技术领域technical field
本发明涉及微流控液滴数字聚合酶链式反应(PCR)领域，特别是涉及一种液滴数字PCR芯片及相应检测方法。此外本发明还涉及一种具有该液滴数字PCR芯片的检测系统。The invention relates to the field of microfluidic droplet digital polymerase chain reaction (PCR), in particular to a droplet digital PCR chip and a corresponding detection method. In addition, the invention also relates to a detection system with the droplet digital PCR chip.
背景技术Background technique
实时荧光定量PCR(以下简称qPCR或qRT-PCR)技术是一种在DNA扩增反应中以荧光化学物质测每次PCR循环后产物总量的方法，其中，通过内参或者外参法对待测样品中的特定DNA序列进行定量分析。由于qPCR技术具有准确度高，线性范围宽等优势，因此，已经广泛地用于分子诊断、疾病研究、临床医学等领域。Real-time fluorescence quantitative PCR (hereinafter referred to as qPCR or qRT-PCR) technology is a method of measuring the total amount of products after each PCR cycle with fluorescent chemical substances in DNA amplification reactions. Quantitative analysis of specific DNA sequences in Due to the advantages of high accuracy and wide linear range, qPCR technology has been widely used in molecular diagnosis, disease research, clinical medicine and other fields.
通常遵循传统PCR的扩增原理，模板的Ct值和该模板的起始拷贝数存在线性关系，所以成为定量检测溶液中模板的原始拷贝数的依据。利用TaqMan探针测定PCR扩增产物的荧光曲线时，需要PCR扩增产物达到1011个拷贝/微升的饱和量，为了使10μl的PCR反应体系在30个循环后达到这样的产物浓度，PCR反应体系至少需要103个起始模板量。如果将PCR的体积减少至10纳升(nl)，则单个模板就可以在经30个PCR循环后被检测到。Generally following the amplification principle of traditional PCR, there is a linear relationship between the Ct value of the template and the initial copy number of the template, so it becomes the basis for quantitative detection of the original copy number of the template in the solution. When using TaqMan probes to measure the fluorescence curve of PCR amplification products, the PCR amplification products need to reach the saturation of1011 copies/microliter. In order to make the 10μl PCR reaction system reach such a product concentration after 30 cycles, PCR The reaction system requires at least 103 starting templates. If the PCR volume is reduced to 10 nanoliters (nl), a single template can be detected after 30 PCR cycles.
专利文献CN103451088A(一种微液滴式PCR芯片及其制作方法)公开一种使用PDMS和玻璃片作为微流控芯片的材料，亦即作为微液滴的产生芯片。其中如此得到的微液滴尺寸具有均匀、界面稳定、PCR反应效率高等优点，但由于使用PDMS作为芯片材料，而PDMS材料的透气性特别好，透水性也会有，因此在这种芯片上直接进行PCR会导致PCR反应液蒸发而造成结果不准确。并且该芯片只具有液滴生成功能，而没有对反应产物进行检测的功能，所以该芯片的功能并不完整。另外，所述芯片还在3个试剂注入口均设置了单相液滴成型微结构，结构较为复杂。Patent document CN103451088A (a micro-droplet PCR chip and its manufacturing method) discloses a microfluidic chip using PDMS and glass as materials, that is, as a micro-droplet generation chip. Among them, the micro-droplet size obtained in this way has the advantages of uniformity, stable interface, high PCR reaction efficiency, etc., but because PDMS is used as the chip material, and the gas permeability of PDMS material is particularly good, and there will be water permeability, so directly on this chip Performing PCR will cause the PCR reaction solution to evaporate and result in inaccurate results. Moreover, the chip only has the function of generating droplets, but does not have the function of detecting reaction products, so the function of the chip is not complete. In addition, the chip is also equipped with a single-phase droplet forming microstructure at the three reagent injection ports, and the structure is relatively complex.
发明内容Contents of the invention
本发明基于以下原理，利用微液滴技术可以对纳升级别的微小体积液体进行操控，具体而言将起始样本反应液分割成皮升至纳升级的微液滴，在这样大小的微液滴中最多只包含一个目的DNA或RNA模板。当完成PCR之后，通过计算阳性液滴的数量就可以推导出起始反应液中目的DNA或RNA的总模板数。微液滴芯片基于传统的单相微流控芯片技术，但相比于单相微流控系统，由于其水/油两相分离的特征，其优点在于：消耗样品和试剂量更少、混合速度更快、不易造成交叉污染、易于操控等。The present invention is based on the following principles. Micro-droplet technology can be used to manipulate micro-volume liquids at the nanoliter level. Specifically, the initial sample reaction solution is divided into micro-droplets ranging from picoliters to nanoliters. Droplets contain at most one DNA or RNA template of interest. After PCR is completed, the total template number of target DNA or RNA in the initial reaction solution can be deduced by counting the number of positive droplets. The micro-droplet chip is based on the traditional single-phase microfluidic chip technology, but compared with the single-phase microfluidic system, due to its water/oil two-phase separation characteristics, its advantages are: less sample and reagent consumption, mixing Faster, less likely to cause cross-contamination, easy to operate, etc.
