CN115382589A - 基于尺寸筛选的外泌体切向分离与富集微流控芯片装置 - Google Patents
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Abstract
本发明公开了一种基于尺寸筛选的外泌体切向分离与富集微流控芯片装置,包括芯片、多孔膜和储液层;所述芯片表面设有蛇形微通道,芯片上设有用于注入或流出液体的开口或孔道;所述多孔膜夹在蛇形微通道相对的两层芯片中间构成一个芯片组,每个芯片组之间设有储液层;所述储液层中设有储液槽。本发明可以根据需求快速分离出不同尺寸范围的纳米粒子,具有高通量、高纯度、尺寸可调、避免使用大型仪器等优势,在外泌体等纳米级颗粒的分离上具有良好的应用潜能。
Description
技术领域
本发明涉及一种微流控芯片装置,尤其涉及一种基于尺寸筛选的外泌体切向分离与富集微流控芯片装置。
背景技术
外泌体(30~150纳米)是一种起源于核内体的细胞外囊泡,由所有细胞产生,广泛分布于人体各种体液中,如尿液、血液等。由于其来源细胞不同,外泌体可携带不同种母细胞独有的分子信息,比如蛋白质、核酸、脂质体、胞内代谢物等。此外,外泌体可介导细胞间通讯,通过外泌体传递到受体的蛋白质、代谢物质、核酸等将影响受体细胞的生物学反应,从而促进或者抑制疾病。除了在治疗方面有着极大的潜能,外泌体对于疾病的诊断、分型、分期、预后等也具有重大意义,只需要一些无创手段即可得到富含外泌体的体液,通过对外泌体的多组分分析可以确定疾病的进展和治疗手段。
生物液体的组成往往非常复杂,其中包括了各种蛋白质、核酸、囊泡以及其他复杂的代谢物,所以从中精确提取所需的外泌体是一项艰巨的挑战。目前常见的外泌体分离方法有超速离心法、密度梯度离心法、共沉淀法、尺寸排阻色谱法等,但无标签分离法大都面临着纯度低、分离效率低、设备或试剂盒昂贵等问题,而特异性好、纯度高的亲和免疫法又不可避免的对外泌体进行标记,在下游检测时难以彻底去除,从而影响检测的结果。
近年来,微流控技术由于其简单、经济、易于自动化、分析时间短、样品和试剂消耗量少等特点逐渐应用于外泌体的分离与纯化。由于外泌体的大小分布相对固定,所以利用基于尺寸筛选的方法结合微流控技术可以相对便捷地实现外泌体的分离与富集。但是,目前的微流控技术还难以通过简单的装置设备来实现对一定范围尺寸的外泌体进行精准的分离与富集。
发明内容
发明目的:本发明旨在提供一种操作便捷、分离纯化效率高的基于尺寸筛选的外泌体切向分离与富集微流控芯片装置。
技术方案:本发明所述的基于尺寸筛选的外泌体切向分离与富集微流控芯片装置,包括芯片、多孔膜和储液层;所述芯片表面设有蛇形微通道,芯片上设有用于注入或流出液体的开口或孔道;所述多孔膜夹在蛇形微通道相对的两层芯片中间,构成一个芯片组,每个芯片组之间设有储液层;所述储液层中设有储液槽。
优选地,该微流控芯片装置还包括用于夹紧装置的夹具;所述夹具按照芯片尺寸开槽,四角留出带有螺纹的通孔,用螺丝固定整个芯片装置。
优选地,所述蛇形微通道的孔道深度为0.05-0.08毫米,孔道宽度与孔道间间隔相同。
优选地,所述多孔膜外径17-24毫米,厚7-22微米,多孔膜孔道直径为30-200纳米。
优选地,所述芯片的材料为PMMA或PDMS中的一种。
优选地,所述多孔膜的材料为PC或PET中的一种。
所述芯片装置对外泌体进行分离富集的方法,是通过调整多孔膜的孔径用于收集目标尺寸范围内的外泌体,用来实现细胞上清液或体液中外泌体的分离与富集。
本发明的微流控芯片,利用尺寸筛选原理,限定待分离物外泌体尺寸的上限与下限,并使用合适孔径的多孔膜控制外泌体的通过与拦截,实现纳米级囊泡外泌体的富集、纯化;
