CN103382434B - 利用含排列成图案的立柱的微通道分离细胞 - Google Patents
利用含排列成图案的立柱的微通道分离细胞 Download PDFInfo
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Abstract
一种用于提取或分离体液或其它液体样品中的细胞的微流体装置(11,71),其所采用的流径中的直线液流被横置立柱(23,81)图案结构打乱。流径中的众立柱占扩大收集室区域(17,75)的整个宽度,并在其上下表面之间延伸,立柱有在横截面上弯曲的直线表面,例如圆形或泪珠形表面,立柱随机排列以破坏流线型液流。该装置的取向为使底面与水平面呈45度角。螯合剂如抗体通过亲水性涂层(优选含异硫氰酸酯基团的可渗透的水凝胶)结合于收集区表面,可有效捕获细胞或其它靶生物分子并水平排出液体样品中的其余物质。
Description
本申请是2006.01.05提交的CN200680002401.4,题为“利用含排列成图案的立柱的微通道分离细胞”的分案申请。
本发明涉及提取或分离进料液体中的靶生物分子,更具体说,涉及从体液等中分离所需靶人细胞的方法和装置。
发明背景
有效分离和收集异质细胞群中的稀有细胞仍然令人很感兴趣,因为对分离细胞群用于疾病诊断和治疗(如基因治疗)以及基础科学研究的需求不断增长。例如,可将正常较大细胞群中的病变细胞(如癌细胞)分离出来、然后将清除后的细胞群再回输给病人。
细胞分离是生物医学和临床发展中的一个迅速成长的领域,分离复杂细胞群中所需细胞亚组方法的改进拓宽了对相对均质和特性明确的细胞的研究和应用。分离的细胞也广泛用于研究,例如测定药物或治疗对靶细胞群的作用,研究生物学通路、分离和研究转化或修饰的细胞群等。目前的临床应用包括,例如,分离造血干细胞以重建血细胞,特别是与损伤性化疗和放疗联用。
已公布的美国专利申请No.2004/038315描述了将可释放接头连接于毛细血管内腔表面来结合所需细胞,然后通过切割试剂释放和回收细胞。已公布的美国专利申请No.2002/132316通过采用移动的梯度光场利用微小通道装置分离细胞群。美国专利No.6,074,827公开了采用微流体(床)装置构成的“富集通道”,利用在该通道中电泳来分离和鉴定样品中的特定核酸。还提到可任选利用抗体或其它结合片段来保留所需的靶生物物质。美国专利No.6,432,630公开了采用一种微流体***,用于引导含生物颗粒的液体流过通道而被选择性偏流(收集),表明可利用该***来分离母血样品中的胎儿细胞。
美国专利No.6,454,924公开的微流体装置可使含分析物的液体样品流经其上安置有直立立柱的表面,立柱上结合有捕获试剂,立柱侧面为疏水性因而有利于限制液体在通道中流动。
K.Takahashi等.,J.Nanobiotechnology.2,5:6(2004,6,13)公开了一种在芯片上分拣细胞的***,此***中有多个微流体入口通道通向固定在玻璃平板上的PDMS板(用主铸模以光阻环氧树脂制作)中形成的中央细胞分拣区。PDMS板中的琼脂凝胶电极通过施加静电作用力促使分离(去掉)不需要的细胞,使这些细胞在流经会聚的短小细胞分拣区时进入平行的废弃缓冲液连续液流中。也显示采用预先过滤法可物理性捕获大的尘埃颗粒。已公布的美国专利申请No.2004/029221公开了结构类似的可用于细胞分离的微流体装置,如通过选择性溶解母体RBC(红细胞)来分离母血中的胎儿细胞。可将含有细胞的样品导入微流体通道装置中分离全血,此装置含有多重栅栏,其表面适当偶联了结合分子(如抗体)而能结合样品中的细胞。美国专利No.5,637,469公开的微流体装置含有多个深100微米或不到的通道,其表面固定有结合分子(如抗体)可捕获感兴趣的生物分子作原位分析。美国专利No.5,147,607描述了将抗体固定在微通道中进行免疫试验(如夹心试验)所用的装置。微通道中的凹陷区域可含有一组从通道底面向上延伸的突出物,抗体即固定在其上。
以上简述的参考文献表明,仍然在不断寻求改进分离体液等中的细胞或其它生物材料的方法。
发明概述
本发明提供一种微流体装置可用于回收较小体积体液等中的稀有细胞或其它靶生物分子,或可在其中安置至少一个专门构建的微通道装置。构建此装置在所用的底材中形成通道样流通途径(以下简称流径),在收集区中横置固定多个立柱。这些立柱与基材整合在一起并在通道的上下表面之间延伸。立柱排列成特定的不规则阵列图案以破坏流过其中的直线液流,至关重要的是破坏流经该阵列的规则性流线型液流,从而确保沿此流径流入收集区的体液或其它液体与立柱碰撞接触和促进其产生旋转和涡流。众立柱的尺寸(如横截面直径)不同。将选择的螯合剂适当结合于整个收集区中横置立柱表面和其它所有表面,来捕获所需的靶分子,将它们收集到微通道的收集区。宜在通向收集区的微通道上游部位安置供应孔。发现该微流体装置的方向与水平面倾斜时,由于重力产生的力矢量作用可获得更完全的细胞分离。
在一具体方面,本发明提供分离体液或其它液体样品中生物分子(如细胞)的方法,该方法包括使这种含靶生物分子的样品沿微流体装置的流径从入口向往下游流向出口,所述流径包括含横截面扩大的收集区的微通道排列,同时该装置的取向为使所述流径收集区与水平面呈约30-60度角,通过以下步骤从流动的样品中分离靶生物分子:(a)打乱流经所述收集区的直线液流,这是安置在所述区域内的多个分离立柱阻断这种液流的结果,这些立柱与所述微通道的上或下表面整合在一起并延伸到其对侧表面,所述立柱横向于所述流径延伸并排列成不规则图案,所述图案侧向延伸横穿所述收集区并防止该流径中的直线液流和流线型液流,包含所述立柱的所述收集区的所有表面上都携带有螯合剂,和(b)由于所述不规则立柱和重力产生的与所述收集区的所述下表面呈锐角的力矢量对液流的破坏作用,通过使靶分子与螯合剂结合将流动的液体样品中的靶分子捕获在收集区表面,以及通过出口排出液体样品的其余部分。
