WO2019080704A1 - 用于核酸合成的微流控芯片 - Google Patents

用于核酸合成的微流控芯片

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Publication number
WO2019080704A1
WO2019080704A1 PCT/CN2018/109278 CN2018109278W WO2019080704A1 WO 2019080704 A1 WO2019080704 A1 WO 2019080704A1 CN 2018109278 W CN2018109278 W CN 2018109278W WO 2019080704 A1 WO2019080704 A1 WO 2019080704A1
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WIPO (PCT)
Prior art keywords
reaction chamber
microchannel
valve
input
reaction
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PCT/CN2018/109278
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English (en)
French (fr)
Inventor
孙宝策
王勇
黄小罗
陈泰
谭宏东
沈玥
徐讯
Original Assignee
深圳华大生命科学研究院
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Application filed by 深圳华大生命科学研究院 filed Critical 深圳华大生命科学研究院
Priority to CN201880068740.5A priority Critical patent/CN111246936B/zh
Publication of WO2019080704A1 publication Critical patent/WO2019080704A1/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
    • B01J14/00—Chemical processes in general for reacting liquids with liquids; Apparatus specially adapted therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the present invention relates to a microfluidic chip for synthesizing nucleic acids, comprising: a reaction chamber, one or more input microchannels connected to an inlet of the reaction chamber, an output microchannel connected to an outlet of the reaction chamber, and At least one valve for controlling fluid input to the input microchannel, at least one valve for controlling fluid flow into and/or out of the reaction chamber, and at least one valve for controlling fluid output of the output microchannel.
  • the invention also relates to a microfluidic system comprising the microfluidic chip, and methods and uses thereof.
  • Nucleic acid is the basic genetic material in life. In vitro artificial synthesis of nucleic acids can replicate any naturally occurring nucleic acid function or create new nucleic acid functions, depending on the needs of the research and application. With the development of genomics, molecular biology, systems biology and synthetic biology, synthetic nucleic acids have a wide range of applications in cell engineering, gene editing, disease diagnosis and treatment, and new material development.
  • a column synthesizer such as the Dr. oligo 192 synthesizer, performs a solid phase synthesis reaction on a porous reaction column of a size of centimeter by the addition of a solenoid valve control reagent.
  • the reaction error rate is low, but the synthesis flux is not It is high and requires a lot of raw materials.
  • Microarray synthesizers such as the CustomArray synthesizer, reduce the synthesis reaction to micron-sized reaction wells. There are tens of thousands of reaction wells in a single cell's synthesis cell, which increases both the synthesis throughput and the consumption of raw materials.
  • the reaction is not easy to control, the error rate is high, and the yield is small, and the product is a mixture, which increases the cost of subsequent operations.
  • DNA synthesis requires more efficient engineering techniques to achieve. From the viewpoint of chemical reaction, in order to improve the reaction efficiency, it is necessary to maintain the concentration of the reagent as much as possible, and remove the residual reagent as soon as possible after the reaction; in order to reduce the by-products and reduce the error rate, it is necessary to shorten the requirement of ensuring sufficient reaction.
  • the time of the reaction, for which the four-step reaction in the DNA synthesis reaction cycle needs to be controlled as precisely as possible.
  • the present invention contemplates a microfluidic chip approach to achieve nucleic acid synthesis.
  • the invention relates to a microfluidic chip for synthesizing nucleic acids, comprising: a reaction chamber, one or more input microchannels connected to an inlet of the reaction chamber, connected to an outlet of the reaction chamber Output microchannel, and at least one valve for controlling fluid input to the input microchannel, at least one valve for controlling fluid flow into and/or out of the reaction chamber, and fluid output for controlling the output microchannel At least one valve.
  • the microfluidic chip can be a two-layer structure comprising a flow channel layer and a control layer covering the flow channel layer.
  • the reaction chamber and microchannels are arranged in a flow channel layer, the valves being arranged in the control layer.
  • the present invention also relates to a microfluidic chip for synthesizing nucleic acids, comprising: a flow channel layer and a control layer covering the fluid layer, the flow channel layer comprising a reaction chamber, and One or more input microchannels connected to the reaction chamber and an output microchannel connected to the reaction chamber, the control layer including at least one valve for controlling fluid input to the input microchannel, for controlling access and/or Or at least one valve flowing out of the fluid of the reaction chamber and at least one valve for controlling the fluid output of the output microchannel.
  • each of the input microchannels and the output microchannels has at least one valve.
  • the reaction chamber has at least one valve that controls outflow of the reaction chamber.
  • the reaction chamber has at least one valve that controls access to the reaction chamber and at least one valve that exits the reaction chamber.
  • the valve of the reaction chamber when opened, allows any reagent or material having a size smaller than the inlet and/or outlet of the reaction chamber to enter and/or exit the reaction chamber.
  • the valve for controlling the reaction chamber does not completely close the inlet and/or outlet of the reaction chamber when closed, which leaves a gap in the inlet and/or outlet of the reaction chamber that allows for a smaller size than the The interstitial reagent or material enters or exits the reaction chamber and prevents reagents or materials having a size greater than the gap from entering or exiting the reaction chamber.
  • the output microchannel can be an output microchannel.
  • the reaction chamber can include multiple reaction chambers.
  • the plurality of reaction chambers are each connected to the one or more input microchannels.
  • the plurality of reaction chambers are each connected to an output microchannel.
  • the invention relates to a microfluidic system for synthesizing nucleic acids comprising a microfluidic chip as described herein.
  • the microfluidic system can also include one or more reservoirs coupled to the microchannels.
  • a reservoir may be used to contain a solution or reagent, such as a solution or reagent for synthesizing nucleic acids, or to receive material output from a reaction chamber.
  • the microfluidic system can also include a pressure drive coupled to the input microchannel and/or the valve that drives the flow of fluid in the microchannel or the closure of the valve by pressure.
  • Pressure-driven devices are generally composed of high-pressure helium and a self-contained control device that controls the pressure output through the control device to drive fluid flow in the microchannel or shut off the valve.
  • the microfluidic chip can also include a thermal regulator for adjusting the temperature of the reaction chamber.
  • the thermal regulator can be any device that regulates the temperature. This includes, for example, electrical resistance wires that heat up when a voltage is applied (such as those used in ovens), electrical resistance heaters, fans for emitting hot or cold air toward the reaction chamber, Peltier devices, IR heat sources such as projection lamps , circulating liquid or gas, and microwave heating.
  • the temperature of the reaction chamber is controlled by a programmed procedure.
  • LabView software can be used to design a program that controls the temperature of the reaction chamber.
  • a "microfluidic chip” is a unit or device that allows manipulation and delivery of a small amount of fluid (eg, microliters or nanoliters) into a substrate that includes microchannels.
  • the apparatus can be configured to allow manipulation of fluids (including reagents and solvents) that need to be transported or transported within the microchannels and reaction chambers using mechanical or non-mechanical pumps.
  • Microfluidic chips can be fabricated from a variety of materials including, but not limited to, glass, quartz, monocrystalline silicon wafers or polymers. Such polymers may include PC (polycarbonate), PDMS (polydimethylsiloxane), DCPD (polydicyclopentadiene), PEEK, and the like.
