WO2020093322A1 - 一种微流控芯片及其制备方法和dna合成方法 - Google Patents

一种微流控芯片及其制备方法和dna合成方法 Download PDF

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WO2020093322A1
WO2020093322A1 PCT/CN2018/114614 CN2018114614W WO2020093322A1 WO 2020093322 A1 WO2020093322 A1 WO 2020093322A1 CN 2018114614 W CN2018114614 W CN 2018114614W WO 2020093322 A1 WO2020093322 A1 WO 2020093322A1
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flow channel
chip
reaction chamber
input
output
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PCT/CN2018/114614
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English (en)
French (fr)
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郭洁
孙宝策
冯利鹤
王勇
黄小罗
沈玥
倪鸣
徐讯
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深圳华大生命科学研究院
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Priority to PCT/CN2018/114614 priority Critical patent/WO2020093322A1/zh
Priority to CN201880099266.2A priority patent/CN112969527A/zh
Publication of WO2020093322A1 publication Critical patent/WO2020093322A1/zh

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    • 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

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  • the present application relates to DNA synthesis technology, in particular to a microfluidic chip, its preparation method and DNA synthesis method.
  • DNA is the basic genetic material in life. Artificial DNA synthesis in vitro can replicate any naturally occurring DNA function or create new DNA functions according to the needs of research and application. With the development of genomics, molecular biology, systems biology and synthetic biology, synthetic DNA has wide application value in the fields of cell engineering, gene editing, disease diagnosis and treatment, new material development and other fields. Since Todd, Khorana and his collaborators first reported DNA synthesis in the 1950s, their synthesis methods have undergone long-term development. The current classic methods include the column synthesis developed in the 1980s and 90s High-throughput synthesis based on microarrays developed in the 1980s.
  • a column synthesizer such as Dr.oligo192, controls the addition of reagents through a solenoid valve, and performs a solid-phase synthesis reaction on a porous reaction column with a size of centimeters.
  • the reaction error rate is low, but the synthesis flux is not high and the Need more raw materials.
  • Microarray synthesizers such as the CustomArray synthesizer, reduce the synthesis reaction to micron-level reaction wells. There are tens of thousands of reaction holes in the synthesis tank of a chip, which not only improves the synthesis flux but also reduces the consumption of raw materials. However, the response is not easy to control and the error rate is high. Further development of DNA synthesis requires more efficient engineering techniques to achieve.
  • Microfluidic technology is a technology that can accurately operate and control microscale fluids. Due to its advantages of precise controllability, high throughput and low cost, microfluidic technology has been widely used in various fields of life sciences. Some of the existing DNA synthesis methods have begun to try to use some microfluidic technologies, such as the etching technology of electrochemical synthesis chips and the process-controlled mask illumination technology in photochemical synthesis, but using microfluidic chips to synthesize DNA The methods and applications are relatively few. As the most basic link in molecular biology and synthetic biology, DNA synthesis puts forward higher requirements for scale and cost. It can be found from theory and previous practice that microfluidic technology has great potential and wide application prospects in the field of DNA synthesis.
  • a column synthesizer such as Dr.oligo 192, controls the addition of reagents through a solenoid valve, and performs a solid-phase synthesis reaction on a porous reaction column with a size of centimeters.
  • the reaction has a high accuracy, but the synthesis flux is not high and More raw materials are required.
  • Microarray synthesizers such as the CustomArray synthesizer, reduce the synthesis reaction to micron-level reaction holes. There are tens of thousands of reaction holes in the synthesis pool of a chip, which not only improves the synthesis flux but also reduces the consumption of raw materials. However, the response is not easy to control and the accuracy is not high.
  • a microfluidic chip which includes a chip body.
  • the chip body is provided with an input flow channel, an output flow channel, and a reaction chamber.
  • One end of the input flow channel extends out of the chip body, and the other end extends to the reaction chamber.
  • the inlet of the chamber is connected, one end of the output flow channel extends out of the sheet body, and the other end extends to connect with the outlet of the reaction chamber.
  • the inlet of the reaction chamber is provided with a first fence-type sieve valve, and the outlet of the reaction chamber is provided with a second
  • the caliber of the first fence-type screen valve is larger than the size of the solid phase carrier
  • the caliber of the second fence-type screen valve is smaller than the size of the solid phase carrier.
  • An embodiment provides a method for manufacturing a microfluidic chip, which is characterized by the following steps:
  • a lower chip with a second input flow channel, a second output flow channel, a reaction chamber, a first fence-type sieve valve and a second fence-type sieve valve is prepared, wherein the second input flow channel passes through the first fence-type sieve valve and reacts The inlet of the chamber is connected, and the second output flow channel is connected to the outlet of the reaction chamber through the second fence-type sieve valve.
  • Solid carrier size
  • the upper chip and the lower chip are combined into a microfluidic chip, wherein one end of the first input flow channel extends to butt with the second input flow channel, the other end extends to the upper surface of the upper chip, and one end of the first output flow channel extends to The second output flow channel is docked, and the other end extends to the upper surface of the upper chip.
  • An embodiment provides a DNA synthesis method, which includes the following steps:
  • the solid phase carrier in the reaction chamber is output from the output channel to the outside of the chip and collected;
  • the solid phase carrier collected for output is subjected to ammonolysis, purification and gene assembly to prepare target DNA or RNA.
  • the first fence-type sieve valve The diameter of the solid phase carrier is larger than the size of the solid phase carrier.
  • the first fence-type sieve valve is smaller than the size of the solid phase carrier. It can effectively control the solid phase carrier after entering the reaction chamber and is limited to the reaction chamber. The accuracy is correct, and a large amount of solid-phase carriers can be accommodated in the reaction chamber for the synthesis reaction, so that the synthesis flux is high.
  • FIG. 1 is a schematic structural diagram of a microfluidic chip in an embodiment
  • FIG. 2 is a schematic structural diagram of a reaction chamber in an embodiment
  • FIG. 3 is a flowchart of a method for preparing a microfluidic chip in an embodiment
  • FIG. 4 is a flowchart of a method for preparing an upper chip in an embodiment
  • FIG. 5 is a flowchart of a method for preparing a lower chip in an embodiment
  • FIG. 6 is a schematic diagram of a method for preparing a lower chip in an embodiment
  • FIG. 7 is a schematic diagram of a reaction chamber template in an embodiment
  • 9 is a detection result of a fluorescent monomer for DNA synthesis in an embodiment.
