U.S. patent application number 16/627344 was filed with the patent office on 2021-01-07 for method for preparing nanocomposite and label-free aptamer electrochemical sensor of gamma-interferon based on the nanocomposite.
This patent application is currently assigned to Qingdao University. The applicant listed for this patent is Qingdao University. Invention is credited to Xiaohui GAO, Rijun GUI, Hui JIN, Zonghua WANG.
Application Number | 20210003566 16/627344 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
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United States Patent
Application |
20210003566 |
Kind Code |
A1 |
JIN; Hui ; et al. |
January 7, 2021 |
Method for Preparing Nanocomposite and Label-free Aptamer
Electrochemical Sensor of Gamma-interferon Based on the
Nanocomposite
Abstract
A method for preparing a label-free aptamer electrochemical
sensor of .gamma.-interferon based on a dendrimer/gold
nanoparticle/molybdenum disulfide nanocomposite is provided. The
nanocomposite is drip-coated on a surface of an electrode to
prepare a modified electrode, and a terminal sulfhydryl group of a
.gamma.-interferon aptamer chain is connected to the gold
nanoparticle via Au--S bond to obtain a nanocomposite-aptamer
modified electrode. When .gamma.-interferon is present, the
.gamma.-interferon specifically binds to the aptamer chain on the
sensor, resulting in the aptamer's hairpin structure being opened
and stretched, which can effectively adsorb methylene blue MB in
the electrolyte, causing significant enhancement of MB redox
signal. A linear relationship between a current intensity of MB
oxidation peak and a concentration of the .gamma.-interferon is
fitted to construct the label-free aptamer electrochemical sensor
of .gamma.-interferon.
Inventors: |
JIN; Hui; (Qingdao, CN)
; GAO; Xiaohui; (Qingdao, CN) ; GUI; Rijun;
(Qingdao, CN) ; WANG; Zonghua; (Qingdao,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Qingdao University |
Qingdao |
|
CN |
|
|
Assignee: |
Qingdao University
Qingdao
CN
|
Appl. No.: |
16/627344 |
Filed: |
March 21, 2019 |
PCT Filed: |
March 21, 2019 |
PCT NO: |
PCT/CN2019/078997 |
371 Date: |
December 30, 2019 |
Current U.S.
Class: |
1/1 |
International
Class: |
G01N 33/543 20060101
G01N033/543; C12N 15/115 20060101 C12N015/115 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2019 |
CN |
201910125057.7 |
Claims
1. A method for preparing a nanocomposite and a label-free aptamer
electrochemical sensor of .gamma.-interferon based on the
nanocomposite, comprising the following steps: (1) adding
molybdenum disulfide (MoS.sub.2) powder to a mixed solvent of
ethanol and distilled water to obtain a first mixed solution,
performing a first ultrasonic treatment on the first mixed solution
in a water bath, then centrifuging the first mixed solution to
separated and remove a precipitate to obtain a MoS.sub.2
homogeneous dispersion for subsequent use; (2) preparing an aqueous
solution of chloroauric acid and adding the aqueous solution of
chloroauric acid to an aqueous solution of poly(amidoamine)
dendrimer (PAMAM) to obtain a second mixed solution, stirring and
mixing the second mixed solution evenly with magnetic stirring,
dropwise adding an aqueous solution of sodium borohydride to the
second mixed solution to obtain a gold nanoparticles
(AuNPs)-supported dendrimers (PAMAM/AuNPs) dispersion, and removing
residual reactants from the PAMAM/AuNPs dispersion by a dialysis
treatment; (3) dropwise adding the PAMAM/AuNPs dispersion to the
MoS.sub.2 homogeneous dispersion to obtain a third mixed solution;
performing a second ultrasonic treatment on the third mixed
solution in a water bath and then performing a magnetic stirring
treatment to the third mixed solution to carry out a reaction to
obtain a PAMAM/AuNPs/MoS.sub.2 nanocomposite dispersion; (4) adding
a cross-linking agent to a surface of a bare glass carbon electrode
subjected to a polishing treatment, and then drip-coating the
PAMAM/AuNPs/MoS.sub.2 nanocomposite dispersion to obtain a
PAMAM/AuNPs/MoS.sub.2 modified electrode; diluting a
.gamma.-interferon nucleic acid aptamer with a buffer solution to
obtain a fourth mixed solution, heating the fourth mixed solution
to a predetermined temperature, and then cooling the fourth mixed
solution to room temperature, allowing the fourth mixed solution to
form an aptamer chain with a secondary hairpin structure, and thus
allowing a sulfhydryl group at a terminal of the aptamer chain to
be connected to a surface of the AuNPs via an Au--S bond to obtain
a PAMAM/AuNPs/MoS.sub.2-aptamer modified electrode; and (5) adding
a redox probe methylene blue (MB) into an electrolyte immersed with
the PAMAM/AuNPs/MoS.sub.2-aptamer modified electrode; wherein, with
an increasing of .gamma.-interferon, an electrochemical signal peak
of the MB enhances gradually; then, fitting a linear relationship
between a current intensity of the electrochemical signal peak of
the MB and a concentration of the .gamma.-interferon to construct
the label-free aptamer electrochemical sensor for detecting the
.gamma.-interferon.
