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CN110624616B - Three-dimensional microfluidic device and method for high-throughput micro-droplet generation - Google Patents

Three-dimensional microfluidic device and method for high-throughput micro-droplet generation Download PDF

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CN110624616B
CN110624616B CN201911031393.1A CN201911031393A CN110624616B CN 110624616 B CN110624616 B CN 110624616B CN 201911031393 A CN201911031393 A CN 201911031393A CN 110624616 B CN110624616 B CN 110624616B
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韦学勇
金少搏
余子夷
秦咸明
任娟
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Guangzhou Qianxiang Biotechnology Co Ltd
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Xian Jiaotong University
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Abstract

A three-dimensional microfluidic device and method for generating high-flux micro-droplets, the device comprises a closed flow channel structure formed by bonding a bottom layer, a middle layer and an upper layer PDMS flow channel, wherein the closed flow channel structure is provided with a dispersed phase inlet joint, a continuous phase inlet joint and a collection outlet joint; fixing a three-dimensional microfluidic device on an objective table of a microscope, starting an injection pump, firstly adjusting a dispersion phase and a continuous phase to corresponding flow velocities through the injection pump, and shearing the dispersion phase into micro-droplets by the continuous phase at positions where T-shaped micro-droplets in the device generate a flow channel array; the invention can overcome the defect of slow micro-droplet generation rate of the current two-dimensional flow channel, realizes the generation of high-flux micro-droplets, has the integral structure of PDMS, is very firm after being mutually bonded, avoids the occurrence of cracking between the flow channels due to overlarge input fluid pressure, and has transparent PDMS and easier observation.

Description

用于高通量微液滴生成的三维微流控装置及方法Three-dimensional microfluidic device and method for high-throughput droplet generation

技术领域technical field

本发明涉及微流控技术领域，特别涉及一种用于高通量微液滴生成的三维微流控装置及方法。The invention relates to the technical field of microfluidics, in particular to a three-dimensional microfluidic device and method for generating high-throughput microdroplets.

背景技术Background technique

液滴微流控平台已经成为生物、化学、医学等领域的重要工具，作为液滴微流控平台中的重要技术，微液滴生成方法至关重要，不同的方法在液滴生成速率方面相差很大。目前，微液滴的生成方法主要有共流法、流式汇聚法、T型流道法以及基于以上方法的衍生方法，通过调节气相(分散相)和液相(连续相)的输入压力，气相可以被液相快速剪成微气泡中，液相通常是油和水溶液。例如，通过聚焦微流控装置和T型微流道装置的并联结构，在不到一小时的时间内就能产生1011个气泡(Jeong H H,Chen Z,Yadavali S,et al.Large-scale production of compound bubbles using parallelized microfluidics forefficient extraction of metal ions[J].Lab on a Chip,2019,19,665-673)。如CN109701430 A公开了一种振动管路控制T型微流控芯片生成微气泡的方法，能够实现单分散微气泡序列的形成。CN 105688721 A公开了一种用于生成球状微气泡的微流控芯片，通过调节液体流量，来生成不同直径大小的微气泡。中国专利(公开号为CN 109908983 A)一种具有三维锥形结构的用于微液滴高比例分裂提取的微流控芯片，涉及一种具有三维锥形结构的微流控芯片。CN107519958A公布了一种二维聚焦微流控装置。但在以上方法中，使用的微流控装置大多为二维流道，其生成速率有一定的限制，不适于大通量微液滴的生成，特别是当需要进行大规模材料制造的时候，生成速率难以满足需要。The droplet microfluidic platform has become an important tool in the fields of biology, chemistry, medicine, etc. As an important technology in the droplet microfluidic platform, the microdroplet generation method is very important. Different methods have different droplet generation rates. very large. At present, the generation methods of microdroplets mainly include co-flow method, flow convergence method, T-channel method and derivative methods based on the above methods. By adjusting the input pressure of gas phase (dispersed phase) and liquid phase (continuous phase), The gas phase can be rapidly sheared into microbubbles by the liquid phase, which is usually oil and aqueous solutions. For example, 10 11 bubbles can be generated in less than an hour by the parallel configuration of a focusing microfluidic device and a T-shaped microfluidic device (Jeong HH, Chen Z, Yadavali S, et al. Large-scale production of compound bubbles using parallelized microfluidics for efficient extraction of metal ions [J]. Lab on a Chip, 2019, 19, 665-673). For example, CN109701430 A discloses a method for generating microbubbles by vibrating pipelines to control a T-shaped microfluidic chip, which can realize the formation of monodisperse microbubble sequences. CN 105688721 A discloses a microfluidic chip for generating spherical microbubbles, which can generate microbubbles with different diameters by adjusting the liquid flow rate. The Chinese patent (publication number CN 109908983 A) is a microfluidic chip with a three-dimensional cone structure for high-proportion splitting and extraction of microdroplets, and relates to a microfluidic chip with a three-dimensional cone structure. CN107519958A discloses a two-dimensional focusing microfluidic device. However, in the above methods, most of the microfluidic devices used are two-dimensional flow channels, and their generation rate is limited to a certain extent, which is not suitable for the generation of large-flux microdroplets, especially when large-scale material manufacturing is required. The generation rate is difficult to meet the needs.

发明内容SUMMARY OF THE INVENTION

为了克服上述现有技术的缺点，本发明的目的在于提供一种用于高通量微液滴生成的三维微流控装置及方法，通过T型流道结构的三维阵列化排布,实现微液滴的高通量生成，微液滴的大小可通过分散相与连续相入口的输入压力进行控制调节，装置为全PDMS材料，通体透明，便于三维观察,且装置各结构间键合牢固，不易开裂，装置不仅适用于微液滴的高通生成，还适用于微气泡的高通生成。In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a three-dimensional microfluidic device and method for the generation of high-throughput microdroplets. For the high-throughput generation of droplets, the size of the microdroplets can be controlled and adjusted by the input pressure of the dispersed phase and continuous phase inlets. The device is made of all PDMS materials, which is transparent throughout, which is convenient for three-dimensional observation, and the structures of the device are firmly bonded. It is not easy to crack, and the device is not only suitable for the high-pass generation of micro-droplets, but also for the high-pass generation of micro-bubbles.

