WO2023070820A1 - Industrial-grade asymmetric droplet generation apparatus and digital nucleic acid amplification detection system - Google Patents

Industrial-grade asymmetric droplet generation apparatus and digital nucleic acid amplification detection system Download PDF

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WO2023070820A1
WO2023070820A1 PCT/CN2021/134672 CN2021134672W WO2023070820A1 WO 2023070820 A1 WO2023070820 A1 WO 2023070820A1 CN 2021134672 W CN2021134672 W CN 2021134672W WO 2023070820 A1 WO2023070820 A1 WO 2023070820A1
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nucleic acid
generating device
droplet generating
droplets
phase unit
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PCT/CN2021/134672
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French (fr)
Chinese (zh)
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何赛灵
程潇羽
张川
郑凯欣
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浙江大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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  • the invention belongs to the field of analysis and detection, and mainly relates to an industrial-grade asymmetric droplet generating device and a digital nucleic acid amplification detection system.
  • Microdroplets as tiny reactors, can greatly accelerate the rate of biochemical reactions, and are widely used in analysis and detection, chemical sensing, medical testing, chemical synthesis and other fields.
  • Traditional droplet generation methods are often based on chip microfluidics. This droplet generation device usually requires expensive chip fabrication and sophisticated peristaltic pumps to adjust the flow rate of the water-oil two-phase to control the size of the droplet.
  • the main problems of using pumps inconvenient operation, high cost, easy waste of reagents.
  • Digital nucleic acid detection technology plays a huge role in biological detection, chemical sensing, medical diagnosis and other fields.
  • the sample is diluted to the single-molecule level by distributing the sample into tens of millions of units using the technical concept of micro-droplets.
  • Each droplet contains nucleic acid or does not contain nucleic acid.
  • the droplet containing nucleic acid has a fluorescent signal, and the number of fluorescent droplets conforms to the Poisson distribution.
  • Absolute quantitative detection of samples such as target nucleic acids can be achieved through counting statistics. Therefore, the micro-droplet generating device is very important for digital PCR technology.
  • CN112522374A “Centrifugal Digital Droplet Generation Method and Device with Low Cost and Wide Adaptability” proposes a centrifugal droplet generation device with simple operation, low cost and wide adaptability.
  • the high molecular polymer used in this device such as PDMS glue, seals the microtubes with uniform size.
  • This operation may cause the following problems: (1) If the high molecular polymer is not tightly sealed, it is easy to cause the upper aqueous phase to pass through The gap between the polymer and the inner pipette tip enters the lower oil phase, and the microtube is not the only channel leading to the lower oil phase, resulting in uneven droplets; (2) high If the molecular polymer is not handled properly during sealing, the extremely fine micron tube may be blocked, and the upper water phase cannot be centrifuged into the lower oil phase, which reduces the separation rate of uniform droplets; (3) Polymerization When the material is sealed with the microtube, if the upper end of the microtube and the solidified upper layer of the polymer are not controlled on the same level, the trace level of the upper water phase will not be completely centrifuged into the lower oil phase. lead to wastage of samples.
  • the purpose of the present invention is to provide an industrial-grade asymmetric droplet generating device and a digital nucleic acid amplification detection system, which uses an asymmetric structure to drain water instead of a symmetrical structure with the same size at both ends to generate liquid. drops, achieving high repetition rates.
  • An industrial-grade asymmetric droplet generating device including an upper water phase unit and a lower oil phase unit.
  • the upper water phase unit is fixed on the lower oil phase unit.
  • the upper water phase in the drainage tube enters the lower oil phase to form water-in-oil droplets of uniform size.
  • the drainage tube is formed by tapering the end of the capillary.
  • the tapered tip of the capillary is cut under a microscope with a razor blade.
  • the external force is centrifugal force.
  • the lower oil phase unit uses a plastic centrifuge tube.
  • the upper aqueous phase unit includes a fixing frame and a drainage tube, the fixing frame is used to fix the drainage tube, and the fixing frame includes one or more nested pipette tips.
  • the diameter of the pipette tip in the fixed frame is cut flat or attached with an O-shaped rubber ring, which is convenient for fixing the drainage tube, or for fixing the upper water phase unit to the lower oil phase unit.
  • the droplet generating device adjusts the size of the generated droplet by controlling the inner diameter and opening size of the drainage tube.
  • the droplet generating device is applied to nucleic acid dPCR and dLamp amplification to realize digital nucleic acid detection.
  • a digital nucleic acid amplification detection system based on the droplet generating device comprising: a high repetition rate asymmetric droplet generating device for generating a large number of uniform droplets; a nucleic acid amplification temperature control device for The nucleic acid amplification reaction is carried out in the droplets generated by the droplet generation device; and the product signal collection device is used to collect the product signals after the nucleic acid amplification reaction in the droplets.
  • the invention breaks through the bottlenecks of the microfluidic chip required for the production of water-in-oil droplets in the existing droplet generating device, such as high production cost and difficulty in use, and achieves the formation of a large amount of high-quality liquid by using conventional centrifugal equipment or similar technologies.
  • the purpose of the drop is to be described.
  • the present invention constructs a droplet generating device that can be mass-produced at an industrial level by tapering the capillary tip and adopting an asymmetrical hollow drainage structure, which has the characteristics of controllable cost, high repetition rate, and simple operation.
  • the industrial-grade asymmetric droplet generating device of the present invention is combined with nucleic acid amplification, and a small amount of aqueous solution can quickly generate high-throughput uniform water-in-oil droplets within a few minutes, realizing digital PCR nucleic acid quantitative detection.
  • the invention can package and distribute samples such as nucleic acid, cells, proteins, nanoparticles, etc., and further combine nucleic acid amplification to complete detection and other applications.
  • FIG. 1 is a schematic structural view of the droplet generating device of the present invention.
  • Fig. 2 is an enlarged view of the tapered asymmetric structure of the capillary in the droplet generating device of the present invention.
  • Fig. 3 is an enlarged view of the tip structure of the tapered capillary in the droplet generating device of the present invention.