  • a microfluidic chip is provided.
  • the microfluidic chip can reduce the material cost through micron-scale reaction control, and can also use the solid-phase synthesis method to ensure the accuracy of the synthesized product.
  • the microfluidic chip also has the characteristics of expansion and easy integration, and the flux can be flexibly adjusted according to needs.
  • this embodiment provides a microfluidic chip.
  • the microfluidic chip includes a chip body.
  • the chip body 10 includes an upper chip 11 and a lower chip 12, and the upper chip 11 and the lower chip 12 are glass and silicon chips. Or polydimethylsiloxane and other materials, the upper chip 11 and the lower chip 12 are bonded and compounded together.
  • the upper chip 11 and the lower chip 12 may be an integrated structure.
  • the lower surface of the upper chip 11 is provided with a first input channel 21 and a first output channel 31, and the upper surface of the lower chip 12 is provided with a second input channel 22, a second output channel 32 and a reaction
  • the chamber 40, the reaction chamber 40 has an inlet and an outlet, the inlet of the reaction chamber 40 is provided with a first fence-type sieve valve 51, the outlet of the reaction chamber 40 is provided with a second fence-type sieve valve 52, and the second input flow path 22 is connected to the inlet of the reaction chamber 40 through the first barrier-type screen valve 51, and the second outflow channel 32 is connected to the outlet of the reaction chamber 40 through the second barrier-type screen valve 52.
  • the first input flow path 21 and the first output flow path 31 on the upper chip 11 and the second input flow path 22, the second output flow path 32, the reaction chamber 40, and the first barrier-type sieve valve 51 on the lower chip 12 And the second barrier-type screen valve 52 are open slots, the first input flow channel 21 and the first output flow channel 31 and the lower chip 12 form a complete flow channel, the second input flow channel 22, the second output flow
  • the channel 32, the reaction chamber 40, the first barrier-type sieve valve 51 and the second barrier-type sieve valve 52 and the upper chip 11 form a complete flow channel, cavity and valve.
  • first input flow channel 21 extends to butt with the second input flow channel 22, and the other end of the first input flow channel 21 has a guide extending out of the upper surface of the upper chip 11
  • the first input flow channel 21 and the second input flow channel 22 are connected to form an input flow channel 20.
  • the first input flow path 21 serves as a control flow path, and a valve is installed on the first input flow path 21, and the first input flow path 21 is thinner than the second input flow path 22.
  • first output flow channel 31 extends to butt with the second output flow channel 32, the other end of the first output flow channel 31 has a guide hole extending out of the upper surface of the upper chip 11, the first output flow channel 31 and The second output channel 32 is connected to the output channel 30.
  • the first output flow path 31 serves as a control flow path, and a valve is installed on the first output flow path 31.
  • the first output flow path 31 is thinner than the second output flow path 32.
  • the first input flow channel 21 and the first output flow channel 31 are provided with valves, which are used to precisely control the import and export and improve the efficiency of synthesis.
  • the input flow channel 20 and the output flow channel 30 can be set into several, and the reaction chamber 40 can be set into several, several input flow channels 20 are collected into one flow channel, and then branched out into several flow channels respectively It is docked with the inlets of several reaction chambers 40, that is, each input flow channel 20 is docked with all reaction chambers 40. Similarly, a plurality of output flow channels 30 are collected into one flow channel, and then a plurality of flow channels are branched out to connect with the outlets of the reaction chambers 40, respectively. In other embodiments, several input flow channels 20 and several output flow channels 30 can also be provided separately, and finally each input flow channel 20 and output flow channel 30 are divided into several flow channels and connected to the reaction chamber 40.
  • a plurality of second input flow channels 22 and a plurality of second output flow channels 23 are collected together and then branched and connected to the reaction chamber 40, while a plurality of first input flow channels 21 and a plurality of first output
  • the flow channels 31 are independent of each other.
  • the first input flow channel 21 corresponds to the second input flow channel 22 one-to-one
  • the first output flow channel 31 corresponds to the second output flow channel 32 one-to-one.
  • the number of input channels 20 and output channels 30 and the number of reaction chambers 40 can be set according to specific requirements, for example, single base synthesis requires four single base monomers and at least four reagents required for chemical synthesis reactions , A total of at least 8 input channels 20 are required.
  • the number of the second output flow channels 32 corresponds to the number of the reaction chambers 40 one by one.
  • reaction chambers 40 there are two reaction chambers 40, preferably a pie-shaped structure, and the reaction chamber 40 may also be a square-shaped structure.
  • the two reaction chambers 40 are arranged side by side.
  • the reaction chamber 40 has one inlet and three outlets. The three outlets are respectively provided with a second barrier-type sieve valve 52.
  • the output flow channel 30 branches out of the flow channel and the reaction chamber 40 respectively. Exit connection.
  • the first fence-type screen valve 51 and the second fence-type screen valve 52 are composed of several long thin pipes arranged side by side.
  • the caliber of the first fence-type sieve valve 51 is slightly larger than the size of the solid phase carrier used for gene synthesis
  • the caliber of the second fence-type sieve valve 52 is slightly smaller than the size of the solid phase carrier, so that the solid phase carrier can be input into the reaction chamber 40 Inside, it is restricted to the inside of the reaction chamber 40 by the second barrier screen valve 52.
  • the specific sizes of the first fence-type sieve valve 51 and the second fence-type sieve valve 52 can be designed according to the size of the actual solid phase carrier, for example, the diameter of the second fence-type sieve valve 52 is designed to be 4 microns, 5 microns, 6 microns, etc. Either way, the height of the pipe section of the second fence-type sieve valve 52 is 20 microns, and the width is 5 microns, which can effectively block the solid phase carrier.
  • the microfluidic chip of this embodiment is also provided with an auxiliary flow channel 60, which is provided on the lower surface of the upper chip 11 and is also an open groove.