2. The method for preparing the nanocomposite and the label-free
aptamer electrochemical sensor of the .gamma.-interferon based on
the nanocomposite according to claim 1, wherein, in the first
ultrasonic treatment of the step (1), an ultrasonic power is
150-200 W, a frequency is 20-50 kHz, an ultrasonic time is 5-10
hours, and a concentration of the MoS2 homogeneous dispersion is
1-2 mg mL.sup.-1.
3. The method for preparing the nanocomposite and-the label-free
aptamer electrochemical sensor of the .gamma.-interferon based on
the nanocomposite according to claim 1, wherein, in the step (2), a
concentration of the aqueous solution of chloroauric acid is 10-50
mM, a mass concentration of the aqueous solution of PAMAM is
0.1-1%, a concentration of the aqueous solution of sodium
borohydride is 0.5-1 M, and a concentration of the PAMAM/AuNPs
dispersion is 1-10 mg mL.sup.-1.
4. The method for preparing the nanocomposite and the label-free
aptamer electrochemical sensor of the .gamma.-interferon based on
the nanocomposite according to claim 1, wherein, in the step (3),
the second ultrasonic treatment is performed for 10-60 minutes, the
magnetic stirring treatment is performed for 6-12 hours, and a mass
concentration ratio of the MoS.sub.2 homogeneous dispersion to the
PAMAM/AuNPs dispersion ranges from (1:10) to (1:2).
5. The method for preparing the nanocomposite and the label-free
aptamer electrochemical sensor of the .gamma.-interferon based on
the nanocomposite according to claim 1, wherein, in the step (4),
the .gamma.-interferon nucleic acid aptamer is diluted to a
concentration of 1-5 .mu.M; the predetermined temperature is
50-100.degree. C.; and a heating time is 1-6 hours.
6. The method for preparing the nanocomposite and the label-free
aptamer electrochemical sensor of the .gamma.-interferon based on
the nanocomposite according to claim 1, wherein, in the step (5), a
concentration of the .gamma.-interferon is 0-1000 .mu.g mL.sup.-1;
and a detection limit of the .gamma.-interferon is 1-3 fg
mL.sup.-1.
Description
CROSS REFERENCE TO THE RELATED APPLICATIONS
[0001] This application is the national phase entry of
International Application No. PCT/CN2019/078997, filed on Mar. 21,
2019, which is based upon and claims priority to Chinese Patent
Application No. 201910125057.7, filed on Feb. 20, 2019, the entire
contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure belongs to the technical field of
electrochemical biosensor and nanocomposite preparation, and more
specifically relates to a method for preparing a dendrimer/gold
nanoparticle/molybdenum disulfide nanocomposite and a label-free
aptamer electrochemical sensor based on the nanocomposite. The
sensor can be used for highly sensitive and selective detection of
interferon gamma (.gamma.-interferon).
