U.S. patent application number 15/269779 was filed with the patent office on 2017-01-12 for membrane phase electrode using printing and bio-molecule detection using same.
This patent application is currently assigned to KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY. The applicant listed for this patent is KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY. Invention is credited to Jun Hyoung AHN, Hyo Arm JOUNG, Min Gon KIM, Yong Beom SHIN, Yun Ju SUNG.
Application Number | 20170010234 15/269779 |
Document ID | / |
Family ID | 46686816 |
Filed Date | 2017-01-12 |
United States Patent
Application |
20170010234 |
Kind Code |
A1 |
KIM; Min Gon ; et
al. |
January 12, 2017 |
MEMBRANE PHASE ELECTRODE USING PRINTING AND BIO-MOLECULE DETECTION
USING SAME
Abstract
A membrane electrode includes a novel sensor combining a
filtering function of a membrane and a signal measuring ability of
an electrode. A target material may be measured by filtration
through the membrane. A small amount of target materials may be
detected with high sensitivity using an amplified electrical signal
by increasing electrical conductivity by reducing metal ions on the
membrane, and thus the target material may be subject to
quantitative analysis. In addition, only a target material
selectively binding to a receptor may be filtrated by passing a
sample through the membrane after a receptor material is fixed to
the electrode, and thus may be used to detect an electrical signal.
In addition, the sensor may measure a signal in various methods
such as electrical conductivity, impedance, etc.
Inventors: |
KIM; Min Gon; (Daejeon,
KR) ; AHN; Jun Hyoung; (Daejeon, KR) ; SUNG;
Yun Ju; (Daejeon, KR) ; SHIN; Yong Beom;
(Daejeon, KR) ; JOUNG; Hyo Arm; (Daejeon,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY |
DAEJEON |
|
KR |
|
|
Assignee: |
KOREA RESEARCH INSTITUTE OF
BIOSCIENCE AND BIOTECHNOLOGY
|
Family ID: |
46686816 |
Appl. No.: |
15/269779 |
Filed: |
September 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13994348 |
Jun 20, 2013 |
9465003 |
|
|
PCT/KR2011/009746 |
Dec 16, 2011 |
|
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15269779 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 33/54366 20130101; A61B 5/1486 20130101; G01N 27/3275
20130101; G01N 33/54346 20130101; B82Y 5/00 20130101; G01N 27/3278
20130101 |
International
Class: |
G01N 27/327 20060101
G01N027/327; G01N 33/543 20060101 G01N033/543; A61B 5/1486 20060101
A61B005/1486 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 16, 2010 |
KR |
10-2010-0128965 |
Dec 16, 2011 |
KR |
10-2011-0136334 |
Claims
1. A sensor, comprising: a filtering membrane for filtering a
reaction solution; and an electrode placed on the filtering
membrane; wherein the reaction solution is prepared by mixing a
ligand-fixed enzyme or ligand-fixed metal nanoparticle and a sample
containing a target material, wherein the ligand-fixed enzyme or
the ligand-fixed metal nanoparticles bound with the target material
remain on the filtering membrane, wherein the electrode generates
an electrical signal by the ligand-fixed enzyme or the ligand-fixed
metal nanoparticles bound with the target material.
2. The sensor according to claim 1, wherein the filtering membrane
is a filter formed of one selected from the group consisting of
nitrocellulose, polycarbonate, nylon, polyester, cellulose acetate,
polysulfone, and polyethanesulfone.
3. The sensor according to claim 1, wherein the electrode is
printed by a screen printing process.
4. The sensor according to claim 3, wherein the electrode is an
interdigitated electrode having a gap of 10 to 1000 .mu.m.
5. The sensor according to claim 1, wherein the ligand is an
antibody, an antigen, an enzyme, a peptide, a protein, DNA, RNA,
PNA, or an aptamer.
6. The sensor according to claim 1, wherein the enzyme is a
peroxidase, an alkali phosphatase, a galactosidase, or a glucose
oxidase.
7. The sensor according to claim 1, wherein the nanoparticle is a
gold, silver, copper or magnetic nanoparticle.
8. The sensor according to claim 1, wherein the target material is
a microorganism, an antigen, a nucleic acid, a cell or an organ of
an animal or plant.
9. The sensor according to claim 1, wherein a receptor is further
fixed on the filtering membrane between the electrodes.
