CN114106978A - 同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法 - Google Patents
同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法 Download PDFInfo
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Abstract
本发明公开了同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法。本发明提供捕获外泌体的微流控芯片,其由键合在一起的基片与盖片组成,盖片上具有微柱阵列区域,微柱阵列区域由微柱组成,微柱与微柱之间形成的间隔区域构成微流控芯片的微通道,微柱上修饰有外泌体捕获适配体,外泌体捕获适配体为用于捕获外泌体的外泌体表面蛋白适配体。还提供一种同时检测外泌体内部microRNA和表面蛋白的试剂盒,其包括所述微流控芯片、根据靶标microRNA序列设计的反应序列H1与H2以及荧光标记的靶标表面蛋白核酸适配体。本发明通过捕获微流控芯片将外泌体捕获在微流控芯片的微结构上,在免疫捕获纯化分离外泌体后,直接进行外泌体microRNA和表面蛋白的检测,避免了样品的损失。
Description
技术领域
本发明属于微流控芯片领域,具体涉及同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法。
背景技术
肺癌是世界范围内人们生命健康的重大威胁之一。数据显示,在2020年,肺癌是全世界死亡率第一(死亡180万人)且新增确诊率第二(新增患者220万人)的癌种。非小细胞肺癌(NSCLC)是最常见的肺癌类型,约80%的肺癌患者是非小细胞肺癌患者。肺癌患者死亡率高的主要原因之一是大多数非小细胞肺癌患者被确诊时,他们的病情已经处于局部晚期或转移期,而晚期非小细胞肺癌的五年存活率只有4%。
在非小细胞肺癌的早期确诊可以显著的提升确诊患者的五年存活率。目前非小细胞肺癌的临床诊断“金标准”方法病理诊断存在着难以忽视的局限:病理诊断需要单针穿刺来取样,穿刺位置的不同会导致诊断结果的不同,可能会造成诊断结果的极大偏差。同时穿刺取样为侵入性取样方法,不能频繁进行,导致无法对患者的病情进行实时监控。液体活检是一项通过对患者体液中肿瘤相关分泌物的检验分析,获取患者肿瘤相关信息的新兴技术。目前主要的液体活检对象有循环肿瘤DNA(ctDNA)、循环肿瘤细胞(CTC)、外泌体。作为一项低侵入性取样的技术,液体活检可以做到连续取样、重复取样,从而可以对患者病情实时监控。这项技术有潜力突破目前病理诊断的局限,对肿瘤的早期诊断、个性化治疗、准确预后等都有重要意义。
近些年,以外泌体为液体活检对象的研究逐年增加。外泌体是一类由细胞分泌,尺寸在50-150nm的细胞外囊泡,这些囊泡都包含有其来源细胞分泌的蛋白质、核酸、脂质等生物分子,同时,膜结构使得这些分子稳定存在于体液中。相较于体液中易于被分解的ctDNA和含量稀少的CTC,在体液中高含量且能稳定存在的外泌体及其包含的生物分子是更优的液体活检的对象。
在众多的外泌体所包含的生物分子中,microRNA和表面蛋白是目前研究者研究最多的两类肿瘤标志物。值得一提的是,microRNA位于外泌体膜内部,表面蛋白嵌于外泌体膜上,这导致对外泌体microRNA和表面蛋白的同时检测难以实现。但是,如果能同时检测外泌体microRNA和表面蛋白,得到更全面的疾病进展相关信息,就可以极大的提高癌症筛查的灵敏度和特异性。Won Jong Rhee课题组在2019年使用外泌体表面蛋白抗体包被的磁珠捕获外泌体后,将microRNA的分子探针与外泌体共孵育,再之后加入了外泌体表面蛋白的荧光标记抗体,使用酶标仪读取信号,首次同时对外泌体microRNA和表面蛋白进行了同时检测,但其检测限高达107/μL。在此之后,并未有新的同时检测外泌体microRNA和表面蛋白的报道,所以开发新的灵敏度高的同时检测外泌体microRNA和表面蛋白的方法,是很有意义的。
