U.S. patent application number 17/051782 was filed with the patent office on 2021-08-12 for method for preparing dual-functional hybrid thin-film for self-calibration detection of tumor-derived exosomes.
This patent application is currently assigned to QINGDAO UNIVERSITY. The applicant listed for this patent is QINGDAO UNIVERSITY. Invention is credited to Rijun GUI, Hui JIN, Yujiao SUN.
Application Number | 20210247350 17/051782 |
Document ID | / |
Family ID | 1000005598246 |
Filed Date | 2021-08-12 |
United States Patent
Application |
20210247350 |
Kind Code |
A1 |
GUI; Rijun ; et al. |
August 12, 2021 |
METHOD FOR PREPARING DUAL-FUNCTIONAL HYBRID THIN-FILM FOR
SELF-CALIBRATION DETECTION OF TUMOR-DERIVED EXOSOMES
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.
Inventors: |
GUI; Rijun; (Qingdao,
CN) ; SUN; Yujiao; (Qingdao, CN) ; JIN;
Hui; (Qingdao, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
QINGDAO UNIVERSITY |
Qingdao |
|
CN |
|
|
Assignee: |
QINGDAO UNIVERSITY
Qingdao
CN
|
Family ID: |
1000005598246 |
Appl. No.: |
17/051782 |
Filed: |
November 8, 2019 |
PCT Filed: |
November 8, 2019 |
PCT NO: |
PCT/CN2019/116519 |
371 Date: |
October 30, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 30/00 20130101;
G01N 27/4163 20130101; B05D 1/00 20130101; G01N 27/3278 20130101;
C12N 2310/16 20130101; G01N 27/3277 20130101; G01N 33/57488
20130101; G01N 2333/70596 20130101; G01N 33/57484 20130101; C09K
11/58 20130101; C07H 21/00 20130101; C12N 15/115 20130101; G01N
33/57415 20130101 |
International
Class: |
G01N 27/327 20060101
G01N027/327; G01N 33/574 20060101 G01N033/574; G01N 27/416 20060101
G01N027/416 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 7, 2019 |
CN |
201911078972.1 |
Claims
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.sub.3).sub.2.6H.sub.2O and 2-methylimidazole and adding the
predetermined amount of Co(NO.sub.3).sub.2.6H.sub.2O 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.sub.3).sub.2.6H.sub.2O 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 mixed solution 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 .mu.M of CD63
transmembrane protein corresponding to a single-stranded DNA
aptamer to obtain a fourth mixture, incubating the fourth mixture
at 37.degree. 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-a 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.sub.Fc/I.sub.MB as a self-calibrated signal output, fitting a
linear relationship between the ratios of the I.sub.Fc/I.sub.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.times.10.sup.2-1.times.10.sup.6 particles .mu.L.sup.-1, and a
detection limit is 50-100 particles .mu.L.sup.-1.
Description
CROSS REFERENCE TO THE RELATED APPLICATIONS
[0001] This application is the national phase entry of
International Application No. PCT/CN2019/116519, filed on Nov. 8,
2019, which is based upon and claims priority to Chinese Patent
Application No. 201911078972.1, filed on Nov. 7, 2019, the entire
contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to the technical field of
preparation of functionalized hybrid thin-film materials and tumor
exosome sensors, and more particularly, relates to a preparation
method of a self-assembled dual-functional hybrid thin-film
material based on a cobalt-based metal-organic framework composite
doped with black phosphorus nanosheets (BPNSs) and ferrocene (Fc).
The thin-film material prepared by the method can be used for
precise capture of CD63 transmembrane protein and self-calibration
detection of breast cancer exosomes.
BACKGROUND
[0003] Exosomes are extracellular vesicles with a size of ranging
between 50-100 nm. They are released from multivesicular bodies via
intracellular lysosomal pathways. Exosomes carry a large number of
macromolecules from parental cells, including transmembrane and
cytoplasmic proteins, mRNA, DNA, and micro-RNA. Exosomes serve as
messengers for mediating intercellular information and play an
important role in detecting diseases, especially cancer-related
conditions. Use of exosomes as biomarkers has shown promise
recently in early cancer detection and diagnosis. The use of
exosomes has helped to alleviate problems like cost prohibition and
low sensitivity that are characteristic of previously dominant
invasive cancer screening and detection processes.
