技术领域technical field
本发明涉及生物芯片技术领域，特别涉及一种全自动样本制备微流控系统及其制备方法与使用方法。The invention relates to the technical field of biochips, in particular to a fully automatic sample preparation microfluidic system and a preparation method and use method thereof.
背景技术Background technique
对于DNA检测技术来说，前置的样本制备过程显得尤为重要。样本制备一般包括细胞裂解、DNA提取、纯化以及DNA扩增这几个步骤。For DNA detection technology, the pre-sample preparation process is particularly important. Sample preparation generally includes the steps of cell lysis, DNA extraction, purification, and DNA amplification.
现有的全集成样本制备系统一般是基于液体工作站式的高通量样本制备仪器。对于液体工作站来说，其原理是将原本需要人工配置、转移的试剂移植到固定程式的机械手中有仪器自动完成，现有的液体工作站主要针对DNA提取、纯化以及PCR体系配置这几个环节来运行，一般不带有细胞裂解以及PCR扩增功能，集成度不高，制备过程中往往还需要其他专用设备如离心机、漩涡震荡仪、移液器、PCR热循环仪等的加入才能完成，因此整个操作过程非常复杂，需要在多台专业仪器间反复切换，耗时耗力，从收集样本到拿到检测结果往往需要等上数天的时间。而且整个仪器十分庞大、笨重，需要在专用环境下由专业人员进行操作，维护起来也不便利。Existing fully integrated sample preparation systems are generally based on liquid workstation-type high-throughput sample preparation instruments. For the liquid workstation, the principle is to transplant the reagents that originally required manual configuration and transfer to the fixed-program manipulator, and the instrument will automatically complete it. The existing liquid workstation is mainly for DNA extraction, purification and PCR system configuration. Generally, it does not have cell lysis and PCR amplification functions, and the integration level is not high. The preparation process often requires the addition of other special equipment such as centrifuges, vortex shakers, pipettes, and PCR thermal cyclers to complete. Therefore, the entire operation process is very complicated, requiring repeated switching between multiple professional instruments, which is time-consuming and labor-intensive. It often takes several days to collect samples and obtain test results. Moreover, the whole instrument is very large and heavy, and needs to be operated by professionals in a special environment, and it is also inconvenient to maintain.
因此，如何提供一种全自动集成化的样本制备系统，使其可接受生物样本输入，并直接输出扩增产物，操作过程不依赖其他实验室设备，操作流程简单，降低对操作人员要求，成为本领域技术人员亟待解决的重要技术问题。Therefore, how to provide a fully automatic and integrated sample preparation system that can accept biological sample input and directly output amplification products, the operation process does not rely on other laboratory equipment, the operation process is simple, and the requirements for operators are reduced. Important technical problems to be solved urgently by those skilled in the art.
发明内容Contents of the invention
有鉴于此，本发明的第一个目的在于提供一种全自动样本制备微流控系统，以使其可接受生物样本输入，并直接输出扩增产物，操作过程不依赖其他实验室设备，操作流程简单，降低对操作人员要求；本发明的第二个目的在于提供一种上述全自动样本制备微流控系统的制备方法及使用方法。In view of this, the first object of the present invention is to provide a fully automatic sample preparation microfluidic system, so that it can accept the input of biological samples and directly output the amplification products. The operation process does not depend on other laboratory equipment, and the operation The process is simple and the requirements for operators are reduced; the second purpose of the present invention is to provide a preparation method and a use method of the above-mentioned fully automatic sample preparation microfluidic system.
为实现上述目的,本发明提供如下技术方案：To achieve the above object, the present invention provides the following technical solutions:
一种全自动样本制备微流控系统，包括：A fully automatic sample preparation microfluidic system, comprising:
芯片主体，其上依次设置有通过微流控管道串联的入口、第一芯片阀、扩增腔室、第二芯片阀、产物输出口以及废液输出口；The main body of the chip is sequentially provided with an inlet through a microfluidic pipeline, a first chip valve, an amplification chamber, a second chip valve, a product output port, and a waste liquid output port;
样本接口模块，与所述芯片主体密封连接，所述样本接口模块包括从上到下依次设置的裂解液存储池以及扩增试剂存储池，所述裂解液存储池与所述扩增试剂存储池之间通过第一密封隔层密封隔离，所述裂解液存储池在所述第一密封隔层破损时与所述扩增试剂存储池连通，所述扩增试剂存储池的下端与所述入口通过第二密封隔层密封隔离，所述扩增试剂存储池的下端在所述第二密封隔层破损时与所述入口连通。The sample interface module is sealed and connected with the chip main body. The sample interface module includes a lysate storage pool and an amplification reagent storage pool arranged sequentially from top to bottom. The room is sealed and isolated by the first sealing interlayer, the lysate storage pool communicates with the amplification reagent storage pool when the first sealing interlayer is damaged, and the lower end of the amplification reagent storage pool passes through the inlet. The second sealing compartment is sealed and isolated, and the lower end of the amplification reagent storage pool communicates with the inlet when the second sealing compartment is damaged.
优选地，所述样本接口模块包括上层模块以及下层模块，所述裂解液存储池开设于所述上层模块，所述扩增试剂存储池开设于所述下层模块，所述上层模块与所述下层模块密封连接。Preferably, the sample interface module includes an upper module and a lower module, the lysate storage pool is set in the upper module, the amplification reagent storage pool is set in the lower module, and the upper module is connected to the lower module. Module sealed connection.
优选地，所述扩增试剂存储池通过排气孔与外部连通。Preferably, the amplification reagent storage pool communicates with the outside through an air vent.
优选地，所述排气孔开设于所述上层模块。Preferably, the exhaust hole is opened in the upper module.
优选地，所述扩增试剂存储池的侧壁上设置有与所述裂解液存储池相接的用于破坏液体表面张力的导流结构。Preferably, the side wall of the amplification reagent storage pool is provided with a flow guide structure connected to the lysate storage pool for breaking the surface tension of the liquid.
优选地，所述导流结构为上下贯通地开设于所述扩增试剂存储池的侧壁上的导流槽。Preferably, the diversion structure is a diversion groove opened up and down on the side wall of the amplification reagent storage pool.
优选地，所述第一密封隔层以及所述第二密封隔层均由脆性材料制成。Preferably, both the first sealing spacer and the second sealing spacer are made of brittle materials.
优选地，所述产物输出口与所述扩增腔室体积相同。Preferably, the product output port has the same volume as the amplification chamber.
