CN111876147B - 银纳米粒/硫量子点双掺杂mof复合物比率荧光外泌体适体探针的制备方法 - Google Patents

银纳米粒/硫量子点双掺杂mof复合物比率荧光外泌体适体探针的制备方法 Download PDF

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CN111876147B
CN111876147B CN202010766015.4A CN202010766015A CN111876147B CN 111876147 B CN111876147 B CN 111876147B CN 202010766015 A CN202010766015 A CN 202010766015A CN 111876147 B CN111876147 B CN 111876147B
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桂日军
姜晓文
金辉
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Qingdao University
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Abstract

本发明公开了银纳米粒/硫量子点双掺杂金属有机骨架(MOF)复合物比率荧光外泌体适体探针的制备方法，银纳米粒(AgNPs)与前驱体反应，AgNPs掺杂在MOF结构中，锌掺杂硫量子点(Zn‑SQDs)吸附在MOF表面，然后附着适体/多巴胺(Aptamer/DA)复合物，形成纳米复合物适体探针；外泌体(exosomes)与适体特异性结合成Aptamer/DA/exosomes复合物；因适体携带DA远离MOF表面，引起Zn‑SQDs蓝荧光恢复，AgNPs红荧光几乎不变；拟合荧光峰强度比值IZn‑SQDs/IAgNPs与外泌体浓度之间的线性关系，构建比率荧光外泌体适体探针。

Description

银纳米粒/硫量子点双掺杂MOF复合物比率荧光外泌体适体探针的制备方法
技术领域
本发明属于金属有机骨架纳米复合物和比率荧光外泌体适体探针的制备技术领域，具体涉及基于银纳米粒和锌-硫量子点双掺杂金属有机骨架复合物的比率荧光外泌体适体探针的制备方法，其制备的探针用于肿瘤外泌体的高效检测。
背景技术
金属有机骨架复合物Metal-organic frameworks(MOF)具备诸多优点，如比表面积大、孔隙率高、晶格结构有序、稳定性好等，在功能纳米载体方面展现出重要的应用前景。MOF与荧光体如有机染料分子、聚合物、有机/无机杂化物、纳米材料等复合，形成荧光体在MOF内腔体或外表面负载的复合体系即荧光体掺杂的MOF复合物，发展为荧光纳米探针，用于生化分析和生物传感。
近年来，研究者证实外泌体是一种新型生物标志物，用于特定肿瘤细胞跨膜蛋白的精准识别，实现对特定肿瘤的早期筛查。外泌体是一种细胞外囊泡，泡体直径为50～100nm，经过內溶体途径从多囊体中释放出来。外泌体含亲本细胞中mRNA、DNA、跨膜和胞质蛋白等生物分子，作为介导细胞之间信息的信使单元，在疾病尤其是癌症有关生理状态及其变化的监测方面发挥关键的作用。
研究者报道了外泌体检测的多种方法，如表面等离子体共振法、流式细胞分析法、颗粒追踪分析法、比色法、发光分析法、电化学分析法等。传统的探针和传感器依赖于单信号强度变化的准确测量，在实际样品的检测中，单一信号强度会受到背景信号、系统误差、传感体系用量、测量环境变化，以及共存组分和溶剂等干扰。相比之下，比率法采用双信号峰值比为信号输出，避免了内在和外在因素的影响，有效提升了传感体系的输出信号对目标物检测的准确性。
