CN110736779B - 用于肿瘤外泌体自校准检测的双功能杂化薄膜的制备方法 - Google Patents

用于肿瘤外泌体自校准检测的双功能杂化薄膜的制备方法 Download PDF

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CN110736779B
CN110736779B CN201911078972.1A CN201911078972A CN110736779B CN 110736779 B CN110736779 B CN 110736779B CN 201911078972 A CN201911078972 A CN 201911078972A CN 110736779 B CN110736779 B CN 110736779B
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桂日军
孙玉娇
金辉
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Qingdao University
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Abstract

本发明公开了用于肿瘤外泌体自校准检测的双功能杂化薄膜的制备方法，基于黑磷纳米片BPNSs、适体和二茂铁Fc掺杂钴基金属有机骨架ZIF‑67复合物在氧化铟锡ITO电极上简易自组装，构建双功能杂化薄膜即适体‑BPNSs/Fc/ZIF‑67/ITO。亚甲基蓝MB标记的适体与CD63蛋白特异性结合，实现精准的蛋白俘获。该蛋白是乳腺癌MCF‑7细胞外泌体携带的特定生物分子，实现对该肿瘤细胞外泌体的检测。以MB为响应信号，Fc为参比，构建肿瘤外泌体定量检测的自校准传感器。与现有技术相比，本发明操作便捷、灵敏度高、成本低、特异性好，可作为一种新的外泌体自校准检测方法，用于生物医学样品中外泌体的定量检测。

Description

用于肿瘤外泌体自校准检测的双功能杂化薄膜的制备方法
技术领域：
本发明属于功能杂化薄膜材料和肿瘤外泌体传感器的制备技术领域，具体涉及一种基于黑磷纳米片和二茂铁掺杂的钴基金属有机骨架复合物自组装的双功能杂化薄膜材料的制备方法，其制备的薄膜材料可用于CD63跨膜蛋白的精准俘获和乳腺癌外泌体的自校准检测。
背景技术：
外泌体是通过內溶体途径从多囊体中释放的尺寸为50～100纳米的细胞外囊泡，它从亲本细胞中携带了大量生物高分子，包括跨膜和胞质蛋白、mRNA、DNA和micro-RNA等。外泌体充当着介导细胞间信息的信使，在探测疾病尤其癌症相关的生理状态改变方面发挥了重要作用。近年来，外泌体作为一种有前景的生物标志物被广泛用于癌症的早期诊断，克服了癌症检测过程存在侵入式筛选的高成本和低敏感度的问题。当前，大量的文献资料报道了外泌体的定量检测，但现有的检测技术依然存在某些挑战，难以实现纳米级外泌体的直接和特异性分析。例如，流式细胞术检测受限于较弱的光散射，粒子追踪方法缺乏特异性。在疾病早期阶段，外泌体浓度较低，需要开发新的方法实现外泌体的高敏感检测。
当前用于外泌体检测的方法主要包括流式细胞术、纳米颗粒追踪分析、表面等离子共振、比色、发光和电化学分析等。例如，Zhu等采用表面等离子共振成像技术实现了对外泌体的定量检测(Ling Zhu,Kun Wang,Jian Cui,Huan Liu,Xiangli Bu,Huailei Ma,Weizhi Wang,He Gong,Christopher Lausted,Leroy Hood,Guang Yang,Zhiyuan Hu,Label-free quantitative detection of tumor-derived exosomes through surfaceplasmon resonance imaging,Analytical Chemistry,2014,86,8857-8864)；Xia等基于DNA包封单臂碳纳米管构建了比色检测外泌体的方法(Yaokun Xia,Mengmeng Liu,Liangliang Wang,An Yan,Wenhui He,Mei Chen,Jianming Lan,Jiaoxing Xu,LunhuiGuan,Jinghua Chen,A visible and colorimetric aptasensor based on DNA-cappedsingle-walled carbon nanotubes for detection of exosomes,Biosensors andBioelectronics,2017,92,8–15)。李智洋等利用G-四连体-Hemin模拟过氧化酶催化H2O2反应产生信号，结合滚环扩增合成大量G-四连体进行信号放大，以实现外泌体的定量检测(李智洋,何农跃,黄蓉蓉.专利名称:一种基于适配体与滚环扩增的外泌体检测方法.国家发明专利.公开号:CN109655512A)；王国胜开发了集外泌体分离纯化和特异性外泌体半定量检测的一种产品，构建了基于外泌体的体外即时检测平台(王国胜.一种基于外泌体的体外即时检测平台及其检测方法.国家发明专利.公开号:CN108872564A)。
