阅读说明：本技术 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 (Preparation method of bifunctional hybrid film for self-calibration detection of tumor exosomes ) 是由 桂日军 孙玉娇 金辉 于 2019-11-07 设计创作，主要内容包括：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。(The invention discloses a preparation method of a bifunctional hybrid film for self-calibration detection of tumor exosomes, which is characterized in that a bifunctional hybrid film, namely an aptamer-BPNSs/Fc/ZIF-67/ITO. methylene blue MB marked aptamer, is specifically combined with CD63 protein on the basis of simple self-assembly of Black Phosphorus Nanosheets (BPNSs), the aptamer and a ferrocene Fc doped cobalt-based metal organic framework ZIF-67 compound on an Indium Tin Oxide (ITO) electrode, so as to realize accurate protein capture, wherein the protein is a specific biomolecule carried by a breast cancer MCF-7 cell exosome, so as to realize detection of the tumor cell exosome, and the MB is taken as a response signal and the Fc is taken as a reference to construct a self-calibration sensor for quantitative detection of the tumor exosomes.)

1. The preparation method of the bifunctional hybrid membrane for self-calibration detection of tumor exosomes is characterized by comprising the following steps:

(1) preparation of ferrocene-doped cobalt-based metal organic framework Fc/ZIF-67 compound, quantitative Co (NO) is weighed₃)₂·6H₂O and 2-methylimidazole are put in a mixed solvent of 47mL of ethanol and 3mL of deionized water and magnetically stirred to form a uniform mixed solution, Co (NO)₃)₂·6H₂The concentrations of O and 2-methylimidazole are respectively adjusted to 0.1-0.5 mol/L and 0.8-1.5 mol/L. Adding the mixed solution into an electrolytic bath, taking Ag/AgCl as a reference electrode, a platinum wire as a counter electrode and Indium Tin Oxide (ITO) as a working electrode, carrying out cyclic voltammetry scanning for 100-500 s at a constant voltage of-5 to-10V, and electrodepositing the Fc/ZIF-67 compound on the surface of the Indium Tin Oxide (ITO) electrode.

(2) Preparing a cobalt-based metal organic framework BPNSs/Fc/ZIF-67 compound doped with black phosphorus nanosheets and ferrocene: weighing 10-30 mg of black phosphorus crystals, adding the black phosphorus crystals into 50mL of 1-methyl-2-pyrrolidone, carrying out ultrasonic treatment in an ultrasonic cleaning machine for 1-6 h, transferring the black phosphorus crystals into a probe type ultrasonic generator for ultrasonic treatment for 1-4 h, centrifuging the product dispersion liquid at 12000rpm for 15min, and centrifuging the upper layer dispersion liquid at 5000rpm for 15 min. And dropwise adding the prepared black phosphorus nanosheet BPNSs dispersion liquid to the surface of the Fc/ZIF-67 compound, naturally drying, and preparing the BPNSs/Fc/ZIF-67 compound on the surface of an Indium Tin Oxide (ITO) electrode.

(3) Preparation of aptamer-BPNSs/Fc/ZIF-67 hybrid film: immersing the BPNSs/Fc/ZIF-67/ITO thin film electrode in phosphate water buffer solution containing 1-10 mu M of CD63 transmembrane protein corresponding single-stranded DNA aptamer, incubating for 30-120 min at 37 ℃, taking out the thin film electrode, naturally drying, and preparing an aptamer BPNSs/Fc/ZIF-67 compound on the surface of the indium tin oxide ITO electrode, namely preparing the aptamer-BPNSs/Fc/ZIF-67 hybrid thin film.

(4) Preparation of hybrid thin film sensor: the method comprises the steps of taking a hybrid film as a working electrode, placing the hybrid film in a three-electrode system of an electrochemical workstation, taking a phosphate water buffer solution as electrolyte, adding exosomes extracted from breast cancer MCF-7 cells, and measuring electrochemical square wave voltammetry curves under different exosome concentrations. Ferrocene doped in ZIF-67 metal organic framework by using methylene blue MB modified on aptamer chain as response signalFc as reference signal, with current peak intensity I_Fc/I_MBThe ratio is the self-calibration signal output, fitting I_Fc/I_MBAnd constructing a self-calibration sensor for quantitative detection of the exosomes according to a linear relation between the ratio and the exosome concentration. Wherein the linear detection range of the tumor exosome concentration is 1 multiplied by 10²～1×10⁶particles μL^–1The detection limit is 50 to 100particles mu L^–1。

The technical field is as follows:

the invention belongs to the technical field of preparation of functional hybrid film materials and tumor exosome sensors, and particularly relates to a preparation method of self-assembled bifunctional hybrid film materials based on cobalt-based metal organic framework compounds doped with black phosphorus nanosheets and ferrocene, wherein the prepared film materials can be used for accurate capture of CD63 transmembrane protein and self-calibration detection of breast cancer exosomes.

