that its characteristic values such as flow path and flow velocity are suitable for subsequent droplet cracking steps.
  • the AC electric field is driven by the power source to perform a cracking operation on the sample liquid, and this operation is expected to be expanded in parallel with high throughput.
  • the particle size of the ultra-small droplets can be adjusted while the droplets are lysed, so that it can be developed for directional applications.
  • the microfluidic chip is provided with a sample channel for injecting discrete and continuous phases, an AC power module for applying two-phase alternating current, and an AC voltage is applied to the sample solution in the sample flow channel.
  • the two immiscible liquids meet at the fluid structure of the sample channel. Due to the difference in liquid surface tension between the “oil phase” sample and the “water phase” sample and the shear force generated by the applied pressure, the “water phase” sample meets The “oil phase” sample is divided from the continuous phase into discrete droplets.
  • the sample solution in the sample flow channel generates droplets under the action of electric field and liquid forces.
  • the droplets are cone-shaped (that is, Taylor cones). In a short distance (the tip of the Taylor cone), ultra-small droplets are generated by high-speed extension of electric field force.
  • the process of generating ultra-small droplets is the dynamic balance of liquid force and electric field force, which can be adjusted by adjusting temperature, humidity, electrode shape, distance between the counter electrode, and liquid conductivity.
  • the characteristics, the shape and size of the flow channel of the microfluidic chip, the two-phase flow rate and the electric field intensity and frequency are controlled to generate ultra-small droplets that meet the expectations.
  • the phenomenon of droplet cracking only occurs under a specific combination of parameters. Determining the effect of different parameters on droplet cracking helps to better control droplet formation.
  • a discrete phase with a suitable ion concentration and a continuous phase mixed with a surfactant are selected to study the influence of different ratios on the stability of the generated droplets.
  • the cleavage of the droplet occurs at the tip of the Taylor cone.
  • the discrete phase (water phase) must have ions in order to conduct electricity.
  • the concentration of ions determines the conductivity, so it has a direct impact on the stability of droplet formation; while the continuous phase (oil phase) is mixed inside
  • the surface active agent directly affects the stability of the droplets after they are formed and during constant temperature amplification. Studying the effects of ion concentration and surfactant concentration can help optimize droplet stability for subsequent biological experiments.
  • the change law of Taylor cone angle under AC electric field conditions is measured, theoretical analysis is performed based on Taylor cone formula, and the generation of droplets is controlled by establishing a mathematical model Size and frequency etc.
  • the opening angle of the Taylor cone is affected by the electric field and the flow field, which directly affects the size and frequency of the subsequent droplets.
  • the establishment of a mathematical model combined with experimental phenomena helps to analyze and understand the mechanism of AC electrospray in oil and water.
  • the flow rate, field strength, frequency and other parameter thresholds when generating ultra-small droplets are determined according to the empirical parameters of droplet cracking, so as to control the AC electrospray to produce droplets of 1-100 microns.
  • the flow rate, field strength, frequency and other parameter thresholds when generating ultra-small droplets are determined according to the empirical parameters of droplet cracking, so as to control the AC electrospray to produce droplets of 1-100 microns.
  • scWGA Ultra-small droplets less than 10 microns.
  • the working range of the parameters of the droplet cracking phenomenon is determined, and the coordinated control model of the droplet size and the AC electric field intensity and frequency can be established.
  • the synergistic relationship between multiple parameters and droplet size can control and stably generate highly uniform ultra-small droplet cracking.
  • the physical mechanism of droplet cleavage under two-phase AC electrospray conditions can be learned, which is beneficial to the adjustment of droplet size.
  • the research on the regulation mechanism of droplets can be carried out by characterizing the particle size characteristics of the droplets and verifying the biological activity of the droplets.
  • a UV-triggered solidification method can be used to solidify ultra-small droplets into solid pellets, and the surface topography can be analyzed by characterization methods such as scanning electron microscope or atomic force microscope.
  • a constant temperature nucleic acid amplification reaction such as LAMP and MDA, is required to determine its biological activity through fluorescent signals.
  • Digital PCR and real-time fluorescence quantitative PCR are used to check whether the amplification has occurred and evaluate the quality of the amplified product.
  • the effects of ultra-small droplets on the uniformity of single-cell whole genome amplification include:
  • the electric field here is extremely strong because all electric field lines pass through this area. Characterizing the activity of biomolecules inside ultra-small droplets helps to understand the law of the action of strong electric fields on biomolecules.