  • Microfluidic chips can be fabricated using a variety of fabrication techniques well known in the art including, but not limited to, hot press molding techniques, injection molding, soft lithography, epoxy casting techniques, three dimensional fabrication techniques (eg, stereolithography), lasers, or Other types of micromachining technology.
  • control layer refers to the structure of a microfluidic chip that is formed by arranging microchannels as described herein and a reaction chamber connected to the microchannels on the materials that make up the chip.
  • Control layer refers to the structure of a microfluidic chip that is formed by arranging a valve as described herein on the materials that make up the chip.
  • the control layer may also include a connecting portion between the valves.
  • the connecting portion between the valves is used to control the opening and closing of the valve.
  • the valve can be a pneumatic valve.
  • the connecting portion between the valves may be a pipe for gas to pass through.
  • the valve can be an electromagnetic valve.
  • the connecting portion between the valves may be an electric circuit.
  • reaction chamber refers to a component in which a reaction can occur on a microfluidic chip.
  • the reaction chamber can be of any shape, such as cylindrical, rectangular, and the like.
  • the reaction chamber has one or more microchannels connected to the reaction chamber that deliver reagents and/or solvents or are designed for product removal (eg, by on-chip valves or equivalent devices).
  • the reaction chamber typically has at least one inlet and at least one outlet.
  • the volume of the reaction chamber depends on the specific application.
  • the reaction chamber can have a diameter to height ratio greater than about 0.5: 10 or greater.
  • the reactor height can be from about 25 microns to about 20,000 microns.
  • microchannel refers to a microfluidic channel through which a fluid, including a solution or gas, can flow.
  • the shape and size of the microchannels are not particularly limited, and generally depend on the particular application required for the reaction process, and can be configured and sized according to the desired application.
  • the microchannels have the same width and depth.
  • the microchannels have different widths and depths.
  • the microchannels in the microfluidic chip can have a width greater than or equal to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 microns.
  • the microchannels have a width of less than or equal to 100, 90, 80, 70, 60, 50, 40, 30 or 20 microns. In certain embodiments, the microchannels can have a depth greater than or equal to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 microns. In certain embodiments, the microchannels can have a depth of less than or equal to 100, 90, 80, 70, 60, 50, 40, 30, or 20 microns. In certain embodiments, the microchannels have side walls that are parallel to each other. In certain other embodiments, the microchannels have tops and bottoms that are parallel to each other. In certain other embodiments, the microchannels comprise regions having different cross sections.
  • connecting means connecting the microchannel to the reaction chamber to enable fluid to flow into or out of the reaction chamber through the microchannel.
  • valve refers to a fluid (including a gas or solution) that can be controlled or actuated to control or regulate between various components of a microfluidic chip.
  • a flowing device comprising a flow between a microchannel, a solution or reagent reservoir, a reaction chamber, a temperature control element, and a device, and the like.
  • valves may include mechanical (or micromechanical valves), (pressure actuated) elastomeric valves, pneumatic valves, solid state valves, solenoid valves, and the like.
  • the valve can be a normally open valve or a normally closed valve.
  • the valve may be constructed by a combination of a flow channel layer and a control layer.
  • valves and their methods of manufacture can be found, for example, in the literature by Felton, The New Generation of Microvalves, Analytical Chemistry, 429-432 (2003), U.S. Patent No. 7,445,926, U.S. Patent Publication No. 2002/0127736, 2006/0073484, 2006/0073484. Found in 2007/0248958, 2008/0014576, 2009/0253181 and PCT Publication No. WO 2008/115626.
  • the valve can be controlled by a programmed control program to effect fluid input (input procedure) to the input microchannel, fluid flow into and/or out of the reaction chamber (reaction procedure), and output micro Control of the fluid output of the channel (output program).
  • the control program in the microfluidic chip of the present invention for synthesizing nucleic acids needs to be designed and optimized according to the specific target nucleic acid sequence to be generated.
  • the control program of the microfluidic chip of the present invention involves a matching optimization algorithm, and may also involve a linear optimization solution or an intelligent algorithm to solve an optimal control flow.
  • LabView software can be used to design control programs.
  • the valve can be controlled by binary addressing.
  • the invention also relates to a method of using a microfluidic chip or microfluidic system of the invention, comprising adding a reagent for nucleic acid synthesis to a reaction chamber through one or more input microchannels, allowing for reaction A nucleic acid synthesis reaction takes place in the chamber, and then the synthesized nucleic acid is output from the reaction chamber through the output microchannel.
  • a reagent for nucleic acid synthesis takes place in the chamber, and then the synthesized nucleic acid is output from the reaction chamber through the output microchannel.
  • different reagents are added to the reaction chamber through different input microchannels.
  • agent for nucleic acid synthesis can be any reagent suitable for use in the synthesis of nucleic acids, including but not limited to reagents for biosynthesis of nucleic acids (eg, polymerase chain reaction), as well as for chemical synthesis of nucleic acids (for example, a reagent for solid phase phosphoramidite synthesis). Methods of nucleic acid synthesis, such as nucleic acid biosynthesis or chemical synthesis, are well known in the art.
  • “reagent for nucleic acid synthesis” includes reagents for solid phase phosphoramidite synthesis. The solid phase phosphoramidite synthesis method comprises four steps: 1.
  • the reagents for nucleic acid synthesis include a phosphoramidite monomer of a nucleotide, a deprotecting agent, a phosphoramidite activator, a capping agent, and an oxidizing agent.
  • the reagent for nucleic acid synthesis further comprises a detergent.
  • the capping step may be omitted if the sequence of the sequence of the oligonucleotide to be synthesized is If the length is long (for example, the number of cycles >25), the capping step is usually not omitted.
  • allowing a nucleic acid synthesis reaction to take place in a reaction chamber means adding a reagent for nucleic acid synthesis to a reaction chamber, allowing the reagent to react under suitable conditions for a sufficient period of time.
  • suitable conditions for nucleic acid synthesis e.g., temperature, e.g., room temperature
  • reaction time depend on the particular method of nucleic acid synthesis employed and can be readily determined by those skilled in the art.
  • the phosphoramidite monomer of the nucleotide comprises a single nucleotide, for example, including adenine nucleotides (adenosine, abbreviated as A), guanine nucleotides (guanosine, abbreviated as G), cytosine nucleotide (cytidine, abbreviated as C) and uracil nucleotide (uridine, abbreviated as U) or thymidine (thymidylate, abbreviated as T).
  • adenine nucleotides adenosine, abbreviated as A
  • guanine nucleotides guanosine, abbreviated as G
  • cytosine nucleotide cytidine, abbreviated as C
  • uridine abbreviated as U
  • thymidine thymidylate
  • the phosphoramidite monomer of the nucleotide comprises a dimer of two nucleotides, including, for example, AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC and GG.
  • the phosphoramidite monomer of the nucleotide comprises a trimer of three nucleotides, a tetramer of four nucleotides, or a multimer of nucleotides.
  • the deprotecting agent comprises any agent capable of 5' deprotection of the 5' protected nucleotide, including but not limited to dichloroacetic acid in dichloromethane or trifluoroacetic acid in acetonitrile.
