WO2021003631A1 - 基于微流控液滴打印系统的数字pcr检测方法及应用 - Google Patents

基于微流控液滴打印系统的数字pcr检测方法及应用 Download PDF

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WO2021003631A1
WO2021003631A1 PCT/CN2019/094987 CN2019094987W WO2021003631A1 WO 2021003631 A1 WO2021003631 A1 WO 2021003631A1 CN 2019094987 W CN2019094987 W CN 2019094987W WO 2021003631 A1 WO2021003631 A1 WO 2021003631A1
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printing
microfluidic
droplets
droplet
substrate
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PCT/CN2019/094987
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English (en)
French (fr)
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门涌帆
潘挺睿
敖婷婷
李致昊
吴碧珠
陈艳
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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
    • C12M1/36—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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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  • the present invention relates to the technical field of PCR, in particular to a digital PCR detection method and application based on a microfluidic droplet printing system.
  • Digital PCR (polymerase chain reaction) technology is an absolute quantification technology of nucleic acid molecules based on the Poisson distribution principle, and has broad application prospects in the field of absolute counting/quantification of nucleic acid molecules.
  • the dispersion of nucleic acid samples is the most important step of digital PCR.
  • the DNA template should be completely dispersed in a separate reaction system, such as micropores or droplets. These tiny reaction systems do not interfere with each other, and the instrument detects the fluorescence change of each micro system to determine whether there is a PCR reaction in it.
  • microfluidic chip method such as the Thermo Quant Studio TM 3d digital PCR system, which combines basic sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes.
  • the operating unit is integrated on a micron-scale chip to automatically complete the entire analysis process. In this way, the work flow is cumbersome, the chip manufacturing process is complex, and the manufacturing process requirements are high. There are problems of different droplet sizes and overlap between droplets, which increases the cost of detection.
  • the other is the droplet method, such as the BIO-RAD QX200 TM digital PCR system. This system uses two-phase flow technology to disperse the sample in a large number of droplets. The number and size of the droplets can be controlled.
  • the three functions of sample division, amplification and detection are independent, and the degree of integration and automation is not high. It is easy to cause droplet fusion, breakage and cross-contamination during pipetting, and the droplet generation speed is not constant No change, the time of droplet generation cannot be accurately grasped.
  • the above two methods are both performed in a closed centrifuge tube, and there is currently no report of directly separating the template solution into small droplets in an open environment.
  • the number of droplets currently generated is about 1000-6000, which needs to be improved. There is a problem of aerosol pollution during the droplet generation process, and the biological safety is low.
  • the purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide a digital PCR detection method and application based on a microfluidic droplet printing system, which uses the microfluidic printing system to automatically control the process of droplet printing, digital PCR detection, etc. .
  • a digital PCR detection method based on a microfluidic droplet printing system comprising:
  • Step S1 using a microfluidic printing system in a non-closed environment to print the sample solution to be tested from the nozzle of the microfluidic printing chip onto the substrate by air pressure, and form a preset droplet array on the substrate;
  • Step S2 Place the substrate carrying the droplet array on the heating plate of the PCR machine for thermal cycling;
  • Step S3 Take a fluorescent photo of the droplet array on the substrate, analyze and identify the light and dark droplets, and count the proportion of positive droplets.
  • the microfluidic printing chip is provided with a gas pressure channel for receiving gas and a fluid channel for receiving the sample solution to be tested.
  • the gas pressure cuts the sample solution to be tested into droplets, and passes through the microfluidic printing chip.
  • the nozzles provided on the fluid control printing chip are printed on the substrate under the microfluidic printing chip.
  • the microfluidic printing system presets droplet printing related parameters, and controls the relative movement of the microfluidic printing chip with respect to the substrate based on the set printing related parameters, so that A preset array of droplets is formed on the substrate.
