WO2021087945A1 - 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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WO2021087945A1
WO2021087945A1 PCT/CN2019/116519 CN2019116519W WO2021087945A1 WO 2021087945 A1 WO2021087945 A1 WO 2021087945A1 CN 2019116519 W CN2019116519 W CN 2019116519W WO 2021087945 A1 WO2021087945 A1 WO 2021087945A1
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zif
bpnss
self
exosomes
aptamer
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桂日军
孙玉娇
金辉
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青岛大学
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  • the invention belongs to the technical field of preparation of functional hybrid film materials and tumor exosomes sensors, and specifically relates to a bifunctional hybrid film based on self-assembly of black phosphorus nanosheets and ferrocene-doped cobalt-based metal-organic framework composites
  • the preparation method of the material, the prepared film material can be used for the precise capture of CD63 transmembrane protein and the self-calibration detection of breast cancer exosomes.
  • Exosomes are extracellular vesicles with a size of 50-100 nanometers that are released from multivesicles through endolysates. They carry a large number of biopolymers from parent cells, including transmembrane and cytoplasmic proteins, mRNA, and DNA. And micro-RNA etc. Exosomes act as messengers that mediate information between cells and play an important role in detecting changes in the physiological state of diseases, especially cancer. In recent years, as a promising biomarker, exosomes have been widely used in the early diagnosis of cancer, which overcomes the high cost and low sensitivity of invasive screening in the cancer detection process.
  • the current methods used for the detection of exosomes mainly include flow cytometry, nanoparticle tracking analysis, surface plasmon resonance, colorimetry, luminescence, and electrochemical analysis.
  • Zhu et al. used surface plasmon resonance imaging technology to achieve quantitative detection of exosomes (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 surface plasmon resonance imaging,Analytical Chemistry,2014,86,8857-8864); Xia et al.
  • Li Zhiyang and others used G-tetrajoint-Hemin to simulate peroxidase catalyzed H 2 O 2 reaction to generate signals, combined with rolling circle amplification to synthesize a large number of G-tetrajoint for signal amplification, in order to realize the quantitative detection of exosomes (Li Zhiyang, He Nongyue, Huang Rongrong. Patent name: A method for detecting exosomes based on aptamer and rolling circle amplification. National invention patent.
  • the present invention discloses a bifunctional hybrid film based on the simple self-assembly of the cobalt-based metal-organic framework ZIF-67 composite doped with black phosphorous nanosheets BPNSs and ferrocene Fc on the indium tin oxide ITO film electrode
  • the preparation method of material BPNSs/Fc/ZIF-67, this thin film material can be used for precise capture of CD63 transmembrane protein and self-calibration detection of breast cancer MCF-7 cell exosomes.
  • the purpose of the present invention is to overcome the above-mentioned problems in the prior art and design a new method for detecting tumor exosomes with convenient operation, high sensitivity, low cost and good specificity.
  • the present invention relates to a bifunctional hybrid film for self-calibration detection of tumor exosomes, and its preparation method includes the following steps:
  • a method for preparing a bifunctional hybrid film for self-calibration and detection of tumor exosomes characterized in that the method specifically includes the following steps:
  • hybrid thin film sensor the hybrid thin film is used as the working electrode, placed in the three-electrode system of the electrochemical workstation, the phosphate water buffer is used as the electrolyte, and the exocytosis extracted from breast cancer MCF-7 cells is added. Measure the electrochemical square wave voltammetry curve of different exosomes concentration.
  • the linear detection range of tumor exosomes concentration is 1 ⁇ 10 2 ⁇ 1 ⁇ 10 6 particles ⁇ L -1
  • the detection limit is 50-100 particles ⁇ L -1 .
  • the effect of the present invention is: a kind of ZIF-67 composite based on black phosphorous nanosheets BPNSs, aptamers and ferrocene Fc doped with cobalt-based metal organic framework, simple and self-assembled on indium tin oxide ITO film electrode is reported.
  • Functional hybrid thin film material namely aptamer-BPNSs/Fc/ZIF-67/ITO.
  • the methylene blue MB-labeled single-stranded DNA aptamer binds specifically to the CD63 transmembrane protein by aptamer-ligand to achieve precise capture of the CD63 transmembrane protein.
  • CD63 transmembrane protein is a specific biopolymer carried by breast cancer MCF-7 cell exosomes.
  • the method of the invention has the advantages of convenient operation, high sensitivity, low cost and good specificity, and can be used as a new exosomes self-calibration detection method for the quantitative detection of exosomes in biomedical samples.
  • Figure 1 shows the preparation of a bifunctional hybrid thin film material based on the self-assembly of black phosphorus nanosheets and ferrocene-doped cobalt-based metal-organic framework complexes, and its use for precise capture of CD63 transmembrane proteins and breast cancer exocrine Schematic diagram of the principle of self-calibration testing;
  • Figure 2(a) shows the electrochemical square wave voltammetry curve measured with the hybrid film material as the working electrode in the presence of different exosomes concentrations
  • Figure 2(b) shows the ratio of redox current peak intensity I Fc /I MB of ferrocene and methylene blue corresponding to different exosomal concentrations, fitting the linear relationship between the different I Fc /I MB ratios and the concentration of exosomes .
