U.S. patent application number 13/081177 was filed with the patent office on 2011-07-28 for method for sensing a substance to be detected in a sample.
This patent application is currently assigned to Japan Science and Technology Agency. Invention is credited to Hirotaka Hosoi, Atsushi Ishii, Hiroshi Kida, Kazuhiko MATSUMOTO, Koichi Mukasa, Yoshihiro Sakoda, Makoto Sawamura, Agus Subagyo, Kazuhisa Sueoka, Seiji Takeda.
Application Number | 20110183438 13/081177 |
Document ID | / |
Family ID | 33478987 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110183438 |
Kind Code |
A1 |
MATSUMOTO; Kazuhiko ; et
al. |
July 28, 2011 |
Method for Sensing a Substance to be Detected in a Sample
Abstract
A single-electron transistor comprising at least a substrate, a
source electrode and a drain electrode formed on top of the
substrate opposing to each other, and a channel arranged between
the source electrode is disclosed wherein the channel is composed
of ultra fine fibers. By having such a constitution, a sensor can
have excellent sensitivity.
Inventors: |
MATSUMOTO; Kazuhiko;
(Tsukuba-shi, JP) ; Mukasa; Koichi; (Sapporo-shi,
JP) ; Ishii; Atsushi; (Sapporo-shi, JP) ;
Takeda; Seiji; (Sapporo-shi, JP) ; Sawamura;
Makoto; (Sapporo-shi, JP) ; Subagyo; Agus;
(Sapporo-shi, JP) ; Hosoi; Hirotaka; (Sapporo-shi,
JP) ; Sueoka; Kazuhisa; (Sapporo-shi, JP) ;
Kida; Hiroshi; (Sapporo-shi, JP) ; Sakoda;
Yoshihiro; (Sapporo-shi, JP) |
Assignee: |
Japan Science and Technology
Agency
Kawaguchi-shi
JP
National Institute of Advanced Industrial Science and
Technology
Chiyoda-ku
JP
|
Family ID: |
33478987 |
Appl. No.: |
13/081177 |
Filed: |
April 6, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10558063 |
Aug 15, 2006 |
7935989 |
|
|
PCT/JP2004/007300 |
May 21, 2004 |
|
|
|
13081177 |
|
|
|
|
Current U.S.
Class: |
436/501 |
Current CPC
Class: |
G01N 27/4146 20130101;
H01L 51/0545 20130101; B82Y 10/00 20130101; H01L 51/0048 20130101;
B82Y 30/00 20130101 |
Class at
Publication: |
436/501 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
May 23, 2003 |
JP |
2003-146480 |
Feb 16, 2004 |
JP |
2004-037866 |
Claims
1. A method for sensing a substance to be detected in a sample by
means of a sensor at least provided with a substrate, and a channel
having a source electrode and a drain electrode formed with a
certain interval and opposite one another on a first insulating
membrane formed on top of the substrate, the method comprising the
steps of: composing the channel of ultra fine fibers; forming a
second insulating membrane in a portion of the substrate on an
opposite side to a position where the channel is located; forming a
back gate electrode on an outer side of the second insulating
membrane; modifying the second insulating membrane by
hemagglutinin; intervening the sample between the modified portion
and the back gate electrode; applying a gate voltage to the back
gate electrode; and detecting a substance that interacts with the
hemagglutinin in the sample by change in current value between the
source electrode and the drain electrode.
2. The method for sensing a substance to be detected in a sample
according to claim 1, wherein the hemagglutinin modifies the second
insulating membrane with which
{N-[5-(3'-Maleimidopropylamino)-1-carboxypentyl]iminodiacetic acid}
has been bonded.
3. The method for sensing a substance to be detected in a sample
according to claim 2, wherein the hemagglutinin modifies the
{N-[5-(3'-Maleimidopropylamino)-1-carboxypentyl]iminodiacetic acid}
with which a divalent positive ion has been reacted.
4. A method for sensing a substance to be detected in a sample by
means of a sensor at least provided with a substrate, and a channel
having a source electrode and a drain electrode formed with a
certain interval and opposite one another on a first insulating
membrane formed on top of the substrate, the method comprising the
steps of: composing the channel of ultra fine fibers; forming a
second insulating membrane in a portion of the substrate on an
opposite side to a position where the channel is located; forming a
back gate electrode on an outer side of the second insulating
membrane; modifying the second insulating membrane by calmodulin;
intervening the sample between the modified portion and the back
gate electrode; applying a gate voltage to the back gate electrode;
and detecting a substance that interacts with the calmodulin in the
sample by change in current value between the source electrode and
the drain electrode.
5. The method for sensing a substance to be detected in a sample
according to claim 4, wherein the calmodulin modifies the second
insulating membrane with which
{N-[5-(3'-Maleimidopropylamino)-1-carboxypentyl]iminodiacetic acid}
has been bonded.
6. The method for sensing a substance to be detected in a sample
according to claim 4, wherein the calmodulin modifies the
{N-[5-(3'-Maleimidopropylamino)-1-carboxypentyl]iminodiacetic acid}
with which a divalent positive ion has been reacted.
7. The method for sensing a substance to be detected in a sample
according to claim 1, wherein, subsequent to the sample is dropped
between the modified portion and the back gate electrode, and a
solvent of the sample is then evaporated, thereby, the substance to
be detected is detected.
8. The method for sensing a substance to be detected in a sample
according to claim 1, wherein, subsequent to the sample is dropped
between the modified portion and the back gate electrode, and a
solvent of the sample is then frozen, thereby, the substance to be
detected is detected.
9. The method for sensing a substance to be detected in a sample
according to claim 4, wherein, subsequent to the sample is dropped
between the modified portion and the back gate electrode, and a
solvent of the sample is then evaporated, thereby, the substance to
be detected is detected.
10. The method for sensing a substance to be detected in a sample
according to claim 4, wherein, subsequent to the sample is dropped
between the modified portion and the back gate electrode, and a
solvent of the sample is then frozen, thereby, the substance to be
detected is detected.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of application Ser. No.
10/558,063, filed Aug. 15, 2006, which is a national stage of
PCT/JP2004/007300, filed May 21, 2004, which claims priority under
35 U.S.C. .sctn.119 to Japanese Patent Application Nos. JP
2003-146480, filed May 23, 2003 and JP 2004-037866, filed Feb. 16,
2004.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for sensing a
substance to be detected in a sample by means of a sensor, and
particularly relates to a method for sensing a substance to be
detected in a sample by means of a sensor such as a biosensor
having a structure of a field-effect transistor (hereinafter
abbreviated to FET) or a single-electron transistor (hereinafter
abbreviated to SET).
