CN109679842B - 微流控芯片 - Google Patents

微流控芯片 Download PDF

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CN109679842B
CN109679842B CN201910158935.5A CN201910158935A CN109679842B CN 109679842 B CN109679842 B CN 109679842B CN 201910158935 A CN201910158935 A CN 201910158935A CN 109679842 B CN109679842 B CN 109679842B
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droplet
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CN109679842A (zh
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於林芬
阳巍
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Shenzhen Borui Biotechnology Co ltd
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Priority to PCT/CN2019/077082 priority patent/WO2020177088A1/zh
Priority to US17/292,716 priority patent/US12030048B2/en
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Abstract

本发明公开一种微流控芯片，所述微流控芯片包括芯片上盖、芯片下层、薄膜、密封垫、密封圈，所述微流控芯片设置样本储存区、液滴生成区、液滴储存区、液滴检测区和废液储存区，所述样本储存区、液滴生成区、液滴储存区、液滴检测区和废液储存区之间均是通过小孔或微通道连通。所述液滴生成区用于将样本相转变为数万个至数百万个液滴，所述液滴在液滴储存区进行PCR反应，所述液滴检测区用于对PCR反应后的液滴进行光学检测，所述废液储存区用于收集和存储检测后的所述液滴和连续相。

Description

微流控芯片
技术领域
本发明涉及数字PCR技术领域，特别涉及一种微流控芯片。
背景技术
现有微滴式数字PCR系统多采用分体式技术路线，即液滴生成、PCR反应和液滴检测分别在不同仪器上完成。该技术路线操作步骤繁琐，难以做到全流程封闭操作，不符合临床诊断分析要求，制约了该技术的临床应用。
发明内容
本发明提供一种微流控芯片，用于实现样本储存及转移、液滴生成、液滴储存、PCR热循环和液滴检测等全流程操作步骤。
本发明所述微流控芯片，其包括芯片上盖、芯片下层、薄膜、密封垫和密封圈，所述芯片上盖的下表面与所述芯片下层的上表面贴合，所述芯片下层的下表面与所述薄膜的上表面贴合。贴合采用粘接、焊接、键合等方式，以保证贴合牢固、紧密。
优选地，本发明所述微流控芯片按功能划分为样本储存区、液滴生成区、液滴储存区、液滴检测区和废液储存区。所述样本储存区和所述废液储存区设于所述芯片上盖的下表面，所述液滴生成区、所述液滴储存区和所述液滴检测区设于所述芯片下层的下表面。所述样本储存区与所述液滴生成区、所述液滴生成区与所述液滴储存区、所述液滴储存区与所述液滴检测区、所述液滴检测区与所述废液储存区均是通过微通道或微孔连通。
优选地，样本储存区用于储存样本相；液滴生成区用于将所述样本相转变为数万个至数百万个液滴，例如，将水相样本转变为油包水液滴。所述液滴在液滴储存区进行PCR反应。完成反应后，液滴检测区用于对PCR反应后的液滴进行光学检测。废液储存区用于收集和存储完成检测后的液滴和连续相。