[0004] The scientific literature is replete with reports relating
to the quantitative detection of exosomes. Existing detection
technology, however, remains challenging in application, and it is
difficult to achieve direct and specific analysis of nanoscale
exosomes. For example, flow cytometry (FCM) detection is limited by
weak light scattering, while nanoparticle tracking analysis (NTA)
lacks specificity. In the early stages of the disease, exosomal
concentration is low, and new and improved methods are needed to
achieve super-sensitive detection of exosomes. Current methods for
exosomal detection include FCM, NTA, surface plasmon resonance
(SPR), colorimetry, luminescence, and electrochemical analysis. For
example, Zhu et al. used SPR 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, 2014, Analytical Chemistry, 86,
8857-8864). Xia et al. constructed a colorimetric detection method
of exosomes based on DNA-capped single-walled carbon nanotubes
(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, 2017,
Biosensors and Bioelectronics, 92, 8-15). L I Zhiyang et al. used
G-quadruplex-Hemin to simulate a H202 reaction catalyzed by
peroxidase to generate signals, combined with rolling circle
amplification to synthesize a large number of G-quadruplexes for
signal amplification to achieve quantitative detection of exosomes
(L I Zhiyang, H E Nongyue and HUANG Rongrong, Chinese National
Invention Patent Publication No. CN109655512A, titled Method for
detecting exosome based on aptamer and rolling circle
amplification). WANG Guosheng developed a product integrating of
isolation and purification of exosomes with specific
semi-quantitative detection of exosomes, and constructed an
exosome-based in vitro real-time detection platform (WANG Guosheng,
Exosome-based in vitro real-time detection platform and detection
method thereof, Chinese National Invention Patent Publication No.
CN108872564A).
[0005] Although works relating to quantitative detection of
exosomes have been published internationally, achieving direct and
specific, super-sensitive detection of nanoexosomes efficiently and
at a low cost remains elusive. Based on this need, the present
invention provides a method for preparing a BPNSs/Fc/ZIF-67
dual-functional hybrid thin-film material based on a cobalt-based
metal-organic framework (ZIF-67) composite doped with black
phosphorus nanosheets (BPNSs) and ferrocene (Fc), which is easily
self-assembled on an indium tin oxide (ITO) film electrode. The
thin-film material can be used for precise capture of CD63
transmembrane protein and self-calibration detection of breast
cancer MCF-7 cell-secreted exosomes. As of the filing date of this
patent application, Chinese and overseas literature and patent
publications germane to the preparation of the BPNSs/Fc/ZIF-67
dual-functional hybrid thin-film material, and the use of the
thin-film material applied in the self-calibration detection of
tumor-derived exosomes have not been uncovered.
SUMMARY
[0006] The objective of the present invention is to overcome the
problems existing in the prior art mentioned above and design a
method for detecting tumor-derived exosomes with convenient
operation, high sensitivity, low cost and excellent
specificity.
[0007] To achieve the above-mentioned objective, the present
invention provides a dual-functional hybrid thin-film for
self-calibration detection of tumor-derived exosomes, and a
preparation method of the hybrid thin-film includes the following
steps:
[0008] (1) preparation of a Fc/ZIF-67 composite: weighing a
predetermined amount of Co(NO.sub.3).sub.2.6H.sub.2O and
2-methylimidazole into a mixed solvent containing 47 mL of ethanol
and 3 mL of deionized water, magnetically stirring to form an
uniform mixed solution, and adjusting concentrations of
Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole to 0.1-0.5 mol/L
and 0.8-1.5 mol/L, respectively; adding the mixed solution to an
electrolytic cell; with Ag/AgCl as a reference electrode, platinum
wire as a counter electrode, and indium tin oxide (ITO) as a
working electrode, electrodepositing an Fc/ZIF-67 composite on the
surface of the ITO electrode through cyclic voltammetry at a
constant voltage of -5 to -10 V for 100-500 s;
[0009] (2) preparation of a BPNSs/Fc/ZIF-67 composite: weighing
10-30 mg of black phosphorus crystals and adding into 50 mL of
1-methyl-2-pyrrolidone; after sonicating in an ultrasonic cleaner
for 1-6 h, transferring to a probe-type ultrasonic generator and
sonicating for 1-4 h; centrifuging a product dispersion for 15 min