一种全自动样本制备微流控系统的制备方法，包括步骤：A method for preparing a fully automatic sample preparation microfluidic system, comprising the steps of:
a1)将样品接口模块密封连接于芯片主体；a1) sealingly connecting the sample interface module to the chip main body;
a2)添加扩增试剂体系溶液至样品接口模块的扩增试剂存储池，然后对扩增试剂体系溶液进行干燥以形成扩增体系粉末；a2) adding the amplification reagent system solution to the amplification reagent storage pool of the sample interface module, and then drying the amplification reagent system solution to form the amplification system powder;
a3)将裂解液添加至样品接口模块的裂解液存储池中；a3) adding the lysate to the lysate storage pool of the sample interface module;
a4)将样品接口模块以及芯片主体上的各接口封闭。a4) Seal the sample interface module and the interfaces on the chip main body.
优选地，所述样品接口模块包括上层模块以及下层模块，所述步骤a1)具体为将样品接口模块的下层模块密封连接于芯片主体，然后进入所述步骤a2)。Preferably, the sample interface module includes an upper layer module and a lower layer module, and the step a1) specifically includes sealing and connecting the lower layer module of the sample interface module to the chip main body, and then entering into the step a2).
优选地，所述步骤a3)具体为：Preferably, the step a3) is specifically:
a3.1)将上层模块密封连接于下层模块，使第一密封隔层将裂解液存储池及扩增试剂存储池隔离；a3.1) The upper module is sealed and connected to the lower module, so that the first sealing compartment isolates the lysate storage pool and the amplification reagent storage pool;
a3.2)将裂解液添加至样品接口模块的裂解液存储池中。a3.2) Add the lysate to the lysate storage pool of the sample interface module.
优选地，进入所述步骤a3)之前，捅破扩增试剂存储池与芯片主体的入口之间的第二密封隔层使两者连通。Preferably, before step a3), the second sealed interlayer between the amplification reagent storage pool and the inlet of the chip main body is pierced to communicate with the two.
一种全自动样本制备微流控系统的使用方法，包括步骤：A method for using a fully automatic sample preparation microfluidic system, comprising steps:
b1)提取样本，并在裂解液存储池的裂解液中涮洗，将样本DNA分子释放到裂解液中；b1) extracting the sample, and rinsing it in the lysate of the lysate storage tank, releasing the sample DNA molecules into the lysate;
b2)若第二密封隔层在全自动样本制备微流控系统的制备过程中被捅破，则关闭第一芯片阀后，再捅破第一密封隔层，使含有样本DNA分子的裂解液进入扩增试剂存储池中溶解扩增体系粉末形成完整的PCR扩增体系；若第二密封隔层在全自动样本制备微流控系统的制备过程中未被捅破，则捅破第一密封隔层，使含有样本DNA分子的裂解液进入扩增试剂存储池中溶解扩增体系粉末形成完整的PCR扩增体系后再捅破第二密封隔层；b2) If the second sealing interlayer is pierced during the preparation process of the fully automatic sample preparation microfluidic system, after closing the first chip valve, the first sealing interlayer is pierced again, so that the lysate containing sample DNA molecules Enter the amplification reagent storage pool to dissolve the amplification system powder to form a complete PCR amplification system; if the second sealing compartment is not pierced during the preparation process of the fully automatic sample preparation microfluidic system, the first sealing layer will be pierced The interlayer allows the lysate containing sample DNA molecules to enter the amplification reagent storage pool to dissolve the amplification system powder to form a complete PCR amplification system, and then break through the second sealed interlayer;
b3)在废液输出口处连接负压源并开启第一芯片阀以及第二芯片阀，将PCR扩增体系引流至扩增腔室；b3) Connect a negative pressure source at the waste liquid output port and open the first chip valve and the second chip valve to drain the PCR amplification system to the amplification chamber;
b4)关闭第一芯片阀以及第二芯片阀，使PCR扩增体系在扩增腔室进行温度循环扩增；b4) closing the first chip valve and the second chip valve, so that the PCR amplification system performs temperature cycle amplification in the amplification chamber;
b5)扩增完成后，开启第一芯片阀以及第二芯片阀，将扩增产物引流至产物输出口以进行提取；b5) After the amplification is completed, open the first chip valve and the second chip valve, and drain the amplification product to the product output port for extraction;
b6)提取扩增产物后的废液经废液输出口输出。b6) The waste liquid after extracting the amplified product is output through the waste liquid output port.
为实现上述目的，本发明提供的全自动样本制备微流控系统，包括芯片主体以及样本接口模块，其中，芯片主体上依次设置有通过微流控管道串联的入口、第一芯片阀、扩增腔室、第二芯片阀、产物输出口以及废液输出口；样本接口模块与芯片主体密封连接，样本接口模块包括从上到下依次设置的裂解液存储池以及扩增试剂存储池，裂解液存储池与扩增试剂存储池之间通过第一密封隔层密封隔离，裂解液存储池在第一密封隔层破损时与扩增试剂存储池连通，以使裂解液能够进入扩增试剂存储池中，扩增试剂存储池的下端与入口通过第二密封隔层密封隔离，扩增试剂存储池的下端在第二密封隔层破损时与入口连通；In order to achieve the above purpose, the fully automatic sample preparation microfluidic system provided by the present invention includes a chip body and a sample interface module, wherein the chip body is sequentially provided with an inlet connected in series through a microfluidic pipeline, a first chip valve, an amplification The chamber, the second chip valve, the product output port and the waste liquid output port; the sample interface module is in sealing connection with the chip main body, and the sample interface module includes a lysate storage pool and an amplification reagent storage pool arranged sequentially from top to bottom, and the lysate The storage pool and the amplification reagent storage pool are sealed and isolated by the first sealing compartment, and the lysate storage pool communicates with the amplification reagent storage pool when the first sealing compartment is damaged, so that the lysate can enter the amplification reagent storage pool , the lower end of the amplification reagent storage pool is sealed and isolated from the entrance by the second sealing compartment, and the lower end of the amplification reagent storage pool communicates with the entrance when the second sealing compartment is damaged;
在制备上述全自动样本制备微流控系统时，首先将样品接口模块密封连接于芯片主体；然后添加扩增试剂体系溶液至样品接口模块的扩增试剂存储池，并对扩增试剂体系溶液进行干燥以形成扩增体系粉末，且在扩增试剂体系溶液干燥形成扩增体系粉末之前，保证扩增试剂存储池与芯片样本的入口之间为密封隔离状态；其后使裂解液存储池与扩增试剂存储池处于密封隔离状态，将裂解液添加至样品接口模块的裂解液存储池中；最后将样品接口模块以及芯片主体上的各接口封闭以便存储及运输；When preparing the above-mentioned fully automatic sample preparation microfluidic system, firstly, the sample interface module is sealed and connected to the chip main body; then the amplification reagent system solution is added to the amplification reagent storage pool of the sample interface module, and the amplification reagent system solution is Dry to form the amplification system powder, and before the amplification reagent system solution is dried to form the amplification system powder, ensure that the amplification reagent storage pool and the entrance of the chip sample are in a sealed and isolated state; The reagent storage pool is in a sealed and isolated state, and the lysate is added to the lysate storage pool of the sample interface module; finally, the sample interface module and the interfaces on the chip main body are sealed for storage and transportation;
