CN112269023A - 微流控拉曼芯片及基于微流控拉曼芯片检测血液中外泌体的方法 - Google Patents
微流控拉曼芯片及基于微流控拉曼芯片检测血液中外泌体的方法 Download PDFInfo
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Abstract
本发明公开了一种微流控拉曼芯片及基于微流控拉曼芯片检测血液中外泌体的方法,技术领域,该微芯片由四个入口、一个出口、一个阀门、一个混合室和一个拉曼检测区组成。血清样本和CD63抗体磁珠分别通过入口1和3泵入芯片,两种溶液流经交错的三角形微柱混合室时,两者充分混合并发生免疫反应。CD63磁珠‑外泌体复合物通过磁力固定在拉曼检测区。从入口4加入EpCAM功能化的拉曼球与CD63磁珠‑外泌体复合物混合反应后,最后进行拉曼检测。该芯片可以快速、简单、高灵敏、定量检测血清样本中的外泌体,检测专一性强,操作简单,进而有助于更加准确评估各种疾病诊断和指导治疗。
Description
技术领域
本研究设计的阵列型微流控芯片,用于血清样品中外泌体的检测,该芯片可用于应用于 临床癌症检测分析技术领域。
背景技术
***癌(PCa)是世界范围内男性最常见的癌症之一。PSA是***癌筛查、诊断中使用 最广泛的生物标志物,然而,PSA并不是***癌所特有的标记物,在非癌性疾病如良性前 列腺增生(BPH)和***炎中PSA水平也会升高。因此,迫切需要特异性的生物标志物来提 高PCa诊断的特异性和敏感性。外泌体直径在30-200nm之间,由大多数细胞所释放,存在 于不同的生物液体中,如血液、唾液和尿液。外泌体的反映其亲代细胞的特定生理条件和功 能。肿瘤来源的外泌体由于其含量高(1011mL-1)和在血液循环中的稳定性,成为癌症患者液体 活检中很有前途的生物标志物。拉曼散射(Raman Scattering)是一种具有单分子灵敏度的振 动光谱技术。目前,拉曼光谱的低信号强度严重限制了其直接应用。自20世纪90年代以来, 科学家们发现被分析物吸附在粗糙的金属表面,特别是Au和Ag上时,拉曼信号的强度可以 大大增强。然而,基于增强拉曼的免疫分析的稳定性和重复性较低,并且生物分子,如脂质、 蛋白质和核酸是拉曼活性,能表现出强烈的拉曼信号,为了能有效地消除血液中这些复杂的成 分的干扰,近年来生物沉默区内聚合物拉曼探针受到了人们广泛关注。我们合成了高密度的氰 基拉曼微珠在生物拉曼沉默区(1800-2800cm-1)具有强烈而独特的拉曼振动,可用于血清中 外泌体定量检测的探针。
微流控技术以其高通量、低消耗、低污染和所需样品量低等优点被认为是一种很有前途 的技术。拉曼散射与微流控的结合为***小型化和集成提供了新的机会,带来了两种技术的 优势。一方面,在纳升体积内以自动化和可重复性的方式进行拉曼测量,使得信号更加稳定, 减少很多人为因素干扰;另一方面,微流控是在小体积平台上检测的方法,拉曼检测不受水 的干扰且灵敏度高,是这项工作的理想选择。于是,我们开发了一种结合芯片上连续混合流 和免疫磁分离的原位拉曼检测分析的芯片。首先,使用抗CD63磁珠,通过交错三角柱阵列 混合区充分混合并发生免疫捕获;接着,捕获的外泌体通过磁性被固定在拉曼检测区;最后, 加入EpCAM功能化高密度的氰基拉曼微珠作为探针,通过监测其特征峰2230cm-1峰的强度, 定量检测外泌体含量。
发明内容
为了克服现有技术的不足,本发明设计了一种阵列型微流控芯片,用于血清样品中外泌 体。本发明公开了微流控芯片,CD63修饰的磁珠和EpCAM功能化拉曼微珠的制备方法,并 基于该芯片实现血清中外泌体的快速检测。