  • Fig. 4 is a schematic diagram of uniform droplets generated by the droplet generating device of the present invention.
  • Fig. 5 is a graph showing the relationship between the outer diameter of the tip of the tapered capillary used in the droplet generating device of the present invention and the outer diameter of the generated uniform droplet.
  • Fig. 6 is an experimental diagram of the use of the droplet generating device of the present invention for the Lamp amplification of the nucleic acid of the new coronavirus.
  • Figure 7 is a diagram of the new coronavirus fluorescent quantitative PCR amplification and other digital PCR amplification experiments.
  • Fig. 8 is an experimental diagram of the use of the droplet generating device of the present invention for PCR amplification of the nucleic acid of the new coronavirus.
  • drainage tube 1 drainage tube 1 , upper water phase unit 2 , fixing frame 3 , centrifuge tube 4 , and lower oil phase unit 5 .
  • the invention constructs an industrial-grade asymmetric droplet generating device.
  • the device can be assembled using a common micropipette tip (or a similar structure for large-scale production) and a centrifuge tube in a biochemical laboratory, and the tapered capillary is fixed in the micropipette tip.
  • the upper water phase enters the lower oil phase to generate uniform-sized, high-quality water-in-oil droplets, and the size of the generated water-in-oil droplets can be changed by adjusting the outer diameter of the tapered capillary tip.
  • this device comprises two parts unit of upper layer water phase unit 2 and lower floor oil phase unit 5, described upper layer water phase unit comprises fixed frame 3 and drainage tube 1 (here is capillary), and fixed frame 3 is used for fixing the drainage tube 1, the fixing frame 3 includes one or more nested pipette tips.
  • the upper layer of the aqueous phase unit is assembled from three types of micropipette tips and capillaries.
  • the outermost layer of the pipette tip is made by cutting and refitting the 1000 ⁇ l pipette tip at the protrusion of the tip.
  • the middle layer of the pipette tip is The tip is modified by cutting a 200 ⁇ l pipette tip at the protrusion of the tip and fixed on the inside of the outermost pipette tip with an O-ring.
  • the innermost pipette tip is made of a 10 ⁇ l pipette tip at a distance The bottom 0.5cm is cut and refitted.
  • Three micropipette tips of different specifications are nested with each other, and the capillary with an inner diameter of 690 ⁇ m is just fixed in the innermost micropipette tip.
  • the lower oil phase unit 5 consists of a 1.5ml centrifuge tube 4 .
  • the outermost pipette tip in the upper aqueous phase unit 2 can be fixed in the 1.5ml centrifuge tube of the lower oil phase system, and placed together in a common laboratory centrifuge to generate water-in-oil droplets.
  • the pipette tip models are as follows, 1000 ⁇ l pipette tip: Axygen T-1000-B; 200 ⁇ l pipette tip: Axygen T-200-Y; 10 ⁇ l pipette tip: Axygen T-300.
  • Common centrifuge tubes can be used as the centrifuge tubes, which can be matched with the pipette tip of the upper aqueous phase.
  • the capillary used in this device is an ordinary capillary with uniform inner diameter, and the tip is tapered by methods such as microelectrode needle pulling instrument, so that the tip forms a tapered structure.
  • the use of this structure significantly increases the generation rate of uniformly sized, high-quality water-in-oil droplets.
  • the capillary used in this example has an inner diameter of 690 ⁇ m (outer diameter of 1200 ⁇ m) and a length of 10 cm. After pulling the needle using a capillary needle-pulling instrument, the tip of the capillary forms a tapered structure ( Figure 2). In the process of use, the temperature of the pulling needle is too high, which often leads to partial collapse of the tip position after the tapered structure is formed, resulting in the tip being sealed.
  • the tip of the capillary is cut under a microscope with a blade to determine the outer diameter of the tip, so as to ensure that the size of the centrifuged droplets can be controlled.
  • a capillary with a tip outer diameter of 10 ⁇ m (Figure 3)
  • FIG. 4 After centrifugation, uniform droplets with a diameter of about 50 ⁇ m can be obtained ( Figure 4).
  • the device in the present invention uses instruments such as a micro sample loading needle to directly add the sample to the inside of the capillary when loading the sample on the upper aqueous phase, completely avoiding the generation of uneven liquid droplets due to the leakage problem caused by poor sealing; It ensures that the upper aqueous phase injected into the capillary can be completely centrifuged to generate droplets, which increases the number of droplets generated and avoids waste of reagents. For example, 10 ⁇ l of the upper aqueous phase can produce more than 70,000 uniform droplets.
  • the device among the present invention and the droplet generating device of CN112522374A respectively make 10, and the result of the successful generation rate of its uniform droplet can be known from the following table 1, and the droplet generating device of CN112522374A has only 1 successfully generated uniform droplet, Its success rate is 10%, while 8 devices of the device of the present invention have successfully generated uniform droplets, and its success rate is 80%. If the broken device is excluded, the repetition rate is 100%.
  • the device of the present invention can change the size of the generated water-in-oil droplets by adjusting the outer diameter of the tapered capillary tip.
  • the present invention cuts 52 tapered capillaries with different tip outer diameters to generate uniform droplets, and the other conditions are kept the same except that the tip outer diameters are different.
  • the relationship between the outer diameter of the tip of the tapered capillary and the diameter of the uniform droplet thus obtained is shown in FIG. 5 .
  • the outer diameter range of the tapered capillary tip is 1.833-17.277 ⁇ m, and the diameter range of the uniform droplet is 15.169-52.038 ⁇ m.
  • the device of the present invention can be assembled using common micropipette tips (or scale production similar structures), centrifuge tubes and tapered capillaries in biochemical laboratories, compared with the centrifugal droplet generating device of CN112522374A (Table 1),
  • the invention uses a tapered capillary to increase the separation rate of uniform droplets, increase the number of droplets generated, and avoid waste of the trace-level upper water phase.
  • the industrial-grade asymmetric droplet generating device constructed in the present invention is combined with a nucleic acid lamp to realize digital lamp nucleic acid detection.