  • the ultra-small droplets generated by the AC electrospray in the embodiments of the present application can successfully perform nucleic acid amplification. By characterizing the activity of biomolecules inside the ultra-small droplets, the influence of the AC electric field on the nucleic acid activity inside the droplets can be further determined.
  • microfluidic chip After the above-mentioned microfluidic chip generates ultra-small droplets, and studies the effect of ultra-small droplets on the uniformity of single-cell genome amplification, on the one hand, it can stably generate droplets and accurately regulate the size of the droplets; on the other hand, , To ensure that the droplets undergo constant temperature MDA nucleic acid amplification.
  • the sample is pre-amplified, further, gene sequencing is performed on the single-cell whole-genome amplification result, the uniformity of the genome amplification is analyzed, and the inhibitory effect on amplification bias is evaluated.
  • the library is routinely built and sent for sequencing.
  • the sequencing results are analyzed and compared with historical experimental results to study the effect of ultra-small droplets on improving the uniformity of amplification. Through bioinformatics analysis, it is verified that the ultra-small droplets generated in the embodiment of the present invention have an effect on improving the uniformity of amplification. Through combining experiments of different genome fragments and droplet sizes, the optimized parameters are found to improve the accuracy of the sequencing results.
  • system of the embodiment of the present invention also includes the integration of a mature single cell capture and separation module, a lysis chamber, and a microvalve into the microfluidic chip, thereby realizing a one-piece high-throughput cell sequential capture and lysis , And generate ultra-small droplets through electrospray and perform whole genome amplification operation.
  • the system of the present invention uses alternating current to generate droplets, combines the comprehensive adjustment of liquid control and electrical control, integrates the micro-droplet generation module into the microelectromechanical chip, and uses the stable control advantages of the fluid method and the speed and speed of the electrical modulation method.
  • a large number of ultrafine droplets such as less than 5 microns
  • the system of the invention has the characteristics of high flux, simple operation, and high uniformity of droplet size.
  • the embodiment of the present invention also provides a single-cell whole-genome amplification method based on microfluidic electrospray. As shown in FIG. 3, the method includes the following steps:
  • step S310 the microfluidic chip is used to generate ultra-small droplets with a desired target particle size through AC driving.
  • the temperature of the sample solution, the shape of the electrode, the distance to the electrode, the conductivity of the sample solution, the viscosity of the sample solution, the shape of the sample flow channel, the size of the sample flow channel, the flow rate of the continuous phase, the dispersion The flow rate of the phase, the amount of surfactant mixed in the continuous phase, the voltage and frequency of the applied alternating current, etc. are controlled to generate ultra-small droplets with a desired particle size and internal activity.
  • Step S320 Determine the correlation between the size of the ultra-small droplet, the size of the nucleic acid fragment and the amplification efficiency.
  • Step S330 Perform gene sequencing on the single-cell whole genome amplification result, and further adjust the parameters for controlling droplet generation according to the sequencing result.
  • the amplification After the amplification is completed, send it to sequencing, and based on the sequencing results, determine the influence of ultra-small droplets on the improvement of the amplification uniformity, and further optimize the parameters that affect the generation of ultra-small droplets, such as the voltage, frequency, and continuity of the AC electric field The amount of surfactant mixed inside the phase, etc.
  • the present invention uses a microfluidic chip to realize droplet pyrolysis in oil-water two phases under the action of an AC electric field, realizes the electrospray emulsification phenomenon for the first time in the oil phase, and overcomes the traditional electrospray microdroplets in the air. Almost evaporate and make the droplet size uniform and adjustable; according to the changes in the properties of nucleic acids, enzymes and other biomolecules after passing through an AC electric field, explain the influence of the electric field on biomolecules. Based on the generated droplets, nucleic acid constant temperature can be performed.
  • the fact of amplification proposes to apply microdroplets to single-cell whole-genome amplification to reduce amplification preference.
  • the AC electrospray droplet lysis method of the present invention is a powerful supplement to the field of electrospray and even the entire microfluidic droplet generation theoretical system, solves the problem of amplification bias in single-cell sequencing, and helps to achieve more accurate Quantitative sequencing and analysis of single-cell whole genomes can help individualized medical treatment such as early tumor screening and prenatal diagnosis.