  • the microfluidic droplet printing system includes a drive control module, an electric translation stage, an air valve device, a camera microscope system, and a data analysis and processing module, wherein the air valve device is used to direct the micro
  • the fluidic printing chip provides air pressure
  • the electric translation stage is used to place the microfluidic printing chip
  • the camera microscope system is used to take fluorescent photos of the droplets
  • the data analysis processing module is used to identify light and dark droplets and The proportion of positive droplets is counted
  • the drive control module is used to control the electric translation stage, the air valve device and the camera microscope system based on the set printing related parameters.
  • the printing-related parameters set by the drive control module include the number of print droplets, the number of print drop rows, the number of print drop columns, the print mode, the channel number of the air valve device, and the number of print droplets.
  • the denaturation parameter in step S2 is set to 90°C, 60s; the annealing parameter is set to 95°C, 5s, 60°C, and 15s; the extension parameter is set to 72°C, 120s; and the number of thermal cycles is set to 40.
  • the substrate is made of 75*50*1mm ordinary glass and polydimethylsiloxane wall bonded by plasma cleaning method.
  • the following steps are used to manufacture the microfluidic printing chip:
  • a soft etching method is used to fabricate polydimethylsiloxane chips to form the microfluidic printing chip.
  • the nozzle of the microfluidic printing chip is a sealing film.
  • the application of the method of the present invention in digital PCR detection such as genetic engineering, medical diagnosis and environmental engineering.
  • the present invention has the advantages that: droplets can be generated in a non-closed space, and the droplets do not touch or interfere with each other, and the droplet array, droplet size, and number of droplets are all accurate.
  • the microfluidic printing chip is easy to manufacture, low cost, and the sample solution loss during the printing process is small.
  • Fig. 1 is a flowchart of a digital PCR detection method based on a microfluidic droplet printing system according to an embodiment of the present invention
  • Figure 2 is an effect diagram of a microfluidic printing chip according to an embodiment of the present invention.
  • FIG. 3 is a process schematic diagram of a digital PCR detection method of a microfluidic droplet printing system according to an embodiment of the present invention
  • Fig. 4 is a fluorescent effect diagram of a droplet array according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram of the proportion of positive droplets according to an embodiment of the present invention.
  • Fig. 6 is a simulation diagram of a microfluidic printing platform according to an embodiment of the present invention.
  • Fig. 7 is a physical diagram of a microfluidic droplet printing platform according to an embodiment of the present invention.
  • a digital PCR detection method uses a microfluidic droplet printing system (or printing platform) to directly print the sample solution to be tested on a substrate,
  • the droplets on the substrate can form droplet arrays of different sizes; then, perform a digital PCR reaction to obtain a fluorescence effect map of the droplet array; and then analyze the relationship between the initial concentration of the sample solution and the proportion of positive droplets.
  • the digital PCR detection method based on the microfluidic droplet printing system includes the following steps:
  • Step S110 preparing a microfluidic printing chip with fluid channels and air pressure channels.
  • the microfluidic printing chip is used to generate liquid droplets, and is provided with a fluid channel for receiving the sample solution to be tested, a gas pressure channel for accessing gas, and a nozzle for delivering the generated liquid droplets.
  • the microfluidic printing chip is divided into a thick layer channel and a thin layer channel.
  • the thick layer is a fluid channel and an air pressure channel.
  • the thin layer provides droplet ejection ports.
  • the sample solution is inserted from one end of the thick layer channel. Connected from the other end of the thick channel, the sample solution and the air meet at the cross of the thick channel, and under the action of pressure and shear force, droplets are formed, and the generated droplets can be output through the nozzle, see Figure 3(b) )
  • FIG. 2 Multiple channels can be set on the microfluidic printing chip, for example, 12 channels can be set, which can simultaneously access 12 valve channels and print simultaneously.
  • the thin layer of the microfluidic printing chip is a sealing film.
  • the preparation process of the microfluidic printing chip includes: first, use the computer commercial software AutoCAD to design the chip pattern, and print the chip design drawing on the transparent film; then, use the transparent film as a photolithography mask and use The photolithography technology transfers the mask pattern to the silicon wafer coated with photoresist; then uses the soft etching method to make polydimethylsiloxane (PDMS) chips; the prepared thick and thin layers are cleaned by plasma ( PLASMA) is pasted and baked in an oven at 80°C for more than 2 hours to complete the production of microfluidic printing chips.