  • Phosphoramidite activators include, but are not limited to, tetrazole, S-ethylthiotetrazole, dicyanoimidazole or pyridinium salts (eg, pyridinium chloride).
  • the capping agent includes any agent capable of adding a protecting group 5' to the nucleotide, including but not limited to an acid anhydride in the presence of a base, such as acetic anhydride or isobutyric anhydride, or an acid chloride such as acetyl chloride or isobutyryl chloride.
  • the oxidizing agent includes, but is not limited to, an I 2 solution, or a peroxide in an organic solvent such as t-butyl hydroperoxide.
  • the solid support is any medium suitable for solid phase oligonucleotide synthesis, including but not limited to pore size controllable glass spheres (also known as "CPG"), polystyrene, microporous polyamides, For example, polydimethylacrylamide, polyethylene glycol coated polystyrene, and polyethylene glycol supported on polystyrene, such as those sold under the tradename Tentagel.
  • the solid support is a CPG bearing an amino-modified reactive functional group.
  • the particle diameter of the CPG may be less than or equal to 5 ⁇ m, less than or equal to 25 ⁇ m, less than or equal to 50 ⁇ m, less than or equal to less than or equal to 200 ⁇ m, less than or equal to 500 ⁇ m or more; and the pore diameter may be less than or equal to less than or equal to less than or equal to less than or equal to less than or equal to Or bigger.
  • the linking molecule of the modified solid phase carrier may have an ester group, a lipid group, a thioester group, an o-nitrobenzyl group, a coumarin group, a hydroxyl group, a thiol group, an anthracene ether group, a carboxyl group, an aldehyde group, an amino group, an amine group, A compound of any one or more of an amide group, an alkenyl group, or an alkynyl group.
  • Oligonucleotides or nucleotides can be attached to a solid support via a reactive functional group for solid phase oligonucleotide synthesis.
  • the invention relates to a method of using a microfluidic chip or microfluidic system of the invention, comprising a) attaching a 5' protected oligonucleotide or nucleotide thereto
  • the solid support is added to the reaction chamber through the input microchannel
  • the deprotecting agent is added to the reaction chamber through the input microchannel so that the 5' of the attached oligonucleotide or nucleotide on the solid support Protection
  • d) adding the phosphoramidite activator to the reaction chamber through the microchannel, and making the phosphoramidite of the nucleotide
  • the monomer is coupled to a 5' deprotected nucleoside or oligonucleotide, e) optionally adding a capping agent to the reaction chamber through a microchannel and rendering the step d) unreacted with the phosphorami
  • different reagents use different input microchannels in the method.
  • the capping step may be omitted if the sequence of the sequence of the oligonucleotide to be synthesized is If the length is long (for example, the number of cycles >25), the capping step is usually not omitted.
  • the solid support used in the present invention is larger in size than the gap left in the inlet and/or outlet of the reaction chamber when the valve for controlling the reaction chamber is closed.
  • the valve for controlling the reaction chamber allows the solid support to enter or exit the reaction chamber when opened, preventing the solid support from entering or exiting the reaction chamber when closed, but does not prevent step a when closed Other reagents or compounds to f enter or exit the reaction chamber.
  • the method can further comprise the step of separating the synthesized nucleic acid from the solid support after step h).
  • the oligonucleotide can be removed from the solid support using an aminolysis process.
  • the reagent for the aminolysis method may be selected from any one of ammonia water, ammonia gas, and methylamine; the aminolysis temperature may be 25, 60, 90 ° C or any temperature therebetween; the aminolysis time is usually from about 0.5 hour to about 18 Hours or longer, such as 2h, 5h, 10h, 18h or 24h.
  • the method can further comprise purifying the synthetic oligonucleotide using a purification method selected from the group consisting of desalting, MOP, PAGE, PAGE Plus or HPLC.
  • the number of input microchannels connected to the reaction chamber depends on the number of different nucleomonomers used in nucleic acid synthesis and the number of reagents required for the synthesis reaction.
  • a single nucleotide is used as a synthetic monomer, and a nucleic acid is synthesized by a four-step cycle solid phase synthesis method based on phosphoramidite.
  • Base synthesis requires four nucleomonomers (ie, A, T, C, G).
  • the nucleic acid is synthesized by a four-step cycle solid phase synthesis method based on phosphoramidite using a dimer of two nucleotides as a synthetic monomer.
  • 16 nucleotide dimers are required for the synthesis of monomers (AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, GG).
  • the microfluidic chip of the present invention further includes a binary addressing system that controls the valve to control fluid flow into and/or out of the reaction chamber and The fluid flow in the input microchannel and the output microchannel.
  • the specific control method can be: each microchannel has a separate binary address code (consisting of a series of 0 and 1), and the two valves in the control system are a group, which respectively represent 0 and a specific binary bit. 1, the valve group can be in one-to-one correspondence with the binary code of the channel.
  • the valve control system inputs a set of address codes (ie, opens the corresponding valve), the corresponding channel can be controlled to be turned on.
  • the reaction is precisely controlled.
  • the microfluidic chip can be used to control the characteristics of trace fluids. Combined with the binary addressing system, the chip can precisely control the reaction from time (more than 50 microseconds) and space (nano upgrade). In contrast, column synthesizers such as the Dr. Oligo 192 synthesizer control the reaction at the second and milliliter levels. In comparison, the chip can control the reaction more accurately, improve the reaction efficiency, and reduce the error rate.
  • the chip can control the reagents required for each cycle within one milliliter, while the same amount of reagents consumed by the column synthesizer is in the tens of milliliters. Obviously, the chip of the present invention greatly reduces the consumption of reagents and reduces the cost of synthesis.
  • the chip can integrate one to ten thousand reaction units (such as reaction chambers), that is, the synthesis flux is between one and ten thousand, which can meet different needs.
  • the chip of the present invention has more flexible configuration parameters and a wider range of applications.
  • the chip material is physically isolated between the reaction chambers of the chip, and the synthesized product can be separately output. Unlike the mixed product of the CustomArray synthesizer, the chip can directly output high-purity synthetic products, saving the cost of subsequent amplification and gene assembly.
  • FIG. 1 shows a schematic view (left image) and a corresponding physical photograph (right image) of an exemplary microfluidic chip according to Embodiment 1.
  • FIG. 2 shows a reagent input portion of an exemplary microfluidic chip according to embodiment 1.
  • Example 3 shows a reaction chamber portion of an exemplary microfluidic chip according to Example 1.
  • Figure 4 shows an exemplary flow diagram for nucleic acid synthesis using the microfluidic chip of the present invention.
  • Figure 5 shows a top cross-sectional schematic view of an exemplary microfluidic chip in accordance with one embodiment of the present invention.
  • Figure 6 shows a two-layer schematic view of an exemplary microfluidic chip in accordance with one embodiment of the present invention.
  • Figure 7 shows a schematic diagram of a flow channel layer of an exemplary microfluidic chip in accordance with one embodiment of the present invention.
  • Figure 8 shows a schematic diagram of a control layer of an exemplary microfluidic chip in accordance with one embodiment of the present invention.
  • Figure 9 shows a physical photograph of an exemplary microfluidic chip in accordance with one embodiment of the present invention.
  • Figure 10 is a graph showing the results of synthesizing nucleic acids using the microfluidic chip of the present invention.