优选地，本发明所述微流控芯片设有数组独立的、并排布置的样本储存区、液滴生成区、液滴储存区、液滴检测区和废液储存区，分别对应于数个样本。每一组样本储存区、液滴生成区、液滴储存区、液滴检测区、废液储存区形成一个样本的全流程处理通路，所述微流控芯片能对所述数个样本相互独立地进行样本储存、液滴生成、液滴储存、PCR热循环反应、液滴检测、废液储存。
优选地，加样时本发明所述微流控芯片水平放置，后续生成液滴、PCR反应、液滴检测时，所述微流控芯片竖直或倾斜放置，液滴生成区位于所述微流控芯片的下端，废液储存区位于所述微流控芯片的上端。微流控芯片竖直或按一定角度倾斜放置可以保证液滴快速上浮，不影响后续的液滴生成过程，并在检测过程可以保证液滴顺利，无损转移。
优选地，芯片上盖设有穿透芯片上盖上下表面的样本注入孔、密封垫安装孔、排气孔和窗口。窗口用于透过光线，方便光学检测设备对液滴进行检测。芯片上盖的上表面设有加样柱，所述样本注入孔设于所述加样柱的中心。芯片上盖的下表面设有加样微通道、样本储存池、排气通道和废液储存池，所述加样微通道连接样本注入孔和样本储存池，所述排气通道连接所述废液储存池和所述排气孔。
优选地，密封垫对称地间隔设有生成连续相注入孔和检测连续相注入孔，即密封垫两端的两个孔为生成连续相注入孔，与之相邻的为检测连续相注入孔，再依次为生成连续相注入孔，如此两端对称地间隔设置生成连续相注入孔和检测连续相注入孔。所述生成连续相注入孔和所述检测连续相注入孔之间由连接段连接。所述生成连续相注入孔和所述检测连续相注入孔的上下两端设有第一密封环，所述密封环为单圈或多圈圆环结构。
优选地，密封圈包括由连接段连接的圆环，所述圆环的上下两端设置有单圈或多圈第二密封环，所述圆环的内壁套设在所述加样柱上。
优选地，薄膜的厚度小于1毫米。为了使PCR反应时的热量传导更迅速，薄膜应尽量薄。薄膜作用为密封芯片下层下表面的孔、通道、液滴储存区，并起到与液滴储存区传递热量的作用。
优选地，液滴储存区设有液滴储存池，所述微流控芯片竖直放置时，所述液滴储存池的上端呈尖角状，尖角顶端通向液滴检测区，可保证液滴的快速无损转移。
优选地，液滴生成区包括生成连续相入口、与所述生成连续相入口连通的生成连续相通道、样本入口、以及与所述样本入口连通的样本通道。其中，所述生成连续相入口与所述密封垫的生成连续相注入孔连通；所述样本通道连接至少一个样本分支通道，所述样本分支通道通过喇叭口连接所述液滴储存池。生成液滴时，先将一定体积的生成连续相注入液滴储存池，然后样本相由样本分支通道进入液滴储存池，在喇叭口处生成液滴并进入液滴储存池。所述生成连续相主要用于辅助生成液滴，检测连续相的主要作用是推动液滴运动。
优选地，所述生成连续相通道设有生成连续相过滤区，所述样本通道设有样本过滤区。所述样本相过滤区和所述生成连续相过滤区均密布有微柱，微柱之间的距离为10～100微米，密布的微柱作用是拦截杂质。喇叭口是两侧对称开口的“<”形或者是单斜边开口的“∠”形，所述喇叭口角度为5°～120°。样本相分支通道的数量优选为1～40个。
优选地，液滴储存池的深度大于或等于2倍的喇叭口的深度，且所述液滴储存池的深度大于或等于2倍的样本相分支通道的深度，所述喇叭口的深度与所述样本相分支通道的深度相同。
优选地，样本相分支通道的宽度与深度比大于或等于1，所述样本相分支通道的宽度为10～200微米，所述样本相分支通道的深度为2～100微米，所述液滴储存池的深度大于50微米。
优选地，液滴检测区包括检测连续相入口、与所述检测连续相入口连通的检测连续相通道、液滴入口、与所述液滴入口连通的液滴通道、检测通道。其中，所述检测连续相入口与密封垫的检测连续相注入孔连通；所述检测连续相通道设有检测连续相过滤区，所述检测连续相过滤区密布有微柱，所述微柱之间的距离为10～100微米。密布的微柱作用是拦截杂质。所述检测连续相通道与所述液滴通道交汇后连接检测通道，所述检测通道与废液通道连通，所述废液通道连接废液出口。所述检测通道的宽度为1～1.5倍的液滴直径，所述检测通道的深度为1～1.5倍的液滴直径。