  • the size of the sample flow channel is designed to match the size of the droplet.
  • the size of the droplet is generally less than 100 microns, and the sample flow channel is also about 100 microns.
  • the two-phase liquid may need to flow through a section of the main pipeline from the inlet to the junction.
  • the width of the main pipe is generally set to be greater than 100 microns (for example, 200 microns to 500 microns), so as to reduce the flow resistance of fluid movement.
  • the ultra-small droplets, ultra-micro droplets, or ultra-micro droplets described herein have the same meaning and refer to ultra-small droplets that are split by an AC electric field, unless otherwise indicated by the context.

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Abstract

本发明提供一种基于微流控电喷雾的单细胞全基因组扩增系统和方法。该系统包括微流控芯片和交流电源模块，其中所述微流控芯片设有用于注入离散相和连续相的样品流道，所述交流电源模块用于对样品流道内的样品溶液施加交流电，样品流道内的样品溶液在电场力和液体力的作用下生成液滴，液滴在一段距离内经过交流电场的拉伸裂解成超小液滴。本发明能够利用微流控芯片通过电喷雾方式生成超小液滴，提高了测序的准确性。

Description

基于微流控电喷雾的单细胞全基因组扩增系统和方法 技术领域
本发明涉及微流控技术领域，尤其涉及一种基于微流控电喷雾的单细胞全基因组扩增系统和方法。
背景技术
油包水的乳化液滴生成技术是当前生命科学及工业生产研究的重要方向，更是国内外主流生物实验、机构检测及工业生产中的关键环节，例如数字PCR、单细胞测序、制药及日用化学品生产等。基于微滴的微流体技术，作为微流控技术的重要分支，具有试剂消耗量远低于传统实验操作所需体积、在保持装置小型化的同时执行大量反应等优势，因此具有更广阔的前景。该微流体技术通过在微器件内部产生和操纵离散微滴，进行单个液滴的独立控制，从而产生可以单独运输、混合并分析的微反应器。此外，该技术可在短时间内形成多个相同的微反应器单元，有效简化后续处理及实验过程，进而提升大数据的收集效率。该技术更为液滴通量的增加和液滴可扩展性提供支撑。
在国内外微流控领域，目前传统的液滴生成方法是依赖通道几何形状控制液滴生成，以T形接头(T-junction)和流动聚焦(flow-focusing)为代表，而传统技术的共性缺点是调试慢、不稳定、通量低。新兴技术则采用电流体动力学(EHD)方法，通过被集成到微器件中的电极实现对液滴的电控制，代表性的两个例子是介电电泳(DEP)和介电电润湿(EWOD)，而目前这类方法存在控制复杂、对液滴粒径调控范围有限等问题。
因此，需要对现有技术进行改进，提供新型的液滴生成方法和液滴调节机制。
发明内容
本发明的目的在于克服上述现有技术的缺陷，提供一种基于微流控电 喷雾的单细胞全基因组扩增系统和方法。
