WO2014101575A1 - 一种具有皮升级精度的自动化微液滴阵列筛选系统的使用方法 - Google Patents

一种具有皮升级精度的自动化微液滴阵列筛选系统的使用方法 Download PDF

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WO2014101575A1
WO2014101575A1 PCT/CN2013/086729 CN2013086729W WO2014101575A1 WO 2014101575 A1 WO2014101575 A1 WO 2014101575A1 CN 2013086729 W CN2013086729 W CN 2013086729W WO 2014101575 A1 WO2014101575 A1 WO 2014101575A1
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sample
capillary
droplet
reagent
liquid
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方群
祝莹
张云霞
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Zhejiang University ZJU
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    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
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    • B01L3/5085—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates
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    • G01N2035/1039—Micropipettes, e.g. microcapillary tubes

Definitions

  • the invention relates to the field of high-throughput screening, and in particular to a method for using an automated micro-droplet array screening system with skin upgrade accuracy.
  • High-throughput screening systems originate from drug screening studies, which are mainly 96 or 384
  • the orifice plate is an array reactor.
  • the liquid distribution and sample mixing are performed by an automated robot.
  • the high-sensitivity, high-speed detection device and data processing software are used to analyze and process the experimental results to achieve at least 10,000 per day.
  • kind of screening flux Due to its powerful screening and analysis capabilities, the application of high-throughput screening technology has expanded from drug screening to multiple scientific fields such as biology, medicine, and chemistry.
  • commercial high-throughput liquid handling and screening systems based on multiwell plates are facing significant challenges.
  • the compounds used for screening are mainly derived from artificial synthesis or isolated and purified from natural products, and the cost is high.
  • the sample volume of the high-throughput screening system based on multiwell plates is 1-100 microliters. If you can manipulate the liquid at the level of the skin lift to the nano upgrade, it is possible to reduce the cost by 1000-100000. Times. Therefore, whether in industry or in academia, a large amount of research has focused on the miniaturization of high-throughput screening systems. For example, the UK Douglas company developed OryxNano The minimum liquid handling volume for a series of liquid handling units is 100 nanoliters (http://www.douglas.co.uk/oryxnano.htm).
  • Droplet-based microfluidic technology is one of the hotspots in the field of high-throughput screening miniaturization research in recent years. It realizes the formation of large-volume water-in-oil or oil-in-water droplet microreactors, sample mixing, reaction, and analysis and identification through the control of multiphase fluids in micron-sized channels.
  • the size of the droplet reactor can be flexibly adjusted from fly upgrade to nano upgrade, making it possible to achieve ultra-low throughput high throughput screening. Due to the encapsulation and protection of the oil phase, solvent evaporation and dilution of the droplet reactor and cross-contamination between the samples are effectively suppressed.
  • the self-assembly effect of biocompatible surfactant molecules can provide a mild and uniform microenvironment for biochemical screening reactions, which is beneficial to improve the accuracy of analysis and screening.
  • the tiny volume of the droplet reactor is beneficial to accelerate mass transfer between substances and improve reaction efficiency. Therefore, droplet-based microfluidic technology is likely to become a new generation of high-throughput screening technology due to its excellent properties.
  • droplet-based microfluidic screening methods there are mainly three types of droplet-based microfluidic screening methods, namely a droplet reservoir method, a SlipChip method, and a droplet assembly method.
  • the droplet reservoir firstly uses different extraction methods to load different sample solutions to be filtered into a capillary to form a droplet, and then connects the capillary to the microfluidic chip channel through the chip.
  • the T-type interface injects the target solution into the droplets to trigger the reaction, and finally collects the formed droplet reactor into another capillary for incubation reaction and detection (Zheng B., Ismagilov R. F. Angew. Chem., Int. Ed., 2005, 44, 2520 ).
  • the SlipChip method firstly inserts a sample to be screened into a droplet in the array micropore of the lower sheet, and then slides the upper sheet to mix the target reagent solution in the microchannel of the upper sheet with the droplet of the lower sheet to perform trigger reaction and screening ( Du W. B., Li L., Nichols K. P., Ismagilov R. F. Lab Chip, 2009, 9, 2286 ).
  • both methods require manual droplet loading, capillary and chip channel connections, and precision chip sliding operations, making it difficult to apply to large-scale sample screening.
  • the droplet assembly method utilizes rapid switching of automated sample tubes and reagent tubes to complete the mixing of samples and reagents to be screened during droplet formation, and then store the droplets on capillaries and chips for reaction and detection ( Du W. B., Sun M., Gu S. Q., Zhu Y., Fang Q. Anal. Chem., 2010, 82, 9941 ;Fang Qun, Du Wenbin, Sun Meng, Microfluidic droplet generation system and method based on droplet sequential assembly technology, Chinese invention patent, application number: 201010250945.0 ).
  • the droplet sequential assembly technique solves the problem of automated droplet screening and large-scale sample screening, since the generation of droplets containing samples and reagents adopts an assembly-by-assembly method, the screening speed is difficult to increase. In addition, due to the size effect brought about by miniaturization, it is difficult for the above several droplet screening methods to perform biochemical screening determination in the skin upgrade volume.
  • the parallelization reagent addition methods can be mainly divided into two categories.
  • the first category uses a T
  • the type of branching channel injects the same reagent into different droplets in the main channel (Zheng B., Ismagilov R. F. Angew. Chem., Int. Ed., 2005, 44, 2520).
  • Such methods are often used in conjunction with the droplet reservoir method described above for microscreening based on droplets.
  • the main problem with this type of method is in T There is a large droplet sample residue at the intersection of the type channel, which causes cross-contamination between the droplets.
  • Another type of method uses a droplet fusion technique to add reagents that are parallelized.
  • the system allows for the automated measurement of skin upgraded liquids, the formation of different sample droplet arrays, the parallelization of target reagents, and the determination of reactions in microvolumes.
  • the system can be used for both high-throughput drug screening as well as for catalyst screening, enzyme kinetic analysis, disease diagnosis, and single-cell and single-molecule analysis.