优选地，样本储存区包括加样微通道、样本储存池以及样本出口，所述样本储存池的下端呈倾斜状；所述样本出口设于所述样本储存池的底端尖角处，所述样本出口与液滴生成区的样本入口连通。废液储存区包括排气通道和废液储存池，所述排气通道连接废液储存池和排气孔。
优选地，所述样本相优选为水相，所述生成连续相和检测连续相优选为油相，所述液滴优选为油包水液滴。
本发明所述微流控芯片，加样时，所述微流控芯片水平放置，样本相进入样本储存区。随后，将所述微流控芯片竖直或者倾斜放置，液滴生成区位于下端，废液储存区位于上端，通过外部压力驱动样本储存区中的样本至液滴生成区。液滴生成区将样本相分散为几万至几百万个液滴，所得液滴存储于所述液滴储存区。结合外部温控设备，可对所述液滴储存区内的液滴进行PCR热循环。完成PCR反应的液滴进入液滴检测区，逐个检测光学信号。完成检测后的液滴存储于所述废液储存区的废液储存池。
本发明提供的微流控芯片集成了液滴生成、液滴储存、PCR热循环和液滴检测等液滴数字PCR全流程操作步骤，极大减少人工手动操作，降低操作难度，满足临床检测自动化操作的需求，可同时处理多个相互独立的样本，显著提高检测效率。
附图说明
为了更清楚地说明本发明的技术方案，下面将对实施方式中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方式，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，还可以从这些附图获得其他的附图。
图1是本发明所述微流控芯片的示意图；
图2是图1所述微流控芯片的组成示意图；
图3是图1所述微流控芯片的各功能区域分布示意图；
图4是图1所述微流控芯片的剖面示意图；
图5是图1所述微流控芯片的芯片上盖上表面的示意图；
图6是图1所述微流控芯片的芯片上盖下表面的示意图；
图7是图1所述微流控芯片的芯片下层下表面的示意图；
图8是图1所述微流控芯片的局部放大示意图；
图9是图1所述微流控芯片液滴生成区的局部放大示意图；
图10是图1所述微流控芯片液滴检测区的局部放大示意图；
图11是图1所述微流控芯片的密封垫示意图；
图12是图1所述微流控芯片的密封垫的剖面示意图；
图13是图1所述微流控芯片的密封圈示意图；
图14是图1所述微流控芯片的密封圈剖面示意图。
图中标号说明：10、芯片上盖；20、芯片下层；30、薄膜；40、密封垫；50、密封圈；60、液滴生成区；70、液滴储存区；80、液滴检测区；90、废液储存区；100、样本储存区；11、芯片上盖上表面；12、芯片上盖下表面；13、加样柱；131、样本注入孔；14、密封垫安装孔；15、排气孔；16、窗口；21、芯片下层上表面；22、芯片下层下表面；31、薄膜上表面；32、薄膜下表面；41、生成连续相注入孔；42、检测连续相注入孔；43、第一密封环；44、连接段；51、内壁；52、第二密封环；61、样本相入口；62、样本相过滤区；621、微柱；63、样本相通道；631、样本相分支通道；632、喇叭口；64、生成连续相入口；65、生成连续相过滤区；66、生成连续相通道；71、液滴储存池；81、检测连续相入口；82、检测连续相过滤区；83、检测连续相通道；84、液滴入口；85；液滴通道；86、检测通道；87、废液通道；88、废液出口；91、排气通道；92、废液储存池；101、加样微通道；102、样本储存池；103、样本出口。
具体实施方式
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作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。
请参阅图1～14，本发明提供一种微流控芯片，用于实现样本储存及转移、液滴生成、液滴储存、PCR热循环、液滴检测的全流程操作步骤。其中，加样后，样本相储存于样本储存区100，液滴生成在液滴生成区60完成，液滴储存和PCR热循环在液滴储存区70完成，液滴检测在液滴检测区80完成，液滴检测完成后所得废液收集于废液储存区90。
