WO2023213266A1 - 微流控芯片及基于微流控技术的高通量纳米颗粒合成系统 - Google Patents

微流控芯片及基于微流控技术的高通量纳米颗粒合成系统 Download PDF

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WO2023213266A1
WO2023213266A1 PCT/CN2023/091999 CN2023091999W WO2023213266A1 WO 2023213266 A1 WO2023213266 A1 WO 2023213266A1 CN 2023091999 W CN2023091999 W CN 2023091999W WO 2023213266 A1 WO2023213266 A1 WO 2023213266A1
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pressure
microfluidic
chip
micro
pressure distribution
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PCT/CN2023/091999
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English (en)
French (fr)
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王雅琦
夏亦秋
凌云峰
梁玉婷
张华�
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苏州锐讯生物科技有限公司
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Publication of WO2023213266A1 publication Critical patent/WO2023213266A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

  • This application belongs to the field of microfluidic nanosynthesis technology, and specifically relates to a microfluidic chip and a high-throughput nanoparticle synthesis system based on microfluidic technology.
  • Nanoparticle synthesis technology is the technological frontier in the rapidly developing field of nanotechnology. Its unique size-dependent characteristics make these materials show great advantages in many fields and are in an irreplaceable position. This technology has been widely used in many industries, such as drug delivery, energy and electronics. Nanoparticle synthesis technology is one of the key steps to realize nanoparticle applications.
  • nanoparticle synthesis Since the size characteristics of nanoparticles are required in most applications, the size distribution, yield, and size repeatability between batches of nanoparticle synthesis are very important parameters in the evaluation of nanoparticle synthesis.
  • One of the traditional methods of nanoparticle synthesis is based on the principle of batch mixing. Specifically, the raw materials for preparing nanoparticles are dissolved in an organic phase or aqueous phase, then added to another phase of aqueous phase or organic phase that is not compatible with it, and quickly mixed in by stirring. beaker or other device.
  • Microreactors based on microfluidic technology can achieve rapid mixing of reagents, temperature control, and precise spatiotemporal control during reactions.
  • controlled and uniform mixing can produce nanoparticles of uniform size.
  • the repeatability of the physical and chemical properties of the nanoparticles can also be precisely controlled.
  • the size uniformity and reproducibility of nanoparticles can be further improved, thereby improving the yield of the nanoparticle preparation process.
  • the instruments currently on the market for nanoparticle synthesis based on microfluidic technology mainly come from Canadian Precision Nanosystems and Precigenome.
  • Precision Nanosystems' instrument mainly uses a syringe pump to push the organic phase and aqueous phase solutions into the microfluidic chip for mixing. Together, the synthesized nanoparticles are collected at the outlet of the microfluidic chip.
  • This instrument achieves very high nanoparticle size controllability, uniformity and high reproducibility.
  • it uses a syringe pump as the driving force to push the fluid it has some insurmountable shortcomings.
  • the syringe pump has a large volume when adjusting the flow rate, slow response speed, low adjustment accuracy, pulsating flow rate, low sample use efficiency, and easy to use. pollution and other issues.
  • the pulsation of flow and low adjustment accuracy will affect the uniformity of mixing during nanoparticle synthesis, thereby reducing the uniformity of nanoparticles.
  • the syringe pump needs to load the reagent into the syringe first, which is complicated to operate and can easily cause contamination.
  • the capacity of the syringe is limited, and the instrument requires a lot of engineering design and improvement during the process of expanding production, making it inconvenient to be directly used in high-throughput production.
  • Precigenome's instrument is an integrated device based on pressure control and microfluidic mixing technology that can achieve fully automated nanoparticle synthesis.
  • This instrument partially makes up for the many shortcomings caused by the syringe pump used in Precision Nanosystems' instruments, and can realize a highly automated and continuous production process of nanoparticles to increase the yield of nanoparticles.
  • this equipment uses a large number of valves, flow meters and other fluid control components. These fluid control components and the pipelines connecting them will bring a large dead volume and cannot meet the formula used in nanoparticle synthesis.
  • the high-throughput, small-volume requirements required for R&D optimization. Therefore, developing a nanoparticle synthesis system that can achieve high throughput and small volume has high market application prospects.