10. The sensor according to claim 1, wherein a metal is further
placed on a surface of the electrode, wherein the electrical signal
is amplified by increased electrical conductivity due to the
metal.
11-17. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and the benefit of
Korean Patent Application Nos. 10-2010-0128965 and 10-2011-0136334,
filed on Dec. 16, 2010 and Dec. 16, 2011, respectively, the
disclosure of which are incorporated herein by reference in their
entirety.
BACKGROUND
[0002] 1. Field of the Invention
[0003] The present invention relates to a method of detecting a
biomaterial using a combination of a filtering characteristic of a
membrane and signal measurement of an electrode by forming the
electrode on the filtering membrane by screen printing.
[0004] 2. Discussion of Related Art
[0005] Quantitative analysis of a biomaterial is an important
technique used in foods, medicine and diagnosis. In particular,
rapid detection of microorganisms is a very important technique in
diagnosis of food poisoning bacteria, detection of environmentally
harmful bacteria and infections, and diagnosis of pathogenic
viruses. As a generally and widely used method of examining for the
presence and concentration of pathogenic materials (microorganisms,
proteins, etc.), a colony method, a DNA probe method, and an
immunoassay method are widely used (Jay J M. Modern Food
Microbiology, 1986, 3rd, ed., p 95, Van Nostrand Reinhold Co., New
York; Tenover F C., DNA Probes for Infectious Diseases, 1989, p 53
CRC Press, Boca Raton). The colony method is a method of counting
the number of colonies formed by microorganisms cultured in a
selective medium having ingredients allowing only microorganisms
from a sample to survive and be detected. This is a very accurate
but time-consuming method, and there is difficulty in selecting a
medium with respect to a microorganism. The DNA probe method is a
method including a real time polymerase chain reaction (PCR) and
nucleic acid hybridization, and is used to detect DNA in a cell by
nucleic acid conjugation after microorganisms are disrupted by a
physicochemical method. This is faster than the colony method in
detecting microorganisms. However, it needs expensive PCR equipment
and requires separate incubation to obtain high sensitivity when a
small amount of microorganisms is detected (Ninet, B et al., Appl
Environ Microbiol 58:4055-4059, 1992). If incubation is not
included, dead cells may be detected, which leads to inaccurate
results. In addition, when PCR is performed, a range of detection
errors becomes wide due to frequent false-positive results, and
thus it is possible to decrease reliability of the analysis. The
immunoassay method uses an antigen-antibody binding reaction. An
enzyme-linked immunosorbent assay (ELISA) using an antibody
specifically reacting with a surface antigen of a microorganism to
be detected is widely used. This is considered as an alternative to
the above-described two methods since it achieves very high
sensitivity in a short time.
[0006] The present invention is directed to providing a sensor that
combines a measurement capability of an electrode and a filtering
characteristic of a membrane, by forming the electrode on the
filtering membrane.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to providing a sensor for
detecting a biomaterial by combining a measurement capability of a
sensor of an electrode and a separation ability of a filtering
membrane, by forming an electrode on the filtering membrane, a
method of manufacturing the same, and a method of detecting a
target material using the same.
[0008] One aspect of the present invention provides a sensor having
an electrode printed on a filtering membrane. A reaction solution
prepared by mixing a ligand-fixed enzyme or ligand-fixed metal
nanoparticles and a sample containing a target material passes
through the filtering membrane, thereby generating an electrical
signal in the electrode.
[0009] Another aspect of the present invention provides a method of
manufacturing a sensor, which includes: (a) printing an electrode
on a filtering membrane; and (b) passing a reaction solution
prepared by mixing a ligand-fixed enzyme or ligand-fixed metal
nanoparticles and a sample containing a target material through the
filtering membrane.
[0010] Still another aspect of the present invention provides a
method of detecting a target material, which includes: (a) mixing a
ligand-fixed enzyme or ligand-fixed metal nanoparticles and a
sample containing a target material to react; (b) filtering only a
target material-ligand-enzyme complex or target
material-ligand-metal nanoparticle complex by passing the reaction
solution through a filtering membrane having an electrode; and (c)
measuring an electrical signal generated in the electrode by a
target material-ligand-enzyme complex or target
material-ligand-metal nanoparticle complex remaining on the
filtering membrane.