微流控芯片技术通过微米甚至纳米尺度微通道结构将样品处理、生物与化学反应、分离、检测这些基本操作单元集成在一块微流控芯片上,再通过对微流体的操纵,完成了复杂的分析流程。这项技术具有样品和试剂消耗量小、分析速度快、通量高、易大规模平行测试等优点,同时给分析系统的小型化、集成化和便携化带来了便利。同时,设计并优化微流控芯片的微阵列结构,可以提高微纳尺度下的传质效率,进而提高了检测方法的灵敏度。越来越多的研究将拥有上述优势的微流控芯片作为外泌体分析的平台,这些研究根据其外泌体分离原理可以分为两大类,一类是基于物理性质(尺寸、密度、介电常数等)的分离方法,一类是基于免疫捕获(外泌体表面蛋白CD63、CD9、CD81等)的分离方法,这两类方法都可以快速高效的得到高纯度的外泌体。此外,一些研究在微流控芯片上免疫捕获外泌体后,可以直接进行外泌体表面蛋白的检测,将外泌体的分离、提纯和检测结合,极大的提高了外泌体检测的效率。
此外,在不破坏外泌体的前提下,高灵敏度的检测外泌体膜内microRNA是同时检测外泌体microRNA和表面蛋白的关键。目前原位检测外泌体膜内microRNA的方法主要有两类,第一类是将microRNA的分子探针与外泌体样品一起孵育,分子探针进入外泌体后产生信号,达到不破坏外泌体且检测外泌体microRNA的目的;第二类是使用载有microRNA分子探针的阳离子脂质体,阳离子脂质体会与外泌体融合,从而让microRNA的分子探针进入外泌体,达到检测的目的。由于上述检测方法存在着一个靶标只能产生一个信号的局限,原位检测外泌体microRNA的灵敏度难以提升。催化发卡自组装反应(CHA),作为高灵敏原位检测活细胞内部RNA的常用方法,其拥有和分子探针一样大小的尺寸,同时有着数倍于分子探针的信号强度,近期有研究将催化发卡自组装反应用于外泌体microRNA的原位检测,可以有效降低检测的检测限。然而,上述研究大多使用传统平台进行检测,受限于复杂、耗时的加样清洗步骤,难以进行外泌体microRNA与表面蛋白的同时检测。
发明内容
为了解决现有技术中的不足,本发明的目的是提供同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法。具体方案如下:
本发明第一方面提供一种捕获外泌体的微流控芯片,其由键合在一起的基片与盖片组成,盖片上具有微柱阵列区域,所述微柱阵列区域由微柱组成,所述微柱与微柱之间形成的间隔区域构成微流控芯片的微通道,所述微柱上修饰有外泌体捕获适配体,所述外泌体捕获适配体为用于捕获外泌体的外泌体表面蛋白适配体。
进一步地,所述外泌体表面蛋白适配体为特异性结合外泌体表面蛋白的DNA单链;
优选地,所述外泌体表面蛋白选自CD63、CD9或CD81;
优选地,所述外泌体表面蛋白选自CD63;
优选地,所述结合CD63的DNA单链具有如SEQ ID NO.5所示的核苷酸序列(TTTTTTTTTTTTCACCCCACCTCGCTCCCGTGACACTAATGCTA)。
进一步地,所述外泌体捕获适配体上具有生物素标记,所述微柱上具有链霉亲和素修饰,通过生物素与链霉亲和素的结合获得修饰外泌体捕获适配体的微柱;
优选地,所述外泌体捕获适配体进行荧光标记,该荧光标记用来来验证外泌体捕获适配体的成功修饰,正式实验使用无荧光标记的外泌体捕获适配体。
进一步地,所述微柱阵列区域的长为4.7-4.9mm,宽为2.4-2.6mm,所述微柱的高为50-60μm,半径为48-52μm,微柱与微柱的间距为98-102μm,以更大程度上增加外泌体与微柱的碰撞几率,增加外泌体捕获的效率;
优选地,所述盖片采用的材料为聚二甲基硅氧烷;
优选地,所述微流控芯片还包括进样口和出样口。
本发明第二方面提供所述微流控芯片在外泌体分离以及外泌体内部microRNA和/或表面蛋白检测中的应用。
本发明第三方面提供一种分离外泌体的试剂盒,包括上述所述的微流控芯片。
本发明第四方面提供一种同时检测外泌体内部microRNA和表面蛋白的试剂盒,包括:所述的微流控芯片、根据靶标microRNA序列设计的反应序列H1与H2以及荧光标记的靶标表面蛋白核酸适配体;所述的靶标为待测的外泌体;