本实施例中，本发明的微流控芯片具有8组独立的、并排布置的液滴生成区60、液滴储存区70、液滴检测区80、废液储存区90、样本储存区100，分别对应于8个样本，每一组样本储存区100、液滴生成区60、液滴储存区70、液滴检测区80、废液储存区90构成一个样本的全流程处理通路。本发明的微流控芯片能同时对8个样本相互独立地进行液滴生成、液滴储存、PCR热循环和液滴检测等操作。以下描述主要阐明单个样本的全流程处理通路，显而易见的是，每一个样本的全流程处理通路的原理和结构是一样的。
本发明的微流控芯片包括芯片上盖10、芯片下层20、薄膜30、密封垫40和密封圈50。其中，废液储存区90、样本储存区100设于芯片上盖10，液滴生成区60、液滴储存区70、液滴检测区80设于芯片下层20。样本储存区100与液滴生成区60、液滴生成区60与液滴储存区70、液滴储存区70与液滴检测区80、液滴检测区80与废液储存区90均是通过微通道或微孔连通。
样本储存区100用于暂存加入的样本相。加样时，微流控芯片水平放置，样本相由样本注入孔131进入样本储存区100中的样本储存池102。随后，将微流控芯片竖直或者倾斜放置，液滴生成区60位于下端，废液储存区90位于上端，通过外部压力将样本储存区100中的样本驱动至液滴生成区60。
本实施例中，液滴生成区60用于将水相样本分散为几万～几百万个油包水液滴，所得油包水液滴储存于液滴储存区70。结合外部温控设备，可对液滴储存区70内的所述油包水液滴进行PCR热循环。完成PCR反应的液滴进入液滴检测区80，逐个检测光学信号。完成检测后的液滴储存于废液储存区90的废液储存池93。
芯片上盖10包括芯片上盖上表面11、芯片上盖下表面12，芯片下层20包括芯片下层上表面21、芯片下层下表面22，薄膜30包括薄膜上表面31、薄膜下表面32。芯片上盖下表面12与芯片下层上表面21相贴合，芯片下层下表面22与薄膜上表面31相贴合。贴合采用粘接、焊接、键合等方式，以保证贴合牢固、紧密。
如图5、图6所示，样本储存区100包括8组加样微通道101和样本储存池102，废液储存区90包括8组排气通道91和废液储存池92。芯片上盖10设有穿透上下两面的样本注入孔131、密封垫安装孔14、排气孔15和窗口16。芯片上盖上表面11设有加样柱13，样本注入孔131设于加样柱13中心。芯片上盖下表面12设有加样微通道101、样本储存池102、排气通道91和废液储存池92，加样微通道101连接样本注入孔131和样本储存池102，排气通道91连接废液储存池92和排气孔15。窗口16用于透过光线，方便光学检测设备对液滴进行检测。
如图7、图8、图9、图10所示，液滴生成区60、液滴储存区70、液滴检测区80设于芯片下层下表面22。液滴生成区60包括8组样本相入口61、样本相过滤区62、微柱621、样本相通道63、样本相分支通道631、喇叭口632、生成连续相入口64、生成连续相过滤区65、微柱621、生成连续相通道66。液滴储存区70包括8个液滴储存池71。液滴检测区80包括8组检测连续相入口81、检测连续相过滤区82、微柱621、检测连续相通道83、液滴入口84、液滴通道85、检测通道86、废液通道87、废液出口88。
如图7、8、9、10所示，生成连续相从生成连续相入口64进入，经过生成连续相过滤区65后通过连续相通道66进入液滴储存池71。样本相从样本相入口61进入，经过样本过滤区62后通过样本相通道63进入样本相分支通道631。样本相分支通道631与液滴储存池71通过喇叭口632连接。喇叭口632是两侧对称开口的“<”形或者是单斜边开口的“∠”形，喇叭口角度为5°～120°，样本相分支通道631的数量为1～40个，数量越多则液滴生成的效率越高。样本相在经过喇叭口632进入储存有生成连续相的液滴储存池71的过程中由于压差、表面张力的作用而断裂形成一个一个的液滴，液滴被包裹在生成连续相中。