  • Preparation of Fc/ZIF-67 complex Weigh a certain amount of Co(NO 3 ) 2 ⁇ 6H 2 O and 2-methylimidazole in a mixed solvent of 47 mL ethanol and 3 mL deionized water, and magnetically stir to form a uniform mixture , The concentration of Co(NO 3 ) 2 ⁇ 6H 2 O and 2-methylimidazole were adjusted to 0.1 mol/L and 0.8 mol/L, respectively. Put this mixed solution into the electrolytic cell, use Ag/AgCl as the reference electrode, platinum wire as the counter electrode, and indium tin oxide ITO as the working electrode. At a constant voltage of -5V, cyclic voltammetry scans for 100s, and the Fc/ZIF-67 The composite is electrodeposited on the surface of the indium tin oxide ITO electrode.
  • BPNSs/Fc/ZIF-67 complex Preparation of BPNSs/Fc/ZIF-67 complex: Weigh 10 mg of black phosphorus crystals and add 50 mL of 1-methyl-2-pyrrolidone, sonicate it in an ultrasonic cleaner for 1 hour, and then transfer it to a probe-type ultrasonic generator for 1 hour. , The product dispersion was centrifuged at 12000rpm for 15min, and the upper dispersion was centrifuged at 5000rpm for 15min. The prepared BPNSs dispersion was added dropwise to the surface of the Fc/ZIF-67 composite, and dried naturally to prepare the BPNSs/Fc/ZIF-67 composite on the surface of the indium tin oxide ITO electrode.
  • Preparation of the aptamer-BPNSs/Fc/ZIF-67 hybrid film immerse the BPNSs/Fc/ZIF-67/ITO film electrode in a phosphate water buffer containing 1 ⁇ M CD63 transmembrane protein corresponding to the single-stranded DNA aptamer, Incubate at 37°C for 30 minutes, take out the film electrode, and dry naturally, and prepare the aptamer BPNSs/Fc/ZIF-67 complex on the surface of the indium tin oxide ITO electrode, that is, the aptamer-BPNSs/Fc/ZIF-67 hybrid ⁇ The film.
  • hybrid thin film sensor the hybrid thin film is used as the working electrode, placed in the three-electrode system of the electrochemical workstation, phosphate water buffer is used as the electrolyte, and the exosomes extracted from breast cancer MCF-7 cells are added to determine Electrochemical square wave voltammetry curves at different concentrations of exosomes (as shown in Figure 2a).
  • the self-assembled bifunctional hybrid thin film material based on black phosphorus nanosheets and ferrocene-doped cobalt-based metal-organic framework composites involved in this embodiment has the same preparation method and detection principle as in Example 1.
  • Other specific preparation steps are as follows :
  • Preparation of Fc/ZIF-67 complex Weigh a certain amount of Co(NO 3 ) 2 ⁇ 6H 2 O and 2-methylimidazole in a mixed solvent of 47 mL ethanol and 3 mL deionized water, and magnetically stir to form a uniform mixture , The concentration of Co(NO 3 ) 2 ⁇ 6H 2 O and 2-methylimidazole were adjusted to 0.3mol/L and 1.2mol/L, respectively. Put this mixed solution into an electrolytic cell, use Ag/AgCl as the reference electrode, platinum wire as the counter electrode, and indium tin oxide ITO as the working electrode. Use a constant voltage of -8V and scan the cyclic voltammetry for 200s. The Fc/ZIF-67 The composite is electrodeposited on the surface of the indium tin oxide ITO electrode.
  • BPNSs/Fc/ZIF-67 complex Preparation of BPNSs/Fc/ZIF-67 complex: Weigh 20 mg of black phosphorus crystals and add 50 mL of 1-methyl-2-pyrrolidone, sonicate it in an ultrasonic cleaner for 3 hours, and then transfer it to a probe-type ultrasonic generator for 2 hours. , The product dispersion was centrifuged at 12000rpm for 15min, and the upper dispersion was centrifuged at 5000rpm for 15min. The prepared BPNSs dispersion was added dropwise to the surface of the Fc/ZIF-67 composite, and dried naturally to prepare the BPNSs/Fc/ZIF-67 composite on the surface of the indium tin oxide ITO electrode.
  • BPNSs/Fc/ZIF-67/ITO film electrode is immersed in phosphate water buffer containing 4 ⁇ M CD63 transmembrane protein corresponding to single-stranded DNA aptamer, Incubate at 37°C for 50 minutes, take out the film electrode, and dry naturally, prepare the aptamer BPNSs/Fc/ZIF-67 complex on the surface of the indium tin oxide ITO electrode, that is, the aptamer-BPNSs/Fc/ZIF-67 hybrid ⁇ The film.