在另一具体方面,本发明提供分离体液或其它液体样品中生物分子(如细胞)的装置,该装置包括主体及密封板,主体中具有可使含有靶生物分子的样品在其中流动的流径,该主体有一通向所述流径的入口通路、从其发出的出口通路、和在所述入口与出口通路之间延伸的微通道排列,所述微通道排列包括具有上下表面的收集区和众多横置分离立柱,所述上下表面之一由所述密封板提供,所述立柱与所述收集区的上下表面之一整合在一起并横穿所述流径侧向延伸到由所述密封板所提供的另一侧表面,所述立柱排列成不规则图案以打乱流经所述区域的直线液流和流线型液流,所述收集区的所述表面,包括所述立柱,带有将与靶生物分子结合的螯合剂,和所述入口与通过所述收集区的所述流径呈约120-150度角,从而可通过所述入口基本垂直地向下加入样品,而所述主体的排列为使所述流径与水平面呈约30-60度角,从而所述立柱的不规则图案和重力产生的力矢量将靶生物分子有效捕获在所述收集区,尤其是其下表面。
在另一具体方面,本发明提供分离体液或其它液体样品中生物分子(如细胞)的微流体装置,该装置包括:主体,主体中具有可使含有靶生物分子的样品在其中流动的被定义为平坦表面中的腔的流径,此流径具有入口装置、出口装置和在所述入口与出口装置之间延伸的微通道排列,所述微通道排列包括收集区,该收集区中有众多横置分离立柱,和密封板装置,所述密封板装置的平坦表面毗邻所述主体的平坦表面并靠近所述流径腔,所述立柱与所述收集区的底面整合在一起并从其伸出延伸到所述密封板装置的表面,所述立柱以不规则图案排列并横穿所述收集区内的所述流径侧向延伸以打乱流经所述区域的直线液流和流线型液流,和所述收集区的所有表面都包含用可渗透性亲水水凝胶涂布并携带有将结合靶生物分子的螯合剂的所述立柱,从而所述立柱的不规则图案破坏流经所述收集区的流线型液流从而将靶生物分子有效捕获在所述收集区的所述表面上。
附图简述
图1是微流体装置基材的透视图,显示在微流体通道中制作的简化含立柱收集区。
图2是局部放大图,显示位于图1收集区部分的图案立柱。
图3是图1基材沿直线3-3横截面的前视图,有一盖板附着在其底面。
图4是图1一般所示包括二个阀门的基材的示意性透视图,其中有一中间板。
图5是沿图4直线5-5的横截面视图。
图6是图1所示类型基材的示意性平视图,其中制作的泵是该流体装置的一部分。
图7是供应区中加入了微量混合器的基材部分的示意图。
图8是通过加入的亲水性涂层使抗体结合于整个收集区的代表性示意图。
图9和10是利用亲水性涂层使螯合剂(如抗体)结合于整个收集区所用化学试剂的代表性示意图,以及描述了捕获所需的靶细胞。
图11是流程图,说明利用图案立柱回收细胞的操作步骤,及细胞装置。
图12是微流体装置另一种实施方式的透视图,其中设计制作的微通道装置中含立柱收集区操作时与水平面倾斜。
图13是图12微流体装置主体的底视图。
图14是尺寸放大的沿图12直线14-14横截面的前视图。
图15是显示图12装置使用时的示意图。
优选实施方式的详细说明
本发明提供的装置基本上包括一个主体或基材11,其中界定的流通途径(以下简称流径)包括至少一个含收集区17的流体通道13,该流径与一个样品入口15和液体出口19相连。如本文以下所述,该流径包括连续排列的几个微通道,各通道有一个收集区。或者,如该领域熟知的那样,一个微通道可有一个以上的连续排列的收集区,及多个入口和多个出口。然而,该主体可以是构建在芯片、园盘等上的集成微流体(床)装置的一部分;此类装置中,可以掺入用于细胞回收和/或分离样品中的生物分子诊断所需的基本上整个MEMS(微电子机械***)或组件,作为一个紧凑的易操作单元。
图1是主体或基材11的透视图,基材中形成的流径包括微通道13,通过作为入口的开口或孔15向其提供液体样品,而开口19作为出口。收集区17的横截面大于通向入口15的进入区18的横截面。入口区有一对轴向排列的分流柱/支撑柱21,它们位于拓宽的收集区17上游的进入区18端。这些位于中央的分流柱使液体分流进入二流径,作用是更均匀地分配液流将其输送至收集区17的入口处。收集区17内有许多直立立柱23,它们横向排列在液体流径中,而且不规则,通常是以随机方式排列横跨流体通道收集区部分的整个纵深。众立柱的排列方式可使流体以非直线流经收集区,而破坏流线型液流,以确保沿此流径流动的液体与立柱表面之间良好接触。立柱与收集区平底面22整合在一起并从其垂直延伸,产生的表面垂直于液体水平流径,引导液体流经基材11中的流体通道。优选液体延伸流过结合的封闭平板27的朝向表面而粘附于其游离表面,如以下所详述,封闭平板27与底面22平行,作用是封闭流体通道。可在封闭平板中钻孔产生入口和出口孔24a和24b,但优选在基材11中形成出入孔。另一液体分流柱/支撑柱21a位于收集区的出口处。
如该领域所熟知的那样,可在基材中形成包括一对平行的、各含有收集区的微通道流径。这种流径可用于连续液体流动,或可用于平行流动操作。可用泵,如注射器泵等推动液流,或用真空泵通过大直径入口孔24a抽吸贮存池的液体通过。优选这种孔能容纳约50-500微升的液体样品。
设计流体通道,使流经该装置的流速在合理范围内,如采用标准的Harward灌注注射器泵注射母血于收集区产生约为0.05-5毫米/秒的流速,这基本上破坏了流经该区的流线型液流而不会产生紊流,这是由于整个收集区中随机排列着大小不同的立柱和立柱的相对空间位置安排所致。通过抽吸大小限定的入口孔,可实现平均流速宜约为0.1-2毫米/秒之间的较为平缓的非流线型液流而无死腔,更优选平均流速维持在0.2-1毫米/秒之间。
一般而言,基材11可用实验室可接受的任何合适材料,如硅、熔融二氧化硅、玻璃和聚合物制作。理想的是采用光学透明的材料,特别是当需要用于诊断时可任选采用。在其最简单实施方式中,将携带有制作的微通道的基材密封在板27中,如图3所示,板27的平坦表面朝向紧靠基材11的表面。可用相同的材料制作此板,或是简单地覆盖在玻璃板上的固体材料;然而,如下文所述。可包含一个中间流体调节板25。可采用的不可渗透的合适固体塑料包括:聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、聚碳酸酯、聚苯乙烯、聚对苯二甲酸乙二酯以及其它熟知的实验室应用可接受的聚合树酯。这类图案的基材可用常规方法,如选自常规模铸和浇铸技术的方法制作。