BACKGROUND ART
[0003] In a biosensor proposed in the background art, a membrane
having a reactive group selectively reacting to a specific molecule
is formed on an electrode so as to measure a change in potential
when the membrane adsorbs the aforementioned specific molecule.
Specifically, the biosensor uses a system in which a membrane
having glucose oxidase is formed on an electrode, and a change in
current value caused by oxidation reaction with glucose is measured
to detect the amount of glucose.
[0004] As for such biosensors, for example, refer to Japanese
Patent Laid-Open No. 260156/1998; Aizawa, Chemical Communications,
p. 945 (1989); Alexander Star, Jean-Christophe P, Gabriel. Keith
Bradley, and George Gruner, Vol. 3, No. 4, 459-463 (2003); etc.
[0005] The biosensors in the background art adopt a method for
directly detecting a current value caused by chemical reaction as
described above. Therefore, the detectability is so low that it is
difficult to detect low-concentration glucose. In such a manner,
the biosensors have a problem that they cannot show their own
feature of high selectivity effectively.
[0006] An object of the present invention is to solve the foregoing
problem in the background art and to provide a sensing method for
sensing a substance to be detected in a sample, having sensitivity
much more excellent than that in the background art.
DISCLOSURE OF THE INVENTION
[0007] In order to attain the foregoing object, a first means of
the present invention is a single-electron transistor including at
least a substrate, a source electrode and a drain electrode formed
on top of the substrate opposing to each other, and a channel
arranged between the source electrode and the drain electrode, the
single-electron transistor being characterized in that the channel
is composed of ultra fine fibers.
[0008] A second means of the present invention is the first means
characterized in that a gate electrode is formed in a site of the
substrate other than the positions where the source electrode and
the drain electrode are placed. For example, the gate electrode is
provided in an opposite surface of the substrate to the surface
where the source electrode and the drain electrode are placed, or
in the same surface as the surface where the source electrode and
the drain electrode are placed but in a position far from the
source electrode and the drain electrode.
[0009] A third means of the present invention is the first means
characterized in that a membrane having a functional group is
provided on a surface of the substrate on the side where the
channel is provided.
[0010] A fourth means of the present invention is the first or
third means characterized in that an air gap is provided between a
top surface of the substrate side and the channel.
[0011] A fifth means of the present invention is the first means
characterized in that the ultra fine fibers are nanotube-like
structures.
[0012] A six means of the present invention is the fifth means
characterized in that the nanotube-like structures are carbon
nanotubes.
[0013] A seventh means of the present invention is the fifth or
sixth means characterized in that a defect is introduced into the
nanotube-like structures.
[0014] An eighth means of the present invention is a field-effect
transistor including at least a substrate, a source electrode and a
drain electrode formed on top of the substrate opposing to each
other, and a channel arranged between the source electrode and the
drain electrode, the field-effect transistor being characterized in
that the channel is composed of ultra fine fibers.
[0015] A ninth means of the present invention is the eighth means
characterized in that a gate electrode is formed in a site of the
substrate other than the positions where the source electrode and
the drain electrode are placed.
[0016] A tenth means of the present invention is the eighth means
characterized in that a membrane made of dielectric and having a
functional group is provided on a surface of the substrate on the
side where the channel is provided.
[0017] An eleventh means of the present invention is the eighth or
tenth means characterized in that an air gap is provided between a
top surface of the substrate side and the channel.
[0018] A twelfth means of the present invention is the eighth means
characterized in that the ultra fine fibers are nanotube-like
structures.
[0019] A thirteenth means of the present invention is the twelfth
means characterized in that the nanotube-like structures are carbon
nanotubes.
[0020] A fourteenth means of the present invention is the twelfth
or thirteenth means characterized in that a defect is introduced
into the nanotube-like structures.
[0021] A fifteenth means of the present invention is a sensor
including at least a substrate, a source electrode and a drain
electrode formed on top of the substrate opposing to each other,
and a channel arranged between the source electrode and the drain
electrode, the sensor being characterized in that the channel is
composed of ultra fine fibers.
[0022] A sixteenth means of the present invention is the fifteenth
means characterized in that a gate electrode is formed in a site of
the substrate other than the positions where the source electrode
and the drain electrode are placed.
[0023] A seventeenth means of the present invention is the
sixteenth means characterized in that at least one electrode of the
source electrode, the drain electrode and the gate electrode is
composed of a titanium layer and a gold layer covering the surface
of the titanium layer.
[0024] An eighteenth means of the present invention is the
fifteenth means characterized in that a membrane having a
functional group is provided on a surface of the substrate on the
side where the channel is provided.
[0025] A nineteenth means of the present invention is the
eighteenth means characterized in that the membrane is made of
silicon oxide.
[0026] A twentieth means of the present invention is the fifteenth
means characterized in that the ultra fine fibers are nanotube-like
structures.
[0027] A twenty-first means of the present invention is the
twentieth means characterized in that the nanotube-like structures
are carbon nanotubes.
[0028] A twenty-second means of the present invention is the
twentieth or twenty-first means characterized in that a defect is
introduced into the nanotube-like structures.
[0029] A twenty-third means of the present invention is the
fifteenth means characterized in that opposite end portions of the
channel are welded with the source electrode and the drain
electrode respectively.
[0030] A twenty-fourth means of the present invention is the
fifteenth means characterized in that a surface of the channel is
modified directly by a specific substance interacting with a
substance to be detected.
[0031] A twenty-fifth means of the present invention is the
fifteenth means characterized in that an insulating membrane is
formed on a surface of the channel, and the insulating membrane is
modified by a specific substance interacting with a substance to be
detected.
[0032] A twenty-sixth means of the present invention is the
sixteenth means characterized in that the gate electrode is
modified by a specific substance interacting with a substance to be
detected.
[0033] A twenty-seventh means of the present invention is any one
of the twenty-fourth through twenty-sixth means characterized in
that the substance to be detected and the specific substance are
biopolymers interacting with each other.
[0034] A twenty-eighth means of the present invention is the
twenty-seventh means characterized in that the substance to be
detected is an antigen or an antibody, and the specific substance
is an antibody or an antigen.
[0035] A twenty-ninth means of the present invention is the
twenty-fourth means characterized in that portions which are not
covered with a coat of the modifying substance are formed in a
surface of the drain electrode and a surface of the gate
electrode.