  • PLASMA polydimethylsiloxane
  • Step S120 preparing a substrate for carrying the microfluidic droplet array.
  • the substrate is used to carry liquid droplets output from the nozzle of the microfluidic printing chip.
  • the substrate can be made of 75*50*1mm ordinary glass and PDMS wall pasted by PLASMA.
  • Step S130 preparing a PCR sample solution to be tested.
  • Step S140 using the microfluidic droplet printing system to print the sample solution to be tested from the nozzle of the microfluidic printing chip onto the substrate by air pressure.
  • the microfluidic droplet printing system includes a drive control module, an electric translation stage, a gas valve device, a camera microscope system, and a data analysis and processing module.
  • the air valve device is used to provide air pressure to the microfluidic printing chip, such as connecting an air pump and the air pressure channel of the microfluidic printing chip, so as to provide appropriate air pressure for the microfluidic printing chip.
  • the electric translation stage is used to place the microfluidic printing chip and the substrate, and the substrate is located under the microfluidic printing chip.
  • the camera microscope system is used to take fluorescence photos of liquid droplets, and includes, for example, a camera, a microscope lens, a light source, and a light source controller.
  • the data analysis and processing module is used to identify light and dark droplets and count the proportion of positive droplets, for example, automatically stitch scanned images to form droplet images and further identify the images.
  • the drive control module is used to set printing-related parameters and correspondingly control the electric translation stage, the air valve device and the camera microscope system based on the set parameters.
  • the drive control module controls the movement speed of the electric translation stage, the single step step length, and automatically moves to different positions of the microfluidic printing chip, so as to realize the free movement of the electric translation stage on the X-Y axis and the Z axis.
  • the drive control module is also used to control the camera, including controlling exposure time, brightness, contrast, white balance, and taking pictures.
  • the drive control module can also control the channel and air pressure of the air valve device.
  • the drive control module and the data analysis and processing module can be implemented by software and provide a human-computer interaction interface to facilitate the user to set printing parameters and visually display data analysis results. It should be noted that the drive control module and the data analysis and processing module can also be integrated into one software module.
  • the process of microfluidic droplet printing includes: before use, the microfluidic droplet printing system detects the connection status with equipment (such as electric translation stage, air valve device, camera, etc.), and then opens the software, The system first automatically tests the connection with the device and initializes the position of the translation table. Specifically, first, select the template for printing (including the number of print droplets, the number of rows, and the number of columns, etc.), and then set the printing mode (such as intermittent printing or continuous printing) and the valve channel according to the different properties of the liquid.
  • equipment such as electric translation stage, air valve device, camera, etc.
  • the system first automatically tests the connection with the device and initializes the position of the translation table. Specifically, first, select the template for printing (including the number of print droplets, the number of rows, and the number of columns, etc.), and then set the printing mode (such as intermittent printing or continuous printing) and the valve channel according to the different properties of the liquid.
  • the microfluidic printing system can control the relative movement of the microfluidic printing chip and the substrate, separate the reaction units by translation, and form an array of droplets on the substrate, and the microfluidic printing system can adjust the size and generation of the generated droplets. Stable and highly uniform droplets, with faster adjustment speed and droplet generation speed, and the adjustment process will not affect the generation of droplets, and can be adjusted continuously.
  • the air pump, camera, microscope lens, etc. in the embodiments of the present invention may all adopt commercially available or customized products, and the connection between these devices and the computer may be realized by using existing interfaces and circuit boxes.
  • step S150 the substrate is placed on the heating plate of the PCR machine for thermal cycling.
  • Step S160 taking a fluorescent photo of the generated droplet array.
  • the glass substrate covered with droplets is placed on a fluorescent inverted microscope, and the local fluorescent photos of the droplets are taken through a motorized translation stage until all the droplet arrays are taken complete.
  • Step S170 Analyze the fluorescence photo of the identified droplets to obtain the proportion of positive droplets in the sample solution to be tested.
  • the local droplet fluorescence photos are spliced through a data analysis and processing module (such as computer software) to form a complete droplet fluorescence photo.
