CN114540182A - 一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控系统及其使用方法 - Google Patents
一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控系统及其使用方法 Download PDFInfo
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Abstract
本发明属于微流控技术领域，具体涉及了一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控芯片系统。所述系统包微流控芯片、微液滴孵育设备、光源设备、检测设备。其中，芯片由一级螺旋结构和二级微液滴形成结构构成；一级螺旋结构包含血液样品入口、周期性扩张的螺旋通道、血细胞出口、将目标肿瘤细胞输出到二级结构的连接通道；二级结构包含分泌物检测底物入口、蛇形混合通道、油相入口、十字形液滴生成结构、微液滴出口。以本发明建立集成循环肿瘤细胞分选、封装单个循环肿瘤细胞于微液滴的微流控芯片系统可以实现循环肿瘤细胞分离和分泌物检测连续进行的目的。
Description
技术领域
本发明属于微流控芯片技术领域，尤其涉及一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控系统及其使用方法。
背景技术
CTCs具有转移活性，进而引发肿瘤转移，而肿瘤转移是肿瘤患者致死的主要原因。因此，探究CTCs与肿瘤转移的相关性是目前CTCs研究重点关注的问题。CTCs从肿瘤原发灶脱落向周围组织侵袭、穿越血管壁和形成远端转移灶的过程中都需要细胞外基质的降解，而基质金属蛋白酶（MMPs）作为胞外水解酶在肿瘤转移过程中具有核心作用，因此分析CTCs的MMPs分泌功能将对肿瘤转移的预测具有重要意义。然而，CTCs的异质性决定并非所有CTCs均会引起肿瘤转移，因此有必要从单细胞水平研究CTCs分泌MMPs的异质性。
对CTCs进行的单细胞水平分泌物检测存在两个技术关键点：一是从血液中有效富集CTCs，二是富集CTCs的分泌物保证可以实现微量检测。由于血液中的 CTCs数量是极其稀少的，每毫升血液中大约有109个血细胞而CTCs数量仅为1~100个，这就需要对其进行有效富集以确保有足量可供分析的CTCs。另外，单个细胞的分泌物含量极少，而常规体系中微量的分泌物将被过度稀释导致无法灵敏检测，故针对于单细胞水平CTCs分泌物的检测体系也提出挑战。
微流控芯片具有操作简单、通量高、成本低、易于自动化、微型化和集成化等优点，被公认为是富集、分析CTCs的有效平台。同时，液滴微流控，是基于微流控技术发展起来的一种操纵微小体积液体的技术，可以短时间内产生大量尺寸均一、微米级别尺寸的细胞微培养室，在高通量、微量化等方面比常规微流控技术更有优势，在单细胞水平分析领域已多有应用。
然而目前，基于微流控技术可以将多种细胞研究功能集成的优势，实现CTCs的富集及单细胞水平分泌物分析的工作仍具有挑战。该类研究平台的构建，可以对CTCs单细胞水平分泌物，如MMPs进行有效分析，进而对CTCs的转移活性实现分子水平的分析，为也将为深入认识CTCs与肿瘤的发生、发展，尤其是CTCs与肿瘤转移的相关性，进而准确预测肿瘤转移提供了新的研究思路。
发明内容
针对上述现有技术的缺陷，本发明的目的是提供一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控系统及其使用方法。该分析系统首先基于螺旋芯片结构对血液中的CTCs进行富集，经富集获得的CTCs进入下游液滴形成微流控芯片结构，经与分泌物检测底物混合后被油相剪切形成微液滴，进一步实现CTCs单细胞水平分泌物的检测。
为了实现上述目的，本发明提供了如下技术方案。
本发明提供了一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控芯片，其特征在于，所述芯片包括一级螺旋结构和二级微液滴形成结构。
进一步地，所述的一级螺旋结构包含样品入口（1），周期性扩张的螺旋通道（2），两个血细胞出口（3）和一个将目标肿瘤细胞输出到二级结构的连接通道（4）。
进一步地，所述的二级微液滴形成结构包含分泌物检测底物入口（5），蛇形混合通道（6），油相入口（7），十字形液滴生成结构（8）和微液滴出口（9）。