BACKGROUND
[0003] .gamma.-interferon is a cell-secreted cytokine that is the
only member of the type II class of interferons, also known as a
macrophage-activating factor. Aberrant .gamma.-interferon
expression is associated with a number of diseases, such as
inflammatory bowel disease, genital herpes simplex virus infection,
Alzheimer's disease, and others. Sensitive detection of
.gamma.-interferon may be used to study the activity of immune
response and the diagnosis of infectious diseases. Qualitative or
quantitative detection of .gamma.-interferon by enzyme-linked
immunosorbent assay can determine whether the human body is
infected with Mycobacterium tuberculosis. Cytokines are usually
detected by antibody immunoassays, however, these antibody
immunoassays are time-consuming, require multiple washes, and
require multiple-step processing to obtain the results. It is
difficult to achieve the dynamic monitoring of cell secretions by
the detection of antibodies due to complex washing and labeling.
Additionally, the efficiency of the enzyme-catalyzed reaction may
cause fluctuations of the output signal, resulting in an
unsatisfactory reproducibility of the detection results and
prolonged analysis time.
[0004] Nucleic acid aptamers are an effective substitute of
antibodies, because of thermal and chemical stability,
reproducibility, easy modification, and others. Currently, a series
of aptamer sensors have been developed based on the technology of
nucleic acid aptamer specific binding target molecules. These
sensors are unique in that an oligonucleotide is designed as a
signal mark, and when a target analyte binds to the
oligonucleotide, a signal is generated without the need for the
labeling and washing steps. The adaptive isomer biosensor's simple
detection processes is particularly suitable for real-time and
dynamic detection of biological samples, and may be broadly applied
elsewhere.
[0005] For the nucleic acid aptamer electrochemical sensor, an
aptamer is used as a molecular recognition element, which is fixed
to a signal converter by a specific method, and then connected by
electronic wires to form a device. Combined with electrochemical
methods, the nucleic acid aptamer electrochemical sensor can be
used for qualitative and quantitative detection of the object to be
measured. Compared with traditional electrochemical analysis
methods, electrochemical aptamer sensors have the advantages of
high sensitivity, wide detection range, easy production, good
selectivity, accuracy, and reproducibility in biomolecule
detection. Researchers have developed a nucleic acid aptamer-based
immunoglobulin detection strategy, where the .gamma.-interferon
aptamer labeled with a redox probe (methylene blue or ferrocene) is
assembled on an electrode through a series of electrode surface
modifications, and the changes of the electrochemical signal at the
electrode are measured to quantify the concentration of
.gamma.-interferon. Liu et al. prepared an aptamer-functionalized
electrode for the detection of cell-secreted cytokines, including
.gamma.- and .alpha.-interferon (Y. Liu, Y. Liu, Z. Matharu, A.
Rahimian, A. Revzin, Detecting multiple cell-secreted cytokines
from the same aptamer-functionalized electrode, Biosensors and
Bioelectronics 2015, 64: 43-50). Abnous et al. developed a
triple-helix molecular switch-based electrochemical aptasensor for
the detection of .gamma.-interferon using Methylene Blue as a redox
probe (K. Abnous, N. M. Danesh, M. Ramezani, M. Alibolandi, K. Y.
Hassanabad, A. S. Emrani, A. Bahreyni, S. M. Taghdisi, A
triple-helix molecular switch-based electrochemical aptasensor for
interferon-gamma using a gold electrode and Methylene Blue as a
redox probe, Microchimica Acta 2017, 184: 4151-4157).
[0006] Patents related to sensors for detecting interferon have
been reported. For example, Rui Zhao et al. disclosed methods for
preparing a biosensor for detecting human .beta.-interferon and a
special polypeptide thereof, where the recognition element is a
polypeptide containing a specific amino acid sequence or a
derivative thereof (Rui Zhao, Jia Luo, Qundan Zhang, Guoquan Liu.
Biosensor for detecting human .beta.-interferon and special peptide
thereof. Chinese Invention Patent. Publication No. CN101221185).
Zhanjun Yang et al. developed an impedance immunosensor based on
zinc oxide nanomaterials for label-free electrochemical immunoassay
of bovine gamma interferon (Zhanjun Yang, Piya Qin, Xiang Chen.