[0011] Yet another aspect of the present invention provides a
method of detecting a target material, which includes: (a) mixing a
ligand-fixed enzyme or ligand-fixed metal nanoparticles and a
sample containing a target material to react; (b) filtering only a
target material-ligand-enzyme complex or target
material-ligand-metal nanoparticle complex specifically binding to
a receptor by passing the reaction solution through a filtering
membrane between electrodes; and (c) measuring an electrical signal
generated in the electrodes by a target material-ligand-enzyme
complex or target material-ligand-metal nanoparticle complex
remaining on the filtering membrane.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The above and other objects, features, and advantages of the
present invention will become more apparent to those of ordinary
skill in the art by describing in detail exemplary embodiments
thereof with reference to the attached drawings, in which:
[0013] FIG. 1 shows a diagram of membrane electrodes formed with a
gap of 100.mu.m using a silk screening printing process (A), and
images of a membrane silver electrode (B, left) and a membrane
carbon electrode (B, right);
[0014] FIG. 2 is a diagram showing a process of forming a silver
ion reduction precipitate using an enzyme on a membrane silver
electrode according to the present invention;
[0015] FIGS. 3 and 4 are graphs showing a change in current
according to the presence of an enzyme and silver ion reduction on
a membrane silver electrode according to the present invention
(0.1M PB--AgGSH: control group without enzyme; STA/HRP--AgGSH: test
group having enzyme);
[0016] FIG. 5 is a diagram showing a process of forming a gold ion
reduction precipitate using gold nanoparticles on a membrane silver
electrode according to the present invention;
[0017] FIGS. 6 and 7 are graphs showing a change in current
according to the presence of gold nanoparticles and gold ion
reduction on a silver micro-electrode according to the present
invention (0.1M PB--Au Enh: control group without gold
nanoparticles; AuNP--Au Enh: test group having gold
nanoparticles);
[0018] FIG. 8 is a diagram showing a process of forming a gold ion
reduction precipitate using a food poisoning bacteria-antibody-gold
nanoparticle complex on a membrane silver electrode according to
the present invention;
[0019] FIGS. 9 and 10 are graphs showing a change in current
according to the presence of a food poisoning
bacteria-antibody-gold nanoparticle complex and reduction of a gold
ion reduction on a membrane silver electrode according to the
present invention (Control: control group only having antibody-gold
nanoparticle conjugate; 10.sup.2 cell: test group having complex;
bare-PBS: addition of PBS to initial electrode; control-PBS:
addition of PBS after gold nanoparticles are added; cell-PBS:
addition of PBS after food poisoning bacteria-antibody-gold
nanoparticle complex is added; Au enh-5 min: treatment with gold
reducing solution for 5 minutes; Au enh 10 min: treatment with gold
reducing solution for 10 minutes; Au enh-PBS: washing with PBS
after gold reducing solution is treated); and
[0020] FIG. 11 is a graph showing a change in concentration
according to a concentration of Staphylococcus aureus on a membrane
silver electrode according to the present invention (Au enh-5 min:
treatment with gold reducing solution for 5 minutes; Au enh 10 min:
treatment with gold reducing solution for 10 minutes; Au enh-PBS:
washing with PBS after gold reducing solution is treated).
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0021] Hereinafter, exemplary embodiments of the present invention
will be described in detail. However, the present invention is not
limited to the exemplary embodiments disclosed below, but can be
implemented in various forms. The following embodiments are
described in order to enable those of ordinary skill in the related
art to embody and practice the present invention.
[0022] Although the terms first, second, etc. may be used to
describe various elements, these elements are not limited by these
terms. These terms are only used to distinguish one element from
another. For example, a first element could be termed a second
element, and, similarly, a second element could be termed a first
element, without departing from the scope of exemplary embodiments.
The term "and/or" includes any and all combinations of one or more
of the associated listed items.
[0023] It will be understood that when an element is referred to as
being "connected" or "coupled" to another element, it can be
directly connected or coupled to the other element or intervening
elements may be present. In contrast, when an element is referred
to as being "directly connected" or "directly coupled" to another
element, there are no intervening elements.
