U.S. patent application number 15/675953 was filed with the patent office on 2018-02-15 for capacitive biosensor for identifying a microorganism or determining antibiotic susceptibility.
This patent application is currently assigned to INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY. The applicant listed for this patent is INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY, PROTEOMETECH INC. Invention is credited to Nal Ae HAN, Bong Jun KIM, Sun Mi LEE, Kook Jin LIM, Jeseung OH, Kyung Hwa YOO.
Application Number | 20180045725 15/675953 |
Document ID | / |
Family ID | 59926144 |
Filed Date | 2018-02-15 |
United States Patent
Application |
20180045725 |
Kind Code |
A1 |
YOO; Kyung Hwa ; et
al. |
February 15, 2018 |
CAPACITIVE BIOSENSOR FOR IDENTIFYING A MICROORGANISM OR DETERMINING
ANTIBIOTIC SUSCEPTIBILITY
Abstract
An apparatus for inspecting an antibiotic and a method for
determining antibiotic sensitivity using the same is provided. The
antibiotic susceptibility inspection time which has conventionally
taken longer than 24 hours is shortened to about 2 hours or less,
the efficacy of the target substance is monitored in real time, the
identification of the microorganism, the kind of the antibiotic
capable of treating the microorganism, and the minimum dosage
thereof are quickly confirmed. Microbial infections requiring
prompt diagnosis and treatment can be effectively treated.
Inventors: |
YOO; Kyung Hwa; (Seoul,
KR) ; HAN; Nal Ae; (Busan, KR) ; KIM; Bong
Jun; (Gyeonggi-do, KR) ; LEE; Sun Mi; (Seoul,
KR) ; LIM; Kook Jin; (Seoul, KR) ; OH;
Jeseung; (Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY
PROTEOMETECH INC |
Seoul
Seoul |
|
KR
KR |
|
|
Assignee: |
INDUSTRY-ACADEMIC COOPERATION
FOUNDATION, YONSEI UNIVERSITY
Seoul
KR
PROTEOMETECH INC.
Seoul
KR
|
Family ID: |
59926144 |
Appl. No.: |
15/675953 |
Filed: |
August 14, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/569 20130101;
G01N 33/00 20130101; G01N 27/227 20130101; G01N 33/15 20130101;
G01N 33/54366 20130101; G01N 33/5438 20130101; C12Q 1/18 20130101;
C12Q 1/04 20130101; G01N 33/56911 20130101; G01N 27/226 20130101;
C12Q 1/00 20130101; G01N 2500/10 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/543 20060101 G01N033/543; G01N 33/15 20060101
G01N033/15; C12Q 1/04 20060101 C12Q001/04; C12Q 1/18 20060101
C12Q001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 2016 |
KR |
1020160103015 |
Claims
1. A copacitive bioseneor for identifying a microorganism or
determining an antibiotic susceptibility of the microorganism, the
copacitive bioseneor comprising: a substrate including an anodic
aluminum oxide; an electrode layer formed on the substrate and
including an interdigitated first electrode and an interdigitated
second electrode; and an aptamer fixed to the substrate and
specifically bound to the microorganism.
2. The copacitive bioseneor of claim 1, wherein the anodic aluminum
oxide is formed by: i) anodizing an aluminum surface with acid
treatment; and ii) extending a porous nanostructure.
3. The copacitive bioseneor of claim 1, wherein the fixing is
accomplished through a bond between a --COOH group introduced on a
surface of the anodic aluminum oxide and a --NH.sub.2 group of the
aptamer.
4. The copacitive bioseneor of claim 3, wherein the --COOH group
introduced on the surface of the anodic aluminum oxide is formed
by: i) introducing a --OH group by treating the surface of the
anodic aluminum oxide with an O.sub.2 plasma; ii) introducing a
--NH.sub.2 group by treating 3-aminopropyltriethoxysilane (APTES)
on the surface of the anodic aluminum oxide on which the --OH group
is introduced; and iii) introducing a --COOH group by treating on
the surface of the anodic aluminum oxide on which the --NH.sub.2
group with succinic anhydride.
5. The copacitive bioseneor of claim 1, wherein a distance between
the first electrode and the second electrode is 1 .mu.m to 100
.mu.m.
6. The copacitive bioseneor of claim 1, wherein the aptamer is
fixed between the first electrode and the second electrode.
7. The copacitive bioseneor of claim 1, the copacitive bioseneor
further comprises a storage portion capable of receiving at least
one of an electrode layer, an antibiotic, and a microorganism
therein.
8. The copacitive bioseneor of claim 1, wherein the microorganism
is a bacterium.
9. The copacitive bioseneor of claim 8, wherein the bacterium is a
gram-positive bacterium, a gram-negative bacterium, or an
antibiotic resistant bacterium of at least one thereof.
10. The copacitive bioseneor of claim 1, wherein the antibiotic is
selected from the group consisting of a Gentamicin, a Tetracycline,
an Ampicillin, an Erythromycin, a Vancomycin, a Linezolid, a
Methicillin, an Oxacillin, a Cefotaxime, a Rifampicin, an Amikacin,
a Kanamycin, a Tobramycin, a Neomycin, an Ertapenem, a Doripenem, a
Imipenem/a Cilastatin, a Meropenem, a Ceftazidime, a Cefepime, a
Ceftaroline, a Ceftobiprole, an Aztreonam, a Piperacillin, a
Polymyxin B, a Colistin, a Ciprofloxacin, a Levofloxacin, a
Moxifloxacin, a Gatifloxacin, a Tigecycline, and combinations and
derivatives thereof.