  • the computer software identifies the light and dark droplets and counts the positive droplets. Ratio to calculate the initial concentration of the sample.
  • the splicing of fluorescent photos adopts the direct splicing method, that is, the parts that do not overlap between the two images are directly connected side to side.
  • the recognition process of light and dark droplets is: converting the captured droplet image into a grayscale image, separating the adhesion between the droplets, between the droplets and the background, between the droplets and the impurities, and then identify the droplets.
  • the embodiment of the present invention uses seven concentration gradient human c-DNA samples as the PCR reaction system. After printing on the microfluidic digital printing platform, the PCR reaction is carried out, and then each system solution is obtained by splicing fluorescent images. The complete image of the droplet array, where the white dots are positive droplets, the black dots are negative droplets, and there are few bright droplets with dilute concentration. The proportion of positive droplets is positively correlated with the concentration gradient, as shown in Figure 5, which proves that the droplet printing platform can achieve efficient, stable and accurate printing effects, and the droplet array can exhibit digital PCR effects.
  • the present invention generates droplets of controllable size through droplet printing, and then places the substrate covered with the droplets on a microscope to detect and count fluorescent signals. This method can avoid sample contamination, improve the simplicity of experimental operation, and provide a new direction for fully automated digital PCR.
  • the present invention uses a microfluidic droplet printing system to perform digital PCR detection.
  • the droplet size and array can be generated in a non-closed environment by precisely controlling the movement of the electric mobile stage and the pressure of the valve.