例如，Yujiao Sun等设计了黑磷纳米片与MOF组装的功能杂化薄膜用于比率电化学传感外泌体(Yujiao Sun,Hui Jin,Xiaowen Jiang,Rijun Gui.Assembly of blackphosphorous nanosheets and MOF to form functional hybrid thin–film forprecise protein capture,dual–signal and intrinsic self–calibration sensing ofspecific cancer–derived exosomes.Analytical Chemistry,2020,92,2866–2875)，LiZhao等制备了3D DNA步行器用于外切酶III-辅助电化学比率生物传感外泌体(Li Zhao,Ruijiao Sun,Peng He,Xiaoru Zhang.Ultrasensitive detection of exosomes bytarget-triggered three-dimensional DNA walking machine and exonuclease III-assisted electrochemical ratiometric biosensing.Analytical Chemistry,2019,91,14773–14779)，肖义等开发了基于两种荧光染料的比率定量用于外泌体的测定(肖义；李宁；张新富；陈令成.一种利用荧光比率进行外泌体快速定量的方法.国家发明专利.公布号CN108169199A)。
当前比率法检测外泌体主要是比率电化学法和比率荧光法，相对比率电化学法，比率荧光法可实现溶液相生化传感，可视化半定量检测以及生物成像检测等，应用前景广阔。比率荧光法检测外泌体已有文献报道，使用染料标记的适体，染料分子掺杂或与纳米材料复合，构建基于染料的复合传感体系，用于比率荧光检测外泌体。先前文献报导了外泌体的检测，但这些检测方法依然存在不足，如执行纳米级肿瘤外泌体的高灵敏性、特异性、可重复性定量检测，低成本和高效率检测等。本发明公开了银纳米粒(AgNPs)和锌-硫量子点(Zn-SQDs)双掺杂的MOF纳米复合物，即AgNPs/Zn-SQDs/MOF，构建基于该无机纳米材料双掺杂且荧光双发射的MOF纳米复合物适体探针，实现比率荧光检测特定肿瘤纳米外泌体。目前，尚未有基于纳米材料双掺杂且荧光双发射MOF纳米复合物适体探针AgNPs/Zn-SQDs/MOF，用于比率荧光检测外泌体的国内外文献和专利报道。
发明内容
本发明的目的在于克服上述现有技术存在的不足，发展一种设计新颖、制备简单和多功能化的纳米探针，用于高灵敏性和特异性比率荧光检测乳腺癌外泌体的新方法。
为实现上述目的，本发明涉及的一种银纳米粒/硫量子点双掺杂MOF复合物比率荧光外泌体适体探针的制备方法，其制备方法包括以下步骤：
(1)制备锌掺杂硫量子点(Zn-SQDs)：在搅拌下向75mL二次蒸馏水中加入1.5g升华硫，3mL聚乙二醇PEG-400和4g氢氧化钠，形成均质混合液；转入含100mL聚四氟乙烯内衬的微型高压反应釜中，在70℃下搅拌反应5h；冷却产物溶液至室温，取5mL产物溶液，在搅拌下加入5mL浓度为5wt％的硝酸锌水溶液，继续搅拌30min，制得Zn-SQDs水分散液；
(2)制备银纳米粒(AgNPs)：在搅拌下向50mL二次蒸馏水中加入50mg硫辛酸粉末，加入0.3mL浓度为1mol L-1的硼氢化钠水溶液，搅拌反应30min，形成均质混合液；加入0.3mL浓度为0.1mol L-1的硝酸银，再加入0.8mL浓度为1mol L-1的硼氢化钠水溶液，继续搅拌2h，制得AgNPs水分散液；