11. The copacitive bioseneor of claim 1, wherein the copacitive
bioseneor identifies the microorganism by determining a change in
capacitance in real time caused by coupling of the microorganism to
the copacitive bioseneor, or determines a change in capacitance in
real time caused by separation or deformation of the microorganism
from the aptamer by the antibiotic.
12. A method for determining an antibiotic susceptibility of a
microorganism, the method comprising: binding the microorganism to
the copacitive bioseneor as claimed in claim 1; treating the
microorganism-bound copacitive bioseneor with an antibiotic; and
determining a change in capacitance after the antibiotic
treatment.
13. A method for identifying a microorganism, the method
comprising: treating a sample containing a microorganism in the
copacitive bioseneor as claimed in claim 1; confirming whether the
microorganism in the sample and the aptamer are bound to each other
by determining a capacitance change after the sample treatment; and
identifying the microorganism.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and the benefit of
Korean Patent Application No. 10-2016-0103015, filed on Aug. 12,
2016, the disclosure of which is incorporated herein by reference
in its entirety.
BACKGROUND
1. Field of the Disclosure
[0002] The present disclosure relates to a capacitive biosensor,
and more particularly, relates to a biosensor able to determine
antibiotic susceptibility of a microorganism or identify a
microorganism according to the result of determining a level of
capacitance, changed by microorganisms, in real time.
2. Discussion of Related Art
[0003] An inspection to detect antibiotics that can inhibit
bacterial growth is known as an antibiotic susceptibility
inspection. An antibiotic susceptibility inspection is a direct and
important inspection allowing a type of antibiotic to counter a
bacterium to be selected. In addition, when prescribing appropriate
antibiotics to a patient, such an inspection may enable a
customized prescription, considering the prescription method,
frequency, cost, and side effects to be made. Using antibiotic
susceptibility inspection results, it is possible to reduce
increases in treatment costs and the disappointment of patient
caregivers in the case that empiric antibiotics are prescribed,
providing opportunities for patients to acquire bacterial
resistance, reducing complications, and reducing patient recovery
periods.
[0004] The most common antibiotic susceptibility inspections are
the disk diffusion method and the broth dilution method. However,
these methods require a process of culturing a bacterium for
several days, identifying the bacterium, and measuring turbidity,
which may take a long time and requires an excessive amount of
labor.
[0005] Therefore, there is a need to develop methods to overcome
the problems of conventional antimicrobial susceptibility
inspection methods by enabling real-time measurement, shortening
test times and reducing labor requirements.
SUMMARY OF THE DISCLOSURE
[0006] In order to solve the above problems, an object of the
present disclosure is to provide a capacitive biosensor capable of
measuring antibiotic susceptibility of microorganisms or
identifying microorganisms by real-time measurement of capacitance
change corresponding to the growth of microorganisms, and methods
for measuring antibiotic susceptibility of a microorganism and
identifying a microorganism using a biosensor.
[0007] In order to achieve the above object, the present disclosure
provides a copacitive bioseneor for identifying a microorganism or
determining an antibiotic susceptibility, the copacitive bioseneor
for identifying a microorganism or inspecting an antibiotic
susceptibility includes, a substrate including anodic aluminum
oxide; an electrode layer formed on the substrate and including an
interdigitated first electrode and an interdigitated second
electrode; and an aptamer fixed to the substrate and specifically
bound to the microorganism.
[0008] In addition, the present disclosure provides a method for
determining an antibiotic susceptibility of the microorganism, the
method includes, binding microorganisms to the biosensor; treating
the microorganism-bound biosensor with an antibiotic; and
determining a change in capacitance after the antibiotic
treatment.
[0009] In addition, the present disclosure provides a method for
identifying a microorganism, the method includes, treating a sample
containing a microorganism in the biosensor; confirming whether the
microorganism in the sample and aptamer are bound to each other by
determining a capacitance change after the sample treatment; and
identifying the microorganism.
[0010] Using the biosensor and the method for measuring antibiotic
susceptibility, an antibiotic susceptibility inspection time which
has conventionally taken longer than 24 hours can be shortened to
about 2 hours or less, since the time required for identifying
microorganisms can be shortened, microorganisms can be identified
quickly, a type of antibiotic and a minimum antibiotic
concentration necessary for administration can be quickly
confirmed, whereby microbial infections requiring prompt diagnosis
and treatment can be effectively treated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is an electron microscope image illustrating the
process of widening (c) using phosphoric acid immediately after
anodic oxidation (b) using an oxalic acid on a film-like aluminum
surface (a).
[0012] FIG. 2(a) is a schematic view illustrating a process of
introducing a-COOH group to the surface of anodic aluminum oxide
and bonding it to an --NH2 group of the aptamer, while (b) is an
optical image (left) and a fluorescence image (right) illustrating
an anodic aluminum oxide substrate and a gold electrode fixed to an
anodic aluminum oxide substrate fixed to a fluorescence-coupled
aptamer prepared according to an embodiment of the present
disclosure and a gold electrode.