  • the auxiliary flow channel 60 and the lower chip 12 form a complete flow channel structure
  • There is one auxiliary flow channel 60 the auxiliary flow channel 60 extends across the outlet of the reaction chamber 40, the auxiliary flow channel 60 is connected to the six outlets of the two reaction chambers 40, one end of the auxiliary flow channel 60 has It extends to the upper surface guide hole of the upper chip 11.
  • the auxiliary flow channel 60 can not only be used to add reactants, but also facilitate the collection and backwashing of substances in the reaction chamber 40, and can be used to check the role of faults during the operation of the chip.
  • the microfluidic chip of this embodiment is used to connect with an automatic control device, and the automatic control device is used to control the import and export of reactants and reagents.
  • the microfluidic chip provided in this embodiment has a first fence-type sieve valve 51 and a second fence-type sieve valve 52 at the inlet and the outlet of the reaction chamber 40, respectively.
  • the size of the phase carrier, the first barrier-type sieve valve 52 is smaller than the size of the solid phase carrier, which can effectively control the solid phase carrier into the reaction chamber 40 after being limited to the reaction chamber 40 for synthesis-based reaction, which improves the correctness of the reaction
  • the reaction chamber 40 can accommodate a large amount of solid-phase carriers for the synthesis reaction, so that the synthesis flux is high.
  • multiple input and output pipes and multiple reaction chambers 40 are provided to further improve the synthesis flux and efficiency.
  • This embodiment provides a method for preparing a microfluidic chip, which is used to prepare the microfluidic chip of the foregoing embodiment.
  • This preparation method mainly prepares the upper chip and the lower chip by photolithography, dry etching or reverse mold process.
  • the upper chip and the lower chip can be prepared by combining photolithography and reverse mold, or dry etching and reverse mold Combined preparation of molds, or a combination of photolithography, dry etching and inverted molds.
  • the combination of photolithography, dry etching, and mold inversion is used as an example for description.
  • the preparation method of the microfluidic chip of this embodiment mainly includes the following steps:
  • the preparation of the upper chip mainly includes the following sub-steps:
  • the substrate is placed on the platform, and a layer of photoresist is coated on the upper surface of the substrate, and a mask with a first input flow channel, a first output flow channel, and an auxiliary flow channel pattern is attached to the photoresist membrane.
  • UV ultraviolet
  • the part of the photoresist that is not blocked by the mask will undergo a chain reaction.
  • the part of the photoresist after the reaction is dissolved in the dissolving agent and blocked.
  • the part is insoluble in the dissolving agent.
  • the irradiated part of the photoresist is dissolved away by the dissolving agent, and only the part of the photoresist that is blocked is left.
  • This part is a pattern of the first input flow channel, the first output flow channel, and the auxiliary flow channel.
  • Ion etching technology will be used to etch the substrate into the same pattern as the photoresist to prepare an upper chip template with the opposite structure of the upper chip.
  • the upper chip is inverted and molded according to the upper chip template by using the inverted mold technology, and then punched to prepare the final upper chip.
  • the preparation of the chip mainly includes the following sub-steps:
  • the substrate 2 is placed on the platform 1, and a layer of photoresist 3 is coated on the upper surface of the substrate 2, and a mask 4 with a reaction chamber pattern is attached to the photoresist 3.
  • UV ultraviolet
  • the irradiated part of the photoresist 3 is dissolved by a dissolving agent, and only the part of the photoresist 3 that is blocked is left, and this part is the pattern of the reaction chamber.
  • the substrate 2 will be etched into the same pattern as the photoresist 3 using ion etching technology.
  • the photoresist 3 is coated on the platform 1 with the substrate 2, the photoresist 3 covers the substrate 2, and the second input flow path, the second output flow path are attached to the area outside the reaction chamber area, Mask 4 of the first fence-type screen valve and the second fence-type screen valve pattern.
  • the platform 1 with the mask 4 is vertically irradiated with UV (ultraviolet rays), and the irradiated photoresist 3 undergoes a chain reaction.
  • UV ultraviolet
  • the lower chip template corresponding to the lower chip is shown in FIG. 7.
  • the lower chip is inverted by the lower die template according to the lower die template.
  • the second input flow channel in the prepared lower chip is connected to the inlet of the reaction chamber through a first fence-type sieve valve, and the second output flow channel is connected to the outlet of the reaction chamber through a second fence-type sieve valve.
  • the first fence type The caliber of the sieve valve is slightly larger than the size of the solid phase carrier, and the caliber of the second fence-type sieve valve is slightly smaller than the size of the solid phase carrier.
  • the prepared upper chip and lower chip are bonded together to prepare a microfluidic chip with a flow channel and a reaction chamber, and a valve is installed on the first input flow channel, the first output flow channel and the auxiliary flow channel .
  • one end of the first input flow path extends to butt with the second input flow path, the other end extends to the upper surface of the upper chip, and one end of the first output flow path extends to the second output flow path
  • the channel is docked, and the other end extends to the upper surface of the upper chip.
  • One end of the auxiliary flow channel extends to butt with the outlet of the reaction chamber, and the other end extends to the upper surface of the upper chip.
  • the preparation method of the microfluidic chip provided by this embodiment adopts photolithography, dry etching and inverted mold processes, and the preparation precision is high and the efficiency is high.
  • This embodiment provides a gene synthesis method.
  • the synthesis method is based on the microfluidic chip of the above embodiment, and is suitable for DNA synthesis and RNA synthesis.
  • This example uses DNA synthesis as an example.
  • the DNA synthesis method of this embodiment includes the following steps:
  • a microfluidic chip with sufficient flow channels and reaction chambers.
  • single-base synthesis requires four single-base monomers and at least four reagents required for chemical synthesis reactions. At least 8 input flow channels are required; in order to meet the synthetic flux and output, several reaction chambers with different sizes and shapes can be designed.
  • the preparation method is as described in the above examples.
  • the fixed carrier is CPG with an amino-modified active functional group
  • the diameter of the fixed carrier is 5 ⁇ m, 25 ⁇ m, 50 ⁇ m, 100 ⁇ m, 200 ⁇ m, 500 ⁇ m Any of them.
  • the pore size of the fixed carrier is Any of them.