本发明的技术方案具体介绍如下。
本发明提供一种交错阵列型的微流控拉曼检测芯片,所述检测芯片包括四个入口、一个 出口、一个阀门、一个混合室和一个拉曼检测区,其中一个入口泵入血清样本,一个入口泵 入CD63抗体修饰的磁珠,血清样本和抗体修饰的磁珠进入混合室充分混合并发生免疫反应, 得到CD63磁珠-外泌体复合物;一个入口泵入PBS缓冲液,用于洗涤通道中残留的CD63磁 珠-外泌体复合物,一个入口泵入拉曼微球,在拉曼检测区,拉曼微球与CD63磁珠-外泌体复 合物发生反应。
进一步,所述混合室由交错的三角形微柱阵列所构成。
进一步,所述CD63修饰的磁珠为羧基修饰的磁珠与CD63抗体偶联。
进一步,,所述拉曼微球,其制备方法如下:
1)制备聚合物单体:将4-氰基苯甲胺溶于二氯甲烷中,加入三甲胺,接着加入甲基丙烯 酰氯,反映后得到单体;
2)拉曼微珠的合成:采用乳液聚合的方法合成拉曼微珠,将单体、二甲基丙烯酸乙二醇 酯、偶氮二异丁腈和十六烯溶解于二氯甲烷中,含十二烷基硫酸钠的去离子水,净化后加入 油相中,然后在聚合得到拉曼微珠,离心后重新分散在PBS中,为拉曼微珠容液;
3)将拉曼微珠溶液加入Tris-HCl缓冲液和多巴胺溶液,结合EpCAM抗体反应得到拉曼 微球。
本发明还提供基于微流控拉曼检测芯片检测样本中外泌体的方法,包括如下步骤:
1)从两个入口分别泵入血清样本和CD63抗体修饰的磁珠,两种溶液流经混合室时,两 者充分混合并发生免疫反应;
2)PBS缓冲液从第三个入口进入,将通道中残留的CD63磁珠-外泌体复合物洗涤出;
3)接着从第四个入口泵入拉曼微球,在拉曼检测区,拉曼微球与CD63磁珠-外泌体复 合物发生反应;
4)最后将该芯片直接进行拉曼检测,定量外泌体的含量。
进一步,步骤1)中,所述混合室由交错的三角形微柱阵列所构成。
进一步,步骤1)中,所述CD63修饰的磁珠为羧基修饰的磁珠与CD63抗体偶联。
进一步,步骤3)中,拉曼微球,其制备方法如下:
1)制备聚合物单体:将4-氰基苯甲胺溶于二氯甲烷中,加入三甲胺,接着加入甲基丙烯 酰氯,反映后得到单体;
2)拉曼微珠的合成:采用乳液聚合的方法合成拉曼微珠,将单体、二甲基丙烯酸乙二醇 酯、偶氮二异丁腈和十六烯溶解于二氯甲烷中,含十二烷基硫酸钠的去离子水,净化后加入 油相中,然后在聚合得到拉曼微珠,离心后重新分散在PBS中,为拉曼微珠容液;
3)将拉曼微珠溶液加入Tris-HCl缓冲液和多巴胺溶液,结合EpCAM抗体反应得到拉曼 微球。
本发明一个实施例中提供一种基于微流控拉曼芯片检测血清中外泌体的方法,包括如下
步骤:
1)通过计算机模拟设计了阵列式微流控芯片,模拟得到最佳效果后,采用硅片上制做图 案,SU8-2075负光胶光刻,PDMS浇筑,从硅片上剥离下PDMS层,最后与玻璃片键合组装 成完整的芯片。
2)用恒流泵以0.6μL min-1的速度分别从入口1和3泵入20μL血清样本和5μL CD63抗体修饰的磁珠,两种溶液流经交错的三角形柱混合室时,两者充分混合并发生免疫反应;
3)PBS缓冲液从入口2以3μL min-1的速度进入,将通道中残留的CD63磁珠-外泌体复 合物洗涤出;
4)接着从进口4以3μL min-1的速度泵入5μL拉曼微球,该拉曼聚合物球含有高密度的 氰基,在拉曼沉默区(1800-2800cm-1)中2230cm-1处具有高灵敏的信号,修饰上EpCAM的拉曼微球能特异性地识别肿瘤来源的外泌体,在拉曼检测区,拉曼微球与CD63磁珠-外泌体复合物发生反应;
5)最后将该芯片直接进行拉曼检测,以2230cm-1处的峰值来定量外泌体的含量。