本发明还提供了一种微流控芯片系统，其特征在于，所述系统包括权利要求1至3任意一项所述的微流控芯片、微液滴孵育设备、光源设备、检测设备。
本发明还提供了一种上所述的微流控芯片系统在单细胞水平上检测循环肿瘤细胞分泌物中的应用。
进一步地，所述应用采用的样品包括外周血。
进一步地，所述循环肿瘤细胞分泌物包括MMPs。
本发明还提供了一种如权利要求4所述微流控芯片系统的使用方法，其特征在于，所述方法包括以下步骤：
步骤1、样品制备：将肿瘤患者血液与红细胞裂解液置于离心管内混合均匀，室温孵育，期间轻轻颠倒混匀数次，待裂解完全后离心弃上清，获取白细胞与CTCs；向离心管内加入 DMEM培养基，混合均匀制备细胞悬液；
步骤2、血液中肿瘤细胞的富集：将步骤1中制得的细胞悬液通过固定流速从血液样品入口（1）注入芯片，细胞悬液中的肿瘤细胞和血细胞在螺旋通道（2）中会受到涡旋升力和迪恩阻力两种力的作用，使得尺寸大的肿瘤细胞将从螺旋结构中心出口（4）流出，而尺寸小的血细胞将聚集成细胞束并从两个侧面出口（3）流出；
步骤3、包裹单个CTCs的微液滴的形成：将分泌物的检测底物从入口（4）进入芯片，步骤2中所述的螺旋结构中心出口（4）流出的细胞与上述检测底物经蛇形混合通道（6）混合，混合流体在十字形液滴生成结构（8）处被从油相入口（7）进入芯片的油相剪切成微液滴；
步骤4、微液滴的孵育反应：收集步骤3中生成的微液滴，并在细胞培养箱进行培养，使所述微液滴中的细胞能够产生待检测的细胞分泌物，并且所述细胞分泌物可与微液滴中的检测底物发生反应；
步骤5、分泌物检测：将步骤4中的微液滴置于倒置荧光显微镜下即实现对CTCs单细胞水平分泌物的检测。
进一步地，所述步骤2中所述固定流速为100~300μL/min。
进一步地，所述方法可以用于在单细胞水平上检测循环肿瘤细胞分泌物。
与现有技术相比本发明的有益效果。
本发明构建的将CTCs富集与微液滴形成过程集成于一体的级联式微流控芯片，简化操作流程且使CTCs不易丢失。结合微液滴体系分析单细胞水平CTCs分泌物，相比于群体细胞分泌物的检测，将为CTCs异质性分析提供新的分析体系。
附图说明
图1为基于单细胞水平检测CTCs分泌物的微流控系统结构。
具体实施方式
下面结合具体实施例对本发明做详细的说明。以下实施例将有助于对本发明的了解，但这些实施例仅为了对本发明加以说明，本发明并不限于这些内容。在实施例中的操作方法均为本技术领域常规操作方法。
实施例1基于PDMS材料的微流控芯片的设计和制备。
如图1所示，使用AutoCAD软件绘制出基于单细胞水平检测循环肿瘤细胞分泌物的微流控芯片通道结构图，所述芯片由一级螺旋结构和二级微液滴形成结构构成；所述的一级螺旋结构包含血液样品入口1，周期性扩张的5环螺旋通道2，两个血细胞出口3和一个将目标肿瘤细胞输出到二级结构的连接通道4；所述的二级微液滴形成结构包含分泌物检测底物入口5，蛇形混合通道6，油相入口7，十字形液滴生成结构8和微液滴出口9。根据设计好的芯片通道结构图形制作掩膜版。
在单晶硅片上甩一层厚度为40-100 μm的SU-8胶，经过前烘、曝光、后烘、显影、硬烘过程，在单晶硅片上制备出通道凸起的SU-8模板；将PDMS与固化剂以质量比10:1比例混合均匀，浇注于SU-8模板上，70℃热板加热固化2小时后，剥离凝固的PDMS层，切割打孔；将芯片结构面朝上和玻璃片一起放入等离子清洗机中清洗1分钟，取出后迅速居中贴合在一起，得到目标微流控芯片。
实施例2.单细胞水平检测循环肿瘤细胞分泌物MMPs的微流控芯片系统的使用方法。
根据该系统，可实现一种单细胞水平检测循环肿瘤细胞分泌物的检测方法，该方法包括：
阶段一：利用微流控芯片从细胞悬液中富集CTCs及后续微液滴的形成；
阶段二：采用孔板收集微液滴，并置于细胞培养条件下进行一定时间的培养，使目标液滴中的细胞能够产生待检测的目标分泌物MMPs，该目标分泌物与MMPs检测底物发生反应，显现荧光，并且荧光强度与分泌物含量正相关；
阶段三：将微液滴置于倒置荧光显微镜下实现对CTCs单细胞水平分泌物MMPs的检测。
实施例3. 以MMPs为例，对本发明所述微流控芯片系统及使用方法进行详细说明。