  • Controllable and adjustable droplets, and no additional digital PCR detection device is needed, which can realize the integrated operation of droplet generation, amplification and detection, avoiding sample contamination and droplets caused by the existing split multi-step operation Fusion problem;
  • the fluorescent signal can be detected in time and effectively, avoiding the problem that the fluorescent signal cannot be detected normally due to the passage of time. Realizing the automation of droplet generation and droplet recognition through digital PCR detection reduces the recognition time and error rate. This automated control and processing is beneficial to the industrialization of digital PCR detection.
  • the digital PCR detection method of the present invention can be applied to genetic engineering, medical diagnosis, environmental engineering, etc., such as tumor research, single cell analysis, virus microbial analysis, gene sequencing verification, etc.

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Abstract

本发明提供一种基于微流控液滴打印系统的数字PCR检测方法及应用。该方法包括：在非封闭环境中利用微流控打印系统控制将待检测样本溶液通过气压方式从微流控打印芯片喷口打印到基底上并在该基底上形成预设的液滴阵列；将承载液滴阵列的基底放置在PCR仪加热板上进行热循环；拍摄基底上液滴阵列的荧光照片，分析识别明暗液滴并统计阳性液滴占比。本发明的方法能够在非封闭空间内产生液滴，并且液滴互不接触，互不干扰，通过自动化地控制液滴打印进程、打印效果和PCR检测，有利于数字PCR检测的产业化。

Description

基于微流控液滴打印系统的数字PCR检测方法及应用 技术领域
本发明涉及PCR技术领域，尤其涉及一种基于微流控液滴打印系统的数字PCR检测方法及应用。
背景技术
数字PCR(聚合酶链式反应)技术是一种基于泊松分布原理的核酸分子绝对定量技术，在核酸分子的绝对计数/定量领域具有广阔的应用前景。核酸样品的分散是数字PCR的最重要的一步。理想情况下，DNA模板应该完全分散在单独的反应体系中，如微孔或液滴。这些微小的反应系统互不干扰，仪器检测每个微系统的荧光变化，以确定其中是否存在PCR反应。目前，主要有两种样品分散方式，一种是微流控芯片法，如Thermo Quant Studio TM3d数字PCR系统，其是将生物、化学、医学分析过程的样品制备、反应、分离、检测等基本操作单元集成到一块微米尺度的芯片上，自动完成分析全过程。这种方式工作流程繁琐，芯片制作过程复杂，对制作工艺要求较高，存在液滴大小不一、液滴间有重叠的问题，从而增加了检测成本。另一种是液滴法，如BIO-RAD QX200 TM数字PCR系统，这种系统采用两相流技术将样品分散在大量的液滴中，液滴数量和大小可控，但在这种液滴式数字PCR系统中，样品划分、扩增和检测三个功能独立，集成化和自动化程度不高，移液过程中易造成液滴融合、破碎以及交叉污染等问题，并且液滴产生速度不是恒定不变，不能精确的把握液滴产生时间。
此外，上述两种方法都是在封闭的离心管中进行的，目前没有在敞开环境中直接将模板溶液分离成小液滴的报道。并且，目前生成的液滴个数1000-6000左右，还有待提高，在液滴生成过程存在气溶胶污染问题，生物安全性较低。