样品与磁珠流经三角形微柱时,增大了样本与磁珠两者碰撞接触的机会使其充分混合并 在该过程中该发生免疫捕获。
优选的,步骤1)中,阵列式微流控芯片,其制备方法如下:
采用在硅片上制做图案,通过微流控软光刻技术,硅片模板使用SU8-2075负光胶光刻, 按照5000rpm/10s,a=100m/s2,2000rpm/30s,a=300m/s2程序匀胶,使得光刻的高度为120 μm。接着进行微通道的刻蚀,依次通过浇铸,曝光,显影来制造硅片模板。表面疏水处理后, 将按照PDMS前驱体与固化剂的重量比为10:1的比例搅拌混合均匀,小心地倒在刻有通道 的硅片模板上,将其抽真空约20min以消除气泡,并在80℃下固化4h。然后,将固化的PDMS复制品从硅片上轻轻剥离。手动使用打孔器和手术刀在成形的入口,出口上钻孔。通过等离子处理将PDMS层和玻璃板粘合在一起,以形成完整的生物微流体***。在显微镜下检查该装置以重新确认入口/出口和微通道。
整个芯片长70mm,宽25mm,微通道的宽度为0.5mm,高度为120μm,混合区的三角 型微柱的间距为200μm,检测区的直径为5mm,阀门的直径为2mm,4个入口和一个出口 的孔径为0.8mm。实验中所有的液体都是通过微型流动注射泵在恒定流量下精确注入的。为 防止非特异性吸附,用阻断缓冲液(2.5w/w%BSA和0.01w/w%Tween-20 in PBS)以3μL min-1的流速先预处理PDMS微通道表面15min。
优选的,步骤2)中,CD63修饰的磁珠,其制备方法如下:
将商业化羧基修饰的磁珠(1mL,10mg mL-1,500nm)用PBST缓冲液洗涤两次,分散在2ml PBST缓冲液中(0.05%Tween-20 in PBS)。接下来,加入1ml EDC(10mg mL-1)和1ml NHS(10mg mL-1),在25℃下孵育30min,然后与CD63抗体(10μg)在室温下偶联2h,磁 性分离后再用包含1%BSA的PBST缓冲液封闭,最后,用PBS洗涤三次去除杂质,用PBST 缓冲液重悬,4℃保存备用。
优选的,步骤4)中,EpCAM修饰的拉曼微球,其制备方法如下:
1)制备聚合物单体:将4-氰基苯甲胺溶于二氯甲烷中,加入干燥的三甲胺,接着慢慢加 入甲基丙烯酰氯,搅拌10min,反应6h后,粗产物经硅胶柱层析得到纯单体;
2)拉曼微珠的合成:采用乳液聚合的方法合成了拉曼微珠,将单体(50mg)、二甲基丙 烯酸乙二醇酯(EGDMA)(10%mol单体)、偶氮二异丁腈(AIBN)(5%w/w)和十六烯(3.1mg)溶解于500μL二氯甲烷中,取5mL含5mg SDS的去离子水,用鼓泡氮气净化5min, 加入油相中。然后在冰浴中超声处理5分钟,在80℃下聚合18h,离心后重新分散在5mL PBS 中,拉曼微珠的最终浓度为10mg mL-1;
3)将拉曼微珠溶液(1ml,10mgmL-1)加入Tris-HCl缓冲液(100μL,50mM)和多巴胺溶液 (100μL,5mg mL-1),结合10μg EpCAM抗体反应1h,反应完成后离心,沉淀再分散,最终 溶液保存在4℃备用。
与现有技术相比,本发明具有如下有益效果:
本发明提供一种一种用于外泌体原位分离分析的新型微流控拉曼生物芯片及利用该芯片 检测血液中外泌体的方法,在纳升体积内以自动化和可重复性的方式进行拉曼测量,使得信 号更加稳定,减少很多人为因素干扰;并且选择的拉曼检测不受水的干扰且灵敏度高。该芯 片主要由两部分组成:快速混合和免疫反应区、拉曼检测区。我们设计了一种交错的三角形微 柱用于有效的混合和捕获外泌体,然后基于***癌外泌体表面特异性蛋白EpCAM,合成一 种新型的聚合物纳米材料用于定量检测***癌的外泌体,其位于拉曼沉默区2230cm-1处的 信号能有效消除细胞内其他复杂成分的干扰,实现***癌特异性检测。检测时间在1h内, 检测灵敏度可达1.6×102particles mL-1,该方法稳定性好、操作简便、成本低。因此,该新芯 片可能成为检测血样中外泌体的一种潜在方法。