[0013] FIG. 3 is a schematic view illustrating a capacitive
biosensor array for identifying a microorganism or measuring
antibiotic susceptibility of a microorganism manufactured according
to an embodiment of the present disclosure.
[0014] FIG. 4 is a schematic view illustrating a capacitive
biosensor array for identifying a microorganism or measuring
antibiotic susceptibility of a microorganism manufactured according
to an embodiment of the present disclosure, and a process for
identifying a microorganism using the same.
[0015] FIG. 5 is a graph illustrating results of performance
differences of a biosensor according to a type of a substrate. As a
result of measuring a change in capacitance after inserting 105
CFU/ml of an Escherichia coli (E. Cole) into the sensor, the blue
line represents the biosensor including the AAO substrate+aptamer,
the red line represents the biosensor with the aptamer unfixed, and
the black line represents the measurement result of the change in
capacitance of the culture medium only, without an E. coli
bacterium.
[0016] FIG. 6(a) is a schematic view illustrating a biosensor in
which an aptamer is not fixed, or a biosensor in which an aptamer
is fixed for each type, and (b) to (e) are a graphs illustrating
results of measuring a capacitance change rate in real-time, when
E. coli, acinetobacter baumani, Staphylococcus aureus, and
endorocococcus pecalis are bound to the biosensor in which an
aptamer is not fixed or a biosensor in which an aptamer is fixed
for each type, respectively.
[0017] FIG. 7 is a graph illustrating a fluorescent optical image
(a) after fluorescence is applied to a bacterium and a graph (b)
illustrating the normalized strength thereof, after bonding E.
coli, acinetobacter baumani, Staphylococcus aureus, and
endorocococcus pecalis to the biosensor in which an aptamer is not
fixed or the biosensor in which an aptamer is fixed for each type,
respectively.
[0018] FIG. 8 is a graph illustrating a result of measuring the
change in real-time capacitance when various types of antibiotics
are treated after E. coli is bound to a biosensor to which an
aptamer specifically bound to E. coli is fixed.
[0019] FIG. 9 is a graph illustrating a result of measuring the
change in real-time capacitance when various concentrations of
ceftriaxone are treated after E. coli is bound to a biosensor to
which an aptamer specifically bound to E. coli is fixed.
[0020] FIG. 10(a) is a schematic view illustrating a capacitive
biosensor for measuring Gentamicin susceptibility of a
microorganism, manufactured according to an embodiment of the
present disclosure; (b) is a graph illustrating a result of
measuring the change in real-time capacitance when various
concentrations of Gentamicinare provided or not provided after E.
coli is bound to a biosensor to which an aptamer specifically bound
to E. coli is fixed, and; (c) is a graph illustrating a result of
measuring the change in real-time capacitance when various
concentrations of Gentamicinare provided or not provided after
Staphylococcus aureus is bound to a biosensor to which an aptamer
specifically bound to Staphylococcus aureus is fixed.
[0021] FIG. 11(a) is a graph illustrating a result of measuring the
change in real-time capacitance when an antibiotic (Tetracycline,
gentamycin or Ampicillin) is provided or not provided after E. coli
is bound to a biosensor to which an aptamer specifically bound to
Tetracycline-resistant E. coli is fixed, and (b) is a graph
illustrating a result of measuring the change in real-time
capacitance when an antibiotic (Tetracycline, gentamycin or
Ampicillin) is provided or not provided after Ampicillin-resistant
E. coli is bound to a biosensor to which an aptamer specifically
bound to Tetracycline-resistant E. coli is fixed.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0022] The present disclosure provides a capacitive biosensor and a
method for measuring antibiotic susceptibility using the biosensor
and a method for identifying a microorganism.
[0023] According to the conventional antibiotic susceptibility
inspection method, since susceptibility can be confirmed by
spreading the bacterium on an entire culture medium, and culturing
the bacterium while being treated with antibiotics for about one
day, the time required to culture the microorganism to obtain a
sufficient amount of microorganism, the time required to confirm
reactivity, and a consumption of labor power are relatively large.
However, when using the biosensor of the present disclosure, since
it is possible to confirm a result in real time by measuring the
change in capacitance, the preparation time for
measuring/identifying can be shortened, and the sensitivity is
high, so that low level changes can be detected in a small amount
of sample.
[0024] Hereinafter, the present disclosure will be described in
detail.
[0025] The present disclosure, however, is capable of having
numerous iterations and various forms, and the specific embodiments
and descriptions set forth below are merely intended to assist in
gaining an understanding of the disclosure, while not being
intended to limit the disclosure to the specific forms of
disclosure. It is to be understood that the scope of the present
disclosure includes all changes, equivalents, and alternatives
falling within the spirit and scope of the present disclosure.
[0026] The capacitive biosensor of the present disclosure is for
identifying a microorganism by measuring a change in capacitance in
real time caused by coupling of a microorganism to a biosensor, or
measuring susceptibility of a microorganism to an antibiotic,
measured by a change in capacitance of the microorganism in real
time, caused by separation or deformation of a microorganism from
an aptamer by an antibiotic. Preferably, the microorganism may be
combined with an aptamer of the biosensor of the present
disclosure.