可用聚合材料制作主铸模或阴铸模结构的常规基材,可用光蚀刻法用厚的负光阻材料产生所述阴铸模结构,这是该领域熟知的,见JNanobiotechnology文章中所述,该文内容纳入本文作参考。例如,可用商品化购得的标准等级的环氧树酯(EPONSU-8)光阻材料和硬化剂(SU-82025)的混合物形成此结构层,在二氧化硅晶片基材上以2000rpm旋转形成(例如40或50微米厚的)光阻膜。此厚度确定了收集区流径的高度。将此膜预先在精准水平面的热平板上60℃烘烤3分钟,然后95℃烘烤7分钟以确保整体厚度均匀,室温冷却得到的样品。在终末装置中用KarlSussContactMaskAligner使膜暴光于理想流径图案。然后60℃烘烤膜2分钟,再95℃烘烤5分钟,接着在购得的SU-8暴光室中暴光5分钟,暴光时实施光搅动。这样产生了光阻环氧树酯图案的阴模具,将其用于制作主模具,复制产生PDMA或其它合适的聚合物树酯的图案立柱基材。
一个例子是,用重量比为10:1的PDMA预聚物和熟化剂混合物(Sylgard184试剂盒,Corining)制作PDMA组合物。抽真空该组合物去除混合时可能形成的气泡,然后将其倒入放在所需深度槽中的环氧树酯主模具上形成所需厚度的基材。主模具任选涂有一薄层(约50nm)合适的金属(如金)以有利于固化后取出PDMS复制物。可用80℃90分钟固化PDMS基材,然而先要使PDMS欠固化,这样才可能有利于收集区(包括立柱表面)的后续功能化。
微通道13和图案立柱的布局和尺寸取决于制作主模具时暴光步骤所用的掩蔽物。微通道13的深度受主模具SU-8层厚度的控制,取决于旋涂条件。图2提供了微通道13的俯视图,显示收集区17中放大的立柱23以通常优选的随机方式排布。
在另一实施方式中,可在平板中钻孔或产生孔洞24而不破裂取出的PDMS模板基材表面,或在盖板中钻孔以提供入口和出口连接。前者,可与一张显微镜盖玻片或其它合适的平板(如一薄片PDMS)配对,为基材提供无孔盖板或底板。使此二组件经历等离子(共振)清洗2分钟,将清洗后的二表面立即置于表面接触,而不触及其朝向表面,通过该领域熟知的表面反应方法密封此二朝向表面,形成永久性密封,并封闭微流体装置的流径。
应当想到在此类装置的整合芯片上处理液流,可类似地制作具有可调节流动性能(如气动阀门)的掺有槽穴的SU-8分离主模具。先将此主模具产生的流体调节板或调节层25薄片叠压成微通道基材11(见图4和图5),进而叠压成密封平板27。微流体装置中采用的这种流体调节组件和其它MEMS见美国专利No.6,074,827和6,454,924,其内容纳入本文作参考。小心地排列对齐此流体调节板25与携带有微通道的基材11,然后80℃退火过夜,制成组合结构。然后用先前描述的相同技术以一平板或载玻片封闭流体调节板25中的槽穴。作为进一步选择,可用同样技术在第一块板25上叠压第二块流体调节板。应想到这样可加入更复杂精致的控制和任选的处理。
例如,可在密封的基材流体调节层25中安置通道,形成多通道***,从而提供芯片流体调节机制。图4和图5说明了一种简单的***,其中通路24a和24b连接于入口和出口。气动阀门29供应的空气可通过在基材11中钻通的孔或适当形成的孔30进入板25中。流体调节板25和基材11可任选含有其它供应通路,将液体输送给入口15,也可包含该领域熟知的另一出口或释放通路。
如上所述,提供了二个连续连接的收集区这种安排导致其自身可用于不同的操作方法和应用。例如,当要处理可能含有两种不同亚群靶生物分子或感兴趣细胞(样品)时,可将一种类型的多价螯合试剂结合于一个收集区或室中的立柱,另一种类型的多价螯合试剂结合于下游收集室中的立柱。或者,当靶细胞极其稀少时,可能需要将同样的多价螯合试剂结合于二个收集室,以提高捕获液体样品中的细胞可能达到约100%。
从结构观点上说,可任选加入此装置中的某些其它组件见图6和图7说明。图6显示,在类似于图1的微通道中,在连接收集室入口通路和出口通路的流径中加入了一组蠕动泵。图解说明了微通道13’包括一个入口15’、一个收集室17’和一个出口19’,室中整合的泵组41含有加入的专门设计的三个膜阀门,这些阀门位于通向收集室的入口通路18’中。此图提供的安排类似于图4和图5,将空气或其它高压气体施加于通路30’,导致流体调节层或板的各阀门膜侧面受压,引起膜膨胀,挤压与微通道相连的毗邻区中的液体。通过编程控制单元自左到右依次操纵三个阀门,产生的波动将入口区18’中的液体泵送到右侧流入收集装置17’。如果需要,也可在收集室17’下游的出口区45中加入一组相似类型的蠕动泵。
作为另一可能的替代方法,图7说明了一种微量混合安排。阐述的微混合器51包括通向供应通道55的环形通路53,通道55可以是通向上述基材收集室的进入通道。一对入口通道57a和57b向环形通路53提供液体,通过气动阀门59可控制液体流经通道55、57a和57b。三个附加的气动阀门61位于该通道本身中,构成了上述类型的蠕动泵63。这种安排提供了微量混合基材自身中两种液体的有效方法,然后将其输送至收集室等。例如,要将一个入口通道57a中的一些液体和入口通道57b中的一些缓冲液充填环形通路53,可通过顺序操纵三个阀门61进行混合将液体泵送至环形通路沿环道流动而彻底混合,再将混合液通过输送通道55释放。