at 12,000 rpm, and centrifuging an upper dispersion for 15 min at
5,000 rpm; adding a prepared BPNSs dispersion dropwise to a surface
of the Fc/ZIF-67 composite, drying naturally, to obtain the
BPNSs/Fc/ZIF-67 composite on the surface of the ITO electrode;
[0010] (3) preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid
thin-film: immersing a BPNSs/Fc/ZIF-67/ITO film electrode in
phosphate buffered saline (PBS) containing 1-10 .mu.M of CD63
transmembrane protein corresponding to single-stranded DNA aptamer,
incubating at 37.degree. C. for 30-120 min, taking out the film
electrode, drying naturally, to obtain an aptamer-BPNSs/Fc/ZIF-67
composite on the surface of the ITO electrode, i.e., the
aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film; and
[0011] (4) preparation of a hybrid thin-film sensor: placing a
hybrid thin-film as a working electrode in a three-electrode system
of an electrochemical workstation, adding exosomes extracted from
breast cancer MCF-7 cells in the PBS 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.sub.Fc/I.sub.MB as self-calibrated
signal output, fitting a linear relationship between ratios of
I.sub.Fc/I.sub.MB and concentrations of the exosomes to construct a
self-calibrating sensor for quantitative detection of the exosomes,
in which a linear detection range of concentrations of the
tumor-derived exosomes is 1.times.10.sup.2-1.times.10.sup.6
particles .mu.L.sup.-1, and a detection limit is 50-100 particles
.mu.L.sup.-1.
[0012] The advantages of the present invention are as follows: The
present invention provides a dual-functional hybrid thin-film
material based on a ZIF-67 composite doped with BPNSs, an aptamer
and Fc, which is facilely self-assembled on an ITO film electrode,
i.e., aptamer-BPNSs/Fc/ZIF-67/ITO. Methylene blue (MB)-labeled
single-stranded DNA aptamer specifically binds to CD63
transmembrane protein to achieve precise capture of CD63
transmembrane protein. CD63 transmembrane protein is a specific
macromolecule carried by breast cancer MCF-7 cell-secreted exosomes
and can be used as a biomarker to detect breast cancer MCF-7
cell-secreted exosomes. Using MB as a response signal, ferrocene
(Fc) doped in ZIF-67 as a reference signal, and the ratios of peak
current intensities I.sub.Fc/I.sub.MB as self-calibrated signal
output, a linear relationship between I.sub.Fc/I.sub.MB and
concentrations of the exosomes is fitted to construct a
self-calibrating sensor for quantitative detection of tumor-derived
exosomes. Compared with the prior art, the method of the present
invention features convenient operation, high sensitivity, low
cost, and excellent specificity, can be used as a novel
self-calibration detection method of the exosomes, and is used for
quantitative detection of the exosomes in biomedical samples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic diagram showing a preparation of a
self-assembled dual-functional hybrid thin-film material based on a
cobalt-based metal-organic framework composite doped with BPNSs and
Fc, and a principle of application thereof in precise capture of
CD63 transmembrane protein and self-calibration detection of breast
cancer exosomes;
[0014] FIG. 2A is a diagram showing electrochemical square wave
voltammetry curves determined with the hybrid thin-film material as
a working electrode at different concentrations of the exosomes;
and
[0015] FIG. 2B is a diagram showing a linear relationship between
different ratios of I.sub.Fc/I.sub.MB and concentrations of the
exosomes fitted by ratios of redox peak current intensities
I.sub.Fc/I.sub.MB of ferrocene versus methylene blue corresponding
to different concentrations of the exosomes.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0016] The present invention will be described in detail below with
reference to the drawings.
Embodiment 1
[0017] A schematic diagram of the preparation method and detection
principle of a self-assembled dual-functional hybrid thin-film
based on a cobalt-based metal-organic framework composite doped
with BPNSs and Fc according to embodiment 1 is shown in FIG. 1, and
the specific preparation steps are as follows:
[0018] Preparation of a Fc/ZIF-67 composite: A predetermined amount
of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were weighed
in a mixed solvent containing 47 mL of ethanol and 3 mL of
deionized water, and magnetically stirred to form an uniform mixed
solution. Concentrations of Co(NO.sub.3).sub.2.6H.sub.2O and
2-methylimidazole were adjusted to 0.1 and 0.8 mol/L, respectively.