步骤1、血液中肿瘤细胞的富集：将2 mL肿瘤患者血液与10 mL红细胞裂解液置于离心管内混合均匀，室温孵育，期间轻轻颠倒混匀数次，待裂解完全后500 g/min离心力离心5 min弃上清，获取白细胞与CTCs；向离心管内加入2 mLDMEM培养基，混合均匀制备细胞悬液；将细胞悬液通过300 µL min-1 从血液样品入口（1）注入芯片，细胞悬液中的肿瘤细胞和血细胞在螺旋通道（2）中会受到涡旋升力和迪恩阻力两种力的作用，使得尺寸大的肿瘤细胞将从螺旋结构中心出口（4）流出，而尺寸小的血细胞将聚集成细胞束并从两个侧面出口（3）流出。
步骤2、包裹单个CTCs的微液滴的形成：分泌物MMPs的检测底物从入口（4）进入芯片，与螺旋结构中心出口（4）流出的细胞与检测底物经蛇形混合通道（6）混合，混合流体在十字形液滴生成结构（8）处被从油相入口（7）进入芯片的油相剪切成微液滴。
步骤3、微液滴的孵育反应：收集步骤2中生成的微液滴，并在细胞培养箱中孵育48h，使所述微液滴中的细胞能够产生待检测的细胞分泌物，并且所述细胞分泌物可与微液滴中的检测底物发生反应。
步骤4、分泌物检测：将步骤3中的微液滴置于倒置荧光显微镜下即实现对CTCs单细胞水平分泌物MMPs的检测。
可以理解的是，以上关于本发明的具体描述，仅用于说明本发明而并非受限于本发明实施例所描述的技术方案，本领域的普通技术人员应当理解，仍然可以对本发明进行修改或等同替换，以达到相同的技术效果；只要满足使用需要，都在本发明的保护范围之内。
Claims (10)
1.一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控芯片，其特征在于，所述芯片包括一级螺旋结构和二级微液滴形成结构。
2.根据权利要求1所述的一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控芯片，其特征在于，所述的一级螺旋结构包含样品入口（1），周期性扩张的螺旋通道（2），两个血细胞出口（3）和一个将目标肿瘤细胞输出到二级结构的连接通道（4）。
3.根据权利要求1所述的一种基于单细胞水平检测循环肿瘤细胞分泌物的微流控芯片，其特征在于，所述的二级微液滴形成结构包含分泌物检测底物入口（5），蛇形混合通道（6），油相入口（7），十字形液滴生成结构（8）和微液滴出口（9）。
4.一种微流控芯片系统，其特征在于，所述系统包括权利要求1至3任意一项所述的微流控芯片、微液滴孵育设备、光源设备、检测设备。
5.一种如权利要求4所述的微流控芯片系统在单细胞水平上检测循环肿瘤细胞分泌物中的应用。
6.根据权利要求5所述的微流控芯片系统的应用，其特征在于，所述应用采用的样品包括外周血。
7.根据权利要求5所述的微流控芯片系统的应用，其特征在于，所述循环肿瘤细胞分泌物包括MMPs。
8.一种如权利要求4所述微流控芯片系统的使用方法，其特征在于，所述方法包括以下步骤：
步骤1、样品制备：将肿瘤患者血液与红细胞裂解液置于离心管内混合均匀，室温孵育，期间轻轻颠倒混匀数次，待裂解完全后离心弃上清，获取白细胞与CTCs；向离心管内加入DMEM培养基，混合均匀制备细胞悬液；
步骤2、血液中肿瘤细胞的富集：将步骤1中制得的细胞悬液通过固定流速从血液样品入口（1）注入芯片，细胞悬液中的肿瘤细胞和血细胞在螺旋通道（2）中会受到涡旋升力和迪恩阻力两种力的作用，使得尺寸大的肿瘤细胞将从螺旋结构中心出口（4）流出，而尺寸小的血细胞将聚集成细胞束并从两个侧面出口（3）流出；
步骤3、包裹单个CTCs的微液滴的形成：将分泌物的检测底物从入口（4）进入芯片，步骤2中所述的螺旋结构中心出口（4）流出的细胞与上述检测底物经蛇形混合通道（6）混合，混合流体在十字形液滴生成结构（8）处被从油相入口（7）进入芯片的油相剪切成微液滴；
步骤4、微液滴的孵育反应：收集步骤3中生成的微液滴，并在细胞培养箱进行培养，使所述微液滴中的细胞能够产生待检测的细胞分泌物，并且所述细胞分泌物可与微液滴中的检测底物发生反应；
步骤5、分泌物检测：将步骤4中的微液滴置于倒置荧光显微镜下即实现对CTCs单细胞水平分泌物的检测。
9.根据权利要求8所述的微流控系统的使用方法，其特征在于，所述步骤2中所述固定流速为100~300μL/min。
10.根据权利要求8所述的的微流控系统的使用方法，其特征在于，所述方法可以用于在单细胞水平上检测循环肿瘤细胞分泌物。
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