有益效果:与现有技术相比,本发明具有如下显著优点:(1)本发明微流控芯片装置针对纳米级的囊泡分离提出了一种无标签无损伤的分离方案,蛇形孔道增大了待分离溶液与多孔膜的接触面积,切向过膜的设计也减小了液体流通过程中产生的压力,保障外泌体结构与功能的完整;(2)本发明微流控芯片装置对于分离富集的外泌体可实现清洗的功效,生物体液中组分复杂,要得到纯净的外泌体,往往需采取多次重复离心或者复杂耗时的蔗糖密度梯度离心方法,本发明只需要在入口处通入纯净的缓冲溶液,即可清洗富集在小孔径膜上的外泌体,减少杂质蛋白的干扰;(3)本发明微流控芯片装置的优势不仅在于对外泌体的分离,将芯片简单调整,被拦截在200nm多孔膜上的大尺寸囊泡也可收集,用于后续的检测与研究;(4)本发明不需要依赖大型设备以及专业操作人员,也不需要考虑特异性分子标记对下游检测带来的干扰,对外泌体的回收率高,纯度高,便于后续的分析以及下游检测;(5)本发明微流控芯片装置的可调整性强,可以根据需求更换不同孔径的多孔膜,实现不同尺寸纳米级粒子的高效分离;(6)本发明微流控芯片装置加工成本低廉,操作简单,不需要大型仪器的辅助,适用于各种场景下对于纳米级颗粒的分离。
附图说明
图1为芯片蛇形图案面结构示意图;
图2为芯片三维结构示意图;
图3为标注芯片组成编号的三维结构示意图;
图4为芯片分离两种尺寸荧光纳米粒子的回收率柱状图;
图5为不同流速下芯片分离外泌体的回收率柱状图。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
如图3所示,图中的标记为:11-芯片1入口;21-芯片2出口;23-芯片2侧边开口;31-储液层入口;32-储液层出口;33-储液槽侧边开口;41-芯片3入口;43-芯片3侧边开口;51-芯片4出口。
如图2、3所示,所述的装置结构从上到下分别为Ⅰ组芯片、储液层、Ⅱ组芯片;芯片入口位于Ⅰ组芯片中的芯片1,出口位于Ⅱ组芯片中的芯片4;Ⅰ组和Ⅱ组芯片构造完全相同,区别仅在于内部所夹多孔膜的孔尺寸,芯片表面设有蛇形微通道(如图1所示),每组芯片由蛇形微通道相对的两层芯片和夹在其中的多孔膜构成;芯片上设有用于注入或流出液体的开口或孔道;蛇形微通道的孔道总长15厘米,孔道深度0.08毫米,孔道宽度与孔道间间隔均为0.5毫米,进样腔体口的通孔尺寸0.5毫米;芯片本身尺寸29.3×24×6毫米。Ⅰ组和Ⅱ组芯片之间设有储液层;所述储液层中设有储液槽。
所述Ⅰ组芯片中所夹多孔膜外径19毫米,厚7-22微米,多孔膜孔道直径为200纳米,材质为聚碳酸酯;所述Ⅱ组芯片中所夹多孔膜外径19毫米,厚7-22微米,多孔膜孔道直径为30纳米,材质为聚碳酸酯。所述的储液层为与芯片同等尺寸的PMMA,在左侧打通一个圆柱形的空槽作为储液槽,深度覆盖上下两组芯片的液体流通口,并在上下两组芯片流出、流入口对应的位置开孔,使得上层芯片流出的液体能够流入储液槽,并且储液槽流出的液体能够流入下层芯片。
所述的储液层侧边的开口直径3毫米,配合同等尺寸聚四氟乙烯堵头可以控制储液槽的开合。
芯片组装完成后,待处理的细胞上清液样品从芯片1入口11处注入,流经第一组装载大孔径膜(200纳米)的蛇形芯片时,尺寸大于多孔膜的囊泡与细胞碎片被留在膜的上方,尺寸小于多孔膜(200纳米)的外泌体随着液体一起向下进入储液槽,流入第二组装载小孔径膜(30纳米)的蛇形芯片,目标尺寸范围内的外泌体被拦截在小孔径膜(30纳米)表面,杂质蛋白、生物小分子和多余的液体从芯片4出口51排出;注入一定体积的PBS缓冲溶液,将通道内所有的外泌体富集在小孔径膜(30纳米)表面并进行清洗;将芯片倒置,从芯片4出口51注入缓冲溶液,将富集在小孔径膜(30纳米)上的外泌体冲回储液槽,利用注射器抽取收集,即得富集纯化后的外泌体。
所述的样品溶液浓度不宜过高,常规细胞上清等液体浓度均可,进样速度为1-100微升每分钟。
实施例1
组装芯片装置,芯片表面的蛇形微通道孔道总长15厘米,孔道深度0.08毫米,孔道宽度与孔道间间隔均为0.5毫米,进样腔体口的通孔尺寸0.5毫米;芯片尺寸为29.3×24×6毫米;Ⅰ组芯片中所夹多孔膜(膜1)外径19毫米,厚7-22微米,多孔膜孔道直径为200纳米,材质为聚碳酸酯;所述Ⅱ组芯片中所夹多孔膜(膜2)外径19毫米,厚7-22微米,多孔膜孔道直径为30纳米,材质为聚碳酸酯。
将100纳米荧光小球(FNPs,λex/λem=542/612nm)与270纳米荧光小球(FNPs,λex/λem=468/508nm)混合分散在PBST(0.5%Tween80)缓冲溶液中,超声分散均匀,得到模拟体液环境的混合粒子样品,粒子浓度为0.001%固体,pH为7.4。