  • a method for using an automated microdroplet array screening system with skin upgrade accuracy comprising a capillary, a liquid drive system, a microwell array chip, a sample/reagent storage tube and an automatic translation stage, the specific steps are:
  • the capillary sampling end is moved into the oil phase above the micropores of the microwell array chip, and the sample solution in the capillary is pushed out into the micropores to form droplets of the sample.
  • step 4) described in the present invention further includes the following specific steps:
  • a large amount of reagent is extracted in the capillary at a time, and the capillary sampling end is inserted into the droplet of each sample separately, a certain volume of the reagent is injected, a droplet reactor is formed, and the mixing, reaction, detection and screening of the sample and the reagent are completed. .
  • step 4) described in the present invention further includes the following specific steps:
  • the sample solution to be screened is separately injected into the droplets of each reagent to form a droplet reactor, and the mixing, reaction, detection and screening of the reagent and the sample are completed.
  • the liquid drive system of the present invention has a liquid drive accuracy of a few nanoliters per minute, and the flow rate of the liquid to be driven is in the range of 1 nanoliter / Minutes to 500 nanoliters / minute.
  • the invention eliminates the mechanical hysteresis of the liquid drive system during the conversion of the liquid driving direction, that is, from the extraction to the ejection or the transition from the ejection to the extraction, thereby ensuring the accuracy of the liquid extraction of the skin upgrade, and extracting the water phase sample. Or extract an extra volume of oil phase into the capillary before the reagent solution; when the sample or reagent solution is pushed out of the capillary, the additional extracted oil phase is also pushed out of the capillary.
  • the liquid driving system and the capillary filled with the low thermal expansion coefficient of the present invention act as a current carrying current to prevent the influence of temperature fluctuation on the liquid driving precision during the experiment, and the thermal expansion coefficient of the current carrying current is 0.00001/ Celsius to 0.0005 / Celsius.
  • the capillary tube of the present invention adopts a thinner tube wall, which is advantageous for the liquid amount of the skin upgrade and the residual of the liquid at the sampling end of the capillary; the wall thickness of the capillary tube ranges from 1 micrometer to 100 micrometers.
  • the method for using the automated micro-droplet array screening system with skin upgrade accuracy according to the present invention, before use, degassing the liquid carrier and oil phases to prevent bubbles from being generated during the liquid driving process.
  • the microporous array chip is above the micropores, and the sample /
  • the reagent storage tube is covered with an oil phase that is immiscible with the aqueous phase to prevent traces, samples and reagents from being volatilized or contaminated by exposure to air.
  • the thickness of the oil phase ranges from 0.1 mm to 10 mm.
  • the concentration of the surfactant is 0.01% to 10%.
  • the advantages of the invention are mainly: (1) Quantitative measurement of liquid volume and droplet generation have volumetric accuracy of skin upgrade, which effectively reduces sample/reagent consumption in high-throughput screening and saves experimental cost; (2) The capillary is inserted into the droplets of the sample to be screened, and the reagents are continuously injected to complete the mixing, reaction detection and screening of the sample and the reagent, thereby effectively improving the screening flux and reducing the risk of cross-contamination; (3) The processes of liquid metering, introduction, droplet formation, and reagent injection are completely automated, effectively reducing human error and error, and facilitating the industrialization and widespread use of the system.
  • Figure 1 is a schematic diagram of a droplet array screening system with skin upgrade accuracy and a method of use
  • FIG. 2 is a schematic diagram of a process of sequentially injecting a reagent into a droplet of a sample to be screened by a method of inserting a droplet into a capillary;
  • Figure 3 is a top view fluorescence image of a Caspase-1 enzyme inhibitor screen using a droplet array screening system
  • Figure 4 is a graph showing the results of semi-inhibitory concentration determination of the screened inhibitor 28;
  • Figure 5 is a graph showing the results of a droplet array screening system for nanoscale protein crystallization screening
  • Figure 6 is a graph of the results of a drop array array screening system for cell-based drug activity studies.
  • the invention relates to a micro-droplet array screening system with skin upgrade resolution, which comprises a capillary, a liquid drive system, a micro-hole array chip, a liquid drive system, a sample /
  • the reagent storage tube and the automatic translation stage are composed.
  • the liquid drive system is connected to the capillary for quantitative extraction and extraction of trace liquids.
  • the sample/reagent storage tube and microwell array chip are mounted on a three-dimensional automatic translation stage, sample /
  • the reagent storage tube is used to store the samples and reagents required for the experiment, and the microwell array chip is used for storage, reaction, detection and screening of trace droplets.
  • the automated micro-droplet array screening system with skin upgrade accuracy is used by first using a liquid drive system and a liquid filled with a low coefficient of thermal expansion in the capillary as a carrier current, and completely emptying the capillary. Bubble; then immersing the capillary sampling end in an oil phase that is immiscible with the aqueous phase sample, extracting an oil phase into the capillary for isolating the aqueous phase sample and current carrying; then immersing the capillary sampling end into the sample / A certain volume of the aqueous phase sample solution is extracted into the capillary in the reagent storage tube; finally, the capillary sampling end is moved into the oil phase above the micropores of the microwell array chip, and the sample solution in the capillary is pushed out into the micropores to form a sample droplet.
  • a plurality of droplets of the sample to be screened having different chemical compositions or concentrations are formed on the microwell array chip, and then a large amount of reagents are extracted in the capillary at a time, and the capillary sampling ends are respectively inserted.
  • a certain volume of reagent is injected to form a droplet reactor, which completes the mixing, reaction, detection and screening of the sample and the reagent.
  • a plurality of reagent droplets of the same chemical composition and concentration may be first formed on the microwell array chip, and then the sample solutions of different chemical compositions and concentrations to be screened are separately injected into the droplets of each reagent. Forming a droplet reactor to complete the mixing, reaction, detection and screening of reagents and samples.
  • the liquid drive system has a liquid drive capability for forward push-out and reverse pull, with a flow rate of 1 picoliter per minute to 100 In microliters / minute, measure the volume of the liquid in 1 picoliter to 100 microliters.