根据本发明的第一方面，提供了一种基于微流控电喷雾的单细胞全基因组扩增系统。该系统包括微流控芯片和交流电源模块，其中所述微流控芯片设有用于注入离散相和连续相的样品流道，所述交流电源模块用于对样品流道内的样品溶液施加交流电，样品流道内的样品溶液在电场力和液体力的作用下生成液滴，液滴在一段距离内经过交流电场的拉伸裂解成超小液滴。
在一个实施例中，所述微流控芯片上固定有第一电极和第二电极，所述第一电极的一端电性连接连续相的样品流道，另一端用于接入交流电压，所述第二电极的一端电性连接生成液滴的样品流道，另一端用于接入交流电压。
在一个实施例中，将所述样品流道的形状设置为液滴生成发生于Taylor锥的尖端，注入的离散相带有离子并具有设定的电导率，注入的连续相内部混合有表面活性剂。
在一个实施例中，根据所述微流控芯片上施加的电场力和样品流道内的液体力控制Taylor锥的张角，进而控制所生成液滴的粒径和频率。
在一个实施例中，通过控制下列项中的一项或多项来控制超小液滴的生成：样品溶液的温度、电极的形状、第一电极和第二电极之间的距离、样品溶液的电导率、样品溶液的粘满性、样品流道的形状、样品流道的尺寸、连续相的流速、离散相的流速、施加的交流电的电压和频率。
在一个实施例中，本发明的系统还包括对于生成的超小液滴，采用紫外光触发固化方式将其固化成固体小球，并用扫描电镜或原子力显微镜表征方式进行表面形貌分析获得超小液滴的粒径特征，以及进行恒温核酸扩增反应，通过荧光信号来判断超小液滴内部的生物活性。
在一个实施例中，基于所获得的超小液滴的粒径特征和超小液内部的生物活性对扩增反应的影响来控制优化超小液滴的生成。
在一个实施例中，所生成的超小液滴的粒径小于5微米。
根据本发明的第二方面，提供了一种基于微流控电喷雾的单细胞全基因组扩增方法。该方法包括：利用本发明实施例提供的基于微流控电喷雾的单细胞全基因组扩增系统生成超小液滴；确定超小液滴粒径，核酸片段大小和扩增效率之间的关联关系；对单细胞全基因组扩增结果进行基因测 序，根据测序结果来控制调节生成的超小液滴的特性。
在一个实施例中，通过控制施加在所述微流控芯片上的交流电的电压和频率来控制超小液滴生成的粒径和频率。
与现有技术相比，本发明的优点在于：通过开发基于在油相中的交流电喷雾方法的微流控系统，实现高效率、高通量、小体积液滴的生成，通过对实验现象观察，研究多参数对液滴生成的协同调节规律，进而掌握实现超高通量的微小液滴生成的调节方法；并利用所生成的液滴单分散和高通量的特点，将其应用于单细胞全基因组扩增和测序文库的建立方面，有助于消除扩增偏见，实现更均匀的稀有核酸扩增。
附图说明
以下附图仅对本发明作示意性的说明和解释，并不用于限定本发明的范围，其中：
图1是根据本发明一个实施例的基于微流控电喷雾的单细胞全基因组扩增系统的示意图；
图2是根据本发明一个实施例的超小液滴生成过程示意图；
图3示出了根据本发明一个实施例的基于微流控电喷雾的单细胞全基因组扩增方法的流程图。
具体实施方式
为了使本发明的目的、技术方案、设计方法及优点更加清楚明了，以下结合附图通过具体实施例对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅用于解释本发明，并不用于限定本发明。
在本文示出和讨论的所有例子中，任何具体值应被解释为仅仅是示例性的，而不是作为限制。因此，示例性实施例的其它例子可以具有不同的值。
对于相关领域普通技术人员已知的技术、方法和设备可能不作详细讨论，但在适当情况下，所述技术、方法和设备应当被视为说明书的一部分。
根据本发明的一个实施例，提供一种基于微流控电喷雾的单细胞全基因组扩增系统作为高度集成化的超小液滴发生实验平台，参见图1所示，该系统包括微流控芯片110和交流电源120，交流电源120通过电极121 和121与微流控芯片110电性连接。该系统整体上包括液体样品注入过程、利用微流控芯片生成目标粒径液滴过程、微液滴收集过程和测序检验过程，其中微流控芯片120集成多个功能单元，包括液体样品注入、液鞘流生成、微液滴裂解、微液滴收集等，能够完成将液体样品裂解成目标粒径微液滴的功能。
参见图1所示，在该系统中，将液体样品a通入到本发明实施例的超小液滴发生微流控芯片b(也标记为微流控芯片110)中；在微流控芯片上自动化完成数十万至数百万个液滴的裂解，获得超小液滴；收集裂解后的超小液滴样品；将收集到的超小液滴样品进行单细胞全基因组扩增；最后，送到测序仪c上机进行测序检验。