  • Sample selection New coronavirus positive standard sample. Use RNA-free-water to dilute to 1-100cp/ ⁇ l.
  • Reverse transcription The reverse transcription operation uses the HiFiScript cDNA Synthesis Kit kit from Shanghai Kangwei Century Biotechnology Co., Ltd., and the operation steps are operated according to the instructions.
  • the DNA obtained by reverse transcription is stored at -20°C and long-term stored at -80°C .
  • Lamp system Harbin Xinhai Gene Detection Co., Ltd. uses the new crown constant temperature fluorescent probe method nucleic acid detection kit for Lamp reaction.
  • the 25 ⁇ l lamp system includes: 5 ⁇ l new coronavirus DNA positive standard, 10 ⁇ l nCoV ⁇ 2 3IsoAmp Solution A in the kit and 10 ⁇ l nCoV-2 3IsoAmp Solution B.
  • the lower oil phase system consists of (isopropyl hexadecanoate containing surfactant EM180) 1.5ml centrifuge tube.
  • the outermost pipette tip in the upper aqueous phase system can be fixed in the 1.5ml centrifuge tube of the lower oil phase system, and placed together in a common laboratory centrifuge (6000rcf, 3min) to generate water-in-oil droplets.
  • Lamp amplification Transfer the generated droplets to a PCR tube and place them in a fluorescent quantitative PCR instrument. Set the program to 65°C for 10s, 65°C for 50s as the cycle unit, and cycle 35 times.
  • the industrial-grade asymmetric droplet generation device constructed by the present invention is combined with nucleic acid PCR amplification, and a small amount of aqueous phase solution can quickly generate high-throughput uniform water-in-oil droplets within a few minutes, realizing digital PCR nucleic acid detection.
  • Sample selection New coronavirus positive standard DNA sample.
  • PCR system 20 ⁇ l PCR system includes: 10 ⁇ l SYBR Green I PCR Mix, 2 ⁇ l new coronavirus DNA positive standard, 1 ⁇ l upstream and downstream primers, 5 ⁇ l glycerol and 1 ⁇ l ddH 2 O.
  • the lower oil phase system consists of a 1.5ml centrifuge tube (containing ePCR droplet generating oil).
  • the outermost pipette tip in the upper aqueous phase system can be fixed in the 1.5ml centrifuge tube of the lower oil phase system, and placed together in a common laboratory centrifuge (5000rcf, 3min) to generate water-in-oil droplets.
  • PCR amplification transfer the generated droplets to a PCR tube and place them in a fluorescent quantitative PCR instrument, and then perform a PCR reaction.
  • the reaction program is: 94°C, 3min; 10min; 95°C, 20s, 60°C, 30s, 72°C 30s, 20cycle; 72°C, 3min.

Abstract

An industrial-grade asymmetric droplet generation apparatus and a digital nucleic acid amplification detection system. The industrial-grade asymmetric droplet generation apparatus comprises an upper-layer water phase unit (2) and a lower-layer oil phase unit (5), wherein the upper-layer water phase unit (2) is fixed on the lower-layer oil phase unit (5); the upper-layer water-phase unit (2) is provided with a drainage tube (1) which is larger at the top and smaller at the bottom; and under the action of an external force, an upper-layer water phase in the drainage tube (1) enters a lower-layer oil phase, so as to form water-in-oil droplets with uniform sizes. The drainage tube (1) is formed by tapering an end part of a capillary tube. The digital nucleic acid amplification detection system comprises a high-repetition-rate asymmetric droplet generation apparatus, which is used for generating a large number of uniform droplets; a nucleic acid amplification temperature control apparatus, which is used for performing a nucleic acid amplification reaction in the droplets generated by the droplet generation apparatus; and a product signal collection apparatus, which is used for collecting product signals after the nucleic acid amplification reaction in the droplets. The apparatus and the system have the remarkable advantages of a controllable cost, a high repetition rate, etc.

Description

工业级非对称液滴发生装置及数字核酸扩增检测系统Industrial-grade asymmetric droplet generating device and digital nucleic acid amplification detection system 技术领域technical field
本发明属于分析检测领域，主要涉及一种工业级非对称液滴发生装置及数字核酸扩增检测系统。The invention belongs to the field of analysis and detection, and mainly relates to an industrial-grade asymmetric droplet generating device and a digital nucleic acid amplification detection system.
背景技术Background technique
液滴(Microdroplet)作为微小反应器，能够大大加快生化反应速率，在分析检测、化学传感、医疗检测、化学合成等领域被广泛应用。传统液滴生成的方法往往基于芯片微流控，这种液滴生成的装置通常需要昂贵的芯片制作以及精密的蠕动泵来调节水油两相的流速从而控制液滴的生成尺寸。使用泵的主要问题：操作不便、成本高昂、易浪费试剂。Microdroplets, as tiny reactors, can greatly accelerate the rate of biochemical reactions, and are widely used in analysis and detection, chemical sensing, medical testing, chemical synthesis and other fields. Traditional droplet generation methods are often based on chip microfluidics. This droplet generation device usually requires expensive chip fabrication and sophisticated peristaltic pumps to adjust the flow rate of the water-oil two-phase to control the size of the droplet. The main problems of using pumps: inconvenient operation, high cost, easy waste of reagents.
数字化核酸检测技术在生物检测、化学传感、医疗诊断等领域发挥具有巨大的作用。作为第三代核酸扩增方法，通过利用微液滴的技术理念将样品分配到上千万个单位中，以此将样品稀释至单分子水平。每个液滴中含有核酸或者不含核酸，经过扩增后，含有核酸的液滴有荧光信号，荧光液滴在数量上符合泊松分布。通过计数统计，可实现目标核酸等的样品的绝对定量检测。因此微液滴发生装置对于数字PCR技术至关重要。Digital nucleic acid detection technology plays a huge role in biological detection, chemical sensing, medical diagnosis and other fields. As a third-generation nucleic acid amplification method, the sample is diluted to the single-molecule level by distributing the sample into tens of millions of units using the technical concept of micro-droplets. Each droplet contains nucleic acid or does not contain nucleic acid. After amplification, the droplet containing nucleic acid has a fluorescent signal, and the number of fluorescent droplets conforms to the Poisson distribution. Absolute quantitative detection of samples such as target nucleic acids can be achieved through counting statistics. Therefore, the micro-droplet generating device is very important for digital PCR technology.