附图说明
图1为三明治式纳米材料的表征;其中a为500nm磁珠的TEM图;b为CD63修饰磁 珠的TEM图;c为拉曼微球的TEM图;d为拉曼微球切片的TEM图;e为LnCap细胞分泌 的外泌体的TEM图;f为外泌体的粒径测试图,平均粒径为100nm;g为LNCap和PrEc分 离的外泌体CD9、CD63和EpCAM的Western blot图;h为拉曼微球的DLS粒径测试图,平 均粒径为300nm;i为PrEC外泌体、CD63-Mag/PrEC外泌体/拉曼微珠、LNCaP外泌体和 CD63-Mag/LNCaP外泌体/拉曼微珠的荧光图像;j为该三明治材料对来源于LNCaP和PrEc 的外泌体的捕获效率,其中对来源于***癌细胞LNCaP分泌的外泌体的捕获效率高达 76.68%。
图2为聚合物单体的NMR图,1H NMR(400MHz,CDCl3)δ7.61,7.59,7.41,7.39,5.29,5.19,4.85,4.74,4.65,1.97,1.86。
图3为拉曼微珠的制备路线,单体通过乳液聚合成拉曼微球然后修饰上EpCAM抗体。
图4为芯片优化图,a为微流控拉曼芯片的实物图,宽25mm,长75mm;b为混合区域的光学显微镜图像;c为混合区的微柱间距的优化图,为防止磁珠聚集导致微通道堵塞,采用200μm作为微柱的最佳间距;d为流速优化图,为了能快速检测目标物,选用以0.6μL min-1为最佳流速,在1h内外泌体的捕获效率高达72.5%。
图5为外泌体检测图。A为不同LNCaP的外泌体浓度的拉曼光谱(1800-2800cm-1);b为 不同外泌体浓度在2230cm-1处拉曼强度的曲线拟合图;c为不同外泌体浓度与拉曼强度的线 性关系。外泌体浓度:1.6×102-1.6×109particles mL-1,每个浓度重复3次。
图6为从临床血清样本中定量检测外泌体。A为采用微流体拉曼芯片检测结果图;b为 临床样本t-检验分析图。
图7为本发明一个实施例中微流控拉曼芯片的结构示意。
具体实施方式
下面结合实施例及附图对本发明作进一步详细描述,但本发明法的实施方式不限于此。
实施例中所采用的试剂及仪器如下:
1.试剂
羧基化的磁珠(10mg/mL,200nm)购自南京东纳有限公司,EpCAM,anti-CD63抗体,4-氰基苯甲胺,EGDMA,AIBN,十六烯,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride(EDC)(≥98.0%),N-hydroxysuccinimide(NHS)(98%),多巴胺,Tween 20,柠购 自Sigma;PBS缓冲液(pH 7.4),Tris-HCl(pH 8.0),4-morpholineethanesulfonicacid(MES, ≥99%),MEST溶液是由100mM MES加入50μL0.05%Tween 20),PBST溶液(19990μL PBS 缓冲液加入10μL Tween 20)。
2、仪器
透射电镜200KV(型号:JEM 2011,厂商:日本JEOL公司),Horiba Jobin YvonXploRA 显微共聚焦拉曼光谱仪,用功率为0.5mw的532nm激光对样品进行激发,共激发出样品每 个SERS谱的采集时间为1秒。每个样品收集3个光谱,以确保信号的重现性。