所述反应序列H1与H2为核苷酸序列,H1与靶标microRNA有n个互补配对碱基,H1与H2有m个互补配对碱基,其中2n≤m;
所述反应序列H1退火后可形成发夹结构;
所述反应序列H2退火后可形成发夹结构;
所述反应序列H2一端修饰有荧光基团,另一端修饰有猝灭基团;
优选地,所述反应序列H2一端修饰有FAM荧光基团,另一端修饰有Dabcyl猝灭基团;
优选地,所述荧光标记的靶标表面蛋白核酸适配体为5’端标记Cy5且能与靶标表面蛋白特异性结合的DNA单链,该序列由指数富集配体进化技术(SELEX)得到,具有与抗体媲美的特异性和亲和性。
本发明的上述技术方案中,所述反应序列H1与靶标microRNA有n个互补配对碱基,H1与H2有m个互补配对碱基,其中2n≤m,使得H2与H1的结合能力远大于靶标microRNA与H1的结合能力,H2可以将结合在H1上的靶标microRNA序列竞争下来;所述反应序列H1、反应序列H2分别含有多对互补碱基对,退火后会自发形成稳定发卡结构,使得二者不会相互反应;所述反应序列H2两端修饰有FAM荧光基团与Dabcyl猝灭基团,形成发卡结构会导致二者接近,使FAM荧光猝灭。反应时,靶标microRNA打开H1的发卡结构,二者结合并使H1成为单链,单链H1会打开发卡H2,发卡H2将靶标microRNA从H1上竞争下来,形成了稳定的双链H1-H2结构,FAM荧光基团和Dabcyl猝灭基团位于双链两端,FAM荧光基团荧光恢复,实现了信号的产生,同时被竞争下来的靶标microRNA会引发新一轮上述反应,周而复始,达到信号放大的目的。
进一步地,当用于同时检测非小细胞癌外泌体内部microRNA-200和表面蛋白EpCAM时,所述H1具有如SEQ ID NO.1所示的核苷酸序列(GCCTGGTAATGATGATAATACTGCCTGTCATCATTACCAGGCAGTATTA),所述H2具有如SEQ ID NO.2所示的核苷酸序列(ATGATAATACTGCCTGGTAATGATGACAGGCAGATTATCATCATTACC),所述荧光标记的靶标表面蛋白核酸适配体具有如SEQ ID NO.3所示的核苷酸序列(AGTGACGCAGCATGCGGCACACACTTCTATCTTTGCGGAACTCCTGCGG)。
所述非小细胞癌外泌体内部microRNA-200的核苷酸序列如SEQ ID NO.4所示(UAAUACUGCCUGGUAAUGAUGA)。
本发明第五方面提供一种外泌体的分离方法,包括步骤,将待分离样品注入到所述微流控芯片的微通道中,静置,捕获待测样品中的外泌体,清洗去除样品。
本发明第六方面提供一种外泌体内部microRNA和表面蛋白的同时检测方法,包括如下步骤:
(1)将待测样品注入到权利要求1所述微流控芯片的微通道中,静置,捕获待测样品中的外泌体;
(2)向步骤(1)的微通道中加入退火后的反应序列,静置,之后清洗通道;
(3)向步骤(2)的微通道中加入荧光标记的靶标表面蛋白核酸适配体,静置,之后清洗通道;
(4)使用共聚焦荧光显微镜拍摄微通道的荧光照片,使用软件对荧光强度进行量化,得到外泌体内部microRNA和表面蛋白的含量;
优选地,步骤(1)中静置时间为2.8-3.2h,静置温度为37℃;
优选地,步骤(2)中静置时间为1.8-2.2h,静置温度为37℃;
优选地,步骤(3)中静置时间为1.8-2.2h,静置温度为37℃;
优选地,步骤(2)微通道中还加入链球菌溶血素和核酸酶抑制剂。
步骤(2)加入反应序列后,靶标microRNA打开H1的发卡结构,二者结合并使H1成为单链,单链H1会打开发卡H2,发卡H2将靶标microRNA从H1上竞争下来,形成了稳定的双链H1-H2结构,FAM荧光基团和Dabcyl猝灭基团位于双链两端,FAM荧光基团荧光恢复,实现了信号的产生,同时被竞争下来的靶标microRNA会引发新一轮上述反应,周而复始,达到信号放大的目的。