下文将具体介绍本发明实施例的微流控芯片、液滴调节机制以及单细胞全基因组扩增和测序的内容。
1)、关于微流控芯片
在本发明实施例中，采用微液滴发生芯片(或称为微流控芯片)通过电喷雾方式将样品溶液有效裂解成目标粒径，例如裂解为超微液滴(小于5微米)。
参见图2所示，在微流控芯片中，液滴生成机制是：在液滴发生单元中，流动聚焦结构来生成液体样品的鞘流，通过流体力学分析，计算模拟微流控芯片内的流场分布，优化设计生成鞘流的形状和特性，使其流径和流速等特征值适用于后续的液滴裂解步骤。在整个鞘流结构通过电极位置以后，通过电源驱动交流电场，对样品液体进行裂解操作，且此操作后续有望进行高通量的并行扩展。在该微流控芯片中，结合流速和场强控制，可以在裂解液滴的同时进行超小液滴粒径的调节，使其可以进行定向应用开发。
具体地，仍参见图2所示，微流控芯片设有用于注入离散相和连续相的样品通道，用于施加两相交流电的交流电源模块，对所述样品流道内的样品溶液施加交流电压，两种不相溶的液体在样品通道的流体结构处交汇，由于“油相”样品和“水相”样品的液体表面张力差和外加压力所产生的剪切力，“水相”样品交汇处被“油相”样品从连续相分割为离散的液滴，样品流道内的样品溶液在电场力和液体力的作用下生成液滴，液滴为锥状(即泰勒锥)，液滴在一个较短距离内(泰勒锥的尖端)经过电场力 的高速拉伸生成超小液滴。
在本发明的微流控芯片中，超小液滴生成的过程是液体力与电场力的动态平衡，可通过调节例如温度，湿度，电极形状，对电极的距离，液体的电导率，粘满性，微流控芯片流道形状和尺寸，两相流速以及电场强度和频率等参数控制生成符合期望的超小液滴。液滴裂解现象只在特定的参数组合下发生，确定不同参数对液滴裂解的作用，有助于更好地控制液滴生成。
在一个实施例中，选择合适的离子浓度离散相和混有表面活性剂的连续相，研究不同配比对所生成液滴的稳定性的影响。液滴裂解发生于Taylor锥的尖端，要求离散相(水相)必须有离子以便导电，离子的浓度决定电导率，因此对液滴生成的稳定有直接影响；而连续相(油相)内部混合的表面活性剂，又直接影响液滴在生成后以及恒温扩增中的稳定性。研究离子浓度和表面活性剂浓度的影响，有助于优化液滴稳定性，以便进行后续生物学实验。
在一个实施例中，根据微流控芯片中超小液滴裂解和调控规律，测量Taylor锥角在交流电场条件下的变化规律，基于Taylor锥公式进行理论分析，通过建立数学模型来控制生成液滴的尺寸和频率等。Taylor锥的张角受电场和流场的双重作用，直接影响后续生成液滴的尺寸和频率，通过建立数学模型并结合实验现象，有助于分析和理解油水两相的交流电喷雾机理。
在一个实施例中，根据液滴裂解的经验参数确定产生超小液滴裂解时的流速、场强、频率等参数阈值，从而控制交流电喷雾可以产生1～100微米的液滴，对于scWGA，需要小于10微米的超小液滴。通过实验，能够总结出稳定生成超小液滴的条件规律，从而有利于区分关键参数和次关键参数。
综上，对于本发明的微流控芯片，通过调节微流管道参数和电学参数，确定发生液滴裂解现象的参数工作范围，建立液滴尺寸与交流电场强度和频率的协同调控模型，能够获得在交流电场条件下，多个参数与液滴尺寸的协同关系，从而控制稳定生成高度均一的超小液滴裂解。此外，通过建立基于Taylor锥的变化规律模型，能够获知在两相交流电喷雾条件下液滴裂解的物理机制，有利于液滴粒径的调节。
2)、关于液滴调节机制
在实际应用中，在微流控芯片初步定性地生成超小液滴之后，为了进一步对液滴的粒径进行调节以获得准确的测序结果，需要把各个重要关键参数进行一一定量测试，将对应的样品溶液的粒径调节的参数组合进行进一步优化。
对于液滴调节机制的研究可通过表征液滴的粒径特征和验证液滴的生物活性进行。例如，为了表征所生成液滴的粒径特性，可以采用紫外光触发固化的方法，将超小液滴固化成固体小球，并用扫描电镜或原子力显微镜等表征方式进行表面形貌分析。同时，为验证超小液滴的生物活性(即经过强电场之后其内的生物分子仍然保持活性)，需要进行恒温核酸扩增的反应，例如LAMP和MDA，通过荧光信号来判断其生物活性，通过数字PCR和实时荧光定量PCR来检验扩增是否发生，并评估扩增产物的质量。