尽管有关外泌体定量检测的工作已有国内外文献和专利报道，但实现对纳米外泌体的直接和特异性、高灵敏及低成本的高效检测，依然是当前亟待解决的重要科学问题之一。基于此，本发明公开了一种基于黑磷纳米片BPNSs和二茂铁Fc掺杂的钴基金属有机骨架ZIF-67复合物在氧化铟锡ITO薄膜电极上简易自组装的双功能杂化薄膜材料BPNSs/Fc/ZIF-67的制备方法，该薄膜材料可用于CD63跨膜蛋白的精准俘获和乳腺癌MCF-7细胞外泌体的自校准检测。截止本专利申请提交之日为止，尚未检索到有关BPNSs/Fc/ZIF-67双功能杂化薄膜材料的制备，以及基于该薄膜材料用于肿瘤外泌体自校准检测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问题，设计一种操作便捷、灵敏度高、成本较低、特异性好的肿瘤外泌体检测新方法。
为实现上述目的，本发明涉及的一种用于肿瘤外泌体自校准检测的双功能杂化薄膜，其制备方法包括以下步骤：
1.用于肿瘤外泌体自校准检测的双功能杂化薄膜的制备方法，其特征在于，该方法具体包括以下步骤：
(1)Fc/ZIF-67复合物的制备：称取一定量的Co(NO3)2·6H2O和2-甲基咪唑，放置于47mL乙醇和3mL去离子水的混合溶剂中，磁力搅拌下形成均匀的混合液，Co(NO3)2·6H2O和2-甲基咪唑的浓度分别调节至0.1～0.5mol/L和0.8～1.5mol/L。将此混合液加入电解槽中，以Ag/AgCl为参比电极，铂丝为对电极，氧化铟锡ITO为工作电极，以-5至-10V恒定电压，循环伏安扫描100～500s，将Fc/ZIF-67复合物电沉积在氧化铟锡ITO电极表面。
(2)BPNSs/Fc/ZIF-67复合物的制备：称取10～30mg黑磷晶体加入50mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中超声1～6h后，转入探头式超声波发生器中超声1～4h，产物分散液在12000rpm下离心15min，取上层分散液在5000rpm下离心15min。将制得的BPNSs分散液，逐滴添加至Fc/ZIF-67复合物表面，自然干燥，在氧化铟锡ITO电极表面制得BPNSs/Fc/ZIF-67复合物。
(3)适体-BPNSs/Fc/ZIF-67杂化薄膜的制备：将BPNSs/Fc/ZIF-67/ITO薄膜电极浸没在含有1～10μM的CD63跨膜蛋白对应单链DNA适体的磷酸盐水缓冲液中，在37℃下孵育30～120min，取出该薄膜电极，自然干燥，在氧化铟锡ITO电极表面制得适体BPNSs/Fc/ZIF-67复合物，即制得适体-BPNSs/Fc/ZIF-67杂化薄膜。
(4)杂化薄膜传感器的制备：以杂化薄膜为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，加入从乳腺癌MCF-7细胞提取的外泌体，测定不同外泌体浓度下的电化学方波伏安曲线。以修饰在适体链上的亚甲基蓝MB为响应信号，掺杂在ZIF-67金属有机骨架内的二茂铁Fc为参比信号，以电流峰强度IFc/IMB比值为自校准信号输出，拟合IFc/IMB比值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自校准传感器。其中，肿瘤外泌体浓度的线性检测范围为1×102～1×106particlesμL–1，检测限为50～100particlesμL–1。
本发明的效果是：报道了一种基于黑磷纳米片BPNSs、适体和二茂铁Fc掺杂钴基金属有机骨架ZIF-67复合物，在氧化铟锡ITO薄膜电极上简易自组装的双功能杂化薄膜材料，即适体-BPNSs/Fc/ZIF-67/ITO。亚甲基蓝MB标记的单链DNA适体与CD63跨膜蛋白发生适-配体特异性结合，实现对CD63跨膜蛋白的精准俘获。CD63跨膜蛋白是乳腺癌MCF-7细胞外泌体携带的特定生物高分子，可作为生物标志物，实现对乳腺癌MCF-7细胞外泌体的检测。以MB为响应信号，掺杂在ZIF-67内的二茂铁Fc为参比信号，以电流峰强度IFc/IMB比值为自校准信号输出，拟合IFc/IMB与外泌体浓度间的线性关系，构建用于肿瘤外泌体定量检测的自校准传感器。与现有技术相比，本发明方法操作便捷、灵敏度高、成本较低、特异性好，可作为一种新的外泌体自校准检测方法，用于生物医学样品中外泌体的定量检测。
附图说明：
图1为基于黑磷纳米片和二茂铁掺杂的钴基金属有机骨架复合物自组装的双功能杂化薄膜材料的制备，及其用于CD63跨膜蛋白的精准俘获和乳腺癌外泌体自校准检测的原理示意图；