  • Example 1 Microfluidic chip for single base DNA synthesis
  • this embodiment provides a microfluidic chip, which is divided into two parts: a reagent input part (top) and a reaction chamber part (below). ). Both parts are composed of a three-layer structure, from top to bottom, a reagent flow layer (also referred to herein as a flow channel layer), a control layer, and a substrate.
  • the material selected for the flow layer and the control layer is polydimethylsiloxane, which is prepared by pre-forming a template to mold the substrate; the substrate is selected as a clean-processed physiological grade slide.
  • the three layers are combined by a plasma bond and a method to form a completed microfluidic chip.
  • the microfluidic chip in this embodiment includes a substrate layer, those skilled in the art will understand that such a substrate layer is not essential.
  • the reagent input portion design structure includes a flow layer (left) and a control layer (right).
  • the flow layer 1-10 is a flow layer reagent input microchannel
  • 0 is a reagent output microchannel
  • the control layers A-E and 0-8 are control channels and valves, and the switch of the flow layer channel is controlled in a binary manner.
  • the reaction chamber section also includes a flow layer (left) and a control layer (right).
  • the wider part is the reaction chamber, and the control valve of the control layer cooperates to form a screen valve which is not completely closed, thereby ensuring the circulation of the reagent and restricting the reaction of the solid phase carrier inside.
  • the above microfluidic chip can be used for DNA synthesis and RNA synthesis. This example is described by taking the synthesis of DNA as an example.
  • the method for synthesizing DNA of the present embodiment includes the following steps:
  • microfluidic chips with sufficient flow channels and reaction chambers according to the target DNA to be synthesized.
  • single base synthesis requires four kinds of single base monomers, and at least four kinds of reagents required for chemical synthesis reaction, thus requiring At least 8 input microchannels; in order to meet the throughput and throughput of the synthesis, several reaction chambers of different sizes can be designed.
  • the preparation method is as described in the above examples.
  • a solid phase carrier modified for synthesis is designed according to the target DNA or RNA sequence to be synthesized; the solid phase carrier is a CPG having an amino group-modified active functional group.
  • the modified solid support is input from the input microchannel of the microfluidic chip into the reaction chamber.
  • the solid phase carrier After the solid phase carrier is input into the reaction chamber, it is controlled by positive pressure or negative pressure and valve control, and a reagent is added from the input microchannel to the reaction chamber in which the solid phase carrier is stored for synthesis reaction.
  • the capping step in the experimental operation can be omitted, and if a longer strand length DNA sequence is to be synthesized (cycle number >25), a capping step is required. Get enough target DNA to reduce the error rate.
  • the solid phase carrier in the reaction chamber is independently output from the output microchannel to the off-chip container by positive pressure or negative pressure driving and valve control.
  • the collected solid phase carrier is subjected to aminolysis, purification and gene assembly in sequence to obtain target DNA.
  • the synthesis method of the present embodiment can be accurately and automatically controlled by a controller that controls the drive and the valve to accurately input and output reactants and reagents to achieve a high throughput and high accuracy synthesis reaction.
  • Example 2 Microfluidic chip for double base DNA synthesis
  • the present embodiment adopts the following scheme to design a microfluidic chip and realize single-strand DNA fragment synthesis:
  • the number of double base monomers required for double base synthesis is sixteen, and the number of single base monomers is four, so a total of twenty monomers, plus at least four reagents required for chemical synthesis reaction, therefore need At least twenty-four input microchannels; as shown in Figure 7, marker 1 represents the input channel.
  • reaction chambers may be provided depending on the requirements of synthesis flux and throughput; as shown in Figure 7, reference numeral 2 denotes a reaction chamber.
  • valve for each microchannel and each microreaction chamber, at least one valve is provided for control; the valve may be a normally open valve or a normally closed valve, which needs to be formed by alignment of the flow channel layer and the control layer; using binary addressing The method is to achieve control of a plurality of microchannels or microreaction chambers; the control layer is as shown in Fig. 8, wherein reference numeral 4 denotes a valve, and reference numeral 5 denotes a connection portion between the valves.
  • the capping step in the experimental operation can be omitted, and if a longer strand length DNA sequence is to be synthesized (cycle number >25), a capping step is required. Get enough target DNA at a reduced error rate
  • the solid phase carrier in the reaction chamber is independently output to the off-chip container by pressure driving and outputting the microchannel and corresponding control valve (ie opening the outlet valve of the reaction chamber);
  • the corresponding 16 (4 ⁇ 4) double base deoxynucleotides (AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA) were synthesized in advance.
  • GT, GC, GG) are synthetic monomers, plus the original four A, T, C, G single base monomers (when the number of bases of the target DNA synthesis sequence is an odd number), these 20 monomers are New DNA synthesis monomer.
  • FIG. 10A shows the results of detecting fluorescence of the purified DNA synthesis product, and the results showed that DNA having a fluorescently labeled base was successfully synthesized.
  • Figure 10B shows the synthesis of the product by HPLC. The results show that there is a distinct main peak in the product signal indicating successful synthesis of DNA.