在应用时，先打开裂解液存储池的密封，通过棉棒或者其他取样设备提取样本，并在裂解液存储池的裂解液中涮洗，将样本DNA分子释放到裂解液中；其后，若第二密封隔层在全自动样本制备微流控系统的制备过程中被捅破，则为避免裂解液不能充分溶解扩增体系粉末的情况，则应当关闭第一芯片阀后，再捅破第一密封隔层，使含有样本DNA分子的裂解液进入扩增试剂存储池中溶解扩增体系粉末形成完整的PCR扩增体系，利用液体的表面张力以及微流控管道内的气压避免裂解液直接进入微流控管道；若第二密封隔层在全自动样本制备微流控系统的制备过程中未被捅破，则捅破第一密封隔层，使含有样本DNA分子的裂解液进入扩增试剂存储池中溶解扩增体系粉末形成完整的PCR扩增体系后再捅破第二密封隔层；在裂解液与扩增体系粉末相互溶解后，开启第一芯片阀以及第二芯片阀，并在废液输出口处连接负压源，将PCR扩增体系引流至扩增腔室；当PCR扩增体系到达扩增腔室后，关闭第一芯片阀以及第二芯片阀，使PCR扩增体系在扩增腔室进行温度循环扩增；扩增完成后，开启第一芯片阀以及第二芯片阀，将扩增产物引流至产物输出口以进行提取；When applying, first open the seal of the lysate storage tank, extract the sample through a cotton swab or other sampling equipment, and rinse in the lysate in the lysate storage tank to release the sample DNA molecules into the lysate; then, if The second sealing compartment is pierced during the preparation process of the fully automatic sample preparation microfluidic system. In order to avoid the situation that the lysate cannot fully dissolve the powder of the amplification system, the valve of the first chip should be closed before piercing the second chip. A sealed compartment, so that the lysate containing sample DNA molecules enters the amplification reagent storage pool to dissolve the amplification system powder to form a complete PCR amplification system. The surface tension of the liquid and the air pressure in the microfluidic pipeline prevent the lysate from directly Enter the microfluidic pipeline; if the second sealed compartment is not pierced during the preparation process of the fully automatic sample preparation microfluidic system, the first sealed compartment is pierced to allow the lysate containing sample DNA molecules to enter the amplification The amplification system powder is dissolved in the reagent storage pool to form a complete PCR amplification system, and then the second sealing compartment is pierced; after the lysate and the amplification system powder dissolve each other, the first chip valve and the second chip valve are opened, and Connect the negative pressure source at the outlet of the waste liquid to drain the PCR amplification system to the amplification chamber; when the PCR amplification system reaches the amplification chamber, close the first chip valve and the second chip valve to allow PCR amplification The system performs temperature cycle amplification in the amplification chamber; after the amplification is completed, open the first chip valve and the second chip valve, and drain the amplification product to the product output port for extraction;
由此可见，上述全自动样本制备微流控系统，结构简单，便于制备，且具备独特的加样模块以及储液模式，能够对口腔拭子、唾液、脱落细胞等生物样本进行集成式的自动化处理，直接给出扩增产物，整个过程完全不需要用移液器、振荡仪、离心机等额外的实验室专用设备或仪器，使得该制备流程可以脱离实验室环境进行，操作简单，可以由稍加培训的非专业人士完成，使DNA的现场检测成为可能。It can be seen that the above-mentioned fully automatic sample preparation microfluidic system has a simple structure, is easy to prepare, and has a unique sample adding module and liquid storage mode, which can perform integrated automation on biological samples such as oral swabs, saliva, and exfoliated cells. processing, directly giving the amplification product, the whole process does not need to use additional laboratory equipment or instruments such as pipettes, oscillators, centrifuges, etc., so that the preparation process can be carried out out of the laboratory environment, the operation is simple, and can be performed by It can be done by non-professionals with a little training, making the on-site detection of DNA possible.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.
图1为本发明实施例提供的全自动样本制备微流控系统的轴测图；Figure 1 is an axonometric view of a fully automatic sample preparation microfluidic system provided by an embodiment of the present invention;
图2为本发明实施例提供的全自动样本制备微流控系统的俯视图；Fig. 2 is a top view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention;
图3为本发明实施例提供的全自动样本制备微流控系统的上层模块的轴测图；3 is an axonometric view of the upper module of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention;
图4为本发明实施例提供的全自动样本制备微流控系统的下层模块的轴测图；4 is an axonometric view of the lower module of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention;
图5为本发明实施例提供的全自动样本制备微流控系统的制备过程中芯片半成品的剖视图；Fig. 5 is a cross-sectional view of a chip semi-finished product during the preparation process of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention;
图6为本发明实施例提供的全自动样本制备微流控系统的制备完成的剖视图；6 is a cross-sectional view of the fully-automatic sample preparation microfluidic system provided by the embodiment of the present invention after preparation;
图7为本发明实施例提供的全自动样本制备微流控系统在加样操作时的剖视图；Fig. 7 is a cross-sectional view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention during the sample loading operation;
图8为本发明实施例提供的全自动样本制备微流控系统形成PCR扩增体系时的剖视图；8 is a cross-sectional view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention when forming a PCR amplification system;
图9为本发明实施例提供的全自动样本制备微流控系统在进行PCR扩增时的剖视图。Fig. 9 is a cross-sectional view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention during PCR amplification.
图中：In the picture:
1为上层模块；2为下层模块；3为裂解液存储池；4为排气孔；5为扩增试剂存储池；6为导流结构；7为芯片主体；8为扩增腔室；9为第一芯片阀；10为产物输出口；11为废液输出口；12为入口；13为微流控管道；14为第二芯片阀；15为第二密封隔层；16为扩增体系粉末；17为裂解液；18为第一密封隔层；19为棉棒。1 is the upper module; 2 is the lower module; 3 is the lysate storage pool; 4 is the air vent; 5 is the amplification reagent storage pool; 6 is the diversion structure; 7 is the chip main body; 8 is the amplification chamber; 9 10 is the product output port; 11 is the waste liquid output port; 12 is the inlet; 13 is the microfluidic pipeline; 14 is the second chip valve; 15 is the second sealing compartment; 16 is the amplification system powder; 17 is lysate; 18 is the first sealing compartment; 19 is cotton swab.
具体实施方式Detailed ways
本发明的第一个目的在于提供一种全自动样本制备微流控系统，该用于全自动样本制备微流控系统的结构设计使其可接受生物样本输入，并直接输出扩增产物，操作过程不依赖其他实验室设备，操作流程简单，降低对操作人员要求；本发明的第二个目的在于提供一种上述全自动样本制备微流控系统的制备方法及使用方法。The first object of the present invention is to provide a fully automatic sample preparation microfluidic system. The structure design of the fully automatic sample preparation microfluidic system allows it to accept the input of biological samples and directly output the amplification products. The process does not rely on other laboratory equipment, the operation process is simple, and the requirements for operators are reduced; the second purpose of the present invention is to provide a preparation method and a use method of the above-mentioned fully automatic sample preparation microfluidic system.