可以各种方法衍生图案立柱区的聚合物表面,使所需靶细胞或其它生物分子的特异性多价螯合试剂能结合于所有柱表面。例如,用等离子(共振)处理密封携带微通道的基材后,可向微通道中注入1-50%容量的氨基功能化硅烷(如10%DowCorningZ-6020溶液),或硫代功能化硅烷的乙醇溶液,充填开口15和19之间的区域17,然后将灌满溶液的微通道13放入室温孵箱30分钟。可对没有完全固化的聚合物(如PDMS)进行衍生,然后密封板中的微通道区。此时,如上所述,一种替代方法是先略微欠固化PDMS基材,在印制该板和用取代的硅烷或其它功能化试剂处理后再完全固化。例如,用Z-6020处理后可采用最后约50-90℃加热约90分钟步骤来完成固化。或者放置室温1或2天也可完成固化。也可在密封微通道前作衍生处理,因为平坦表面的衍生处理不是真正需要的结局。用乙醇灌洗该流径后,微通道已准备好(接受)结合生物分子的多价螯合试剂。
术语多价螯合试剂用于指能以特异性方式与靶生物分子相互反应而物理螯合该靶分子的物质。这些多价螯合剂可包括:核酸如DNA、RNA和结合有蛋白质的PNA,通常采用的非杂交螯合剂包括生物材料,如蛋白质,例如受体、肽、酶、酶抑制剂、酶底物、免疫球蛋白(具体是抗体)、抗原、凝集素、修饰的蛋白质、修饰的肽、双链DNA、生物胺和复杂的碳水化合物。也可采用合成分子,如药物,和设计的具有此种特异性结合活性的合成配体。“修饰的”蛋白质或多肽指蛋白质或肽分子中所含的一个或多个氨基酸由于加入了新的化学基团而改变,或去除了原有的某些化学基团。或这种去除和加入二者的某种组合。这种改变可包括天然和合成修饰。天然修饰可包括但不限于:磷酸化、硫酸酯化、糖基化、加入核苷酸和脂化。合成性修饰可包括但不限于:加入化学接头以利于与水凝胶、微米结构、纳米结构(如量子点)材料或其它合成材料结合。此外,修饰可包括去除现有的功能基团,如羟基、巯基或苯基,或去除或改变天然侧链或多肽的酰胺骨架。复杂碳水化合物的例子包括但不限于:天然或合成的线性或分支寡糖、修饰的多糖(如糖脂)、肽聚糖、葡糖氨基聚糖或乙酰化糖,及异质性寡糖如N-乙酰基葡糖胺或硫酸酯化糖。天然产生的复杂碳水化合物的例子是:壳多糖、透明质酸、硫酸角蛋白、硫酸软骨素、肝素、纤维素和见于修饰的蛋白质(如白蛋白和IgG)上的糖部分。可将此类试剂的两种或多种联合固定在立柱上,可作为两种成分的混合物联合加入,或可依次加入。
可将多价螯合剂直接或间接固定在立柱上。可预先处理和/或涂布立柱以利于结合。间接固定显然是优选的,考虑采用中介试剂或底物先连接于立柱;然而可能需要采用一对偶联剂来连接螯合剂与中介试剂。例如,可将链霉亲和素或针对另一种抗体(Ab)的抗体与中介试剂连接,中介试剂再与生物素化Ab或针对其它Ab的抗体偶联。这种安排使得微流体装置的基因产物可用于捕获不同样品中的各种细胞,或进行减除富集。
细胞分离和粘附优选利用Ab作为螯合剂,描述见美国专利No.5,646,404和4,675,286及原有技术。例如,美国专利No.4,528,267描述了非共价结合方法。IchiroChibata在IMMOBILIZEDENZYMES;HalsteadPress:NewYork(1978)中和在A.Cuatrecasas,JBioChem.245:3059,1970中也描述了抗体共价结合固相支持物的方法,此二文内容纳入本文作参考。Kawata等,JExpMed.160:653,1984描述了采用细胞特异性Ab,如抗人滋养层细胞单克隆抗体(抗-Trop-1和抗Trop-2)检测靶细胞来分离胎盘细胞群的方法。美国专利No.5,503,981鉴定了可用于此目的的其它三种单克隆抗体。
优选用间接法,如利用含有可结合Ab的长接头的表面层或涂布层使抗体结合于立柱的固相表面。例如,可先用双功能或多功能试剂(如蛋白质)涂布表面,然后用偶联剂(如戊二醛)偶联该试剂与抗体。也可将抗体水溶液加到已涂布了一层游离异硫氰酸酯或等价基团(如异硫氰酸聚酯)的表面而有效结合抗体,或可用溴化氰使抗体与羟基化材料偶联。特别优选采用下文与图9结合描述的亲水性聚脲烷水凝胶层(含游离异硫氰酸酯基团),或采用下文与图10结合描述的具有适当长度的亲水性接头,如PEG、聚甘氨酸。
所选的螯合剂应能特异性捕获感兴趣的生物分子,靶分子可以是各种细胞,以及蛋白质、病毒、糖等;然而认为本发明分离细胞显示特别有效和有特别优点。虽然本申请书中采用了术语“细胞”,但应理解其包括携带有螯合剂特异性表面配体的细胞片段和/或碎片。如该领域所知,选择合适的具有特异性高亲和力的螯合剂可获得对靶生物分子所需的特异性。如上所述,也可利用此微流体装置通过靶向已知的污染细胞进行减除富集。
采用抗体时,可用该领域熟知的机制使之适当地,优选通过中介试剂结合。例如,用2-氨基硫烷处理Ab使之硫醇化,将得到的硫醇化抗体与已用PEG-马来酰亚胺处理的立柱偶联;或者,可使抗体直接结合于合适的反应活性亲水异硫氰酸酯基团或硫氰酸酯基团。
当将抗体或其它螯合试剂安置在图案的整个立柱收集区中后,微通道装置即已准备好使用。通过小心地将含有靶细胞群的体液(如血液,尿液样品)或某些其它经预处理的液体从标准的注射器泵排放到通向微通道装置入口15的进入通道24a中,或用真空泵等抽吸,使其沿流径流过收集区17,直径较大入口通道24a提供了样品贮存孔,可保留测试所需体积的样品。使用时,开口24a可含与注射器泵相连管道相配的附件(未显示)。可操作此泵产生约0.5-10微升/分的液流通过该装置。根据所要处理和/或分析的体液或其它含细胞液体,如该领域所知,可采用预处理步骤减少其体积和/或删除不需要的生物分子。
为了大大提高细胞分离方法的整体效率,需要收集流出出口19的样品,使之(反复)流过微通道一次以上;当所述细胞特别罕见而在样品中数量非常少时这种反复处理可能特别有用。然而,由于该装置实现的捕获效率高,预计这种反复流动偶而才需要。或者,如以上所述将二个收集室串联使用。然而,如果要处理的体液样品体积较大,可利用基材中的二个或多个平行微通道。
可使螯合剂结合于微通道收集室的底面、朝向表面、立柱和侧壁。然而侧壁破坏液流不如底面、朝向表面和立柱那样有效。业已测定到含细胞或其它生物分子的液体均匀流过收集腔时,细胞主要存在于中央液流区中,此处的剪切力最小;因此,与横置有立柱从而破坏流线型液流的直接区域表面的捕获量相比,带有螯合剂的侧壁捕获量相当少。这些区域中,由于正确偶联,螯合剂可确保其天然的三维构象,而效果令人惊奇。