[0036] A thirtieth means of the present invention is a method for
manufacturing a sensor including at least a substrate, a source
electrode and a drain electrode formed on top of the substrate
opposing to each other, and a channel arranged between the source
electrode and the drain electrode, the method for manufacturing a
sensor being characterized by providing catalysts in lines in the
positions where the source electrode and the drain electrode are
placed, opposing the two catalyst lines, and growing up ultra fine
fibers from the source electrode to the drain electrode due to
catalysis of the catalysts so as to arrange the channel.
[0037] A thirty-first means of the present invention is the
thirtieth means characterized in that the catalysts are composed of
a base layer, an intermediate layer made of a transition metal
layer formed on the base layer, and a top layer made of a
transition metal layer formed on the intermediate layer.
[0038] A thirty-second means of the present invention is the
thirtieth or thirty-first means characterized in that the catalysts
are patterned like dots so as to arrange the catalyst lines.
[0039] A thirty-third means of the present invention is the
thirtieth means characterized in that the ultra fine fibers are
nanotube-like structures.
[0040] A thirty-fourth means of the present invention is the
thirty-third means characterized in that the nanotube-like
structures are carbon nanotubes.
[0041] A thirty-fifth means of the present invention is the
thirty-third or thirty-fourth means characterized in that a defect
is introduced into the nanotube-like structures.
[0042] A thirty-sixth means of the present invention is a method
for sensing a substance to be detected in a sample solution by
means of a sensor including at least a substrate, a source
electrode and a drain electrode formed on top of the substrate
opposing to each other, and a channel arranged between the source
electrode and the drain electrode, the sensing method being
characterized in that the channel is composed of ultra fine fibers,
the sample solution is dropped onto the channel, and a solvent of
the sample solution is then evaporated.
[0043] A thirty-seventh means of the present invention is a method
for sensing a substance to be detected in a sample solution by
means of a sensor including at least a substrate, a source
electrode and a drain electrode formed on top of the substrate
opposing to each other, and a channel arranged between the source
electrode and the drain electrode, the sensing method being
characterized in that the channel is composed of ultra fine fibers,
the sample solution is dropped onto the channel, and a solvent of
the sample solution is then frozen.
[0044] A thirty-eighth means of the present invention is the
thirty-sixth or thirty-seventh means characterized in that the
ultra fine fibers are nanotube-like structures.
[0045] A thirty-ninth means of the present invention is the
thirty-eighth means characterized in that the nanotube-like
structures are carbon nanotubes.
[0046] A fortieth means of the present invention is the
thirty-eighth or thirty-ninth means characterized in that a defect
is introduced into the nanotube-like structures.
[0047] The present invention is configured as described above.
Since ultra fine fibers such as carbon nanotubes are used for the
channel, it is possible to provide a single electron transistor, a
field-effect transistor, a sensor, a method for producing sensor,
and a sensing method, which are supersensitive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is a perspective view of a sensor according to an
embodiment of the present invention.
[0049] FIG. 2 is a schematic configuration view of the sensor.
[0050] FIG. 3 is a schematic view showing a state where the sensor
is applied to detection.
[0051] FIG. 4 is a schematic view showing another state where the
sensor according to the embodiment of the present invention is
applied to detection.
[0052] FIG. 5 is an enlarged schematic view of the sensor between
an insulating substrate and a gate electrode.
[0053] FIG. 6 is a schematic configuration view showing a state
where carbon nanotubes are grown and formed in the embodiment of
the present invention.
[0054] FIG. 7 is a view showing a room temperature Coulomb diamond
characteristics with a carbon-nanotube single-electron
transistor.
[0055] FIG. 8 is a schematic perspective view showing a state where
a carbon nanotube is grown and formed by the background-art
technique.
[0056] FIG. 9 is a schematic perspective view showing a state where
carbon nanotubes are grown and formed by the technique of the
present invention.
[0057] FIG. 10 is a schematic perspective view showing an example
of the layout of catalysts according to the technique of the
present invention.
[0058] FIG. 11 is an enlarged perspective view of the catalyst.
[0059] FIG. 12 are a plan view (a) and a sectional view (b) of a
sensor to which a second technique is not applied.
[0060] FIG. 13 are a plan view (a) and a sectional view (b) of the
sensor showing a state where a solution has been dropped onto the
sensor.
[0061] FIG. 14 are a plan view (a) and a sectional view (b) of the
sensor according to the present invention.
[0062] FIG. 15 are a plan view (a) and a sectional view (b) of the
sensor showing a state where a solution has been dropped onto the
sensor.
[0063] FIG. 16 is a sectional view showing a state where a back
gate electrode is modified in the sensor according to the present
invention.
[0064] FIG. 17 is a sectional view showing a state where carbon
nanotubes are modified directly by a molecule in the sensor
according to the present invention.
[0065] FIG. 18 is a sectional view showing a state where the carbon
nanotubes are modified indirectly by a molecule in the sensor
according to the present invention.
[0066] FIG. 19 is a schematic configuration view showing another
structure of the sensor according to the present invention.
[0067] FIG. 20 is a schematic configuration view showing further
another structure of the sensor according to the present
invention.
[0068] FIG. 21 is an I-V characteristic curve graph when FITC was
detected by the sensor according to the present invention.
[0069] FIG. 22 is an I-V characteristic curve graph when an Ni ion
was detected by the sensor according to the present invention.
[0070] FIG. 23 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction of the
sensor according to the present invention.
[0071] FIG. 24 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction of the
sensor according to the present invention.
[0072] FIG. 25 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction of the
sensor according to the present invention.
[0073] FIG. 26 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction of the
sensor according to the present invention.
[0074] FIG. 27 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction of the
sensor according to the present invention.
[0075] FIG. 28 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction of the
sensor according to the present invention.
[0076] FIG. 29 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction in a
sol-gel method of the sensor according to the present
invention.
[0077] FIG. 30 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction in a
sol-gel method of the sensor according to the present
invention.
[0078] FIG. 31 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction in a
sol-gel method of the sensor according to the present
invention.
[0079] FIG. 32 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction in a
sol-gel method of the sensor according to the present
invention.
[0080] FIG. 33 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction in a
sol-gel method of the sensor according to the present
invention.
[0081] FIG. 34 is an I-V characteristic curve graph when
hemagglutinin was detected by antigen-antibody reaction in a
sol-gel method of the sensor according to the present
invention.
[0082] FIG. 35 is an I-V characteristic curve graph when calmodulin
was detected by antigen-antibody reaction of the sensor according
to the present invention.