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
请参阅图1和图2，图1为本发明实施例提供的全自动样本制备微流控系统的轴测图，图2为本发明实施例提供的全自动样本制备微流控系统的俯视图。Please refer to Figure 1 and Figure 2, Figure 1 is an isometric view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention, and Figure 2 is a top view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention.
本发明实施例提供的一种全自动样本制备微流控系统，包括芯片主体7以及样本接口模块。A fully automatic sample preparation microfluidic system provided in an embodiment of the present invention includes a chip body 7 and a sample interface module.
其中，芯片主体7上依次设置有通过微流控管道13串联的入口12、第一芯片阀9、扩增腔室8、第二芯片阀14、产物输出口10以及废液输出口11，废液输出口11后端连接负压源，比如精密注射泵或是蠕动泵以精密控制微流控管道13内的液体位置，上述产物输出口10以及废液输出口11在芯片运作的整个过程中，其上方的开口处均由胶带粘接密封，使用时再打开；样本接口模块与芯片主体7密封连接，所述密封连接可以为密封胶粘接、超声焊接或是热封接等形式，样本接口模块包括从上到下依次设置的裂解液存储池3以及扩增试剂存储池5，裂解液存储池3与扩增试剂存储池5之间通过第一密封隔层18密封隔离，裂解液存储池3在第一密封隔层18破损时与扩增试剂存储池5连通，以使裂解液17能够进入扩增试剂存储池5中，扩增试剂存储池5的下端与入口12通过第二密封隔层15密封隔离，扩增试剂存储池5的下端在第二密封隔层15破损时与入口12连通；第一密封隔层18以及第二密封隔层15均采用易损材料制作；第一芯片阀9以及第二芯片阀14可以是现有的任意芯片阀结构，如经典的气动阀，或是微流控芯片中常用的PDMS阀体等。Wherein, the chip main body 7 is sequentially provided with an inlet 12, a first chip valve 9, an amplification chamber 8, a second chip valve 14, a product output port 10, and a waste liquid output port 11 connected in series through a microfluidic pipeline 13. The back end of the liquid output port 11 is connected to a negative pressure source, such as a precision syringe pump or a peristaltic pump to precisely control the position of the liquid in the microfluidic pipeline 13. , the opening above it is sealed by adhesive tape, and then opened when in use; the sample interface module is sealed and connected with the chip main body 7, and the sealed connection can be in the form of sealant bonding, ultrasonic welding or heat sealing, etc., the sample The interface module includes a lysate storage pool 3 and an amplification reagent storage pool 5 arranged sequentially from top to bottom. The lysate storage pool 3 and the amplification reagent storage pool 5 are sealed and isolated by a first sealing compartment 18, and the lysate storage pool 5 is sealed and isolated. Pond 3 communicates with amplification reagent storage pool 5 when the first sealing interlayer 18 is damaged, so that lysate 17 can enter in the amplification reagent storage pool 5, and the lower end of amplification reagent storage pool 5 and inlet 12 pass through the second seal. The compartment 15 is sealed and isolated, and the lower end of the amplification reagent storage pool 5 communicates with the inlet 12 when the second seal compartment 15 is damaged; the first seal compartment 18 and the second seal compartment 15 are made of fragile materials; the first The chip valve 9 and the second chip valve 14 can be any existing chip valve structure, such as a classic pneumatic valve, or a PDMS valve body commonly used in microfluidic chips.
每个全自动样本制备微流控系统的芯片主体7上可设置多条微流控管道13，并且每条微流控管的上游均设置样本接口模块，以使上述全自动样本制备微流控系统能够同时对多个样本进行检测，提高效率，本发明图示的实施例中，全自动样本制备微流控系统包括两个样本接口模块以及分别与之连接的两条微流控管道13。A plurality of microfluidic pipelines 13 can be set on the chip body 7 of each fully automatic sample preparation microfluidic system, and a sample interface module is arranged upstream of each microfluidic tube, so that the above-mentioned fully automatic sample preparation microfluidic The system can detect multiple samples at the same time to improve efficiency. In the illustrated embodiment of the present invention, the fully automatic sample preparation microfluidic system includes two sample interface modules and two microfluidic pipelines 13 respectively connected thereto.
与现有技术相比，本发明提供的用于现场DNA检测的全自动样本制备微流控系统，制备时在扩增试剂存储池5中存储扩增体系粉末16，裂解液存储池3中存储裂解液17，且扩增试剂存储池5与裂解液存储池3之间通过一易损的第一密封隔层18隔离，在使用时，将样本与裂解液17混合，使样本DNA分子释放到裂解液17中，然后捅破第一密封隔层18，使含有样本DNA分子的裂解液17进入扩增试剂存储池5中溶解扩增体系粉末16形成PCR扩增体系，且在此过程中，利用第二密封隔层15或者液体表面张力及气压保证形成的PCR扩增体系处于扩增试剂存储池5中，而不进入芯片本体的入口12；然后开启第一芯片阀9以及第二芯片阀14，并在废液输出口11处连接负压源，将PCR扩增体系引流至扩增腔室8；当PCR扩增体系到达扩增腔室8后，关闭第一芯片阀9以及第二芯片阀14，使PCR扩增体系在扩增腔室8进行温度循环扩增；扩增完成后，开启第一芯片阀9以及第二芯片阀14，将扩增产物引流至产物输出口10以进行提取；Compared with the prior art, the fully automatic sample preparation microfluidic system for on-site DNA detection provided by the present invention stores the amplification system powder 16 in the amplification reagent storage pool 5 during preparation, and stores the powder 16 in the lysate storage pool 3. The lysate 17, and the amplification reagent storage pool 5 and the lysate storage pool 3 are isolated by a fragile first sealing interlayer 18. When in use, the sample is mixed with the lysate 17, so that the sample DNA molecules are released into the in the lysate 17, and then break through the first sealing interlayer 18, so that the lysate 17 containing the sample DNA molecules enters the amplification reagent storage pool 5 to dissolve the amplification system powder 16 to form a PCR amplification system, and in the process, Utilize the second sealing interlayer 15 or liquid surface tension and air pressure to ensure that the PCR amplification system formed is in the amplification reagent storage pool 5, and does not enter the inlet 12 of the chip body; then open the first chip valve 9 and the second chip valve 14, and connect the negative pressure source at the waste liquid output port 11, and drain the PCR amplification system to the amplification chamber 8; when the PCR amplification system reaches the amplification chamber 8, close the first chip valve 9 and the second The chip valve 14 enables the PCR amplification system to perform temperature cycle amplification in the amplification chamber 8; after the amplification is completed, the first chip valve 9 and the second chip valve 14 are opened to drain the amplified product to the product output port 10 to to extract;
由此可见，上述全自动样本制备微流控系统，结构简单，便于制备，且具备独特的加样模块以及储液模式，能够对口腔拭子、唾液、脱落细胞等生物样本进行集成式的自动化处理，直接给出扩增产物，整个过程完全不需要用移液器、振荡仪、离心机等额外的实验室专用设备或仪器，使得该制备流程可以脱离实验室环境进行，操作简单，可以由稍加培训的非专业人士完成，使DNA的现场检测成为可能。It can be seen that the above-mentioned fully automatic sample preparation microfluidic system has a simple structure, is easy to prepare, and has a unique sample adding module and liquid storage mode, which can perform integrated automation on biological samples such as oral swabs, saliva, and exfoliated cells. processing, directly giving the amplification product, the whole process does not need to use additional laboratory equipment or instruments such as pipettes, oscillators, centrifuges, etc., so that the preparation process can be carried out out of the laboratory environment, the operation is simple, and can be performed by It can be done by non-professionals with a little training, making the on-site detection of DNA possible.