Preparation method of impedance immunosensor for bovine gamma
interferon based on zinc oxide nanomaterials. Chinese Invention
Patent. Publication No. CN104090116A). In recent years, label-free
aptasensors reasearch has attracted widespread attention. Compared
with the traditional aptamer labeled sensor, the label-free
aptasensor has certain advantages such as easy production,
label-free aptamer and inexpensive. Based on this, in the present
disclosure, a label-free aptamer electrochemical sensor based on a
novel dendrimer/gold nanoparticle/molybdenum disulfide
nanocomposite is designed for efficient .gamma.-interferon
detection. Thus far, there are no domestic and foreign literatures
and patent reports on the dendrimer/gold nanoparticle/molybdenum
disulfide nanocomposite, and the nanocomposite-based label-free
aptamer electrochemical sensor.
SUMMARY
[0007] The objective of the present disclosure is to overcome the
drawbacks of the prior art mentioned above, and to design a
label-free aptamer electrochemical sensor based on a dendrimer/gold
nanoparticle/molybdenum disulfide nanocomposite with the benefits
of simple preparation, economical, high sensitivity, good
selectivity, and others. The prepared sensor can be used for highly
sensitive and selective detection of .gamma.-interferon.
[0008] In order to achieve the above objective, the present
disclosure relates to a preparation process of a label-free aptamer
electrochemical sensor of .gamma.-interferon based on a
dendrimer/gold nanoparticle/molybdenum disulfide nanocomposite,
including the steps as follows.
[0009] 1. A method for preparing a nanocomposite and a label-free
aptamer electrochemical sensor of .gamma.-interferon based on the
nanocomposite, characterized in that, the method specifically
includes the following steps:
[0010] (1) adding molybdenum disulfide (MoS.sub.2) powder to a
mixed solvent of ethanol and distilled water, performing an
ultrasonic treatment in a water bath, then centrifuging to
separate, and removing the precipitate to obtain a MoS.sub.2
homogeneous dispersion for subsequent use;
[0011] (2) preparing an aqueous solution of chloroauric acid and
adding the aqueous solution of chloroauric acid to an aqueous
solution of poly(amidoamine) dendrimer (PAMAM), stirring and mixing
evenly with magnetic stirring, dropwise adding an aqueous solution
of sodium borohydride to prepare gold nanoparticle-supported
dendrimers (PAMAM/AuNPs), and removing residual reactants by
dialysis treatment;
[0012] (3) dropwise adding the PAMAM/AuNPs dispersion to the
MoS.sub.2 homogeneous dispersion; performing an ultrasonic
treatment in a water bath, and then performing a magnetic stirring
treatment to carry out a reaction to obtain a PAMAM/AuNPs/MoS.sub.2
nanocomposite;
[0013] (4) adding a cross-linking agent Nafion to a surface of a
bare glass carbon electrode subjected to a polishing treatment, and
then drip-coating the nanocomposite dispersion to obtain a
PAMAM/AuNPs/MoS.sub.2 modified electrode; diluting a
.gamma.-interferon nucleic acid aptamer with a buffer solution,
heating to a certain temperature, and then cooling to room
temperature, allowing the aptamer to form a secondary hairpin
structure, and a sulfhydryl group at the terminal of the aptamer
chain to be connected to a surface of AuNPs via Au--S bond; and
[0014] (5) adding a redox probe methylene blue (MB) into an
electrolyte immersed with the PAMAM/AuNPs/MoS.sub.2-aptamer
modified electrode; with an increasing of a target molecule
.gamma.-interferon, an electrochemical signal peak of the MB
enhances gradually; then, fitting a linear relationship between a
current intensity of MB oxidation peak and a concentration of the
.gamma.-interferon to construct a label-free aptamer
electrochemical sensor for detecting the .gamma.-interferon.
[0015] In step (1), an ultrasonic power is 150-200 W, a frequency
is 20-50 kHz, an ultrasonic time is 5-10 h, and a concentration of
the MoS2 homogeneous dispersion is 1-2 mg mL.sup.-1.
[0016] In step (2), the concentration of the chloroauric acid is
10-50 mM, the mass concentration of the PAMAM is 0.1-1%, the
concentration of the sodium borohydride is 0.5-1 M, and the
concentration of the PAMAM/AuNPs dispersion is 1-10 mg
mL.sup.-1.