近年来提出的液滴生成装置中，CN112522374A“低成本广适应离心式数字液滴发生方法及装置”提出了一种操作简单、低成本、适应广泛的离心式液滴发生装置。然而，此装置中使用的高分子聚合物如PDMS胶等对尺寸均匀的微米管进行密封，该操作可能会引起以下问题：(1)高分子聚合物若未密封严实，易导致上层水相经过高分子聚合物与内移液枪吸头之间的缝隙进入到下层油相中，这时的微米管则不是唯一通向下层油相的通道，导致生成的液滴不均一；(2)高分子聚合物在密封时若处理不当，可能会将极细的微米管堵住，上层水相不能被离心到下层油相中，这使得均一液滴的离出率降低；(3)高分子聚合物在与微米管密封时，微米管的上端与高分子聚合物凝固后的上层若不控制在同一个水平面上，这就会出现微量级的上层水相无法完全被离心入下层油相中，导致样品的浪费。Among the droplet generation devices proposed in recent years, CN112522374A "Centrifugal Digital Droplet Generation Method and Device with Low Cost and Wide Adaptability" proposes a centrifugal droplet generation device with simple operation, low cost and wide adaptability. However, the high molecular polymer used in this device, such as PDMS glue, seals the microtubes with uniform size. This operation may cause the following problems: (1) If the high molecular polymer is not tightly sealed, it is easy to cause the upper aqueous phase to pass through The gap between the polymer and the inner pipette tip enters the lower oil phase, and the microtube is not the only channel leading to the lower oil phase, resulting in uneven droplets; (2) high If the molecular polymer is not handled properly during sealing, the extremely fine micron tube may be blocked, and the upper water phase cannot be centrifuged into the lower oil phase, which reduces the separation rate of uniform droplets; (3) Polymerization When the material is sealed with the microtube, if the upper end of the microtube and the solidified upper layer of the polymer are not controlled on the same level, the trace level of the upper water phase will not be completely centrifuged into the lower oil phase. lead to wastage of samples.
以上各种原因导致发明CN112522374A或类似技术的重复率不足，无法达到工业化生产标准。Above-mentioned various reasons cause the repetition rate of invention CN112522374A or similar technology to be insufficient, can't reach industrialized production standard.
发明内容Contents of the invention
为了克服现有技术中的不足，本发明的目的是提供一种工业级非对称液滴发生装置及数字核酸扩增检测系统，利用非对称结构引流，而非两端同样大小的对称结构生成液滴，实现很高的重复率。In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide an industrial-grade asymmetric droplet generating device and a digital nucleic acid amplification detection system, which uses an asymmetric structure to drain water instead of a symmetrical structure with the same size at both ends to generate liquid. drops, achieving high repetition rates.
一种工业级非对称液滴发生装置，包括上层水相单元和下层油相单元，上层水相单元固定在下层油相单元上，上层水相单元设有上大下小的引流管，在外力的作用下，引流管内的上层水相进入到下层油相中，形成尺寸均一的油包水液滴。An industrial-grade asymmetric droplet generating device, including an upper water phase unit and a lower oil phase unit. The upper water phase unit is fixed on the lower oil phase unit. Under the action of the fluid, the upper water phase in the drainage tube enters the lower oil phase to form water-in-oil droplets of uniform size.
所述的引流管由毛细管经端部拉锥而成。The drainage tube is formed by tapering the end of the capillary.
所述的毛细管的锥部尖端通过刀片在显微镜下进行切割。The tapered tip of the capillary is cut under a microscope with a razor blade.
所述的外力为离心力。The external force is centrifugal force.
所述的下层油相单元采用塑料离心管。The lower oil phase unit uses a plastic centrifuge tube.
所述的上层水相单元包括固定架和引流管，固定架用于固定引流管，所述中的固定架包括一个或者多个嵌套的移液枪吸头。The upper aqueous phase unit includes a fixing frame and a drainage tube, the fixing frame is used to fix the drainage tube, and the fixing frame includes one or more nested pipette tips.
所述的固定架中的移液枪吸头，口径通过平切或者附加O型橡胶圈，便于固定引流管，或者便于上层水相单元固定于下层油相单元。The diameter of the pipette tip in the fixed frame is cut flat or attached with an O-shaped rubber ring, which is convenient for fixing the drainage tube, or for fixing the upper water phase unit to the lower oil phase unit.
所述的液滴发生装置，通过控制引流管的内径和开口大小调节生成液滴的尺寸。The droplet generating device adjusts the size of the generated droplet by controlling the inner diameter and opening size of the drainage tube.
所述的液滴发生装置，应用于核酸dPCR和dLamp扩增，实现数字化核酸检测。The droplet generating device is applied to nucleic acid dPCR and dLamp amplification to realize digital nucleic acid detection.
一种基于所述的液滴发生装置的数字核酸扩增检测系统，包括：高重复率非对称液滴发生装置，用于产生大量均一性的液滴；核酸扩增控温装置，用于在所述的液滴发生装置生成的液滴中进行核酸扩增反应；和产物信号采集装置，用于采集液滴中核酸扩增反应后的产物信号。A digital nucleic acid amplification detection system based on the droplet generating device, comprising: a high repetition rate asymmetric droplet generating device for generating a large number of uniform droplets; a nucleic acid amplification temperature control device for The nucleic acid amplification reaction is carried out in the droplets generated by the droplet generation device; and the product signal collection device is used to collect the product signals after the nucleic acid amplification reaction in the droplets.