  • this application provides a microfluidic chip and a high-throughput nanoparticle synthesis system based on microfluidic technology.
  • a microfluidic chip based on microfluidic technology includes a chip body, and the chip body is provided with There is at least one flow channel for nanoparticle synthesis.
  • a liquid reservoir is provided at both the inlet end and the synthesis end of the flow channel. The central axis of the liquid reservoir is perpendicular to the flow channel.
  • the liquid reservoir and the chip body are integrally formed or detachably connected.
  • the liquid reservoir and the microfluidic chip are detachably connected through mutually matched Luer connectors.
  • a high-throughput, small-volume nanoparticle synthesis system based on microfluidic technology includes a pressure controller, a pressure distribution component connected to the output end of the pressure controller, and a pressure output end of the pressure distribution component.
  • the microfluidic chip is provided with at least one.
  • a sealing component is provided between the pressure distribution component and the microfluidic chip to form an airtight connection between the pressure distribution component and the microfluidic chip.
  • the pressure distribution component includes a pressure distribution plate, and the sealing component is a sealing gasket.
  • the sealing gasket is provided with an opening and covers at least the top opening of the liquid reservoir to ensure that the pressure distribution component is in contact with the microflow. airtight connection between control chips.
  • the sealing gasket is selectively connected to the pressure distribution plate or to the liquid reservoir port.
  • the volume of the liquid reservoir is 20 ⁇ l-1 ml.
  • the pressure controller is a multi-channel pressure controller, and the number of channels is greater than or equal to 2.
  • a control valve is provided between the pressure controller and the pressure distribution component.
  • the beneficial effects of this application are reflected in: it can accurately control the size, distribution and consistency of nanoparticles.
  • the reagent raw materials are directly synthesized through the flow channel, which avoids the waste of reagents caused by the dead volume caused by redundant connections.
  • By increasing the micro-flow Controlling the number of chips or the number of parallel channels on the same chip can well meet high-throughput requirements.
  • Figure 1 Schematic diagram of the principle structure of the synthesis system of this application.
  • Figure 2 Schematic diagram of the front cross-sectional structure of Figure 1.
  • Figure 3 Schematic structural diagram of one example of this application.
  • Figure 4 Structural diagram of the air pressure distribution plate in Figure 3 when it is opened.
  • Figure 5 Measurement histogram of the size and distribution of lipid nanoparticles synthesized by this system.
  • Figure 6 Particle size and distribution diagram of different batches of nanoliposomes synthesized using a low-throughput flow control device.
  • Figure 7 Comparison of particle size and size distribution of DNA-encapsulated nanoliposomes synthesized in different devices.
  • Figure 8 Comparison of DNA encapsulation efficiency of nanoliposomes synthesized in different devices.
  • Figure 9 Fluorescence and brightfield images of HepG2 (top) and K562 (bottom) after treatment with synthetic GFP DNA-encapsulated nanoliposomes for 48 hours.
  • the microfluidic chip 3 includes a chip body, a flow channel 31 provided on the chip body for nanoparticle synthesis, an inlet end 32 and a synthesis end of the flow channel 31 33 are provided with a liquid reservoir 34.
  • the central axis of the liquid reservoir 34 is arranged perpendicularly to the flow channel.
  • the liquid reservoir 34 and the microfluidic chip 3 are integrally injection molded or detachably connected. Detachable connection means that the liquid reservoir 34 and the microfluidic chip 3 are connected through mutually matched Luer connectors.
  • the flow channel 31 is directly connected to the liquid reservoir 34, which can effectively avoid the connection of multi-flow valves or pipelines in the prior art, and will not cause waste of reagents due to dead volume.
  • This application also discloses a high-throughput nanoparticle synthesis system based on microfluidic technology using the microfluidic chip, including a pressure controller 4 and a pressure distribution connected to the output end of the pressure controller 4 The pressure output end of the component and the pressure distribution component is connected to the microfluidic chip 3 .
  • the pressure distribution assembly includes a pressure distribution plate 1.
  • the pressure distribution plate 1 is provided with a pressure input end and a pressure output end.
  • a control valve 11 is provided between the pressure input end and the pressure controller 4.