  • the liquid drive system has a number of nanoliters / / for the completion of the measurement of the skin upgrade liquid /
  • the liquid drive accuracy of minutes, the flow rate of the driving liquid ranges from 1 nanoliter / minute to 500 nanoliters / minute, and the volume of the liquid is measured in the range of 1 picoliter to 1000 picolitres.
  • the liquid extraction accuracy of the skin upgrade is ensured, and the water is extracted.
  • An additional volume of oil phase is drawn into the capillary prior to the phase sample or reagent solution; as the sample or reagent solution is pushed out of the capillary, the additional extracted oil phase is also pushed out of the capillary.
  • the liquid drive system and the capillary filled with a low coefficient of thermal expansion as a carrier flow prevent the influence of temperature fluctuation on the liquid drive accuracy during the experiment.
  • the thermal expansion coefficient of the carrier current is 0.00001/ Celsius to 0.0005 / Celsius.
  • the sampling end of the capillary is subjected to a tipping treatment to reduce the diameter and cross-sectional area of the tip end of the sampling end, and preferably, the diameter of the tip end of the sampling end Range is 1 micron to 100 micron; at the same time, the inner wall of the capillary and the outer wall of the sampling end are hydrophobized.
  • the use of a capillary tube having a thin tube wall is advantageous for realizing the liquid amount of the skin upgrade and reducing the residual of the liquid at the sampling end of the capillary, and reducing the cross-contamination generated when the liquid is measured.
  • the wall thickness of the capillary is in the range of 1 micron to 100 microns.
  • the liquid carrier and oil phases are subjected to a (vacuum or ultrasonic) degassing treatment prior to use to prevent the generation of bubbles during the liquid driving process.
  • a (vacuum or ultrasonic) degassing treatment prior to use to prevent the generation of bubbles during the liquid driving process.
  • the presence of air bubbles can significantly reduce the accuracy of the measurement of the micro-skin upgrade liquid.
  • an oil phase which is immiscible with the sample (or reagent) solution is preliminarily extracted from the capillary to isolate the sample (or reagent) and the liquid carrier having a low coefficient of thermal expansion; preferably, The length of the oil phase ranges from 50 microns to 20 mm.
  • a plurality of pits for holding a trace amount of liquid are processed on the microwell array chip.
  • the volume of each pit ranges from 1 pico to 100 Microliters.
  • the microwell array on the chip and the sample/reagent storage tube sample are used /
  • the reagent tube is covered with an oil phase to prevent traces and samples from being volatilized or contaminated by exposure to air.
  • the thickness of the oil film is 0.1 mm to 10 mm.
  • the oil phase has a thickness ranging from 0.1 mm to 10 Millimeter.
  • a biocompatible surfactant is added to the oil phase, and the self-assembly effect of the surfactant molecule at the oil-water interface is utilized to reduce The adsorption and deactivation of biomolecules at the interface, preferably, the concentration range of the surfactant is 0.01% to 10%.
  • a plurality of liquid samples are simultaneously used with a plurality of capillary tubes or a plurality of liquid driving devices / Reagent extraction, ejection, and droplet generation operations.
  • FIG. 1 is a schematic diagram of an automated microdroplet array screening system with a skin upgrade accuracy and a method of use. It is used as follows: using capillary 1 As a sampling probe, the tail is connected to the liquid drive system 2. Both the capillary 1 and the liquid drive system 2 are filled with a liquid having a low coefficient of thermal expansion as the carrier 4 and in the capillary 1 The sampling end introduces an oil phase 6 that is immiscible with the aqueous phase to separate the aqueous phase sample 5 and the carrier stream 4 .
  • capillary 1 or sample / reagent storage tube 7 to immerse capillary 1 sampling end in oil phase 6 Extract a certain extra volume of oil phase into the capillary 1 sampling end; then immerse the capillary 1 sampling end into sample 5 to quantitatively extract a volume of sample 5 into the capillary. Then move the capillary again 1 Alternatively, the microwell array chip 3 is such that the sampling end of the capillary 1 is placed in the oil phase 6 above the microwell 8 on the chip 3. Start the liquid drive system 2 Place the sample of the liquid in capillary 1 5 And an additional extracted oil phase 6 is introduced into the micropores 8 to form droplets 9 of the trace phase 5 of the aqueous phase.
  • the sampling end of the capillary 1 is subjected to a tipping process, and the capillary 1 is The inner and outer walls, and the surface of the microwell array chip 3 are subjected to a hydrophobic surface treatment. Vacuum degassing or ultrasonic degassing of liquid carrier 4 and oil phase 6 to prevent air bubbles from being generated during liquid driving.
  • Fig. 2 is a schematic view showing a process of sequentially injecting a reagent into a droplet of a sample to be screened by a method of inserting a droplet by a capillary.
  • the method described produces an array of sample droplets 9 having different chemical compositions or concentrations on the microwell array chip 3. Extract a large volume of reagent 10 from the sampling capillary 1 and then move the capillary 1 Or the microwell array chip 3, insert the sampling end of the capillary 1 into the sample droplet 9, start the liquid drive system 2, and push out a certain volume of the reagent 10 into the sample droplet 9 to complete the sample /
  • the reagents are mixed to form a droplet reactor 11 .
  • Figure 3 is a droplet array screening system and method of use according to Figures 1 and 2, to 32 A small molecule compound is used as a sample to be screened, and a fluorescent picture obtained by screening an enzyme inhibitor using a Caspase-1 enzyme as a screening target.
  • First load 100 ⁇ M small molecule compound into the sample / Reagent storage tube.
  • the droplet array generation method described in Figure 1 was used to generate droplets of 32 small molecule compounds on a microwell array chip, each droplet having a volume of 180 picoliters.
  • the target reagent injection method injects 180 picoliters of Caspase-1 enzyme solution (6 mU/ ⁇ L) and 180 picoliters of substrate into each droplet ( Z-YVAD-R110) solution (20 ⁇ M) to trigger the reaction. Incubate the microwell array chip at 35 degrees Celsius 1 In the hour, the experimental results were obtained by fluorescence imaging. Analysis of experimental results, compound versus Caspase-1 The stronger the inhibition ability of the enzyme, the weaker the enzyme activity, and the lower the reaction rate of the catalytic substrate under the same reaction conditions, the weaker the fluorescence signal corresponding to Fig. 3. Therefore, numbers 28, 30, and 32 The compound is an inhibitor identified by screening.