本发明的有益效果：Beneficial effects of the present invention:
本发明突破现有的液滴发生装置中，油包水液滴生产所需要的微流控芯片，生产成本高，使用困难等瓶颈，达到使用常规离心设备或类似技术即可实现形成大量优质液滴的目的。The invention breaks through the bottlenecks of the microfluidic chip required for the production of water-in-oil droplets in the existing droplet generating device, such as high production cost and difficulty in use, and achieves the formation of a large amount of high-quality liquid by using conventional centrifugal equipment or similar technologies. The purpose of the drop.
本发明通过对毛细管尖端进行拉锥处理，采用非对称空心引流结构，构建了一种可以工业级量产的液滴生成装置，具有成本可控、重复率高、操作简单等特点。The present invention constructs a droplet generating device that can be mass-produced at an industrial level by tapering the capillary tip and adopting an asymmetrical hollow drainage structure, which has the characteristics of controllable cost, high repetition rate, and simple operation.
另外，本发明工业级非对称液滴发生装置与核酸扩增相结合，微量的水相溶液在几分钟内即可快速产生高通量的均一油包水液滴，实现数字化PCR核酸定量检测。In addition, the industrial-grade asymmetric droplet generating device of the present invention is combined with nucleic acid amplification, and a small amount of aqueous solution can quickly generate high-throughput uniform water-in-oil droplets within a few minutes, realizing digital PCR nucleic acid quantitative detection.
本发明可对样品如核酸、细胞、蛋白、纳米颗粒等进行封装分配，进而结合核酸扩增完成检测等应用。The invention can package and distribute samples such as nucleic acid, cells, proteins, nanoparticles, etc., and further combine nucleic acid amplification to complete detection and other applications.
附图说明Description of drawings
图1为本发明液滴发生装置的结构示意图。FIG. 1 is a schematic structural view of the droplet generating device of the present invention.
图2为本发明液滴发生装置中毛细管的锥形非对称结构放大图。Fig. 2 is an enlarged view of the tapered asymmetric structure of the capillary in the droplet generating device of the present invention.
图3为本发明液滴发生装置中锥形毛细管尖端结构放大图。Fig. 3 is an enlarged view of the tip structure of the tapered capillary in the droplet generating device of the present invention.
图4为本发明液滴发生装置生成的均一液滴示意图。Fig. 4 is a schematic diagram of uniform droplets generated by the droplet generating device of the present invention.
图5为本发明液滴发生装置使用的锥形毛细管尖端外径与生成均一液滴外径的关系图。Fig. 5 is a graph showing the relationship between the outer diameter of the tip of the tapered capillary used in the droplet generating device of the present invention and the outer diameter of the generated uniform droplet.
图6为本发明液滴发生装置用于新冠病毒核酸Lamp扩增的实验图。Fig. 6 is an experimental diagram of the use of the droplet generating device of the present invention for the Lamp amplification of the nucleic acid of the new coronavirus.
图7为新冠病毒荧光定量PCR扩增与其他数字PCR扩增实验图。Figure 7 is a diagram of the new coronavirus fluorescent quantitative PCR amplification and other digital PCR amplification experiments.
图8为本发明液滴发生装置用于新冠病毒核酸PCR扩增的实验图。Fig. 8 is an experimental diagram of the use of the droplet generating device of the present invention for PCR amplification of the nucleic acid of the new coronavirus.
附图标记说明：引流管1、上层水相单元2、固定架3、离心管4、下层油相单元5。Explanation of reference numerals: drainage tube 1 , upper water phase unit 2 , fixing frame 3 , centrifuge tube 4 , and lower oil phase unit 5 .
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图对本发明做进一步阐述。In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further elaborated below in conjunction with the accompanying drawings.
实施例1Example 1
本发明构建了一种工业级非对称液滴发生装置。The invention constructs an industrial-grade asymmetric droplet generating device.
该装置可使用生化实验室常见的微量移液枪头(或规模生产类似的结构)和离心管进行组装，将锥形的毛细管固定在微量移液枪头中，在高速离心作用下，微量级的上层水相进入到下层油相中生成尺寸均一、高质量的油包水液滴，并且通过调节锥形毛细管尖端的外径可改变生成的油包水液滴的尺寸。The device can be assembled using a common micropipette tip (or a similar structure for large-scale production) and a centrifuge tube in a biochemical laboratory, and the tapered capillary is fixed in the micropipette tip. The upper water phase enters the lower oil phase to generate uniform-sized, high-quality water-in-oil droplets, and the size of the generated water-in-oil droplets can be changed by adjusting the outer diameter of the tapered capillary tip.
如图1所示，该装置包括上层水相单元2和下层油相单元5两部分单元，所述的上层水相单元包括固定架3和引流管1(此处为毛细管)，固定架3用于固定引流管1，所述中的固定架3包括一个或者多个嵌套的移液枪吸头。上层水相单元由三种微量移液枪头和毛细管组装而成，最外层的移液枪头由1000μl的移液枪头在枪头凸起处切割改装而成，中间层的移液枪头由200μl的移液枪头在枪头凸起处切割改装而成并使用O形项圈固定在最外层移液枪头内侧，最内侧的移液枪头由10μl的移液枪头在距最下端0.5cm处切割改装而成，三种不同规格的微量移液枪头相互嵌套，内径为690μm毛细管则刚好被固定在最内侧的微量移液枪头中。下层油相单元5则由1.5ml离心管4组成。上层水相单元2中最外层的移液枪头可以固定在下层油相体系的1.5ml离心管中，共同置于实验室常见离心机中进行油包水液滴的生成。本例中，移液枪头的型号如下，1000μl移液枪头：Axygen T‐1000‐B；200μl移液枪头：Axygen T‐200‐Y；10μl移液枪头：Axygen T‐300。离心管可以使用普通的离心管，与上层水相的移液枪头匹配即可。对于本领域技术人员来说，显然可以使用生化实验室常见的其它微量移液枪头，或者采用市场上类似的结构和产品。As shown in Fig. 1, this device comprises two parts unit of upper layer water phase unit 2 and lower floor oil phase unit 5, described upper layer water phase unit comprises fixed frame 3 and drainage tube 1 (here is capillary), and fixed frame 3 is used for For fixing the drainage tube 1, the fixing frame 3 includes one or more nested pipette tips. The upper layer of the aqueous phase unit is assembled from three types of micropipette tips and capillaries. The outermost layer of the pipette tip is made by cutting and refitting the 1000μl pipette tip at the protrusion of the tip. The middle layer of the pipette tip is The tip is modified by cutting a 200μl pipette tip at the protrusion of the tip and fixed on the inside of the outermost pipette tip with an O-ring. The innermost pipette tip is made of a 10μl pipette tip at a distance The bottom 0.5cm is cut and refitted. Three micropipette tips of different specifications are nested with each other, and the capillary with an inner diameter of 690 μm is just fixed in the innermost micropipette tip. The lower oil phase unit 5 consists of a 1.5ml centrifuge tube 4 . The outermost pipette tip in the upper aqueous phase unit 2 can be fixed in the 1.5ml centrifuge tube of the lower oil phase system, and placed together in a common laboratory centrifuge to generate water-in-oil droplets. In this example, the pipette tip models are as follows, 1000μl pipette tip: Axygen T-1000-B; 200μl pipette tip: Axygen T-200-Y; 10μl pipette tip: Axygen T-300. Common centrifuge tubes can be used as the centrifuge tubes, which can be matched with the pipette tip of the upper aqueous phase. For those skilled in the art, it is obvious that other common micropipette tips in biochemical laboratories can be used, or similar structures and products on the market can be adopted.