为了实现上述目的，本发明采用以下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:

一种用于高通量微液滴生成的三维微流控装置，包括由底层PDMS流道400、中层PDMS流道500和上层PDMS流道600键合形成的封闭式流道结构，封闭式流道结构设有分散相入口接头100、连续相入口接头300和收集出口接头200；A three-dimensional microfluidic device for high-throughput microdroplet generation, comprising a closed flow channel structure formed by bonding a bottom PDMS flow channel 400, a middle PDMS flow channel 500 and an upper PDMS flow channel 600. The channel structure is provided with a dispersed phase inlet joint 100, a continuous phase inlet joint 300 and a collection outlet joint 200;

所述的底层PDMS流道400包括两组T型微液滴生成流道阵列，其中第一组T型微液滴生成流道阵列包括第一分散相流道402、第一连续相流道401、第二连续相流道405和第二分散相流道406，第一分散相流道402的入口端与第二分散相流道406入口端交汇后与第一分散相入口407连接，第一分散相流道402的出口端与第一连续相流道401的中部连通，第一连续相流道401的入口端与第二连续相流道405的入口端交汇后与第一连续相入口414连接，第一连续相流道401的出口端与第一输送出口403连接；第二分散相流道406出口端与第二连续相流道405的中部连通，第二连续相流道405的出口端与第二输送出口404连接；The bottom PDMS flow channel 400 includes two groups of T-shaped micro-droplet generation flow channel arrays, wherein the first group of T-shaped micro-droplet generation flow channel arrays includes a first dispersed phase flow channel 402 and a first continuous phase flow channel 401. , the second continuous phase flow channel 405 and the second dispersed phase flow channel 406, the inlet end of the first dispersed phase flow channel 402 and the inlet end of the second dispersed phase flow channel 406 meet and then connect to the first dispersed phase inlet 407, the first dispersed phase flow channel 407 The outlet end of the dispersed phase flow channel 402 is communicated with the middle of the first continuous phase flow channel 401 , and the inlet end of the first continuous phase flow channel 401 and the inlet end of the second continuous phase flow channel 405 merge with the first continuous phase inlet 414 . connected, the outlet end of the first continuous phase flow channel 401 is connected to the first delivery outlet 403; the outlet end of the second dispersed phase flow channel 406 is connected to the middle of the second continuous phase flow channel 405, and the outlet of the second continuous phase flow channel 405 The end is connected with the second delivery outlet 404;

第二组T型微液滴生成流道阵列包括第三分散相流道408、第三连续相流道410、第四分散相流道412和第四连续相流道413，第三分散相流道408的入口端与第四分散相流道412入口端交汇后与第一分散相入口407连接，第三分散相流道408的出口端与第三连续相流道410的中部连通，第三连续相流道410的入口端与第四连续相流道413的入口端交汇后与第一连续相入口414连接，第三连续相流道410的出口端与第三输送出口409连接；第四分散相流道412的出口端与第四连续相流道413的中部连通，第四连续相流道413的出口端与第四输送出口411连接。The second group of T-shaped droplet generation flow channel arrays includes a third dispersed phase flow channel 408, a third continuous phase flow channel 410, a fourth dispersed phase flow channel 412 and a fourth continuous phase flow channel 413. The third dispersed phase flow channel The inlet end of the channel 408 meets the inlet end of the fourth dispersed phase channel 412 and is connected to the first dispersed phase inlet 407, the outlet end of the third dispersed phase channel 408 is connected to the middle of the third continuous phase channel 410, and the third The inlet end of the continuous phase flow channel 410 meets the inlet end of the fourth continuous phase flow channel 413 and is connected to the first continuous phase inlet 414, and the outlet end of the third continuous phase flow channel 410 is connected to the third delivery outlet 409; The outlet end of the dispersed phase flow channel 412 is communicated with the middle of the fourth continuous phase flow channel 413 , and the outlet end of the fourth continuous phase flow channel 413 is connected with the fourth delivery outlet 411 .

所述的中层PDMS流道500包括两组T型微液滴生成流道阵列，其中第一组T型微液滴生成流道阵列包括第五分散相流道503、第五连续相流道502、第六连续相流道501和第六分散相流道507，第五分散相流道503的入口端与第五分散相流道507入口端交汇后与第二分散相入口508连接，第五分散相流道503的出口端与第五连续相流道502的中部连通，第五连续相流道502的入口端与第六连续相流道501的入口端交汇后与第二连续相入口516连接，第五连续相流道502的出口端与第五输送出口504连接；第六分散相流道507的出口端与第六连续相流道501的中部连通，第六连续相流道501的出口端与第六输送出口507连接，第五输送出口504与第六输送出口506经流道在第七输送出口505处汇合；The middle-layer PDMS flow channel 500 includes two groups of T-shaped micro-droplet generation flow channel arrays, wherein the first group of T-shaped micro-droplet generation flow channel arrays includes a fifth dispersed phase flow channel 503 and a fifth continuous phase flow channel 502. , the sixth continuous phase flow channel 501 and the sixth disperse phase flow channel 507, the inlet end of the fifth dispersed phase flow channel 503 and the inlet end of the fifth dispersed phase flow channel 507 are connected to the second dispersed phase inlet 508 after the intersection. The outlet end of the dispersed phase flow channel 503 is communicated with the middle of the fifth continuous phase flow channel 502, and the inlet end of the fifth continuous phase flow channel 502 and the inlet end of the sixth continuous phase flow channel 501 meet with the second continuous phase inlet 516. connected, the outlet end of the fifth continuous phase flow channel 502 is connected with the fifth delivery outlet 504; the outlet end of the sixth dispersed phase flow channel 507 is communicated with the middle of the sixth continuous phase flow channel 501, and the The outlet end is connected to the sixth delivery outlet 507, and the fifth delivery outlet 504 and the sixth delivery outlet 506 converge at the seventh delivery outlet 505 through the flow channel;