  • the linking molecules of the fixed carrier are ester group, lipid group, thioester group, o-nitrobenzyl group, coumarin group, hydroxyl group, mercapto group, mercapto ether group, carboxyl group, aldehyde group, amino group, amine group, amide group, alkene Any one or more functional groups in the group, alkynyl group.
  • the modified solid-phase carrier is input into the reaction chamber from the input flow channel of the microfluidic chip.
  • the solid-phase carrier After the solid-phase carrier is input into the reaction chamber, it is driven by positive or negative pressure and controlled by a valve, and a reagent is added from the input flow channel to the reaction chamber in which the solid-phase carrier is stored to perform a synthesis reaction.
  • the capping step in the experimental operation can be omitted. If a DNA sequence with a longer chain length is synthesized (cycle number> 25), the capping step To reduce the error rate to get enough target DNA.
  • the solid phase carrier in the reaction chamber is independently output from the output flow channel to the container outside the chip through positive pressure or negative pressure drive and valve control.
  • the solid phase carrier collected by the output is successively subjected to ammonolysis, purification and gene assembly to obtain the target DNA. As shown in FIG. 9, the detection result of the synthetic target DNA fluorescent monomer.
  • the reagent used for the ammonialysis is any one of ammonia water, ammonia gas and methylamine
  • the temperature of the ammonialysis is any one of 25 ° C, 60 ° C and 90 ° C
  • the time of the ammonialysis is 2h, 5h and 10h , 18h, 24h
  • the purification method is desalination, MOP, PAGE, PAGE Plus, HPLC.
  • the synthesis method of this embodiment can be accurately and automatically controlled by the controller.
  • the controller controls the drive and the valve to accurately input and output the reactants and reagents to achieve a high-throughput and high-accuracy synthesis reaction.
  • This embodiment provides a gene synthesis method, which adopts multi-channel, multi-reaction chamber and valve control, can reduce the material cost through micron-scale reaction control, and can also use the solid-phase synthesis method to ensure the correct rate of the synthesis product.
  • the chip used in this synthesis also has the characteristics of scalability and easy integration, and the flux can be flexibly adjusted according to the demand.
  • the microfluidic-based DNA synthesis method proposed by the present invention improves the synthesis efficiency, but also technically avoids the shortcomings of the current commercial synthesizers, and provides a new approach with high feasibility for the development of future synthesizers .