The mixed solution was added to an electrolytic cell; with Ag/AgCl
as a reference electrode, platinum wire as a counter electrode, and
ITO as a working electrode. An Fc/ZIF-67 composite was
electrodeposited on the surface of the ITO electrode through cyclic
voltammetry at a constant voltage of -5 V for 100 s.
[0019] Preparation of a BPNSs/Fc/ZIF-67 composite: 10 mg of black
phosphorus crystals were weighed and added into 50 mL of
1-methyl-2-pyrrolidone, sonicated in an ultrasonic cleaner for 1 h,
and then transferred to a probe-type ultrasonic generator and
sonicated for 1 h. A product dispersion was centrifuged for 15 min
at 12,000 rpm, and an upper dispersion was centrifuged for 15 min
at 5,000 rpm. A prepared BPNSs dispersion was added dropwise to the
surface of the Fc/ZIF-67 composite, followed by natural drying, and
the BPNSs/Fc/ZIF-67 composite was prepared on the surface of the
ITO electrode.
[0020] Preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film:
A BPNSs/Fc/ZIF-67/ITO film electrode was immersed in phosphate
buffered saline (PBS) containing 1 .mu.M of CD63 transmembrane
protein corresponding to single-stranded DNA aptamer, and incubated
at 37.degree. C. for 30 min. The film electrode was taken out and
dried naturally, and an aptamer-BPNSs/Fc/ZIF-67 composite was
prepared on the surface of the ITO electrode, i.e., the
aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film was prepared.
[0021] Preparation of a hybrid thin-film sensor: A hybrid thin-film
as a working electrode was placed in a three-electrode system of an
electrochemical workstation, exosomes extracted from breast cancer
MCF-7 cells were added in PBS as an electrolyte, and
electrochemical square wave voltammetry curves were measured at
different concentrations of the exosomes (as shown in FIG. 2A).
With 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.sub.Fc/I.sub.MB as
self-calibrated signal output, a linear relationship between
I.sub.Fc/I.sub.MB ratio and concentrations of the exosomes (as
shown in FIG. 2B) was fitted to construct a self-calibrating sensor
for quantitative detection of the exosomes. A linear detection
range of concentrations of the tumor-derived exosomes was
1.3.times.10.sup.2-2.6.times.10.sup.5 particles .mu.L.sup.-1, and a
detection limit was 60 particles .mu.L.sup.-1.
Embodiment 2
[0022] A self-assembled dual-functional hybrid thin-film based on a
cobalt-based metal-organic framework composite doped with BPNSs and
Fc according to the embodiment had the same preparation method and
detection principle as embodiment 1, and other specific preparation
steps are as follows:
[0023] Preparation of a Fc/ZIF-67 composite: A predetermined amount
of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were weighed
in a mixed solvent containing 47 mL of ethanol and 3 mL of
deionized water, and magnetically stirred to form an uniform mixed
solution. Concentrations of Co(NO.sub.3).sub.2.6H.sub.2O and
2-methylimidazole were adjusted to 0.3 and 1.2 mol/L, respectively.
The mixed solution was added to an electrolytic cell; with Ag/AgCl
as a reference electrode, platinum wire as a counter electrode, and
ITO as a working electrode, an Fc/ZIF-67 composite was
electrodeposited on the surface of the ITO electrode through cyclic
voltammetry at a constant voltage of -8 V for 200 s.
[0024] Preparation of a BPNSs/Fc/ZIF-67 composite: 20 mg of black
phosphorus crystals were weighed and added into 50 mL of
1-methyl-2-pyrrolidone, sonicated in an ultrasonic cleaner for 3 h,
and then transferred to a probe-type ultrasonic generator and
sonicated for 2 h. A product dispersion was centrifuged for 15 min
at 12,000 rpm, and an upper dispersion was centrifuged for 15 min
at 5,000 rpm. A prepared BPNSs dispersion was added dropwise to the
surface of the Fc/ZIF-67 composite, followed by natural drying, and
the BPNSs/Fc/ZIF-67 composite was prepared on the surface of the
ITO electrode.
[0025] Preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film:
A BPNSs/Fc/ZIF-67/ITO film electrode was immersed in PBS containing
4 .mu.M of CD63 transmembrane protein corresponding to
single-stranded DNA aptamer, and incubated at 37.degree. C. for 50
min; the film electrode was taken out and dried naturally, and an
aptamer-BPNSs/Fc/ZIF-67 composite was prepared on the surface of
the ITO electrode, i.e., the aptamer-BPNSs/Fc/ZIF-67 hybrid
thin-film was prepared.