因此，需要对现有技术进行改进，以提供液滴精确可控、互不干扰、检测成本低的数字PCR检测方法。
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液滴打印系统的数字PCR检测方法及应用，利用微流控打印系统自动化地控制液滴打印、数字PCR检测等过程。
根据本发明的第一方面，提供了一种基于微流控液滴打印系统的数字PCR检测方法，该方法包括：
步骤S1：在非封闭环境中利用微流控打印系统控制将待检测样本溶液通过气压方式从微流控打印芯片喷口打印到基底上并在该基底上形成预设的液滴阵列；
步骤S2：将承载液滴阵列的基底放置在PCR仪加热板上进行热循环；
步骤S3：拍摄基底上液滴阵列的荧光照片，分析识别明暗液滴并统计阳性液滴占比。
在一个实施例中，所述微流控打印芯片上设有用于接收气体的气压通道和用于接收待检测样本溶液的流体通道，气压将待检验样品溶液剪切为液滴，通过所述微流控打印芯片上设置的喷口打印到位于所述微流控打芯片下方的基底上。
在一个实施例中，所述微流控打印系统预先设置液滴打印相关参数，并基于所设置的打印相关参数来控制所述微流控打印芯片相对于所述基底的相对移动，从而在所述基底上形成预设的液滴阵列。
在一个实施例中，所述微流控液滴打印系统包括驱动控制模块、电动平移台、气阀装置、摄像显微镜系统和数据分析处理模块，其中，所述气阀装置用于向所述微流控打印芯片提供气压；所述电动平移台用于放置所述微流控打印芯片；所述摄像显微镜系统用于拍摄液滴的荧光照片，所述数据分析处理模块用于识别明暗液滴并统计阳性液滴占比，所述驱动控制模块用于基于所设置的打印相关参数控制所述电动平移台、所述气阀装置和所述摄像显微镜系统。
在一个实施例中，所述驱动控制模块设置的打印相关参数包括打印液滴的个数、打印液滴行数、打印液滴列数、打印模式、气阀装置的通道编号、打印液滴之间的间距、打印频率、打印脉宽、打印头与电动平移台之间距离中的一项或多项。
在一个实施例中，步骤S2中的变性参数设置为90℃，60s；退火参数设置为95℃，5s，60℃，15s；延伸参数设置为72℃，120s；热循环次数 设置为40。
在一个实施例中，所述基底由75*50*1mm普通玻璃和聚二甲基硅氧烷墙经等离子清洗方法粘贴而成。
在一个实施例中，采用以下步骤制造所述微流控打印芯片：
将所述微流控打印芯片的设计图打印在透明胶片上；
将透明胶片作为光刻掩膜并将光刻掩模转移到涂有光刻胶的硅片上；
采用软刻蚀方法制作聚二甲基硅氧烷芯片，形成所述微流控打印芯片。
在一个实施例中，所述微流控打印芯片喷口是封口膜。
根据本发明的第二方面，提供了本发明的方法在数字PCR检测中的应用，例如应用于基因工程、医学诊断和环境工程等。
与现有的PCR技术相比，本发明的优点在于：可以在非封闭空间内产生液滴，并且液滴互不接触，互不干扰，液滴阵列、液滴大小、液滴数量都精确可控，液滴打印进程能够随停随开；此外，微流控打印芯片制造简单，成本低，打印过程样本溶液损耗少。
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液滴打印系统的数字PCR检测方法的流程图；
图2是根据本发明一个实施例的微流控打印芯片的效果图；
图3是根据本发明一个实施例的微流控液滴打印系统的数字PCR检测方法的过程示意；
图4是根据本发明一个实施例的液滴阵列的荧光效果图；
图5是根据本发明一个实施例的阳性液滴占比的示意图；
图6是根据本发明一个实施例的微流控打印平台的模拟图；
图7是根据本发明一个实施例的微流控液滴打印平台的实物图。
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，提供了一种数字PCR检测方法，简言之，该方法利用微流控液滴打印系统(或称打印平台)，将待检测的样品溶液直接打印在基板上，基板上液滴能够形成不同大小的液滴阵列；然后，进行数字PCR反应得出液滴阵列荧光效果图；进而分析样品溶液的初始浓度和阳性液滴占比之间的关系。
具体地，结合图1和图3所示，本发明实施例的基于微流控液滴打印系统的数字PCR检测方法包括以下步骤：
步骤S110，制备具有流体通道和气压通道的微流控打印芯片。
在本发明中，微流控打印芯片用于产生液滴，其上设有用于接收待检测样品溶液的流体通道、用于接入气体的气压通道以及用于输送所生成液滴的喷口。
在一个实施例中，微流控打印芯片分为厚层通道和薄层通道，其中厚层是流体通道和气压通道，薄层提供液滴喷口，样品溶液从厚层通道的一端接入，气压从厚层通道的另一端接入，样品溶液和空气在厚层通道交叉相遇，在压力和剪切力的作用下，形成液滴，所产生的液滴可通过喷口输出，参见图3(b)示意的微流控打印芯片的结构和图2示意的微流控打印芯片效果。微流控打印芯片上可设置多个通道，例如设置12个通道，能够同时接入12个气阀通道同时打印。