该装置中使用的毛细管为普通内径均一的毛细管，经过如微电极拉针仪等方法对尖端进行拉锥，使尖端形成一个锥形结构。这一结构的使用显著提高了尺寸均一、高质量油包水液滴的生成率。本例中使用的毛细管内径为690μm(外径1200μm)，长度10cm，使用毛细管拉针仪等仪器对其进行拉针后，毛细管的尖端形成锥形结构(图2)，但由于在拉针仪使用的过程中，拉针使用的温度过高而往往导致在形成锥形结构后尖端位置会出现部分塌陷，导致尖端被密封。因此，毛细管经过拉锥后，使用刀片等在显微镜下对毛细管尖端进行切割确定尖端的外径，以确保离心出来的液滴尺寸可以控制。例如：尖端外径为10μm的毛细管(图3)，经过离心处理后，即可得到直径为50μm左右的均一液滴(图4)。The capillary used in this device is an ordinary capillary with uniform inner diameter, and the tip is tapered by methods such as microelectrode needle pulling instrument, so that the tip forms a tapered structure. The use of this structure significantly increases the generation rate of uniformly sized, high-quality water-in-oil droplets. The capillary used in this example has an inner diameter of 690 μm (outer diameter of 1200 μm) and a length of 10 cm. After pulling the needle using a capillary needle-pulling instrument, the tip of the capillary forms a tapered structure (Figure 2). In the process of use, the temperature of the pulling needle is too high, which often leads to partial collapse of the tip position after the tapered structure is formed, resulting in the tip being sealed. Therefore, after the capillary is tapered, the tip of the capillary is cut under a microscope with a blade to determine the outer diameter of the tip, so as to ensure that the size of the centrifuged droplets can be controlled. For example: a capillary with a tip outer diameter of 10 μm (Figure 3), after centrifugation, uniform droplets with a diameter of about 50 μm can be obtained (Figure 4).
本发明中的装置在上层水相上样时使用如微量上样针等仪器直接将样品加入到毛细管内部，完全避免了因密封不严出现漏液问题而导致生成不均匀的液滴；同时也确保了注射进入毛细管内部的上层水相可以完全被离心生成液滴，提高了液滴生成的数量，同时也避免了试剂的浪费。例如，10μl的上层水相可产生7万个以上的均一液滴。The device in the present invention uses instruments such as a micro sample loading needle to directly add the sample to the inside of the capillary when loading the sample on the upper aqueous phase, completely avoiding the generation of uneven liquid droplets due to the leakage problem caused by poor sealing; It ensures that the upper aqueous phase injected into the capillary can be completely centrifuged to generate droplets, which increases the number of droplets generated and avoids waste of reagents. For example, 10 μl of the upper aqueous phase can produce more than 70,000 uniform droplets.
本发明中的装置与CN112522374A的液滴发生装置分别各制作10个，其均一液滴的成功生成率结果由下表1可知，CN112522374A的液滴发生装置只有1个成功生成了均一的液滴，其成功率为10％，而本发明的装置有8个装置成功生成了均一的液滴，其成功率为80％。若排除切坏器件，重复率为100％。The device among the present invention and the droplet generating device of CN112522374A respectively make 10, and the result of the successful generation rate of its uniform droplet can be known from the following table 1, and the droplet generating device of CN112522374A has only 1 successfully generated uniform droplet, Its success rate is 10%, while 8 devices of the device of the present invention have successfully generated uniform droplets, and its success rate is 80%. If the broken device is excluded, the repetition rate is 100%.
本发明的装置可以通过调节锥形毛细管尖端的外径改变生成的油包水液滴的尺寸。本发明切割了52个不同尖端外径的锥形毛细管进行均一液滴的生成，除尖端外径不同外，其余条件均保持一致。由此得到的锥形毛细管尖端外径与生成均一液滴直径的关系如图5所示。锥形毛细管尖端外径范围为1.833‐17.277μm，生成均一液滴的直径范围为15.169‐52.038μm。The device of the present invention can change the size of the generated water-in-oil droplets by adjusting the outer diameter of the tapered capillary tip. The present invention cuts 52 tapered capillaries with different tip outer diameters to generate uniform droplets, and the other conditions are kept the same except that the tip outer diameters are different. The relationship between the outer diameter of the tip of the tapered capillary and the diameter of the uniform droplet thus obtained is shown in FIG. 5 . The outer diameter range of the tapered capillary tip is 1.833-17.277 μm, and the diameter range of the uniform droplet is 15.169-52.038 μm.