堵塞出/入口芯片2侧边开口23、储液槽侧边开口33、芯片3侧边开口43,将混合液从芯片1入口11处以50微升每分钟通入芯片,在两层多孔膜上进行尺寸筛选。
从芯片1入口11处以50微升每分钟通入缓冲溶液,将游离在各个孔道以及储液槽中的粒子全部富集到对应尺寸的多孔膜上。
堵塞芯片2出口21,移除储液槽侧边开口33处塞子,将储液槽内液体抽出,此时储液槽内液体均为缓冲溶液。
从芯片4出口51以5微升每分钟注入500微升缓冲溶液,用注射器从储液槽侧边开口33处收集浓缩的100纳米荧光纳米粒子。
堵塞储液槽侧边开口33,恢复芯片2出口21流通,从芯片4出口51以5微升每分钟注入500微升缓冲溶液,在芯片1入口11处可收集270纳米荧光纳米粒子。
将收集到的溶液分别在对应激发光下测试荧光发射强度,与分离前原溶液对应荧光强度进行对比,即可算得芯片对于混合荧光纳米粒子的回收效率,如图4所示。
实施例2
组装芯片装置,芯片表面的蛇形微通道孔道总长15厘米,孔道深度0.08毫米,孔道宽度与孔道间间隔均为0.5毫米,进样腔体口的通孔尺寸0.5毫米;芯片尺寸为29.3×24×6毫米;Ⅰ组芯片中所夹多孔膜(膜1)外径19毫米,厚7-22微米,多孔膜孔道直径为200纳米,材质为聚碳酸酯;所述Ⅱ组芯片中所夹多孔膜(膜2)外径19毫米,厚7-22微米,多孔膜孔道直径为30纳米,材质为聚碳酸酯。
取无牛血清细胞上清液样本,在4℃下,分别在500g下离心10分钟,2000g下离心10分钟,以去除残留的细胞及细胞碎片。
堵塞芯片2侧边开口23、储液槽侧边开口33、芯片3侧边开口43,将处理后的细胞上清液从芯片1入口11处以10-50微升每分钟通入芯片,在两层多孔膜上进行尺寸筛选。
从芯片1入口11处以20微升每分钟通入PBS缓冲溶液,将游离在各个孔道以及储液槽中的囊泡全部富集到对应尺寸的多孔膜上,并对膜上富集囊泡进行冲洗。
堵塞芯片2出口21,移除储液槽侧边开口33处塞子,将储液槽内液体抽出,此时储液槽内液体均为缓冲溶液。
从芯片4出口51以5微升每分钟注入500微升缓冲溶液,用注射器从储液槽侧边开口33处收集纯净的细胞上清外泌体,不同流速下的回收率如图5所示。
堵塞储液槽侧边开口33,恢复芯片2出口21流通,从芯片4出口51以5微升每分钟注入500微升缓冲溶液,在芯片1入口11处可收集大尺寸囊泡,进行后续表征与实验。
Claims (8)
1.一种基于尺寸筛选的外泌体切向分离与富集微流控芯片装置,其特征在于,包括芯片、多孔膜和储液层;所述芯片表面设有蛇形微通道,芯片上设有用于注入或流出液体的开口或孔道;所述多孔膜夹在蛇形微通道相对的两层芯片中间,构成一个芯片组,每个芯片组之间设有储液层;所述储液层中设有储液槽。
2.根据权利要求1所述的基于尺寸筛选分离外泌体的微流控芯片装置,其特征在于,还包括用于夹紧装置的铝制夹具。
3.根据权利要求2所述的基于尺寸筛选分离外泌体的微流控芯片装置,其特征在于,所述铝制夹具按照芯片尺寸开槽,四角留出带有螺纹的通孔,用螺丝固定整个芯片装置。
4.根据权利要求1所述的基于尺寸筛选分离外泌体的微流控芯片装置,其特征在于,所述蛇形微通道的孔道深度为0.05-0.08毫米,孔道宽度与孔道间间隔相同。
5.根据权利要求1所述的基于尺寸筛选分离外泌体的微流控芯片装置,其特征在于,所述多孔膜外径17-24毫米,厚7-22微米,多孔膜孔道直径为30-200纳米。
6.根据权利要求1所述的基于尺寸筛选分离外泌体的微流控芯片装置,其特征在于,所述芯片的材料为PMMA或PDMS中的一种。
7.根据权利要求1所述的基于尺寸筛选分离外泌体的微流控芯片装置,其特征在于,所述多孔膜的材料为PC或PET中的一种。
8.一种利用权利要求1所述芯片装置对外泌体进行分离富集的方法,其特征在于,通过调整多孔膜的孔径用于收集目标尺寸范围内的外泌体,实现细胞上清液或体液中外泌体的分离与富集。
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