可选地，微流控打印芯片的薄层是封口膜。
在一个实施例中，微流控打印芯片的制备过程包括：首先，利用计算机商业软件AutoCAD设计芯片图案，将芯片设计图打印在透明胶片上；然后，将该透明胶片作为光刻掩膜，利用光刻技术将掩膜图案转移到涂有光刻胶的硅片上；再采用软刻蚀方法制作聚二甲基硅氧烷(PDMS)芯片；将制备好的厚层和薄层用等离子清洗方法(PLASMA)进行粘贴，并在80℃烘箱烘2h以上，从而完成微流控打印芯片的制作。
步骤S120，制备用于承载微流控液滴阵列的基底。
在本发明中，基底用于承载从微流控打印芯片的喷口输出的液滴。例如，基底可由75*50*1mm普通玻璃和PDMS墙经PLASMA粘贴而成。
步骤S130，配制待检测的PCR样品溶液。
在此步骤中，制备待检测的PCR样品溶液，置于离心管中，例如，参见图3(a)所示，将人源c-DNA模板、PCR MIX、探针、水按比例制成20ul混合溶液，震荡混匀离心，置于离心管中备用。
步骤S140，利用微流控液滴打印系统将待检测的样品溶液用气压方式从微流控打印芯片喷口打印到基底上。
参见图6和图7所示，在本发明实施例中，微流控液滴打印系统包括驱动控制模块、电动平移台、气阀装置、摄像显微镜系统和数据分析处理模块。
气阀装置用于向微流控打印芯片提供气压，例如连接气泵和微流控打印芯片的气压通道，从而为微流控打印芯片提供适当的气压。
电动平移台用于放置微流控打印芯片和基底，其中基底位于微流控打印芯片的下方。
摄像显微镜系统用于拍摄液滴的荧光照片，例如包括相机、显微镜头、光源以及光源控制器等。
数据分析处理模块用于识别明暗液滴并统计阳性液滴占比，例如，将扫描图像自动拼接形成液滴图像并进一步识别图像。
驱动控制模块用于设置打印相关参数并基于所设置的参数相应地控制电动平移台、气阀装置和摄像显微镜系统等。
例如，驱动控制模块控制电动平移台的运动速度、单步步长、自动移动到微流控打印芯片的不同位置等，实现电动平移台在X-Y轴和Z轴的自由运动。
例如，驱动控制模块还用于实现对摄像头的控制功能，包括控制曝光时间、亮度、对比度、白平衡和拍照等。
例如，驱动控制模块还可以控制气阀装置的通道和气压大小等。
在本发明实施例中，驱动控制模块和数据分析处理模块可采用软件实现并提供人机交互界面，以便于用户设置打印参数以及直观显示数据分析结果等。需说明的是，驱动控制模块和数据分析处理模块也可以集成为一个软件模块。
在一个实施例中，微流控液滴打印的过程包括：使用前，微流控液滴 打印系统检测与设备(例如电动平移台、气阀装置、摄像机等)的连接状况，然后打开软件，系统先自动测试与设备的连接状况，并初始化平移台的位置。具体地，首先，选择打印的模板(包括打印液滴的个数、行数和列数等)，再根据液体的不同性质设定好打印模式(如间断打印或连续打印)、气阀通道channel的编号、打印液滴与液滴之间的间距以及打印的频率、脉宽等参数；接着，调整气泵的气压并按按钮将打印头自动就位(也可以通过软件中的控制面板来调整打印头与平移台的距离)；最后，也可增加手动摇杆，通过摇杆挑选合适的起始打印位置，加液之后便可以开始打印。在打印期间，可以随时暂停，重新开始，结束打印。
利用微流控打印系统能够控制微流控打印芯片和基底的相对移动，通过平移方式分隔反应单元，在基底上形成液滴阵列，并且微流控打印系统能够调节生成的液滴的尺寸、生成稳定且均一度较高的液滴，并具有较快的调节速度和液滴生成速度，且调节过程不会影响液滴的生成，可连续化调整。
应理解的是，本发明实施例的气泵、相机、显微镜头等均可采用市售的或定制的产品，这些设备与计算机的连接可利用现有的接口和电路盒实现。
步骤S150，将基底放置在PCR仪加热板上进行热循环。
如图3(c)、图3(d)所示，将2至3滴矿物油滴在加热板上，以减少PCR扩增期间的液滴蒸发；然后，将承载液滴阵列的基底(或称衬底)放在平板PCR仪加热板上进行热循环反应，其中反应参数设置为：变性：90℃，60s；退火：95℃，5s，60℃，15s，延伸：72℃，120s；40个热循环。
通过热循环反应，使样品液滴性状发生改变，从而改变被检测样本的受激发光强度。
步骤S160，拍摄所生成的液滴阵列的荧光照片。
如图3(e)所示，在跑完PCR热循环后，将铺满液滴的玻璃基底置于荧光倒置显微镜上，通过电动平移台拍摄液滴局部荧光照片，直至所有液滴阵列全部拍摄完毕。
步骤S170，分析识别液滴荧光照片，获得待测样品溶液的阳性液滴占比。
如图3(f)所示，将局部液滴荧光照片通过数据分析处理模块(如计 算机软件)进行拼接，形成一张完整的液滴荧光照片，计算机软件识别明暗液滴并统计阳性液滴占比，以此推算样品初始浓度。