  • hybrid thin film sensor the hybrid thin film is used as the working electrode, placed in the three-electrode system of the electrochemical workstation, phosphate water buffer is used as the electrolyte, and the exosomes extracted from breast cancer MCF-7 cells are added to determine Electrochemical square wave voltammetry curves at different exosomes concentrations.
  • the linear detection range of tumor exosomes concentration is 1.0 ⁇ 10 2 ⁇ 1.0 ⁇ 10 5 particles ⁇ L -1 , and the detection limit is 50 particles ⁇ L -1 .
  • the self-assembled bifunctional hybrid thin film material based on black phosphorus nanosheets and ferrocene-doped cobalt-based metal-organic framework composites involved in this embodiment has the same preparation method and detection principle as in Example 1.
  • Other specific preparation steps are as follows :
  • Preparation of Fc/ZIF-67 complex Weigh a certain amount of Co(NO 3 ) 2 ⁇ 6H 2 O and 2-methylimidazole in a mixed solvent of 47 mL ethanol and 3 mL deionized water, and magnetically stir to form a uniform mixture , The concentration of Co(NO 3 ) 2 ⁇ 6H 2 O and 2-methylimidazole were adjusted to 0.5 mol/L and 1.5 mol/L, respectively. Put this mixed solution into the electrolytic cell, use Ag/AgCl as the reference electrode, platinum wire as the counter electrode, and indium tin oxide ITO as the working electrode. With a constant voltage of -10V, cyclic voltammetry scans for 400s, and the Fc/ZIF-67 The composite is electrodeposited on the surface of the indium tin oxide ITO electrode.
  • BPNSs/Fc/ZIF-67 complex Preparation of BPNSs/Fc/ZIF-67 complex: Weigh 30 mg of black phosphorus crystals into 50 mL of 1-methyl-2-pyrrolidone, sonicate it in an ultrasonic cleaner for 5 hours, and then transfer it to a probe-type ultrasonic generator for 4 hours. , The product dispersion was centrifuged at 12000rpm for 15min, and the upper dispersion was centrifuged at 5000rpm for 15min. The prepared BPNSs dispersion was added dropwise to the surface of the Fc/ZIF-67 composite, and dried naturally to prepare the BPNSs/Fc/ZIF-67 composite on the surface of the indium tin oxide ITO electrode.
  • Preparation of aptamer-BPNSs/Fc/ZIF-67 hybrid film immerse the BPNSs/Fc/ZIF-67/ITO film electrode in a phosphate water buffer containing 8 ⁇ M CD63 transmembrane protein corresponding to single-stranded DNA aptamer, Incubate for 100 minutes at 37°C, take out the film electrode, and dry naturally, prepare the aptamer BPNSs/Fc/ZIF-67 complex on the surface of the indium tin oxide ITO electrode, that is, the aptamer-BPNSs/Fc/ZIF-67 hybrid ⁇ The film.
  • hybrid thin film sensor the hybrid thin film is used as the working electrode, placed in the three-electrode system of the electrochemical workstation, phosphate water buffer is used as the electrolyte, and the exosomes extracted from breast cancer MCF-7 cells are added to determine Electrochemical square wave voltammetry curves at different exosomes concentrations.
  • the linear detection range of tumor exosomes concentration is 1.0 ⁇ 10 3 ⁇ 1.0 ⁇ 10 6 particles ⁇ L -1 , and the detection limit is 80 particles ⁇ L -1 .

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Abstract

一种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，该方法操作便捷、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 surface plasmon 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,Lunhui Guan,Jinghua Chen,A visible and colorimetric aptasensor based on DNA-capped single-walled carbon nanotubes for detection of exosomes,Biosensors and Bioelectronics,2017,92,8–15)。李智洋等利用G-四连体-Hemin模拟过氧化酶催化H 2O 2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(NO 3) 2·6H 2O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(NO 3) 2·6H 2O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I Fc/I MB比值为自校准信号输出，拟合I Fc/I MB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1×10 2～1×10 6particles μL –1，检测限为50～100particles μL –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I Fc/I MB比值为自校准信号输出，拟合I Fc/I MB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I Fc/I MB，拟合不同I Fc/I MB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(NO 3) 2·6H 2O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(NO 3) 2·6H 2O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I Fc/I MB比值为自校准信号输出，拟合I Fc/I MB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1.3×10 2～2.6×10 5particles μL –1，检测限60particles μL –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(NO 3) 2·6H 2O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(NO 3) 2·6H 2O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I Fc/I MB比值为自校准信号输出，拟合I Fc/I MB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1.0×10 2～1.0×10 5particles 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(NO 3) 2·6H 2O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(NO 3) 2·6H 2O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I Fc/I MB比值为自校准信号输出，拟合I Fc/I MB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1.0×10 3～1.0×10 6particles 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(NO 3) 2·6H 2O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(NO 3) 2·6H 2O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I Fc/I MB比值为自校准信号输出，拟合I Fc/I MB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1×10 2～1×10 6particles μL –1，检测限为50～100particles μL –1。
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