  • Figure 4 shows Caspase-1 for Compound No. 28 obtained from the screening experiment described in Figure 3. Record of the results of enzyme semi-inhibitory concentration determination. First, load the sample / reagent storage tube to a concentration of 0.1 nM, 1 nM, 10 nM, 100 nM, 1 ⁇ M, 10 ⁇ M and 100 ⁇ M solution of compound No. 28. According to the droplet array screening system and the method of use described in FIG. 1 and FIG. 2, different concentrations are generated on the microwell array chip.
  • Figure 5 is a graph showing the results of the screening of the droplet array screening system for nanoscale protein crystallization conditions.
  • the reagent storage tube is filled with 50mg/mL lysozyme sample solution and precipitant solution containing 51 different chemical components (Crystal Corporation, Crystal Company, USA) Crystallization kit).
  • 51 droplets of 2 nL of different precipitant were generated on the microwell array chip, and each precipitant condition was repeated. Times.
  • 2 nL of the lysozyme sample solution was separately injected into each of the droplets to form a crystallization reactor. Place the chip at a temperature of 16 The incubation reaction was carried out in an incubator in Celsius.
  • paraffin oil or mineral oil with poor gas permeability was selected as the oil phase.
  • the droplet array was imaged by microscope to identify the precipitant conditions suitable for lysozyme crystallization.
  • FIG. 6 is a graph showing the results of the assay using the droplet array screening system for cell-based drug activity studies.
  • the perfluoro oil with strong gas permeability was selected.
  • FC40 acts as an oil phase to meet the gas exchange required for cell culture.
  • the non-small lung cancer cell A549 suspension was spotted into the array chip using the droplet array generation method described in Figure 1, each droplet volume being 500 nL containing 80 ⁇ 20 cells.
  • the droplet array chip is then placed in a cell culture chamber. In hours, the cells grow adherently. After the cell-attached droplet array chip was taken out, the old culture solution was removed, and the residual culture solution in the cell culture droplet was washed with PBS, and 500 nL was added to each droplet.

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Abstract

一种具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，通过将液体驱动系统（2）和毛细管（1）中充满低热膨胀系数的液体作为载流（4），并完全排空毛细管（1）内的气泡，然后将毛细管（1）取样端浸入与水相样品（5）不互溶的油相（6）中抽取一段油相（6）至毛细管（1）中，用于隔离水相样品（5）和载流（4），再将毛细管（1）取样端浸入样品/试剂储存管（7）中抽取一定体积的水相样品（5）溶液进入毛细管（1），最后将毛细管（1）取样端移入微孔阵列芯片（3）的微孔（8）上方的油相（6）中，将毛细管（1）中的样品（5）溶液推出至微孔（8）中形成样品（5）的液滴（9）。

Description

一种具有皮升级精度的自动化微液滴阵列筛选系统的使用方法