[0024] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
exemplary embodiments. The singular forms "a," "an," and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. It will be further understood that the
terms "comprises," "comprising," "includes," and/or "including,"
when used herein, specify the presence of stated features,
integers, steps, operations, elements, components, and/or groups
thereof, but do not preclude the presence or addition of one or
more other features, integers, steps, operations, elements,
components, and/or groups thereof.
[0025] With reference to the appended drawings, exemplary
embodiments of the present invention will be described in detail
below. To aid in understanding the present invention, like numbers
refer to like elements throughout the description of the figures,
and the description of the same elements will be not
reiterated.
[0026] The present invention provides a sensor having an electrode
printed on a filtering membrane. Here, a reaction solution prepared
by mixing a ligand-fixed enzyme or ligand-fixed metal nanoparticles
and a sample containing a target material passes through the
filtering membrane, thereby generating an electrical signal in the
electrode.
[0027] Specifically, the sensor has electrodes printed on a
filtering membrane. When a reaction solution prepared by mixing a
ligand-fixed enzyme or ligand-fixed metal nanoparticles and a
sample containing a target material passes through the filtering
membrane, a reaction solution not bound with the target material
may pass through the membrane and thus only a target
material-ligand-enzyme complex or target material-ligand-metal
nanoparticle complex bound with the target material may remain,
thereby generating an electrical signal in the electrodes.
[0028] In the present invention, the "filtering membrane" refers to
a filter having small pores having a diameter of 100 nm to 10.mu.m.
Accordingly, when a reaction solution prepared by reacting a
mixture of a ligand-fixed enzyme or ligand-fixed metal
nanoparticles and a sample containing a target material passes
through the filtering membrane, a filtering function in which a
ligand-fixed enzyme or ligand-fixed metal nanoparticles that are
not bound with a target material may pass through the membrane, and
thus only a target material-ligand-enzyme complex or target
material-ligand-metal nanoparticle complex bound with the target
material may remain on the membrane, thereby generating a signal by
the electrodes formed on the membrane may be performed.
[0029] The filtering membrane may be, for example, a
nitrocellulose, polycarbonate, nylon, polyester, cellulose acetate,
polysulfone or polyethanesulfone filter, but the present invention
is not limited thereto.
[0030] According to an exemplary embodiment of the present
invention, the electrode may be printed by screen printing,
specifically, a silk screen printing process. For example, the
electrode may be formed by directly printing a paste of an
electrode material (platinum, gold, silver, carbon, etc.) on a
membrane through a screen having a predetermined pattern, and
drying the printed result at high temperature (generally,
100.degree. C. or more). This process may be repeatedly
performed.
[0031] The electrode may be, for example, an interdigitated
electrode (IDE). In the IDE, two comb-shaped electrodes face each
other. The IDE is used to detect food poisoning bacteria since a
microorganism can be detected by an impedance method when
microorganisms are bound between insulation of the two electrodes
(Yang, L. et al. Anal. Chem., 76:1107-1113, 2004). In addition, the
IDE is used as various sensors such as a non-labeled biosensor, a
gas sensor, etc. (Dharuman, V. et al. Biosens. Bioelectron.,
21:645-654, 2005, Hermans E. C. M., Sensor. Actuat. 5:181-186,
1984).
[0032] The interdigitated electrode may have a gap between
electrodes of, for example, 10 to 1000, 10 to 900, 10 to 800, 10 to
700, 10 to 600, 10 to 500, 10 to 450, 10 to 400, 10 to 350, 1 to
300, 10 to 250, 10 to 200 or 10 to 150.mu.m, but the present
invention is not limited thereto.
[0033] In the present invention, the "ligand" is a material having
specific binding affinity for a target material, which may be, for
example, an antibody, an antigen, an enzyme, a peptide, a protein,
DNA, RNA, peptide nucleic acid (PNA) or an aptamer, but the present
invention is not limited thereto. The kind of the ligand may be
suitably selected by one of ordinary skill in the art according to
the kind of the target material to be detected.
[0034] When the ligand-fixed enzyme or ligand-fixed metal
nanoparticles are mixed with the sample containing the target
material to react, a "target material-ligand-enzyme complex" or
"target material-ligand-metal nanoparticle complex", in which a
target material and an enzyme, or a target material and metal
nanoparticles, are bound with each other by the ligand in the mixed
reaction solution, may be prepared. Here, a ligand-fixed enzyme or
ligand-fixed metal nanoparticles that are not bound with the target
material pass through the membrane, and only the target
material-ligand-enzyme complex or target material-ligand-metal
nanoparticle complex that is bound with the target material remains
on the membrane, thereby generating an electrical signal in the
electrode formed on the membrane. By measuring the electrical
signal, the target material may be detected. Particularly, since
the measured electrical signal is proportional to a concentration
of the metal nanoparticles remaining on the membrane (that is, a
concentration of the target material), the target material can be
quantitatively detected.