第二组T型微液滴生成流道阵列包括第七分散相流道509、第七连续相流道515、第八连续相流道514和第八分散相流道513，第七分散相流道509的入口端与第八分散相流道513入口端交汇后与第二分散相入口508连接，第七分散相流道509的出口端与第七连续相流道515的中部连通，第七连续相流道515的入口端与第八连续相流道514的入口端交汇后与第二连续相入口516连接，第七连续相流道515的出口端与第八输送出口510连接；第八分散相流道513的出口端与第八连续相流道514的中部连通，第八连续相流道514的出口端与第九输送出口512连接，第八输送出口510与第九输送出口512经流道在第十输送出口511处汇合。The second group of T-shaped droplet generation flow channel arrays includes a seventh dispersed phase flow channel 509, a seventh continuous phase flow channel 515, an eighth continuous phase flow channel 514 and an eighth dispersed phase flow channel 513. The seventh dispersed phase flow channel The inlet end of the channel 509 and the inlet end of the eighth disperse phase channel 513 meet and are connected to the second dispersed phase inlet 508, the outlet end of the seventh dispersed phase channel 509 is communicated with the middle of the seventh continuous phase channel 515, and the seventh The inlet end of the continuous phase flow channel 515 meets the inlet end of the eighth continuous phase flow channel 514 and is connected to the second continuous phase inlet 516, and the outlet end of the seventh continuous phase flow channel 515 is connected to the eighth delivery outlet 510; The outlet end of the dispersed phase flow channel 513 is communicated with the middle of the eighth continuous phase flow channel 514, the outlet end of the eighth continuous phase flow channel 514 is connected with the ninth delivery outlet 512, and the eighth delivery outlet 510 and the ninth delivery outlet 512 are passed through. The flow channels meet at the tenth delivery outlet 511 .

所述的上层PDMS流道600包括第三分散相入口604、第三连续相入口601和微液滴收集口605，第三分散相入口604连接分散相入口接头100，第三连续相入口601连接连续相入口接头300；微液滴收集口605的入口端通过第一输送流道602、第二输送流道607与第十一输送出口603、第十二输送出口606的出口端连接。The upper PDMS flow channel 600 includes a third dispersed phase inlet 604, a third continuous phase inlet 601 and a droplet collection port 605, the third dispersed phase inlet 604 is connected to the dispersed phase inlet connector 100, and the third continuous phase inlet 601 is connected to Continuous phase inlet joint 300; the inlet end of the droplet collection port 605 is connected to the outlet ends of the eleventh delivery outlet 603 and the twelfth delivery outlet 606 through the first delivery channel 602 and the second delivery channel 607.

所述的封闭式流道结构的流道高度均为60微米，除第五输送出口504、第六输送出口506、第七输送出口505、第八输送出口510、第九输送出口512、第二分散相入口508、第十输送出口511，第二连续相入口516、第三分散相入口604、微液滴收集口605、第三连续相入口601均为通孔，且孔径相同，其他各入口出口均为非通孔，其高度与流道等高。The height of the flow channel of the closed flow channel structure is all 60 microns, except for the fifth delivery outlet 504, the sixth delivery outlet 506, the seventh delivery outlet 505, the eighth delivery outlet 510, the ninth delivery outlet 512, the second delivery outlet 512, and the second delivery outlet 504. The dispersed phase inlet 508 , the tenth delivery outlet 511 , the second continuous phase inlet 516 , the third dispersed phase inlet 604 , the droplet collection port 605 , and the third continuous phase inlet 601 are all through holes with the same diameter, and the other inlets are The outlets are all non-through holes, and their height is the same as that of the flow channel.

所述的底层PDMS流道400、中层PDMS流道500和上层PDMS流道600之间的相对位置关系：中层PDMS流道500无流道的下表面键合在底层PDMS流道400上有流道的上表面，上层PDMS流道600的下表面键合在中层PDMS流道500上有流道的上表面；第一分散相入口407、第二分散相入口508、第三分散相入口604、分散相入口接头100中轴线同轴，相互贯通；第一连续相入口414、第二连续相入口516、第三连续相入口601、连续相入口接头300中轴线同轴，相互贯通；第一输送出口403与第五输送出口504、第二输送出口404与第六输送出口506、第三输送出口409与第八输送出口510、第四输送出口411与第九输送出口512、第七输送出口505与第十一输送出口603、第十输送出口511与第十二输送出口606分别中轴线同轴，相互贯通。The relative positional relationship between the bottom PDMS flow channel 400, the middle PDMS flow channel 500 and the upper PDMS flow channel 600: the lower surface of the middle PDMS flow channel 500 without flow channel is bonded to the bottom PDMS flow channel 400 with a flow channel The upper surface of the upper PDMS flow channel 600 of the upper layer is bonded to the upper surface of the middle PDMS flow channel 500 with the flow channel; the first dispersed phase inlet 407, the second dispersed phase inlet 508, the third dispersed phase inlet 604, the dispersed phase inlet The central axis of the phase inlet joint 100 is coaxial and penetrates each other; the first continuous phase inlet 414, the second continuous phase inlet 516, the third continuous phase inlet 601, and the continuous phase inlet joint 300 have coaxial central axes and penetrate each other; the first delivery outlet 403 and the fifth delivery outlet 504, the second delivery outlet 404 and the sixth delivery outlet 506, the third delivery outlet 409 and the eighth delivery outlet 510, the fourth delivery outlet 411 and the ninth delivery outlet 512, and the seventh delivery outlet 505 and the The eleventh delivery outlet 603 , the tenth delivery outlet 511 and the twelfth delivery outlet 606 are respectively coaxial with their central axes and penetrate each other.