[0083] FIG. 36 is an I-V characteristic curve graph when calmodulin
was detected by antigen-antibody reaction of the sensor according
to the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0084] Next, an embodiment of the present invention will be
described with reference to the drawings. FIG. 1 is a perspective
view of an SET type biosensor according to an embodiment of the
present invention. FIG. 2 is a schematic configuration diagram of
the SET type biosensor.
[0085] In these drawings, the reference numeral 1 represents a
chip-like insulating substrate; 2, a membrane applied on the
insulating substrate 1 and having a surface provided with a
functional group such as a hydroxyl group, an amino group, a
carboxylic group, etc. (membrane made of SiO2 with a hydroxyl group
in this embodiment); and 3 and 4, a source electrode and a drain
electrode formed at a predetermined interval on the membrane 2.
Apical portions 5 and 6 are formed in opposed portions of the two
electrodes 3 and 4 (see FIG. 1). Carbon nanotubes (hereinafter
abbreviated to CNT) with a defect introduced therein are grown and
formed between the apical portions 5 and 6 of the two electrodes 3
and 4. Agate electrode 8 is formed in a surface of the substrate 1
on the opposite side to the membrane 2.
[0086] For example, an inorganic compound such as silicon oxide,
silicon nitride, aluminum oxide, titanium oxide, etc. or an organic
compound such as acrylic resin, polyimide, etc. is used for the
insulating substrate 1. For example, metal such as gold, platinum,
titanium, etc. is used for the electrodes 3, 4 and 8. The
electrodes 3, 4 and 8 have an electric connection relationship as
shown in FIG. 8.
[0087] CNTs are used as nanotube-like structures in this
embodiment. Due to use of the nanotube-like structures, a very
minute channel can be formed. Thus, a high sensitive sensor can be
obtained.
[0088] Incidentally, as shown in FIG. 2, an air gap G is formed
under the CNTs 7. A sensor having an SET structure is formed thus.
SET and FET have the same fundamental structure, but are different
from each other in a channel serving a current passageway. That is,
a channel of SET has a quantum dot structure while a channel of FET
does not have a quantum dot structure.
[0089] In this transistor (SET or FET), a current value between the
source electrode 3 and the drain electrode 4 changes sensitively to
a change of charges (more strictly spin electronic states) on the
gate electrode 8 or the CNTs 7. SET is generally more sensitive
than FET. However, SET properties are rare observed directly after
CNTs are produced. When FET-like CNTs are heated to the temperature
(high temperature of about 900.degree. C.) in which the CNTs were
produced, the CNTs are broken partially to form islands and show
the current characteristic of SET. Alternatively, when a current
(up to several mA) larger than an operating current (up to several
.mu.A) is applied, a similar result can be obtained.
[0090] According to the present invention, the spin electronic
states on the CNTs change indirectly or directly when a molecule
adheres to the gate electrode 8 or the CNTs 7 of the transistor.
Thus, the adhering molecule can be detected from a change in
current generated between the source electrode 3 and the drain
electrode 4 in this event. A modifying molecule or a reaction
between the modifying molecule and another molecule can be detected
from a change in current when the gate electrode 8 or the CNTs 7
themselves are modified by the molecule.
[0091] Particularly when the gate electrode 8 or the CNTs 7 are
modified by an antibody (or antigen), a specific antigen (or
antibody) can be detected by use of antibody-antigen reaction.
Accordingly, a microorganism such as a virus or a bacterium of
infection can be detected supersensitively and fast in this
technique. This technique can be effectively applied to early
detection and prevention of infection or researches of
microorganisms. In addition, a device (sensor) itself can be
extremely miniaturized so that the device (sensor) can be brought
out to the field and applied to detection of infectious viruses or
researches of these.
[0092] FIG. 3 is a schematic view showing the state where the
sensor is applied to detection. As shown in FIG. 3, the sensor has
a molecule detection portion 18 and a signal conversion portion 19
closely related to each other. In FIG. 3, the reference numeral 12
represents a protective film made of SiO2; 13, a specific substance
(e.g. antibody) selectively reacting or sticking (interacting) to a
substance to be detected; 14, a to-be-detected substance (e.g.
antigen) selectively reacting or sticking (interacting) to the
specific substance 13; and 15, a sample solution containing the
to-be-detected substance 14.
[0093] FIG. 4 and FIG. 5 are schematic views showing another state
where the sensor according to the present invention is applied to
detection. FIG. 5 is a schematic enlarged view of the sensor
between the insulating substrate 1 and the gate electrode 8. In the
case of this example, the sample solution 15 containing the
to-be-detected substance 14 is put between the insulating substrate
1 and the gate electrode 8 so as to detect the to-be-detected
substance 14. In FIG. 5, the reference numeral 20 represents a
molecule of the specific substance (e.g. antibody) having an
orientation; and 21, a substance other than the to-be-detected
substance present in the sample solution 15. FIG. 5 shows the state
where the specific substance (e.g. antibody) 13 selectively reacts
or sticks to the to-be-detected substance (e.g. antigen) 14.
[0094] Next, description will be made on control of the fundamental
electric transport properties of the CNTs. [0095] (1) The
application of an electric field or a magnetic field, the kind and
shape of a catalyst to be used for growing up the CNTs, and so on,
are optimized to desirably design the growth position, direction,
number, chirality, properties, etc. of the CNTs serving as a base
element of the biosensor device.
[0096] FIG. 6 is a schematic configuration view showing a technique
for patterning the catalyst and controlling the position and
direction of CNTs while applying an electric field thereto. In FIG.
6, the reference numeral 1 represents an insulating substrate; 2, a
membrane made of SiO2 applied onto the insulating substrate 1; 9a
and 9b, catalyst layers patterned on the SiO2 membrane 2 and made
of iron or the like; 7, CNTs formed between the catalyst layers 9a
and 9b by application of the electric field. The growth position,
direction, number, chirality, properties, etc. of the CNTs 7 are
controlled desirably. The reference numeral 10 represents a
reaction chamber; and 11, hydrocarbon gas such as methane gas or
the like, which is a raw material of the CNTs. The grown CNTs are
formed into an ultrafine fibrous aggregate measuring about several
.mu.m (e.g. about 3 .mu.m) in length and about several nm in
diameter. [0097] (2) The CNTs whose position, direction,
properties, etc. have been controlled are used as a noninvasive
electrode to make up a shape of a four-probe method.
[0098] The four-probe method is a method using four needle-like
electrodes (e.g. electrodes A, B, C and D) placed in a straight
line on a specimen. A constant current is applied between the outer
two (e.g. electrodes A and D) of the probes. A potential difference
appearing between the inner two (e.g. electrodes B and C) of the
probes is measured to obtain a resistance value. The obtained
resistance value is multiplied by the thickness of the specimen and
a correction coefficient RCF. Thus, a volume resistance value of
the specimen is calculated. [0099] (3) Each electrode and the
channel (CNTs) are welded in their lapping portion by a locally
impressed current using a high-electric-field electron beam, or STM
(Scanning Tunneling Microscopy)/AFM (Atomic Force Microscope).