本发明提出一种液滴数字PCR芯片，具有一个单元或者两个或多个相同单元，单元包括：油相储液池、PCR起始反应液储液池和液滴储存池，连接油相储液池的流道和连接PCR起始反应液储液池的流道汇合，成为通向液滴储存池的液滴导流流道，其中液滴储存池部分地或全部位于在油相储液池和PCR起始反应液储液池的下方，用于收集所生成的液滴并进行PCR扩增反应。The present invention proposes a droplet digital PCR chip, which has one unit or two or more identical units, and the unit includes: an oil phase storage pool, a PCR initial reaction liquid storage pool and a droplet storage pool, connected to the oil phase storage pool The flow channel of the liquid pool and the flow channel connected to the PCR initial reaction liquid storage pool merge to become a droplet diversion channel leading to the droplet storage pool, wherein the droplet storage pool is partially or completely located in the oil phase storage liquid Below the pool and the PCR initial reaction solution storage pool, it is used to collect the generated droplets and perform PCR amplification reaction.
按照本发明，由油相储液池流出的油相与由起始反应液储液池流出的起始反应液交汇，生成油包水型液滴。所生成的液滴经流道汇入液滴储存池并在此进行PCR扩增反应。According to the present invention, the oil phase flowing out from the oil phase liquid storage tank and the initial reaction liquid flowing out from the initial reaction liquid storage tank meet to form water-in-oil droplets. The generated droplets flow into the droplet storage pool through the flow channel, where the PCR amplification reaction is carried out.
按照本发明，所述单元可以包括两个或更多个PCR起始反应液储液池，以容纳不同的PCR反应物。在这种情况下，来自所有PCR起始反应液储液池的液体首先汇合形成混合物，然后与由油相储液池流出的油相交汇，生成油包水型液滴。According to the present invention, the unit may comprise two or more PCR starter reaction solution reservoirs to accommodate different PCR reactions. In this case, the liquids from all the PCR starter reaction reservoirs first join to form a mixture, and then meet the oil phase flowing from the oil phase reservoir to form water-in-oil droplets.
按照本发明，可以通过在芯片上设置的单层液滴平铺腔，将液滴储存池中经过PCR扩增反应后的液滴引入单层液滴平铺腔，对位于单层液滴平铺腔中的液滴进行PCR扩增产物的检测。因此，按照本发明的芯片的每个单元还包括用于检测PCR扩增产物的单层液滴平铺腔和废液储液池，单层液滴平铺腔通过流道与液滴储存池相通，废液储液池通过流道与单层液滴平铺腔和液滴储存池相通。其中有利地，所述芯片可以具有上中下三层式结构，其中上层结构包括所述油相储液池、PCR起始反应液储液池、废液储液池；中层结构包括所述流道和液滴导流流道以及单层液滴平铺腔；下层结构包括液滴储存池。According to the present invention, the droplets after PCR amplification reaction in the droplet storage pool can be introduced into the single-layer droplet-spreading cavity through the single-layer droplet-spreading cavity provided on the chip, The droplets in the paving chamber are used for detection of PCR amplification products. Therefore, each unit of the chip according to the present invention also includes a single-layer droplet tiling chamber and a waste liquid storage tank for detecting PCR amplification products, and the single-layer droplet tiling chamber passes through the flow channel and the droplet storage tank. In communication, the waste liquid storage tank communicates with the single-layer droplet tiling cavity and the droplet storage tank through the flow channel. Advantageously, the chip can have an upper, middle and lower three-layer structure, wherein the upper layer structure includes the oil phase reservoir, the PCR initial reaction solution reservoir, and the waste liquid reservoir; the middle layer structure includes the flow reservoir. The channel and the droplet diversion channel and the single-layer droplet tiling cavity; the lower structure includes the droplet storage pool.
按照本发明，在另一实施形式中，位于液滴储存池中的液滴在经过PCR扩增反应后，也可以使用吸液针，从液滴储存池中吸出液滴，对液滴进行PCR扩增产物的检测。因此，在本发明一种有利的实施形式中，所述芯片可以具有上中下三层式结构，其中上层结构包括所述油相储液池、PCR起始反应液储液池；中层结构包括所述流道和液滴导流流道；所述液滴储存池贯穿所述上中下三层，以便插入吸液针。According to the present invention, in another implementation form, after the droplets located in the droplet storage tank undergo a PCR amplification reaction, a liquid suction needle can also be used to suck out the droplets from the droplet storage tank, and perform PCR on the droplets. Detection of amplification products. Therefore, in an advantageous implementation form of the present invention, the chip can have an upper, middle and lower three-layer structure, wherein the upper layer structure includes the oil phase reservoir and the PCR initial reaction solution reservoir; the middle layer structure includes The flow channel and the droplet diversion channel; the droplet storage tank runs through the upper, middle and lower layers, so as to insert a liquid suction needle.
按照本发明提出的微流控芯片集成了液滴生成和PCR扩增的功能以及任选的PCR扩增产物检测功能，从而可极大的提高利用液滴进行低拷贝基因的数字PCR检测。此外，按照本发明提出的芯片无需在各试剂注入口设置单相液滴成型微结构，结构更为简单。The microfluidic chip proposed according to the present invention integrates the functions of droplet generation, PCR amplification and optional detection of PCR amplification products, thereby greatly improving the digital PCR detection of low-copy genes by using droplets. In addition, the chip proposed in accordance with the present invention does not need to arrange a single-phase liquid droplet forming microstructure at each reagent injection port, and the structure is simpler.
此外有利地，在按照本发明芯片的应用中采用具有缺口、例如米字或十字缺口的硅胶塞对所述各储液池进行密封。通过采用具有米字或十字缺口的硅胶塞进行密封，从而有效避免交叉污染。Furthermore, it is advantageous for the application of the chip according to the invention to seal the reservoirs with silicone plugs having notches, for example Pozi or cross notches. By adopting a silicone plug with a Pozi or cross notch for sealing, cross-contamination can be effectively avoided.