图2(a)为不同外泌体浓度存在下，以该杂化薄膜材料为工作电极测定的电化学方波伏安曲线；
图2(b)为不同外泌体浓度对应的二茂铁和亚甲基蓝的氧化还原电流峰强度比值IFc/IMB，拟合不同IFc/IMB比值与外泌体浓度之间的线性关系。
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：
本实施例涉及的基于黑磷纳米片和二茂铁掺杂的钴基金属有机骨架复合物自组装的双功能杂化薄膜材料，其制备方法和检测原理的示意图如图1所示，具体制备步骤如下：
Fc/ZIF-67复合物的制备：称取一定量Co(NO3)2·6H2O和2-甲基咪唑放置47mL乙醇和3mL去离子水的混合溶剂中，磁力搅拌形成均匀的混合液，Co(NO3)2·6H2O和2-甲基咪唑的浓度分别调节至0.1mol/L和0.8mol/L。将此混合液加入电解槽中，以Ag/AgCl为参比电极，铂丝为对电极，氧化铟锡ITO为工作电极，以-5V恒定电压，循环伏安扫描100s，将Fc/ZIF-67复合物电沉积在氧化铟锡ITO电极表面。
BPNSs/Fc/ZIF-67复合物的制备：称取10mg黑磷晶体加入50mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中超声1h后，转入探头式超声波发生器中超声1h，产物分散液在12000rpm下离心15min，取上层分散液在5000rpm下离心15min。将制得的BPNSs分散液，逐滴添加至Fc/ZIF-67复合物表面，自然干燥，在氧化铟锡ITO电极表面制得BPNSs/Fc/ZIF-67复合物。
适体-BPNSs/Fc/ZIF-67杂化薄膜的制备：将BPNSs/Fc/ZIF-67/ITO薄膜电极浸没在含有1μM的CD63跨膜蛋白对应单链DNA适体的磷酸盐水缓冲液中，在37℃下孵育30min，取出该薄膜电极，自然干燥，在氧化铟锡ITO电极表面制得适体BPNSs/Fc/ZIF-67复合物，即制得适体-BPNSs/Fc/ZIF-67杂化薄膜。
杂化薄膜传感器的制备：以杂化薄膜为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，加入从乳腺癌MCF-7细胞提取的外泌体，测定不同外泌体浓度下的电化学方波伏安曲线(如图2a所示)。以修饰在适体链上的亚甲基蓝MB为响应信号，掺杂在ZIF-67金属有机骨架内的二茂铁Fc为参比信号，以电流峰强度IFc/IMB比值为自校准信号输出，拟合IFc/IMB比值与外泌体浓度之间的线性关系(如图2b所示)，构建用于外泌体定量检测的自校准传感器。肿瘤外泌体浓度线性检测范围1.3×102～2.6×105particlesμL–1，检测限60particlesμL–1。
实施例2：
本实施例涉及的基于黑磷纳米片和二茂铁掺杂的钴基金属有机骨架复合物自组装的双功能杂化薄膜材料，其制备方法和检测原理同实施例1，其它具体制备步骤如下：
Fc/ZIF-67复合物的制备：称取一定量Co(NO3)2·6H2O和2-甲基咪唑放置47mL乙醇和3mL去离子水的混合溶剂中，磁力搅拌形成均匀的混合液，Co(NO3)2·6H2O和2-甲基咪唑的浓度分别调节至0.3mol/L和1.2mol/L。将此混合液加入电解槽中，以Ag/AgCl为参比电极，铂丝为对电极，氧化铟锡ITO为工作电极，以-8V恒定电压，循环伏安扫描200s，将Fc/ZIF-67复合物电沉积在氧化铟锡ITO电极表面。
BPNSs/Fc/ZIF-67复合物的制备：称取20mg黑磷晶体加入50mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中超声3h后，转入探头式超声波发生器中超声2h，产物分散液在12000rpm下离心15min，取上层分散液在5000rpm下离心15min。将制得的BPNSs分散液，逐滴添加至Fc/ZIF-67复合物表面，自然干燥，在氧化铟锡ITO电极表面制得BPNSs/Fc/ZIF-67复合物。
适体-BPNSs/Fc/ZIF-67杂化薄膜的制备：将BPNSs/Fc/ZIF-67/ITO薄膜电极浸没在含有4μM的CD63跨膜蛋白对应单链DNA适体的磷酸盐水缓冲液中，在37℃下孵育50min，取出该薄膜电极，自然干燥，在氧化铟锡ITO电极表面制得适体BPNSs/Fc/ZIF-67复合物，即制得适体-BPNSs/Fc/ZIF-67杂化薄膜。
杂化薄膜传感器的制备：以杂化薄膜为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，加入从乳腺癌MCF-7细胞提取的外泌体，测定不同外泌体浓度下的电化学方波伏安曲线。以修饰在适体链上的亚甲基蓝MB为响应信号，掺杂在ZIF-67金属有机骨架内的二茂铁Fc为参比信号，以电流峰强度IFc/IMB比值为自校准信号输出，拟合IFc/IMB比值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自校准传感器。肿瘤外泌体浓度线性检测范围1.0×102～1.0×105particles μL–1，检测限50particles μL–1。