本发明涉及的领域为高通量筛选领域，特别涉及一种具有皮升级精度的自动化微液滴阵列筛选系统的使用方法。
高通量筛选系统起源于药物筛选的研究，它主要以 96 或者 384 孔板为阵列化反应器，通过自动化机器人进行液体分配和试样混合，采用高灵敏、高速的检测装置和数据处理软件对实验结果进行分析和处理，实现每天至少 10,000 样的筛选通量。由于其强大的筛选和分析功能，高通量筛选技术的应用范围已从药物筛选扩展到生物学、医学、化学等多个科学领域。然而，随着新的靶标和样本量的迅速增加，基于多孔板的商品化高通量液体处理与筛选系统逐渐面临巨大的挑战。用于筛选的化合物主要来源于人工合成或从天然产物中分离纯化得到，成本较高。目前，基于多孔板的高通量筛选系统的试样消耗体积在 1-100 微升。如果能在皮升至纳升级的水平上操纵液体，来对样品进行筛选，则有可能将其成本降低 1000-100000 倍。因此，无论是在工业界还是在学术界，大量的研究均集中在高通量筛选系统的微型化。比如，英国 Douglas 公司研制 OryxNano 系列液体处理装置的最小液体处理体积是 100 纳升（ http://www.douglas.co.uk/oryxnano.htm ）。英国 TTP LabTech 公司研制的 Mosquito 系列液体处理装置的最小液体操纵体积达到 25 纳升（ www.ttplabtech.com/products/mosquito/ ）。这些仪器的应用显著的降低了筛选和研发的成本。然而，目前仍然缺少可在几个纳升甚至皮升级体积上进行液体操纵和高通量筛选的技术和装置。
皮升级高通量筛选的研究难点主要体现在： 1 ）现有仪器设备难以在皮升级进行可靠的液体操纵，比如准确液体量取、液体转移、试样混合等； 2 ）随着液体体积的缩小，蒸发效应显著增加。比如在典型实验室条件下，一个皮升级的水相液滴会在 1 秒内完全挥发； 3 ）微体系下的液体具有极大的比表面积，由于水 / 气界面和水 / 固界面上的分子自组装或者非特异性作用，容易引起生物活性分子的失活、损失以及交叉污染，从而导致筛选结果的假阳性或者假阴性。
基于液滴的微流控技术是近年来高通量筛选微型化研究领域中的热点之一。它通过微米级通道中多相流体的控制，实现大批量油包水或水包油型液滴微反应器的生成、试样混合、反应、以及分析鉴定。液滴反应器的尺寸可在飞升级至纳升级灵活调节，从而可能实现超低消耗的高通量筛选。由于油相的包裹和保护作用，有效抑制了液滴反应器的溶剂蒸发和稀释作用，以及样品间的交叉污染。利用生物兼容性表面活性剂分子的自组装效应，可以给生化筛选反应提供一个温和均一的微环境，有利于提高分析筛选的准确性。同时，液滴反应器微小的体积有利于加速物质间的传质，提高反应效率。因此，基于液滴的微流控技术因其优异的特性有可能成为新一代的高通量筛选技术。
目前，基于液滴的微流控筛选方法主要有三种，分别为液滴储存器方法、滑动芯片方法、以及液滴组装方法。液滴储存器是首先采用逐个抽取的方法将不同的待筛选样品溶液装载至毛细管中形成液滴，然后将该毛细管与微流控芯片通道连接，通过芯片上的 T 型接口将靶标溶液注入到液滴中触发反应，最后将形成的液滴反应器收集至另外一个毛细管中进行孵育反应和检测（ Zheng B., Ismagilov R. F. Angew. Chem., Int. Ed., 2005, 44, 2520 ）。滑动芯片方法则首先在下片的阵列化微孔中装入待筛选样品形成液滴，然后滑动上片使上片微通道中的靶标试剂溶液与下片的液滴混合，进行触发反应和筛选（ Du W. B., Li L., Nichols K. P., Ismagilov R. F. Lab Chip, 2009, 9, 2286 ）。然而，上述两种方法均需要手动的液滴装载、毛细管和芯片通道的连接，以及精密的芯片滑动操作，难以应用于大规模样品的筛选。液滴组装方法利用自动化的样品管和试剂管的快速切换，在液滴形成的过程中完成待筛选样品和试剂的混合，然后将液滴储存至毛细管和芯片上进行反应和检测（ Du W. B., Sun M., Gu S. Q., Zhu Y., Fang Q. Anal. Chem., 2010, 82, 9941 ；方群，杜文斌，孙蒙，基于液滴顺序组装技术的微流控液滴生成系统及使用方法，中国发明专利，申请号： 201010250945.0 ）。尽管液滴顺序组装技术解决了液滴筛选的自动化和大规模样品筛选的难题，但是由于含有样品和试剂的液滴的生成采用了逐个组装方法，其筛选速度难以提高。另外，由于微型化带来的尺寸效应，上述几种液滴筛选方法都难以在皮升级体积进行生化筛选测定。
在微流控液滴系统中，平行化的试剂加入方法主要可分为两类。第一类采用一个 T 型分支通道将一相同试剂注入到主通道中的不同的液滴中（ Zheng B., Ismagilov R. F. Angew. Chem., Int. Ed., 2005, 44, 2520 ）。这类方法经常与上述的液滴储存器方法进行联用，用于基于液滴的微量筛选。然而，这类方法的主要问题是在 T 型通道交叉口存在较大的液滴样品残留，从而引起液滴间的交叉污染。另外一类方法采用液滴融合技术进行平行化的试剂加入。首先为微通道中生成互相配对的样品和试剂的液滴，然后利用流体动力辅助或者介电辅助的方法使配对好的液滴融合形成独立的微反应器（ Teh S Y, Lin R. Hung L. H., Lee A. P., Lab Chip, 2008, 8:198 ）。然而此类方法结构较为复杂，加工难度大，并且难以用于具有大量不同样品的筛选系统中。并且，上述两类方法的实现都需要复杂的液体流速调节、液滴频率、液滴大小的控制、液滴位置的反馈等手动调节手段，难以实现可靠的自动化，在仪器产业化方面存在较大的困难。
本发明的目的是提供一种具有皮升级分辨率的自动化微液滴阵列筛选系统的使用方法。该系统可进行全自动的皮升级液体的量取、不同样品液滴阵列的形成、靶标试剂的平行化定量加入、以及微量体积下的反应测定。该系统既可用于高通量药物筛选，也可用于催化剂筛选、酶动力学分析、疾病诊断、以及单细胞和单分子分析中。
本发明的具体技术方案如下：
一种具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，系统包括毛细管、液体驱动系统、微孔阵列芯片、样品 / 试剂储存管和自动平移台，具体步骤为：