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Abstract

一种微流控芯片及制备方法和DNA合成方法，微流控芯片包括片体，片体内设有输入流道（21、22）、输出流道（31、32）和反应腔室（40），反应腔室（40）的入口设有第一栅栏式筛阀（51），反应腔室（40）的出口设有第二栅栏式筛阀（52），第一栅栏式筛阀（51）的口径大于固相载体的尺寸，第二栅栏式筛阀（52）的口径小于固相载体的尺寸。由于在反应腔室（40）的入口和出口分别设有第一栅栏式筛阀（51）和第二栅栏式筛阀（52），第一栅栏式筛阀（51）的口径大于固相载体的尺寸，第二栅栏式筛阀（52）的口径小于固相载体的尺寸，能够有效控制固相载体进入反应腔室（40）内部后被限制在反应腔室（40）内部进行基于合成反应，提高了反应的正确率，并且反应腔室（40）内可容纳大量的固相载体进行合成反应，从而合成通量高。

Description

一种微流控芯片及其制备方法和DNA合成方法 技术领域
本申请涉及DNA合成技术，具体涉及一种微流控芯片及其制备方法和DNA合成方法。
背景技术
DNA是生命体内的基础遗传物质。人工体外合成DNA能够根据研究和应用的需要，复制出任何天然存在的DNA功能或者创造出新的DNA功能。随着基因组学，分子生物学，系统生物学以及合成生物学的发展，人工合成的DNA在细胞工程改造，基因编辑，疾病诊断与治疗，新材料开发等领域都具有广泛的应用价值。自二十世纪五十年代Todd，Khorana及其合作者第一次报道了DNA合成以来，其合成方法经历了长期的发展，目前经典的方法包括八十年代发展起来的柱式合成，以及九十年代发展起来的基于微阵列的高通量合成。柱式合成仪，例如Dr.oligo192,是通过电磁阀控制试剂的添加，在尺寸为厘米级别的多孔反应柱上进行固相合成反应，该反应错误率较低，但是合成通量不高而且所需原料也较多。微阵列合成仪，例如CustomArray合成仪，是将合成反应缩小到微米级别的反应孔内，一张芯片的合成池上有上万个反应孔，这样既提高了合成通量也减少了原料的消耗，然而反应不易控制，错误率高。DNA合成进一步的发展需要更高效的工程技术手段去实现。从化学反应的角度考虑，为了提高反应效率，需要尽可能维持试剂的浓度在一定水平，并且反应过后尽快去除残留试剂；为了减少副产物和降低错误率，需要在保证反应充分的前提下，缩短反应的时间，为此DNA合成反应循环中的四步反应需要被尽可能精确地控制。从合成成本的角度出发，为了减少原料的消耗，需要在保证合理产出的情况下，严格控制原料的使用；为了在保证通量的情况下，缩短整个目标合成的时间，也需要优化设计合成的流程，比如优化组合单体和试剂添加的顺序，尽快得到目标产物。综合而言，目前尚未有一种能够在保证低错误率的情况下，低成本高通量地实现DNA合成的技术。如何通过高水平的技术手段实现高效低成本的DNA合成是急需解决的一个问题。
微流控技术是一种能够精确操作控制微尺度流体的技术。由于其具有精确可控性、高通量和低成本的优势，微流控技术已经广泛应用于生 命科学各个领域。一些现有的DNA合成方法中，已经开始尝试使用一些微流控技术，例如电化学合成芯片的刻蚀技术和光化学合成中的程序控制的掩模光照技术，但是利用微流控芯片来合成DNA的方法和应用相对较少。DNA合成作为分子生物学和合成生物学等领域最基础的一环，对于规模和成本提出了较高的要求。从理论上和以前的实践中可以发现，微流控技术在DNA合成领域具有极大的潜力和广泛的应用前景。
现有的单碱基DNA合成方法，包括多孔玻璃的化学合成、电化学合成以及光化学合成，都是基于亚磷酰胺化学合成四步方法，按照预定的序列，逐个增加DNA单体。基于单碱基DNA合成方法，一些商业化的合成仪已经进入市场。这些合成仪大体可以分为两种：柱式合成仪和微阵列合成仪。柱式合成仪，例如Dr.oligo 192,是通过电磁阀控制试剂的添加，在尺寸为厘米级别的多孔反应柱上进行固相合成反应，该反应正确率较高，但是合成通量不高而且所需原料也较多。微阵列合成仪，例如CustomArray合成仪,是将合成反应缩小到微米级别的反应孔内，一张芯片的合成池上有上万个反应孔，这样既提高了合成通量也减少了原料的消耗，然而反应不易控制，正确率不高。
发明内容
一种实施例中提供一种微流控芯片,包括片体，片体内设有输入流道、输出流道和反应腔室，输入流道的一端延伸出片体，另一端延伸至与反应腔室的入口连接，输出流道的一端延伸出片体，另一端延伸至与反应腔室的出口连接，反应腔室的入口设有第一栅栏式筛阀，反应腔室的出口设有第二栅栏式筛阀，第一栅栏式筛阀的口径大于固相载体的尺寸，第二栅栏式筛阀的口径小于固相载体的尺寸。
一种实施例中提供一种微流控芯片的制备方法，其特征在于，包括如下步骤：
制备具有第一输入流道和第一输出流道的上芯片；
制备具有第二输入流道、第二输出流道、反应腔室、第一栅栏式筛阀和第二栅栏式筛阀的下芯片，其中第二输入流道通过第一栅栏式筛阀与反应腔室的入口连接，第二输出流道通过第二栅栏式筛阀与反应腔室的出口连接，第一栅栏式筛阀的口径大于固相载体的尺寸，第二栅栏式筛阀的口径小于固相载体的尺寸；
将上芯片和下芯片复合成微流控芯片，其中第一输入流道的一端延 伸至与第二输入流道对接，另一端延伸至上芯片的上表面，第一输出流道的一端延伸至与第二输出流道对接，另一端延伸至上芯片的上表面。
一种实施例中提供一种DNA合成方法，其特征在于，包括如下步骤：
根据待合成的目标DNA或RNA序列，制备具有对应流道和反应腔室的微流控芯片；
根据待合成的目标DNA或RNA序列，设计修饰用于合成的固相载体；
将修饰后的固相载体从上述实施例微流控芯片的输入流道输入到反应腔室内；
再将试剂从微流控芯片的输入流道输入到反应腔室内进行合成反应；
合成反应完成后，将反应腔室内的固相载体从输出流道输出到芯片外并收集；
将输出收集的固相载体进行氨解、纯化及基因组装，制得目标DNA或RNA。
依据上述实施例的微流控芯片及其制备方法和DNA合成方法，由于在反应腔室的入口和出口分别设有第一栅栏式筛阀和第二栅栏式筛阀，第一栅栏式筛阀的口径大于固相载体的尺寸，第一栅栏式筛阀小于固相载体的尺寸，能够有效控制固相载体进入反应腔室内部后被限制在反应腔室内部进行基于合成反应，提高了反应的正确率，并且反应腔室内可容纳大量的固相载体进行合成反应，从而合成通量高。