[0027] The capacitive biosensor of the present disclosure may
comprise a substrate including anodic aluminum oxide; an electrode
layer formed on the substrate and including an interdigitated first
electrode and an interdigitated second electrode; and an aptamer
fixed to the substrate and specifically bound to the
microorganism.
[0028] The biosensor of the present disclosure refers to a device
or an apparatus for examining a property of a substance using a
function of an organism, the electrode is specifically formed on a
substrate, particularly, and refers to a sensor in which an
electrode is formed on a substrate, and detects whether
binding/separation of substances has occurred by measuring the
change in capacitance caused by the binding of the target material
between the electrodes using an electrical method, and more
particularly, may be a device or an apparatus for measuring
capacitance according to the ability of the microorganism to grow
in an antibiotic environment, confirming the susceptibility of the
microorganism to the antibiotic according to the result.
Alternatively, it may be a device or an apparatus capable of
identifying a microorganism according to whether an unknown
microorganism is bound in a sample using characteristics of an
aptamer that specifically binds to the specific microorganism.
[0029] In the present disclosure, a substance identified by a
biosensor or capable of measuring antibiotic susceptibility
includes both microorganisms and biomolecules thereof and the like,
particularly, the type of the microorganism is not particularly
limited.
[0030] Hereinafter, in an embodiment of the present disclosure, it
was confirmed that microorganisms can be identified by measurement
of capacitance changes and antibiotic susceptibility of bacterium
can be detected in real time by using aptamer specific to E. coli
and Salmonella, respectively (FIG. 8 to FIG. 11).
[0031] Antibiotic susceptibility is also referred to as antibiotic
sensitivity, and the microorganism (for example, a strain, or the
like) is affected by the inhibition of growth by the specific
antibiotic. According to a method of inspecting typical antibiotic
susceptibility, it is said that when an antibiotic is provided and
a bacterium does not grow around the location provided with the
antibiotic, the bacterium has sensitivity. A result of antibiotic
susceptibility inspection can be divided into, for example,
susceptibility, intermediate susceptibility (intermediate
tolerance) and resistance (tolerance). A susceptible antibiotic is
used to treat an infection caused by microorganisms, an infection
of a susceptible read-out strain means that the strain can be
treated with a recommended amount of an antimicrobial agent for the
infection of the species and its site, an infection of the
intermediate susceptible (or intermediate tolerance) read-out
strain means that the minimum inhibitory concentration of the
antimicrobial agent against the inspection strain is similar to
that of the blood or tissue, and therefore the treatment effect is
lower than that for the susceptible strains, this means that there
is a therapeutic effect, when the infected area of the
antimicrobial agent, such as the urine, is concentrated, or when
the maximum amount of the drug capable of administering a large
amount of the antimicrobial agent is administered, and an infection
of the resistant (or tolerant) read-out strain means that the blood
concentration of the antimicrobial agent when administered at a
normal dose has no therapeutic effect.
[0032] In the present disclosure, an identification (or
identifying) means performing confirmation of species, properties,
and the like of a microorganism contained in a sample requiring
inspection or analysis, and an identification (or identifying) of a
microorganism means identifying the species or the like of the
microorganism contained in the sample.
[0033] In the present disclosure, a sample is a substance, presumed
to contain or contain a target substance, to be analyzed, and may
be in the form of a composition, and may be collected from any one
of liquid, soil, air, food, waste, plant and animal intestines and
animal and plant tissues, blood, urine, tears, saliva, the animal
or plant includes a human body.
[0034] A biosensor according to an embodiment of the present
disclosure can identify a microorganism using an aptamer that
specifically binds to a microorganism, and can directly measure an
antibiotic sensitivity and a minimum concentration of the
identified microorganism. Thus, it is possible to carry out
identifying a microorganism and measuring susceptibility thereof,
simultaneously. In addition, the biosensor according to an
embodiment of the present disclosure has an advantage of being able
to provide data on a more precise dosage/concentration of an
antibiotic to treat an infection, since minute levels of change can
be detected through measuring a change in capacitance.
[0035] In the present disclosure, an aptamer refers to a small
single-stranded oligonucleotide capable of specifically recognizing
a target substance with high affinity or specific affinity.
[0036] The biosensor of the present disclosure includes a substrate
comprising an anodized aluminum (or ananodic aluminum oxide, AAO),
preferably, a substrate made of AAO. The anodic aluminum oxide
(AAO) refers to porous alumina in which nano-sized pores having
regularity on a surface are formed using anodic oxidation to
electrochemically oxidize aluminum. An anodic aluminum oxide may be
prepared by: i) anodizing an aluminum surface with an acid
treatment; and ii) extending a porous nanostructure. Particularly,
the acid is preferably oxalic acid, but is not limited thereto.
[0037] When an aptamer is bonded to a nanometer-sized porous
nanostructure on the surface of the thin film layer and used as a
sensor, it is possible to bind more aptamers than in the case of
using other substrates, and it is possible to measure capacitance
with higher sensitivity when the same amount of samples are used,
therefore, it is confirmed that the performance of the biosensor
can be remarkably improved through an embodiment of the present
disclosure.