步骤(3)加入荧光标记的靶标表面蛋白核酸适配体后,靶标表面蛋白核酸适配体与靶标表面蛋白结合,从而实现对表面蛋白的标记。
本发明的有益效果为:
1、本发明提供的捕获外泌体的微流控芯片进行外泌体分离时,与常规的外泌体分离方法(如超速离心法、磁珠分离法)相比,所需样品、试剂量大幅降低,速度更快,纯度更高。
进一步设置微柱阵列区域长为4.7-4.9mm,宽为2.4-2.6mm;所述微柱的高为50-60μm,半径为48-52μm,微柱与微柱的间距为98-102μm,在流体力学上能更大程度上增加外泌体与微柱的碰撞几率,增加外泌体捕获的效率。
微流控芯片的材料采用聚二甲基硅氧烷(PDMS),其生物兼容性好,透光性强,适用于生物标志物的检测。
2、本发明提供的同时检测外泌体内部microRNA和表面蛋白的方法,通过免疫捕获微流控芯片将外泌体捕获在微流控芯片的微结构上,在免疫捕获纯化分离外泌体后,直接进行外泌体microRNA和表面蛋白的检测,避免了样品的损失,并且使用催化发卡自组装反应来检测外泌体microRNA,凭借微尺度下高效率传质和催化发卡自组装自身的高信号强度,可以克服传统外泌体microRNA检测方法低灵敏度的问题,实现更灵敏的microRNA检测。本发明可以同时原位检测外泌体microRNA和表面蛋白两类肿瘤标志物,得到更全面的疾病进展的信息,提高了癌症早期诊断的灵敏度和特异性。整个检测过程简单快捷,并且微流控芯片平台样品、试剂消耗量都非常少,芯片制造成本低,易于批量制作,可以同时进行多个靶标组合和多个样品检测,具有较好的液体活检应用前景。该发明为非小细胞肺癌的早期诊断提供了新的技术平台。
附图说明
图1为实施例1的微流控芯片的立体结构图,其中,1-微柱阵列区域,2-微柱,3-进样口,4-出样口;
图2为实施例2的盖片通道掩膜尺寸设计图;
图3为实施例2的微通道修饰结果图;
图4为实施例3的外泌体microRNA与表面蛋白检测的标准曲线图。
具体实施方式
为了更清楚地理解本发明,现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中,各原始试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
实施例1:微流控芯片的结构
本实施例提供的同时检测外泌体内部microRNA和表面蛋白的微流控芯片的立体结构如图1所示。微流控芯片由键合在一起的玻璃基片与盖片组成。盖片上具有微柱阵列区域1,微柱阵列区域由微柱2组成,所述微柱与微柱之间形成的间隔区域构成微流控芯片的微通道,所述微柱上修饰有外泌体捕获适配体,所述外泌体捕获适配体为用于捕获外泌体的外泌体表面蛋白适配体。微流控芯片还包括进样口3与出样口4。微柱阵列区域长为4.7-4.9mm,宽为2.4-2.6mm;所述微柱的高为50-60μm,半径为48-52μm,微柱与微柱的间距为98-102μm,这样在流体力学上更大程度上增加外泌体与微柱的碰撞几率,增加外泌体捕获的效率。
进一步地,所述外泌体表面蛋白适配体为特异性结合外泌体表面蛋白的特定序列DNA单链。
优选地,所述外泌体表面蛋白选自CD63、CD9或CD81。在一个优选的方案中,所述外泌体表面蛋白选自CD63。
进一步地,所述外泌体捕获适配体上具有生物素标记,所述微柱上具有链霉亲和素修饰,通过生物素与链霉亲和素的结合获得修饰外泌体捕获适配体的微柱。
优选地,所述外泌体捕获适配体进行荧光标记,该荧光标记用来验证外泌体捕获适配体的成功修饰,正式实验使用无荧光标记的外泌体捕获适配体。
实施例2:微流控芯片的制备
将硅片先用乙醇超声5分钟后,再用去离子水超声5分钟,进行三次后,在加热板上加热120分钟。
在硅片上甩涂一薄层SU-82050负性光刻胶,通过控制转速使胶层厚度大约为50μm。将具有图2结构的掩膜与硅片贴合,紫外线通过光掩模使光刻胶曝光,未曝光的部分用显影液溶解。硅片表面上凸起的SU-8结构作为PDMS盖片的阳模。然后将硅阳模硅烷化过夜。
PDMS预聚体(单体/固化剂=10/1混合)浇注在硅阳模上并真空脱气,然后放入75℃烘箱中固化2小时左右,然后将PDMS从阳模剥离,形成了PDMS的盖片。将PDMS盖片切割,并进行打孔。
