CN113462520A - 一种循环肿瘤外泌体富集芯片及其应用 - Google Patents
一种循环肿瘤外泌体富集芯片及其应用 Download PDFInfo
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Abstract
本发明涉及到一种循环肿瘤外泌体检测芯片及其应用。该微流控芯片结合侧向位移原理及静电亲和识别原理,实现了外周血中循环肿瘤外泌体的协同高效率、高纯度捕获检测。在微芯片内,设计了修饰有正电荷的聚合物材料、具有特定几何排列方式的“Y”形微柱阵列。通过调控微柱阵列的距离、密度等参数,可控制外泌体在微通道的流动行为,从而提高循环肿瘤外泌体的捕获效率和纯度。
Description
技术领域
本发明涉及一种循环肿瘤外泌体富集芯片设计、理论模拟及应用,涉及微流控芯片及临床诊断领域。
背景技术
目前,很多疾病的诊断依然面临着严峻的挑战。肿瘤是导致病死的主要原因之一,也是目前研究的热点和难点。随着人们对肿瘤转移机制研究的深入,越来越多人认识到肿瘤的早期诊断、及时治疗以及预后检测,在降低癌症死亡率,减少和控制癌症发病率方面的重要性。
外泌体,是直径30-150nm的双层脂质膜包被的小囊泡,是细胞外环境的重要成分之一。目前普遍认为,外泌体存在于血液、尿液、腹水等人体大多数体液中。外泌体可携带多种生物活性物质,包括蛋白质、脂质、及遗传物质如信使RNA(messenger RNA,mRNA),微小RNA(microRNA,miRNA)及长链非编码RNA(long non-coding RNA,lncRNA)等。外泌体的稳定性极高,在各种冷冻冷藏和解冻条件下都可保存长达数年,含量也极为丰富,每毫升血浆中就含有108-1013个外泌体。这些优点赋予了外泌体成为新型肿瘤标志物的可能性,近年来的一些研究也很好地证明了这点。如2015年《Nature》报道在胰腺癌研究中,可检测到细胞表面糖蛋白(glypican-1,GPC1)特异性地表达于胰腺癌细胞来源的外泌体中。通过检测胰腺癌患者血清中GPC1+外泌体,可以将胰腺癌患者同胰腺炎患者,健康人等分开,且可以用于不同进程癌症的分期,特异度和敏感度都接近100%。动物学发明证明了GPC1+外泌体可以比MRI更早地发现胰腺上皮内瘤变,具有胰腺癌早期诊断的价值。由此可见,外泌体具有成为肿瘤新型生物标志物的潜能。
目前的循环肿瘤外泌体分离技术主要分为两类:基于免疫亲和的方法和超速离心(UC)。基于免疫亲和性的方法利用抗体包被的磁珠捕获体液中含有特定标记物的外泌体。该方法允许分离外泌体的特定亚群,但通常不适于从大量生物样品中分离外泌体。离心方法是一种无标记、高通量的分离方法,根据外泌体的大小差异来分离外泌体。可以使用标准离心(<20 000g)分离大细胞或囊泡,而必须使用超速离心(>100 000g)从蛋白质污染物中纯化外泌体。这种复杂的程序耗时(约5小时),所需设备昂贵,需要严格训练的技术人员操作,且回收率相对较低(5-25%)。尽管通过增加梯度离心步骤,如OptiPrep™碘克沙醇梯度离心,可以获得更高的外泌体纯度,但样品处理方案的进一步复杂化导致分离时间更长。考虑到现有常规方法的缺点,需要一种无标记、快速、高通量的外泌体分离方法。
微流控芯片具有高通量、高比表面积、体积小和消耗少等特点,因此可以设计成具有低成本、高富集效率、高选择性等优点的循环肿瘤外泌体富集微流控芯片。目前利用微流控芯片进行循环肿瘤外泌体检测的方法主要有两种:1)利用亲和性识别捕获;2)利用物理特性差异,比如利用微孔过滤、体积排阻方式或结合电场、声场等外力辅助的方式,根据囊泡大小对外泌体进行分离。滤膜过滤法虽然已经有产品推出,但分离的时候会产生蛋白质堵塞膜孔,外泌体破裂等一系列问题。其他方法则需要专门的仪器,并且效率低,不适合实际应用。总之,目前肿瘤癌症外泌体的高性能捕获及检测仍然较为困难,且费时费力。因此需要寻求一种有效的肿瘤癌症外泌体富集策略。
发明内容
本发明的第一目的在于提供一种超高捕获效率的循环肿瘤外泌体富集。
本发明的第二目的在于提供一种基于微流控芯片上实现循环肿瘤外泌体捕获的方法。
本发明的目的通过以下的技术方案实现:
一种循环肿瘤外泌体捕获芯片,其特征在于:芯片设有一个进样孔和一个出样孔;进样孔和出样孔之间为“Y”形微柱阵列,“Y”形微柱阵列按照确定性侧向位移原理排列;所述微阵列表面修饰带正电荷的聚合物。
在所述的三角形微阵列表面修饰带正电荷的聚合物材料,所述的聚合物包括壳聚糖、3-氨基丙基三乙氧基硅烷、聚乙烯亚胺、聚甲基丙烯酸N,N-二甲基氨基乙酯、其他通过烷基化形成季铵盐或带有吡啶基、咪唑盐、季磷盐等侧基的带正电荷聚合物;2.如权利要求1所述的一种特异性循环肿瘤外泌体捕获芯片,其特征在于:“Y”形微柱阵列中,每个“Y”形临界宽度为0-500μm。
本发明的芯片在特定旋转角度的“Y”形微柱阵列上修饰带正电聚合物并以基于确定性侧向位移原理排列。结合了确定性侧向位移及静电识别两种捕获原理,既利用物理性质差异,同时也利用循环肿瘤外泌体表面电荷性质不同。调控了用于循环肿瘤外泌体捕获确定性侧向位移芯片的参数,以提高了循环肿瘤外泌体与“Y”形微柱阵列的碰撞概率。旋转“Y”形呈特定角度,使“Y”形微柱阵列的三面呈现梯度剪应力,增加靶标外泌体和微阵列上聚合物分子的接触时间,提高捕获效率和纯度。
本发明的又一技术方案为:
循环肿瘤外泌体富集的方法,包括如下步骤:
1)将含有循环肿瘤外泌体的血液样品或者缓冲溶液通过进样孔注入,当外泌体进入捕获微阵列,由于其表面电荷与微阵列表面修饰聚合物电荷相吸,循环肿瘤外泌体和微阵列实现多次接触,最终进行捕获;
在所述的循环肿瘤外泌体捕获芯片体系中,可通过改变流经微阵列洗脱液的pH值对捕获的循环肿瘤外泌体进行释放,并检测;
在本发明的较佳实施例中,三角形阵列的旋转角度0-180度、临界直径0-500μm、流速0-10mL/h之前,包括以下的模拟和优化步骤:基于计算流体动力学对不同尺寸囊泡通过微阵列的路径模拟。
本发明的优点如下:
1)调控临界宽度0-500μm,结合确定性侧向位移实现空间的分离,确定尺寸大于临界直径尺寸的循环肿瘤外泌体按“Y”形微柱阵列偏移方向运动,而其他尺寸囊泡按液流方向移动而降低了碰微概率;
2)“Y”形微柱阵列旋转为带θ角度(0-180度)的DLD阵列,旋转后可以降低剪应力,增加外泌体和微阵列上聚合物的接触时间,并且“Y”形微柱阵列的三面呈现梯度剪应力,均利于提高外泌体捕获效率以及捕获纯度;
3)结合静电亲和识别原理进行循环肿瘤外泌体捕获,结合确定性侧向位移原理对靶标外泌体进行高效捕获,既利用了外泌体与其他细胞分泌囊泡尺寸大小的差别,同时也利用循环肿瘤外泌体表面电荷的差别;
4)调控三角形阵列的旋转角度(0-180度)、临界宽度(0-500μm)、流速(0-10mL/h)等参数为特定值,使得基于两种捕获原理的芯片设计捕获效率最优。
附图说明
图1 外泌体电镜图;
图2为外泌体粒径分布检测结果;
图3为基于高通量“Y”型微柱阵列、微漩涡碰撞、侧面高效捕获的外泌体捕获芯片及外泌体流动速度模拟结果图;
图4 外泌体捕获与释放模拟图和示意图;
图5 APTES修饰、穿梭流模式及“Y”型微阵列结构对外泌体芯片捕获的影响。
具体实施方式
本发明结合结合静电亲和识别和尺寸原理进行循环肿瘤外泌体捕获,结合确定性侧向位移原理对靶标外泌体进行高效捕获,既利用了外泌体与其他细胞分泌囊泡尺寸大小的差别,同时也利用循环肿瘤外泌体表面电荷的差别,利用了两种捕获肿瘤外泌体的方法以提高捕获效率,高纯度地有效实现外泌体分离的要求。
实施例1
循环肿瘤外泌体捕获芯片及捕获装置
制作微流控芯片,芯片包括两层PDMS和一层玻璃芯片,将PDMS、PDMS、玻璃芯片依次键和;芯片设有一个进样孔和一个出样孔;进样孔和出样孔之间为“Y”形微柱阵列,“Y”形微柱阵列按照确定性侧向位移原理排列。
在本实施例中,一个进样孔设于芯片中间,一个出样孔设于芯片外侧。
微流控芯片捕获循环肿瘤外泌体的驱动控制进样装置,包括:芯片进样口与一个两头分流阀相连,分流阀的两个口分别连接血清样品容器和缓冲液容器,之间通过管道连接。芯片外侧的出样孔与一个两头分流阀,出样口分流阀的两个口分别连接外泌体收集容器和缓冲液容器。