此外有利地，当按照本发明的芯片包括单层液滴平铺腔时，在用于引导液滴进入液滴储存池的流道中的出口部分设有向下引流通道，以便这样生成的液滴流体直接汇入所述液滴储存池。通过这样的设计，可以有效避免微腔道的虹吸现象产生，从而可以防止液滴直接流入单层液滴平铺腔。Furthermore advantageously, when the chip according to the invention comprises a single-layer droplet tiling cavity, a downward drainage channel is provided at the outlet portion of the flow channel for guiding the droplets into the droplet reservoir, so that the droplets thus generated Fluid flows directly into the droplet reservoir. Through such a design, the siphon phenomenon of the microcavity can be effectively avoided, thereby preventing the droplet from directly flowing into the single-layer droplet flattening cavity.
在一种实施形式中，所述油相储液池、PCR起始反应液储液池、单层液滴平铺腔、废液储液池和所述的流道形成上半芯片，所述液滴储存池形成下半芯片，所述上半芯片和下半芯片封接为一个整体。在另一实施形式中，所述油相储液池、PCR起始反应液储液池、所述流道和所述液滴储存池的上部分形成上半芯片，所述液滴储存池下部分形成下半芯片，所述上半芯片和下半芯片封接为一个整体。In one embodiment, the oil phase reservoir, the PCR initial reaction solution reservoir, the single-layer droplet tiling chamber, the waste liquid reservoir and the flow channel form the upper half of the chip, and the The droplet storage pool forms the lower half chip, and the upper half chip and the lower half chip are sealed as a whole. In another embodiment, the upper part of the oil phase storage tank, the PCR initial reaction solution storage tank, the flow channel and the droplet storage tank forms an upper half chip, and the lower part of the droplet storage tank A lower half chip is formed, and the upper half chip and the lower half chip are sealed as a whole.
有利地，所述上半芯片和下半芯片经热压键合封接为一个整体。Advantageously, the upper half chip and the lower half chip are sealed as a whole through thermocompression bonding.
此外，按照本发明的芯片可以由具有良好光学性和能耐受PCR反应温度的材料制成。作为实例，所述材料可以为聚碳酸酯(PC)塑料或环烯烃共聚物(COC)材料。Furthermore, the chip according to the present invention can be made of a material that has good optics and can withstand the temperature of the PCR reaction. As an example, the material may be polycarbonate (PC) plastic or cycloolefin copolymer (COC) material.
当按照本发明的芯片由聚碳酸酯(PC)塑料或环烯烃共聚物(COC)材料制成时，可采用热压键合实现芯片的封接，从而在PCR整个过程中都处于完全封闭的环境中，因此这样可以有效的避免PCR过程产生DNA气溶胶的问题，为实际的应用提供有效的解决方案。When the chip according to the present invention is made of polycarbonate (PC) plastics or cyclic olefin copolymer (COC) material, thermocompression bonding can be used to realize the sealing of the chip, so that it is in a completely closed environment during the entire process of PCR. environment, so this can effectively avoid the problem of DNA aerosol generated during the PCR process, and provide an effective solution for practical applications.
此外有利地，按照本发明的芯片由聚碳酸酯(PC)塑料或环烯烃共聚物(COC)材料注塑成型。由于按照本发明的芯片是采用注塑成型并且在应用中一般是一次性使用的，因此按照本发明的芯片相比于采用PDMS材料的芯片是在成本上是非常有利的。此外由于采用耐高温材质PC或COC制作芯片，那么无需取出所生成的液滴，通过薄壁的锥形孔(液滴储存池)就可以直接进行PCR温控反应(升温和降温)。Furthermore advantageously, the chip according to the invention is injection molded from polycarbonate (PC) plastic or cycloolefin copolymer (COC) material. Since the chips according to the invention are injection-molded and generally disposable in application, the chips according to the invention are very cost-effective compared with chips using PDMS material. In addition, since the high temperature resistant material PC or COC is used to make the chip, there is no need to take out the generated droplets, and the PCR temperature control reaction (heating and cooling) can be directly performed through the thin-walled tapered hole (droplet storage pool).
作为一个实例，按照本发明的芯片中的所述油相储液池的内腔具有0.5cm的直径、0.8cm的深度以及1.2mm的壁厚；所述PCR起始反应液储液池的内腔具有0.6cm的直径、0.8cm的深度、1.2mm的壁厚；所述流道具有200μm的宽度、50μm的深度；所述单层液滴平铺腔具有0.9cm的宽度、150μm的深度；所述液滴储存池的形状为锥形，其具有0.6cm的上圈直径、0.3cm的下圈直径、18度的锥度、1cm的内部深度以及0.5mm的壁厚。As an example, according to the inner chamber of the oil phase reservoir in the chip of the present invention has a diameter of 0.5cm, a depth of 0.8cm and a wall thickness of 1.2mm; The cavity has a diameter of 0.6 cm, a depth of 0.8 cm, and a wall thickness of 1.2 mm; the flow channel has a width of 200 μm and a depth of 50 μm; the single-layer droplet tiling cavity has a width of 0.9 cm and a depth of 150 μm; The shape of the droplet reservoir is conical with an upper ring diameter of 0.6 cm, a lower ring diameter of 0.3 cm, a taper of 18 degrees, an internal depth of 1 cm, and a wall thickness of 0.5 mm.