[0035] Particularly, when the target material is a microorganism, a
cell or an organ of an animal or plant, which has a large size, the
target material may be easily detected without a separate receptor
equipped on the membrane. In the present invention, the "target
material" may be a microorganism, an antigen, a nucleic acid, a
cell or an organ of an animal or plant, and here, the
"microorganism" may be, but is not limited to, for example, a
virus, a bacterium or a fungus.
[0036] In the present invention, the ligand-fixed "enzyme" may be,
but is not limited to, for example, a peroxidase, an alkaline
phosphatase, a galactosidase, or a glucose oxidase, and the
ligand-fixed "nanoparticle" may be, but is not limited to, for
example, gold, silver, copper or a magnetic nanoparticle.
[0037] In the present invention, the "nanoparticle" refers to a
superfine particle having a diameter of approximately 1 to 100 nm.
In one embodiment of the present invention, nanoparticles having a
size of 20 nm are used, but the present invention is not limited
thereto. Nanoparticles may have a size of 0.5 to 100, 0.5 to 90,
0.5 to 80, 0.5 to 70, 0.5 to 60, 0.5 to 50, 0.5 to 40, 0.5 to 30,
0.5 to 20 or 1 to 20 nm.
[0038] According to another embodiment of the present invention, a
sensor including a receptor additionally fixed on a filtering
membrane between electrodes may be manufactured.
[0039] According to the exemplary embodiment, the sensor of the
present invention includes the receptor additionally fixed on the
filtering membrane between electrodes, and thus only a target
material-ligand-enzyme complex or target material-ligand-metal
nanoparticle complex specifically binding to the receptor remains
on the membrane, thereby generating an electrical signal in the
electrode.
[0040] The "receptor" is a material having specific affinity for
the target material, and has a binding site different from the
ligand binding a target material with an enzyme or ligand-fixed
metal nanoparticles. After electrodes are formed on the membrane,
the receptor may be fixed on the membrane between the electrodes.
The receptor may be fixed by a method known in the art, and any one
of physical adsorbing methods and chemical methods may be used. A
specific fixing method may be suitably selected by one of ordinary
skill in the art according to the kinds of the receptor and
membrane.
[0041] When a reaction solution prepared by mixing a ligand-fixed
enzyme or ligand-fixed metal nanoparticles and a sample containing
a target material is added to the sensor having the additionally
fixed receptor, the target material specific to the receptor is
bound with the receptor on the membrane, thereby forming a
receptor-target material-ligand-enzyme complex or receptor-target
material-ligand-metal nanoparticle complex. Accordingly, the
complex is fixed on the membrane, and the target material specific
to the receptor may be detected by an electrical signal.
[0042] The receptor may be, but is not limited to, for example, an
antibody, an antigen, an enzyme, a peptide, a protein, DNA, RNA,
PNA, or an aptamer.
[0043] The target material may be, but is not limited to, for
example, an antibody, an antigen, an enzyme, a peptide, a protein,
DNA, RNA, a microorganism, a cell or an organ of an animal or
plant.
[0044] According to still another embodiment of the present
invention, a sensor without a receptor or having an additionally
fixed receptor, in which metal ions are reduced and precipitated on
a surface of an electrode by adding a metal reducing solution on a
filtering membrane, may be manufactured.
[0045] According to the exemplary embodiment, the sensor of the
present invention may have an electrode printed on the filtering
membrane, and metal ions may be reduced and precipitated on a
surface of the electrode.
[0046] According to an exemplary embodiment of the present
invention, as described above, the metal ions are reduced by the
metal reducing solution to be precipitated on a surface of the
electrode, but the present invention is not limited thereto. Metal
ions may be reduced and precipitated on a surface of the electrode
by a method that is suitably selected by one of ordinary skill in
the art. An electrical signal generated by a target
material-ligand-enzyme complex, a material-ligand-metal
nanoparticle complex, a receptor-target material-ligand-enzyme
complex or a receptor-target material-ligand-metal nanoparticle
complex remaining on the membrane may be amplified by increased
electrical conductivity due to the addition of a metal reducing
solution to reduce and precipitate metal ions on a surface of the
electrode.