  • This pressure distribution plate 1 is provided with a pressure input end and a pressure output end.
  • the control valve 11 is a three-way valve.
  • the pressure control valve 11 can also be installed in the pressure controller. When the pressure controller 4 is adjusting the pressure, the control valve 11 is used to disconnect the pressure controller and the pressure distribution component. When the pressure reaches stability, the corresponding air path is connected through the control valve 11 .
  • the function of the pressure distribution plate 1 is to more effectively connect the pressure of the pressure controller 4 with the microfluidic chip to form a gas path connection. It has a pressure input end and a pressure output end, and inputs the pressure. The path connecting the pressure output end and the pressure output end.
  • the pressure output end of the pressure distribution plate 1 is connected to the liquid reservoir 34 on the microfluidic chip 3 , and further, is connected to the liquid reservoir 34 on the inlet end 32 of the microfluidic chip 3 .
  • a complete flow channel 31 consists of two inlet ends and a synthesis end 33 .
  • the liquid reservoirs at the two inlet ends are reagent reservoirs, and the one at the synthesis end is a nanoparticle collection reservoir.
  • the microfluidic chip 3 can be provided with multiple independent flow channels 31 to synthesize multiple nanoparticles to achieve high throughput.
  • the pressure controller 4 is a multi-channel pressure controller, the number of channels is greater than or equal to 2, and the channels refer to the output control pressure channels.
  • the output pressure range of the pressure controller is 0-30 psi or higher, and the accuracy and precision of the pressure value is less than 0.05 psi.
  • multiple microfluidic chips 3 can also be arranged in parallel.
  • the number of pressure output terminals on the pressure controller 4 is equal to the number of inlets on the microfluidic chip 3.
  • the number of reagent reservoirs is equivalent.
  • the capacity of the liquid reservoir determines the output of nanosynthesis. In this embodiment, the volume of the liquid reservoir is 20 ⁇ l-1 ml.
  • a sealing component 2 is provided between the pressure distribution plate 1 and the microfluidic chip 3.
  • the sealing component 2 is a sealing gasket.
  • the sealing gasket is provided with an opening and at least covers the top opening of the liquid storage tank to ensure that there is a gap between the pressure distribution plate 1 and the liquid storage tank 34 on the microfluidic chip 3. Airtight connection.
  • the sealing gasket may be made of rubber or silicone, and the sealing gasket is selectively connected to the pressure distribution plate 1 or to the port of the liquid reservoir 34 . When connected to the pressure distribution plate, it is tightly connected to the bottom of the pressure distribution plate 1.
  • the sealing gasket is provided with a vent hole, and the vent hole is connected to the liquid reservoir and the pressure output end of the pressure distribution plate. They form a connection without hindering the passage of air pressure.
  • the sealing gasket can also be directly placed on the top open end of the liquid reservoir 34 , as long as both of them achieve an airtight connection between the air pressure distribution plate 1 and the liquid reservoir 34 .
  • the pressure output of the pressure controller 4 is transmitted to the liquid in the liquid reservoir 34 almost instantaneously ( ⁇ 1s) ,
  • the fluid flow rate and pressure output in the microfluidic channel are completely one-to-one correspondence.
  • the fluid in the microfluidic channel is laminar flow, so the flow rate can be predicted and calculated through simple fluid calculations. Therefore, this application can realize the flow rate control of the fluid in the flow channel of the microfluidic chip through precise control of the pressure, thereby achieving precise control of the size of the synthesized nanoparticles.
  • microfluidic chip described in this application can also be used in patent application number 2021110609590.
  • the advantage is that the nanosynthesis formula optimized by the user in this system can be directly used in the low-throughput but high-yield continuous production device of the aforementioned invention application.
  • 12.5mM lipid (mixture) dissolved in ethanol is loaded into one reservoir 34 as a reagent, and nucleic acid is dissolved in an aqueous buffer and loaded into another reservoir 34 as another reagent.
  • the pressure output of the pressure controller 4 the flow rate of the water-oil phase solution can reach 4:1, and the total flow rate is 3mL/min.
  • the pressure output can be stored in the controller as a fixed variable for later use. In order to prevent the reagent in the reservoir from being empty, it is usually necessary to add a certain amount of reagent to the reservoir.