所述的一种用于高通量微液滴生成的三维微流控装置的微液滴生成方法，包括以下步骤：The described microdroplet generation method for a three-dimensional microfluidic device for high-throughput microdroplet generation includes the following steps:

1)将用于高通量微液滴生成的三维微流控装置固定在显微镜的载物台上，通过物镜观察确保各中层PDMS流道500的两组T型微液滴生成流道阵列部位处于显微镜视场内并且无倾斜；1) Fix the three-dimensional microfluidic device for high-throughput microdroplet generation on the stage of the microscope, and observe through the objective lens to ensure that two sets of T-shaped microdroplet generation flow channel array positions in each PDMS flow channel 500 in the middle layer within the microscope field of view and without tilt;

2)将分散相入口接头100、连续相入口接头300、收集出口接头200通过特氟龙导管分别与氮气压力注射泵上的分散相溶液储液瓶、连续相溶液储液瓶、微液滴收集容器相连接；2) The disperse phase inlet joint 100, the continuous phase inlet joint 300, and the collection outlet joint 200 are respectively connected to the dispersed phase solution storage bottle, continuous phase solution storage bottle, and microdroplet collection on the nitrogen pressure syringe pump through a Teflon conduit. container is connected;

3)开启注射泵，通过注射泵将分散相与连续相调整到相应的流速，在各T型微液滴生成流道阵列处，连续相将分散相剪切成微液滴。3) Turn on the syringe pump, adjust the disperse phase and the continuous phase to the corresponding flow rates through the syringe pump, and at each T-shaped microdroplet generation channel array, the continuous phase shears the dispersed phase into microdroplets.

相对于现有技术，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

(1)本发明可以克服目前二维流道生成微液滴速率慢的缺点，装置可累加扩增微液滴生成的T型微液滴生成流道阵列，实现高通量的微液滴的生成。(1) The present invention can overcome the shortcoming of the current two-dimensional flow channel that generates micro-droplets at a slow rate, and the device can accumulate and amplify the T-shaped micro-droplets generated by the generate.

(2)本发明各层流道结构均为PDMS，相互键合后非常牢固，避免了流道之间因输入流体压力过大开裂的发生，且PDMS通体透明，更易观察。(2) The structure of each layer of the flow channel of the present invention is PDMS, which is very strong after bonding with each other, which avoids the occurrence of cracking between the flow channels due to excessive input fluid pressure, and the PDMS body is transparent and easier to observe.

(3)本发明装置不仅适用于微液滴的高通生成，当把分散相换成气相时，也可进行微气泡的高通生成。(3) The device of the present invention is not only suitable for high-pass generation of micro-droplets, but also can be used for high-pass generation of micro-bubbles when the dispersed phase is changed to gas phase.

附图说明Description of drawings

图1是本发明用于高通量微液滴生成的三维微流控装置的等轴侧视图。Figure 1 is an isometric view of the three-dimensional microfluidic device of the present invention for high-throughput droplet generation.

图2中图(a)是底层PDMS流道400的等轴侧视图，图(b)是底层PDMS流道400的后视图。In FIG. 2 , (a) is an isometric side view of the underlying PDMS flow channel 400 , and FIG. 2 is a rear view of the underlying PDMS flow channel 400 .

图3中图(a)是中层PDMS流道500的等轴侧视图,图(b)是中层PDMS流道500的后视图。Figure (a) in FIG. 3 is an isometric side view of the middle-layer PDMS flow channel 500 , and Figure (b) is a rear view of the middle-layer PDMS flow channel 500 .

图4是上层PDMS流道600的等轴侧视图。FIG. 4 is an isometric view of the upper PDMS flow channel 600 .

图5是用于高通量微液滴生成的三维微流控装置的微液滴生成原理图。FIG. 5 is a schematic diagram of microdroplet generation of a three-dimensional microfluidic device for high-throughput microdroplet generation.

具体实施方式Detailed ways

下面结合附图对本发明作详细叙述。The present invention will be described in detail below in conjunction with the accompanying drawings.

参照图1，一种用于高通量微液滴生成的三维微流控装置，包括由底层PDMS流道400、中层PDMS流道500和上层PDMS流道600键合形成的封闭式流道结构，封闭式流道结构设有分散相入口接头100、连续相入口接头300和收集出口接头200；所述的封闭式微流道系统用于容纳分散相溶液和连续相溶液样品，为微液滴的生成提供环境并且将生成的液滴输送至液滴收集口处；Referring to FIG. 1, a three-dimensional microfluidic device for high-throughput microdroplet generation includes a closed flow channel structure formed by bonding a bottom PDMS flow channel 400, a middle PDMS flow channel 500 and an upper PDMS flow channel 600. , the closed flow channel structure is provided with a disperse phase inlet joint 100, a continuous phase inlet joint 300 and a collection outlet joint 200; the closed micro flow channel system is used to accommodate the disperse phase solution and the continuous phase solution samples, which are microdroplets. generating an environment and delivering the generated droplets to a droplet collection port;

参照图2，所述的底层PDMS流道400包括两组T型微液滴生成流道阵列，其中第一组T型微液滴生成流道阵列包括第一分散相流道402、第一连续相流道401、第二连续相流道405和第二分散相流道406，第一分散相流道402的入口端与第二分散相流道406入口端交汇后与第一分散相入口407连接，第一分散相流道402的出口端与第一连续相流道401的中部连通，第一连续相流道401的入口端与第二连续相流道405的入口端交汇后与第一连续相入口414连接，第一连续相流道401的出口端与第一输送出口403连接；第二分散相流道406出口端与第二连续相流道405的中部连通，第二连续相流道405的出口端与第二输送出口404连接；Referring to FIG. 2, the bottom PDMS flow channel 400 includes two groups of T-shaped micro-droplet generation flow channel arrays, wherein the first group of T-shaped micro-droplet generation flow channel arrays includes a first dispersed phase flow channel 402, a first continuous flow channel The phase flow channel 401, the second continuous phase flow channel 405 and the second dispersed phase flow channel 406, the inlet end of the first dispersed phase flow channel 402 and the inlet end of the second dispersed phase flow channel 406 meet with the first dispersed phase inlet 407 connected, the outlet end of the first dispersed phase flow channel 402 is connected to the middle of the first continuous phase flow channel 401, and the inlet end of the first continuous phase flow channel 401 and the inlet end of the second continuous phase flow channel 405 intersect with the first continuous phase flow channel 405. The continuous phase inlet 414 is connected, the outlet end of the first continuous phase flow channel 401 is connected with the first delivery outlet 403; the outlet end of the second dispersed phase flow channel 406 is connected with the middle of the second continuous phase flow channel 405, and the second continuous phase flow The outlet end of the channel 405 is connected with the second delivery outlet 404;