将玻璃基片与PDMS盖片一起放入氧等离子体真空管中,抽真空90秒,打开高频电源,90秒后取出基片和盖片,立即键合。
键合后在室温下用4%(v/v)MPTS的无水乙醇溶液处理通道,静置30min。再用无水乙醇进行洗涤,重复三次或至完全洗涤干净。洗涤干净后将芯片放置于100℃的烘箱中干燥1h。
在室温下将芯片用新鲜制备的0.01μmol/mL GMBS的无水乙醇溶液在室温下处理,同样静置30min。再用无水乙醇进行洗涤,重复三次或至完全洗涤干净。洗涤干净后将芯片放置于100℃的烘箱中干燥1h。
在室温下用10μg/mL链霉亲和素的PBS溶液填充通道,静置1h,之后引入浓度为1μM的生物素标记的外泌体捕获核酸适配体溶液,并在室温下孵育30min,然后用PBS缓冲液洗涤以除去未结合的捕获适配体。修饰完成后将该芯片保存在4℃,以便之后使用。
在一个具体的实施方案中,使用修饰有FAM基团的生物素标记的外泌体捕获适配体修饰微通道,并用PBS缓冲液洗涤除去了未结合的捕获适配体。该荧光标记用来来验证外泌体捕获适配体的成功修饰,正式实验使用无荧光标记的外泌体捕获适配体。如图3,使用普通荧光显微镜即可观察到大量的外泌体捕获适配体成功修饰在微柱上。
实施例3
本实施例进行非小细胞肺癌外泌体microRNA-200与表面蛋白EpCAM的同时检测。通过实施例2制备微流控芯片,根据microRNA-200序列设计反应序列H1与H2,根据表面蛋白EpCAM设计荧光标记的EpCAM核酸适配体。
其中,微流控芯片的微柱上修饰的外泌体捕获适配体为用于捕获外泌体的外泌体表面蛋白CD63适配体,该外泌体表面蛋白适配体为特异性结合CD63的特定序列DNA单链(TTTTTTTTTTTTCACCCCACCTCGCTCCCGTGACACTAATGCTA,SEQ ID NO.5),该序列5’端修饰有生物素,与微柱上修饰的链霉亲和素结合。
H1序列为GCCTGGTAATGATGATAATACTGCCTG TCATCATTACCAGGCAGTATTA(SEQ IDNO.1);
H2序列为ATGATAATACTGCCTGGTAATGATGACAGGCAGATTATCATCATTACC(SEQ IDNO.2),H2序列5’端修饰有FAM荧光基团,3’端修饰有Dabcyl猝灭基团;
Cy5荧光标记的EpCAM核酸适配体的序列为AGTGACGCAGCATGCGGCACACACTTCTATCTTTGCGGAACTCCTGCGG(SEQ ID NO.3),该序列5’端修饰有Cy5;
非小细胞癌外泌体内部microRNA-200的核苷酸序列为UAAUACUGCCUGGUAAUGAUGA(SEQ ID NO.4)。
具体步骤如下:
(1)将2μL待测样品用移液枪注入修饰有外泌体捕获适配体的微通道中,37℃静置3h,使微柱捕获样品中的外泌体,然后进行清洗,将样品冲出。
(2)向步骤(1)微通道中加入退火后的根据microRNA-200设计的H1(100μM)与H2(400μM)混合液2μL,混合液中含有链球菌溶血素和核酸酶抑制剂,浓度分别为0.4U/mL和4U/ml,37℃静置2h后用PBS缓冲液进行清洗,将未进入外泌体的H1、H2清除。
(3)向步骤(2)微通道中加入荧光标记的EpCAM核酸适配体,静置1.8-2.2h后进行清洗,将未与外泌体表面蛋白结合的核酸适配体用PBS缓冲液清洗,此时完成了对microRNA-200和表面蛋白EpCAM的荧光标记。
(4)使用共聚焦荧光显微镜拍摄微通道的荧光照片,使用imageJ软件将得到的照片转化为8-bit图像,再测量每张图片的平均灰度值,得到了待测靶标的信号强度。
结果如图4,左边为不同浓度外泌体样品EpCAM测量结果量化并处理后得到的标准曲线图,右边为不同浓度外泌体样品microRNA-200测量结果量化并处理后得到的标准曲线图。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
SEQUENCE LISTING
<110> 清华大学深圳国际研究生院