Thus, the electrodes and the channel (CNTs) are integrated. [0100]
(4) Next, the transport properties of the CNTs are evaluated.
Electric transport properties to be evaluated include the ballistic
transport properties, the spin injection probability, the spin
transport probability, etc. [0101] (5) By pilot studies of the
present inventors, it has been confirmed that the electric
properties of the CNTs change on a large scale due to a defect
introduced into the CNTs (it has been confirmed by the pilot
studies that Coulomb energy up to 5,000 K is provided due to a
defect introduced into the CNTs, so that SET acting in a room
temperature can be formed).
[0102] Accordingly, when a defect is desirably introduced into the
CNTs by STM/AFM processing or by an electron beam, CNTs having
controllable electric transport properties can be obtained.
[0103] As a specific example of the method for introducing a defect
into the CNTs, there is a method in which the CNTs are, for
example, annealed at almost the same temperature (e.g. about
800.degree. C.) as the CNTs were produced, and then cooled
naturally. The defect of the CNTs means that a part of carbon atoms
fly out due to heat, with the result that the CNTs are changed in
shape or the like so that the CNTs are nearly broken into pieces
narrowly connected to one another. However, it is not obvious as of
now what structure the CNTs have actually. [0104] (6) The
correlation between the defect in the CNTs and the electric
properties of the CNTs is investigated. For example, the density,
distribution and magnitude (size, energy barrier, etc.) of the
defect are evaluated by a scanning probe method (a Kelvin probe
method, a Maxwell probe method, etc.) so as to clarify the
correlation between the defect and the electric properties of the
CNTs. When the correlation between the defect in the CNTs and the
electric properties of the CNTs is grasped thus, it is possible to
manufacture SET having properties excellent in reproducibility and
uniformity. [0105] (7) The electric properties of the carbon
nanotubes can be controlled by controlling the introduction of the
defect in the aforementioned process (6).
[0106] SET acting in a room temperature can be manufactured using
the CNTs having a defect introduced therein according to the
present invention. Here, description has been made on the case
where the CNTs having a defect introduced therein are used.
However, CNTs having no defect introduced therein may be used.
[0107] Floating charges or moving charges have leaded to problems
in SET in the background art. In order to avoid malfunction caused
by such floating charges or moving charges, according to the
present invention, two SETs using CNTs are produced to be close to
each other, and the output characteristics (room temperature) of
the two SETs are ANDed when a single charge is detected. As a
result, the two SETs operate only when there is a true charge.
Thus, malfunction caused by floating charges or moving charges can
be avoided.
[0108] Further, not a background-art DC system but an AC drive
system using a resonance circuit by use of the aforementioned
technique is used to increase the measurement speed. Thus, a single
charge distribution can be measured at a room temperature, at a
high speed and without malfunction.
[0109] FIG. 7 is a view showing room temperature Coulomb diamond
characteristics with SET using CNTs. From the room temperature
Coulomb diamond characteristics, it can be proved that the SET
using CNTs according to the present invention can operate at a room
temperature.
[0110] As shown in FIG. 1, SET using CNTs is formed on the
substrate 1, while the chip is coated with the protective film 12
made of SiO2 in order to be operated in a solution as shown in FIG.
3, and the specific substance 13 such as an antibody is fixed onto
the SiO2 protective film 12. Although the protective film 12 is
provided in this embodiment, there may be a case where the
protective film 12 does not have to be provided.
[0111] The biosensor according to this embodiment is installed in
the sample solution 15 in which the to-be-detected substance 14
such as DNA or the like has been dissolved. The biosensor is
operated by AC using a resonance circuit, so as to measure a single
electron interaction between the specific substance 13 and the
to-be-detected substance 14. Thus, the to-be-detected substance 14
can be detected (surface charge distribution characteristics can be
evaluated).
[0112] Next, description will be made in detail on production of
the signal conversion portion of the sensor using CNTs. An FET or
SET type transistor is produced using the semiconductor properties
of CNT. The production method is constituted by the processes of
depositing catalysts by general lithography, growing CNT by thermal
CVD, and producing electrodes.
[0113] However, this has problems as follows. First, it is not easy
to control the growth of CNT. Some CNT growing methods have been
proposed. When a device in which electrodes in a signal conversion
portion are connected through a single CNT is formed, yield and
structural stability of the CNT bridging catalysts are important.
Therefore, conditioning of the catalysts (mutual positions,
structures, sizes, etc.) and conditioning of the thermal CVD method
(temperature, kind of gas, flow rate, introduction of electric
field or magnetic field, etc.) are important.
[0114] Further, the electrodes are produced after the growth of the
CNT on the catalysts. However, it is likely that there appears such
a phenomenon that the electrodes are separated from the substrate
or the electrodes are cracked. It is also likely that the contact
potential with the CNT affects the characteristic or strength of
the device. In order to obtain a stable current characteristic, it
is necessary to investigate the electrode materials.
[0115] In the embodiment of the present invention, a novel
technique (first technique which will be described later) is used
particularly for elements of the catalysts. When CNT is modified
directly by a molecule, the electrodes may be covered with a
solvent including the electrodes and the molecule so that the
solvent covering the surfaces of the electrodes may affect the
connection between the electrodes and a measuring device such as a
prober. Therefore, a technique (second technique which will be
described later) for preventing this is used.
[0116] Further, even when a back gate electrode or a side gate
electrode is used in the device, a specimen, vapor containing the
specimen, or the like, may affect the gate electrode. This can be
avoided by protecting the CNT (third technique which will be
described later). In fact, of results of detection of reactions
using a back gate electrode or a side gate electrode, there was an
example in which it could be believed that a change in current
value was caused by a to-be-detected substance evaporated and
attached to not only the gate electrode but also the surface of
CNT.
[0117] Specifically, the aforementioned first technique is to
deposit catalysts onto an SiO2 film by use of electron-beam
lithography in order to form nuclei of growth of CNTs. In this
embodiment, the first technique relates to a technique in which
each of the opposite surfaces of an Si substrate 380 .mu.m thick is
covered with an SiO2 film about 300 nm, and catalysts containing
transition metal such as iron, nickel, cobalt, molybdenum,
tungsten, etc. or particles of such a transition metal are
deposited on the SiO2 film as nuclei of growth of CNTs.