第二组T型微液滴生成流道阵列包括第三分散相流道408、第三连续相流道410、第四分散相流道412和第四连续相流道413，第三分散相流道408的入口端与第四分散相流道412入口端交汇后与第一分散相入口407连接，第三分散相流道408的出口端与第三连续相流道410的中部连通，第三连续相流道410的入口端与第四连续相流道413的入口端交汇后与第一连续相入口414连接，第三连续相流道410的出口端与第三输送出口409连接；第四分散相流道412的出口端与第四连续相流道413的中部连通，第四连续相流道413的出口端与第四输送出口411连接。The second group of T-shaped droplet generation flow channel arrays includes a third dispersed phase flow channel 408, a third continuous phase flow channel 410, a fourth dispersed phase flow channel 412 and a fourth continuous phase flow channel 413. The third dispersed phase flow channel The inlet end of the channel 408 meets the inlet end of the fourth dispersed phase channel 412 and is connected to the first dispersed phase inlet 407, the outlet end of the third dispersed phase channel 408 is connected to the middle of the third continuous phase channel 410, and the third The inlet end of the continuous phase flow channel 410 meets the inlet end of the fourth continuous phase flow channel 413 and is connected to the first continuous phase inlet 414, and the outlet end of the third continuous phase flow channel 410 is connected to the third delivery outlet 409; The outlet end of the dispersed phase flow channel 412 is communicated with the middle of the fourth continuous phase flow channel 413 , and the outlet end of the fourth continuous phase flow channel 413 is connected with the fourth delivery outlet 411 .

参照图3，所述的中层PDMS流道500包括两组T型微液滴生成流道阵列，其中第一组T型微液滴生成流道阵列包括第五分散相流道503、第五连续相流道502、第六连续相流道501和第六分散相流道507，第五分散相流道503的入口端与第五分散相流道507入口端交汇后与第二分散相入口508连接，第五分散相流道503的出口端与第五连续相流道502的中部连通，第五连续相流道502的入口端与第六连续相流道501的入口端交汇后与第二连续相入口516连接，第五连续相流道502的出口端与第五输送出口504连接；第六分散相流道507的出口端与第六连续相流道501的中部连通，第六连续相流道501的出口端与第六输送出口507连接，第五输送出口504与第六输送出口506经流道在第七输送出口505处汇合；Referring to FIG. 3 , the middle-layer PDMS flow channel 500 includes two groups of T-shaped micro-droplet generation flow channel arrays, wherein the first group of T-shaped micro-droplet generation flow channel arrays includes a fifth dispersed phase flow channel 503 , a fifth continuous The phase flow channel 502, the sixth continuous phase flow channel 501 and the sixth dispersed phase flow channel 507, the inlet end of the fifth dispersed phase flow channel 503 and the inlet end of the fifth dispersed phase flow channel 507 meet with the second dispersed phase inlet 508 connected, the outlet end of the fifth disperse phase flow channel 503 is communicated with the middle of the fifth continuous phase flow channel 502, and the inlet end of the fifth continuous phase flow channel 502 and the inlet end of the sixth continuous phase flow channel 501 intersect with the second continuous phase flow channel 501. The continuous phase inlet 516 is connected, the outlet end of the fifth continuous phase flow channel 502 is connected with the fifth delivery outlet 504; the outlet end of the sixth dispersed phase flow channel 507 is connected with the middle of the sixth continuous phase flow channel 501, and the sixth continuous phase The outlet end of the flow channel 501 is connected to the sixth delivery outlet 507, and the fifth delivery outlet 504 and the sixth delivery outlet 506 converge at the seventh delivery outlet 505 through the flow channel;

第二组T型微液滴生成流道阵列包括第七分散相流道509、第七连续相流道515、第八连续相流道514和第八分散相流道513，第七分散相流道509的入口端与第八分散相流道513入口端交汇后与第二分散相入口508连接，第七分散相流道509的出口端与第七连续相流道515的中部连通，第七连续相流道515的入口端与第八连续相流道514的入口端交汇后与第二连续相入口516连接，第七连续相流道515的出口端与第八输送出口510连接；第八分散相流道513的出口端与第八连续相流道514的中部连通，第八连续相流道514的出口端与第九输送出口512连接，第八输送出口510与第九输送出口512经流道在第十输送出口511处汇合。The second group of T-shaped droplet generation flow channel arrays includes a seventh dispersed phase flow channel 509, a seventh continuous phase flow channel 515, an eighth continuous phase flow channel 514 and an eighth dispersed phase flow channel 513. The seventh dispersed phase flow channel The inlet end of the channel 509 and the inlet end of the eighth disperse phase channel 513 meet and are connected to the second dispersed phase inlet 508, the outlet end of the seventh dispersed phase channel 509 is communicated with the middle of the seventh continuous phase channel 515, and the seventh The inlet end of the continuous phase flow channel 515 meets the inlet end of the eighth continuous phase flow channel 514 and is connected to the second continuous phase inlet 516, and the outlet end of the seventh continuous phase flow channel 515 is connected to the eighth delivery outlet 510; The outlet end of the dispersed phase flow channel 513 is communicated with the middle of the eighth continuous phase flow channel 514, the outlet end of the eighth continuous phase flow channel 514 is connected with the ninth delivery outlet 512, and the eighth delivery outlet 510 and the ninth delivery outlet 512 are passed through. The flow channels meet at the tenth delivery outlet 511 .