  • lipid ethanol solution For example, if 200 ⁇ L of mRNA-coated lipid nanoparticles are synthesized, 190 ⁇ L of aqueous buffer containing mRNA needs to be added to one reservoir and 70 ⁇ L of lipid ethanol solution to another reservoir. After the synthesis is completed, 160 ⁇ L of aqueous solution and 40 ⁇ L of lipid ethanol solution will be consumed, leaving 30 ⁇ L of each solution in each reservoir, and finally 200 ⁇ L of nanoliposome particles are collected.
  • the nanoparticles synthesized using the flow rate control device The size and particle size distribution are very close to the nanoparticles obtained by the device (pressure control) of the present invention.
  • the average particle diameter of the nanoparticles synthesized by this system is slightly larger (5%). This difference is probably due to the fact that the actual flow rate and flow rate ratio under pressure control are slightly different from the preset ones, but this difference is acceptable. within the range.
  • Nanoliposomes coated with green fluorescent protein (GFP) DNA synthesized by this system were used for in vitro cell transfection studies in HepG2 (human liver cancer cell line) and K562 (human immortalized myeloid leukemia cell line). 24 hours before transfection, HepG2 and K562 cells were seeded in 96-well plates at a concentration of 2–4x 10 cells/well. On the day of transfection, nanoliposomes encapsulating GFP DNA plasmid were added to HepG2 or K562 cell culture medium at a concentration of 400ng DNA/well.
  • GFP green fluorescent protein
  • this system can achieve small volume ( ⁇ 0.2mL) and high-throughput (two or more samples can be made at the same time) synthesis of nanoparticles, thus saving users a lot of raw materials and reducing experimental time. times, suitable for research on the selection and optimization of nanosynthesis formulas.