扩增体系粉末16可直接添加于扩增试剂存储池5中，也可先加入扩增试剂体系溶液，然后对其进行干燥形成扩增体系粉末16，扩增试剂体系溶液的干燥可以在真空干燥箱或冻干机中进行，在本发明实施例中，采用加入扩增试剂体系溶液然后干燥的方式获得扩增体系粉末16，为便于操作，样本接口模块采用上下层的结构，包括上层模块1以及下层模块2，裂解液存储池3开设于上层模块1，扩增试剂存储池5开设于下层模块2，上层模块1与下层模块2密封连接，上层模块1与下层模块2之间可使用生物兼容性好的双面胶粘接固定或者采用其他的方式密封连接固定。The amplification system powder 16 can be directly added in the amplification reagent storage pool 5, or the amplification reagent system solution can be added first, and then it is dried to form the amplification system powder 16, and the drying of the amplification reagent system solution can be performed in a vacuum. In the embodiment of the present invention, the amplification system powder 16 is obtained by adding the amplification reagent system solution and then drying. For the convenience of operation, the sample interface module adopts the structure of the upper and lower layers, including the upper layer module 1 And the lower module 2, the lysate storage pool 3 is set in the upper module 1, the amplification reagent storage pool 5 is set in the lower module 2, the upper module 1 and the lower module 2 are sealed and connected, and the upper module 1 and the lower module 2 can be used. Adhesive and fixed with good compatibility double-sided tape or sealed connection and fixed in other ways.
这样，在制备时，可先将下层模块2固定于芯片本体，加入扩增试剂体系溶液，干燥后在扩增试剂存储池5中形成扩增体系粉末16，然后再将上层模块1连接到下层模块2上，进行下一步的操作。In this way, during preparation, the lower layer module 2 can be fixed on the chip body first, the amplification reagent system solution is added, and after drying, the amplification system powder 16 is formed in the amplification reagent storage pool 5, and then the upper layer module 1 is connected to the lower layer On module 2, proceed to the next step.
在上述干燥过程中，由于表面张力的作用，扩增体系粉末16会均匀地分布在扩增试剂存储池5底部边缘地带，如图5所示，因此可在获得扩增体系粉末16将第二密封隔层15捅破以方便基因检测过程中的操作，当然，也可以保留第二密封隔层15，在后面基因检测操作时再行破坏。在本发明实施例中，为简化基因检测过程中的操作流程，在制备过程中得到扩增体系粉末16后即将第二密封隔层15破坏掉。In the above-mentioned drying process, due to the effect of surface tension, the amplification system powder 16 will be evenly distributed on the edge of the bottom of the amplification reagent storage pool 5, as shown in Figure 5, so the second amplification system powder 16 can be obtained after the amplification system powder 16 is obtained. The sealing interlayer 15 is pierced to facilitate the operation in the genetic testing process. Of course, the second sealing interlayer 15 can also be retained and destroyed again during the subsequent genetic testing operation. In the embodiment of the present invention, in order to simplify the operation process in the genetic detection process, the second sealing interlayer 15 is destroyed after the amplification system powder 16 is obtained in the preparation process.
由于第二密封隔层15破坏掉在制备过程中即被破坏，因此，在应用过程中，裂解液17进入扩增试剂存储池5与扩增体系粉末16混合形成PCR扩增体系溶液的过程中，只能依靠微流控管道13以及液体的表面张力保证PCR扩增体系溶液处于扩增试剂存储池5而不提前进入微流控管道13中，从而保证裂解液17对扩增体系粉末16的充分溶解，但由于扩增试剂存储池5与裂解液存储池3上下布置，裂解液17从上方流入会导致扩增试剂存储池5气压升高，容易导致液体的表面张力与微流控管道13中气压对PCR扩增体系溶液的支撑，从而使裂解液17过早进入微流控管道13，为解决上述问题，在本发明实施例中，扩增试剂存储池5通过排气孔4与外部连通，如图1和图3所示，进一步地，排气孔4开设在上层模块1中，并沿厚度方向贯穿上层模块1。当然，排气孔4的开设位置及形状并不唯一，比如，其可以开设在上层模块1与下层模块2的配合面中的至少一个上，也可以开设在上层模块1中但为L形等等，只要能够实现扩增试剂存储池5的排气即可。Since the second sealing interlayer 15 is destroyed during the preparation process, during the application process, the lysate 17 enters the amplification reagent storage pool 5 and mixes with the amplification system powder 16 to form the PCR amplification system solution. , can only rely on the microfluidic pipeline 13 and the surface tension of the liquid to ensure that the PCR amplification system solution is in the amplification reagent storage pool 5 without entering the microfluidic pipeline 13 in advance, so as to ensure that the lysate 17 has the same effect on the amplification system powder 16. Fully dissolved, but because the amplification reagent storage pool 5 and the lysate storage pool 3 are arranged up and down, the inflow of the lysate 17 from above will cause the air pressure in the amplification reagent storage pool 5 to rise, which will easily cause the surface tension of the liquid to interfere with the microfluidic pipeline 13 The medium air pressure supports the PCR amplification system solution, so that the lysate 17 enters the microfluidic pipeline 13 prematurely. In order to solve the above problems, in the embodiment of the present invention, the amplification reagent storage pool 5 communicates with the external Communication, as shown in FIG. 1 and FIG. 3 , further, the exhaust hole 4 is opened in the upper module 1 and penetrates the upper module 1 along the thickness direction. Of course, the location and shape of the air vent 4 are not unique, for example, it can be set on at least one of the mating surfaces of the upper module 1 and the lower module 2, or it can be set in the upper module 1 but is L-shaped, etc. etc., as long as the exhaust of the amplification reagent storage pool 5 can be realized.