[0118] FIG. 8 is a view for explaining a background-art technique.
In FIG. 8, the reference numeral 1 represents an Si insulating
substrate having an SiO2 film formed on each of the opposite
surfaces thereof; 7, a CNT; 9a and 9b, catalysts; and 22a and 22b,
positions where electrodes will be formed later. In the
background-art technique, as shown in FIG. 8, the catalysts 9a and
9b are formed at a predetermined interval one by one by vapor
deposition so that the catalysts 9a and 9b can be connected by the
CNT 7 as soon as the CNT 7 grown from one catalyst 9a reaches the
paired catalyst 9b.
[0119] FIG. 9 is a view for explaining the embodiment (first
technique) of the present invention. As shown in FIG. 9, a
plurality of dot-like catalysts (9a-1, 9a-2, . . . 9a-n) are formed
and arranged in a position 22a where one electrode will be formed,
and a plurality of dot-like catalysts (9b-1, 9b-2, . . . 9b-n) are
also formed in a position 22b where the other electrode will be
formed, so that the catalysts (9b-1, 9b-2, . . . 9b-n) are opposed
to the aforementioned catalysts (9a-1, 9a-2, . . . 9a-n). In such a
manner, the number of the catalysts 9 placed, that is, the number
of nuclei of growth of CNTs is increased so that the catalysts 9
are arrayed thickly. Thus, it is possible to extremely increase the
probability that the CNTs easy to grow up at random from the
catalysts 9 in themselves will reach the paired catalysts 9. Due to
this technique, the yield can be made 10 or more times as high as
that in the background art.
[0120] FIG. 10 is a view showing an example of the layout of the
catalysts 9 according to this embodiment. Six catalysts are
arranged thickly in each array so that an interval L1 between
adjacent catalysts is 2 .mu.m. An interval L2 between one catalyst
array (9a-1, 9a-2, . . . 9a-n) and the other catalyst array (9b-1,
9b-2, . . . 9b-n) is 4 .mu.m. Incidentally, the number of the
catalysts 9 placed, the interval L1 and the interval L2 can be set
desirably.
[0121] FIG. 11 is an enlarged perspective view of the catalyst 9.
As shown in FIG. 11, the catalyst 9 has a three-layer structure of
a base layer 25, an intermediate layer 26 and a top layer 27. The
base layer 25 is made of Si or the like and has a thickness of 50
nm. The intermediate layer 26 is formed on the base layer 25, made
of transition metal such as Mo, Ta, W, etc. and has a thickness of
10 nm. The top layer 27 is formed on the intermediate layer 26,
made of transition metal such as Fe, Ni, Co, etc. and has a
thickness of 3 nm. Accordingly, the total height of the catalyst 9
is 63 nm, and the diameter D thereof is 2 .mu.m. The catalysts 9
each having such a multilayer structure are patterned by a thin
film formation technique such as vapor deposition, sputtering, ion
plating, etc.
[0122] The insulating substrate 1 where the catalysts 9 have been
formed is placed in a reaction chamber 10 of a thermal CVD
apparatus as shown in FIG. 6. After that, hydrocarbon gas 11 such
as methane, ethane or the like is injected to grown the CNTs 7 on
the catalysts 9.
[0123] In this embodiment, the growth of the CNTs 7 is performed in
the following procedure. The insulating substrate 1 where the
catalysts 9 have been formed is heated from the room temperature to
900.degree. C. for 15 minutes. In this event, Ar is injected into
the chamber 10 at a flow rate of 1,000 sccm (gas flow rate per
minute). Methane and hydrogen are injected at flow rates of 1,000
sccm and 500 sccm respectively for 10 minutes while keeping the
temperature. After that, the inside of the reaction chamber 10 is
cooled down to the room temperature for 120 minutes. In this event
again, Ar gas is injected at 1,000 sccm.
[0124] After the CNTs are produced thus, electrodes (source
electrode 3 and drain electrode 4) are deposited. Au is deposited
on the electrodes. Alternatively, Ti is deposited on the
electrodes, and the surfaces thereof are then coated with Au.
Particularly the latter method is characterized in that it can
suppress the separation of the electrodes from the substrate or the
occurrence of cracks in the electrodes. The width of the electrodes
covering the catalysts is about 10 .mu.m.
[0125] Next, description will be made on the aforementioned second
technique. A large number of electrodes (about 50-400 electrodes)
are formed concurrently. For example, when CNT is modified
directly, a solution containing a modifying molecule may be dropped
down onto the CNT. In this event, the solution may cover the whole
of an electrode depending on some amount of the solution. Once the
surface of the electrode is covered with the solution, a coat may
be formed between a probe of a measuring device such as a prober
and the electrode when a current between electrodes connected by
the CNT is measured. Thus, it is likely that a correct current
value cannot be obtained.
[0126] FIG. 12 and FIG. 13 are views for explaining a sensor to
which the second technique is not applied. FIG. 12 are views
showing a state before a solution is dropped. FIG. 13 are views
showing a state after the solution is dropped. In FIGS. 12 and 13,
(a) shows a plan view, and (b) shows a sectional view. The
electrodes 3 and 4 are small in size in the background-art sensor.
Therefore, the electrode 3, 4 is often entirely covered with a coat
28 formed by the solution dropped, as shown in FIG. 13. A value of
a current flowing between the electrodes 3 and 4 is about 1 .mu.A,
which is so minute that the current cannot be measured correctly if
the coat 28 is present between the probe of the measuring apparatus
and the electrode 3, 4.
[0127] Therefore, according to the present invention, as shown in
FIG. 14 and FIG. 15, length L3 of each electrode 3, 4 [see FIG.
14(a)] is made about 1.5-3 times as long as that in FIG. 12. Thus,
since length L3 of the electrode 3, 4 is made longer thus, a
portion 29 (see FIG. 15) which is not covered with the coat 28 can
be formed in an end portion of the electrode 3, 4 even if the coat
28 of the molecule modifying the CNTs 7 is formed. By use of an
optical microscope, the probe of the measuring apparatus such as a
prober is applied to this portion 29 which is not covered with the
coat 28. Thus, the current flowing between the electrodes 3 and 4
can be measured correctly.
[0128] In this embodiment, width w1 of the tip portion, width W2 of
the portion the probe will be applied to, and length L3 are made 10
.mu.m, 150 .mu.m and 500 .mu.m respectively in each electrode 3, 4
in FIG. 14(a). As shown in FIG. 14(b), the CNTs 7 are slightly bent
between the electrodes 3 and 4 so that an air gap G is provided
between the CNTs 7 and the top surface of the substrate 1 side.