例如，荧光照片的拼接采用直接拼接方式，即将两副图像之间没有重叠的部分进行边与边直接连接。
例如，明暗液滴的识别过程是：将所拍的液滴图像转换为灰度图像，将液滴间、液滴与背景间、液滴与杂质间的黏连分离，进而识别液滴。
如图4所示，本发明实施例采用七个浓度梯度的人源c-DNA样本作为PCR反应体系，经过微流控数字打印平台打印后进行PCR反应，再通过拼接荧光图像得到每个体系液滴阵列的完整图像，其中白点是阳性液滴，黑点是阴性液滴，浓度稀的亮液滴少。阳性液滴占比与浓度梯度成正相关性，如图5所示，由此证明液滴打印平台可以做到高效稳定精准的打印效果，液滴阵列能呈现数字PCR效果。
本发明通过液滴打印产生可控大小的液滴，随后将铺满液滴的基板置于显微镜上进行荧光信号的检测和计数。这种方式能够避免发生样品污染，提高了实验操作的简易性，为全自动化的数字PCR提供了新的方向。
综上，本发明利用微流控液滴打印系统进行数字PCR检测，在液滴生成过程通过精确地控制电动移动台的移动、气阀压力等能够在非封闭环境下产生液滴大小和阵列可控可调的液滴，并且不需要额外的数字PCR检测装置，即能实现液滴生成、扩增和检测的一体化操作，避免了现有分体式多步操作带来的样品污染和液滴融合问题；此外，由于实现了在线检测，能够及时有效的检测出荧光信号，避免由于时间推移而导致的无法正常检测出荧光信号的问题。通过对数字PCR检测实现液滴生成和液滴识别的自动化，减小了识别时间和错误率，这种自动化的控制和处理有利于数字PCR检测的产业化。
本发明的数字PCR检测方法可应用于基因工程，医学诊断和环境工程等，例如肿瘤研究、单细胞分析，病毒微生物分析、基因测序验证等。
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. 一种基于微流控液滴打印系统的数字PCR检测方法，包括以下步骤：
    步骤S1：在非封闭环境中利用微流控打印系统控制将待检测样本溶液通过气压方式从微流控打印芯片喷口打印到基底上并在该基底上形成预设的液滴阵列；
    步骤S2：将承载液滴阵列的基底放置在PCR仪加热板上进行热循环；
    步骤S3：拍摄基底上液滴阵列的荧光照片，分析识别明暗液滴并统计阳性液滴占比。
  2. 根据权利要求1所述的方法，其特征在于，所述微流控打印芯片上设有用于接收气体的气压通道和用于接收待检测样本溶液的流体通道，气压将待检验样品溶液剪切为液滴，通过所述微流控打印芯片上设置的喷口打印到位于所述微流控打芯片下方的基底上。
  3. 根据权利要求1所述的方法，其特征在于，所述微流控打印系统预先设置液滴打印相关参数，并基于所设置的打印相关参数来控制所述微流控打印芯片相对于所述基底的相对移动，从而在所述基底上形成预设的液滴阵列。
  4. 根据权利要求1所述的方法，其特征在于，所述微流控液滴打印系统包括驱动控制模块、电动平移台、气阀装置、摄像显微镜系统和数据分析处理模块，其中，所述气阀装置用于向所述微流控打印芯片提供气压；所述电动平移台用于放置所述微流控打印芯片；所述摄像显微镜系统用于拍摄液滴的荧光照片；所述数据分析处理模块用于识别明暗液滴并统计阳性液滴占比，所述驱动控制模块用于基于所设置的打印相关参数控制所述电动平移台、所述气阀装置和所述摄像显微镜系统。
  5. 根据权利要求4所述的方法，其特征在于，所述驱动控制模块设置的打印相关参数包括打印液滴的个数、打印液滴行数、打印液滴列数、打印模式、气阀装置的通道编号、打印液滴之间的间距、打印频率、打印脉宽、打印头与电动平移台之间距离中的一项或多项。
  6. 根据权利要求1所述的方法，其特征在于，在步骤S2中，变性参数设置为90℃，60s；退火参数设置为95℃，5s，60℃，15s；延伸参数设置为72℃，120s；热循环次数设置为40。
  7. 根据权利要求1至6任一项所述的方法，其特征在于，所述基底由75*50*1mm普通玻璃和聚二甲基硅氧烷墙经等离子清洗方法粘贴而成。
  8. 根据权利要求1至6任一项所述的方法，其特征在于，采用以下步骤制造所述微流控打印芯片：
    将所述微流控打印芯片的设计图打印在透明胶片上；
    将透明胶片作为光刻掩膜并将光刻掩模转移到涂有光刻胶的硅片上；
    采用软刻蚀方法制作聚二甲基硅氧烷芯片，形成所述微流控打印芯片。
  9. 根据权利要求1至6任一项所述的方法，其特征在于，所述微流控打印芯片喷口是封口膜。
  10. 一种根据权利要求1至9任一项所述的方法在数字PCR检测中的应用。
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