(3)制备AgNPs和Zn-SQDs双掺杂的MOF复合物(AgNPs/Zn-SQDs/MOF)：在搅拌下向10mL的2-甲基咪唑甲醇溶液中加入1mL浓度为1～10mg mL-1的AgNPs水分散液，搅拌10min形成混合液；再加入10mL六水合硝酸锌水溶液，搅拌30min形成沉淀物，用乙醇和蒸馏水洗涤三次，在5000rpm转速下离心10min，制得AgNPs/MOF复合物水分散液；在搅拌下加入1mL浓度为1～10mg mL-1的Zn-SQDs水分散液，搅拌反应2h；产物经离心得沉淀物，沉淀物经洗涤和干燥制得AgNPs/Zn-SQDs/MOF水分散液；
(4)制备比率荧光外泌体适体探针：配制浓度为1～10μmol L-1的CD63跨膜蛋白对应单链DNA适体Aptamer的磷酸盐水(PBS)缓冲液，在搅拌下加入多巴胺(DA)，在37℃下孵育30～120min，DA浓度为0.1～1μmol L-1，形成Aptamer/DA复合物；在搅拌下将此复合物加入AgNPs/Zn-SQDs/MOF水分散液中，在37℃下孵育1～6h，制得AgNPs/Zn-SQDs/MOF/Aptamer/DA纳米复合物水分散液；加入从乳腺癌MCF-7细胞提取的外泌体的PBS缓冲液，形成均质混合液；测量混合液的荧光发射光谱，拟合荧光峰强度比值IZn-SQDs/IAgNPs与外泌体浓度之间的线性关系，构建用于外泌体检测的比率荧光探针；外泌体浓度的线性检测范围为5×101～1×106particles μL–1，检测限为10～50particlesμL–1。
本发明的效果是：公开了一种基于银纳米粒和锌-硫量子点双掺杂金属有机骨架复合物AgNPs/Zn-SQDs/MOF/Aptamer/DA的比率荧光外泌体适体探针的制备方法，AgNPs与MOF前驱体反应，将AgNPs掺杂在MOF结构中，然后Zn-SQDs吸附在MOF结构表面；通过静电吸附、氢键、分子间作用力等，Aptamer/DA复合物附着在AgNPs/Zn-SQDs/MOF结构表面；在光激发下，电子受体分子DA与MOF表面Zn-SQDs接触，引起光电子转移导致Zn-SQDs荧光淬灭；加入从乳腺癌MCF-7细胞提取的外泌体(exosomes)，该外泌体与CD63跨膜蛋白对应单链DNAAptamer发生特异性结合，形成Aptamer/DA/exosomes复合物；因Aptamer携带DA远离MOF结构表面的Zn-SQDs，引起Zn-SQDs荧光恢复；在外泌体的添加过程中，由于AgNPs掺杂在MOF结构中，其荧光几乎不变，将其作为参比信号；拟合荧光峰强度比值IZn-SQDs/IAgNPs与外泌体浓度间的线性关系，构建基于AgNPs/Zn-SQDs/MOF/Aptamer/DA纳米复合物的比率荧光外泌体适体探针。与现有技术相比，本发明设计新颖，制备简单，纳米探针具备高灵敏性、特异性、比率荧光内置校准和定量检测的多功能性，应用于生物学流体样品中乳腺癌纳米外泌体的高效检测。
附图说明
图1为基于AgNPs/Zn-SQDs/MOF/Aptamer/DA纳米复合物的新型比率荧光外泌体适体探针的制备过程示意图。
具体实施方式
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1
本实施例涉及的银纳米粒/硫量子点双掺杂MOF复合物比率荧光外泌体适体探针的制备方法，该适体探针的制备示意图如图1所示，具体制备步骤如下：
在搅拌下向75mL二次蒸馏水中加入1.5g升华硫，3mL PEG-400和4g NaOH，形成均质混合液；转入含100mL聚四氟乙烯内衬的微型高压反应釜中，在70℃下搅拌反应5h；冷却产物溶液至室温，取5mL产物溶液，在搅拌下加入5mL浓度为5wt％的硝酸锌水溶液，继续搅拌30min，制得Zn-SQDs水分散液。