在组装上层模块1与下层模块2时，务必保证裂解液存储池3以及排气孔4均能够与扩增试剂存储池5相通。When assembling the upper module 1 and the lower module 2, it must be ensured that the lysate storage pool 3 and the air vent 4 can communicate with the amplification reagent storage pool 5.
进一步优化上述技术方案，如图1和图6所示，裂解液存储池3从上到下截面尺寸渐缩，其大口端开设在上层模块1的顶部，小口端开设在上层模块1的底部。更进一步地，如图6所示，裂解液存储池3的纵截面为直角梯形。To further optimize the above technical solution, as shown in Figure 1 and Figure 6, the cross-sectional size of the lysate storage tank 3 is tapered from top to bottom. Furthermore, as shown in FIG. 6 , the longitudinal section of the lysate storage pool 3 is a right-angled trapezoid.
第一密封隔层18设置在上层模块1的底部以密封裂解液存储池3的小口端。The first sealing spacer 18 is arranged at the bottom of the upper module 1 to seal the small opening of the lysate storage tank 3 .
由于裂解液存储池3的下端开口较小，在应用过程中，当第一密封隔层18被破坏时，容易由于液体表面张力的作用，导致裂解液17无法在自身重力的作用下进入扩增试剂存储池5，为此，在本发明实施例中，扩增试剂存储池5的侧壁上设置有与裂解液存储池3相接的用于破坏液体表面张力的导流结构6，在应用时，通过该导流结构6破坏裂解液17在裂解液存储池3小口端的液面张力，使其能够顺利流入扩增试剂存储池5中，导流结构6可采用多种方式实现，比如可使扩增试剂存储池5与裂解液存储池3相接的部分侧壁的粗糙度与其他部分不同，或者，如图4所示，在本发明一种具体实施例中，导流结构6为上下贯通地开设于扩增试剂存储池5的侧壁上的导流槽，该导流槽设置有多个，在扩增试剂存储池5的侧壁上平行布置。Due to the small opening of the lower end of the lysate storage pool 3, during application, when the first sealing interlayer 18 is destroyed, it is easy to cause the lysate 17 to fail to enter the amplification under the action of its own gravity due to the effect of the surface tension of the liquid. Reagent storage pool 5, for this reason, in the embodiment of the present invention, the side wall of the amplification reagent storage pool 5 is provided with a flow guide structure 6 connected with the lysate storage pool 3 for destroying the surface tension of the liquid. At this time, the liquid surface tension of the lysate 17 at the small mouth end of the lysate storage pool 3 is destroyed by the flow guide structure 6, so that it can flow smoothly into the amplification reagent storage pool 5. The flow guide structure 6 can be realized in various ways, such as The roughness of the part of the side wall where the amplification reagent storage pool 5 is connected to the lysate storage pool 3 is different from that of other parts, or, as shown in Figure 4, in a specific embodiment of the present invention, the flow guide structure 6 is There are a plurality of flow diversion grooves penetrating up and down on the side wall of the amplification reagent storage pool 5 , and they are arranged in parallel on the side wall of the amplification reagent storage pool 5 .
在本发明实施例中，第一密封隔层18以及第二密封隔层15均由脆性材料制成，脆性材料包括但不限于铝、铜、塑料等，即第一密封隔层18以及第二密封隔层15可以为铝箔、铜箔以及塑料薄膜中的任意一种，且第一密封隔层18与第二密封隔层15所使用的脆性材料可一致，或者可根据使用需求各自采用不同的材料。In the embodiment of the present invention, both the first sealing spacer 18 and the second sealing spacer 15 are made of brittle materials, including but not limited to aluminum, copper, plastic, etc., that is, the first sealing spacer 18 and the second sealing spacer The sealing spacer 15 can be any one of aluminum foil, copper foil and plastic film, and the brittle materials used in the first sealing spacer 18 and the second sealing spacer 15 can be consistent, or different materials can be used according to the requirements of use. Material.
进一步优化上述技术方案，在本发明实施例中，为便于扩增腔室8内形成的扩增产物全部输出，产物输出口10与扩增腔室8体积相同。To further optimize the above technical solution, in the embodiment of the present invention, in order to facilitate the output of all amplification products formed in the amplification chamber 8 , the volume of the product output port 10 is the same as that of the amplification chamber 8 .
本发明实施例还提供了一种制备上述全自动样本制备微流控系统的方法，该方法包括步骤：The embodiment of the present invention also provides a method for preparing the above-mentioned fully automatic sample preparation microfluidic system, the method includes the steps of:
a1：将样品接口模块密封连接于芯片主体7；、a1: sealingly connect the sample interface module to the chip main body 7;,
样品接口模块与芯片主体7之间必须密封连接以避免漏液，具体地，密封连接可采用密封胶粘接、超声焊接或是热封接等形式，下面的步骤a2以及步骤a3可以在样品接口模块密封连接于芯片主体7上之前完成，也可以在样品接口模块连接于芯片主体7上之后完成，本发明实施例中，步骤a2以及步骤a3在样品接口模块密封连接于芯片主体7之后进行。The connection between the sample interface module and the chip main body 7 must be sealed to avoid liquid leakage. Specifically, the sealed connection can be in the form of sealant bonding, ultrasonic welding, or heat sealing. The following steps a2 and a3 can be performed on the sample interface. It is completed before the module is sealed and connected to the chip body 7, or it can be completed after the sample interface module is connected to the chip body 7. In the embodiment of the present invention, step a2 and step a3 are performed after the sample interface module is sealed and connected to the chip body 7.
a2：添加扩增试剂体系溶液至样品接口模块的扩增试剂存储池5，然后对扩增试剂体系溶液进行干燥以形成扩增体系粉末16；a2: Add the amplification reagent system solution to the amplification reagent storage pool 5 of the sample interface module, and then dry the amplification reagent system solution to form the amplification system powder 16;
在步骤a2进行过程中，保证扩增试剂体系溶液仅存在于扩增试剂存储池5，因此应当对扩增试剂存储池5下端进行密封，将其与芯片主体7的入口12密封隔离。During step a2, ensure that the amplification reagent system solution only exists in the amplification reagent storage pool 5, so the lower end of the amplification reagent storage pool 5 should be sealed to isolate it from the entrance 12 of the chip body 7.