附图说明
图1为一种实施例中微流控芯片的结构示意图；
图2为一种实施例中反应腔室的结构示意图；
图3为一种实施例中微流控芯片的制备方法的流程图；
图4为一种实施例中上芯片的制备方法的流程图；
图5为一种实施例中下芯片的制备方法的流程图；
图6为一种实施例中下芯片的制备方法的示意图；
图7为一种实施例中反应腔室模板的示意图；
图8为一种实施例中DNA合成方法的流程图；
图9为一种实施例中DNA合成荧光单体检测结果。
具体实施方式
在本发明实施例中提供了一种微流控芯片，本微流控芯片可以通过微米尺度的反应控制降低物料成本，也可以利用固相合成的方法确保合成产品的正确率。另外，本微流控芯片也具有扩展和易集成的特性，可根据需求灵活调节通量。
实施例1：
如图1所示，本实施例提供了一种微流控芯片，微流控芯片包括片体，片体10包括上芯片11和下芯片12，上芯片11和下芯片12为玻璃、硅片或聚二甲基硅氧烷等材质，上芯片11和下芯片12粘接复合在一起。在其他实施例种，上芯片11和下芯片12可为一体式结构。
本实施例中，上芯片11的下表面设有第一输入流道21和第一输出流道31，下芯片12的上表面设有第二输入流道22、第二输出流道32和反应腔室40，反应腔室40具有入口和出口，反应腔室40的入口设有第一栅栏式筛阀51，反应腔室40的出口设有第二栅栏式筛阀52，第二输入流道22通过第一栅栏式筛阀51与反应腔室40的入口连接，第二流出流道32通过第二栅栏式筛阀52与反应腔室40的出口连接。
上芯片11上的第一输入流道21和第一输出流道31及下芯片12上的第二输入流道22、第二输出流道32、反应腔室40、第一栅栏式筛阀51和第二栅栏式筛阀52均是开放式的槽，第一输入流道21和第一输出流道31与下芯片12围合成完整的流道，第二输入流道22、第二输出流道32、反应腔室40、第一栅栏式筛阀51和第二栅栏式筛阀52与上芯片11围合成完整的流道、腔体和阀门。
上芯片11和下芯片12复合在一起后，第一输入流道21的一端延伸至与第二输入流道22对接，第一输入流道21的另一端具有延伸出上芯片11上表面的导孔，第一输入流道21和第二输入流道22对接成输入流道20。第一输入流道21作为控制流道，在第一输入流道21上安装有阀门，第一输入流道21比第二输入流道22细。
同样的，第一输出流道31的一端延伸至与第二输出流道32对接，第一输出流道31的另一端具有延伸出上芯片11上表面的导孔，第一输出流道31和第二输出流道32对接成输出流道30。第一输出流道31作为控制流道，在第一输出流道31上安装有阀门，第一输出流道31比第二输出流道32细。第一输入流道21和第一输出流道31设有阀门，阀门 用于精确控制导入和导出，提高合成的效率。
本实施例中，输入流道20和输出流道30可设置成若干条，反应腔室40可设置成若干个，若干条输入流道20汇集到一条流道中，再分支出若干个流道分别与若干个反应腔室40的入口对接，即每条输入流道20都与所有的反应腔室40对接。同样的，若干个输出流道30汇集到一条流道中，再分支出若干个流道分别与若干个反应腔室40的出口对接。在其他实施例中，若干个输入流道20和若干个输出流道30也可分别单独设置，最终每个输入流道20和输出流道30分出若干个流道与反应腔室40连接。
本实施例中，若干个第二输入流道22和若干个第二输出流道23分别汇集在一起再分支与反应腔室40连接，而若干个第一输入流道21和若干个第一输出流道31相互之间独立，第一输入流道21与第二输入流道22一一对应，第一输出流道31与第二输出流道32一一对应。
输入流道20和输出流道30的条数和反应腔室40的个数可根据具体需求设置，例如单碱基的合成需要单碱基单体四种和化学合成反应所需试剂至少四种，共至少需要8条输入流道20。第二输出流道32的条数与反应腔室40的个数一一对应。
如图1和图2所示，本实施例中，反应腔室40具有两个，优选为圆饼状结构，反应腔室40也可为方形状结构。两个反应腔室40并排设置，反应腔室40具有一个入口和三个出口，三个出口分别设有第二栅栏式筛阀52，输出流道30分支出流道分别与反应腔室40的出口连接。
第一栅栏式筛阀51和第二栅栏式筛阀52由并排的若干个长条细管道组成。第一栅栏式筛阀51的口径略大于用于合成基因的固相载体的尺寸，第二栅栏式筛阀52的口径略小于固相载体的尺寸，使得固相载体能够输入到反应腔室40内部，又被第二栅栏式筛阀52限制在反应腔室40内部。第一栅栏式筛阀51和第二栅栏式筛阀52的具体尺寸可根据实际固相载体的尺寸设计，例如设计第二栅栏式筛阀52的直径长度为4微米、5微米、6微米等均可，第二栅栏式筛阀52的管道截面的高度为20微米，宽度为5微米，可有效隔档固相载体。
本实施例的微流控芯片还设有辅助流道60，辅助流道60设置在上芯片11的下表面，同样为开放式槽，辅助流道60与下芯片12围合成完整的流道结构，辅助流道60具有一条，辅助流道60的延伸横跨在反应腔室40的出口处，辅助流道60与两个反应腔室40的六个出口均连接， 辅助流道60的一端具有延伸至与上芯片11的上表面导孔。辅助流道60不仅能用于添加反应物，还能够更方便的进行反应腔室40内物质的收集和进行反向冲洗，并且在芯片工作过程中可用于检验故障作用。
本实施例的微流控芯片用于与自动控制设备连接，自动控制设备用于控制反应物和试剂的导入和导出。
本实施例提供的微流控芯片，由于在反应腔室40的入口和出口分别设有第一栅栏式筛阀51和第二栅栏式筛阀52，第一栅栏式筛阀51的口径大于固相载体的尺寸，第一栅栏式筛阀52小于固相载体的尺寸，能够有效控制固相载体进入反应腔室40内部后被限制在反应腔室40内部进行基于合成反应，提高了反应的正确率，并且反应腔室40内可容纳大量的固相载体进行合成反应，从而合成通量高。并且，设有多条输入和输出的管道，及多个反应腔室40，进一步提高了合成的通量和效率。
实施例2：
本实施例提供了一种微流控芯片的制备方法，用于制备上述实施例的微流控芯片。本制备方法主要通过光刻、干刻或倒模工艺制备上芯片和下芯片，在将上芯片和下芯片复合在一起，可采用光刻和倒模的结合制备，也可以采用干刻和倒模的结合制备，或者采用光刻、干刻和倒模的结合制备。本实施例以光刻、干刻和倒模的结合为例进行说明。
如图3所示，本实施例的微流控芯片的制备方法主要包括如下步骤：
S100：制备上芯片；