将含有循环肿瘤外泌体的血液样品或者缓冲溶液通过进样孔注入,当外泌体进入捕获微阵列,由于其表面电荷与微阵列表面修饰2% APTES聚合物电荷相吸,循环肿瘤外泌体和微阵列实现多次接触,最终进行捕获;
在所述的循环肿瘤外泌体捕获芯片体系中,可通过改变流经微阵列洗脱液的pH值对捕获的循环肿瘤外泌体进行释放,并检测;
在本发明的较佳实施例中,三角形阵列的旋转角度60度,通道层高度为100微米,“Y”型主的宽度30微米,间距40微米,施加流速3mL/h时,包括以下的模拟和优化步骤:基于计算流体动力学对不同尺寸囊泡通过微阵列的路径模拟。如附图3所示。
实施例2
血清外泌体的鉴定
将乳腺癌病人血清标本(约200ul/份)自-80℃冰箱取出化冻后,3000g离心15分钟;将含有循环肿瘤外泌体的血液样品或者缓冲溶液通过进样孔注入,当外泌体进入捕获微阵列,由于其表面电荷与微阵列表面修饰2% APTES聚合物电荷相吸,循环肿瘤外泌体和微阵列实现多次接触,最终进行捕获;在所述的循环肿瘤外泌体捕获芯片体系中,可通过改变流经微阵列洗脱液的pH值对捕获的循环肿瘤外泌体进行释放,用透射显微电镜对所分离的血清外泌体进行鉴定,如附图1所示,可见电镜下所分离到的外泌体表现为圆形形态,大小较一致的颗粒,直径在30-150nm,通过动态光散射(DLS)技术对外泌体粒径分布情况进行分析,如附图2所示,可见其峰值为140 nm左右。随后,进行Western blot,可检测到外泌体表面标志性蛋白CD63,CD9,EpCAM的表达。以上结果表明本发明成功提取了血清中的外泌体。
实施例3
血清外泌体的富集和释放
本发明结合结合静电亲和识别原理进行循环肿瘤外泌体捕获,如附图4所示,简单地说,在酸性溶液中,APTES会暴露其阳离子基团。在该操作中,将样品与捕获缓冲液以1∶4的比例混合,以将ph调节至约6。0.然后使用注射泵以穿梭流将样品注射到微流体芯片中。通过静电吸附捕获外泌体。捕获的外泌体体可以直接分析或分解,以在芯片上原位获得核酸和蛋白质。此外,它们可以通过PBS或Tris–HCl碱性缓冲液(pH 7.5–8.0)释放以供进一步分析。
我们还研究了孵育策略(流动环境)是否会影响外泌体捕获效率。在本实验中,我们首先研究了静态孵育条件下外泌体的捕获率。为此,将20μl样品注入芯片中,每10分钟检测一次外来体的浓度。在静态孵育条件下,外泌体的捕获率持续增加40分钟,捕获率在40分钟时达到约40%。另一方面,在动态孵育中,将50μl样品以50μl min-1的流速灌注到芯片中,然后在1分钟后,将溶液再次抽回到芯片中以产生穿梭流,循环周期为2分钟。如附图5所示。在这些动态流动条件下,外泌体的捕获率在12分钟内快速增加,然后捕获率稳定在55–60%。通过比较静态孵育和动态孵育,我们发现穿梭流可以提高捕获效率,减少分离时间,捕获效率高达60%,但静态孵育的捕获效率为40%。
以上仅为本发明的较佳实施例而已,不能以此限定本发明的保护范围,即大凡依本发明权利要求书及发明内容所做的简单的等效变化与修改,皆仍属于本发明专利申请的保护范围。
Claims (4)
1.一种循环肿瘤外泌体捕获芯片,其特征在于:所述芯片设有一个进样孔和一个出样孔;所述进样孔和出样孔之间为“Y”形微柱阵列,所述“Y”形微柱阵列按照确定性侧向位移原理排列;所述微阵列表面修饰带正电荷的聚合物。
2.如权利要求1所述的一种特异性循环肿瘤外泌体捕获芯片,其特征在于:所述“Y”形微柱阵列中,每个“Y”形的临界宽度为0-500μm。
3.一种分离检测循环肿瘤外泌体的方法,其特征在于,包括如下步骤:
1)制作如权利要求1或2所述的芯片;
2)在微阵列表面修饰带正电荷的聚合物材料,所述的聚合物选自壳聚糖、3-氨基丙基三乙氧基硅烷、聚乙烯亚胺、聚甲基丙烯酸N,N-二甲基氨基乙酯、或其他通过烷基化形成季铵盐或带有吡啶基、咪唑盐、季磷盐等侧基的带正电荷聚合物;
3)调控“Y”形阵列的旋转角度0-180度、临界直径0-500μm、流速0-10mL/h,使得基于两种捕获原理的芯片设计捕获效率最优;
4)将样品加入到芯片中进行循环肿瘤外泌体的捕获;
5)通过改变流经微阵列洗脱液的pH值对捕获的循环肿瘤外泌体进行释放,并检测。