[0017] In step (3), the time for the ultrasonic treatment is 10-60
minutes, the time for the magnetic stirring treatment is 6-12
hours, and the mass concentration ratio of the MoS.sub.2 to the
PAMAM/AuNPs ranges from (1:10) to (1:2).
[0018] In step (4), the concentration of the aptamer is diluted to
1-5 .mu.M; the heating temperature is 50-100.degree. C.; and the
heating time is 1-6 hours.
[0019] In step (5), the concentration of the .gamma.-interferon is
0-1000 pg mL.sup.-1; and the detection limit of the
.gamma.-interferon is 1-3 fg mL.sup.-1.
[0020] The advantages of the present invention are as follows. The
PAMAM/AuNPs/MoS.sub.2 composite is drip-coated on the surface of a
bare glass carbon electrode to prepare a modified electrode, and
the thiol terminal of the .gamma.-interferon aptamer chain is
connected to the surface of AuNPs via Au--S bond to prepare a
PAMAM/AuNPs/MoS.sub.2-aptamer modified electrode. MoS.sub.2 is
selected as a substrate for electrochemical reactions, and AuNPs
grown on the PAMAM are used as binding sites for the label-free
aptamer chains, and then a label-free electrochemical aptamer
sensor based on the nanocomposite is constructed. When
.gamma.-interferon is present, the .gamma.-interferon specifically
binds to the aptamer chain on the sensor, resulting in the
destruction of the aptamer's hairpin structure and the aptamer
chain being opened and stretched, which can effectively adsorb the
MB in the electrolyte, causing significant enhancement of MB redox
signal. The linear relationship between the current intensity of
the MB oxidation peak and the concentration of the
.gamma.-interferon is fitted to construct a label-free aptamer
electrochemical sensor for detecting the .gamma.-interferon.
Compared with the prior art, the method of the present disclosure
has the advantages of easy operation, low cost, high sensitivity
and good selectivity, and may be developed into a novel label-free
aptamer electrochemical sensor for highly sensitive and selective
detection of .gamma.-interferon in biological samples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic diagram showing a preparation process
of a dendrimer/gold nanoparticle/molybdenum disulfide
nanocomposite, a label-free aptamer electrochemical sensor based on
the nanocomposite, and a principle of .gamma.-interferon detection
according to the present invention;
[0022] FIG. 2A is an electrochemical square wave voltammetry curve
corresponding to different concentrations of .gamma.-interferon by
a label-free aptamer electrochemical sensor of the present
invention; and
[0023] FIG. 2B is a diagram showing a fitted linear relationship
between different intensities of oxidative current peaks and
concentrations of .gamma.-interferon, corresponding to oxidation
current peak intensities of methylene blue, in the presence of
different concentrations of .gamma.-interferon.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0024] The present invention is described in detail below with
reference to the drawings and specific embodiments.
Embodiment 1
[0025] The present invention relates to a preparation of a
dendrimer/gold nanoparticle/molybdenum disulfide nanocomposite and
a label-free aptamer electrochemical sensor based on the
nanocomposite and an electrochemical detection of
.gamma.-interferon. The schematic diagram of the preparation
process and principle are shown in FIG. 1. The specific process
steps are as follows.
[0026] 30 mg of MoS.sub.2 powder was added to a mixed solvent of
ethanol/distilled water (with a ratio of 1:1 by volume), subjected
to an ultrasonic treatment in a water bath for 8 h (180 W, 40 kHz),
then centrifugated at 3000 rpm for 20 min, and a precipitate was
removed to obtain a MoS.sub.2 homogeneous dispersion (1.5 mg
mL.sup.-1) for subsequent use. 25 mM aqueous solution of
chloroauric acid was prepared, 10 mL of the aqueous solution of
chloroauric acid was added to an aqueous solution of
poly(amidoamine) dendrimer (PAMAM, 1 wt %, 15 mL), magnetic
stirring was performed for 1 hour for evenly mixing, and then an
aqueous solution of sodium borohydride (0.5 M) was added dropwise
until the solution changed from pale yellow to reddish-brown. The
products were subjected to dialysis to remove residual reactants,
and gold nanoparticle-supported dendrimers (PAMAM/AuNPs, 1 mg
mL.sup.-1) were obtained for subsequent use. The PAMAM/AuNPs
dispersion was added dropwise to the MoS.sub.2 dispersion with a
mass ratio of 1:2. An ultrasonic treatment in a water bath was
performed for 30 min, and then a magnetic stirring treatment was
performed for 12 hours to carry out a reaction to obtain a
PAMAM/AuNPs/MoS.sub.2 nanocomposite.