在搅拌下向50mL二次蒸馏水中加入50mg硫辛酸粉末，加入0.3mL浓度为1mol L-1的NaBH4水溶液，搅拌反应30min，形成均质混合液；加入0.3mL浓度为0.1mol L-1的硝酸银，再加入0.8mL浓度为1mol L-1的NaBH4水溶液，继续搅拌2h，制得AgNPs水分散液。
在搅拌下向10mL的2-甲基咪唑甲醇溶液中加入1mL浓度为2mg mL-1的AgNPs水分散液，搅拌10min形成混合液；再加入10mL六水合硝酸锌水溶液，搅拌30min形成沉淀物，用乙醇和蒸馏水洗涤三次，在5000rpm转速下离心10min，制得AgNPs/MOF复合物水分散液；在搅拌下加入1mL浓度为3mg mL-1的Zn-SQDs水分散液，搅拌反应2h；产物经离心得沉淀物，沉淀物经洗涤和干燥制得AgNPs/Zn-SQDs/MOF水分散液。
配制浓度为2μmol L-1的CD63跨膜蛋白对应单链DNA适体Aptamer的PBS缓冲液，在搅拌下加入DA，在37℃下孵育60min，DA浓度为0.2μmol L-1，形成Aptamer/DA复合物；在搅拌下将此复合物加入AgNPs/Zn-SQDs/MOF水分散液中，在37℃下孵育3h，制得AgNPs/Zn-SQDs/MOF/Aptamer/DA纳米复合物水分散液；加入从乳腺癌MCF-7细胞提取的外泌体的PBS缓冲液，形成均质混合液；测量混合液的荧光发射光谱，拟合荧光峰强度比值IZn-SQDs/IAgNPs与外泌体浓度之间的线性关系，构建用于外泌体定量检测的比率荧光探针；外泌体浓度的线性检测范围为1×102～1×106particlesμL–1，检测限为50particlesμL–1。
实施例2
本实施例涉及的银纳米粒/硫量子点双掺杂MOF复合物比率荧光外泌体适体探针的制备方法，该适体探针的制备示意图如图1所示，Zn-SQDs和AgNPs水分散液的制备同实施例1，其它具体制备步骤如下：
在搅拌下向10mL的2-甲基咪唑甲醇溶液中加入1mL浓度为5mg mL-1的AgNPs水分散液，搅拌10min形成混合液；再加入10mL六水合硝酸锌水溶液，搅拌30min形成沉淀物，用乙醇和蒸馏水洗涤三次，在5000rpm转速下离心10min，制得AgNPs/MOF复合物水分散液；在搅拌下加入1mL浓度为6mg mL-1的Zn-SQDs水分散液，搅拌反应2h；产物经离心得沉淀物，沉淀物经洗涤和干燥制得AgNPs/Zn-SQDs/MOF水分散液。
配制浓度为5μmol L-1的CD63跨膜蛋白对应单链DNA适体Aptamer的PBS缓冲液，在搅拌下加入DA，在37℃下孵育90min，DA浓度为0.5μmol L-1，形成Aptamer/DA复合物；在搅拌下将此复合物加入AgNPs/Zn-SQDs/MOF复合物水分散液中，在37℃下孵育4h，制得AgNPs/Zn-SQDs/MOF/Aptamer/DA纳米复合物水分散液；加入从乳腺癌MCF-7细胞提取的外泌体的PBS缓冲液，形成均质混合液；测量混合液的荧光发射光谱，拟合荧光峰强度比值IZn-SQDs/IAgNPs与外泌体浓度之间的线性关系，构建用于外泌体定量检测的比率荧光探针；外泌体浓度的线性检测范围为5×101～5×105particlesμL–1，检测限为20particlesμL–1。
实施例3
本实施例涉及的银纳米粒/硫量子点双掺杂MOF复合物比率荧光外泌体适体探针的制备方法，该适体探针的制备示意图如图1所示，Zn-SQDs和AgNPs水分散液的制备同实施例1，其它具体制备步骤如下：