本发明装置可使用生化实验室中常见的微量移液枪头(或规模生产类似的结构)、离心管和锥形毛细管进行组装，与CN112522374A的离心式液滴发生装置相比(表1)，本发明使用锥形毛细管提高了均一液滴的离出率、提高了液滴的生成数量以及避免了微量级上层水相的浪费。The device of the present invention can be assembled using common micropipette tips (or scale production similar structures), centrifuge tubes and tapered capillaries in biochemical laboratories, compared with the centrifugal droplet generating device of CN112522374A (Table 1), The invention uses a tapered capillary to increase the separation rate of uniform droplets, increase the number of droplets generated, and avoid waste of the trace-level upper water phase.
表1Table 1
表1Table 1
Figure PCTCN2021134672-appb-000001
Figure PCTCN2021134672-appb-000001
实施例2Example 2
本发明构建的工业级非对称液滴发生装置与核酸lamp结合，实现数字化lamp核酸检测。The industrial-grade asymmetric droplet generating device constructed in the present invention is combined with a nucleic acid lamp to realize digital lamp nucleic acid detection.
样品的选择：新冠病毒阳性标准样品。利用RNA‐free‐water稀释至1‐100cp/μl。Sample selection: New coronavirus positive standard sample. Use RNA-free-water to dilute to 1-100cp/μl.
反转录：逆转录操作使用上海康为世纪生物科技股份有限公司的HiFiScript cDNA Synthesis Kit试剂盒，操作步骤按照说明书进行操作，反转录得到的DNA于‐20℃保存,长期保存于‐80℃。Lamp体系：Lamp反应采哈尔滨新海基因检测有限公司用新冠恒温荧光探针法核酸检测试剂盒，25μl的lamp体系内包括：5μl新冠病毒DNA阳性标准品，试剂盒内的10μl nCoV‐2 3IsoAmp Solution A和10μl nCoV‐2 3IsoAmp Solution B。微液滴生成：同实施例1，在本发明中的装置上层水相上样时采用如微量上样针等仪器直接将配置好的lamp体系样品加入到毛细管内部。下层油相体系则由(内含表面活性剂EM180的十六烷酸异丙酯)1.5ml离心管组成。上层水相体系中最外层的移液枪头可以固定在下层油相体系的1.5ml离心管中，共同置于实验室常见离心机中(6000rcf,3min)进行油包水液滴的生成。Reverse transcription: The reverse transcription operation uses the HiFiScript cDNA Synthesis Kit kit from Shanghai Kangwei Century Biotechnology Co., Ltd., and the operation steps are operated according to the instructions. The DNA obtained by reverse transcription is stored at -20°C and long-term stored at -80°C . Lamp system: Harbin Xinhai Gene Detection Co., Ltd. uses the new crown constant temperature fluorescent probe method nucleic acid detection kit for Lamp reaction. The 25 μl lamp system includes: 5 μl new coronavirus DNA positive standard, 10 μl nCoV‐2 3IsoAmp Solution A in the kit and 10 μl nCoV-2 3IsoAmp Solution B. Generation of microdroplets: Same as in Example 1, when the upper aqueous phase of the device in the present invention is loaded with samples, instruments such as micro-sampling needles are used to directly add the prepared lamp system samples into the capillary. The lower oil phase system consists of (isopropyl hexadecanoate containing surfactant EM180) 1.5ml centrifuge tube. The outermost pipette tip in the upper aqueous phase system can be fixed in the 1.5ml centrifuge tube of the lower oil phase system, and placed together in a common laboratory centrifuge (6000rcf, 3min) to generate water-in-oil droplets.
Lamp扩增：将生成的液滴转移至PCR管中并放置于荧光定量PCR仪中，设置程序65℃10s，65℃50s为循环单元，循环35次。Lamp amplification: Transfer the generated droplets to a PCR tube and place them in a fluorescent quantitative PCR instrument. Set the program to 65°C for 10s, 65°C for 50s as the cycle unit, and cycle 35 times.
结果检测：lamp扩增结束后，用移液管尖端小心地将微滴转移到玻璃盖玻片或48孔板上用于荧光显微镜观察。结果如图6所示。Result detection: After the lamp amplification is completed, carefully transfer the droplet to a glass coverslip or a 48-well plate with a pipette tip for fluorescence microscopy observation. The result is shown in Figure 6.
此外利用同一样品(1‐100cp/μl新冠病毒DNA)分别采用荧光定量PCR系统检测和市面上的商业化数字PCR系统(DropDx‐2044数字PCR系统)进行检测，结果如图7所示。In addition, the same sample (1-100cp/μl new coronavirus DNA) was tested by fluorescent quantitative PCR system and commercial digital PCR system (DropDx-2044 digital PCR system) on the market, and the results are shown in Figure 7.
实施例3Example 3
本发明构建的工业级非对称液滴发生装置与核酸PCR扩增结合，微量的水相溶液在几分钟内即可快速产生高通量的均一油包水液滴，实现数字化PCR核酸检测。The industrial-grade asymmetric droplet generation device constructed by the present invention is combined with nucleic acid PCR amplification, and a small amount of aqueous phase solution can quickly generate high-throughput uniform water-in-oil droplets within a few minutes, realizing digital PCR nucleic acid detection.
样品的选择：新冠病毒阳性标准DNA样品。Sample selection: New coronavirus positive standard DNA sample.
PCR体系：20μl PCR体系内包括：10μl SYBR Green I PCR Mix，2μl新冠病毒DNA阳性标准品，1μl上游和下游引物，5μl甘油以及1μl ddH 2O。 PCR system: 20 μl PCR system includes: 10 μl SYBR Green I PCR Mix, 2 μl new coronavirus DNA positive standard, 1 μl upstream and downstream primers, 5 μl glycerol and 1 μl ddH 2 O.