使液体样品完全流过该装置后,靶细胞如果存在即被捕获在收集区中,先用缓冲液清洗收集区,去除所有无关生物物质,这些物质是样品的一部分但不能被抗体或其它螯合剂牢固捕获。用有效的缓冲液清洗后预计只留下结合在微通道装置收集区中的靶细胞,而除去了所有非特异性结合物质。
一旦完成缓冲液清洗,如果该处理方法的目的只是收集细胞,随后可适当地释放细胞。如以下所述,某些情况时,可能需要原位进行某些分析。例如,计数收集室或下游区域中结合的细胞,或可能要裂解细胞作PCR。
当要释放细胞时,可采用该领域已知的方法,如机械法(如高速液流)、化学法(如改变pH),或利用酶切割剂等。例如,可加入试剂切除螯合剂,或切断螯合剂与细胞之间的连接键将靶细胞从收集区释放出来。例如,可采用胰蛋白酶或特异性聚焦酶来降解Ab和/或细胞表面抗原。结合Ab等然后有效除去捕获配体的特定方法描述见美国专利No.5,378,624。例如,如果细胞已通过所用的罕见细胞表面特异性抗体而结合,可用含胰蛋白酶或其它合适的蛋白酶(如蛋白酶K)处理而释放。或者可采用胶原酶释放其它螯合剂,或利用特异性可切割接头来结合螯合剂。切割时,将微通道出口与贮存器或其它收集器相连,收集含有释放细胞的流出液作进一步分析。制作的微通道可有一个以上的排出通路和出口,及调节出口开放的阀门;从而可在初步实验步骤中用一个出口通路来释放废物,用另一出口通路使靶细胞液流入收集容器。
已发现可工程改造图案立柱收集区17中的立柱23排列和形状,通过其表面特性来优化流体动力学和增进对靶细胞的捕获。非常一般地说,在大多数情况下,横置固定立柱23的水平横截面优选形状应避免尖角,困其可能促进各柱横向表面产生非特异性结合。立柱23可为直线形外表面,宜为通用的圆形横截面或6边或更多边的规则多角形。或者,可用的形状是泪珠形,尖端朝向下游略弯曲,或卵圆形;然而若需要更具冲击性可采用正方形。立柱的图案在液流中所产生的流动模式应能增进结合于柱表面、底面和朝向面的螯合剂对靶细胞的捕获。为实现此目的,已发现立柱应有不同尺寸和随机图案排列。令人惊奇的是,横截面大小不同,例如至少3或4种不同尺寸、直径约70-130微米的圆形横截面,收集区高约100微米,宽约2-4微米的立柱23随机图案,看来能特别有效地捕获流动液体样品中的细胞,立柱之间的最小间隔是50-70微米,优选约60微米。
特别优选立柱区的横截面积,即由垂直于底面的平行线所形成的侧壁所围的面积约占收集区容积的15-25%。优选立柱图案占收集区容积的约20%,留下给液流的空容积约为80%。图2所显示立柱位置的具体随机图案看来能特别增进细胞被螯合剂捕获的倾向,因这些区域中的流线型液流已受到有效破坏。
在较大立柱下游的较小立柱可能产生涡流区,因而产生此流动模式,显示邻近区的表面,特别是收集区的底面捕获靶细胞特别有效。如图2所示,所述流径纵向直线延伸距离侧壁约100微米以上,横贯多个立柱。如上所述,将众立柱与基材底面20整合在一起,优选将它们的相对端或游离端固定于朝向表面,即固定于流体调节板25或密封平板27。
如上所述,可在整个收集区中结合有螯合剂(如抗体),结合方法是用一层专门的亲水性水凝胶物质或(b)亲水接头,如分子量至少约1000道尔顿、优选分子量约2000-100000道尔顿、更优选3000-50000道尔顿之间的PEG、聚甘氨酸等来涂布此表面。特别优选采用(a)可渗透的亲水性水凝胶涂布,它们是一种含异硫氰酸酯功能基团聚合物的PEG、PPG,或它们的经脲烷键聚合的共聚物,含有反应活性异硫氰酸酯基团。优选的水凝胶采用分子量约6000的三支链PEG,可通过将环氧乙烷加入甘油而产生。使得到的多元醇与二异硫氰酸异佛尔酮和三甲醇丙烷反应制备预聚物。使该预聚物与适当缓冲液、溶剂和特定用途的其它组分的混合物原位交联在微流体装置收集区的表面。图8显示的示意图提供了微通道收集区,区中有多个直径不同的立柱61,它们随机排列破坏了通过该室的流线型液流,各个立柱61和朝向平坦表面都有外涂层63。抗体形式的螯合剂65结合于立柱上的可渗透性亲水水凝胶涂层;因此螯合剂保留了其天然三维构象,不会因结合于主要成分为水的水凝胶而改变,几乎不变形。
图9提供了采用图8的性能优选可渗透性亲水水凝胶涂层49时,可用的化学试剂的示意图。显示了结合于收集区所有表面的螯合剂(即抗体)的代表性序列。图9中的点1表示用氨基硅烷等进行氨基衍生后的表面。此步之后采用脱脂奶(封闭)酪蛋白涂布的表面,见点2。点3代表实施涂布后的涂布表面。含分子量约3400的PEG预聚物用溶于水可混溶的有机溶剂(优选质子隋性溶剂如NMP与CH3CN的混合物)的二异硫氰酸甲苯封闭末端。优选该聚合物含功能性三元醇或更高级醇,如PEG和PPG,可含有三功能异硫氰酸酯(基团)。然后制备含水98.5%的水溶液,将该溶液泵送通过微通道,由于与氨基衍生表面某些封端异硫氰酸酯基团的反应,收集区立柱表面和朝向表面被此亲水水凝胶涂层涂布。图9点3表示最后结果,产生的水凝胶是先与水反应然后形成脲键所致。
点4表示加入含表面氨基的抗体。如点5所示,这种抗体可通过Ab的氨基与亲水涂层所含异硫氰酸酯或硫氰酸酯基团共价结合,直接结合于立柱的可渗透性亲水水凝胶涂层。或者,可如图9点6所示先硫醇化抗体,然后供给收集室这些硫醇化抗体水溶液,它们进而易于共价结合涂层聚合物的异硫氰酸酯基团,见图7。
如图8和图9所示,当使液体样品中的细胞流经收集室时,由于流线型液流受到破坏,细胞接触立柱和/或朝向表面,细胞表面上该抗体的特异性抗原与之结合,细胞被有效地捕获。