此外，本发明提出一种使用按照本发明的芯片的相应检测方法，包括以下步骤：Furthermore, the invention proposes a corresponding detection method using the chip according to the invention, comprising the following steps:
将油相和PCR起始反应液分别添加到油相储液池和PCR起始反应液储液池中并且施加具有预留通气孔的硅胶帽；Add the oil phase and the PCR initial reaction solution to the oil phase reservoir and the PCR initial reaction solution reservoir respectively and apply a silica gel cap with a reserved vent hole;
分别在所述油相储液池和PCR起始反应液储液池施加气压并持续特定时间，以便使分别来自油相储液池和PCR起始反应液储液池的液体汇合而生成大量大小均匀的小液滴；和Apply air pressure to the oil phase liquid storage pool and the PCR initial reaction liquid storage pool respectively and continue for a specific time, so that the liquids from the oil phase liquid storage pool and the PCR initial reaction liquid storage pool are combined to generate a large number of uniform small droplets; and
当所述生成的液滴都汇入所述液滴储存池后进行PCR扩增反应；和Performing a PCR amplification reaction after the generated droplets are all merged into the droplet storage pool; and
当PCR扩增反应结束后，读取PCR反应结果。After the PCR amplification reaction is finished, read the PCR reaction result.
有利地，该方法还可以包括以下步骤：Advantageously, the method may also include the following steps:
当PCR扩增反应结束后，经所述油相储液池和/或PCR起始反应液储液池给所述液滴储存池注入比重大于液滴比重的重油，使得所述液滴能流入所述单层液滴平铺腔；After the PCR amplification reaction is over, inject heavy oil with a specific gravity greater than that of the droplet into the droplet storage tank through the oil phase storage tank and/or the PCR initial reaction solution storage tank, so that the droplet can flow into The monolayer droplet tiling cavity;
对位于单层液滴平铺腔中的液滴，读取PCR反应结果。For the droplets located in the monolayer droplet tiling chamber, the PCR reaction results are read.
其中，利用按照本发明的芯片采用油相抬升的方法在实现PCR反应后驱动液滴流入单层平铺腔内。由于所述油的比重大于液滴的比重，注入液滴储存池的油位于液滴之下，从而使液滴储存池的液面抬高，使液滴能从出口流出，平铺成单层的液滴，方便对PCR结果的记录。Wherein, the chip according to the present invention adopts the method of lifting the oil phase to drive the droplet to flow into the single-layer tiling cavity after realizing the PCR reaction. Since the specific gravity of the oil is greater than that of the droplets, the oil injected into the droplet storage tank is located under the droplets, thereby raising the liquid level of the droplet storage tank, so that the droplets can flow out from the outlet and spread into a single layer Droplets are convenient for recording PCR results.
在此优选地，对位于单层液滴平铺腔中的液滴，使用带有特定滤镜的荧光显微镜读取PCR反应结果。Preferably, for the droplets located in the monolayer droplet tiling chamber, the PCR reaction results are read using a fluorescence microscope with a specific filter.
有利地，在按照本发明提出的方法中，在PCR扩增反应结束后经油相储液池给液滴储存池注入油的步骤中使用100mbars气压并持续5分钟，从而液滴储存池的液面持续抬高，以至于实现液滴能从出口流入单层液滴平铺腔中。Advantageously, in the method proposed according to the present invention, the step of injecting oil into the droplet storage tank through the oil phase liquid storage tank after the PCR amplification reaction is completed uses an air pressure of 100 mbars and lasts for 5 minutes, so that the liquid in the droplet storage tank The surface is continuously raised, so that the droplets can flow from the outlet into the single-layer droplet tiling cavity.
在另一有利实施形式中，该方法还可以包括以下步骤：当PCR扩增反应结束后，通过吸液针把经反应后的液滴从所述液滴储存池吸到透明检测装置中；对位于所述透明检测装置中的液滴，读取PCR反应结果。In another advantageous implementation form, the method may also include the following steps: after the PCR amplification reaction is completed, suck the reacted droplet from the droplet storage pool into the transparent detection device through a pipette needle; The liquid droplet located in the transparent detection device reads the PCR reaction result.
其中优选地，使用PMT光电倍增管模块读取PCR反应结果。Among them, preferably, a PMT photomultiplier tube module is used to read the PCR reaction result.
此外，本发明还提出一种检测系统，包括：In addition, the present invention also proposes a detection system, including:
按照本发明的PCR芯片；PCR chip according to the present invention;
用于PCR扩增反应的循环加热装置；和Circulator heating means for PCR amplification reactions; and
PCR结果读取装置。PCR result reading device.
在一个实施形式中，PCR结果读取装置可以包括：In one embodiment, the PCR result reading device may include:
激光光源，其位于在芯片区域正上方并且具有45°入射方向；A laser light source, which is located directly above the chip area and has a 45° incident direction;
可变焦镜头和CCD相机，它们位于在所述芯片区域正上方；A zoom lens and a CCD camera located directly above the chip area;
带通荧光滤光片，其位于在所述可变焦镜头与CCD相机之间。A bandpass fluorescence filter is located between the variable focus lens and the CCD camera.