微液滴生成：同实例1，在本发明中的装置上层水相上样时采用如微量上样针等仪器直接将配置好的PCR体系样品加入到毛细管内部。下层油相体系则由(含有ePCR液滴发生油)1.5ml离心管组成。上层水相体系中最外层的移液枪头可以固定在下层油相体系的1.5ml离心管中，共同置于实验室常见离心机中(5000rcf,3min)进行油包水液滴的生成。Generation of micro-droplets: Same as Example 1, when the upper aqueous phase of the device in the present invention is sampled, instruments such as micro-loading needles are used to directly add the configured PCR system samples into the capillary. The lower oil phase system consists of a 1.5ml centrifuge tube (containing ePCR droplet generating oil). The outermost pipette tip in the upper aqueous phase system can be fixed in the 1.5ml centrifuge tube of the lower oil phase system, and placed together in a common laboratory centrifuge (5000rcf, 3min) to generate water-in-oil droplets.
PCR扩增：将生成的液滴转移至PCR管中并放置于荧光定量PCR仪中，然后进行PCR反应,反应程序为:94℃,3min；10min；95℃，20s,60℃30s,72℃30s，20cycle；72℃，3min。PCR amplification: transfer the generated droplets to a PCR tube and place them in a fluorescent quantitative PCR instrument, and then perform a PCR reaction. The reaction program is: 94°C, 3min; 10min; 95°C, 20s, 60°C, 30s, 72°C 30s, 20cycle; 72°C, 3min.
结果检测：PCR扩增结束后，用移液管尖端小心地将微滴转移到玻璃盖玻片或48孔板上用于荧光显微镜观察。结果如图8所示。Result detection: After PCR amplification, use a pipette tip to carefully transfer the droplet to a glass coverslip or 48-well plate for fluorescence microscopy. The result is shown in Figure 8.
上述描述中的实施方案可以进一步组合或者替换，且实施方案仅仅是对本发明的优选实施例进行描述，并非对本发明的构思和范围进行限定，在不脱离本发明设计思想的前提下，本领域普通技术人员对本发明的技术方案做出的各种变化和改进，均属于本发明的保护范围。本发明的保护范围由所附权利要求及其任何等同物给出。The implementations in the above description can be further combined or replaced, and the implementations are only descriptions of preferred embodiments of the present invention, and are not intended to limit the concept and scope of the present invention. Various changes and improvements made by technicians to the technical solution of the present invention belong to the protection scope of the present invention. The scope of protection for the present invention is given by the appended claims and any equivalents thereof.

Claims (10)

  1. 一种工业级非对称液滴发生装置，其特征在于：An industrial-grade asymmetric droplet generating device, characterized in that:
    包括上层水相单元和下层油相单元，上层水相单元固定在下层油相单元上，上层水相单元设有上大下小的引流管，在外力的作用下，引流管内的上层水相进入到下层油相中，形成尺寸均一的油包水液滴。It includes the upper water phase unit and the lower oil phase unit. The upper water phase unit is fixed on the lower oil phase unit. The upper water phase unit is provided with a drainage tube with a large top and a small bottom. Under the action of external force, the upper water phase in the drainage tube enters into the lower oil phase to form water-in-oil droplets of uniform size.
  2. 根据权利要求1所述的液滴发生装置，其特征在于：所述的引流管由毛细管经端部拉锥而成。The droplet generating device according to claim 1, characterized in that: the drainage tube is formed by tapering a capillary through an end.
  3. 根据权利要求2所述的液滴发生装置，其特征在于：所述的毛细管的锥部尖端通过刀片在显微镜下进行切割。The droplet generating device according to claim 2, characterized in that: the tapered tip of the capillary is cut by a blade under a microscope.
  4. 根据权利要求1所述的液滴发生装置，其特征在于：所述的外力为离心力。The droplet generating device according to claim 1, wherein the external force is centrifugal force.
  5. 根据权利要求1所述的液滴发生装置，其特征在于：所述的下层油相单元采用塑料离心管。The droplet generating device according to claim 1, characterized in that: said lower oil phase unit adopts a plastic centrifuge tube.
  6. 根据权利要求1所述的液滴发生装置，其特征在于：所述的上层水相单元包括固定架和引流管，固定架用于固定引流管，所述中的固定架包括一个或者多个嵌套的移液枪吸头。The droplet generating device according to claim 1, characterized in that: the upper water phase unit includes a fixing frame and a drainage tube, the fixing frame is used to fix the drainage tube, and the fixing frame includes one or more embedded Set of pipette tips.
  7. 根据权利要求6所述的液滴发生装置，其特征在于：所述的固定架中的移液枪吸头，口径通过平切或者附加O型橡胶圈，便于固定引流管，或者便于上层水相单元固定于下层油相单元。The droplet generating device according to claim 6, characterized in that: the tip of the pipette in the fixed frame has a caliber through a flat cut or an O-shaped rubber ring, which is convenient for fixing the drainage tube, or for the upper water phase The unit is fixed to the lower oil phase unit.
  8. 根据权利要求1所述的液滴发生装置，其特征在于：通过控制引流管的内径和开口大小调节生成液滴的尺寸。The droplet generating device according to claim 1, characterized in that the size of the generated droplets is adjusted by controlling the inner diameter and opening size of the drainage tube.
  9. 根据权利要求1所述的液滴发生装置，其特征在于：应用于核酸dPCR和dLamp扩增，实现数字化核酸检测。The droplet generating device according to claim 1, characterized in that it is applied to nucleic acid dPCR and dLamp amplification to realize digital nucleic acid detection.
  10. 一种基于根据权利要求1所述的液滴发生装置的数字核酸扩增检测系统，其特征在于：包括：高重复率非对称液滴发生装置，用于产生大量均一性的液滴；核酸扩增控温装置，用于在所述的液滴发生装置生成的液滴中进行核酸扩增反应；和A digital nucleic acid amplification detection system based on the droplet generating device according to claim 1, characterized in that: comprising: a high repetition rate asymmetric droplet generating device for generating a large number of uniform droplets; A temperature-increasing device for performing a nucleic acid amplification reaction in the droplets generated by the droplet generating device; and
    产物信号采集装置，用于采集液滴中核酸扩增反应后的产物信号。The product signal collecting device is used for collecting the product signal after the nucleic acid amplification reaction in the droplet.
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