图10提供了当采用伸长的PEG或PPG线性聚合物代替水凝胶将螯合剂(特别是抗体)连接于收集区表面时,可采用的代表性化学试剂示意图。选择的线性聚合物长度应使抗体在捕获(细胞)时的水环境中能保持其天然三维构象。图10的点1显示了用氨基硅烷等处理而氨基衍生的表面。此步后也如上所述用脱脂奶(封闭)酪蛋白涂布的表面。洗涤后用分子量至少约2000、优选约3000的线性PEG或PPG(其一端为NHS基团,另一端为马来酰亚胺基团)处理所有表面。N-羟基琥珀酰亚胺酯基团易与表面上的氨基起反应,产生至少厚约1微米的涂层,经适当培育后,用合适的缓冲液灌洗微通道,图10的点3表示留下的马来酰亚胺-PEG涂布的表面。点4表示滋养层细胞特异性抗体和固有的表面氨基,抗体宜用合适的试剂(如Traut试剂)硫醇化,与图10点5所示点反应。将纯化的巯基抗体缓冲溶液导入微通道作适当培育,使巯基抗体与马来酰亚胺-PEG涂布的立柱偶联。用适当缓冲液洗涤微通道,点6处即为获得的偶联安排。
图10提供的点7示意图,显示通过线性PEG偶联剂连接于表面的抗体对滋养层细胞的捕获。
微流体装置的另一种更优选结构如图12-15所示。所示的微流体装置类似于图1-3所示,但结构上有优点,收集区中即靠重力产生的均匀流动改进了待处理液体与装置内表面之间的接触,从而最大程度减少了收集室区域外(如入口和出口)靶细胞的粘附。此方面,设计该装置时倾向采用与水平面呈约30-60度角,优选45度角。因此,该装置包括主体或基材73,其中所含的微通道75从入口通路77延伸至出口通路79,和位于二通路之间的收集区81,收集区81包括上述进入区83和排出区85。收集区81在主体的平坦表面87中形成凹陷或接受(液体的)腔。收集区自身有一个与主体表面87基本上平行的平底面89。多个立柱如上所述沿底面89扩展,垂直于此底面,与主体平面87平行,一起分隔发送进入和排出区中的液流。
除了进入和排出通路83、85的朝向外,装置71的结构基本上与上述图1-5所示装置相同。如图12和14所示,构成微通道75的腔用尺寸宜大于主体的固体平板93密封,以利于液体样品处理和分析期间整个装置的操作。封闭平板93可用玻璃或上述合适的不可渗透聚合物材料制备。板93可涂布一层浇铸、模铸主体所用的同样聚合物,或经适当构建。更优选采用PDMS聚合物,如果采用标准的25mmX75mm玻片,可用图12所示的一层PDMS或薄膜95涂布玻片。将封闭板93的平面粘结于上述立柱91的末端表面,或简单地使其基本上毗邻。
如图14最佳所示,入口通路77的安排与平行于整个流径的微通道平坦表面呈30-60度锐角,更优选呈40-50度角,最优选呈45度角。这种安排如图15所示,使进料样品可垂直向下流入微流体装置71,板98将该装置(中的液体)限制于沿其上缘流向底96的倾斜表面,使整个流径倾向(例如45度角)垂直。这样进料样品可充填宽进入区83至收集区81,重力帮助其保持在含立柱的收集区中,充满和促进了在该区中所需的缓慢均匀流动。更重要的是,因为液体以平行于上下表面的方向呈线性流经该室,和因为细胞比运载它们的缓冲水溶液重,重力对这些细胞产生的力矢量不同于对运载液体产生的力矢量。因此,除了收集区中不同尺寸立柱的随机图案可对流线型液流产生破坏作用外,该力矢量可使细胞离开液流,通过结合螯合剂而粘附表面。发现此作用使通过收集区的流速较慢,而能大大改善细胞收集。
优选将入口和出口通路77、79安置在同一垂直面上,出口通路的朝向宜约90度。这样可如图15所示将装置夹在底座96上,入口通路77垂直,出口通路79水平。这使样品中所含的细胞和其它生物物质不会被Ab结合而水平流出,有利于将它们从流体通道中除去和最大程度减少非特异性粘附,并沉降在出口区中。
图15也显示将样品垂直加入到通向微流体装置入口通路进行处理的优选方法。出口通路79通过合适管道与注射器泵抽吸侧相连,有利于使样品以所需流速流经该装置。小心控制流速,使液体样品排出的速度等于液体流入收集区的平均速度,在约0.1-1毫米/秒之间,优选在约0.2-0.8毫米/秒之间,更优选在约0.25-0.5毫米/秒之间,发现此流速时由于立柱对液流的破坏和重力产生的力矢量可最大捕获靶细胞。发现高流速效果较差,还可能损伤所要寻求的娇嫩细胞。虽然图中所示的是最简单形式的该装置,但可将这些MEMS装置中所知的和本文上述的各种阀门和辅助部件与微流体(床)装置71组装在一起。
以下实施例说明了此型的原型微通道装置可有效用于螯合(捕获)***提取物中的滋养层细胞。图11所附流程概括了整个方法。当然,这些实施例应理解只是为了阐述本发明的某些实施方式,不构成对本发明范围的限制,本发明的范围由本说明书末尾的权利要求书所定义。
实施例1
用一般如图1所示的原型基材构建用于分离生物分子的微流体装置。使由PDMS形成的基材与玻璃平板粘合以密封流体通道。室温下与10体积%的DowCorningZ-6020溶液培育30分钟以衍生整个收集区的内表面。用乙醇洗涤后室温以脱脂奶处理约1小时,产生酪蛋白薄涂层。用10%乙醇水溶液洗涤后,将平均分子量6000的异硫氰酸酯封端的PEG三醇预聚物,用一份重量的此预预物和6份有机溶剂(即乙腈和DMF)与水混合形成可渗透水凝胶,使其流经图9所述的通道涂布其内表面,进行这种处理。
为进行此试验,需要分离***样品中的滋养层细胞,选用对胚胎来源滋养层细胞外表面所携带配体具有特异性的抗Trop-1和Trop-2抗体。将抗体溶于100μl含5mMEDTA(pH8.3)的0.2M硼酸钠/0.15MNaCl中,室温与5μl40mMTraut试剂反应1小时以进行硫醇化。过量的Traut试剂与10μl100mM甘氨酸反应,然后在Centricon-30TM膜上纯化硫醇化的抗体。用标准实验室方法验证硫醇化。
将总量约5微克的浓度约0.5mg/ml的硫醇化的Trop-1和-2抗体水溶液加入到经预处理的流体装置中,25℃培育该溶液2小时,培育后,用1%PBS/BSA冲洗流体通道产生抗体涂布的表面,用于分离胚胎滋养层细胞。