实施例1
基于微流控拉曼芯片检测血液中外泌体的方法该方法包括以下步骤:
1.外泌体标准样品的制备
细胞培养:LNCaP和正常***上皮细胞PrEC在37℃含RPMI 1640(10%(v/v)胎牛血 清和1%(v/v)青霉素链霉素)的培养基中培养。生长到100%时,去除培养基,用PBS(2X)洗涤细胞,采用血清饥饿培养(RPMI 1640,无胎牛血清)72小时后收集培养基。
外泌体提取:收集细胞培养液,4℃离心(3000g,20min),去除细胞和细胞碎片,然后 用0.22μm过滤器过滤,将大的细胞碎片彻底去除,接下来在4℃,120000g离心60min, 经过两次离心去除上清液。最后,加入100μL的PBS,得到制备好的外泌体溶液,保存在-20℃以便进一步分析。
2.CD63修饰的磁珠的制备
1)商业化羧基修饰的磁珠(500nm,10mg/mL)超声3min混匀后,取200μL
2)磁珠置于干净的EP管中,用PBST溶液洗涤3次,加入100μL EDC(10mg/mL)和 100μL NHS(10mg/mL)溶液25℃震荡孵育30min后磁性分离;
3)加入10μg CD63,25℃震荡偶联2h后磁性分离,用200μL PBST缓冲液(包含 1%BSA)封闭1h,其中BSA被用来减少非特异性吸附。
4)最后,用PBS洗涤三次去除杂质,用PBST缓冲液重悬,4℃保存备用,最终CD63 浓度为50μg/mL。
3.硼酸化的SERS标签的制备EpCAM修饰的拉曼微球,其制备方法如下:
1)制备聚合物单体:将4-氰基苯甲胺溶于二氯甲烷中,加入干燥的三甲胺,接着慢慢加 入甲基丙烯酰氯,搅拌10min,反应6h后,粗产物经硅胶柱层析得到纯单体;
2)拉曼微珠的合成:采用乳液聚合的方法合成了拉曼微珠,将单体(50mg)、EGDMA(10%mol单体)、AIBN(5%w/w)和十六烯(3.1mg)溶解于500μL二氯甲烷中,取5mL含 5mgSDS的去离子水,用鼓泡氮气净化5min,加入油相中。然后在冰浴中超声处理5分钟, 在80℃下聚合18h,离心后重新分散在5mL PBS中,拉曼微珠的最终浓度为10mg mL-1;
3)将拉曼微珠溶液(1ml,10mg mL-1)加入Tris-HCl缓冲液(100μL,50mM)和多巴胺溶液 (100μL,5mg mL-1),结合10μg EpCAM抗体反应1h,反应完成后离心,沉淀再分散,最终溶液保存在4℃备用,最终EpCAM修饰的拉曼微球浓度为10mg mL-1。
实施例2芯片通道以及流速的优化
为了优化混合区的微柱间距以及流速,设计了150μm,200μm,250μm,300μm四个间距 来研究其对捕获效率影响,采用PKH26染色标记的外泌体流经芯片,计算流入和流出的荧光 强度来计算外泌体的捕获效率。如图4c所示,随着间距的增大外泌体的捕获效率有所降低, 防止磁珠聚集导致微通道堵塞,我们采用200μm作为微柱的最佳间距。4d为流速优化图, 采用6个不同的流速进行研究,为了能快速检测目标物,选用以0.6μL min-1为最佳流速,在 1h内外泌体的捕获效率高达72.5%。
设计如图7所示的芯片。采用在硅片上制做图案,通过微流控软光刻技术,硅片模板使 用SU8-2075负光胶光刻,按照5000rpm/10s,a=100m/s2,2000rpm/30s,a=300m/s2程序匀 胶,使得光刻的高度为120μm。接着进行微通道的刻蚀,依次通过浇铸,曝光,显影来制造 硅片模板。表面疏水处理后,将按照PDMS前驱体与固化剂的重量比为10:1的比例搅拌混合均匀,小心地倒在刻有通道的硅片模板上,将其抽真空约20min以消除气泡,并在80℃ 下固化4h。然后,将固化的PDMS复制品从硅片上轻轻剥离。手动使用打孔器和手术刀在 成形的入口,出口上钻孔。通过等离子处理将PDMS层和玻璃板粘合在一起,以形成完整的 生物微流体***。在显微镜下检查该装置以重新确认入口/出口和微通道。