有利地，激发光源包括发光二极管(LED)和15°透镜以及带通激发光滤光片，其中带通激发光滤光片具有473nm的中心波长和10nm的带宽。其中，15°透镜和带通激发光滤光片分别用于发光二极管发出的光的聚焦和过滤，而45°斜射角度可以确保在芯片的单层液滴平铺区域上的均匀照射，从而可有效降低激发光散射背景，提高荧光检测的灵敏度。Advantageously, the excitation light source comprises a light emitting diode (LED) and a 15° lens and a bandpass excitation filter having a center wavelength of 473nm and a bandwidth of 10nm. Among them, the 15° lens and the band-pass excitation light filter are used to focus and filter the light emitted by the light-emitting diode, and the 45° oblique angle can ensure uniform irradiation on the single-layer droplet tiling area of the chip, so that Effectively reduce the excitation light scattering background and improve the sensitivity of fluorescence detection.
有利地，带通荧光滤光片具有535nm的中心波长和40nm的带宽。具体地，在应用中在芯片上液滴内部的荧光被激发并被上方的可变焦镜头收集，经该带通荧光滤光片过滤后进入CCD相机采集荧光图片，从而实现PCR结果的获取。Advantageously, the bandpass fluorescence filter has a center wavelength of 535nm and a bandwidth of 40nm. Specifically, in the application, the fluorescence inside the droplet on the chip is excited and collected by the upper zoom lens, filtered by the band-pass fluorescence filter, and then enters the CCD camera to collect fluorescence pictures, thereby realizing the acquisition of PCR results.
在另一有利实施形式中，PCR结果读取装置可以包括：吸液针和PMT光电倍增管模块。In another advantageous embodiment, the PCR result reading device can comprise: a pipetting needle and a PMT photomultiplier tube module.
有利地，PCR结果读取装置还可以包括透明检测装置。Advantageously, the PCR result reading device may also include a transparent detection device.
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描述本发明的一部分实施例。这些附图对于本发明来说并不是限制性的，而是起示例性的作用。其中：In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art. It is obvious that the drawings in the following description only depict some embodiments of the present invention. The drawings are not limiting to the invention, but are illustrative. in:
图1示意地示出按照本发明的液滴数字PCR芯片的正面和对应截面图；Fig. 1 schematically shows the front and corresponding cross-sectional views of the droplet digital PCR chip according to the present invention;
图2示意地示出按照本发明的相应检测方法200流程图。FIG. 2 schematically shows a flow chart of a corresponding detection method 200 according to the invention.
图3示意地示出按照本发明的另一种液滴PCR芯片的正面和对应截面图。Fig. 3 schematically shows a front view and a corresponding cross-sectional view of another droplet PCR chip according to the present invention.
具体实施方式Detailed ways
图1示意地示出按照本发明的液滴数字PCR芯片的正面和对应截面图。在图1的上部分是芯片的正面图，下部分是相应的截面图。Fig. 1 schematically shows a front view and a corresponding cross-sectional view of a droplet digital PCR chip according to the present invention. The upper part of FIG. 1 is the front view of the chip, and the lower part is the corresponding cross-sectional view.
如图1上部分的芯片正面图所示，该芯片作为一种液滴PCR芯片具有上下两个相同单元，每个单元包括：油相储液池1、流道2、PCR起始反应液储液池3、液滴导流流道4、液滴储存池5、单层液滴平铺腔6、支撑线7以及废液储液池8。此外，在应用时还包括施加在各储液池上用于密封的硅胶塞9。其中，PCR起始反应液储液池3、废液储液池8、流道2和4、单层液滴平铺腔6形成上半芯片，而液滴储存池5形成下半芯片，上半芯片和下半芯片均由PC塑料经注塑并且经热压键合封接为一个整体。As shown in the front view of the upper part of the chip in Figure 1, the chip, as a droplet PCR chip, has two identical units above and below, and each unit includes: an oil phase liquid storage pool 1, a flow channel 2, and a PCR initial reaction liquid storage tank. A liquid pool 3 , a droplet diversion channel 4 , a droplet storage pool 5 , a single-layer droplet flattening cavity 6 , a support line 7 and a waste liquid storage pool 8 . In addition, when applied, it also includes a silicone plug 9 applied to each liquid reservoir for sealing. Among them, the PCR initial reaction liquid storage tank 3, the waste liquid storage tank 8, the flow channels 2 and 4, and the single-layer droplet tiling chamber 6 form the upper half of the chip, while the droplet storage tank 5 forms the lower half of the chip, and the upper half of the chip is formed by the upper half of the chip. Both the half-chip and the lower half-chip are injection molded from PC plastic and sealed as a whole by thermocompression bonding.
从图1中下部分的截面图来看，该芯片具有上中下三层结构。上层结构包括油相储液池1、PCR起始反应液储液池3、废液储液池8。中层结构包括各储液池相连的流道例如流道2和液滴导流流道4以及单层液滴平铺腔6。下层结构只包括液滴储存池5。From the cross-sectional view of the lower part in Figure 1, the chip has a three-layer structure of upper, middle and lower. The superstructure includes an oil phase storage tank 1 , a PCR initial reaction solution storage tank 3 , and a waste liquid storage tank 8 . The middle layer structure includes flow channels connected to each liquid reservoir, such as the flow channel 2 and the droplet guide flow channel 4 , and a single-layer droplet flattening chamber 6 . The lower structure only includes the droplet storage tank 5 .