Thus, the difference in thermal expansion coefficient from the
substrate 1 is absorbed by the slack of the CNTs 7.
[0129] Next, description will be made on the aforementioned third
technique. It is said that CNT has strength 2,000 times as high as
iron when they have same size. In fact, CNT is hardly damaged when
the CNT is cleansed after the CNT is modified directly by a
molecule. However, CNT easily interacts with various molecules
including water so as to change its spin electronic states. The
change appears as a change in current value. This can be positively
used as a gas sensor. At the same time, when a back gate electrode,
a side gate electrode, or the like, is used in a sensor, CNT may be
a noise source.
[0130] Therefore, according to the present invention, the CNTs and
the electrodes are partially covered with an insulating protective
film so as to reduce the noise. An insulating adhesive agent can be
used for forming the insulating film. A passivation film used
broadly for spin coating can be also used. Particularly the
increase of a current which would appear when water was given to
the back gate electrode is not observed due to the formation of the
insulating protective film. In addition, due to the formation of
this insulating protective film, ultrasonic cleaning can be applied
to the device as a whole, or the back gate electrode and so on can
be cleansed with detergent more powerful than ever.
[0131] The gate electrode of the sensor can be formed in various
positions. The gate electrode can have various structures in
accordance with the application of the sensor or the easiness to
manufacture the sensor. Next, description will be made on each
structure.
(A) Structure of Gate Electrode Modified by Molecule
[0132] When a molecule adheres onto the SiO2 film formed on the
substrate, there appears a change in value of the current flowing
between the source electrode and the drain electrode. For example,
the current value changes when a fluorescent molecule FITC
(Fluorescein isothiocyanate) is given to the gate electrode. As an
example of antibody-antigen reaction, the SiO2 film is
molecule-modified by an antibody (or antigen) so as to react to a
corresponding antigen (or antibody) and detect a change in electric
signal. Molecule modification can be attained in a larger area than
that in CNT. Thus, this molecule modification is suitable for
detection aimed at more molecules. In addition, since CNT is not
modified directly, damage of the CNT caused by cleaning after use
can be avoided.
[0133] FIG. 16 is a view showing this structure. In this structure,
as shown in FIG. 16, the SiO2 film on the insulating substrate 1 on
the opposite side to the CNTs 7 is molecule-modified by a specific
substance (e.g. antibody) 13, while a sample solution 15 containing
a to-be-detected substance (e.g. antigen) is put between the
insulating substrate 1 and the gate electrode 8.
(B) Structure of CNTs Modified Directly by Molecule
[0134] FIG. 17 is a view showing a structure in which the CNTs 7
are modified directly by a molecule. Since the CNTs 7 are modified
directly by a molecule, a change in spin electronic states on the
CNTs 7 caused by the modifying molecule is larger than that in the
case where the back gate electrode 8 is modified by a molecule.
Thus, high sensitivity is provided.
(C) Structure of CNTs Modified Indirectly by Molecule
[0135] FIG. 18 is a view showing a structure of the CNTs 7 modified
indirectly by a molecule. In order to modify the CNTs 7 indirectly
by a molecule, the CNTs 7 are coated with an insulating membrane 30
made of an organic compound such as an adhesive agent or the like
as shown in FIG. 18. A change in spin electronic states in the
membrane 30 caused by the modifying molecule or a molecule adhering
thereto leads to a change in spin electronic states in the CNTs 7.
As a result, there occurs a change in current. This structure has
both the feature as the structure in which the back gate electrode
8 is modified by a molecule and the feature as the structure in
which the CNTs 7 are modified directly by a molecule. Thus, the
structure has high sensitivity and stability.
(D) Structure using Sol-Gel
[0136] In each of the aforementioned structures (A) to (C), sol-gel
containing a to-be-detected substance is used in place of the
solution 15. A change in electric signal can be detected in the
same manner as in the case of the solution.
(E) Structure using Side Gate
[0137] An island is built near the CNTs on the substrate, and this
is used as a gate. This structure is characterized in that the CNTs
7 themselves can be prevented from being damaged by direct
modification of the CNTs 7 without any effort such as modification
of the back surface (back gate electrode) by a molecule. Thus, this
is a structure suitable for SET.
[0138] In the aforementioned structure (A) of the back gate
electrode modified by a molecule, it is preferable that the CNTs
and the electrodes are partially covered with a protective film so
as to stabilize the current characteristic. In the aforementioned
structure (B) of the CNTs modified directly by a molecule and in
the aforementioned structure (C) of the CNTs modified indirectly by
a molecule, it is preferable that a portion 29 which is not covered
with a coat is formed on each electrode 3, 4 as described with
reference to FIG. 15.
[0139] FIG. 19 is a schematic configuration view for explaining
further another structure. In this structure, the substrate 1
itself is used as a channel (back channel), and the electrodes 3
and 4 are provided on the substrate 1 so as to put the CNTs 7
therebetween. A recess portion 16 serving as a channel is formed in
the back surface of the substrate 1. When the recess portion 16 is
wet with a liquid containing a to-be-detected substance, the
to-be-detected substance can be detected by the back surface of the
substrate 1.
[0140] FIG. 20 is a schematic configuration view for explaining
further another structure. Also in this structure, the substrate 1
itself is used as a channel (back channel), and a probe 17 made of
CNTs or the like is provided in the channel of the substrate 1.
This integrated combination of the back channel and the probe 17
can be, for example, used as a probe of a scanning probe microscope
or the like.
[0141] Next, description will be made on specific examples of the
present invention. As a pilot study, a solution containing a
fluorescent molecule FITC was dropped onto an SiO2 film back gate
electrode, and a change in current characteristic was measured.
FIG. 21 shows the I-V characteristic when the gate voltage was set
at -20 V and the concentration of the fluorescent molecule FITC was
set at 0.64 nM. In FIG. 21, the abscissa designates a value (A) of
a current flowing between the source electrode and the drain
electrode, and the ordinate designates a value of a voltage (V)
between the source electrode and the drain electrode. In FIG. 21,
the broken line designates the I-V characteristic curve before the
fluorescent molecule FITC was attached, and the solid line
designates the I-V characteristic curve after the fluorescent
molecule FITC was attached. As is apparent from FIG. 21, there is a
large change between the I-V characteristics before and after the
fluorescent molecule FITC was attached.