实施例3：
本实施例涉及的基于黑磷纳米片和二茂铁掺杂的钴基金属有机骨架复合物自组装的双功能杂化薄膜材料，其制备方法和检测原理同实施例1，其它具体制备步骤如下：
Fc/ZIF-67复合物的制备：称取一定量Co(NO3)2·6H2O和2-甲基咪唑放置47mL乙醇和3mL去离子水的混合溶剂中，磁力搅拌形成均匀的混合液，Co(NO3)2·6H2O和2-甲基咪唑的浓度分别调节至0.5mol/L和1.5mol/L。将此混合液加入电解槽中，以Ag/AgCl为参比电极，铂丝为对电极，氧化铟锡ITO为工作电极，以-10V恒定电压，循环伏安扫描400s，将Fc/ZIF-67复合物电沉积在氧化铟锡ITO电极表面。
BPNSs/Fc/ZIF-67复合物的制备：称取30mg黑磷晶体加入50mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中超声5h后，转入探头式超声波发生器中超声4h，产物分散液在12000rpm下离心15min，取上层分散液在5000rpm下离心15min。将制得的BPNSs分散液，逐滴添加至Fc/ZIF-67复合物表面，自然干燥，在氧化铟锡ITO电极表面制得BPNSs/Fc/ZIF-67复合物。
适体-BPNSs/Fc/ZIF-67杂化薄膜的制备：将BPNSs/Fc/ZIF-67/ITO薄膜电极浸没在含有8μM的CD63跨膜蛋白对应单链DNA适体的磷酸盐水缓冲液中，在37℃下孵育100min，取出该薄膜电极，自然干燥，在氧化铟锡ITO电极表面制得适体BPNSs/Fc/ZIF-67复合物，即制得适体-BPNSs/Fc/ZIF-67杂化薄膜。
杂化薄膜传感器的制备：以杂化薄膜为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，加入从乳腺癌MCF-7细胞提取的外泌体，测定不同外泌体浓度下的电化学方波伏安曲线。以修饰在适体链上的亚甲基蓝MB为响应信号，掺杂在ZIF-67金属有机骨架内的二茂铁Fc为参比信号，以电流峰强度IFc/IMB比值为自校准信号输出，拟合IFc/IMB比值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自校准传感器。肿瘤外泌体浓度线性检测范围1.0×103～1.0×106particles μL–1，检测限80particles μL–1。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (1)

1.用于肿瘤外泌体自校准检测的双功能杂化薄膜的制备方法，其特征在于，该方法具体包括以下步骤：
(1)二茂铁掺杂的钴基金属有机骨架Fc/ZIF-67复合物的制备：称取一定量的Co(NO3)2·6H2O和2-甲基咪唑，放置于47mL乙醇和3mL去离子水的混合溶剂中，磁力搅拌下形成均匀的混合液，Co(NO3)2·6H2O和2-甲基咪唑的浓度分别调节至0.1～0.5mol/L和0.8～1.5mol/L；将此混合液加入电解槽中，以Ag/AgCl为参比电极，铂丝为对电极，氧化铟锡ITO为工作电极，以-5至-10V恒定电压，循环伏安扫描100～500s，将Fc/ZIF-67复合物电沉积在氧化铟锡ITO电极表面；
(2)黑磷纳米片和二茂铁掺杂的钴基金属有机骨架BPNSs/Fc/ZIF-67复合物的制备：称取10～30mg黑磷晶体加入50mL的1-甲基-2-吡咯烷酮中，在超声波清洗机中超声1～6h后，转入探头式超声波发生器中超声1～4h，产物分散液在12000rpm下离心15min，取上层分散液在5000rpm下离心15min；将制得的黑磷纳米片BPNSs分散液，逐滴添加至Fc/ZIF-67复合物表面，自然干燥，在氧化铟锡ITO电极表面制得BPNSs/Fc/ZIF-67复合物；
(3)适体-BPNSs/Fc/ZIF-67杂化薄膜的制备：将BPNSs/Fc/ZIF-67/ITO薄膜电极浸没在含有1～10μM的CD63跨膜蛋白对应单链DNA适体的磷酸盐水缓冲液中，在37℃下孵育30～120min，取出该薄膜电极，自然干燥，在氧化铟锡ITO电极表面制得适体BPNSs/Fc/ZIF-67复合物，即制得适体-BPNSs/Fc/ZIF-67杂化薄膜；