其中，油相储液池1的内腔具有0.5cm的直径、0.8cm的深度以及1.2mm的壁厚；PCR起始反应液储液池3的内腔具有0.6cm的直径、0.8cm的深度、1.2mm的壁厚；流道2、4具有200μm的宽度、50μm的深度；单层液滴平铺腔6具有0.9cm的宽度、150μm的深度；液滴储存池5的形状为锥形，其具有0.6cm的上圈直径、0.3cm的下圈直径、18度的锥度、1cm的内部深度以及0.5mm的壁厚。Wherein, the inner chamber of the oil phase reservoir 1 has a diameter of 0.5 cm, a depth of 0.8 cm and a wall thickness of 1.2 mm; the inner chamber of the PCR initial reaction liquid reservoir 3 has a diameter of 0.6 cm and a depth of 0.8 cm , a wall thickness of 1.2mm; the flow channels 2 and 4 have a width of 200 μm and a depth of 50 μm; the single-layer droplet tiling cavity 6 has a width of 0.9 cm and a depth of 150 μm; the shape of the droplet storage pool 5 is conical, It has an upper ring diameter of 0.6 cm, a lower ring diameter of 0.3 cm, a taper of 18 degrees, an internal depth of 1 cm, and a wall thickness of 0.5 mm.
从功能上来讲，油相储液池1和PCR起始反应液储液池3用于将起始反应液分割成体积在0.5-1纳升的液滴反应液，即来自油相储液池1的油相和PCR起始反应液储液池3的起始反应液汇合，生成0.5-1纳升的油包水型液滴反应液；液滴存储池5用于收集液滴并进行PCR；当PCR完成后，单层液滴平铺腔6用于接收从液滴存储池5导入的液滴，以便随后实现采用适合的方式读取相关PCR结果，例如利用特定滤镜的荧光显微镜。From a functional point of view, the oil phase reservoir 1 and the PCR initial reaction solution reservoir 3 are used to divide the initial reaction solution into droplet reaction solutions with a volume of 0.5-1 nanoliters, that is, from the oil phase reservoir The oil phase of 1 and the initial reaction solution of PCR initial reaction solution storage tank 3 are combined to generate 0.5-1 nanoliter water-in-oil droplet reaction solution; droplet storage tank 5 is used to collect droplets and perform PCR ; When the PCR is completed, the single-layer droplet tiling chamber 6 is used to receive the droplets imported from the droplet storage pool 5, so that the relevant PCR results can be read in a suitable way, such as using a fluorescent microscope with a specific filter.
特别地，由于液滴导流流道4专门用于引导液滴汇入液滴储存池5，在此可设有向下引流通道，以便这样生成的液滴流体直接汇入所述液滴储存池。该设计的目的在于，有效防止在狭小的流道腔内比较显著的液体虹吸作用。In particular, since the droplet guide channel 4 is specially used to guide the droplets into the droplet storage pool 5, a downward drainage channel may be provided here so that the droplet fluid thus generated directly flows into the droplet storage tank 5. pool. The purpose of this design is to effectively prevent the relatively significant liquid siphon effect in the narrow channel cavity.
图2示意地示出按照本发明的相应检测方法200的流程图。方法200包括以下步骤：FIG. 2 schematically shows a flow chart of a corresponding detection method 200 according to the invention. Method 200 includes the following steps:
将油相和PCR起始反应液分别添加到油相储液池和PCR起始反应液储液池中并且施加具有预留通气孔的硅胶帽201；Adding the oil phase and the PCR initial reaction solution to the oil phase reservoir and the PCR initial reaction solution reservoir respectively, and applying a silica gel cap 201 with a reserved vent hole;
分别在所述油相储液池和PCR起始反应液储液池施加气压并持续特定时间，以便使分别来自油相储液池和PCR起始反应液储液池的液体汇合而生成大量大小均匀的小液滴202；和Apply air pressure to the oil phase liquid storage pool and the PCR initial reaction liquid storage pool respectively and continue for a specific time, so that the liquids from the oil phase liquid storage pool and the PCR initial reaction liquid storage pool are combined to generate a large number of uniform small droplets 202; and
当所述生成的液滴都汇入所述液滴储存池后进行PCR扩增反应203；和Perform PCR amplification reaction 203 after the generated droplets are all merged into the droplet storage pool; and
当PCR扩增反应结束后，读取PCR反应结果205。When the PCR amplification reaction is over, read the PCR reaction result 205 .
其中有利地，该方法还可以包括以下步骤：当PCR扩增反应结束后，经所述油相储液池和/或PCR起始反应液储液池给所述液滴储存池注入比重重于液滴的重油，使得所述液滴能流入所述单层液滴平铺腔204；Advantageously, the method may also include the following steps: after the PCR amplification reaction is finished, inject the droplet storage tank with a specific gravity greater than the heavy oil of the droplets so that the droplets can flow into the single layer droplet flattening cavity 204;
随后对位于单层液滴平铺腔中的液滴，读取PCR反应结果205。Then, for the droplets located in the monolayer droplet tiling chamber, the PCR reaction result is read 205 .
其中，利用按照本发明的芯片采用油相抬升的方法在实现PCR反应后驱动液滴流入单层平铺腔内。由于所述油的比重大于液滴的比重，注入液滴储存池的油位于液滴之下，从而使液滴储存池的液面抬高，使液滴能从出口流出，平铺成单层的液滴，方便对PCR结果的记录。Wherein, the chip according to the present invention adopts the method of lifting the oil phase to drive the droplet to flow into the single-layer tiling cavity after realizing the PCR reaction. Since the specific gravity of the oil is greater than that of the droplets, the oil injected into the droplet storage tank is located under the droplets, thereby raising the liquid level of the droplet storage tank, so that the droplets can flow out from the outlet and spread into a single layer Droplets are convenient for recording PCR results.