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Abstract

用于合成核酸的微流控芯片，其包括：反应室，与所述反应室的入口连接的一个或多个输入微通道，与所述反应室的出口连接的输出微通道，以及用于控制输入微通道的流体输入的至少一个阀门、用于控制进入和/或流出所述反应室的流体流动的至少一个阀门和用于控制输出微通道的流体输出的至少一个阀门。以及微流控芯片的微流控系统、使用其的方法和用途。

Description

用于核酸合成的微流控芯片 技术领域
本发明涉及用于合成核酸的微流控芯片，其包括：反应室，与所述反应室的入口连接的一个或多个输入微通道，与所述反应室的出口连接的输出微通道，以及用于控制输入微通道的流体输入的至少一个阀门、用于控制进入和/或流出所述反应室的流体流动的至少一个阀门和用于控制输出微通道的流体输出的至少一个阀门。本发明还涉及包含所述微流控芯片的微流控系统，以及使用其的方法和用途。
发明背景
核酸是生命体内的基础遗传物质。人工体外合成核酸能够根据研究和应用的需要，复制出任何天然存在的核酸功能或者创造出新的核酸功能。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，人工合成的核酸在细胞工程改造，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核酸合成以来，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。这些方法以单个核酸为单位进行合成，依赖的主要原理都是基于亚磷酰胺四步循环固相合成法：包括：1.碱基单体脱保护；2.基于亚磷酰胺化学的单体偶联；3.未反应单体保护；4.氧化亚磷酸到磷酸。
柱式合成仪，例如Dr.oligo 192合成仪,是通过电磁阀门控制试剂的添加，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，并且产量小，产物为混合物，增加了后续操作的成本。为了满足快速增长的DNA合成需求，DNA合成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核酸合成的技术。如何通过高水平的技术手段实现高效低成本的核酸合成是急需解决的一个问题。
发明内容
本发明设计了一种微流控芯片方案来实现核酸合成。
在一个方面，本发明涉及一种用于合成核酸的微流控芯片，其包括：反应室，与所述反应室的入口连接的一个或多个输入微通道，与所述反应室的出口连接的输出微通道，以及用于控制输入微通道的流体输入的至少一个阀门、用于控制进入和/或流出所述反应室的流体流动的至少一个阀门和用于控制输出微通道的流体输出的至少一个阀门。
在优选的实施方案中，所述微流控芯片可以是双层结构，其包括流道层和覆盖所述流道层的控制层。所述反应室和微通道被布置在流道层中，所述阀门被布置在控制层中。因此，在一个实施方案中，本发明还涉及一种用于合成核酸的微流控芯片，其包括：流道层和覆盖所述流动层的控制层，所述流道层包括反应室、与所述反应室连接的一个或多个输入微通道和与所述反应室连接的输出微通道，所述控制层包括用于控制输入微通道的流体输入的至少一个阀门、用于控制进入和/或流出所述反应室的流体流动的至少一个阀门和用于控制输出微通道的流体输出的至少一个阀门。在优选的实施方案中，每个输入微通道和输出微通道各自具有至少一个阀门。在优选的实施方案中，反应室具有控制流出所述反应室的至少一个阀门。在优选的实施方案中，反应室具有控制进入所述反应室的至少一个阀门和流出所述反应室的至少一个阀门。在优选的实施方案中，反应室的阀门在打开时，允许尺寸小于反应室的入口和/或出口的任何试剂或材料进入和/或流出反应室。在优选的实施方案中，用于控制反应室的阀门在关闭时未完全关闭反应室的入口和/或出口，这使得反应室的入口和/或出口留有间隙，所述间隙允许尺寸小于该间隙的试剂或材料进入或流出反应室，并阻止尺寸大于该间隙的试剂或材料进入或流出反应室。
在一些实施方案中，所述输出微通道可以是一个输出微通道。
在一些实施方案中，所述反应室可以包括多个反应室。优选地，所述多个反应室各自连接至所述一个或多个输入微通道。优选地，所述多个反应室各自连接至一个输出微 通道。
在另一个方面，本发明涉及一种用于合成核酸的微流控系统，其包括如本文所述的微流控芯片。
在一些实施方案中，微流控系统还可以包括与微通道连接的一个或多个贮存器。这样的贮存器可以用来包含溶液或试剂，例如用于合成核酸的溶液或试剂，或者用来接收从反应室中输出的材料。
在某些实施方案中，微流控系统还可以包括与输入微通道和/或所述阀门连接的压力驱动装置，其通过压力来驱动微通道中的流体流动或者阀门的关闭。压力驱动装置一般由高压氦气和自制的控制装置组成，通过控制装置控制压力输出，进而驱动微通道中的流体流动或者阀门的关闭。
在某些实施方案中，微流控芯片还可以包括用于调节所述反应室的温度的热调节器。热调节器可以是调节温度的任何装置。这包括例如在被施加电压时发热的电阻丝(例如在烤箱中使用的那些)、电阻加热器、用于朝向反应室发出热空气或冷空气的风扇、珀尔帖装置、IR热源例如投射灯、循环液体或气体、以及微波加热。在优选的实施方案中，通过经编程的程序来控制所述反应室的温度。作为示例但非限制，可以使用LabView软件来设计控制所述反应室的温度的程序。
如本文所用，“微流控芯片”是允许操控和传送少量的流体(例如微升或纳升)到包括微通道的衬底中的单元或设备。该设备可以被配置成允许利用机械的或非机械的泵对需要在微通道和反应室内传送或运送的流体(包括试剂和溶剂)进行操控。微流控芯片可以由不同的材料制造，包括但不限于玻璃、石英、单晶硅片或聚合物。这样的聚合物可以包括PC(聚碳酸酯)、PDMS(聚二甲基硅氧烷)、DCPD(聚双环戊二烯)、PEEK等。微流控芯片可以使用本领域熟知的各种制造技术来制造，包括但不限于热模压成型技术、注射成型、软光刻、环氧浇铸技术、三维制造技术(例如立体光刻)、激光或其他类型的微加工技术。
如本文所用，“流动层”是指微流控芯片的结构，其通过在构成芯片的材料上布置如本文所述的微通道和与微通道连接的反应室来形成。“控制层”是指微流控芯片的结构，其通过在构成芯片的材料上布置如本文所述的阀门来形成。在优选的实施方案中，控制层还可以包括阀门之间的连接部分。所述阀门之间的连接部分用于控制阀门的开关。例如，如下文详细描述的，在一个实施方案中，阀门可以是气动阀门。在这样的情况下， 阀门之间的连接部分可以是用于气体通过的管道。又例如，在一个实施方案中，阀门可以是电磁阀门。在这样的情况下，阀门之间的连接部分可以是电路。
如本文所用，“反应室”(或称作“反应器”或“微反应室”)是指在微流体芯片上可以在其中发生反应的部件。反应室可以是任何形状，例如圆柱形、长方形等。该反应室具有连接到该反应室的一个或多个微通道，所述微通道递送试剂和/或溶剂或者被设计成用于产物去除(例如通过芯片上的阀门或等同的设备来控制)。反应室通常具有至少一个入口和至少一个出口。反应室的体积取决于具体的应用。作为示例而非限制，该反应室可以具有大于大约0.5:10或更大的直径与高度比。作为示例而非限制，该反应器高度可以是大约25微米到大约20,000微米。