[0038] Since types of microorganism which specifically bind are
different, depending on a type of the aptamer, a microorganism
bound according to the type of the aptamer that specifically binds
to the specific microorganism can be identified. For example, when
an aptamer specifically bound to Escherichia coli (E. coli) is
immobilized on the substrate and a sample containing a
microorganism is treated on the sensor, and when the sample
contains E. coli, then since the change in capacitance will be
sensed by the electrode due to a binding of the aptamer and E. coli
and a proliferation of bound E. coli, it is possible to promptly
detect the presence of E. coli in the sample, a type of
microorganism in the sample can be identified through the target
specific binding characteristics of the aptamer.
[0039] The fixing may be performed through bonding between a --COOH
group introduced on the surface of the anodic aluminum oxide and an
--NH.sub.2 group of the aptamer, particularly, the --COOH group
introduced on the surface of the anodic aluminum oxide is formed
by: i) introducing a --OH group by treating the surface of the
anodic aluminum oxide with an O.sub.2 plasma; ii) introducing a
--NH.sub.2 group by treating 3-aminopropyltriethoxysilane (APTES)
on the surface of the anodic aluminum oxide on which the --OH group
is introduced; and iii) introducing a --COOH group by treating the
surface of the anodic aluminum oxide on which the --NH.sub.2 group
is introduced with succinic anhydride.
[0040] The biosensor of the present disclosure includes an
electrode layer, and may particularly include an interdigitated
microelectrode. The interdigitated microelectrodes, a bar arranged
in a line form one electrode, and two electrodes (the first
electrode and the second electrode) may have opposing structures
while being engaged with each other. The two electrodes function as
an impedance measuring electrodes of a classical form. In a sensor
using an interdigitated microelectrode, a spacing distance between
the first electrode and the second electrode may be 0.1 .mu.m to
1000 .mu.m, 1 .mu.m to 500 .mu.m, or 10 .mu.m to 100 .mu.m. By
adjusting the spacing distance between the first electrode and the
second electrode to the above-mentioned range, it is possible to
precisely measure even micro-level biomolecules. In addition, a
height of each electrode is in a range of 50 nm to 5,000 nm, and a
width of each electrode may be in a range of 1 .mu.m to 500 .mu.m,
or 10 .mu.m to 100 .mu.m. Each of the electrodes may be
independently selected from a group consisting of gold, platinum,
carbon, a conductive polymer, and indium tin oxide (ITO).
[0041] The biosensor of the present disclosure may include various
types of interdigitated microelectrodes, and may include a
plurality of the electrodes. The microelectrode may be formed on
the substrate.
[0042] The aptamer may be fixed to the space between the first
electrode and the second electrode, that is, a spaced apart area.
The antimicrobial susceptibility of the microorganism is confirmed
by measuring the change in the capacitance of the aptamer fixed
between the first electrode and the second electrode when the
microorganism to be identified binds to the aptamer, or when the
bound microorganism is separated or transformed by the antibiotic.
The type of microorganism can also be identified.
[0043] The biosensor of the present disclosure may further include
a storage portion. The storage portion may contain an electrode
layer, an antibiotic, and a microorganism therein. In addition, the
storage portion may be formed in a direction perpendicular to the
substrate. Particularly, when an identification of a microorganism
or antibiotic susceptibility is measured using a biosensor, the
microorganism or the antibiotic or the like can be treated inside
the storage section. The storage portion may have an open top
portion.
[0044] The storage portion may be made of one or more materials
selected from a group consisting of glass, polypropylene (PP),
polyethylene terephthalate (PET) and polycarbonate (PC). As a
commercially available example, it may be a plastic well. The
capacity of the storage portion may be from 10 ul to 1 ml. The
microorganism may be treated together with a culture medium, and as
such, the storage unit may also include a culture medium for the
microorganism.
[0045] The culture medium is also referred to as a medium or a
nutrient broth, a liquid or a solid material used for
proliferation, preservation, or the like of a microorganism. The
specific constitution of the culture medium may be different,
depending on a type of microorganism. Any liquid form is preferred,
and the culture medium may be blood or may include blood.
[0046] The microorganism may be a bacterium, and the bacterium is
preferably a gram-positive bacterium, a gram-negative bacterium and
an antibiotic-resistant bacterium thereof, but is not limited
thereto. Particularly, the gram-positive bacterium may be one or
more selected from a group consisting of Bacillus subtilis,
Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus
epidermidis, the gram-negative bacterium may be one or more
selected from a group consisting of E. coli, Psedomonas aeruginosa,
Acinetobacter baumannii, and Salmonella typhimurium.
[0047] In the present disclosure, the antibiotic is not
particularly limited by type, and any antibiotic can be used as
long as susceptibility is able to be measured using the biosensor
of the present disclosure. Particularly, the antibiotic is selected
from a group consisting of Gentamicin, Tetracycline, Ampicillin,
Erythromycin, Vancomycin, Linezolid, Methicillin, Oxacillin,
Cefotaxime, Rifampicin, Amikacin, Kanamycin, Tobramycin, Neomycin,
Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Ceftazidime,
Cefepime, Ceftaroline, Ceftobiprole, Aztreonam, Piperacillin,
Polymyxin B, Colistin, Ciprofloxacin, Levofloxacin, Moxifloxacin,
Gatifloxacin, Tigecycline, as well as combinations and a
derivatives thereof.