[0027] A cross-linking agent Nafion was added dropwise on a surface
of a bare glass carbon electrode subjected to a polishing
treatment, and a nanocomposite dispersion was drip-coated on the
surface of the bare glass carbon electrode to obtain a
PAMAM/AuNPs/MoS.sub.2 modified electrode. A .gamma.-interferon
nucleic acid aptamer was diluted to 5 .mu.M with a buffer solution,
followed by heating to 90.degree. C. and maintaining for 5 hours,
and then cooled to room temperature. The aptamer formed a secondary
hairpin structure, and the sulfhydryl group at the terminal of the
aptamer chain was connected to the surface of AuNPs via Au--S bond.
In an electrolyte immersed with the PAMAM/AuNPs/MoS.sub.2-aptamer
modified electrode, 10 mM Tris-HCl buffer was contained, and a
redox probe methylene blue MB (1 mM) was added. With an increasing
of the target molecule .gamma.-interferon, an electrochemical
signal peak of the MB enhanced gradually. Then, a linear
relationship between a current intensity of MB oxidation peak and a
concentration of the .gamma.-interferon was fitted to construct a
label-free aptamer electrochemical sensor for detecting the
.gamma.-interferon. As shown in FIGS. 2A-2B, the linear detection
range of .gamma.-interferon concentration is 0.01-1000 ng
mL.sup.-1, and the detection limit is 3 fg mL.sup.-1.
Embodiment 2
[0028] In this embodiment, a preparation of a dendrimer/gold
nanoparticle/molybdenum disulfide nanocomposite and a label-free
aptamer electrochemical sensor based on the nanocomposite, and an
electrochemical detection of .gamma.-interferon are provided. The
schematic diagram of the preparation process and principle are the
same as that in Embodiment 1. The specific process steps are as
follows.
[0029] 30 mg of MoS.sub.2 powder was added to a mixed solvent of
ethanol/distilled water (with a ratio of 1:1 by volume), subjected
to an ultrasonic treatment in a water bath for 6 hours (150 W, 30
kHz), then centrifugated at 3000 rpm for 20 min, and a precipitate
was removed to prepare a MoS.sub.2 homogeneous dispersion (1 mg
mL.sup.-1) for subsequent use. 15 mM aqueous solution of
chloroauric acid was prepared, 10 mL of the aqueous solution of
chloroauric acid was added to an aqueous solution of poly
(amidoamine) dendrime (PAMAM, 0.5 wt %, 15 mL), magnetic stirring
was performed for 1 hour for evenly mixing, and then an aqueous
solution of sodium borohydride (0.5 M) was added dropwise until the
solution changed from pale yellow to reddish-brown. The products
were subjected to dialysis to remove residual reactants, and gold
nanoparticle-supported dendrimers (PAMAM/AuNPs, 2 mg mL.sup.-1)
were obtained for subsequent use. The PAMAM/AuNPs dispersion was
added dropwise to the MoS.sub.2 dispersion with a mass ratio of
1:4. An ultrasonic treatment in a water bath was performed for 20
min, and then a magnetic stirring treatment was performed for 6
hours to carry out a reaction to obtain a PAMAM/AuNPs/MoS.sub.2
nanocomposite.
[0030] A cross-linking agent Nafion was added dropwise on a surface
of a bare glass carbon electrode subjected to a polishing
treatment, and a nanocomposite dispersion was drip-coated on the
surface of the bare glass carbon electrode to obtain a
PAMAM/AuNPs/MoS.sub.2 modified electrode. A .gamma.-interferon
nucleic acid aptamer was diluted to 2 .mu.M with a buffer solution,
followed by heating to 80.degree. C. and maintaining for 3 hours,
and then cooled to room temperature. The aptamer formed a secondary
hairpin structure, and the sulfhydryl group at the terminal of the
aptamer chain was connected to the surface of AuNPs via Au--S bond.