进一步的，喇叭口632的深度尺寸与样本相分支通道631的尺寸相同，液滴储存池71的深度尺寸大于或等于2倍的喇叭口632的深度尺寸。样本相分支通道631的宽度为10～200微米，深度为2～100微米，样本相分支通道631宽度与深度比大于或等于1。液滴储存池71的深度大于50微米。
样本相过滤区62、生成连续相过滤区65、检测连续相过滤区82分别密布有微柱621，微柱621之间的距离在10～100微米，密布的微柱作用是拦截杂质。
检测连续相通道83与液滴通道85交汇后连接检测通道86。检测通道86的宽度及深度为1～1.5倍的液滴直径。检测连续相从检测连续相入口81进入检测连续相过滤区82，经微柱621过滤后进入检测连续相通道83，同时液滴从液滴入口84进入液滴通道85，液滴与检测连续相同时进入检测通道86，液滴之间的距离由于检测连续相的挤入而变大，有利于其它光学检测系统对液滴进行检测。
本实施例中，检测通道86一共设有8组，集中并排在一起，有利于其它光学检测系统的检测。检测通道86与废液通道87连通，经过检测的液滴和检测连续相经过废液通道87流向废液出口88。
如图2、4、7、9所示，液滴储存池71设置在芯片下层下表面22，与薄膜30贴合封接后形成封闭的液滴储存空间。
如图2、4、5、6所示，样本储存池102、废液储存池92设置于芯片上盖下表面12，与芯片下层上表面21贴合封接后形成封闭的样本储存空间、废液储存空间。
如图11、12所示，密封垫40包括8个生成连续相注入孔41、8个检测连续相注入孔42，中间由连接段44连接，两端的两个孔为生成连续相注入孔41，与之相邻的为检测连续相注入孔42，再依次为生成连续相注入孔41，如此两端对称地间隔设置生成连续相注入孔41、检测连续相注入孔42。在生成连续相注入孔41和检测连续相注入孔42的上下两端设置有第一密封环43，第一密封环43为单圈或者多圈圆环结构。
如图1、2、3所示，生成连续相注入孔41、检测连续相注入孔42，分别与生成连续相入口64、检测连续相入口81对准。废液出口88与废液储存池92对准连通，样本入口61与样本出口103对准。
微流控芯片在加样时水平放置，样本相从样本注入孔131注入，经过加样微通道101进入样本储存池102。随后，微流控芯片竖直或者倾斜放置，废液储存区90处于上端。生成液滴时，生成连续相从密封垫40的生成连续相注入孔41注入，并进入生成连续相入口64，之后再进入液滴储存池71，样本相在压力作用下从样本出口103进入样本相入口61。在检测时，检测连续相从检测连续相注入孔42注入，并进入检测连续相入口81。
检测时，液滴入口84处于液滴储存池71的顶端，当检测连续相进入液滴储存池71并填满时，液滴上浮并被检测连续相推动从液滴入口84进入液滴通道85。液滴储存池71的上端是一个逐渐缩小的尖角形状，有利于液滴集中到液滴入口84，并且有利于液滴迅速、彻底地排出。
样本储存池102的下端为倾斜状，下端尖角处为样本出口103，这样的形状有利于样本相从样本出口103排出干净。
如图13、14所示，本实施例中，密封圈50由8个圆环组成，中间由连接段44连接，圆环上下两端设置有单圈或者多圈的第二密封环52，安装时，密封圈的内壁51套设在加样柱13上。薄膜30的厚度小于1毫米，为了使PCR反应时的热量传导更迅速，薄膜30应尽量薄。
本发明提供的微流控芯片集成了样本储存及转移、液滴生成、液滴储存、PCR热循环和液滴检测等液滴数字PCR全流程操作步骤，极大减少人工手动操作，降低操作难度，满足临床检测自动化操作的需求，可同时处理多个相互独立的样本，显著提高检测效率。

Claims (9)

1.一种微流控芯片，其特征在于，所述微流控芯片包括芯片上盖、芯片下层、薄膜、密封垫和密封圈，所述芯片上盖的下表面与所述芯片下层的上表面贴合，所述芯片下层的下表面与所述薄膜的上表面贴合；