  • the microfluidic chip in this system is compatible with the previously invented low-throughput and high-throughput flow rate control device. Users can directly use the optimized formula on this device to the flow rate control device and obtain similar nanometer Particles.

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Abstract

本申请提供了一种微流控芯片及基于微流控技术的高通量小体积纳米颗粒合成系统，包括压力控制器及与所述压力控制器输出端连接的压力分配组件、所述压力分配组件的压力输出端与微流控芯片连接，所述微流控芯片上设置有用于纳米颗粒合成的流道，所述流道的入口端及合成端均设置有储液池。本申请的有益效果体现在：可以精准的实现纳米颗粒大小、分布和一致性的控制，试剂原料直接经过流道合成，避免了多余的连接造成的死体积带来的试剂浪费，通过增加微流控芯片或者同一芯片上的并行流道的数量可以很好的完成高通量的需求。

Description

微流控芯片及基于微流控技术的高通量纳米颗粒合成系统 技术领域
本申请属于微流控纳米合成技术领域，具体涉及一微流控芯片及基于微流控技术的高通量纳米颗粒合成系统。
背景技术
纳米颗粒合成技术，是高速发展的纳米技术领域的技术前沿，其独特的尺寸依赖特性，使这些材料在许多领域表现出了极大的优势，处于不可替代的位置。此项技术已广泛应用于诸多行业，如药物输送、能源和电子等。纳米颗粒合成技术是实现纳米颗粒应用的关键步骤之一。
由于在多数应用中，都需要应用到纳米颗粒的尺寸特性，因此纳米颗粒合成各批次间的尺寸分布、产量以及尺寸的可重复性，是纳米颗粒合成评估中非常重要的参数。传统的纳米颗粒合成的方法之一是基于批处理混合的原理来进行的。具体来讲，将制备纳米颗粒的原材料溶于有机相或水相中，再将其加入到与之相容性不好的另一相水相或有机相中，并通过搅拌的方式迅速混合在烧杯或其他设备中。但采用传统的批处理合成方法(在本体溶液中混合)进行纳米颗粒大规模生产时，其颗粒合成质量较差，同时还存在一些不可控因素，如聚集和异构混合，导致纳米粒子的尺寸均一性和可重复性比较差。
基于微流控技术的微型反应器，可实现试剂快速混合、温度控制以及反应中的精确时空操控。采用微流控技术进行纳米颗粒合成，混合受控且均匀，可产生尺寸大小均一的纳米颗粒，同时，纳米颗粒的物理化学性质的可重复性也能得到精确控制。此外，通过调控纳米颗粒合成微环境，可进一步提高纳米颗粒的尺寸均一性和可重复性，进而提高纳米颗粒的制备工艺产率。
目前市场上已有的基于微流控技术实现纳米颗粒合成的仪器主要来自于加拿大Precision Nanosystems以及Precigenome。Precision Nanosystems的仪器主要通过注射泵来推动有机相和水相两相溶液进入微流控芯片进行混 合，在微流控芯片的出口端收集合成好的纳米颗粒。这款仪器实现了非常高的纳米颗粒的尺寸可控性、均一性及高度重复性。但由于其用注射泵作为推动流体的动力，所以存在一些自身不可克服的缺点，例如，注射泵调节流量时体积大，响应速度慢，调节精度低，流量有脉动性，样品使用效率低，易污染等问题。流量的脉动性、调节精度低会影响纳米颗粒合成时混合的均匀程度，从而降低纳米颗粒的均一性。注射泵需要先将试剂加载到注射器中，操作比较复杂，也容易造成污染。此外，注射器的容量有限，该仪器在扩大生产的过程中需要做很大的工程设计及改进，不便于直接用于高通量的生产中。此外，采用注射泵在实现高通量(多个样品同时运行)也有不可克服的难点：在同时运行多个样品时，每个样品都需要配置单独注射泵，不可避免的导致系统复杂，非常不利于系统往高通量方向拓展。目前这些设备为了实现从小体积(小于1ml)到大体积的产量提高(大于1ml)，都需要采用不同设计的微流控芯片，不同设计的芯片往往还需要调整不同的生产纳米颗粒的条件来生产出类似的纳米颗粒。这样不仅增加了设计生产成本，同时也增加了为提高量产且又能保持同样性能的技术难度。