Background art:

the exosome is an extracellular vesicle with the size of 50-100 nanometers and released from a polycycle through an endosome pathway, and carries a large number of biological macromolecules including transmembrane and cytoplasmic proteins, mRNA, DNA, micro-RNA and the like from a parent cell, and the exosome serves as a messenger for mediating intercellular information and plays an important role in detecting disease, particularly change of physiological states related to cancer.

Current methods for exosome detection mainly include flow cytometry, nanoparticle tracking analysis, surface plasmon resonance, colorimetric, luminescent, and electrochemical analysis, among others. For example, Zhu et al have implemented quantitative detection of exosomes using surface plasmon resonance imaging techniques (Ling Zhu, Kun Wang, Jian Cui, Huan Liu, Xiangli Bu, Huailei Ma, Weishi Wang, He Gong, Christopher Lausted, Leroy Hood, Guang Yang, Zhuyuan Hu, Label-free qualitative detection of tumor-derived exosomes and tumor surface plasmon resonance imaging, Analytical Chemistry,2014,86, 8857-; xia et al constructed a method for colorimetric detection of exosomes based on DNA-encapsulated single-armed carbon nanotubes (Yaokun Xia, Mengmeng Liu, Liangliang Wang, An Yan, Wenhui He, Mei Chen, Jianming Lan, Jianxing Xu, LunhuiGuan, Jinghua Chen, A visible and colorimetric aptamer based on DNA-captured single-walled carbon nanotubes for detection of exosomes, Biosensors and Biodetectors for DNA analysis, 2017,92, 8-15). Lizhiyang et al use G-tetrad-Hemin to simulate peroxidase to catalyze H₂O₂The reaction generates signals, and a large number of G-tetrads are synthesized by combining rolling circle amplification to carry out signal amplification so as to realize quantitative detection of exosomes (plum-Chilean, Henong, yellow-mashed cistanche. the patent name is exosome detection methods based on aptamer and rolling circle amplification. the patent publication number is CN109655512A), and the Wangsheng develops products integrating exosome separation and purification and specific exosome semi-quantitative detection, and constructs an exosome-based in vitro instant detection platform (Wangsheng. exosome-based in vitro instant detection platform and detection method thereof. the patent publication number is CN 108872564A).

Although the work of quantitative detection of exosomes has been reported in domestic and foreign documents and patents, the realization of direct and specific, high-sensitivity and low-cost high-efficiency detection of nano exosomes is still which is an important scientific problem to be solved at present, based on the above, the invention discloses a preparation method of hybrid bifunctional thin film materials BPNSs/Fc/ZIF-67 based on simple self-assembly of cobalt-based metal organic framework ZIF-67 compounds doped with black phosphorus nanosheets BPNSs and ferrocene Fc on an Indium Tin Oxide (ITO) thin film electrode, wherein the thin film materials can be used for accurate capture of CD63 protein and self-calibration detection of breast cancer MCF-7 cell exosomes.

The invention content is as follows:

the invention aims to overcome the problems in the prior art, and designs novel tumor exosome detection methods which are convenient to operate, high in sensitivity, low in cost and good in specificity.

In order to achieve the purpose, the invention relates to bifunctional hybrid membranes for self-calibration detection of tumor exosomes, which are prepared by the following steps:

1. the preparation method of the bifunctional hybrid membrane for self-calibration detection of tumor exosomes is characterized by comprising the following steps:

(1) preparation of Fc/ZIF-67 Complex:weighing quantitative Co (NO)₃)₂·6H₂O and 2-methylimidazole are put in a mixed solvent of 47mL of ethanol and 3mL of deionized water and magnetically stirred to form a uniform mixed solution, Co (NO)₃)₂·6H₂The concentrations of O and 2-methylimidazole are respectively adjusted to 0.1-0.5 mol/L and 0.8-1.5 mol/L. Adding the mixed solution into an electrolytic bath, taking Ag/AgCl as a reference electrode, a platinum wire as a counter electrode and Indium Tin Oxide (ITO) as a working electrode, carrying out cyclic voltammetry scanning for 100-500 s at a constant voltage of-5 to-10V, and electrodepositing the Fc/ZIF-67 compound on the surface of the Indium Tin Oxide (ITO) electrode.