<120> 同时检测外泌体内部microRNA和表面蛋白的试剂盒和方法
<130> CP121010904C
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Claims (10)
1.一种捕获外泌体的微流控芯片,其特征在于,所述微流控芯片由键合在一起的基片与盖片组成,盖片上具有微柱阵列区域,所述微柱阵列区域由微柱组成,所述微柱与微柱之间形成的间隔区域构成微流控芯片的微通道,所述微柱上修饰有外泌体捕获适配体,所述外泌体捕获适配体为用于捕获外泌体的外泌体表面蛋白适配体。
2.根据权利要求1所述的微流控芯片,其特征在于,所述外泌体表面蛋白适配体为特异性结合外泌体表面蛋白的DNA单链;
优选地,所述外泌体表面蛋白选自CD63、CD9或CD81;
优选地,所述外泌体表面蛋白选自CD63;
优选地,所述结合CD63的DNA单链具有如SEQ ID NO.5所示的核苷酸序列。
3.根据权利要求1所述的微流控芯片,其特征在于,所述外泌体捕获适配体上具有生物素标记,所述微柱上具有链霉亲和素修饰,通过生物素与链霉亲和素的结合获得修饰外泌体捕获适配体的微柱;
优选地,所述外泌体捕获适配体进行荧光标记。
4.根据权利要求1所述的微流控芯片,其特征在于,所述微柱阵列区域的长为4.7-4.9mm,宽为2.4-2.6mm,所述微柱的高为50-60μm,半径为48-52μm,微柱与微柱的间距为98-102μm;
优选地,所述盖片采用的材料为聚二甲基硅氧烷;
优选地,所述微流控芯片还包括进样口和出样口。
5.权利要求1所述微流控芯片在外泌体分离以及外泌体内部microRNA和/或表面蛋白检测中的应用。
6.一种分离外泌体的试剂盒,其特征在于,包括权利要求1所述的微流控芯片。
7.一种同时检测外泌体内部microRNA和表面蛋白的试剂盒,其特征在于,包括:权利要求1所述的微流控芯片、根据靶标microRNA序列设计的反应序列H1与H2以及荧光标记的靶标表面蛋白核酸适配体;
所述反应序列H1与H2为核苷酸序列,H1与靶标microRNA有n个互补配对碱基,H1与H2有m个互补配对碱基,其中2n≤m;
所述反应序列H1退火后可形成发夹结构;
所述反应序列H2退火后可形成发夹结构;
所述反应序列H2一端修饰有荧光基团,另一端修饰有猝灭基团;
优选地,所述反应序列H2一端修饰有FAM荧光基团,另一端修饰有Dabcyl猝灭基团;
优选地,所述荧光标记的靶标表面蛋白核酸适配体为5’端标记Cy5且能与靶标表面蛋白特异性结合的DNA单链。
8.根据权利要求7所述的试剂盒,其特征在于,所述试剂盒用于同时检测非小细胞癌外泌体内部microRNA-200和表面蛋白EpCAM时,所述H1具有如SEQ ID NO.1所示的核苷酸序列,所述H2具有如SEQ ID NO.2所示的核苷酸序列,所述荧光标记的靶标表面蛋白核酸适配体具有如SEQ ID NO.3所示的核苷酸序列。
9.一种外泌体的分离方法,其特征在于,包括步骤,将待分离样品注入到权利要求1所述微流控芯片的微通道中,静置,捕获待测样品中的外泌体,清洗去除样品。
10.一种外泌体内部microRNA和表面蛋白的同时检测方法,其特征在于,包括如下步骤:
(1)将待测样品注入到权利要求1所述微流控芯片的微通道中,静置,捕获待测样品中的外泌体;
(2)向步骤(1)的微通道中加入退火后的反应序列,静置,之后清洗通道;
(3)向步骤(2)的微通道中加入荧光标记的靶标表面蛋白核酸适配体,静置,之后清洗通道;
(4)使用共聚焦荧光显微镜拍摄微通道的荧光照片,使用软件对荧光强度进行量化,得到外泌体内部microRNA和表面蛋白的含量;
优选地,步骤(1)中静置时间为2.8-3.2h,静置温度为37℃;
优选地,步骤(2)中静置时间为1.8-2.2h,静置温度为37℃;
优选地,步骤(3)中静置时间为1.8-2.2h,静置温度为37℃;
优选地,步骤(2)微通道中还加入链球菌溶血素和核酸酶抑制剂。
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