如本文所用，“微通道”或“通道”是指流体(包括溶液或气体)可以流动通过其的微流体通道。微通道的形状和尺寸没有特定限制，通常取决于反应过程所需的特定应用，并且可以根据期望的应用来配置并确定尺寸。例如，在某些实施方案中，微通道具有相同的宽度和深度。在其他的实施方案中，微通道具有不同的宽度和深度。例如，在某些实施方案中，微流体芯片中的微通道可以具有大于或等于50、60、70、80、90、100、110、120、130、140、150微米的宽度。在某些实施方案中，微通道具有小于或等于100、90、80、70、60、50、40、30或20微米的宽度。在某些实施方案中，微通道可以具有大于或等于50、60、70、80、90、100、110、120、130、140、150微米的深度。在某些实施方案中，微通道可以具有小于或等于100、90、80、70、60、50、40、30或20微米的深度。在某些实施方案中，微通道具有彼此平行的侧壁。在某些其他实施方案中，微通道具有彼此平行的顶部和底部。在某些其他实施方案中，微通道包括具有不同横截面的区域。
如本文所使用，微通道与反应室“连接”意指将微通道与反应室连接以使得流体能够通过微通道流动进入或流出反应室。
如本文所使用，“阀”、“阀门”(或“微阀门”)是指可以被控制或被致动以控制或调节在微流体芯片的各个部件之间的流体(包括气体或溶液)的流动的设备，所述流动包括在微通道、溶液或试剂贮存器、反应室、温度控制元件和设备等之间的流动。作为示例而非限制，这样的阀门可以包括机械(或微机械阀门)、(压力致动的)弹性阀门、气动阀门、固态阀门、电磁阀门等等。阀门可以是常开阀门或者常闭阀门。在某些实施方案中，阀门可以由流道层和控制层通过对齐共同构成。阀门和它们的制造方法的实例例如可以在 文献Felton,The New Generation of Microvalves,Analytical Chemistry,429-432(2003)、美国专利号7,445,926、美国专利公布号2002/0127736、2006/0073484、2006/0073484、2007/0248958、2008/0014576、2009/0253181以及PCT公布号WO 2008/115626中找到。
在优选的实施方案中，阀门可以通过经编程的控制程序来控制阀门，从而实现对输入微通道的流体输入(输入程序)、进入和/或流出反应室的流体流动(反应程序)和输出微通道的流体输出的控制(输出程序)。本发明的用于合成核酸的微流控芯片中的控制程序需要根据具体的要生成的目标核酸序列来设计和优化。通常，本发明的微流控芯片的控制程序涉及到匹配优化算法，还可能涉及到线性优化求解或者智能算法求解最优的控制流程。作为示例但非限制，可以使用LabView软件来设计控制程序。在优选的实施方案中，阀门可以通过二进制寻址的方式来控制。
在另一个方面，本发明还涉及使用本发明的微流控芯片或微流控系统的方法，包括将用于核酸合成的试剂通过一个或多个输入微通道加入到反应室中，允许在反应室中发生核酸合成反应，然后通过输出微通道从反应室输出合成的核酸。在优选的实施方案中，不同的试剂通过不同的输入微通道加入到反应室中。
如本文所用，“用于核酸合成的试剂”可以是适合用于合成核酸的任何试剂，包括但不限于用于生物合成核酸(例如聚合酶链式反应)的试剂，以及用于化学合成核酸(例如固相亚磷酰胺合成法)的试剂。核酸合成方法例如核酸的生物合成方法或化学合成方法是本领域熟知的。在优选的实施方案中，“用于核酸合成的试剂”包括用于固相亚磷酰胺合成法的试剂。固相亚磷酰胺合成法包括四个步骤：1.使用脱保护剂将核苷酸的亚磷酰胺单体脱保护；2.使用亚磷酰胺活化剂在固相载体上进行基于亚磷酰胺化学的单体偶联；3.使用封端剂对未反应单体进行保护；4.使用氧化剂氧化亚磷酸到磷酸；重复以上步骤，直到合成所需长度的多聚核苷酸。因此，在一些实施方案中，用于核酸合成的试剂包括核苷酸的亚磷酰胺单体，脱保护剂，亚磷酰胺活化剂，封端剂，和氧化剂。在优选的实施方案中，用于核酸合成的试剂还包括洗涤剂。在本发明的实施方案中，如果待合成的寡核苷酸的序列的链长较短(例如，循环数≤25)，封端步骤可以省略，如果待合成的寡核苷酸的序列的链长较长(例如，循环数>25)，则封端步骤通常不省略。
如本文所用，“允许在反应室中发生核酸合成反应”意指在将用于核酸合成的试剂加入到反应室中，使所述试剂在合适的条件下反应足够的时间。用于核酸合成的合适的条件(例如温度，例如室温)和反应时间取决于具体采用的核酸合成方法，并且是本领域 技术人员能够容易确定的。
在一些实施方案中，核苷酸的亚磷酰胺单体包括单个核苷酸，例如包括腺嘌呤核苷酸(腺苷酸，简称为A)、鸟嘌呤核苷酸(鸟苷酸，简称为G)、胞嘧啶核苷酸(胞苷酸，简称为C)和尿嘧啶核苷酸(尿苷酸，简称为U)或胸腺嘧啶核苷酸(胸苷酸，简称为T)。在一些实施方案中，核苷酸的亚磷酰胺单体包括两个核苷酸的二聚体，例如包括AA，AT，AC，AG，TA，TT，TC，TG，CA，CT，CC，CG，GA，GT，GC和GG。在一些实施方案中，核苷酸的亚磷酰胺单体包括三个核苷酸的三聚体，四个核苷酸的四聚体，或更多个核苷酸的多聚体。
在一些实施方案中，脱保护剂包括能够使5’保护的核苷酸的5’去保护的任何试剂，包括但不限于二氯乙酸的二氯甲烷溶液或三氟乙酸的乙腈溶液。亚磷酰胺活化剂包括但不限于四唑、S-乙硫基四唑、二氰基咪唑或吡啶鎓盐(如氯化吡啶鎓)。封端剂包括能够在核苷酸的5’添加保护基团的任何试剂，包括但不限于在碱存在下的酸酐，例如乙酸酐或异丁酸酐，或酰氯，例如乙酰氯或异丁酰氯。氧化剂包括但不限于I 2溶液，或在有机溶剂中的过氧化物(如叔丁基过氧化氢)。
在一些实施方案中，固相载体是适合固相寡核苷酸合成的任何介质，包括但不限于孔径可控的玻璃球(也称为“CPG”)，聚苯乙烯，微孔聚酰胺，例如聚二甲基丙烯酰胺，聚乙二醇包覆的聚苯乙烯，以及载在聚苯乙烯上的聚乙二醇，例如以Tentagel的商品名称销售的那些固相载体。优选地，固相载体是带有氨基修饰的活性官能团的CPG。CPG的粒径可以是小于或等于5μm，小于或等于25μm，小于或等于50μm，小于或等于小于或等于200μm，小于或等于500μm或更大；孔径可以是小于或等于
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小于或等于
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小于或等于
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小于或等于
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小于或等于
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小于或等于
Figure PCTCN2018109278-appb-000006
或更大。修饰固相载体的连接分子可以是具有酯基、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。寡核苷酸或核苷酸可以通过活性官能团附接到固相载体上，以用于固相寡核苷酸合成。
因此，在优选的实施方案中，本发明涉及使用本发明的微流控芯片或微流控系统的方法，包括a)将其上附接有5’保护的寡核苷酸或核苷酸的固相载体通过输入微通道加入至反应室中，b)将脱保护剂通过输入微通道加入至反应室中，以使得固相载体上附接的寡核苷酸或核苷酸的5’去保护，c)将核苷酸的亚磷酰胺单体通过输入微通道加入至反 应室中，d)将亚磷酰胺活化剂通过微通道加入至反应室中，并使得核苷酸的亚磷酰胺单体与5’去保护的核苷或寡核苷酸发生偶合，e)任选地将封端剂通过微通道加入至反应室中，并使得步骤d)中未与亚磷酰胺单体反应的5’去保护的核苷或寡核苷酸封端；f)将氧化剂通过微通道加入至反应室中，并使得步骤d)中的偶合反应形成的三价磷基团氧化；g)任选地重复步骤b)至d)一或多次，其中最终步骤是步骤e)或f)，由此合成所需的核酸；和h)通过输出微通道输出反应室中的固相载体。在优选的实施方案中，在所述方法中不同的试剂使用不同的输入微通道。在本发明的实施方案中，如果待合成的寡核苷酸的序列的链长较短(例如，循环数≤25)，封端步骤可以省略，如果待合成的寡核苷酸的序列的链长较长(例如，循环数>25)，则封端步骤通常不省略。