(2) Preparation of BPNSs/Fc/ZIF-67 complexes: weighing 10-30 mg of black phosphorus crystals, adding the black phosphorus crystals into 50mL of 1-methyl-2-pyrrolidone, carrying out ultrasonic treatment in an ultrasonic cleaning machine for 1-6 h, transferring the black phosphorus crystals into a probe type ultrasonic generator for ultrasonic treatment for 1-4 h, centrifuging the product dispersion liquid at 12000rpm for 15min, and centrifuging the upper layer dispersion liquid at 5000rpm for 15 min. And dropwise adding the prepared BPNSs dispersion liquid to the surface of the Fc/ZIF-67 compound, naturally drying, and preparing the BPNSs/Fc/ZIF-67 compound on the surface of an Indium Tin Oxide (ITO) electrode.

(3) Preparation of aptamer-BPNSs/Fc/ZIF-67 hybrid film: immersing the BPNSs/Fc/ZIF-67/ITO thin film electrode in phosphate water buffer solution containing 1-10 mu M of CD63 transmembrane protein corresponding single-stranded DNA aptamer, incubating for 30-120 min at 37 ℃, taking out the thin film electrode, naturally drying, and preparing an aptamer BPNSs/Fc/ZIF-67 compound on the surface of the indium tin oxide ITO electrode, namely preparing the aptamer-BPNSs/Fc/ZIF-67 hybrid thin film.

(4) Preparation of hybrid thin film sensor: the method comprises the steps of taking a hybrid film as a working electrode, placing the hybrid film in a three-electrode system of an electrochemical workstation, taking a phosphate water buffer solution as electrolyte, adding exosomes extracted from breast cancer MCF-7 cells, and measuring electrochemical square wave voltammetry curves under different exosome concentrations. Methylene blue MB modified on an aptamer chain is taken as a response signal, ferrocene Fc doped in a ZIF-67 metal organic framework is taken as a reference signal, and the current peak intensity I is taken_Fc/I_MBThe ratio is the self-calibration signal output, fitting I_Fc/I_MBThe linear relation between the ratio and the exosome concentration is constructed for the quantitative detection of exosomesThe sensor is self-calibrated. Wherein the linear detection range of the tumor exosome concentration is 1 multiplied by 10²～1×10⁶particlesμL^–1The detection limit is 50 to 100particles mu L^–1。

The method has the effects that simple self-assembly bifunctional hybrid thin-film materials based on black phosphorus nanosheet BPNSs, aptamers and ferrocene Fc doped cobalt-based metal organic framework ZIF-67 compounds are reported on an indium tin oxide ITO thin-film electrode, namely aptamer-BPNSs/Fc/ZIF-67/ITO. methylene blue MB marked single-chain DNA aptamers and CD63 transmembrane protein are subjected to aptamer-ligand specific combination, so that accurate capture of the CD63 transmembrane protein is realized, the CD63 transmembrane protein is a specific biological polymer carried by breast cancer MCF-7 cell exosome, can be used as a biomarker, the detection of the breast cancer MCF-7 cell exosome is realized, MB is used as a response signal, the ferrocene Fc doped in the ferrocene Fc-67 is used as a reference signal, and the current peak intensity I is used as a ZIF-67 compound_Fc/I_MBThe ratio is the self-calibration signal output, fitting I_Fc/I_MBCompared with the prior art, the method has the advantages of convenient operation, high sensitivity, lower cost and good specificity, can be used as novel exosome self-calibration detection methods, and is used for quantitative detection of exosomes in biomedical samples.

Description of the drawings:

FIG. 1 is a schematic diagram of the preparation of a bifunctional hybrid thin film material based on self-assembly of a cobalt-based metal-organic framework composite doped with black phosphorus nanosheets and ferrocene, and the principle of the bifunctional hybrid thin film material used for the accurate capture of CD63 transmembrane protein and the self-calibration detection of breast cancer exosomes;

FIG. 2(a) is an electrochemical square wave voltammogram measured with the hybrid thin film material as a working electrode in the presence of different exosome concentrations;

FIG. 2(b) shows the ratio of the redox current peak intensities I of ferrocene and methylene blue for different exosome concentrations_Fc/I_MBFitting differences I_Fc/I_MBLinear relationship between ratio and exosome concentration.

The specific implementation mode is as follows:

the present invention will be described in detail below with reference to the accompanying drawings by way of specific embodiments.