In an electrolyte immersed with the PAMAM/AuNPs/MoS.sub.2-aptamer
modified electrode, 10 mM Tris-HCl buffer was contained, and a
redox probe methylene blue MB (1 mM) was added. With an increasing
of the target molecule .gamma.-interferon, an electrochemical
signal peak of the MB enhanced gradually. Then, a linear
relationship between a current intensity of MB oxidation peak and a
concentration of the .gamma.-interferon was fitted to construct a
label-free aptamer electrochemical sensor for detecting the
.gamma.-interferon. As shown in FIGS. 2A-2B, the linear detection
range of .gamma.-interferon concentration is 0.01-500 ng mL.sup.-1,
and the detection limit is 2.5 fg mL.sup.-1.
Embodiment 3
[0031] In this embodiment, a preparation of a dendrimer/gold
nanoparticle/molybdenum disulfide nanocomposite and a label-free
aptamer electrochemical sensor based on the nanocomposite, and an
electrochemical detection of .gamma.-interferon are provided. The
schematic diagram of the preparation process and principle are the
same as that in Embodiment 1. The specific process steps are as
follows.
[0032] 30 mg of MoS.sub.2 powder was added to a mixed solvent of
ethanol/distilled water (with a ratio of 1:1 by volume), subjected
to an ultrasonic treatment in a water bath for 10 hours (200 W, 50
kHz), then centrifugated at 3000 rpm for 20 min, and a precipitate
was removed to obtain a MoS.sub.2 homogeneous dispersion (2 mg
mL.sup.-1) for subsequent use. 50 mM aqueous solution of
chloroauric acid was prepared, 10 mL of the aqueous solution of
chloroauric acid was added to an aqueous solution of
poly(amidoamine) dendrimer (PAMAM, 1 wt %, 15 mL), magnetic
stirring was performed for 1 hour for evenly mixing, and then an
aqueous solution of sodium borohydride (1 M) was added dropwise
until the solution changed from pale yellow to reddish-brown. The
products were subjected to dialysis to remove residual reactants,
and gold nanoparticle-supported dendrimers (PAMAM/AuNPs, 10 mg
mL.sup.-1) were obtained for subsequent use. The PAMAM/AuNPs
dispersion was added dropwise to the MoS.sub.2 dispersion with a
mass ratio of 1:5. An ultrasonic treatment in a water bath was
performed for 60 min, and then a magnetic stirring treatment was
performed for 12 hours to carry out a reaction to obtain a
PAMAM/AuNPs/MoS.sub.2 nanocomposite.
[0033] A cross-linking agent Nafion was added dropwise on a surface
of a bare glass carbon electrode subjected to a polishing
treatment, and a nanocomposite dispersion was drip-coated on the
surface of the bare glass carbon electrode to obtain a
PAMAM/AuNPs/MoS.sub.2 modified electrode. A .gamma.-interferon
nucleic acid aptamer was diluted to 5 .mu.M with a buffer solution,
followed by heating to 90.degree. C. and maintaining for 6 hours,
and then cooled to room temperature. The aptamer formed a secondary
hairpin structure, and the sulfhydryl group at the terminal of the
aptamer chain was connected to the surface of AuNPs via Au--S bond.
In an electrolyte immersed with the PAMAM/AuNPs/MoS.sub.2-aptamer
modified electrode, 10 mM Tris-HCl buffer was contained, and a
redox probe methylene blue MB (1 mM) was added. With an increasing
of a target molecule .gamma.-interferon, an electrochemical signal
peak of the MB enhanced gradually. Then, a linear relationship
between a current intensity of MB oxidation peak and a
concentration of the .gamma.-interferon was fitted to construct a
label-free aptamer electrochemical sensor for detecting the
.gamma.-interferon. As shown in FIGS. 2A-2B, the linear detection
range of .gamma.-interferon concentration is 0.01-800 ng mL.sup.-1,
and the detection limit is 2 fg mL.sup.-1.
[0034] The foregoing descriptions are preferred embodiments of the
present invention. It should be noted that numerous improvements
and modifications may be made by those of ordinary skill in the art
without departing from the principles of the present disclosure,
and such improvements and modifications shall also be considered to
be within the protection scope of the present disclosure.
* * * * *