在优选的实施方案中，在本发明中使用的固相载体的尺寸大于用于控制反应室的阀门在关闭时在反应室的入口和/或出口留有的间隙。因此，在优选的实施方案中，用于控制反应室的阀门在打开时允许固相载体进入或流出反应室，在关闭时阻止固相载体进入或流出反应室，但在关闭时不阻止步骤a至f的其它试剂或化合物进入或流出反应室。
在一些实施方案中，所述方法还可以包括在步骤h)之后，使合成的核酸与固相载体分开的步骤。优选地，可以使用氨解法从固相载体上除掉寡核苷酸。用于氨解法的试剂可以选自氨水，氨气，甲胺中的任意一种；氨解温度可以为25、60、90℃或其间的任何温度；氨解时间通常为约0.5小时至约18小时或更长时间，例如2h，5h，10h，18h或24h。随后，所述方法还可以包括使用选自脱盐、MOP、PAGE、PAGE Plus或HPLC的纯化方式来纯化合成的寡核苷酸。
在本发明的用于合成核酸的微流控芯片中，与反应室连接的输入微通道的数量取决于核酸合成中所使用的不同核苷酸单体的数量和合成反应所需的试剂的数量。例如，以DNA合成为例，在单碱基合成法的一个示例性实施方案中，以单个核苷酸为合成单体，通过基于亚磷酰胺四步循环固相合成法来合成核酸因此，单碱基合成法需要4种核苷酸单体(即A，T，C，G)。而所需要的合成核酸的其它试剂至少为4种(包括脱保护剂，亚磷酰胺活化剂，封端剂，氧化剂)。因此，用于基于亚磷酰胺的单碱基核酸合成的微流控芯片的输入微通道的数量至少为4+4＝8个。
又例如，在双碱基合成法的一个示例性实施方案中，以两个核苷酸的二聚体为合成单体，通过基于亚磷酰胺四步循环固相合成法来合成核酸。在这样的方法中，合成单体需要16种核苷酸二聚体(AA，AT，AC，AG，TA，TT，TC，TG，CA，CT，CC， CG，GA，GT，GC，GG)以及4种单核苷酸单体(A，T，C，G)(当目标DNA合成序列碱基数目为奇数时)。因此，用于基于亚磷酰胺的双碱基核酸合成的微流控芯片的输入微通道的数量至少为16+4+4＝24个。
进一步地，该设计思路还可以扩展至使用更高聚体作为合成单体的多碱基合成。例如，三碱基核酸合成需要64(4 3)种核苷酸三聚体为合成单体，则输入微通道的数量至少为64+16+4+4＝88个；四碱基DNA合成，需要预先合成256(4 4)种核苷酸四聚体为合成单体，则输入微通道的数量至少为256+64+16+4+4＝344个；以此类推。
随着输入微通道的数量增多，微流控芯片内部的阀门的数量需要相应增加，控制复杂度也会大幅提高。因此，在某些实施方案中，本发明的微流控芯片还包括二进制寻址系统，所述二进制寻址系统控制所述阀门，从而控制进入和/或流出所述反应室的流体流动以及所述输入微通道和所述输出微通道中的流体流动。具体控制方式可以是：每个微通道都有一个单独的二进制地址编码(由一系列的0和1组成)，控制系统中的阀门两个为一组，分别代表某一特定二进制位的0和1，这样阀门组即可与通道的二进制编码一一对应，当阀门控制系统输入一组地址编码时(即打开对应阀门)，可控制对应通道的开启。通过采用二进制寻址的方式，可以显著简化对阀门的控制，减少了系统复杂度。
本发明的有益技术效果
本芯片独特的优势包括：
1.反应精确控制。利用微流控芯片可以控制微量流体的特点，结合二进制寻址系统，本芯片可以从时间(50微秒以上)和空间(纳升级别)上精确控制反应的进行。相比之下，柱式合成仪例如Dr.Oligo 192合成仪对于反应的控制在秒和毫升级别。相比较而言，本芯片能够更精确的控制反应的进行，提高反应效率，降低错误率。
2.节省原料。本芯片可以将每个循环所需的试剂可以控制在一毫升以内，而同样的通量下，柱式合成仪消耗的试剂在数十毫升。显然，本发明的芯片极大降低了试剂的消耗，降低了合成成本。
3.通量可调节。本芯片根据不同需求，可集成一至一万个反应单元(例如反应室)，即合成通量在一至一万之间，可满足不同的需求。与柱式合成仪(96-768条)和微阵列合成仪(如CustomArray合成仪为10000条左右)相比，本发明的芯片有更灵活的配置参数和更广泛的应用。
4.具有物理隔离。本芯片的反应室之间均有芯片材料作为物理隔离，合成产物可单独输出。与CustomArray合成仪的混合产物不同，本芯片可直接输出纯度很高的合成产物，节省了后续扩增及基因组装的成本。
附图说明
应理解，本说明书的附图仅是本发明的实施方案的示例性说明，不旨在对本发明的范围进行限制。为了简便起见，相同或相似的部件仅被示例性呈现和标识，并且在一些附图中未对其进行重复性标识。在本说明书的教导下，本领域技术人员能够容易地对示例性呈现的部件进行修改或扩展。
图1示出根据实施例1所示的示例性微流体芯片的示意图(左图)和相应的实物照片(右图)。
图2示出根据实施例1所示的示例性微流体芯片的试剂输入部分。
图3示出根据实施例1所示的示例性微流体芯片的反应室部分。
图4示出使用本发明的微流体芯片进行核酸合成的示例性流程图。
图5示出根据本发明一个实施方案的示例性微流体芯片的俯视剖面示意图。
图6示出根据本发明一个实施方案的示例性微流体芯片的双层结构示意图。
图7示出根据本发明一个实施方案的示例性微流体芯片的流道层示意图。
图8示出根据本发明一个实施方案的示例性微流体芯片的控制层示意图。
图9示出根据本发明一个实施方案的示例性微流体芯片的实物照片。
图10示出使用本发明的微流控芯片合成核酸的结果图。
具体实施方式
实施例1：单碱基DNA合成的微流控芯片
以单碱基DNA合成为例，如图1所示，本实施例提供了一种微流控芯片，微流控芯片分为两部分：试剂输入部分(上图)和反应室部分(下图)。两个部分均有三层结构组成，从上至下依次是试剂流动层(在本文中也称为流道层)、控制层和基底。流动层和控制层选用的材料为聚二甲基硅氧烷，通过预先制作模板倒模的方法制作；基底选用洁净处理的生理级载玻片。三层通过等离子键和方法结合在一起，组成一个完成的微流控芯片。虽然本实施例中的微流控芯片包含基底层，但本领域技术人员能够理解，这 样的基底层不是必需的。
如图2所示，试剂输入部分设计结构包括流动层(左图)和控制层(右图)。其中，流动层1-10为流动层试剂输入微通道，0标记了试剂输出微通道；控制层A-E和0-8为控制通道和阀门，采用二进制的方式排布控制了流动层通道的开关。
如图3所示，反应室部分同样包括流动层(左图)和控制层(右图)。其中，较宽部分为反应室，通过控制层的控制阀门配合，形成不完全关闭的筛阀，进而保证试剂的流通和限制固相载体在内部进行反应。
上述的微流控芯片可用于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4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条输入微通道；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；固相载体为带有氨基修饰的活性官能团的CPG。
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微通道输入到反应室内。
S40：输入试剂；