所述微流控芯片设置样本储存区、液滴生成区、液滴储存区、液滴检测区和废液储存区，所述样本储存区和所述废液储存区设于所述芯片上盖的下表面，所述液滴生成区、所述液滴储存区和所述液滴检测区设于所述芯片下层的下表面，所述样本储存区与所述液滴生成区、所述液滴生成区与所述液滴储存区、所述液滴储存区与所述液滴检测区、所述液滴检测区与所述废液储存区均是通过微通道或微孔连通；
所述液滴储存区设有液滴储存池，所述液滴储存池的上端呈尖角状；所述液滴生成区包括生成连续相入口、与所述生成连续相入口连通的生成连续相通道、样本相入口、以及与所述样本相入口连通的样本相通道，其中，所述生成连续相入口与所述密封垫的生成连续相注入孔连通；所述样本通道连接至少一个样本相分支通道，所述样本相分支通道通过喇叭口连接所述液滴储存池；所述生成连续相通道设有生成连续相过滤区，所述样本相通道设有样本过滤区；
所述样本储存区包括加样微通道、样本储存池以及样本出口，所述样本储存池的下端呈倾斜状；所述样本出口设于所述样本储存池的底端尖角处，所述样本出口与液滴生成区的所述样本入口连通；
加样时，所述微流控芯片水平放置；生成液滴、PCR反应、液滴检测时所述微流控芯片竖直或倾斜放置，且所述液滴生成区位于所述微流控芯片的下端，所述废液储存区位于所述微流控芯片的上端。
2.如权利要求1所述的微流控芯片，其特征在于，所述芯片上盖设有穿透芯片上盖上表面和芯片上盖下表面的样本注入孔、密封垫安装孔、排气孔和窗口；所述芯片上盖上表面设有加样柱，所述样本注入孔设于所述加样柱的中心；所述芯片上盖下表面设有加样微通道、样本储存池、排气通道和废液储存池，所述加样微通道连接样本注入孔和样本储存池，所述排气通道连接所述废液储存池和所述排气孔。
3.如权利要求1所述的微流控芯片，其特征在于，所述密封垫对称地间隔设有生成连续相注入孔和检测连续相注入孔，所述生成连续相注入孔和所述检测连续相注入孔之间由连接段连接，所述密封垫的左右两端为生成连续相注入孔；所述生成连续相注入孔和所述检测连续相注入孔的上下两端设有第一密封环；
所述密封圈包括由连接段连接的圆环，所述圆环的上下两端设置有单圈或多圈第二密封环，所述圆环的内壁套设在所述加样柱上；
所述薄膜的厚度小于1毫米。
4.如权利要求1所述的微流控芯片，其特征在于，所述样本相过滤区和所述生成连续相过滤区均密布有微柱，所述微柱之间的距离为10～100微米；
所述喇叭口是两侧对称开口的“<”形或者是单斜边开口的“∠”形，所述喇叭口角度为5°～120°；
所述样本相分支通道的数量为1～40个。
5.如权利要求4所述的微流控芯片，其特征在于，所述液滴储存池的深度大于或等于2倍的喇叭口的深度，且所述液滴储存池的深度大于或等于2倍的样本相分支通道的深度，所述喇叭口的深度与所述样本相分支通道的深度相同。
6.如权利要求5所述的微流控芯片，其特征在于，所述样本相分支通道的宽度与深度比大于或等于1，所述样本相分支通道的宽度为10～200微米，所述样本相分支通道的深度为2～100微米，所述液滴储存池的深度大于50微米。
7.如权利要求1所述的微流控芯片，其特征在于，所述液滴检测区包括检测连续相入口、与所述检测连续相入口连通的检测连续相通道、液滴入口、与所述液滴入口连通的液滴通道、检测通道，其中，
所述检测连续相入口与密封垫的检测连续相注入孔连通；
所述检测连续相通道设有检测连续相过滤区，所述检测连续相过滤区密布有微柱，所述微柱之间的距离为10～100微米；
所述检测连续相通道与所述液滴通道交汇后连接检测通道，所述检测通道与废液通道连通，所述废液通道连接废液出口；
所述检测通道的宽度为1～1.5倍的液滴直径，所述检测通道的深度为1～1.5倍的液滴直径。
8.如权利要求1所述的微流控芯片，其特征在于，所述废液储存区包括排气通道和废液储存池，所述排气通道连接废液储存池和排气孔。
9.如权利要求1所述的微流控芯片，其特征在于，所述样本相为水相，生成连续相和检测连续相为油相，所述液滴为油包水液滴。
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