用HAM液(Vitrogen)将怀孕母亲(妊娠8-12周)的***稀释到10ml,37℃用DNA酶处理30分钟,用100微米细胞滤器过滤后,1500rpm离心细胞30分钟。用100微升HAM液重悬沉淀细胞,将微流体分离装置连接于Harvard装置注射器泵(其中装有约50μl***提取物的细胞悬液)的输出管使细胞悬液流过Trop-1和Trop-2抗体涂布的微通道。室温推动注射器泵使液体样品缓慢连续流过该微流体装置,流速约10微升/分。此时,已结合于收集区中随机分布图案横置立柱上的Trop-1和Trop-2抗体捕获样品中存在的滋养层细胞。用注射器泵输送所有样品后,用1%PBS/BSA缓冲液缓缓冲洗。将此缓冲液约100μl送入该装置约10分钟,有效除去该装置流体通道中所有非特异结合的生物物质。然后再冲洗二次,每次用约100μl的1%PBS和1%BSA冲洗约10分钟。
此时,如光透明材料制备的装置那样,用光学显微镜检查捕获效果。用对所捕获滋养层来源细胞的特有细胞角蛋白7和细胞角蛋白17(抗体)染色结合细胞。光学显微镜计数细胞,样品中被图案立柱收集区捕获的细胞估计基本上97%为滋养层细胞。认为此结果非常优秀。
重复整个捕获和洗涤过程,不作原位染色,27℃使100μl0.25%的胰蛋白酶溶液缓慢流经流体通道20分钟以释放被捕获的滋养层细胞。该试剂消化Ab后将滋养层细胞释放到水流中,随水流流到出口而收集。用基于PCR和FISH的技术分析收集的细胞,显示的确是所用抗体靶向的滋养层细胞。
实施例2
如实施例1所述,用原型基材构建用于分离生物分子的另一种微流体装置。如实施例1所述衍生基材的内表面,用乙醇洗涤,用脱脂奶处理。用10%乙醇水溶液洗涤后,用预聚物、BSA和Trop-1和Trop-2抗体硼酸缓冲液进行处理。用含BSA的pH8.0100mM硼酸钠配制1mg/ml抗体水溶液。该专用制剂含有100mg溶于Acn/DMF的同一预聚物;含0.25mg/ml抗体的硼酸缓冲液350μl;1mg/mlBSA硼酸缓冲液350μl,含有重量比约20%的聚合物。
抗体不硫醇化,在经过预处理的微流体装置中加入总量5μl的Trop-1和Trop-2水凝胶溶液。25℃培育该溶液约30分钟,培育后冲洗流体通道采用矿物油慢慢推入流体通道以取代水凝胶,并推出过剩的水凝胶。流体通道填入油的结果是使水凝胶薄涂层与PDMS材料分离。3小时后水凝胶完全固化,用1xPBS/0.1%Tween溶液冲洗排出油。然后用1xPBS溶液充满此装置保存Ab。
稀释怀孕母亲的***,处理、过滤、离心,重悬于100μl的HAM液中。如实施例1那样,用Harvard装置中的注射器泵使细胞悬液样品通过Trop-1和Trop-2涂布的微通道。用注射器泵输送所有样品液后,用1%PBS和1%BSA缓冲液缓慢冲洗。使该缓冲液约100μl流经该装置约10分钟,有效除去该装置流体通道中的所有非特异结合的生物物质。然后再冲洗二次,每次用约100μl的1%PBS和1%BSA冲洗约10分钟。
用细胞角蛋白7和细胞角蛋白17(抗体)染色结合细胞后,再用光学显微镜检查捕获效果。用光学显微镜计数细胞,经测定估计达到了对样品中存在的滋养层细胞优异捕获。
实施例3
如实施例1所述,用原型基材构建用于分离生物分子的另一种微流体装置。如实施例1所述衍生基材的内表面,用乙醇洗涤,用脱脂奶处理。
用10%乙醇水溶液洗涤后,将用0.2MOPS/0.5MNaCl,pH7.0配的2.5mMNHS-聚甘氨酸(平均分子量约4500)溶液10μl室温用泵在通道中轻轻来回抽动混合2小时进行处理。用pH7.0的MOPS缓冲液500μl洗涤微通道三次获得马来酰亚胺-聚甘氨酸涂布的通道。
按实施例1所述处理滋养层细胞外表面配体特异性抗体Trop-1和Trop-2使之硫醇化。
在经预处理的微流体通道中加入浓度约0.25mg/ml总量约5微克的硫醇化Trop-1和-2抗体水溶液,25℃培育该溶液2小时,培育后,用1%PBS/BSA冲洗流体通道(三次)产生抗体涂布的表面,然后用于分离胚胎滋养层细胞。
稀释怀孕母亲的***,处理、过滤、离心,重悬于100μl的HAM液中。用Harvard装置中的注射器泵使细胞悬液样品通过Trop-1和Trop-2涂布的微通道。用注射器泵液输送所有样品后,用1%PBS和1%BSA缓冲液缓慢冲洗。使该缓冲液约100μl流经该装置约10分钟,有效除去该装置流体通道中的所有非特异结合的生物物质。然后再冲洗二次,每次用约100μl的1%PBS和1%BSA冲洗约10分钟。
用细胞角蛋白7和细胞角蛋白17(抗体)染色结合细胞后,再用光学显微镜检查捕获效果。用光学显微镜计数细胞,经测定估计达到了对样品中存在的滋养层细胞优异捕获。
实施例4
制备如图3所示多个类似于实施例1所采用的微通道,来检测与水平面呈45度角操作时能否改进所得结果。所加入的液体采用BeWo和Jurkat细胞的混合物,检测了以这种角度安置的微流体装置71的效果有无改进。选择BeWo细胞是因为它们表达Trop-1和Trop-2抗原,而Jurkat细胞二种抗原都不表达,用作阴性对照。预处理微流体通道71的内表面,然后如实施例2所述用可渗透性水凝胶涂布,涂布液采用抗Trop-1和Trop-2(抗体)水溶液。用Ab涂布液充填流体通道内表面,25℃培育约30分钟。如实施例2,用矿物油然后用PBS缓冲液冲洗。
制备6次试验用的足够量的试验溶液。其含有以1%BSA/PBS缓冲液配的约3000个BeWo细胞和约3000个Jurkat细胞。将该溶液分成6等份,各等份含约500个BeWo细胞和约500个Jurkat细胞。水平安置三个同样的微流体装置71,用真空泵抽吸使各一份混合细胞液流过各装置。三个试验装置采用三种不同流速:1微升/分、5微升/分和10微升/分。
流过这三个试验装置后,用PBS缓冲液洗涤,用显微镜检查各装置。镜下分别计数各自二组捕获细胞。对于BeWo靶细胞,发现最低流速时入口区捕获了约47%的BeWo细胞,收集通道只捕获约32%,其余细胞在出口区。5微升/分流速时,收集区捕获的BeWo细胞百分比略降至约27%,而入口区仍捕获较多细胞,出口区收集到最高百分比细胞。以最高流速水平方向通过该装置时,收集区只捕获10%的BeWo细胞,而出口区收集到约65%细胞。对于Jurkat细胞,最低流速时入口和通道区各捕获约20-25%细胞,中等流速时,收集通道区只捕获到约10%的Jurkat细胞。如预期的那样,出口区收集到的这些细胞数量随每次流速提高而提高,混合该收集装置不保留的所有细胞。