Precigenome的仪器(专利申请号2021110609590)是一款基于压力控制以及微流控混合技术可以实现全自动化的纳米颗粒合成的集成设备。该仪器部分弥补了Precision Nanosystems的仪器中采用注射泵带来的诸多缺点，可以实现高度自动化及连续生产纳米颗粒的过程，以提高纳米颗粒的产量。但是该设备中由于采用了较多的阀、流量计等多种流体控制元件，这些流体控制元件以及将其相连的管路会带来很大的死体积，无法满足应用于纳米颗粒合成的配方研发优化所需的高通量小体积的需求。因此开发一种可以实现高通量小体积的纳米颗粒合成系统具有很高的市场应用前景。
发明内容
为了解决现有技术的不足，可以很好的实现高通量小体积的纳米颗粒合成，本申请提供了一种微流控芯片及基于微流控技术的高通量纳米颗粒合成系统。
本申请的目的通过以下技术方案来实现：
基于微流控技术的微流控芯片，包括芯片本体，所述芯片本体上设置有 至少一条用于纳米颗粒合成的流道，所述流道的入口端及合成端均设置有储液池，所述储液池的中心轴与所述流道垂直。
优选地，所述储液池与所述芯片本体一体化成型或可拆卸连接。
优选地，所述储液池与所述微流控芯片之间通过相互配合的鲁尔接头形成可拆卸连接。
优选地，一种基于微流控技术的高通量小体积纳米颗粒合成系统，包括压力控制器，及与所述压力控制器输出端连接的压力分配组件、所述压力分配组件的压力输出端与以上任意一种微流控芯片连接，所述微流控芯片设置有至少一个。
优选地，所述压力分配组件与所述微流控芯片之间设置有密封组件，以形成压力分配组件与微流控芯片之间的气密性连接。
优选地，所述压力分配组件包括一压力分配板，所述密封组件为密封垫，所述密封垫上设置有开口且至少覆盖置于所述储液池的顶端开口处确保压力分配组件与微流控芯片之间的气密性连接。
优选地，所述密封垫选择性的与所述压力分配板连接或与储液池端口连接。
优选地，所述储液池的体积为20μl-1ml。
优选地，所述压力控制器为多通道压力控制器，所述通道数量大于等于2。
优选地，所述压力控制器与所述压力分配组件之间设置有控制阀。
本申请的有益效果体现在：可以精准的实现纳米颗粒大小、分布和一致性的控制，试剂原料直接经过流道合成，避免了多余的连接造成的死体积带来的试剂浪费，通过增加微流控芯片的数量或者同一芯片上并行流道的数量可以很好的完成高通量的需求。
附图说明
图1：本申请的合成系统原理结构示意图。
图2：图1的正视截面结构示意图。
图3：本申请的其中一种示例结构示意图。
图4：图3中气压分配板打开时的结构示意图。
图5：本系统合成的脂质纳米颗粒大小及分布的测量柱状图。
图6：采用低通量流速控制装置合成的不同批次纳米脂质体粒径及分布图。
图7：在不同装置中合成包裹有DNA的纳米脂质体的粒径及大小分布比较图。
图8：在不同装置中合成纳米脂质体对DNA的包裹率比较。
图9：用合成的包裹GFP DNA纳米脂质体处理48小时后的HepG2(上)和K562(下)荧光和明场图像。
具体实施方式
本申请揭示了一种基于微流控技术的微流控芯片，以及应用该芯片进行高通量小体积纳米颗粒合成的合成系统。结合图1-图2所示，所述微流控芯片3包括芯片本体，及所述芯片本体上设置的用于纳米颗粒合成的流道31，所述流道31的入口端32及合成端33均设置有储液池34。所述储液池34中心轴与所述流道呈垂直设置。所述储液池34与所述微流控芯片3之间为一体注塑成型或可拆卸连接。可拆卸连接是指所述储液池34与所述微流控芯片3之间通过相互配合的鲁尔接头进行连接。当然，也可以采用其他相互配合的连接件。本申请中流道31与储液池34直接连接，可以很好的避免现有技术中多流量阀或管道的连接，不会因为死体积而带来试剂的浪费。
本申请还揭示了一种应用所述微流控芯片下，基于微流控技术的高通量纳米颗粒合成系统，包括压力控制器4，及与所述压力控制器4输出端连接的压力分配组件、所述压力分配组件的压力输出端与所述的微流控芯片3连接。
所述压力分配组件包括一压力分配板1，所述压力分配板1上设置有压力输入端及压力输出端，所述压力输入端与所述压力控制器4之间设置有控制阀11，本实施例中，所述的控制阀11为三通阀。压力控制阀11也可以安装在压力控制器中。当压力控制器4在调节压力时，通过控制阀11将压力控制器与压力分配组件之间断开，当压力达到稳定后，通过控制阀11联通相应的气路。
所述压力分配板1作用是更有效的将压力控制器4的压力与微流控芯片上形成气路上的连接，其具有压力输入端和压力输出端，以及将压力输入 端及压力输出端联通的通路。所述压力分配板1的压力输出端与微流控芯片3上的储液池34连接，进一步的，是与微流控芯片3的入口端32上的储液池34连接。通常，完整的一条流道31由两个入口端及一个合成端33。两个入口端的储液池分别为试剂储液池，合成端的为纳米颗粒收集储液池。所述微流控芯片3上可以设置有多条各自独立的流道31，以合成多个纳米颗粒，从而实现高通量。