如图4所示，制备上芯片主要包括如下子步骤：
S101：初始准备；
初始准备将基板置于平台上，并在基板的上表面涂覆一层光刻胶，并在光刻胶上贴附具有第一输入流道、第一输出流道和辅助流道图案的掩膜。
S102：UV光照；
采用UV(紫外线)垂直照射在贴附有掩膜的光刻胶上，光刻胶上没有被掩膜遮挡的部分将进行链式反应，光刻胶反应后的部分溶于溶解剂，被遮挡的部分不溶于溶解剂。
S103：光刻胶剥离；
通过溶解剂将光刻胶被光照的部分溶解掉，光刻胶仅剩下被遮挡的部分，该部分为第一输入流道、第一输出流道和辅助流道的图形。
S104：反应离子刻蚀；
将采用离子刻蚀技术将基板刻蚀成与光刻胶一样的图形，制备成与上芯片结构相反的上芯片模板。
S105：倒模制备；
采用倒模技术根据上芯片模板倒模出上芯片，再对其打孔，制备成最终的上芯片。
S200：制备下芯片；
如图5和图6所示，制备下芯片主要包括如下子步骤：
S201：初始准备；
初始准备将基板2置于平台1上，并在基板2的上表面涂覆一层光刻胶3，并在光刻胶3上贴附具有反应腔室图案的掩膜4。
S202：第一次UV光照；
采用UV(紫外线)垂直照射在贴附有掩膜4的光刻胶3上，光刻胶3上没有被掩膜4遮挡的部分将进行链式反应，光刻胶3反应后的部分溶于溶解剂，被遮挡的部分不溶于溶解剂。
S203：第一次光刻胶剥离；
通过溶解剂将光刻胶3被光照的部分溶解掉，光刻胶3仅剩下被遮挡的部分，该部分为反应腔室的图形。
S204：反应离子刻蚀；
将采用离子刻蚀技术将基板2刻蚀成与光刻胶3一样的图形。
S205：再次涂覆光刻胶；
在光刻胶3涂覆在具有基板2的平台1上，光刻胶3覆盖住基板2，并在反应腔室区域之外的区域贴附具有第二输入流道、第二输出流道、第一栅栏式筛阀和第二栅栏式筛阀图案的掩膜4。
S206：第二次UV光照；
采用UV(紫外线)垂直照射具有掩膜4的平台1，被照射的光刻胶3进行链式反应。
S207：第二次光刻胶剥离；
采用溶解剂溶解被曝光的光刻胶3，光刻胶3剩下与第二输入流道、第二输出流道、第一栅栏式筛阀和第二栅栏式筛阀对应的图形，制备成与下芯片对应的下芯片模板，如图7所示。
S208：倒模制备。
采用倒模技术根据下芯片模板倒模出下芯片。制备后的下芯片中第二输入流道通过第一栅栏式筛阀与反应腔室的入口连接，第二输出 流道通过第二栅栏式筛阀与反应腔室的出口连接，第一栅栏式筛阀的口径略大于固相载体的尺寸，第二栅栏式筛阀的口径略小于固相载体的尺寸。
S300：将上芯片和下芯片复合在一起。
将制备好的上芯片和下芯片粘合在一起，制备成具有流道和反应腔室的微流控芯片，并且第一输入流道、第一输出流道和辅助流道均上安装有阀门。
具体的，制备的微流控芯片中第一输入流道的一端延伸至与第二输入流道对接，另一端延伸至上芯片的上表面，第一输出流道的一端延伸至与第二输出流道对接，另一端延伸至上芯片的上表面。辅助流道的一端延伸至与反应腔室的出口对接，另一端延伸至与上芯片的上表面。
本实施例提供的微流控芯片的制备方法，采用光刻、干刻和倒模工艺制备，制备的精度高、效率高。
实施例3：
本实施例提供了一种基因的合成方法，本合成方法基于上述实施例的微流控芯片，并且适用于DNA的合成和RNA的合成。本实施例以DNA的合成为例进行说明。
如图8所示，本实施例的DNA的合成方法包括如下步骤：
S10：制备微流控芯片；
根据待合成的目标DNA，设计和制备具有足够流道和反应腔室内的微流控芯片，例如单碱基合成需要单碱基单体四种，和化学合成反应所需试剂至少四种，因而需要至少8条输入流道；为了满足合成的通量和产量，可设计尺寸形状不一的反应腔室若干个。制备的方法如上述实施例所述。
S20：设计固相载体；
再根据待合成的目标DNA或RNA序列，设计修饰用于合成的固相载体；固定载体为带有氨基修饰的活性官能团的CPG，固定载体的直径为5μm，25μm，50μm，100μm，200μm，500μm中的任意一种。固定载体的孔径为
Figure PCTCN2018114614-appb-000001
中的任意一种。固定载体的连接分子是酯基、脂基、硫酯基、邻硝基苄基、香豆素基团、羟基、巯基、巯醚基、羧基、醛基、氨基、胺基、酰胺基、烯基、炔基中任意一种或多种官能团的化合物。
S30：输入固相载体；
将修饰后的固相载体从微流控芯片的输入流道输入到反应腔室内。
S40：输入试剂；
固相载体输入到反应腔室后，再通过正压或负压驱动和阀门控制， 从输入流道往存储有固相载体的反应腔室内添加试剂进行合成反应。
合成反应的步骤、所用试剂、溶剂及时间如下表所示：
表1、合成反应步骤流程
Figure PCTCN2018114614-appb-000002
其中，如果待合成的DNA序列的链长较短(循环数≤25)，实验操作中的盖帽步骤可以省略，如若要合成更长链长的DNA序列(循环数>25)，则需要盖帽步骤以降低错误率得到足够多的目标DNA。
S50：输出固相载体；
待合成反应完成后，通过正压或负压驱动和阀门控制，将反应腔室内的固相载体独立从输出流道输出到芯片外的容器中。
S60：氨解、纯化及基因组装。
将输出收集的固相载体先后进行氨解、纯化及基因组装，制得目标DNA。如图9所示，合成目标DNA荧光单体的检测结果。
其中，氨解采用的试剂为氨水、氨气、甲胺中的任意一种，氨解的温度为25℃、60℃、90℃中的任意一种，氨解的时间为2h、5h、10h、18h、24h中的任意一种，纯化的方式为脱盐、MOP、PAGE、PAGE Plus、HPLC中的任意一种。
本实施例的合成方法可通过控制器精确自动化控制，控制器控制驱动和阀门精确输入和输出反应物和试剂，以实现高通量高正确率的合成 反应。
本实施例提供了一种基因的合成方法，采用多通道、多反应腔室及阀门控制，可以通过微米尺度的反应控制降低物料成本，也可以利用固相合成的方法确保合成产物的正确率。另外，本合成所用的芯片也具有可扩展、易集成的特性，可根据需求灵活调节通量。本发明提出的基于微流控的DNA合成方法在提高合成效率的同时，也从技术上避免了目前商业化合成仪的缺点，为未来合成仪的开发提供了一条具有较高可行性的全新途径。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本领域的一般技术人员，依据本发明的思想，可以对上述具体实施方式进行变化。

Claims (26)