[0048] As shown in FIGS. 1 to 4, when AAO substrate is formed, an
electrode is formed thereon and then an aptamer is processed, a
surface area capable of specific binding is widened, as compared
with the case in which a conventional glass substrate is used, a
fixing efficiency of the aptamer is remarkably improved, whereby
the sensitivity of the sensor can be increased (FIG. 5(a)).
[0049] The biosensor of the present disclosure may further include
a wireless transmission unit for transmitting a capacitance
measurement result, and a transmission method may be any commonly
used method, including both data transmissions using a wired
method, such as via USB, or a wireless method, such as a
Bluetooth.RTM. method.
[0050] In addition, the biosensor of the present disclosure can be
connected to an LCR meter capable of measuring capacitance to
monitor a change in concentration of bacterium in real time. The
bacterial sensor may be supplied with a 1 to 100 mV of alternating
current voltage having a frequency of 0.1 to 100 KHz.
[0051] According to another aspect of the present disclosure, a
method for measuring antibiotic susceptibility of a microorganism
is provided, comprising: a preparation step of binding
microorganisms to the capacitive biosensor; a step of treating the
microorganism-bound capacitive biosensor with an antibiotic; and a
step of measuring a change in capacitance after an antibiotic
treatment.
[0052] According to another aspect of the present disclosure, a
method for identifying a microorganism is provided, comprising: a
step of treating a sample containing a microorganism in the
capacitive biosensor; a step of confirming whether the
microorganism in the sample and the aptamer are bound to each other
by measuring a change in capacitance after the sample treatment;
and a step of identifying the microorganism.
[0053] The method may further comprise a step of measuring
capacitance before the treating the antibiotic in the measuring
method or before the treating the sample containing the target
microorganism.
[0054] The antibiotic sensitivity can be determined by measuring
the capacitance value before the antibiotic treatment and the
changed capacitance value after the antibiotic treatment and
determining whether the microorganisms are killed according to
susceptibility in real time, using a change value of the
capacitance, and can be measured more precisely through the change
in capacitance. For example, the sensitivity to the antibiotic
concentration can be measured, so that the minimum amount can be
used in the concentration and dose of the antibiotic to be
administered for treatment, whereby abuse of antibiotics can be
prevented, and side effects can be decreased accordingly.
[0055] The biosensor may further include a storage portion for
storing an electrode layer, an antibiotic, and a microorganism
therein. When the biosensor including the storage portion is used,
the antibiotic, the microorganism, and the aptamer may be processed
in the storage portion. In this case, high sensitivity can be
measured even with a small sample amount.
[0056] When the antibiotic of the microorganism is measured by the
method for measuring antibiotic susceptibility of the present
disclosure, measuring of the selected minimum dose of antibiotics
without resistance can be performed quickly, and it is possible to
prevent the indiscriminate use of antibiotics and to reduce usage
amounts.
[0057] Hereinafter, the present disclosure is described in detail
with reference to manufacturing examples and experimental examples.
The following manufacturing examples and experimental examples are
illustrative in the present disclosure and are not intended to
limit the scope of the present disclosure.
Manufacturing Example 1: Preparation of Experiment
[0058] Cephalothin, chloramphenicol, Gentamicin, Ciprofloxacin,
cefrtiaxone, and Tetracycline antibiotics were purchased from Sigma
Aldrich (US), and various concentrations were prepared.
Chloramphenicol was dissolved in ethanol, Ciprofloxacin was
dissolved in DMSO, gentamycin and cephalothin were dissolved in
distilled water, and Sephritaxone and Tetracycline were dissolved
in ethanol.
[0059] The strains used in the experiment were E. coli,
Acinetobacter baumannii, Staphylococcus aureus, Enterococcus
faecalis, Enterococcus faecalis, as well as Ampicillin-resistant E.
coli and Tetracycline-resistant E. coli.
Manufacturing Example 2: Manufacturing of Capacitive Biosensor
[0060] In order to manufacture a capacitive biosensor for detecting
a microorganism, a sensor (Comparative Example 1) in which
electrodes are formed on a glass substrate, a sensor (Comparative
Example 2) which a glass electrode formatted and a aptamer-treated
sensor, and a sensor (Example 1) treated with an aptamer to AAO
were manufactured.
[0061] Particularly, the porous nanostructured plate (for example,
AAO plate) was formed to have a thickness of Ti/Au/Al (100/20/1000
nm) on a 4-inch Si substrate with a 1 nm thick SiO.sub.2 layer
grown thereon and a thickness of Ti/Au/Al (100/20/1000 nm).
[0062] Then, the porous nanostructured plate was immersed in oxalic
acid while maintaining the temperature at 15.degree. C. using a
water bath and a chiller at a concentration of 0.3M of oxalic acid,
and anodic oxidation was carried out by applying a DC current of
40V. After anodic oxidation, a widening reaction was performed to
produce AAO having porous nanostructures. An electron micrograph of
the AAO prepared above is shown in FIG. 1.
[0063] The formation of the electrode, as shown in FIG. 3, was
produced by depositing Cr with a thickness of 5 nm and Au with a
thickness of 40 nm on the AAO plate, manufactured after the anodic
oxidation, using a photolithographic process of patterning
interdigitated microelectrodes having an electrode width of 70
.mu.m and a spacing distance of 30 .mu.m, with each of bars aligned
in a row forming a pole and different electrodes (the first
electrode and the second electrode) having a pair structure in
which the other poles having opposing polarities face each other.