所述压力控制器4为多通道压力控制器，所述通道数量大于等于2，所述通道是指具有的输出控制压力通道。所述压力控制器的输出压力范围在0-30psi或更高，压力值的准确度及精度小于0.05psi。针对一套压力控制器4，所述微流控芯片3也可以并列设置有多个，一般来说，所述压力控制器4上的压力输出端数量与所述微流控芯片3上的入口试剂储液池数量相当。储液池的容量决定了纳米合成的产量，本实施例中，所述储液池的体积为20μl-1ml。
为了在压力分配板1与微流控芯片3之间形成气密性连接，所述压力分配板1与所述微流控芯片3之间设置有密封组件2。所述密封组件2为密封垫，所述密封垫上设置有开口且至少覆盖置于所述储液池的顶端开口处确保压力分配板1与微流控芯片3上的储液池34之间的气密性连接。所述密封垫可以为橡胶或硅胶材质，所述密封垫选择性的与所述压力分配板1连接或与储液池34端口连接。与所述压力分配板连接时，其紧密连接于所述压力分配板1的底部，相应的，所述密封垫上开设有通气孔，所述通气孔与储液池和压力分配板的压力输出端之间形成联通，而不阻碍气压的通过。当然，所述密封垫也可以采用直接套置于所述储液池34的顶部开口端，两者均达到气压分配板1与储液池34之间的气密性连接即可。
为更好的理解本申请，结合图3-图4所示，图3-图4中展示了其中一种示例结构。使用时，将不同的试剂载入相应的储液池34中，然后将压力分配板1下压关上，密封垫与储液池34上沿紧密贴合形成气密接触，通过机构上的锁扣将压力分配板1固定在气密紧贴储液池34的状态。用户设置所需的压力及生产的纳米颗粒溶液的体积后，启动压力控制器运行。当纳米颗粒合成达到所需的体积后程序自动结束，压力控制器通过三通阀自动将压力快速释放到和大气压平衡，掰动锁紧的锁扣，将气动压板释放。用户将生成的纳米颗粒溶液取出，并将芯片和储液池做适当的回收处理。
由于压力分配组件中的流体阻力远低于微流控芯片的流道阻力(100倍以上)，因此压力控制器4的压力输出传递到储液池34中的液体几乎是瞬时的(<1s),微流控流道中的流体流速和压力输出完全是一一对应关系，同时，微流孔流道中的流体是层流，因此可以通过简单的流体计算进行预测计算流速。所以本申请可以通过对压力的精确控制从而实现对微流控芯片中流道内流体的流速控制，从而实现合成的纳米颗粒大小的精确控制。进一步的，本申请中所述微流控芯片也可以在专利申请号2021110609590中使用。这样的好处是用户在本系统中优化的纳米合成配方直接可以用在前述发明申请的低通量但高产量的连续生产的装置中。
为更好的验证本申请的有效性，以下进行纳米颗粒合成测试：
将12.5mM脂质(混合物)溶于乙醇中作为一种试剂加载入一个储液池34中，将核酸溶于水相缓冲液中作为另一种试剂载入另一储液池34中。通过调节压力控制器4的压力输出可以使水油相溶液的流速达到4:1,以及总流速为3mL/min。调节好后，压力输出可以作为固定变量存储在控制器中，以便以后使用。为避免储液池中的试剂被打空，通常需在储液池中多加一定量的试剂。举例来说，若合成200μL的包裹了mRNA脂质纳米颗粒，需要在一个储液池中加190μL的含mRNA的水相缓冲液，在另外一个储液池中加70μL的脂质乙醇溶液。合成完毕后，160μL的水相溶液以及40μL的脂质乙醇溶液会被消耗掉，每个储液池中会各有30μL的溶液剩下，最后收集到200μL的纳米脂质体颗粒。
结合图5所示，图中显示了四次独立实验(所有的条件均相同)的结果，可以看出四次实验合成的纳米颗粒从大小以及分布来讲都非常接近。说明本系统虽然用压力控制，但每次通过精确的压力控制输出是可以控制芯片流道内的流速，从而精确的控制纳米颗粒的大小、分布和一致性。
我们还比较用本系统和早先开发的纳米颗粒合成装置(低通量流速控制，专利申请号2021110609590)合成的纳米颗粒。值得指出的是，在比较中由于装置对微流控芯片的兼容性，我们可以采用同样的芯片进行实验比较。早先开发的装置是采用的流速控制，用于采用了流量计及控制流体的一些器件，死体积比较大，最小合成量是1mL。图6为用早先流速控制的装置在相同条件下(总流速为3mL/min,水油流速比为4:1)四次合成的纳米颗粒大小及颗粒分布PDI的测量结果。可以看出用流速控制装置合成的纳米颗粒 的大小及颗粒大小分布和本发明装置(压力控制)得到的纳米颗粒非常接近。本系统合成的纳米颗粒的平均颗粒直径略大(5％)，这个区别很有可能是在压力控制下实际的流速及流速比与预设的略有差别，但是这样的差别是在可以接受的范围内。