在搅拌下向10mL的2-甲基咪唑甲醇溶液中加入1mL浓度为8mg mL-1的AgNPs水分散液，搅拌10min形成混合液；再加入10mL六水合硝酸锌水溶液，搅拌30min形成沉淀物，用乙醇和蒸馏水洗涤三次，在5000rpm转速下离心10min，制得AgNPs/MOF复合物水分散液；在搅拌下加入1mL浓度为9mg mL-1的Zn-SQDs水分散液，搅拌反应2h；产物经离心得沉淀物，沉淀物经洗涤和干燥制得AgNPs/Zn-SQDs/MOF水分散液。
配制浓度为8μmol L-1的CD63跨膜蛋白对应单链DNA适体Aptamer的PBS缓冲液，在搅拌下加入DA，在37℃下孵育120min，DA浓度为0.8μmol L-1，形成Aptamer/DA复合物；在搅拌下将此复合物加入AgNPs/Zn-SQDs/MOF复合物水分散液中，在37℃下孵育5h，制得AgNPs/Zn-SQDs/MOF/Aptamer/DA纳米复合物水分散液；加入从乳腺癌MCF-7细胞提取的外泌体的PBS缓冲液，形成均质混合液；测量混合液的荧光发射光谱，拟合荧光峰强度比值IZn-SQDs/IAgNPs与外泌体浓度之间的线性关系，构建用于外泌体定量检测的比率荧光探针；外泌体浓度的线性检测范围为1×102～5×105particlesμL–1，检测限为40particlesμL–1。

Claims (1)

1.一种银纳米粒/硫量子点双掺杂MOF复合物比率荧光外泌体适体探针的制备方法，其特征在于，该方法具体包括以下步骤：
(1)制备锌掺杂硫量子点Zn-SQDs：在搅拌下向75mL二次蒸馏水中加入1.5g升华硫，3mL聚乙二醇PEG-400和4g氢氧化钠，形成均质混合液；转入含100mL聚四氟乙烯内衬的微型高压反应釜中，在70℃下搅拌反应5h；冷却产物溶液至室温，取5mL产物溶液，在搅拌下加入5mL浓度为5wt％的硝酸锌水溶液，继续搅拌30min，制得Zn-SQDs水分散液；
(2)制备银纳米粒AgNPs：在搅拌下向50mL二次蒸馏水中加入50mg硫辛酸粉末，加入0.3mL浓度为1mol L-1的硼氢化钠水溶液，搅拌反应30min，形成均质混合液；加入0.3mL浓度为0.1mol L-1的硝酸银，再加入0.8mL浓度为1mol L-1的硼氢化钠水溶液，继续搅拌2h，制得AgNPs水分散液；
(3)制备AgNPs和Zn-SQDs双掺杂的MOF复合物AgNPs/Zn-SQDs/MOF：在搅拌下向10mL的2-甲基咪唑甲醇溶液中加入1mL浓度为1～10mg mL-1的AgNPs水分散液，搅拌10min形成混合液；再加入10mL六水合硝酸锌水溶液，搅拌30min形成沉淀物，用乙醇和蒸馏水洗涤三次，在5000rpm转速下离心10min，制得AgNPs/MOF复合物水分散液；在搅拌下加入1mL浓度为1～10mg mL-1的Zn-SQDs水分散液，搅拌反应2h；产物经离心得沉淀物，沉淀物经洗涤和干燥制得AgNPs/Zn-SQDs/MOF水分散液；
(4)制备比率荧光外泌体适体探针：配制浓度为1～10μmol L-1的CD63跨膜蛋白对应单链DNA适体Aptamer的磷酸盐水PBS缓冲液，在搅拌下加入多巴胺DA，在37℃下孵育30～120min，DA浓度为0.1～1μmol L-1，形成Aptamer/DA复合物；在搅拌下将此复合物加入AgNPs/Zn-SQDs/MOF水分散液中，在37℃下孵育1～6h，制得AgNPs/Zn-SQDs/MOF/Aptamer/DA纳米复合物水分散液；加入从乳腺癌MCF-7细胞提取的外泌体的PBS缓冲液，形成均质混合液；测量混合液的荧光发射光谱，拟合荧光峰强度比值IZn-SQDs/IAgNPs与外泌体浓度之间的线性关系，构建用于外泌体检测的比率荧光探针；外泌体浓度的线性检测范围为5×101～1×106particles μL–1，检测限为10～50particles μL–1。
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