a3：将裂解液17添加至样品接口模块的裂解液存储池3中；a3: adding the lysate 17 to the lysate storage pool 3 of the sample interface module;
在上述步骤完成后，向与扩增试剂存储池5密封隔离的裂解液存储池3中加入裂解液17，由于本案中的全自动样本制备微流控系统主要针对人体细胞的裂解，针对人体细胞，碱性条件能够较好的使细胞膜破裂释放DNA分子，因此，此处的裂解液17为碱性裂解液17，利用pH条件使细胞裂解；当然，上述碱性裂解液17仅仅是本发明根据全自动样本制备微流控系统的主要用途所做出的优选实施方案，实际并不局限于此，本领域技术人员根据全自动样本制备微流控系统用途的不同，也可加入其它类型的裂解液17，在此不做限定。After the above steps are completed, the lysate 17 is added to the lysate storage pool 3 that is sealed and isolated from the amplification reagent storage pool 5. Since the fully automatic sample preparation microfluidic system in this case is mainly aimed at the lysis of human cells, it is aimed at human cells. , alkaline conditions can better rupture the cell membrane and release DNA molecules. Therefore, the lysate 17 here is an alkaline lysate 17, and the cells are lysed by pH conditions; The preferred embodiment of the main application of the automatic sample preparation microfluidic system is not limited to this, and those skilled in the art can also add other types of lysis according to the different uses of the automatic sample preparation microfluidic system. Liquid 17 is not limited here.
a4：将样品接口模块以及芯片主体7上的各接口封闭。a4: Seal the sample interface module and the interfaces on the chip main body 7 .
待步骤a3完成，裂解液17存储到位后，可以直接将全自动样本制备微流控系统的所有接口密封后进行存储与运输。使用前再将密封外包装拆除即可。After step a3 is completed and the lysate 17 is stored in place, all interfaces of the fully automatic sample preparation microfluidic system can be directly sealed for storage and transportation. Just remove the sealed outer packaging before use.
进一步优化上述技术方案，为便于全自动样本制备微流控系统的制备，在本发明实施例中，如图1所示，样品接口模块采用了双层结构，包括上层模块1以及下层模块2，因此，扩增试剂体系溶液的加入与干燥以及裂解液17的截图可分别在两个模块上进行，互不影响，在此基础上，步骤a1具体为将样品接口模块的下层模块2密封连接于芯片主体7，然后进入步骤a2，步骤a2完成后所形成的芯片半成品的剖视图如图5所示，从图中可以看出，受液体表面张力的影响，扩增体系粉末16均匀分布在扩增试剂存储池5底部边缘地带，而将被第二密封隔层15封闭的扩增试剂存储池5底部中心空出，此时，为便于以后的应用，简化操作流程，在进入步骤a3之前，捅破扩增试剂存储池5与芯片主体7的入口12之间的第二密封隔层15使两者连通。To further optimize the above technical solution, in order to facilitate the preparation of a fully automatic sample preparation microfluidic system, in the embodiment of the present invention, as shown in Figure 1, the sample interface module adopts a double-layer structure, including an upper module 1 and a lower module 2, Therefore, the addition and drying of the amplification reagent system solution and the screenshot of the lysate 17 can be carried out on the two modules respectively without affecting each other. On this basis, step a1 is specifically to seal the lower module 2 of the sample interface module and connect it Chip main body 7, then enter step a2, the cross-sectional view of the chip semi-finished product formed after step a2 is completed is shown in Figure 5, as can be seen from the figure, affected by the surface tension of the liquid, the amplification system powder 16 is evenly distributed in the amplification The bottom edge of the reagent storage pool 5, and the center of the bottom of the amplification reagent storage pool 5 closed by the second sealing interlayer 15 is vacated. At this time, in order to facilitate future applications and simplify the operation process, before entering step a3, poke The second sealing spacer 15 between the amplification reagent storage pool 5 and the inlet 12 of the chip main body 7 connects the two.
如图6所示的实施例中，步骤a3具体为：In the embodiment shown in Figure 6, step a3 is specifically:
a3.1：将上层模块1密封连接于下层模块2，使第一密封隔层18将裂解液存储池3及扩增试剂存储池5隔离；a3.1: The upper module 1 is sealed and connected to the lower module 2, so that the first sealing compartment 18 isolates the lysate storage pool 3 and the amplification reagent storage pool 5;
上层模块1与下层模块2可通过生物兼容性好的双面胶粘接固定，当然也可以通过其他方式固定，且上层模块1的底部设置有第一密封隔层18以将裂解液存储池3与扩增试剂存储池5隔离。The upper module 1 and the lower module 2 can be bonded and fixed by double-sided tape with good biocompatibility, and of course can also be fixed by other methods, and the bottom of the upper module 1 is provided with a first sealing compartment 18 to store the lysate 3 It is isolated from the amplification reagent storage pool 5 .
a3.2：将裂解液17添加至样品接口模块的裂解液存储池3中。a3.2: Add the lysate 17 to the lysate storage pool 3 of the sample interface module.
请参阅图7-图9，图7为本发明实施例提供的全自动样本制备微流控系统在加样操作时的剖视图，图8为本发明实施例提供的全自动样本制备微流控系统形成PCR扩增体系时的剖视图，图9为本发明实施例提供的全自动样本制备微流控系统在进行PCR扩增时的剖视图，本发明实施例还提供了一种全自动样本制备微流控系统的使用方法，包括步骤：Please refer to Figures 7-9, Figure 7 is a cross-sectional view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention during sample loading operation, and Figure 8 is the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention The cross-sectional view of the PCR amplification system is formed. Figure 9 is a cross-sectional view of the fully automatic sample preparation microfluidic system provided by the embodiment of the present invention during PCR amplification. The embodiment of the present invention also provides a fully automatic sample preparation microfluidic system. How to use the control system, including steps:
b1：提取样本，并在裂解液存储池3的裂解液17中涮洗，将样本DNA分子释放到裂解液17中；b1: extract the sample, rinse it in the lysate 17 of the lysate storage pool 3, and release the sample DNA molecules into the lysate 17;
在图示实施例中，采用棉棒19提取样本，如图7所示，棉棒19蘸取样本后将其伸入裂解液存储池3中进行涮洗，涮洗过程中附着在棉棒19头上的样本细胞将会脱落或是破裂从而将样本DNA分子释放到裂解液17中。In the illustrated embodiment, a cotton swab 19 is used to extract the sample. As shown in FIG. The sample cells on the head will be detached or ruptured to release the sample DNA molecules into the lysate 17 .