再用三个相同的各与垂直线呈45度角的流体装置再进行实验。此时用的流速为1、3和5微升/分。令人惊奇的是提高了收集通道区中捕获的所需BeWo细胞。最低流速时,收集通道区捕获了约75%的BeWo细胞。中等流速3微升/分时此值提高到82%,测试的最高流速5微升/分时仍有约60%。另一方面,虽然收集区中非特异结合的Jurkat细胞在最低流速时较高,约45%,但3微升/分流速降至约15%,最高流速时不到5%。因此,当操作流速为3-5微升/分时,与水平面呈45度角时性能提高非常大,收集到约80%细胞而非仅27%。计算显示,通过操纵流速等于通过收集室区域的流速约为0.27毫米/秒时,获得了对靶细胞的优异收集,而非特异性结合细胞的污染最少。
虽然已通过本发明人目前所知的构成实施本发明最佳模式的某些优选实施方式说明了本发明,应理解本领域普通技术人员显然知道,可对其作出各种改变和修饰,但这不脱离以下权利要求书所确定的本发明的范围。例如,虽然已描述了用于制作微通道基材的某些优选材料,但本领域熟知有许多结构材料可采用,它们适合用作这种实验室装置。虽然总体上本文着重讲了从母血样品分离胎儿细胞和从***提取物分离滋养层细胞,但应理解本发明可用于分离各种各样血细胞,如无核红细胞、淋巴细胞、转移癌细胞、干细胞等;也可分离液体样品中的其它生物物质,如蛋白质、糖、病毒等。当样品含有特定细胞亚群时,要捕获的靶细胞可能是不需要与罕见细胞相分离而不要的一组细胞。然而,一旦收集到靶细胞,可原位裂解提供细胞DNA,收集后供下游分析或在收集室作PCR。美国专利申请2003/0153028描述了裂解结合的细胞获得其释放的核酸。如果样品中含二种不同靶细胞亚群,可使不同的螯合剂结合于一对上下游收集室中的立柱上。另一种情况,优选在上游收集室中收集一种细胞,释放,再在下游收集室中筛选、分离细胞亚属。
Claims (9)
1.一种分离体液或其它液体样品中生物分子的微流体装置,所述装置包括:
具有可使含有靶生物分子的样品在其中流动的被定义为平坦表面中的腔的流径的主体,所述流径包括入口装置、出口装置和在所述入口与出口装置之间延伸的微通道排列,
其中所述微通道排列包含收集区,所述收集区具有众多横置分离立柱,其中所述立柱与所述收集区的底面整合在一起,
所述立柱以不规则的随机图案排列并横穿所述收集区内的所述流径侧向延伸,破坏通过所述收集区的直线液流,所述不规则的随机图案具有以下特征:
所述立柱具有至少3种不同的横截面尺寸;立柱随机排列;以及立柱区的横截面积占收集区容积的15-25%;
所述收集区的所有表面都包含携带有将结合靶生物分子的螯合剂的所述立柱,所述螯合剂直接或间接固定在立柱上。
2.如权利要求1所述的装置,其特征在于,所述立柱的排列垂直于所述底面。
3.如权利要求1所述的装置,其特征在于,所述立柱的直径70-130微米。
4.如权利要求1所述的装置,其特征在于,两个所述立柱之间的最小距离为50-70微米。
5.如权利要求1所述的装置,其特征在于,所述入口装置包括与所述底面呈30-60度角的入口通路。
6.如权利要求1所述的装置,其特征在于,所述入口装置和所述出口装置分别包括入口通路和出口通路,且所述入口通路与所述出口通路彼此呈90度角排列。
7.如权利要求1所述的装置,其特征在于,所述出口装置包括水平的出口通路。
8.如权利要求1所述的装置,其特征在于,所述出口装置连接于泵。
9.如权利要求1所述的装置,其特征在于,所述主体有一平坦表面和位于平坦表面下的流径腔中的微通道排列。
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- 2006-01-05 ES ES06717562.0T patent/ES2437845T3/es active Active
- 2006-01-05 JP JP2007551296A patent/JP2008526251A/ja not_active Withdrawn
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WO2006078470A2 (en) | 2006-07-27 |
US20170144156A1 (en) | 2017-05-25 |
US20160320272A1 (en) | 2016-11-03 |
JP5824387B2 (ja) | 2015-11-25 |
JP2012143237A (ja) | 2012-08-02 |
JP2017055775A (ja) | 2017-03-23 |
CN103382434A (zh) | 2013-11-06 |
US10369568B2 (en) | 2019-08-06 |
HK1116720A1 (en) | 2009-01-02 |
IL184345A0 (en) | 2007-10-31 |
JP2015077154A (ja) | 2015-04-23 |
WO2006078470A3 (en) | 2006-09-14 |
EP1838442B1 (en) | 2013-09-11 |
KR20070116585A (ko) | 2007-12-10 |
JP2008526251A (ja) | 2008-07-24 |
ES2437845T3 (es) | 2014-01-14 |
US20240151234A1 (en) | 2024-05-09 |
EP1838442A2 (en) | 2007-10-03 |
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