Method for preparing dual-functional hybrid thin-film for self-calibration detection of tumor-derived exosomes

Patent metadata

Field	Value
Publication number	US-11105767-B1
Application number	US-201917051782-A
Country	US
Kind code	B1
Filing date	Nov 8, 2019
Priority date	Nov 7, 2019
Publication date	Aug 31, 2021
Grant date	Aug 31, 2021

Abstract

A method for preparing a dual-functional hybrid thin-film for self-calibration detection of tumor-derived exosomes is disclosed. A dual-functional hybrid thin-film, aptamer-BPNSs/Fc/ZIF-67/ITO, is constructed by facile self-assembly of a cobalt-based metal-organic framework (ZIF-67) composite doped with black phosphorus nanosheets (BPNSs), an aptamer and ferrocene (Fc) on an indium tin oxide (ITO) electrode. Methylene blue (MB) labeled aptamer specifically binds to CD63 protein to precisely capture protein. The protein is a specific biomolecule carried by breast cancer MCF-7 cell exosome, and realizes the detection of the tumor cell exosome. A self-calibration sensor for quantitative detection of the tumor exosome is constructed by using MB as a response signal and Fc as a reference. Compared with the prior art, the present invention features convenient operation, high sensitivity, low cost and excellent specificity, and can be used as a novel exosome self-calibration detection method for quantitative detection of the exosomes in biomedical samples.

First claim

What is claimed is:

1. A method for preparing a dual-functional hybrid thin-film for a self-calibration detection of tumor-derived exosomes, comprising:
(1) preparation of a ferrocene-doped cobalt-based metal-organic framework (Fc/ZIF-67) composite: weighing a predetermined amount of Co(NO 3 ) 2 ·6H 2 O and 2-methylimidazole and adding the predetermined amount of Co(NO 3 ) 2 ·6H 2 O and 2-methylimidazole into a mixed solvent containing 47 mL of ethanol and 3 mL of deionized water to obtain a first mixture, magnetically stirring the first mixture to form an uniform first mixture, and adjusting a concentration of the Co(NO 3 ) 2 ·6H 2 O to 0.1-0.5 mol/L and adjusting a concentration of the 2-methylimidazole to 0.8-1.5 mol/L, respectively; adding the uniform first mixture to an electrolytic cell; with Ag/AgCl as a reference electrode, platinum wire as a counter electrode, and indium tin oxide (ITO) as a first working electrode, electrodepositing the Fc/ZIF-67 composite on a surface of the first working electrode through a cyclic voltammetry at a constant voltage of −5 to −10 V for 100 to 500 s;
(2) preparation of a black phosphorus nanosheets and ferrocene-doped cobalt-based metal-organic framework (BPNSs/Fc/ZIF-67) composite: weighing 10-30 mg of black phosphorus crystals and adding the black phosphorus crystals into 50 mL of 1-methyl-2-pyrrolidone to obtain a second mixture; sonicating the second mixture in an ultrasonic cleaner for 1-6 h, transferring the second mixture to a probe-type ultrasonic generator and sonicating the second mixture for 1-4 h to obtain a product dispersion; centrifuging the product dispersion for 15 min at 12,000 rpm to obtain an upper dispersion, and centrifuging the upper dispersion for 15 min at 5,000 rpm to obtain a black phosphorus nanosheets (BPNSs) dispersion; adding the black phosphorus nanosheets (BPNSs) dispersion dropwise to a surface of the Fc/ZIF-67 composite to obtain a third mixture, drying the third mixture naturally, to obtain the BPNSs/Fc/ZIF-67 composite on the surface of the first working electrode as a BPNSs/Fc/ZIF-67/ITO film electrode;
(3) preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film: immersing the BPNSs/Fc/ZIF-67/ITO film electrode in phosphate buffered saline (PBS) containing 1-10 μM of CD63 transmembrane protein corresponding to a single-stranded DNA aptamer to obtain a fourth mixture, incubating the fourth mixture at 37° C. for 30-120 min to obtain a resulting film electrode, taking out the resulting film electrode, drying the resulting film electrode naturally, to obtain an aptamer-BPNSs/Fc/ZIF-67 composite on the surface of the first working electrode, wherein the aptamer-BPNSs/Fc/ZIF-67 composite on the surface of the first working electrode is the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film; and
(4) preparation of a hybrid thin-film sensor: placing the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film in a three-electrode system of an electrochemical workstation, wherein the aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film serves as a second working electrode, adding exosomes in PBS, wherein the exosomes are extracted from breast cancer MCF-7 cells and the PBS serves as an electrolyte, and measuring electrochemical square wave voltammetry curves at different concentrations of the exosomes; with methylene blue (MB) modified on an aptamer strand as a response signal, Fc doped in the ZIF-67 metal-organic framework as a reference signal, and ratios of peak current intensities I Fc /I MB as a self-calibrated signal output, fitting a linear relationship between the ratios of the I Fc /I MB and the concentrations of the exosomes to construct a self-calibrating sensor for a quantitative detection of the exosomes, wherein a linear detection range of the concentrations of the exosomes is 1×10 2 -1×10 6 particles μL −1 , and a detection limit is 50-100 particles μL −1 .

Assignees

• Univ Qingdao

Inventors

• Gui Rijun
• Sun Yujiao
• Jin Hui

Classifications

• G01N33/57585

  involving compounds identifiable in body fluids · CPC title

• G01N33/57515

  of the breast · CPC title

• G01N33/5758

  involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites · CPC title

• B82Y15/00

  Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors · CPC title

• B82Y30/00

  Nanotechnology for materials or surface science, e.g. nanocomposites · CPC title