具体地，超小体积液滴对单细胞全基因组扩增均匀度的作用包括：
A)、利用常见的如吸收光谱，荧光寿命光谱、杂交等方法，测量超小液滴中酶、核酸的活性。
由于液滴裂解发生于Taylor锥的尖端，此处的电场为极强，因为所有的电场线均穿过此区域。表征超小液滴内部的生物分子活性，有助于理解强电场对生物分子的作用规律。本申请实施例的交流电喷雾生成的超小液滴可以成功进行核酸扩增，通过表征超小液滴内部的生物分子活性，能够进一步确定交流电场对液滴内部核酸活性的影响。
B)、探索在超小液滴中实现恒温核酸扩增(MDA)的条件，并进行单细胞全基因组扩增反应。
确定液滴大小，核酸片段大小和扩增效率之间的关联关系。由于核酸扩增原料有限制，为了达到一定的扩增比例，并不是越小的液滴越好；考虑到液滴的大小一致，但碎裂的DNA片段大小不尽相同，一般是有一个分布，这样扩增产物的分子数量也会有一定程度的差别；再加上过小的片段由于无法成环，也不能进行MDA，因此片段也有一个下限。这些都决定了在减小液滴体积和增加液滴数量的过程中，需要仔细寻找一个效能最优区间。
3)、关于单细胞全基因组扩增和测序实验
经过上述微流控芯片生成超小液滴，以及研究超小体积液滴对单细胞全基因组扩增均匀度的作用，一方面能够稳定生成液滴，并精确调控液滴大小；另一方面，能够确保液滴进行恒温MDA核酸扩增。
在样品完成预扩增之后，进一步地，对单细胞全基因组扩增结果进行基因测序，分析基因组扩增的均匀度，评估对扩增偏见的抑制效果。例如，在完成预扩增之后，进行常规建库，并送交测序，分析测序结果，与历史的实验结果作对比，研究超小液滴对提高扩增均匀性的影响。通过生物信息学分析，验证本发明实施例生成的超小液滴对提高扩增均匀度的作用。通过进行不同基因组片段、液滴大小的组合实验，找出优化参数，以提高测序结果的准确性。
需说明的是，本发明实施例的系统还包括将成熟的单细胞捕获和分离模块、裂解腔室与微阀门等整合到微流控芯片中，从而实现一片式高通量细胞顺序捕获、裂解、并经过电喷雾生成超小液滴并进行全基因组扩增操作。
综上，本发明的系统使用交流电生成液滴，结合液体控制与电学控制的综合调节，将微液滴发生模块集成到微机电芯片中，利用流体方法稳定的控制优势及电学调制方法的速度和通量优势，最终实现短时间内大量粒径均一的超微液滴(如小于5微米)的生成。本发明的系统具有通量高、操作简单、液滴粒径均一性高等特点。
本发明实施例还提供一种基于微流控电喷雾的单细胞全基因组扩增方法，参见图3所示，该方法包括以下步骤：
步骤S310，利用微流控芯片通过交流电驱动产生期望目标粒径的超小液滴。
例如，通过设定样品溶液的温度、电极的形状、对电极的距离、样品溶液的电导率、样品溶液的粘满性、样品流道的形状、样品流道的尺寸、连续相的流速、离散相的流速、连续相内部混合的表面活性剂的量、施加的交流电的电压和频率等来控制生成具有期望粒径和内部活性的超小液滴。
步骤S320，确定超小液滴粒径，核酸片段大小和扩增效率之间的关联关系。
例如，在超小液滴中实现恒温核酸扩增(MDA)的条件，并进行单细 胞全基因组扩增反应，确定液滴大小，核酸片段大小和扩增效率之间的关联关系，进而确定扩增比例、超小液滴粒径、碎裂的DNA片段之间的平衡关系，寻找一个效能最优区间。
步骤S330，对单细胞全基因组扩增结果进行基因测序，根据测序结果进一步调节控制液滴生成的参数。
在完成扩增之后，送交测序，并基于测序结果确定超小液滴对提高扩增均匀性的影响，进而进一步优化影响超小液滴生成的参数，例如，交流电场的电压、频率、连续相内部混合的表面活性剂的量等。