1 ）将液体驱动系统和毛细管中充满低热膨胀系数的液体作为载流，并完全排空毛细管内的气泡；
2 ）将毛细管取样端浸入与水相样品不互溶的油相中抽取一段油相至毛细管中，用于隔离水相样品和载流；
3 ）将毛细管取样端浸入样品 / 试剂储存管中抽取一定体积的水相样品溶液进入毛细管；
4 ）将毛细管取样端移入微孔阵列芯片的微孔上方的油相中，将毛细管中的样品溶液推出至微孔中形成样品的液滴。
本发明所述的步骤 4 ）后还包括如下具体步骤：
a ）在微孔阵列芯片上形成化学组成或者浓度不同的多个待筛选样品的液滴；
b ）在毛细管中一次抽取大量的试剂，并将毛细管取样端分别插入至每个样品的液滴中，注入一定体积的试剂，形成液滴反应器，完成样品与试剂的混合、反应、检测和筛选。
本发明所述的步骤 4 ）后还包括如下具体步骤：
m ）在微孔阵列芯片上形成大量的试剂的液滴；
n ）在每个试剂的液滴中分别注入待筛选的样品溶液，形成液滴反应器，完成试剂与样品的混合、反应、检测和筛选。
本发明所述的液体驱动系统具有数纳升 / 分钟的液体驱动精度，其驱动液体的流速范围是 1 纳升 / 分钟至 500 纳升 / 分钟。
本发明为了消除液体驱动系统在转换液体驱动方向时，即从抽取转换至推出或者从推出转换至抽取这一期间所存在的机械回差，保证皮升级的液体量取精度，在抽取水相样品或试剂溶液之前先抽取一定额外体积的油相进入毛细管；在将样品或试剂溶液推出毛细管时，也将额外抽取的油相一并推出毛细管。
本发明所述的液体驱动系统和毛细管中充满低热膨胀系数的液体作为载流，来防止实验过程中温度波动对液体驱动精度的影响，载流的热膨胀系数范围为 0.00001/ 摄氏度至 0.0005/ 摄氏度。
本发明所述的毛细管采用较薄的管壁，有利于实现皮升级的液体量取以及降低液体在毛细管取样端的残留；毛细管的管壁厚度范围是 1 微米至 100 微米。
本发明所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，在使用之前，对液体载流和油相进行脱气处理，防止在液体驱动过程中产生气泡。
本发明在使用时，微孔阵列芯片的微孔的上方，与样品 / 试剂储存管的上方均覆盖一层与水相不互溶的油相，以防止微量液滴、样品和试剂暴露在空气中挥发或受到污染，油相的厚度范围为 0.1 毫米至 10 毫米。
本发明在使用时，为了消除筛选反应过程中油相对微量生化反应的干扰，在油相中添加生物兼容性的表面活性剂，利用表面活性剂分子在油水界面的自组装效应，来降低生物分子在界面的吸附和失活，表面活性剂的浓度为 0.01% 至 10% 。
本发明的优点主要在于：（ 1 ）液体的定量量取和液滴生成具有皮升级的体积精度，有效降低了高通量筛选中的样品 / 试剂消耗，节省了实验成本；（ 2 ）采用将毛细管插入待筛选样品液滴中，分别连续注入试剂来完成样品和试剂的混合、反应检测和筛选，有效提高了筛选的通量，并降低了产生交叉污染的风险；（ 3 ）液体量取、推出、液滴生成、以及试剂注入等过程完全自动化，有效降低了人为失误和误差，易于实现系统的产业化和广泛普及。
图 1 是具有皮升级精度的液滴阵列筛选系统及使用方法示意图；
图 2 是采用毛细管插入液滴的方法，在待筛选样品液滴中依次注入试剂的过程示意图；
图 3 是利用液滴阵列筛选系统进行 Caspase-1 酶抑制剂筛选的俯视荧光图片；
图 4 是对筛选得到的抑制剂 28 进行半抑制浓度测定结果记录图；
图 5 是将液滴阵列筛选系统用于纳升级蛋白质结晶条件筛选的测定结果图；
图 6 是将液滴阵列筛选系统用于基于细胞的药物活性研究的测定结果图。
图中：
1- 毛细管， 2- 液体驱动系统， 3- 微孔阵列芯片， 4- 液体载流， 5- 水相样品， 6- 油相， 7- 样品 / 试剂储存管， 8- 微孔， 9- 样品的液滴， 10- 试剂， 11- 液滴反应器。
下面对本发明的技术方案作详细阐述：
本发明涉及一种具有皮升级分辨率的微液滴阵列筛选系统，由毛细管、液体驱动系统、微孔阵列芯片、液体驱动系统、样品 / 试剂储存管和自动平移台组成。液体驱动系统与毛细管相连用于微量液体的定量抽取和推出，样品 / 试剂储存管和微孔阵列芯片固定在可三维移动的自动平移台上，样品 / 试剂储存管用于储存实验所需的样品和试剂，微孔阵列芯片用于微量液滴的储存、反应、检测和筛选。
根据本发明，所述的具有皮升级精度的自动化微液滴阵列筛选系统，其使用方法是：首先将液体驱动系统和毛细管内充满低热膨胀系数的液体作为载流，并完全排空毛细管内的气泡；然后将毛细管取样端浸入与水相样品不互溶的油相中抽取一段油相至毛细管中，用于隔离水相样品和载流；再将毛细管取样端浸入样品 / 试剂储存管中抽取一定体积的水相样品溶液进入毛细管；最后将毛细管取样端移入微孔阵列芯片的微孔上方的油相中，将毛细管中的样品溶液推出至微孔中形成样品液滴。
根据本发明，为了提高筛选的通量，首先在微孔阵列芯片上形成化学组成或者浓度不同的多个待筛选样品液滴，然后在毛细管中一次抽取大量的试剂，并将毛细管取样端分别插入至每个样品液滴中，注入一定体积的试剂，形成液滴反应器，完成样品与试剂的混合、反应、检测和筛选。作为另外一种方案，也可首先在微孔阵列芯片上形成大量的相同化学组成和浓度的试剂液滴，然后在每个试剂的液滴中分别注入待筛选的不同化学组成和浓度的样品溶液，形成液滴反应器，完成试剂与样品的混合、反应、检测和筛选。
根据本发明，液体驱动系统具有正向推出和反向抽取的液体驱动能力，流速为 1 皮升 / 分钟至 100 微升 / 分钟，量取液体的体积在 1 皮升至 100 微升。作为优选，为完成皮升级液体的量取，所述的液体驱动系统具有数纳升 / 分钟的液体驱动精度，其驱动液体的流速范围是 1 纳升 / 分钟至 500 纳升 / 分钟，量取液体的体积范围在 1 皮升至 1000 皮升。
根据本发明，为了消除液体驱动系统在转换液体驱动方向时（从抽取转换至推出状态时或者从推出转换至抽取状态时）存在的机械回差，保证皮升级的液体量取精度，在抽取水相样品或试剂溶液之前先抽取一定额外体积的油相进入毛细管；在将样品或试剂溶液推出毛细管时，也将额外抽取的油相一并推出毛细管。