在方法200中一次性地使用上述按照本发明提出的具有如图1所述结构的芯片，按照本发明的芯片由聚碳酸酯(PC)塑料或环烯烃共聚物(COC)材料注塑成型，因此相比于采用PDMS材料的芯片是在成本上是非常有利的。In method 200, the above-mentioned chip proposed according to the present invention with the structure as shown in FIG. Compared with the chip using PDMS material, it is very advantageous in cost.
具体而言，在步骤201中，在油相储液池1中添加50μl油作为油相，添加25μl PCR起始反应液在储存池3中。完成添加之后，在两个储液池上分别施加如图1所示的专用单向进气硅胶帽9；Specifically, in step 201 , 50 μl of oil is added to the oil phase reservoir 1 as the oil phase, and 25 μl of PCR initial reaction solution is added to the reservoir 3 . After the addition is completed, the special one-way air inlet silica gel cap 9 shown in Figure 1 is respectively applied on the two liquid reservoirs;
随后，在步骤202中，分别给油相储液池2和PCR起始反应液储液池3施加100mbars和125mbars的气压，这样在油相和PCR反应液的交汇处会形成大约20000-30000个大小均匀的微液滴，这些形成的微液滴经液滴导流流道4汇聚到液滴储存池5里面。为了更好地收集所形成的液滴，在液滴流出口设计了一个向下引流的流道，这样生成液滴后流体会随着流道直接汇入液滴储存池。液滴生成的时间大约需要10分钟。Subsequently, in step 202, the air pressure of 100 mbars and 125 mbars is applied to the oil phase reservoir 2 and the PCR initial reaction solution reservoir 3 respectively, so that about 20000-30000 cells will be formed at the intersection of the oil phase and the PCR reaction solution. Micro-droplets of uniform size, these formed micro-droplets converge into the droplet storage tank 5 through the droplet diversion channel 4 . In order to better collect the formed droplets, a downward drainage channel is designed at the droplet outlet, so that after the droplets are generated, the fluid will flow directly into the droplet storage pool along with the flow channel. The droplet generation time takes about 10 minutes.
当液滴生成完毕、生成的液滴全部都汇入液滴储液池5中之后，在步骤203中进行PCR。例如，可以根据用户具体设置好的PCR反应程序进行PCR反应。After the droplet generation is completed and all the generated droplets are merged into the droplet reservoir 5 , PCR is performed in step 203 . For example, the PCR reaction can be performed according to the PCR reaction program specifically set by the user.
当PCR结束后，进入步骤204，其中在油相储存池1中注入50-80μl油，所述油的比重大于液滴，而且所述油与液滴这两相是互不相容的，随后给油相储存池1施加100mbars的气压约5分钟，任选同时给PCR反应液储存池3上施加20mbars的气压。由此，添加的油流入液滴储存池5中位于底部从而抬高液面，使得上层的液滴可以流入单层液滴平铺腔6内。由于在芯片的生产过程中平铺腔高度为150μm，而该芯片在上述气压的控制下形成的液滴直径在100-150μm之间，因此，平铺腔中的液滴只会慢慢流入形成单层的液滴面。After PCR finishes, enter step 204, wherein inject 50-80 μ l oil in oil phase reservoir 1, the specific gravity of described oil is greater than liquid drop, and described oil and liquid drop these two phases are mutually incompatible, then Apply an air pressure of 100 mbars to the oil phase storage tank 1 for about 5 minutes, and optionally apply an air pressure of 20 mbars to the PCR reaction solution storage tank 3 at the same time. As a result, the added oil flows into the droplet storage pool 5 at the bottom to raise the liquid level, so that the upper layer of droplets can flow into the single-layer droplet flattening cavity 6 . Since the height of the tiling cavity is 150 μm during the production process of the chip, and the diameter of the droplets formed by the chip under the control of the above-mentioned air pressure is between 100-150 μm, the droplets in the tiling cavity will only slowly flow into the formation Single-layer droplet surface.
随后，在步骤205中，使用特定检测系统进行荧光成像，再通过相关的软件对检测结果的进行分析，最后可以计算出PCR起始反应液中总的DNA模板数。Subsequently, in step 205, use a specific detection system to perform fluorescence imaging, and then analyze the detection results through relevant software, and finally calculate the total number of DNA templates in the PCR initial reaction solution.
其中该特定检测系统可以设计如下：除了按照本发明提出的PCR芯片之外，该检测系统还可以包括：用于PCR的循环加热装置；激光光源，其位于在芯片区域正上方并且具有45°入射方向；可变焦镜头和CCD相机，它们位于在所述芯片区域正上方；带通荧光滤光片，其位于在所述可变焦镜头与CCD相机之间。Wherein the specific detection system can be designed as follows: in addition to the PCR chip proposed according to the present invention, the detection system can also include: a cycle heating device for PCR; a laser light source, which is positioned directly above the chip area and has a 45° incidence direction; a zoom lens and a CCD camera, which are located directly above the chip area; a bandpass fluorescence filter, which is located between the zoom lens and the CCD camera.
在该例子中，激发光源包括发光二极管(LED)和15°透镜以及带通激发光滤光片，其中所述带通激发光滤光片具有473nm的中心波长和10nm的带宽。此外，所述带通荧光滤光片具有535nm的中心波长和40nm的带宽。In this example, the excitation light source included a light emitting diode (LED) and a 15° lens and a bandpass excitation filter with a center wavelength of 473 nm and a bandwidth of 10 nm. In addition, the bandpass fluorescence filter has a center wavelength of 535nm and a bandwidth of 40nm.