After the produced electrode was sterilized using autoclave and
ethanol, a sensor was manufactured by attaching 16 wells to the
electrode formed on the substrate for culturing bacterium.
[0064] Further, an aptamer for inducing specific binding of E. coli
was deposited on the substrate. The aptamer may be an E.
coli-specific aptamer (5'-GCA ATG GTA CGG TAC TTC CCC ATG AGT GTT
GTG AAA TGT TGG GAC ACT AGG TGG CAT AGA GCC GCA AAA GTG CAC GCT ACT
TTG CTA A-3', Genotech, Daejeon, Korea), an
Asnithobacterbaumannii-specific aptamer (5'-TAC ATG GTC AAC CAA ATT
CTT GCA AAT TCT GCA TTC CTA CTG T-3', Genotech, Daejeon, Korea), a
Staphylococcal aptamer-specific aptamer (5'-GCA ATG GTA CGG TAC TTC
CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA
GTG CAC GCT ACT TTG CTA A-3', Genotech, Daejeon, Korea), and an
Enterococcus faecalis-specific aptamer (5'-ATC CAG AGT GAC GCA GCA
CGA CAC GTT AGG TTG GTT AGG TTG GTT AGT TTC TTG TGG ACA CGG TGG CTT
A-3', Genotech, Daejeon, Korea) was used.
[0065] First, the AAO substrate was treated with O.sub.2 plasma to
generate an --OH group, and the 3-aminopropyltriethoxysilane
(APTES) 10% ethanol solution was treated with the --OH group to
replace the --NH.sub.2 group. Subsequently, the reaction mixture
was treated with a succinic anhydride 0.1 M ethanol solution to
convert it to a --COOH group, then the aptamer was immobilized on
AAO by reacting with the --NH.sub.2 group at the terminal of the
aptamer using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
and N-hydroxysuccinimide (NHS).
Experimental Example 1: Confirmation of Measurement Performance of
Capacitance Change of Sensor According to Substrate
[0066] A measurement performance of a capacitance change of a
sensor was confirmed using the sensor manufactured according to
manufacturing Example 2. Particularly, E. coli was treated for each
of a sensor (Comparative Example 1) in which an electrode was
formed on a glass substrate, a sensor (Comparative Example 2) in
which a glass substrate was treated with an aptamer, and a sensor
(Example 1) in which AAO was treated with an aptamer, and a change
in capacitance was measured.
[0067] As shown in FIG. 5, when the capacitance is measured using
the sensor of the present disclosure, the change in capacitance is
increased as the bacterium binds/propagates, the change in
capacitance of all three treatment groups was measured, in the case
of the AAO+aptamer sensor of Example 1 as compared to the glass
substrate sensor (black) of Comparative Example 1 and the glass
substrate sensor (red) treated with the aptamer of Comparative
Example 2, it was confirmed that the capacitance increased after
0.5 hours. Particularly, in the case of the AAO+aptamer treatment,
the change in capacitance was measured within a short period of
time. In the case of the biosensor having the aptamer bonded to the
AAO substrate of the present disclosure, the change in capacitance
was measured with remarkably high sensitivity.
Experimental Example 2: Identification of a Microorganism
[0068] An experiment was conducted to confirm whether a
microorganism could be identified using the biosensor produced
according to Production Example 2.
[0069] Particularly, each well was treated and fixed with an
aptamer specific to E. coli, Acinetobacter baumannii,
Staphylococcus aureus or Enterococcus faecalis, and media (a
control group or a control) not containing a microorganism, E.
coli, Acinetobacter baumannii, Staphylococcus aureus or
Enterococcus faecalis were treated with a sensor, and then the
change in capacitance was measured after about 2 hours in an
incubator at 37.degree. C.
[0070] As shown in FIG. 6(b), when the E. coli-specific aptamer was
treated, it was confirmed that the change in capacitance was
significantly increased during the treatment with E. coli. When
treating other microorganisms, it was confirmed that the change in
capacitance did not occur significantly. On the other hand, as
shown in FIG. 6(d), it was confirmed that when a Staphylococcus
aureus-specific aptamer was treated, a large capacitance change
occurred during Staphylococcus aureus treatment. When treating
other microorganisms, it was confirmed that the change in
capacitance did not occur significantly.
[0071] As shown in FIG. 7, when each microorganism is treated with
a selective aptamer, when the selective microorganism was treated,
it was verified by fluorescence that microorganisms could be
identified by confirming that they were attached to the capacitive
substrate.
Experimental Example 3: Susceptibility Measurement by Type of
Antibiotic
[0072] The susceptibility inspection for antibiotics was performed,
after identifying an E. coli using the capacitive biosensor
manufactured by treating E. coli-specific aptamer in Experimental
Example 2 on the substrate manufactured according to Manufacturing
Example 2.
[0073] PBS was treated as a control, each treatment was treated
with cephalothin, Gentamicin, Ciprofloxacin and chloramphenicol at
a concentration of 100 ng/ml, the change in capacitance with time
was measured for 6 hours in an incubator at 37.degree. C.