(4)杂化薄膜传感器的制备：以杂化薄膜为工作电极，置于电化学工作站的三电极体系中，以磷酸盐水缓冲液为电解液，加入从乳腺癌MCF-7细胞提取的外泌体，测定不同外泌体浓度下的电化学方波伏安曲线；以修饰在适体链上的亚甲基蓝MB为响应信号，掺杂在ZIF-67金属有机骨架内的二茂铁Fc为参比信号，以电流峰强度IFc/IMB比值为自校准信号输出，拟合IFc/IMB比值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自校准传感器；其中，肿瘤外泌体浓度的线性检测范围为1×102～1×106particlesμL–1，检测限为50～100particles μL–1。
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CN112034172A (zh) * 2020-08-24 2020-12-04 军事科学院军事医学研究院环境医学与作业医学研究所 一种用于诺氟沙星快速检测的黑磷比色/光热双模式可视化免疫层析检测方法
CN113219180B (zh) * 2021-01-29 2022-05-13 厦门大学 一种外泌体pd-l1的研究方法
CN113567206B (zh) * 2021-07-20 2022-09-09 上海交通大学 基于金属有机骨架纳米材料的膜蛋白及膜相关蛋白提取方法
CN114113251A (zh) * 2021-11-16 2022-03-01 上海纳米技术及应用国家工程研究中心有限公司 一种稳定检测铵根离子的钴基电化学传感器的制备方法及其产品和应用
CN114561463B (zh) * 2021-12-03 2023-07-28 济南大学 一种基于滚环与杂交链式反应检测外泌体的生物传感器
CN114184662B (zh) * 2021-12-10 2023-11-03 安徽医科大学第二附属医院 一种外泌体分析用mof电化学传感器及其制备与应用
CN114295694B (zh) * 2022-01-03 2023-06-06 重庆医科大学 一种用于乳腺癌her-2检测的电化学发光适体传感器及其检测方法
CN114563456A (zh) * 2022-03-04 2022-05-31 章毅 以MOFs探针对外泌体实施电化学检测的方法
CN114894871B (zh) * 2022-05-16 2024-01-16 安徽大学 一种高灵敏度亚硝酸还原酶生物电极的制备方法及应用
CN115109268B (zh) * 2022-08-01 2023-05-23 安徽科技学院 一种高效降解土霉素光催化材料的制备方法及其应用
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Family Cites Families (11)

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Publication number Priority date Publication date Assignee Title
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WO2017090050A1 (en) 2015-11-29 2017-06-01 Ramot At Tel-Aviv University Ltd. Sensing electrode and method of fabricating the same
CN109133014B (zh) 2018-06-13 2022-04-12 青岛大学 一种CoN3@N-C复合电催化剂的制备方法
CN108872564A (zh) 2018-09-12 2018-11-23 杭州多泰科技有限公司 一种基于外泌体的体外即时检测平台及其检测方法
CN109239040B (zh) * 2018-10-09 2020-12-15 太原理工大学 一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子检测方法
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CN109655512A (zh) 2018-12-18 2019-04-19 南京鼓楼医院 一种基于适配体与滚环扩增的外泌体检测方法
CN109529064A (zh) 2019-01-04 2019-03-29 北京大学深圳医院 可以实现肿瘤局部定点放射治疗的黑磷微泡药物载体及其制备方法
CN109813786B (zh) 2019-03-13 2021-11-09 郑州轻工业学院 双金属-有机骨架材料、支架材料及其制备方法，电化学免疫传感器及其制备方法、应用
CN110108881B (zh) * 2019-04-30 2022-03-22 江苏大学 一种双功能生物传感器hrp@zif-8/dna的制备方法及其应用

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