[0047] Here, the "metal reducing solution" refers to a solution
including metal ions and a reducing agent capable of reducing the
metal ions. Any solution having such a characteristic may be used
without limitation, and a specific composition may be suitably
selected by one of ordinary skill in the art.
[0048] Here, the metal ions may be, but are not limited to, at
least one selected from the group consisting of gold, silver and
copper. In addition, a reducing agent capable of reducing the metal
ions may be selected from, for example, hydroxylamine (NH.sub.2OH),
ascorbic acid, glucose and a mixture thereof, but the present
invention is not limited thereto.
[0049] As the metal ions are reduced by the metal reducing
solution, and precipitated on a surface of the electrode on the
membrane, electrical conductivity of the electrode is increased,
resulting in amplification of an electrical signal. Here, the
reduction of such metal ions may be considerably stimulated by the
ligand-fixed enzyme or ligand-fixed metal nanoparticles added to
the membrane, and the electrical conductivity may be considerably
enhanced due to the enzyme or ligand-fixed metal nanoparticles,
thereby manufacturing a sensor having excellent detection
sensitivity.
[0050] According to the following embodiment, it can be noted that
the electrical conductivity may be considerably enhanced since a
reduction rate of silver ions in a silver reducing solution is
higher in the case (STA/HRP--AgGSH) that the silver ions are
reduced by adding the silver reducing solution in the presence of a
peroxidase enzyme, compared with a control group (PB--AgGSH) in
which silver ions are reduced by adding only a silver reducing
solution to an electrode (refer to FIGS. 3 and 4).
[0051] According to yet another embodiment, it can be noted that
electrical conductivity is considerably enhanced since a reduction
rate of gold ions in a gold reducing solution is higher in the case
(AuNP--Au Enh) that the gold ions are reduced by treating the gold
reducing solution in the presence of gold nanoparticles, compared
with a control group (PB--Au Enh) in which an electrode is treated
with only a gold reducing solution (refer to FIGS. 6 and 7).
[0052] In addition, the present invention provides a method of
manufacturing a sensor, which includes: (a) printing an electrode
on a filtering membrane; and (b) passing a reaction solution
prepared by mixing a ligand-fixed enzyme or ligand-fixed metal
nanoparticles and a sample containing a target material through the
filtering membrane.
[0053] After operation (a), the method may further include
additionally fixing a receptor on the filtering membrane between
electrodes.
[0054] According to an exemplary embodiment of the present
invention, the method may further include reducing and adding metal
ions on a surface of the electrode by adding a metal reducing
solution on the filtering membrane of the sensor that does not have
the receptor, or has the additionally-fixed receptor.
[0055] In the method of manufacturing a sensor of the present
invention, the electrode, the ligand, the enzyme, the
nanoparticles, the target material, the receptor, the metal
reducing solution, etc. may be the same as in the sensor described
above.
[0056] In addition, the present invention provides a method of
detecting a target material, which includes: (a) reacting a mixture
of a ligand-fixed enzyme or ligand-fixed metal nanoparticles and a
sample containing a target material; (b) passing the reaction
solution through the filtering membrane having an electrode to
filter only a target material-ligand-enzyme complex or target
material-ligand-metal nanoparticle complex; and (c) measuring an
electrical signal generated in the electrode by a target
material-ligand-enzyme complex or target material-ligand-metal
nanoparticle complex remaining on the filtering membrane.
[0057] In addition, the present invention provides a method of
detecting a target material, which includes: (a) reacting a mixture
of a ligand-fixed enzyme or ligand-fixed metal nanoparticles and a
sample containing a target material; (b) passing the reaction
solution through the filtering membrane having a receptor fixed on
a filtering membrane between electrodes to filtrate only a target
material-ligand-enzyme complex or target material-ligand-metal
nanoparticle complex specifically binding to the receptor; and (c)
measuring an electrical signal generated in the electrodes by a
target material-ligand-enzyme complex or target
material-ligand-metal nanoparticle complex remaining on the
filtering membrane.