参照图4，所述的上层PDMS流道600包括第三分散相入口604、第三连续相入口601和微液滴收集口605，第三分散相入口604连接分散相入口接头100，第三连续相入口601连接连续相入口接头300；微液滴收集口605的入口端通过第一输送流道602、第二输送流道607与第十一输送出口603、第十二输送出口606的出口端连接。4, the upper PDMS flow channel 600 includes a third dispersed phase inlet 604, a third continuous phase inlet 601 and a droplet collection port 605. The third dispersed phase inlet 604 is connected to the dispersed phase inlet connector 100, and the third continuous phase inlet 604 is connected to the dispersed phase inlet joint 100. The phase inlet 601 is connected to the continuous phase inlet joint 300; the inlet end of the droplet collection port 605 passes through the first delivery channel 602, the second delivery channel 607 and the outlet ends of the eleventh delivery outlet 603 and the twelfth delivery outlet 606 connect.

四组T型微液滴生成流道阵列的构成，以实现液滴的高通生成。Four groups of T-shaped micro-droplet generation flow channel arrays are constructed to achieve high-pass generation of droplets.

所述的封闭式流道结构的流道高度均为60微米，除第五输送出口504、第六输送出口506、第七输送出口505、第八输送出口510、第九输送出口512、第二分散相入口508、第十输送出口511，第二连续相入口516、第三分散相入口604、微液滴收集口605、第三连续相入口601均为通孔，且孔径相同，其他各入口出口均为非通孔，其高度与流道等高。The height of the flow channel of the closed flow channel structure is all 60 microns, except for the fifth delivery outlet 504, the sixth delivery outlet 506, the seventh delivery outlet 505, the eighth delivery outlet 510, the ninth delivery outlet 512, the second delivery outlet 512, and the second delivery outlet 504. The dispersed phase inlet 508 , the tenth delivery outlet 511 , the second continuous phase inlet 516 , the third dispersed phase inlet 604 , the droplet collection port 605 , and the third continuous phase inlet 601 are all through holes with the same diameter, and the other inlets are The outlets are all non-through holes, and their height is the same as that of the flow channel.

所述的封闭式微流道系统采用具有良好透光性与生物兼容性的聚二甲基硅氧烷(PDMS)制作，便于对微液滴生成过程进行光学监测和记录。The closed microfluidic channel system is made of polydimethylsiloxane (PDMS) with good light transmittance and biocompatibility, which facilitates optical monitoring and recording of the microdroplet generation process.

所述的底层PDMS流道400、中层PDMS流道500和上层PDMS流道600之间的相对位置关系：中层PDMS流道500无流道的下表面键合在底层PDMS流道400上有流道的上表面，上层PDMS流道600的下表面键合在中层PDMS流道500上有流道的上表面；第一分散相入口407、第二分散相入口508、第三分散相入口604、分散相入口接头100中轴线同轴，相互贯通，以实现分散相在三维流道中竖直方向上的传输流动；第一连续相入口414、第二连续相入口516、第三连续相入口601、连续相入口接头300中轴线同轴，相互贯通，以实现连续相在三维流道中竖直方向上的传输流动；第一输送出口403与第五输送出口504、第二输送出口404与第六输送出口506、第三输送出口409与第八输送出口510、第四输送出口411与第九输送出口512、第七输送出口505与第十一输送出口603、第十输送出口511与第十二输送出口606分别中轴线同轴，相互贯通，以实现生成微液滴的传输，并最终输送至微液滴收集口605，以进行微液滴的收集。The relative positional relationship between the bottom PDMS flow channel 400, the middle PDMS flow channel 500 and the upper PDMS flow channel 600: the lower surface of the middle PDMS flow channel 500 without flow channel is bonded to the bottom PDMS flow channel 400 with a flow channel The upper surface of the upper PDMS flow channel 600 of the upper layer is bonded to the upper surface of the middle PDMS flow channel 500 with the flow channel; the first dispersed phase inlet 407, the second dispersed phase inlet 508, the third dispersed phase inlet 604, the dispersed phase inlet The central axis of the phase inlet joint 100 is coaxial and penetrates each other to realize the transmission flow of the dispersed phase in the vertical direction in the three-dimensional flow channel; the first continuous phase inlet 414, the second continuous phase inlet 516, the third continuous phase inlet 601, the continuous The central axis of the phase inlet joint 300 is coaxial and penetrates each other to realize the transmission flow of the continuous phase in the vertical direction in the three-dimensional flow channel; the first delivery outlet 403 and the fifth delivery outlet 504, the second delivery outlet 404 and the sixth delivery outlet 506, the third delivery outlet 409 and the eighth delivery outlet 510, the fourth delivery outlet 411 and the ninth delivery outlet 512, the seventh delivery outlet 505 and the eleventh delivery outlet 603, the tenth delivery outlet 511 and the twelfth delivery outlet The central axes of 606 are respectively coaxial and pass through each other, so as to realize the transmission of the generated micro-droplets, and finally transport them to the micro-droplet collection port 605 to collect the micro-droplets.

所述的一种用于高通量微液滴生成的三维微流控装置的微液滴生成方法，包括以下步骤：The described microdroplet generation method for a three-dimensional microfluidic device for high-throughput microdroplet generation includes the following steps:

1)将用于高通量微液滴生成的三维微流控装置固定在显微镜的载物台上，通过物镜观察确保各中层PDMS流道500的两组T型微液滴生成流道阵列部位处于显微镜视场内并且无倾斜；1) Fix the three-dimensional microfluidic device for high-throughput microdroplet generation on the stage of the microscope, and observe through the objective lens to ensure that two sets of T-shaped microdroplet generation flow channel array positions in each PDMS flow channel 500 in the middle layer within the microscope field of view and without tilt;

2)将分散相入口接头100、连续相入口接头300、收集出口接头200通过特氟龙导管分别与氮气压力注射泵上的分散相溶液储液瓶、连续相溶液储液瓶、微液滴收集容器相连接；2) The disperse phase inlet joint 100, the continuous phase inlet joint 300, and the collection outlet joint 200 are respectively connected to the dispersed phase solution storage bottle, continuous phase solution storage bottle, and microdroplet collection on the nitrogen pressure syringe pump through a Teflon conduit. container is connected;