  1. 一种微流控芯片,其特征在于，包括片体，所述片体内设有输入流道、输出流道和反应腔室，所述输入流道的一端延伸出所述片体，另一端延伸至与所述反应腔室的入口连接，所述输出流道的一端延伸出所述片体，另一端延伸至与所述反应腔室的出口连接，所述反应腔室的入口设有第一栅栏式筛阀，所述反应腔室的出口设有第二栅栏式筛阀，所述第一栅栏式筛阀的口径大于固相载体的尺寸，所述第二栅栏式筛阀的口径小于固相载体的尺寸。
  2. 权利要求1所述的微流控芯片，其特征在于，所述片体包括复合在一起的上芯片和下芯片，所述输入流道包括第一输入流道和第二输入流道，所述输出流道包括第一输出流道和第二输出流道，所述第一输入流道和第一输出流道位于上芯片的下表面，所述第二输入流道、第二输出流道、反应腔室、第一栅栏式筛阀和第二栅栏式筛阀位于下芯片的上表面，所述第一输入流道的一端延伸至与第二输入流道对接，另一端延伸至所述上芯片的上表面，所述第一输出流道的一端延伸至与所述第二输出流道对接，另一端延伸至所述上芯片的上表面。
  3. 权利要求2所述的微流控芯片，其特征在于，所述上芯片的下表面还设有辅助流道，所述辅助流道的一端延伸至与所述反应腔室的出口对接，另一端延伸至与所述上芯片的上表面。
  4. 权利要求3所述的微流控芯片，其特征在于，所述第一输入流道、第一输出流道和辅助流道均上安装有阀门。
  5. 权利要求3所述的微流控芯片，其特征在于，所述第一输入流道、第一输出流道和辅助流道细于所述第二输入流道和第二输出流道。
  6. 权利要求2所述的微流控芯片，其特征在于，所述反应腔室为圆饼状或方块状，所述反应腔室具有一个入口和三个出口，所述第二输出流道延伸至与所述反应腔室的三个出口连接。
  7. 如权利要求2所述的微流控芯片，其特征在于，所述输入流道和输出流道具有若干条，所述反应腔室具有若干个，每条所述输入流道和输出流道分别延伸至所有的反应腔室连接。
  8. 如权利要求2所述的微流控芯片，其特征在于，所述上芯片和下芯片为玻璃、硅片或聚二甲基硅氧烷材质。
  9. 一种微流控芯片的制备方法，其特征在于，包括如下步骤：
    制备具有第一输入流道和第一输出流道的上芯片；
    制备具有第二输入流道、第二输出流道、反应腔室、第一栅栏式筛阀和第二栅栏式筛阀的下芯片，其中所述第二输入流道通过所述第一栅栏式筛阀与所述反应腔室的入口连接，所述第二输出流道通过所述第二栅栏式筛阀与所述反应腔室的出口连接，所述第一栅栏式筛阀的口径大于固相载体的尺寸，所述第二栅栏式筛阀的口径小于固相载体的尺寸；
    将上芯片和下芯片复合成微流控芯片，其中所述第一输入流道的一端延伸至与第二输入流道对接，另一端延伸至所述上芯片的上表面，所述第一输出流道的一端延伸至与所述第二输出流道对接，另一端延伸至所述上芯片的上表面。
  10. 如权利要求9所述的制备方法，其特征在于，所述上芯片制备有辅助流道，所述辅助流道的一端延伸至与所述反应腔室的出口对接，另一端延伸至与所述上芯片的上表面。
  11. 如权利要求10所述的制备方法，其特征在于，所述第一输入流道、第一输出流道和辅助流道均上安装有阀门。
  12. 如权利要求9所述的制备方法，其特征在于，所述上芯片和下芯片通过光刻、干刻和倒模工艺中的一种或几种组合制得。
  13. 一种DNA合成方法，其特征在于，包括如下步骤：
    根据待合成的目标DNA或RNA序列，制备具有对应流道和反应腔室的微流控芯片；
    再根据待合成的目标DNA或RNA序列，设计修饰用于合成的固相载体；
    将修饰后的固相载体从如权利要求1至8任一项所述的微流控芯片的输入流道输入到反应腔室内；
    再将试剂从微流控芯片的输入流道输入到反应腔室内进行合成反应；
    合成反应完成后，将反应腔室内的固相载体从输出流道输出到芯片外并收集；
    将输出收集的固相载体进行氨解、纯化及基因组装，制得目标DNA或RNA。
  14. 如权利要求13所述的DNA合成方法，其特征在于，所述固定载体为带有氨基修饰的活性官能团的CPG。
  15. 如权利要求14所述的DNA合成方法，其特征在于，所述固定载体的直径为5μm，25μm，50μm，100μm，200μm，500μm中的任 意一种。
  16. 如权利要求14所述的DNA合成方法，其特征在于，所述固定载体的孔径为
    Figure PCTCN2018114614-appb-100001
    中的任意一种。
  17. 如权利要求14所述的DNA合成方法，其特征在于，所述固定载体的连接分子是酯基、脂基、硫酯基、邻硝基苄基、香豆素基团、羟基、巯基、巯醚基、羧基、醛基、氨基、胺基、酰胺基、烯基、炔基中任意一种或多种官能团的化合物。
  18. 如权利要求13所述的DNA合成方法，其特征在于，所述固相载体和试剂通过正压或负压驱动输入到芯片内和从芯片内输出。
  19. 如权利要求13所述的DNA合成方法，其特征在于，所述氨解采用的试剂为氨水、氨气、甲胺中的任意一种。
  20. 如权利要求13所述的DNA合成方法，其特征在于，所述氨解的温度为25℃、60℃、90℃中的任意一种。
  21. 如权利要求13所述的DNA合成方法，其特征在于，所述氨解的时间为2h、5h、10h、18h、24h中的任意一种。
  22. 如权利要求13所述的DNA合成方法，其特征在于，所述纯化的方式为脱盐、MOP、PAGE、PAGE Plus、HPLC中的任意一种。
  23. 如权利要求13所述的DNA合成方法，其特征在于，所述合成反应依次包括脱保护、清洗、偶联、清洗、氧化和清洗工艺。
  24. 如权利要求23所述的DNA合成方法，其特征在于，所述清洗和氧化工艺之间还包括盖帽和清洗工艺。
  25. 如权利要求24所述的DNA合成方法，其特征在于，所述脱保护的试剂为体积浓度为10％三氟乙酸的乙腈溶液，所述偶联的试剂为0.1M亚磷酰胺单体的乙腈溶液与0.5M四唑的乙腈溶液以体积比2/3配成混合溶液，所述盖帽的试剂为乙酸酐/吡啶/四氢呋喃以体积比1/1/8配成的混合溶液与17.6％w/v氮-甲基咪唑的乙腈溶液以体积比1/1配成混合溶液，所述氧化的试剂为10％w/v樟脑磺哑嗪的乙腈溶液，所述清洗的试剂为乙腈。
  26. 如权利要求23所述的DNA合成方法，其特征在于，所述脱保护、清洗、偶联、清洗、盖帽、清洗、氧化和清洗工艺的时间分别为60s、60s、60s、30s、90s、60s、60s和60s。
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