如图7所示,检测芯片包括四个入口(分别为第一入口1,第二入口2,第三入口3,第四入口4),一个出口5、一个阀门7、一个混合室6和一个拉曼检测区8。
其中第一入口至第三入口3可互换。
其中一个实施例中,第一入口1泵入血清样本,第三入口3泵入CD63抗体修饰的磁珠, 血清样本和抗体修饰的磁珠进入混合室6充分混合并发生免疫反应,得到CD63磁珠-外泌体 复合物;第二入口2泵入PBS缓冲液,用于洗涤通道中残留的CD63磁珠-外泌体复合物。第 四入口4泵入拉曼微球,在拉曼检测区,拉曼微球与CD63磁珠-外泌体复合物发生反应。
其中一个优选实施例中,整个芯片长70mm,宽25mm,微通道的宽度为0.5mm,高度为120μm,混合区的三角型微柱的间距为200μm,检测区的直径为5mm,阀门的直径为2 mm,4个入口和一个出口的孔径为0.8mm。实验中所有的液体都是通过微型流动注射泵在恒 定流量下精确注入的。为防止非特异性吸附,用阻断缓冲液(2.5w/w%BSA和0.01w/w% Tween-20 in PBS)以3μL min-1的流速先预处理PDMS微通道表面15min。
对外泌体的检测
采用Nano-sight检测超速离心得到的外泌体溶液的浓度,如图1f外泌体的浓度为1.6× 109particles mL-1将其稀释为10个不同浓度的外泌体溶液,采用微流体拉曼芯片进样20μL 检测,得到不同浓度下对应的拉曼谱图,图5a所示。图5b显示了不同浓度的外泌体(6×102-1.6 ×109particles mL-1)与在2230cm-1处拉曼强度关系图,图5c表明Log(外泌体浓度)与Log (拉曼强度)呈现很好的线性关系,R2=0.982。
实施例4血样中外泌体的检测
分别收集8例***癌患者和10例正常人的血清样本。本实验由上海市第九人民医院伦 理委员会批准,并于项目开始时获得书面知情同意。具体如表1所示。
表1 临床血清样本的信息。
Patient ID | TNM | Sex | Age,years | PLT,10<sup>9</sup>/L | Pathology |
1 | NA | M | 53 | - | Control |
2 | NA | M | 56 | - | Control |
3 | NA | M | 52 | - | Control |
4 | NA | M | 52 | - | Control |
5 | NA | M | 51 | - | Control |
6 | NA | M | 53 | - | Control |
7 | NA | M | 55 | - | Control |
8 | NA | M | 54 | - | Control |
9 | T2N0M0 | M | 71 | 127 | Malignant |
10 | T1N0M0 | M | 75 | 223 | Malignant |
11 | T3N0M0 | M | 74 | 118 | Malignant |
12 | T2N0M0 | M | 72 | 188 | Malignant |
13 | T4N0M1 | M | 78 | 180 | Malignant |
14 | T1N0M0 | M | 76 | 197 | Malignant |
15 | T2N0M0 | M | 75 | 198 | Malignant |