4.如权利要求3所述的一种分离检测循环肿瘤外泌体的方法,,其特征在于,所述步骤4)中,调控三角形阵列的旋转角度0-180度、临界直径0-500μm、流速0-10mL/h之前,包括以下的模拟和优化步骤:基于计算流体动力学对不同尺寸囊泡通过微阵列的路径模拟。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114106978A (zh) * | 2021-11-22 | 2022-03-01 | 清华大学深圳国际研究生院 | 同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105861297A (zh) * | 2016-03-29 | 2016-08-17 | 厦门大学 | 一种循环肿瘤细胞检测芯片及其应用 |
CN107597216A (zh) * | 2017-03-17 | 2018-01-19 | 李芳芳 | 一种应用微流体芯片分离外泌体的方法 |
CN109212211A (zh) * | 2017-07-07 | 2019-01-15 | 叶健 | 一种用于检测循环肿瘤细胞的芯片 |
WO2019085388A1 (zh) * | 2017-11-01 | 2019-05-09 | 深圳市瑞格生物科技有限公司 | 一种分离捕获细胞的芯片及其在肿瘤细胞分选中的应用 |
WO2019233303A1 (zh) * | 2018-06-06 | 2019-12-12 | 深圳市瑞格生物科技有限公司 | 一种侧向偏移微柱阵列芯片及其应用 |
WO2020166978A2 (ko) * | 2019-02-14 | 2020-08-20 | 고려대학교 산학협력단 | 생물학적 샘플로부터 미소 소포체를 추출하는 방법 |
CN113008652A (zh) * | 2021-02-25 | 2021-06-22 | 重庆医科大学附属第三医院(捷尔医院) | 一种利用tim-4功能化鱼骨状微流控芯片分离外泌体的方法 |
-
2021
- 2021-07-26 CN CN202110848711.4A patent/CN113462520A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105861297A (zh) * | 2016-03-29 | 2016-08-17 | 厦门大学 | 一种循环肿瘤细胞检测芯片及其应用 |
CN107597216A (zh) * | 2017-03-17 | 2018-01-19 | 李芳芳 | 一种应用微流体芯片分离外泌体的方法 |
CN109212211A (zh) * | 2017-07-07 | 2019-01-15 | 叶健 | 一种用于检测循环肿瘤细胞的芯片 |
WO2019085388A1 (zh) * | 2017-11-01 | 2019-05-09 | 深圳市瑞格生物科技有限公司 | 一种分离捕获细胞的芯片及其在肿瘤细胞分选中的应用 |
WO2019233303A1 (zh) * | 2018-06-06 | 2019-12-12 | 深圳市瑞格生物科技有限公司 | 一种侧向偏移微柱阵列芯片及其应用 |
WO2020166978A2 (ko) * | 2019-02-14 | 2020-08-20 | 고려대학교 산학협력단 | 생물학적 샘플로부터 미소 소포체를 추출하는 방법 |
CN113008652A (zh) * | 2021-02-25 | 2021-06-22 | 重庆医科大学附属第三医院(捷尔医院) | 一种利用tim-4功能化鱼骨状微流控芯片分离外泌体的方法 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114106978A (zh) * | 2021-11-22 | 2022-03-01 | 清华大学深圳国际研究生院 | 同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法 |
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