[0058] Before measurement of the electrical signal, the method may
further include reducing and depositing metal ions on a surface of
the electrodes by adding a metal reducing solution on the filtering
membrane.
[0059] The electrical signal may be amplified into a signal capable
of being electrically measured by means of the enzyme or metal, and
then measured.
[0060] In an embodiment of the present invention, the measured
signal may be an electrical signal measured by an electrode on the
membrane, and for example, by measurement of electrical
conductivity or impedance, but the present invention is not limited
thereto.
[0061] The method of detecting a target material of the present
invention may be performed using the above-described sensor, and
thus may include all the configuration and characteristics of the
above-described sensor.
[0062] The present invention will now be described in further
detail with reference to Examples. The following Examples are
provided to fully describe the present invention, and should not be
construed as limiting its scope.
EXAMPLES
Example 1
Silver Ion Reduction after Enzyme is Fixed to Membrane Silver
Electrode
[0063] Electrodes interdigitated with a gap of 100.mu.m were formed
using a silver paste on an asymmetric super-micron membrane (MMM)
having a pore size of 0.45.mu.m through a silk screen printing
technique.
[0064] A peroxidase enzyme was fixed between the interdigitated
electrodes, and reacted with 0.1 M citrate buffer (pH 8.5, silver
reducing solution) containing 1 mM silver acetate, 1 mM
glutathione, 10 mM hydroquinone and 100 mM hydrogen peroxide to
reduce silver ions between the electrodes. Here, a direct current
voltage was applied, and a current was measured.
[0065] FIG. 2 is a diagram showing a process of fixing a peroxidase
enzyme (HRP) to the membrane silver electrode formed as described
above, and reducing silver ions in the silver reducing solution
prepared as described above on the electrodes by the peroxidase
enzyme.
[0066] FIGS. 3 and 4 show changes in current measured by fixing a
peroxidase enzyme to the membrane silver electrodes formed as
described above, reducing silver using the enzyme and the silver
reducing solution prepared as described above, and applying a
direct current voltage thereto. It could be confirmed that
electrical conductivity was considerably enhanced as silver ions
were reduced (5 min, 10 min) by treating a silver reducing solution
in the test group (STA/HRP--AgGSH) using a peroxidase enzyme,
compared with the control group (PB--AgGSH) without a peroxidase
enzyme.
Example 2
Gold Ion Reduction after Gold Nanoparticles were Fixed to Membrane
Silver Electrode
[0067] Electrodes interdigitated with a gap of 100.mu.m were formed
using a silver paste on an asymmetric super-micron membrane (MMM)
having a pore size of 0.45.mu.m through a silk screen printing
technique.
[0068] Gold nanoparticles having a size of 20 nm were fixed between
the interdigitated electrodes, and reacted with 10 mM citrate
buffer (gold reducing solution, pH 3.0) containing 1 mM
hydroxylamine and 10 mM HAuCl.sub.4 to reduce gold ions between the
electrodes. Here, a direct current voltage was applied, and a
current was measured.
[0069] FIG. 5 is a diagram showing a process of fixing gold
nanoparticles to the membrane silver electrode formed as described
above, and reducing the gold reducing solution prepared as
described above on the electrodes by the gold nanoparticles.
[0070] FIGS. 6 and 7 show changes in current measured by fixing
gold nanoparticles to the membrane silver micro-electrode formed as
described above, reducing gold using the gold reducing solution
prepared as described above, and applying a direct current voltage
thereto. It could be confirmed that electrical conductivity was
considerably enhanced as gold ions were reduced (5 min, 10 min) by
treating a gold reducing solution in the test group (AuNP--Au Enh)
to which gold nanoparticles were fixed, compared with the control
group (PB--Au Enh) without using gold nanoparticles.