b2：若第二密封隔层15在全自动样本制备微流控系统的制备过程中被捅破，则关闭第一芯片阀9后，再捅破第一密封隔层18，使含有样本DNA分子的裂解液17进入扩增试剂存储池5中溶解扩增体系粉末16形成完整的PCR扩增体系；若第二密封隔层15在全自动样本制备微流控系统的制备过程中未被捅破，则捅破第一密封隔层18，使含有样本DNA分子的裂解液17进入扩增试剂存储池5中溶解扩增体系粉末16形成完整的PCR扩增体系后再捅破第二密封隔层15；b2: If the second sealing interlayer 15 is pierced during the preparation process of the fully automatic sample preparation microfluidic system, after the first chip valve 9 is closed, the first sealing interlayer 18 is pierced to make the samples containing DNA molecules The lysate 17 enters the amplification reagent storage pool 5 to dissolve the amplification system powder 16 to form a complete PCR amplification system; , the first sealing interlayer 18 is pierced, and the lysate 17 containing sample DNA molecules enters the amplification reagent storage pool 5 to dissolve the amplification system powder 16 to form a complete PCR amplification system, and then the second sealing interlayer is pierced. 15;
涮洗后，将棉棒19往下按压到裂解液存储池3底部，将第一密封隔层18扎破，之后作为耗材的棉棒19可以丢弃。第一密封隔层18被扎破后，裂解液17由于重力作用会向下运动至扩增试剂存储池5中，在此过程中，如图7所示，第二密封隔层15在制备过程中已经被破坏，因此为使裂解液17向下流动过程可控，有三个条件必须共同满足：首先，第一芯片阀9关闭，阻断微流控管道13以防止液体进入微流控管道13入口12；其次，排气口与扩增试剂存储池5相互连通以保证扩增试剂存储池5与外部气压平衡；最后，裂解液17的下端界面与导流结构6接触以克服其下表面的表面张力；在此三个条件满足的基础上，裂解液17会沿导流结构6向下流动进入扩增试剂存储池5，由于后端第一芯片阀9关闭，扩增试剂存储池5内的空气由排气口排出，裂解液17底部的表面张力以及微流控管道13内部的气压会维持住裂解液17的底部表面，使其不进入入口12。After rinsing, press the cotton swab 19 down to the bottom of the lysate storage tank 3 to puncture the first sealing compartment 18, and then the cotton swab 19 as a consumable can be discarded. After the first sealing interlayer 18 is punctured, the lysate 17 will move down to the amplification reagent storage pool 5 due to gravity, and in this process, as shown in Figure 7, the second sealing interlayer 15 will has been destroyed, so in order to make the downward flow process of the lysate 17 controllable, three conditions must be met together: first, the first chip valve 9 is closed, blocking the microfluidic pipeline 13 to prevent liquid from entering the microfluidic pipeline 13 Inlet 12; secondly, the exhaust port communicates with the amplification reagent storage pool 5 to ensure that the amplification reagent storage pool 5 is balanced with the external air pressure; finally, the lower interface of the lysate 17 is in contact with the flow guide structure 6 to overcome the pressure on the lower surface. Surface tension; on the basis of the satisfaction of these three conditions, the lysate 17 will flow down along the guide structure 6 and enter the amplification reagent storage pool 5. Since the first chip valve 9 at the rear end is closed, the amplification reagent storage pool 5 will The air is discharged from the exhaust port, and the surface tension at the bottom of the lysate 17 and the air pressure inside the microfluidic pipeline 13 will maintain the bottom surface of the lysate 17 so that it does not enter the inlet 12.
当然，上述内容仅仅本发明提供的优选实施方案，实际上，还存在第二密封隔层15在制备过程中未被破坏的情况，在此情况下，由于第二密封隔层15依然能够起到密封隔离的作用，因此第一芯片阀9关闭与否并不重要，可待裂解液17与扩增体系粉末16混合完成后直接破坏第二密封隔层15。Of course, the above-mentioned content is only the preferred embodiment provided by the present invention. In fact, there is also a situation where the second sealing spacer 15 is not damaged during the preparation process. In this case, because the second sealing spacer 15 can still play a role Because of the role of sealing and isolation, it is not important whether the first chip valve 9 is closed or not, and the second sealing interlayer 15 can be directly destroyed after the mixing of the lysate 17 and the amplification system powder 16 is completed.
b3：在废液输出口11处连接负压源并开启第一芯片阀9以及第二芯片阀14，将PCR扩增体系引流至扩增腔室8；b3: connect the negative pressure source at the waste liquid output port 11 and open the first chip valve 9 and the second chip valve 14 to drain the PCR amplification system to the amplification chamber 8;
待步骤b2中的裂解液17将扩增体系粉末16充分溶解后，在废液输出口11处利用负压源真空抽吸将PCR扩增体系溶液引流至扩增腔室8以便进行扩增操作。After the lysate 17 in step b2 fully dissolves the amplification system powder 16, use negative pressure source vacuum suction at the waste liquid output port 11 to drain the PCR amplification system solution to the amplification chamber 8 for amplification operation .
b4：关闭第一芯片阀9以及第二芯片阀14，使PCR扩增体系在扩增腔室8进行温度循环扩增；b4: closing the first chip valve 9 and the second chip valve 14, so that the PCR amplification system performs temperature cycle amplification in the amplification chamber 8;
将第一芯片阀9与第二芯片阀14关闭以保证扩增腔室8的密封性，从而避免高温时在扩增腔室8内生成的气泡扩张，然后在扩增腔室8外部安装热源与冷源对PCR扩增体系溶液进行温度循环。Close the first chip valve 9 and the second chip valve 14 to ensure the sealing of the amplification chamber 8, thereby avoiding the expansion of the air bubbles generated in the amplification chamber 8 at high temperature, and then install a heat source outside the amplification chamber 8 Perform temperature cycling on the PCR amplification system solution with a cold source.
b5：扩增完成后，开启第一芯片阀9以及第二芯片阀14，将扩增产物引流至产物输出口10以进行提取；b5: After the amplification is completed, open the first chip valve 9 and the second chip valve 14, and drain the amplification product to the product output port 10 for extraction;
b6：提取扩增产物后的废液经废液输出口输出。b6: The waste liquid after the amplification product is extracted is output through the waste liquid output port.
本发明实施例提供的全自动样本制备微流控系统及其制备方法与使用方法，具有以下优点：1、操作简便，不需要移液器、离心机等实验室专用设备与仪器即可实现全集成自动化DNA样本制备；2、效率高，细胞裂解、DNA提取与扩增在芯片上自动进行，整个流程可以在两个小时以内完成；3、大众化，操作简单使得未经过专业训练的普通民众也能够操作仪器完成样本制备流程，为基因技术的普及与推广提供便利。The fully automatic sample preparation microfluidic system provided by the embodiment of the present invention and its preparation method and use method have the following advantages: 1. It is easy to operate and can realize full-automatic sample preparation without special laboratory equipment and instruments such as pipettes and centrifuges. Integrated automated DNA sample preparation; 2. High efficiency, cell lysis, DNA extraction and amplification are automatically carried out on the chip, and the whole process can be completed within two hours; 3. Popularity, easy operation makes it easy for ordinary people without professional training Be able to operate the instrument to complete the sample preparation process, and provide convenience for the popularization and promotion of genetic technology.
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。Each embodiment in this specification is described in a progressive manner, each embodiment focuses on the difference from other embodiments, and the same and similar parts of each embodiment can be referred to each other.
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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| CN201810502150.0ACN108728327B (en) | 2018-05-23 | 2018-05-23 | Full-automatic sample preparation micro-fluidic system and preparation method and application method thereof |
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