固相载体输入到反应室后，再通过正压或负压驱动和阀门控制，从输入微通道往存储有固相载体的反应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 PCTCN2018109278-appb-000007
Figure PCTCN2018109278-appb-000008
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，将反应室内的固相载体独立从输出微通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。
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。
实施例2：双碱基DNA合成的微流控芯片
以双碱基DNA合成为例，本实施例采用如下方案设计微流控芯片和实现单链DNA片段合成：
1)设计微流控合成芯片(如图5和6所示。在图6中，上方的图表示流道层，下方的图表示控制层)：
a)双碱基合成所需的双碱基单体为十六种，单碱基单体四种，因此总共为二十种单体，加上化学合成反应所需试剂至少四种，因此需要至少二十四条输入微通道；如图7 所示，标记1表示输入通道。
b)根据合成通量和产量的需求，可设置一个或多个反应室；如图7所示，标记2表示反应室。
c)针对每一个反应室，均设计一条产物输出微通道；如图7所示，标记3表示输出通道。
d)对于每一条微通道和每一个微反应室，设置至少一个阀门进行控制；阀门可以是常开阀门或者常闭阀门，需要由流道层和控制层通过对齐共同构成；使用二进制寻址的方法以实现对多个微通道或者微反应室的控制；控制层如图8所示，其中标记4表示阀门，标记5表示阀门之间的连接部分。
e)通过设计模板，经过刻蚀工艺直接获得包含上述结构的芯片，或者通过倒模间接获得；芯片材料采用PMDS；
2)根据待合成的目标DNA序列，设计单体的输入控制程序、反应控制程序和输出程序；
3)根据具体的需求，设计修饰用于合成的固相载体；
4)将修饰之后的固相载体通过特定的输入微通道输入到反应室中，随后关闭反应室的入口阀门，同时保持出口阀门关闭；
5)根据预先设定的程序，通过压力驱动和阀门控制以及相应微通道，往存储有固相载体的反应室中添加试剂进行合成反应；
下表1示例性地概述了合成反应的一个反应循环。应理解，下面的具体实验条件仅是示例性的，并非旨在限定。本领域技术人员能够根据本说明书的教导和现有技术进行常规的改变和调整。
表1：
Figure PCTCN2018109278-appb-000009
Figure PCTCN2018109278-appb-000010
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
6)在完成合成反应之后，通过压力驱动和输出微通道及相应控制阀门(即打开反应室的出口阀门)，将反应室中的固相载体独立输出到芯片外的容器中；
7)在芯片外的容器中，进行氨解、纯化及基因组装的步骤。
在上述方案中，预先合成相应的16(4×4)种双碱基的脱氧核苷酸(AA，AT，AC，AG，TA，TT，TC，TG，CA，CT，CC，CG，GA，GT，GC，GG)为合成单体，再加上原先的4种A，T，C，G单碱基单体(目标DNA合成序列碱基数目为奇数时)，这20种单体为新的DNA合成单体。
实施例3：使用本发明的微流控芯片合成核酸
使用如实施例1制备的微流控芯片，根据如实施例1所述的DNA的合成方法合成5个核苷酸长度的核酸的结果如图10所示。其中图10A是对纯化后的DNA合成产物检测荧光的结果，结果显示成功合成了具有经荧光标记的碱基的DNA。图10B是利用HPLC对合成的产物进行检测，结果显示产物信号中有一个明显的主峰，表明成功合成了DNA。
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 (12)

  1. 一种用于合成核酸的微流控芯片，其包括：
    反应室，
    与所述反应室的入口连接的一个或多个输入微通道，
    与所述反应室的出口连接的输出微通道，
    以及用于控制输入微通道的流体输入的至少一个阀门、用于控制进入和/或流出所述反应室的流体流动的至少一个阀门和用于控制输出微通道的流体输出的至少一个阀门，
    优选地，用于控制反应室的阀门在关闭时未完全关闭反应室的入口和/或出口。
  2. 权利要求1的微流控芯片，其中所述微流控芯片包括流道层和覆盖所述流道层的控制层，所述反应室和微通道被布置在流道层中，所述阀门被布置在控制层中。
  3. 权利要求1或2的微流控芯片，其中所述输出微通道是一个输出微通道，
    优选地，所述反应室是多个反应室，
    优选地，所述多个反应室各自连接至所述一个或多个输入微通道，
    优选地，所述多个反应室各自连接至一个输出微通道。
  4. 前述权利要求中任一项的微流控芯片，其中输入微通道的数量为至少8个，优选至少24个，优选至少88个，优选至少344个。
  5. 一种用于合成核酸的微流控系统，其包括权利要求1-4中任一项的微流控芯片。
  6. 权利要求5的微流控系统，其还包括通过经编程的控制程序来实现对阀门的控制，
    优选地，所述微流控系统还包括二进制寻址系统，所述二进制寻址系统控制所述阀门，从而控制进入和/或流出所述反应室的流体流动以及所述输入微通道和所述输出微通道中的流体流动。
  7. 权利要求5或6的微流控系统，其还包括与输入微通道和/或阀门连接的压力驱动装置。
  8. 权利要求6-7中任一项的微流控系统，其还包括与一个或多个微通道连接的一个或多个贮存器。
  9. 一种使用权利要求1-4中任一项的微流控芯片或权利要求5-8中任一项的微流控系统的方法，包括将用于核酸合成的试剂通过一个或多个输入微通道加入到反应室中，允许在反应室中发生核酸合成反应，然后通过输出微通道从反应室输出合成的核酸，
    优选地，不同的试剂通过不同的输入微通道加入到反应室中。
  10. 一种使用权利要求1-4中任一项的微流控芯片或权利要求5-8中任一项的微流控系统的方法，包括
    a)将其上附接有5’保护的寡核苷酸或核苷酸的固相载体通过输入微通道加入至反应室中，
    b)将脱保护剂通过输入微通道加入至反应室中，以使得固相载体上附接的寡核苷酸或核苷酸的5’去保护，
    c)将核苷酸的亚磷酰胺单体通过输入微通道加入至反应室中，
    d)将亚磷酰胺活化剂通过微通道加入至反应室中，并使得核苷酸的亚磷酰胺单体与5’去保护的核苷或寡核苷酸发生偶合，
    e)任选地将封端剂通过微通道加入至反应室中，并使得步骤d)中未与亚磷酰胺单体反应的5’去保护的核苷或寡核苷酸封端；
    f)将氧化剂通过微通道加入至反应室中，并使得步骤d)中的偶合反应形成的三价磷基团氧化；
    g)任选地重复步骤b)至d)一或多次，其中最终步骤是步骤e)或f)，由此合成所需的核酸；和
    h)通过输出微通道输出反应室中的固相载体，
    优选地，不同的试剂使用不同的输入微通道，
    优选地，用于控制反应室的阀门在打开时允许固相载体进入或流出反应室，在关闭时阻止固相载体进入或流出反应室，但在关闭时不阻止步骤a至f的其它试剂或化合物进入或流出反应室。
  11. 权利要求10的方法，其中所述核苷酸的亚磷酰胺单体包括单个核苷酸的单体、两个核苷酸的二聚体、三个核苷酸的三聚体、四个核苷酸的四聚体、或更多个核苷酸的多聚体。
  12. 权利要求1-4中任一项的微流控芯片或权利要求5-8中任一项的微流控系统用于合成核酸的用途。
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