根据本发明，液体驱动系统和毛细管中充满低热膨胀系数的液体作为载流，来防止实验过程中温度波动对液体驱动精度的影响，作为优选，载流的热膨胀系数范围为0.00001/ 摄氏度至 0.0005/ 摄氏度。
根据本发明，为实现皮升级的液体量取以及降低液体在毛细管取样端的残留，对毛细管的取样端进行拉尖处理来降低取样端尖端的直径和横截面积，作为优选，取样端尖端的直径范围是 1 微米至 100 微米；同时对毛细管的内壁和取样端外壁进行疏水化处理。
根据本发明，采用管壁较薄的毛细管，有利于实现皮升级的液体量取以及降低液体在毛细管取样端的残留，减小量取不同液体时产生的交叉污染。作为优选，毛细管的管壁厚度范围是 1 微米至 100 微米。
根据本发明，在使用之前，对液体载流和油相进行（真空或者超声）脱气处理，防止在液体驱动过程中产生气泡。气泡的存在会显著降低对微量皮升级液体的量取精度。
根据本发明，在抽取样品（或试剂）溶液之前，毛细管中预先抽取一段与样品（或试剂）溶液不互溶的油相来隔离样品（或试剂）和低热膨胀系数的液体载流；作为优选，油相的长度范围为 50 微米至 20 毫米。
根据本发明，微孔阵列芯片上加工多个用于盛载微量液体的小坑。每个小坑的体积范围是 1 皮升至 100 微升。
根据本发明，使用时，芯片上的微孔阵列与样品 / 试剂储存管的样品 / 试剂管上覆盖一层油相，以防止微量液滴和样品暴露在空气中挥发或受到污染，油膜的厚度为 0.1 毫米至 10 毫米。使用时，微孔阵列芯片上的微孔的上方，与试样样品 / 试剂储存管的上方，均覆盖一层与水相不互溶的油相，以防止微量液滴、样品和试剂暴露在空气中挥发或受到污染，作为优选，油相的厚度范围为 0.1 毫米至 10 毫米。
根据本发明，作为一种优选方案，为了消除筛选反应过程中油相对生化反应的干扰，在油相中添加生物兼容性的表面活性剂，利用表面活性剂分子在油水界面的自组装效应，来降低生物分子在界面的吸附和失活，作为优选，表面活性剂的浓度范围为 0.01% 至 10% 。
根据本发明，采用多个毛细管或者多个液体驱动装置，同时进行多种液体样品 / 试剂的量取、推出和液滴生成操作。
下面结合具体实施例对本发明的技术方案作进一步说明：
参照附图，以下将详细描述根据本发明的优选实施例。
图 1 是具有皮升级精度的自动化微液滴阵列筛选系统及使用方法示意图。其使用方法如下：使用毛细管 1 作为取样探针，将其尾部与液体驱动系统 2 相连。毛细管 1 和液体驱动系统 2 内均充满低热膨胀系数的液体作为载流 4 ，并在毛细管 1 的取样端引入一段与水相不互溶的油相 6 来分隔水相样品 5 和载流 4 。移动毛细管 1 或者样品 / 试剂储存管 7 ，使毛细管 1 取样端浸入油相 6 中抽取一定额外体积的油相 6 进入毛细管 1 取样端；再将毛细管 1 取样端浸入样品 5 中定量抽取一定体积的样品 5 进入毛细管。然后再次移动毛细管 1 或者微孔阵列芯片 3 ，使毛细管 1 取样端置于芯片 3 上微孔 8 上方的油相 6 中。启动液体驱动系统 2 将毛细管 1 中的微量液体样品 5 和额外抽取的油相 6 推出至微孔 8 中，形成微量水相样品 5 的液滴 9 。
在利用该系统生成微液滴阵列前，毛细管 1 的取样端进行拉尖处理，并对毛细管 1 的内外壁、以及微孔阵列芯片 3 的表面进行疏水化表面处理。对液体载流 4 和油相 6 进行真空脱气或者超声脱气处理，防止在液体驱动过程中产生气泡。
图 2 是采用毛细管插入液滴的方法，在待筛选样品液滴中依次注入试剂的过程示意图。根据图 1 所述的方法，在微孔阵列芯片 3 上生成含有不同化学组成或浓度的样品液滴 9 的阵列。在取样毛细管 1 中抽取大量体积的试剂 10 ，然后移动毛细管 1 或者微孔阵列芯片 3 ，使毛细管 1 取样端插入样品液滴 9 中，启动液体驱动系统 2 ，推出一定体积的试剂 10 进入样品液滴 9 中，完成样品 / 试剂的混合，形成液滴反应器 11 。
实施例 1
图 3 是根据图 1 和图 2 所述的液滴阵列筛选系统及使用方法，以 32 个小分子化合物作为待筛选样品，以 Caspase-1 酶作为筛选靶标进行酶抑制剂筛选得到的荧光图片。首先将 100 µM 的小分子化合物装入样品 / 试剂储存管中。采用图 1 所述的液滴阵列生成方法在微孔阵列芯片上生成 32 个小分子化合物的液滴，每个液滴的体积为 180 皮升。然后按照图 2 所述的靶标试剂注入方法，分别向每个液滴中注入 180 皮升的 Caspase-1 酶溶液（ 6 mU/µL ）以及 180 皮升的底物（ Z-YVAD-R110 ）溶液（ 20 µM ）来触发反应。将微孔阵列芯片在 35 摄氏度条件下孵育 1 个小时，采用荧光成像的方法获取实验结果。分析实验结果，化合物对 Caspase-1 酶的抑制能力越强，酶活性就弱，在相同反应条件下，催化底物反应的速度就低，对应图 3 中荧光信号就越弱。因此，编号为 28 、 30 、和 32 化合物为筛选鉴定出的抑制剂。
实施例 2
图 4 是对图 3 所述的筛选实验得到的 28 号化合物进行 Caspase-1 酶半抑制浓度测定结果记录图。首先在样品 / 试剂储存管中分别装入浓度为 0.1 nM 、 1 nM 、 10 nM 、 100 nM 、 1 µM 、 10 µM 、以及 100 µM 的 28 号化合物溶液。根据图 1 和图 2 所述的液滴阵列筛选系统及使用方法，在微孔阵列芯片上生成含有不同浓度 28 号化合物溶液的液滴（ 180 皮升），再向每个液滴中注入 180 皮升的 Caspase-1 酶溶液（ 6 mU/µL ）以及 180 皮升的底物（ Z-YVAD-R110 ）溶液（ 20 µM ）来触发反应。按照实施例 1 的方法获得结果荧光图片，对荧光亮度值进行提取并进行归一化处理。对化合物 28 的浓度进行取对数处理，并将处理得到的结果与浓度对应的归一化荧光亮度值进行作图。利用数据处理软件对结果图进行 Sigmoidal 拟合，得到半抑制浓度为 31.6±3.4 nM 。
实施例 3