通过本系统进行在合成纳米脂质体的同时包裹DNA的实验研究，我们测量了包裹DNA后的纳米脂质体的大小、DNA的包裹率，并和用低通量流速控制的装置合成的包裹有DNA的纳米脂质颗粒进行比较。图7的结果显示在两种装置下合成的包裹了DNA的纳米脂质体的大小及粒径分布PDI都比较接近，平均粒径在80nm左右，PDI在0.2左右。进一步的对包裹率进行研究表明，在两种装置上合成的纳米脂质体对DNA的包裹率都达到了90％左右，如图8所示。
通过本系统合成的包裹了绿色荧光蛋白(GFP)DNA的纳米脂质体进行体外细胞转染研究，分别在HepG2(人类肝癌细胞系)和K562(人类永生化骨髓性白血病细胞系)中进行。转染前24小时，将HepG2和K562细胞以2–4x 104细胞/孔的浓度接种在96孔板中。在转染当天，将包裹了GFP DNA质粒的纳米脂质体以400ngDNA/孔的浓度添加到HepG2或者K562细胞培养基中。
转染48小时后，HepG2细胞用Hoechst 33342(1μg/ml)处理细胞核染色，并通过BioRad Zoe荧光细胞影像仪记录了绿色(细胞质内表达的绿色荧光蛋白GFP)和蓝色(细胞核)荧光图像。选定的绿场/蓝场叠加图像和明场图像如图9(上)所示。K562细胞的绿色荧光场和明场图像如图9(下)所示。实验结果表面，用本系统合成的包裹了DNA的纳米脂质体可以在体外成功地将DNA转染到细胞中并表达。
综上，本系统可以实现小体积(<0.2mL)及高通量(同时可以做两个或两个以上的样品)的纳米颗粒的合成，从而给用户节省了大量的原材料以及减少了实验的次数，适用于对纳米合成配方的甄选及优化的研究。同时本系统中微流控芯片可以兼容之前发明的低通量高产量基于流速控制的装置上使用，用户可以直接将在本装置上优化好的配方使用到流速控制的装置上并得到类似的纳米颗粒。
最后应说明的是：术语“上”、“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位 或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。此外，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性。
且以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。

Claims (10)

  1. 基于微流控技术的微流控芯片，包括芯片本体，所述芯片本体上设置有至少一条用于纳米颗粒合成的流道，所述流道的入口端及合成端均设置有储液池，所述储液池的中心轴与所述流道垂直。
  2. 如权利要求1所述的基于微流控技术的微流控芯片，其中，所述储液池与所述芯片本体一体化成型或可拆卸连接。
  3. 如权利要求2所述的基于微流控技术的微流控芯片，其中，所述储液池与所述微流控芯片之间通过相互配合的鲁尔接头形成可拆卸连接。
  4. 基于微流控技术的高通量纳米颗粒合成系统，其中，包括压力控制器，及与所述压力控制器输出端连接的压力分配组件、所述压力分配组件的压力输出端与权利要求1-3中任意一种微流控芯片连接，所述微流控芯片设置有至少一个。
  5. 如权利要求4所述的基于微流控技术的高通量纳米颗粒合成系统，其中，所述压力分配组件与所述微流控芯片之间设置有密封组件，以形成压力分配组件与微流控芯片之间的气密性连接。
  6. 如权利要求5所述的基于微流控技术的高通量纳米颗粒合成系统，其中，所述压力分配组件包括一压力分配板，所述密封组件为密封垫，所述密封垫上设置有开口且至少覆盖置于所述储液池的顶端开口处确保压力分配组件与微流控芯片之间的气密性连接。
  7. 如权利要求5所述的基于微流控技术的高通量纳米颗粒合成系统，其中，所述密封垫选择性的与所述压力分配板连接或与储液池端口连接。
  8. 如权利要求4所述的基于微流控技术的高通量纳米颗粒合成系统，其中，所述储液池的体积为20μl-1ml。
  9. 如权利要求4所述的基于微流控技术的高通量纳米颗粒合成系统，其中，所述压力控制器为多通道压力控制器，所述通道数量大于等于2。
  10. 如权利要求4所述的基于微流控技术的高通量纳米颗粒合成系统，其中，所述压力控制器与所述压力分配组件之间设置有控制阀。
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