3)开启注射泵，首先通过注射泵将分散相与连续相调整到相应的流速，在各T型微液滴生成流道阵列处，通过连续相的流体剪切力，将分散相连续剪切成微液滴，从而实现微液滴的生成，然后通过各连接口、输送口、流道、收集出口接头将液滴输送至液滴收集容器。3) Turn on the syringe pump, first adjust the disperse phase and the continuous phase to the corresponding flow rates through the syringe pump, and at each T-shaped microdroplet generation channel array, the disperse phase is continuously sheared by the fluid shear force of the continuous phase. The droplets are formed into microdroplets, thereby realizing the generation of microdroplets, and then the droplets are transported to the droplet collection container through each connection port, delivery port, flow channel, and collection outlet joint.

参照图5，微液滴在用于高通量微液滴生成的三维微流控装置中的生成过程为：连续相溶液通过第一连续相入口414、第二连续相入口516、第三连续相入口601的通孔同时分别进入第一连续相流道401、第二连续相流道405、第三连续相流道410、第四连续相流道413、第五连续相流道502、第六连续相流道501、第七连续相流道515、第八连续相流道514，分散相溶液通过第一分散相入口407、第二分散相入口508、第三分散相入口604的通过同时分别进入第一分散相流道402、第二分散相流道406、第三分散相流道408、第四分散相流道412、第五分散相流道503、第六分散相流道507、第七分散相流道509、第八分散相流道513，通过氮气压力注射泵调节分散相溶液和连续相溶液的输入压力，使分散相、连续相充满各流道，将分散相与连续相调整到相应的流速，从而在各T型微通道结构处，通过连续相对分散相的的流体剪切，将分散相连续剪切成微液滴，实现微液滴的高通量生成。Referring to FIG. 5 , the generation process of microdroplets in the three-dimensional microfluidic device for high-throughput microdroplet generation is as follows: the continuous phase solution passes through the first continuous phase inlet 414 , the second continuous phase inlet 516 , and the third continuous phase inlet 414 . The through holes of the phase inlet 601 enter the first continuous phase flow channel 401, the second continuous phase flow channel 405, the third continuous phase flow channel 410, the fourth continuous phase flow channel 413, the fifth continuous phase flow channel 502, the The six continuous phase flow channels 501, the seventh continuous phase flow channel 515, and the eighth continuous phase flow channel 514, the dispersed phase solution passes through the first dispersed phase inlet 407, the second dispersed phase inlet 508, and the third dispersed phase inlet 604 at the same time. Enter the first dispersed phase flow channel 402, the second dispersed phase flow channel 406, the third dispersed phase flow channel 408, the fourth dispersed phase flow channel 412, the fifth dispersed phase flow channel 503, the sixth dispersed phase flow channel 507, The seventh disperse phase flow channel 509 and the eighth disperse phase flow channel 513 adjust the input pressure of the disperse phase solution and the continuous phase solution through a nitrogen pressure syringe pump, so that the disperse phase and the continuous phase are filled with each flow channel, and the disperse phase and the continuous phase are filled with each other. Adjust to the corresponding flow rate, so that at each T-shaped microchannel structure, the dispersed phase is continuously sheared into microdroplets through continuous relative fluid shearing of the dispersed phase, so as to realize the high-throughput generation of microdroplets.

Claims (6)