[0074] As shown in FIG. 8, when a bacterium was cultured in the
sensor and each of the four antibiotics was deposited, there was a
difference in the change in capacitance depending on the type of
the antibiotic. In the case of chloramphenicol (blue line) and
cephalothin (green line), the increase in capacitance after the
addition of antibiotics showed the bacterium resistance (tolerance)
against the two antibiotics, Ciprofloxacin (pink line) and
Gentamicin (red line) showed a decrease in capacitance, and the
bacterium was confirmed to have susceptibility to ciprofloxacin and
Gentamicin.
[0075] Particularly, it can be seen that susceptibility to
ciprofloxacin is high, according to the variation width of
capacitance.
[0076] According to the present disclosure, results can be
confirmed within 6 hours, it is possible to quickly measure
susceptibility, as the results can be confirmed within 6 hours, it
is possible to simultaneously measure several antibiotics, and
susceptibility can be measured quickly and simultaneously for
several antibiotics.
Experimental Example 4: Inspection of Antibioticminimum Inhibitory
Concentration
[0077] To determine the minimum inhibitory concentration of an
antibiotic, E. coli (105 cells/ml) was treated with a capacitive
biosensor and grown at 37.degree. C. for 2 hours, and then treated
with antibiotic ceftriaxone (MIC: .about.30 ng/ml) at
concentrations of 100, 80, 60, 40 and 20 ng/ml, respectively. The
degree of susceptibility was determined based on the results of the
capacitance monitored in real time, and the minimum inhibitory
concentration thereof was confirmed.
[0078] As shown in FIG. 9, it can be seen that there is a
difference in the change in capacitance of E. coli according to the
concentration of antibiotic ceftriaxone, especially, as the change
in capacitance value is shown from 40 ng/ml, it can be confirmed
that the minimum inhibitory concentration for this E. coli is 40
ng/ml. In addition, since the change is remarkably exhibited within
2 hours after the antibiotic treatment, the minimum antibiotic
concentration that can limit tolerance can be quickly confirmed by
using the capacitance value of the sensor of the present
disclosure.
[0079] In addition, E. coli (105 cells/ml) was treated with a
capacitive biosensor and grown at 37.degree. C. for 2 hours, and
then treated with antibiotic Gentamicin (MIC: .about.0.5 .mu.g/ml)
50, 5, 0.5, 0.05, and 0.005 .mu.g/ml, respectively. The degree of
susceptibility was determined based on the results of the
capacitance monitored in real time, and the minimum inhibitory
concentration thereof was confirmed.
[0080] As shown in FIG. 10(a) and FIG. 10(b), it can be seen that
there is a difference in the change in capacitance of E. coli
according to the concentration of antibiotic Gentamicin,
especially, as the change in capacitance value is shown from 0.5
.mu.g/ml, it can be confirmed that the minimum inhibitory
concentration for this E. coli is 0.5 .mu.g/ml. In addition, since
the change is remarkably exhibited within 2 hours after the
antibiotic treatment, the minimum antibiotic concentration that can
limit tolerance can be quickly confirmed by using the capacitance
value of the sensor of the present disclosure.
[0081] In addition, Staphylococcus aureus (105 cells/ml) was
treated with a capacitive biosensor and grown at 37.degree. C. for
2 hours, and then was treated with antibiotic Gentamicin (MIC:
.about.0.5 .mu.g/ml) 50, 5, 0.5, 0.05, and 0.005 .mu.g/ml,
respectively. The degree of susceptibility was determined based on
the results of the capacitance monitored in real time, and the
minimum inhibitory concentration thereof was confirmed.
[0082] As shown in FIG. 10(a) and FIG. 10(b), it can be seen that
there is a difference in the change in capacitance of E. coli and
Staphylococcus aureus according to the concentration of antibiotic
Gentamicin, especially, as the change in capacitance value is shown
from 0.5 .mu.g/ml, it can be confirmed that the minimum inhibitory
concentration for this E. coli is 0.5 .mu.g/ml. In addition, since
the change is remarkably exhibited within 2 hours after the
antibiotic treatment, the minimum antibiotic concentration that can
limit tolerance can be quickly confirmed by using the capacitance
value of the sensor of the present disclosure.
[0083] In addition, Tetracycline-resistant E. coli or
Ampicillin-resistant E. coli (105 cells/ml) was treated with a
capacitive biosensor and grown at 37.degree. C. for 2 hours, and
was then treated with Tetracycline at a concentration of 2
.mu.g/ml, Gentamicin at a concentration of 1 .mu.g/ml, and
Ampicillin at a concentration of 8 .mu.g/ml, respectively. The
degree of susceptibility was determined based on the results of the
capacitance monitored in real time.
[0084] As shown in FIG. 11(a) and FIG. 11(b), E. coli, resistant to
antibiotics, showed an increase in capacitance value after
treatment with each resistant antibiotic, confirming that it did
not affect the growth of E. coli. However, for antibiotics not
resistant to Gentamicin and the like, the decrease in capacitance
value was confirmed to affect the growth of E. coli. The results of
this experiment can be used to evaluate resistant antibiotics and
antibiotics that can be effectively treated within a short period
of time through by measuring AST of antibiotic resistant
bacterium.
* * * * *