Example 3
Gold Ion Reduction after Food Poisoning Bacteria-Antibody-Gold
Nanoparticle Complex was Filtrated Through Membrane Silver
Electrode
Example 3-1
Preparation of Gold Nanoparticle-Antibody Conjugate
[0071] 0.1 mL of borate buffer (0.1 M, pH 8.5) and 10.mu.g of an
antibody (Abcam, ab20002) having selectivity to Staphylococcus
aureus were added to 1 mL of a 20-nm gold nanoparticle solution (BB
International). After 30 minutes, 0.1 mL of a 1% bovine serum
albumin (BSA) solution (pH 8.5, dissolved in 10 mM carbonate
buffer) was added to the mixture, and stayed for 30 minutes. The
solution was subjected to centrifugation at 4.degree. C. and 10,000
rpm for 20 minutes, and a surfactant was removed. After 1 mL of
0.1% BSA (pH 8.5, dissolved in 10 mM carbonate buffer) was added to
and mixed with the mixture, the resulting mixture was subjected to
centrifugation at 10,000 rpm for 20 minutes, and then a surfactant
was removed. The above process was repeated again, and 0.5 ml of a
0.1% BSA solution (dissolved in PBS buffer) was finally added to
and mixed with the resulting mixture, and then stored in a
refrigerator.
Example 3-2
Culture of Staphylococcus Aureus
[0072] A stock of Staphylococcus aureus, which are food poisoning
bacteria, was inoculated into nutrient broth (NB), and incubated at
37.degree. C. in a shaking incubator for 18 to 24 hours. To count
colony forming units (CFU) of the incubated bacteria, the culture
solution was diluted in a range of 10.sup.5 to 10.sup.9, inoculated
by 100.mu.l and plated on a solid plate to be incubated at
37.degree. C. for 16 to 24 hours. Afterward, colonies formed on the
plate were counted and multiplied by a dilution rate, thereby
measuring a vial cell count. In addition, a suitable amount of a
liquid medium containing the incubated bacteria was taken to
measure absorbance at a wavelength of 600 nm using a
spectrophotometer.
Example 3-3
Analysis of Staphylococcus Aureus
[0073] Electrodes interdigitated with a gap of 100.mu.m were formed
using a silver paste on a polysulfone membrane (Pall life science)
having a pore size of 0.45.mu.m through a silk screen printing
technique.
[0074] A Staphylococcus aureus-antibody-god nanoparticle complex
was prepared by reacting Staphylococcus aureus with a gold
nanoparticle-antibody conjugate for 30 minutes, and filtrated
through the membrane silver micro-electrode formed as described
above. Here, gold ions were reduced between electrodes by reacting
10 mM citrate buffer (pH 3.0, gold reducing solution) containing 1
mM hydroxylamine and 10 mM HAuCl.sub.4 with the filtrated result.
Here, a direct current voltage was applied to measure a
current.
[0075] FIG. 8 is a diagram showing fixing a food poisoning
bacteria-gold nanoparticle complex to the membrane silver
micro-electrode formed as described above, and reducing the gold
reducing solution prepared as described above on the electrode by
the gold nanoparticles.
[0076] FIGS. 9 and 10 show changes in current measured by fixing a
food poisoning bacteria-gold nanoparticle complex to the membrane
silver micro-electrode formed as described above, reducing gold
using the gold reducing solution prepared as described above, and
applying a direct current voltage.
[0077] FIG. 11 shows current values at 0.1 V according to
concentrations of Staphylococcus aureus from the experiments shown
in FIGS. 9 and 10. It is seen that a current value is increased
according to the concentration of the bacteria, and may be measured
even at a cell concentration of approximately 10.sup.2 cfu.
[0078] The present invention is a result of the research conducted
as a part of New Technology-Fused Growth Engine Industry by support
from the National Research Foundation of Korea, which was funded by
the Government (Ministry of Education and Science Technology
(MEST)) in 2011 (2011K000910).
[0079] A membrane electrode according to the present invention
provides a novel sensor combining a filtering function of a
membrane and a signal measuring ability of an electrode. According
to the present invention, a target material can be measured by
filtration through the membrane, a small amount of target materials
can be detected with high sensitivity using an amplified electrical
signal by increasing electrical conductivity by reducing metal ions
on the membrane, and thus the target material can be subject to
quantitative analysis. In addition, only a target material
selectively binding to a receptor can be filtrated by passing a
sample through the membrane after a receptor material is fixed to
the electrode, and thus can be used to detect an electrical signal.
In addition, the sensor according to the present invention can
measure a signal by various methods such as electrical
conductivity, impedance, etc.
[0080] While the invention has been shown and described with
reference to certain exemplary embodiments thereof, it will be
understood by those skilled in the related art that various changes
in form and details may be made therein without departing from the
scope of the invention as defined by the appended claims.
* * * * *