16 | T2N0M0 | M | 77 | 140 | Malignant |
17 | T2N0M0 | M | 59 | 268 | Malignant |
18 | T2N0M0 | M | 79 | 177 | Malignant |
血清样本通过0.22μm的过滤器过滤,然后利用实施例1制得的芯片和方法进行检测。 通过微型流动注射泵在恒定流量下精确注入的20μL样本进行检测得到拉曼谱图。根据前面 的外泌体标本样品的检测,计算出实际血样中外泌体的含量,如图6a所示,得到了*** 患者和正常人血样中外泌体含量值,可以看出***患者血样中外泌体浓度大约是正常人的 3倍。通过t检测得到了p<0.0001,如图6b所示。
Claims (8)
1.一种交错阵列型的微流控拉曼检测芯片,其特征在于,所述检测芯片包括四个入口、一个出口、一个阀门、一个混合室和一个拉曼检测区,其中一个入口泵入血清样本,一个入口泵入CD63抗体修饰的磁珠,血清样本和抗体修饰的磁珠进入混合室充分混合并发生免疫反应,得到CD63磁珠-外泌体复合物;一个入口泵入PBS缓冲液,用于洗涤通道中残留的CD63磁珠-外泌体复合物,一个入口泵入拉曼微球,在拉曼检测区,拉曼微球与CD63磁珠-外泌体复合物发生反应。
2.根据权利要求1所述的微流控拉曼检测芯片,其特征在于,所述混合室由交错的三角形微柱阵列所构成。
3.根据权利要求1所述的微流控拉曼检测芯片,其特征在于,所述CD63修饰的磁珠为羧基修饰的磁珠与CD63抗体偶联。
4.根据权利要求1所述的微流控拉曼检测芯片,其特征在于,所述拉曼微球,其制备方法如下:
1)制备聚合物单体:将4-氰基苯甲胺溶于二氯甲烷中,加入三甲胺,接着加入甲基丙烯酰氯,反映后得到单体;
2)拉曼微珠的合成:采用乳液聚合的方法合成拉曼微珠,将单体、二甲基丙烯酸乙二醇酯、偶氮二异丁腈和十六烯溶解于二氯甲烷中,含十二烷基硫酸钠的去离子水,净化后加入油相中,然后在聚合得到拉曼微珠,离心后重新分散在PBS中,为拉曼微珠容液;
3)将拉曼微珠溶液加入Tris-HCl缓冲液和多巴胺溶液,结合EpCAM抗体反应得到拉曼微球。
5.基于微流控拉曼检测芯片检测样本中外泌体的方法,其特征在于,包括如下步骤:
1)从两个入口分别泵入血清样本和CD63抗体修饰的磁珠,两种溶液流经混合室时,两者充分混合并发生免疫反应;
2)PBS缓冲液从第三个入口进入,将通道中残留的CD63磁珠-外泌体复合物洗涤出;
3)接着从第四个入口泵入拉曼微球,在拉曼检测区,拉曼微球与CD63磁珠-外泌体复合物发生反应;
4)最后将该芯片直接进行拉曼检测,定量外泌体的含量。
6.根据权利要求5所述的方法,其特征在于,步骤1)中,所述混合室由交错的三角形微柱阵列所构成。
7.根据权利要求5所述的方法,其特征在于,步骤1)中,所述CD63修饰的磁珠为羧基修饰的磁珠与CD63抗体偶联。
8.根据权利要求5所述的方法,其特征在于,步骤3)中,拉曼微球,其制备方法如下:
1)制备聚合物单体:将4-氰基苯甲胺溶于二氯甲烷中,加入三甲胺,接着加入甲基丙烯酰氯,反映后得到单体;
2)拉曼微珠的合成:采用乳液聚合的方法合成拉曼微珠,将单体、二甲基丙烯酸乙二醇酯、偶氮二异丁腈和十六烯溶解于二氯甲烷中,含十二烷基硫酸钠的去离子水,净化后加入油相中,然后在聚合得到拉曼微珠,离心后重新分散在PBS中,为拉曼微珠容液;
3)将拉曼微珠溶液加入Tris-HCl缓冲液和多巴胺溶液,结合EpCAM抗体反应得到拉曼微球。
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