图 5 是将该液滴阵列筛选系统用于纳升级蛋白质结晶条件筛选的测定结果图。首先在样品样品 / 试剂储存管中分别装入 50mg/mL 的溶菌酶样品溶液和含有 51 种不同化学组分的沉淀剂溶液（美国 Hampton 公司 Crystal I 结晶试剂盒）。采用图 1 所述的液滴阵列生成方法在微孔阵列芯片上生成 51 个体积为 2 nL 不同沉淀剂的液滴，每个沉淀剂条件重复 5 次。然后向每个液滴中分别注入 2 nL 的溶菌酶样品溶液，形成结晶反应器。将芯片置于温度为 16 摄氏度的恒温箱中进行孵育反应。为了防止长时间孵育过程中的微液滴蒸发问题，实验中选取透气性较差的石蜡油或者矿物油作为油相。最后采用显微镜对液滴阵列进行成像检测，鉴定出适合溶菌酶结晶的沉淀剂条件。
实施例 4
图 6 是将该液滴阵列筛选系统用于基于细胞的药物活性研究的测定结果图。实验中选用透气性较强的全氟油 FC40 作为油相，来满足细胞培养需要的气体交换。首先采用图 1 所述的液滴阵列生成方法将非小肺癌细胞 A549 悬浮液点样至阵列芯片中，每个液滴体积为 500 nL ，包含 80 ± 20 个细胞。然后将液滴阵列芯片置于细胞培养箱培养 24 小时，使细胞贴壁生长。将细胞贴壁后的液滴阵列芯片取出，移除旧培养液并用 PBS 对细胞培养液滴中残余的培养液进行清洗后，向每个液滴中加入 500 nL 不同浓度的 5-Fluorouracil 药物溶液。将加药后的液滴阵列芯片重新放入培养箱中继续培养 24 小时，然后利用 PBS 清洗液滴并加入新鲜培养液进行细胞的后培。最后，用毛细管移除液滴中的细胞培养液并用 PBS 对液滴进行清洗，然后加入 500 nL 用于细胞活性检测的混合荧光染料试剂 Calcein AM 与 Ethidium homodimer-1 。将液滴阵列芯片放入培养箱中孵育 30 分钟后，在荧光显微镜下进行成像检测，对每个液滴中活细胞和死细胞的个数进行计数，计算出药物刺激后的细胞存活率。

Claims (10)

  1. 一种具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，所述的系统包括毛细管（ 1 ）、液体驱动系统（ 2 ）、微孔阵列芯片（ 3 ）、样品 / 试剂储存管（ 7 ）和自动平移台，其特征在于，具体步骤为：
    1 ）将液体驱动系统（ 2 ）和毛细管（ 1 ）中充满低热膨胀系数的液体作为载流（ 4 ），并完全排空毛细管（ 1 ）内的气泡；
    2 ）将毛细管（ 1 ）取样端浸入与水相样品（ 5 ）不互溶的油相（ 6 ）中抽取一段油相（ 6 ）至毛细管（ 1 ）中，用于隔离水相样品（ 5 ）和载流（ 4 ）；
    3 ）将毛细管取样端浸入样品 / 试剂储存管（ 7 ）中抽取一定体积的水相样品（ 5 ）溶液进入毛细管（ 1 ）；
    4 ）将毛细管（ 1 ）取样端移入微孔阵列芯片（ 3 ）的微孔（ 8 ）上方的油相（ 6 ）中，将毛细管（ 1 ）中的样品（ 5 ）溶液推出至微孔（ 8 ）中形成样品（ 5 ）的液滴（ 9 ）。
  2. 根据权利要求 1 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，所述的步骤 4 ）后还包括如下具体步骤：
    a ）在微孔阵列芯片（ 3 ）上形成化学组成或者浓度不同的多个待筛选样品的液滴（ 9 ）；
    b ）在毛细管（ 1 ）中一次抽取大量的试剂（ 10 ），并将毛细管（ 1 ）取样端分别插入至每个样品的液滴（ 9 ）中，注入一定体积的试剂（ 10 ），形成液滴反应器（ 11 ），完成样品（ 5 ）与试剂（ 10 ）的混合、反应、检测和筛选。
  3. 根据权利要求 1 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，所述的步骤 4 ）后还包括如下具体步骤：
    m ）在微孔阵列芯片（ 3 ）上形成大量的试剂（ 10 ）的液滴；
    n ）在每个试剂（ 10 ）的液滴中分别注入待筛选的样品（ 5 ）溶液，形成液滴反应器（ 11 ），完成试剂（ 10 ）与样品（ 5 ）的混合、反应、检测和筛选。
  4. 根据权利要求 1 或 2 或 3 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，所述的液体驱动系统（ 2 ）具有数纳升 / 分钟的液体驱动精度，其驱动液体的流速范围是 1 纳升 / 分钟至 500 纳升 / 分钟。
  5. 根据权利要求 1 或 2 或 3 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，在抽取水相样品（ 5 ）或试剂（ 10 ）溶液之前先抽取一定额外体积的油相（ 6 ）进入毛细管（ 1 ）；在将样品（ 5 ）或试剂（ 10 ）溶液推出毛细管（ 1 ）时，也将额外抽取的油相（ 6 ）一并推出毛细管（ 1 ）。
  6. 根据权利要求 1 或 2 或 3 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，所述的液体驱动系统（ 2 ）和毛细管（ 1 ）中充满低热膨胀系数的液体作为载流（ 4 ），所述的载流（ 4 ）的热膨胀系数范围为 0.00001/ 摄氏度至 0.0005/ 摄氏度。
  7. 根据权利要求 1 或 2 或 3 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，所述的毛细管（ 1 ）的管壁厚度范围是 1 微米至 100 微米。
  8. 根据权利要求 1 或 2 或 3 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，在使用之前，对液体载流（ 4 ）和油相（ 6 ）进行脱气处理。
  9. 根据权利要求 1 或 2 或 3 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，使用时，微孔阵列芯片（ 3 ）的微孔（ 8 ）的上方，与样品 / 试剂储存管（ 7 ）的上方均覆盖一层与水相不互溶的油相（ 6 ），所述的油相（ 6 ）的厚度范围为 0.1 毫米至 10 毫米。
  10. 根据权利要求 1 或 2 或 3 所述的具有皮升级精度的自动化微液滴阵列筛选系统的使用方法，其特征在于，使用时，在油相（ 6 ）中添加生物兼容性的表面活性剂，表面活性剂的浓度为 0.01% 至 10% 。
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