1. A three-dimensional microfluidic device for high-throughput microdroplet generation, characterized by: the device comprises a closed flow channel structure formed by bonding a bottom PDMS flow channel (400), a middle PDMS flow channel (500) and an upper PDMS flow channel (600), wherein the closed flow channel structure is provided with a dispersed phase inlet connector (100), a continuous phase inlet connector (300) and a collection outlet connector (200);
the bottom PDMS flow channel (400) comprises two groups of T-shaped micro-droplet generation flow channel arrays, wherein the first group of T-shaped micro-droplet generation flow channel arrays comprises a first dispersed phase flow channel (402), a first continuous phase flow channel (401), a second continuous phase flow channel (405) and a second dispersed phase flow channel (406), the inlet end of the first dispersed phase flow channel (402) is connected with the first dispersed phase inlet (407) after intersecting with the inlet end of the second dispersed phase flow channel (406), the outlet end of the first dispersed phase flow channel (402) is communicated with the middle part of the first continuous phase flow channel (401), the inlet end of the first continuous phase flow channel (401) is connected with the first continuous phase inlet (414) after intersecting with the inlet end of the second continuous phase flow channel (405), and the outlet end of the first continuous phase flow channel (401) is connected with a first conveying outlet (403); the outlet end of the second dispersed phase flow channel (406) is communicated with the middle part of the second continuous phase flow channel (405), and the outlet end of the second continuous phase flow channel (405) is connected with the second conveying outlet (404);
the second group of T-shaped micro-droplet generation flow channel array comprises a third dispersed phase flow channel (408), a third continuous phase flow channel (410), a fourth dispersed phase flow channel (412) and a fourth continuous phase flow channel (413), wherein the inlet end of the third dispersed phase flow channel (408) is connected with the first dispersed phase inlet (407) after meeting with the inlet end of the fourth dispersed phase flow channel (412), the outlet end of the third dispersed phase flow channel (408) is communicated with the middle part of the third continuous phase flow channel (410), the inlet end of the third continuous phase flow channel (410) is connected with the first continuous phase inlet (414) after meeting with the inlet end of the fourth continuous phase flow channel (413), and the outlet end of the third continuous phase flow channel (410) is connected with a third conveying outlet (409); the outlet end of the fourth dispersed phase flow channel (412) is communicated with the middle part of the fourth continuous phase flow channel (413), and the outlet end of the fourth continuous phase flow channel (413) is connected with the fourth conveying outlet (411).
2. The three-dimensional microfluidic device for high throughput microdroplet generation of claim 1, wherein: the middle PDMS flow channel (500) comprises two groups of T-shaped micro-droplet generation flow channel arrays, wherein the first group of T-shaped micro-droplet generation flow channel array comprises a fifth dispersed phase flow channel (503), a fifth continuous phase flow channel (502), a sixth continuous phase flow channel (501) and a sixth dispersed phase flow channel (507), the inlet end of the fifth dispersed phase flow channel (503) is connected with the second dispersed phase inlet (508) after meeting with the inlet end of the fifth dispersed phase flow channel (507), the outlet end of the fifth dispersed phase flow channel (503) is communicated with the middle part of the fifth continuous phase flow channel (502), the inlet end of the fifth continuous phase flow channel (502) is connected with the second continuous phase inlet (516) after meeting with the inlet end of the sixth continuous phase flow channel (501), and the outlet end of the fifth continuous phase flow channel (502) is connected with a fifth conveying outlet (504); the outlet end of the sixth dispersed phase flow channel (507) is communicated with the middle part of the sixth continuous phase flow channel (501), the outlet end of the sixth continuous phase flow channel (501) is connected with a sixth conveying outlet (507), and a fifth conveying outlet (504) and a sixth conveying outlet (506) are converged at a seventh conveying outlet (505) through the flow channels;
the second group of T-shaped micro-droplet generation flow channel array comprises a seventh dispersed phase flow channel (509), a seventh continuous phase flow channel (515), an eighth continuous phase flow channel (514) and an eighth dispersed phase flow channel (513), wherein the inlet end of the seventh dispersed phase flow channel (509) is connected with the second dispersed phase inlet (508) after meeting with the inlet end of the eighth dispersed phase flow channel (513), the outlet end of the seventh dispersed phase flow channel (509) is communicated with the middle part of the seventh continuous phase flow channel (515), the inlet end of the seventh continuous phase flow channel (515) is connected with the second continuous phase inlet (516) after meeting with the inlet end of the eighth continuous phase flow channel (514), and the outlet end of the seventh continuous phase flow channel (515) is connected with the eighth delivery outlet (510); the outlet end of the eighth dispersed phase flow channel (513) is communicated with the middle part of the eighth continuous phase flow channel (514), the outlet end of the eighth continuous phase flow channel (514) is connected with the ninth delivery outlet (512), and the eighth delivery outlet (510) and the ninth delivery outlet (512) are converged at the tenth delivery outlet (511) through the flow channels.
3. The three-dimensional microfluidic device for high throughput microdroplet generation of claim 1, wherein: the upper PDMS flow channel (600) comprises a third dispersed phase inlet (604), a third continuous phase inlet (601) and a micro-droplet collection port (605), wherein the third dispersed phase inlet (604) is connected with a dispersed phase inlet joint (100), and the third continuous phase inlet (601) is connected with a continuous phase inlet joint (300); the inlet end of the micro-droplet collecting port (605) is connected with the outlet ends of the eleventh delivery outlet (603) and the twelfth delivery outlet (606) through the first delivery flow channel (602) and the second delivery flow channel (607).
4. A three-dimensional microfluidic device for high throughput micro-droplet generation according to claim 2 or 3, wherein: the height of the flow channel of the closed flow channel structure is 60 micrometers, except for a fifth conveying outlet (504), a sixth conveying outlet (506), a seventh conveying outlet (505), an eighth conveying outlet (510), a ninth conveying outlet (512), a second dispersed phase inlet (508) and a tenth conveying outlet (511), the second continuous phase inlet (516), the third dispersed phase inlet (604), the micro-droplet collecting port (605) and the third continuous phase inlet (601) are all through holes, the diameters of the through holes are the same, and the other inlets and outlets are all non-through holes, and the heights of the inlets and outlets are equal to the height of the flow channel.
5. A three-dimensional microfluidic device for high throughput micro-droplet generation according to claim 2 or 3, wherein: the relative position relationship among the bottom layer PDMS flow channel (400), the middle layer PDMS flow channel (500) and the upper layer PDMS flow channel (600) is as follows: the lower surface of the middle-layer PDMS runner (500) without the runner is bonded to the upper surface of the bottom-layer PDMS runner (400) with the runner, and the lower surface of the upper-layer PDMS runner (600) is bonded to the upper surface of the middle-layer PDMS runner (500) with the runner; the central axes of the first dispersed phase inlet (407), the second dispersed phase inlet (508), the third dispersed phase inlet (604) and the dispersed phase inlet joint (100) are coaxial and are communicated with each other; the central axes of the first continuous phase inlet (414), the second continuous phase inlet (516), the third continuous phase inlet (601) and the continuous phase inlet joint (300) are coaxial and are communicated with each other; the first conveying outlet (403) and the fifth conveying outlet (504), the second conveying outlet (404) and the sixth conveying outlet (506), the third conveying outlet (409) and the eighth conveying outlet (510), the fourth conveying outlet (411) and the ninth conveying outlet (512), the seventh conveying outlet (505) and the eleventh conveying outlet (603), and the tenth conveying outlet (511) and the twelfth conveying outlet (606) are coaxial in central axis and mutually communicated.
6. A method of generating micro-droplets for a three-dimensional microfluidic device for high throughput micro-droplet generation according to claim 1, comprising the steps of:
1) fixing a three-dimensional microfluidic device for high-flux micro-droplet generation on an objective table of a microscope, and ensuring that two groups of T-shaped micro-droplet generation flow channel array parts of each middle-layer PDMS flow channel (500) are positioned in a microscope field of view and are not inclined through objective observation;
2) respectively connecting a disperse phase inlet connector (100), a continuous phase inlet connector (300) and a collection outlet connector (200) with a disperse phase solution storage bottle, a continuous phase solution storage bottle and a micro-droplet collection container on a nitrogen pressure injection pump through Teflon catheters;
3) and starting the injection pump, adjusting the dispersed phase and the continuous phase to corresponding flow rates through the injection pump, and shearing the dispersed phase into micro droplets by the continuous phase at the positions where the T-shaped micro droplets generate the flow channel array.
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