在按照本发明的方法的具体应用中，在PCR反应结束之后，系统中的发光二极管经由15°透镜以及带通激发光滤光片，从芯片上方45°均匀斜射在芯片的单层液滴平铺区域6。15°透镜和带通激发光滤光片分别用于聚焦和过滤。在此，45°的斜射光路可有效降低激发光散射背景，从而提高荧光检测的灵敏度。由此激发液滴内部的荧光之后，上方的可变焦镜头可进行收集，并经带通荧光滤光片过滤后进入CCD相机，通过CCD相机采集荧光图片从而获取PCR反应结果。In the specific application of the method according to the present invention, after the PCR reaction is completed, the light-emitting diode in the system shoots uniformly obliquely at 45° above the chip on the surface of the single-layer droplet on the chip through a 15° lens and a band-pass excitation filter. Pave area 6. A 15° lens and a bandpass excitation filter are used for focusing and filtering, respectively. Here, the 45° oblique light path can effectively reduce the excitation light scattering background, thereby improving the sensitivity of fluorescence detection. After the fluorescence inside the droplet is thus excited, it can be collected by the zoom lens above, filtered by a band-pass fluorescence filter, and then entered into the CCD camera, and the fluorescence picture is collected by the CCD camera to obtain the PCR reaction result.
图1所示的芯片集成了液滴生成、PCR扩增和PCR扩增产物检测。按照本发明提出的芯片也可以仅集成液滴生成和PCR扩增的功能，如图3所示。The chip shown in Figure 1 integrates droplet generation, PCR amplification, and PCR amplification product detection. The chip proposed according to the present invention can also only integrate the functions of droplet generation and PCR amplification, as shown in FIG. 3 .
图3示意地示出按照本发明的另一种液滴数字PCR芯片的正面和对应截面图。在图3的上部分是芯片的正面图，下部分是相应的截面图。其中1为油相储存池，2为流道，3为PCR起始反应液储存池，4为液滴导流流道，5为液滴储存池，9为硅胶塞，10为PMT光电倍增管，11为吸液针，12为透明检测装置。Fig. 3 schematically shows the front and corresponding cross-sectional views of another droplet digital PCR chip according to the present invention. In FIG. 3 the upper part is the front view of the chip, and the lower part is the corresponding cross-sectional view. Among them, 1 is the oil phase storage tank, 2 is the flow channel, 3 is the PCR initial reaction solution storage tank, 4 is the droplet diversion flow channel, 5 is the droplet storage tank, 9 is the silica gel plug, and 10 is the PMT photomultiplier tube , 11 is a liquid suction needle, and 12 is a transparent detection device.
如图3所示，除了液滴储存池5含有上下两个部分之外，芯片中的油相储液池1、PCR起始反应液储液池3、流道2和液滴导流流道4以及液滴储存池5的设置与图1中相应结构的设置相似。当使用如图3所示的芯片时，在按照上文关于图1所示芯片所述的方法形成油包水型液滴并完成PCR扩增之后，可以使用吸液针11从液滴存储池5中吸取液滴到透明检测装置12中，随后采用适合的方式(例如利用光电倍增管PMT)读取相关PCR结果。因此，如图3所示的芯片无需包括单层液滴平铺腔和废液储液池。As shown in Figure 3, except that the droplet storage pool 5 contains upper and lower parts, the oil phase storage pool 1, the PCR initial reaction solution storage pool 3, the flow channel 2 and the droplet diversion channel in the chip 4 and the settings of the droplet storage pool 5 are similar to those of the corresponding structure in FIG. 1 . When using the chip shown in FIG. 3, after forming water-in-oil droplets and completing PCR amplification as described above for the chip shown in FIG. In step 5, the liquid drop is sucked into the transparent detection device 12, and then a suitable method (for example, using a photomultiplier tube PMT) is used to read the relevant PCR results. Therefore, the chip shown in FIG. 3 does not need to include a single-layer droplet tiling chamber and a waste liquid reservoir.
对所提出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。本发明并不限于以列举的具体实施方式。例如，本领域普通技术人员根据本发明的原理，根据想要获得的油包水液滴的体积，可以对各项参数(例如各储液池、流道单层液滴平铺腔和流道的尺寸、所施加的气压等)做出相应地调整。The above description of the proposed embodiments will enable any person skilled in the art to make or use the invention. The invention is not limited to the specific embodiments exemplified. For example, according to the principle of the present invention, those skilled in the art can adjust various parameters (such as each liquid storage pool, flow channel single-layer droplet tiling chamber and flow channel according to the volume of the water-in-oil droplet to be obtained) size, applied air pressure, etc.) and adjust accordingly.
应当理解，以上实施例中所公开的特征，除了有特别说明的情形外，都可以单独地或者相结合地使用。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，本文所公开的本发明并不局限于所公开的具体实施例，而是意在涵盖如所附权利要求书所限定的本发明的精神和范围之内的修改。It should be understood that the features disclosed in the above embodiments can be used alone or in combination unless otherwise specified. 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. It is therefore intended that the invention disclosed herein not be limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611107369.8ACN108148743A (en) | 2016-12-06 | 2016-12-06 | Liquid drop digital PCR chip and corresponding detection method and detection system |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611107369.8ACN108148743A (en) | 2016-12-06 | 2016-12-06 | Liquid drop digital PCR chip and corresponding detection method and detection system |
| Publication Number | Publication Date |
|---|---|
| CN108148743Atrue CN108148743A (en) | 2018-06-12 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611107369.8APendingCN108148743A (en) | 2016-12-06 | 2016-12-06 | Liquid drop digital PCR chip and corresponding detection method and detection system |
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