综上所述，本发明利用微流控芯片，在交流电场作用下，实现油水两相中的液滴裂解，在油相中首次实现电喷雾乳化现象，克服传统电喷雾微液滴在空气中容易蒸发的问题，并使得液滴粒径均匀、可调；根据核酸、酶等生物分子在经过交流电场后的性状变化，解释电场对生物分子的影响规律，基于所生成液滴可以进行核酸恒温扩增的事实，提出将微液滴应用于单细胞全基因组扩增，以减少扩增偏好。本发明的交流电喷雾的液滴裂解方法，对于电喷雾领域乃至整个微流体液滴生成理论体系的一个强有力的补充，解决了单细胞测序中的扩增偏见问题，有助于实现更精准的单细胞全基因组的定量测序分析，从而为肿瘤早筛、产前诊断等个体化医疗提供帮助。
应理解的是，在本发明实施例中，样品流道的尺寸设计与液滴的尺寸匹配，例如，液滴的尺寸一般小于100微米，样品流道也在100微米左右。在实际应用中，两相液体从进样口到交汇处，可能需要流经一段主管道。主管道的宽度一般设置为大于100微米(例如200微米～500微米)，从而降低流体运动的流阻。此外，本文所述超小液滴、超微小液滴或超微液滴具有相同的含义，表示通过交流电场裂解后的超小液滴，除非根据上下文另有所指。
需要说明的是，虽然上文按照特定顺序描述了各个步骤，但是并不意味着必须按照上述特定顺序来执行各个步骤，实际上，这些步骤中的一些可以并发执行，甚至改变顺序，只要能够实现所需要的功能即可。
以上已经描述了本发明的各实施例，上述说明是示例性的，并非穷尽性的，并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下，对于本技术领域的普通技术人员来说许多修改和变更 都是显而易见的。本文中所用术语的选择，旨在最好地解释各实施例的原理、实际应用或对市场中的技术改进，或者使本技术领域的其它普通技术人员能理解本文披露的各实施例。

Claims (10)

  1. 一种基于微流控电喷雾的单细胞全基因组扩增系统，其特征在于，包括微流控芯片和交流电源模块，其中所述微流控芯片设有用于注入离散相和连续相的样品流道，所述交流电源模块用于对样品流道内的样品溶液施加交流电，样品流道内的样品溶液在电场力和液体力的作用下生成液滴，液滴在一段距离内经过交流电场的拉伸裂解成超小液滴。
  2. 根据权利要求1所述的系统，其特征在于，所述微流控芯片上固定有第一电极和第二电极，所述第一电极的一端电性连接连续相的样品流道，另一端用于接入交流电压，所述第二电极的一端电性连接生成液滴的样品流道，另一端用于接入交流电压。
  3. 根据权利要求1所述的系统，其特征在于，将所述样品流道的形状设置为液滴生成发生于Taylor锥的尖端，注入的离散相带有离子并具有设定的电导率，注入的连续相内部混合有表面活性剂。
  4. 根据权利要求3所述的系统，其特征在于，根据所述微流控芯片上施加的电场力和样品流道内的液体力控制Taylor锥的张角，进而控制所生成液滴的粒径和频率。
  5. 根据权利要求2所述的系统，其特征在于，通过控制下列项中的一项或多项来控制超小液滴的生成：样品溶液的温度、电极的形状、第一电极和第二电极之间的距离、样品溶液的电导率、样品溶液的粘满性、样品流道的形状、样品流道的尺寸、连续相的流速、离散相的流速、施加的交流电的电压和频率。
  6. 根据权利要求1所述的系统，其特征在于，该系统还包括对于生成的超小液滴，采用紫外光触发固化方式将其固化成固体小球，并用扫描电镜或原子力显微镜表征方式进行表面形貌分析获得超小液滴的粒径特征，以及进行恒温核酸扩增反应，通过荧光信号来判断超小液滴内部的生物活性。
  7. 根据权利要求6所述的系统，其特征在于，基于所获得的超小液滴的粒径特征和超小液内部的生物活性对扩增反应的影响来控制优化超小液滴的生成。
  8. 根据权利要求1至7任一项所述的系统，其特征在于，所生成的超 小液滴的粒径小于5微米。
  9. 一种基于微流控电喷雾的单细胞全基因组扩增方法，包括：
    利用权利要求1至8任一项所述的系统生成超小液滴；
    确定超小液滴粒径，核酸片段大小和扩增效率之间的关联关系；
    对单细胞全基因组扩增结果进行基因测序，根据测序结果来控制调节生成的超小液滴的特性。
  10. 根据权利要求9所述的方法，其特征在于，通过控制施加在所述微流控芯片上的交流电的电压和频率来控制超小液滴生成的粒径和频率。
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