[0026] Preparation of a hybrid thin-film sensor: A hybrid thin-film
as a working electrode was placed in a three-electrode system of an
electrochemical workstation, exosomes extracted from breast cancer
MCF-7 cells were added in PBS as an electrolyte, and
electrochemical square wave voltammetry curves were measured at
different concentrations of the exosomes. With 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.sub.Fc/I.sub.MB as self-calibrated signal
output, a linear relationship between I.sub.Fc/I.sub.MB ratio and
concentrations of the exosomes was fitted to construct a
self-calibrating sensor for quantitative detection of the exosomes.
A linear detection range of concentrations of the tumor-derived
exosomes was 1.0.times.10.sup.2-1.0.times.10.sup.5 particles
.mu.L.sup.-1, and a detection limit was 50 particles
.mu.L.sup.-1.
Embodiment 3
[0027] A self-assembled dual-functional hybrid thin-film based on a
cobalt-based metal-organic framework composite doped with BPNSs and
Fc according to the embodiment had the same preparation method and
detection principle as embodiment 1, and other specific preparation
steps are as follows:
[0028] Preparation of a Fc/ZIF-67 composite: A predetermined amount
of Co(NO.sub.3).sub.2.6H.sub.2O and 2-methylimidazole were weighed
in a mixed solvent containing 47 mL of ethanol and 3 mL of
deionized water, and magnetically stirred to form an uniform mixed
solution. Concentrations of Co(NO.sub.3).sub.2.6H.sub.2O and
2-methylimidazole were adjusted to 0.5 and 1.5 mol/L, respectively.
The mixed solution was added to an electrolytic cell; with Ag/AgCl
as a reference electrode, platinum wire as a counter electrode, and
ITO as a working electrode, an Fc/ZIF-67 composite was
electrodeposited on the surface of the ITO electrode through cyclic
voltammetry at a constant voltage of -10 V for 400 s.
[0029] Preparation of a BPNSs/Fc/ZIF-67 composite: 30 mg of black
phosphorus crystals were weighed and added into 50 mL of
1-methyl-2-pyrrolidone, sonicated in an ultrasonic cleaner for 5 h,
and then transferred to a probe-type ultrasonic generator and
sonicated for 4 h. A product dispersion was centrifuged for 15 min
at 12,000 rpm, and an upper dispersion was centrifuged for 15 min
at 5,000 rpm. A prepared BPNSs dispersion was added dropwise to the
surface of the Fc/ZIF-67 composite, followed by natural drying, and
the BPNSs/Fc/ZIF-67 composite was prepared on the surface of the
ITO electrode.
[0030] Preparation of an aptamer-BPNSs/Fc/ZIF-67 hybrid thin-film:
A BPNSs/Fc/ZIF-67/ITO film electrode was immersed in PBS containing
8 .mu.M of CD63 transmembrane protein corresponding to
single-stranded DNA aptamer, and incubated at 37.degree. C. for 100
min. The film electrode was taken out and dried naturally, and an
aptamer-BPNSs/Fc/ZIF-67 composite was prepared on the surface of
the ITO electrode, i.e., the aptamer-BPNSs/Fc/ZIF-67 hybrid
thin-film was prepared.
[0031] Preparation of a hybrid thin-film sensor: A hybrid thin-film
as a working electrode was placed in a three-electrode system of an
electrochemical workstation, exosomes extracted from breast cancer
MCF-7 cells were added in PBS as an electrolyte, and
electrochemical square wave voltammetry curves were measured at
different concentrations of the exosomes. With 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.sub.Fc/I.sub.MB as self-calibrated signal
output, a linear relationship between I.sub.Fc/I.sub.MB ratio and
concentrations of the exosomes was fitted to construct a
self-calibrating sensor for quantitative detection of the exosomes.
A linear detection range of concentrations of the tumor-derived
exosomes was 1.0.times.10.sup.3-1.0.times.10.sup.6 particles
.mu.L.sup.-1, and a detection limit was 80 particles 0.sup.-1.
[0032] The foregoing descriptions are merely preferred embodiments
of the present invention. It should be noted that several
variations and modifications can be made by those skilled in the
art without departing from the principles of the present invention
and should also fall within the protection scope of the
invention.
* * * * *