Example 1
[0142] Next, description will be made on detection of a divalent
ion using ionic reaction. CNTs of a CNT biosensor was modified
directly by pyrene, and
{N-[5-(3'-Maleimidopropylamino)-1-carboxypentyl]iminodiacetic acid:
hereinafter abbreviated to NTA} was bonded with a back gate
electrode. After that, a solution containing Ni ions was dropped,
and the conduction characteristic was examined based on the I-V
characteristic in each case. FIG. 22 shows the I-V characteristics
when no electric field was applied to the gate electrode. The
abscissa designates a value (A) of a current flowing between the
source electrode and the drain electrode, and the ordinate
designates a value (V) of a voltage between the source electrode
and the drain electrode. In FIG. 22, di designates the I-V
characteristic curve after the back gate electrode was cleaned, nta
designates the I-V characteristic curve after NTA was bonded, and
ni designates the I-V characteristic curve after the solution
containing Ni ions was dropped.
[0143] As is apparent from FIG. 22, when the voltage between the
source electrode and the drain electrode was increased, the current
increased, but the current rarely increased near dv=0 V in all the
systems (systems of di, nta and ni). That is, semiconductor-like
properties were observed.
[0144] The I-V characteristic curve after NTA was bonded with the
back gate electrode showed remarkable reduction in current as
compared with the I-V characteristic curve after the back gate
electrode was cleaned. In contrast, when Ni ions were added to the
system, the current increased. NTA reacts with not only Ni ions but
also divalent plus ions. Accordingly, other divalent plus ions can
be detected likewise.
Example 2
[0145] Next, description will be made on detection of H9
hemagglutinin (HA) using antigen-antibody reaction. C-terminus of
HA was cut in various levels (220, 250, 290 and 320), and
expression was attempted. Genes were introduced into a 293T cell,
and expression of HA protein in the cell was confirmed using a
monoclonal antibody E2/3 and a polyclonal antibody. Secretion of
the HA protein in supernatant was confirmed by a western blotting
method. Plenty of HA1-290 was expressed and refined from the
supernatant by Ni2 and a column. A fraction including the intended
HA protein was confirmed by ELISA and SDS-PAGE, and this fraction
was fractionated and dialyzed by PBS so as to obtain the HA.
Expression could be observed as to shorter HA1-220. However, the HA
creased from reaction with the monoclonal antibody. Therefore, the
HA was not used.
[0146] NTA was bonded with the SiO2 film back gate electrode of the
CNT biosensor. After that, Ni ions were added to the system, and HA
antibodies having concentrations ranged from 10-10 to 10-5 in
dilution ratio with respect to a stock solution were applied. Thus,
I-V characteristic curves were obtained. In this event, HA was
absent from the back gate electrode. Therefore, the HA antibodies
were not bonded to have orientation on the back gate electrode.
[0147] Next, the HA was fixed to NTA on the SiO2 film back gate
electrode by His tag attached in advance. HA antibodies were
applied likewise, and I-V characteristic curves were obtained.
These I-V characteristic curve graphs are shown in FIGS. 23-28.
Incidentally, the gate voltage was set to be -20 V.
[0148] FIG. 23 is an I-V characteristic curve graph when the
solution containing Ni ions was applied after NTA was bonded. FIG.
24 is an I-V characteristic curve graph when the HA antibody having
a dilution ratio of 10-10 with respect to a stock solution was
applied. FIG. 25 is an I-V characteristic curve graph when the HA
antibody having a dilution ratio of 10-8 with respect to a stock
solution was applied. FIG. 26 is an I-V characteristic curve graph
when the HA antibody having a dilution ratio of 10-7 with respect
to a stock solution was applied. FIG. 27 is an I-V characteristic
curve graph when the HA antibody having a dilution ratio of 10-6
with respect to a stock solution was applied. FIG. 28 is an I-V
characteristic curve graph when the HA antibody having a dilution
ratio of 10-5 with respect to a stock solution was applied.
[0149] In each of these graphs, the broken line designates the
aforementioned former in which the HA was absent from the SiO2 film
back gate electrode, and the solid line designates the
aforementioned latter in which the HA was fixed to NTA on the SiO2
film back gate electrode by His tag attached in advance.
[0150] As is apparent from these graphs, little difference in
current value between the source electrode and the drain electrode
was observed between the both (solid line and broken line) when the
voltage between the source electrode and the drain electrode was
varied from 0 V to 1 V. However, when the voltage was increased to
1 V or higher, the characteristic in which the current value
increased suddenly was shown in the system (solid line) where the
HA was regarded as fixed.
[0151] From this fact, it is understood that the HA antibody can be
detected even in an area of high dilution ratio as compared with
the background-art method such as ELISA (Enzyme-Linked
ImmunoSorbent Assay).
Example 3
[0152] As for such detection of H9 hemagglutinin (HA) using
antigen-antibody reaction, similar results could be obtained by use
of a sol-gel method. I-V characteristics obtained thus are shown in
FIGS. 29-34. Incidentally, in all the systems before testing of
antigen-antibody reaction, a solution containing Ni ions was
applied after NTA was bonded. The gate voltage was set to be -20
V.
[0153] FIG. 29 is an I-V characteristic curve graph when the HA
antibody having a dilution ratio of 10-7 was applied without
attaching the HA antigen. FIG. 30 is an I-V characteristic curve
graph when the HA antibody having a dilution ratio of 10-6 was
applied without attaching the HA antigen. FIG. 31 is an I-V
characteristic curve graph when the HA antibody having a dilution
ratio of 10-5 was applied without attaching the HA antigen. FIG. 32
is an I-V characteristic curve graph when the HA antibody having a
dilution ratio of 10-6 was applied after attaching the HA antigen.
FIG. 33 is an I-V characteristic curve graph when the HA antibody
having a dilution ratio of 10-5 was applied after attaching the HA
antigen. FIG. 34 is an I-V characteristic curve graph when the HA
antibody having a dilution ratio of 10-4 was applied after
attaching the HA antigen.
[0154] In each of these graphs, ni designates an I-V characteristic
curve when the solution containing Ni ions was applied after NTA
was bonded, and HA designates an I-V characteristic curve in which
the HA was fixed to NTA on the SiO2 film back gate electrode by His
tag attached in advance.
[0155] As is apparent from these graphs, there appeared a great
change in current value between the source electrode and the drain
electrode particularly when the dilute ratio with respect to the
stock solution was 10-5 and 10-4. The detection sensitivity was
almost as high as that in ELISA.
Example 4
[0156] Next, description will be made on detection of calmodulin
(CaM) using antigen-antibody reaction. A DNA fragment containing
rat calmodulin gene cDNA was inserted into a Sacl-Xbal site of an
expression vector pBAD/gIII (made by Invitrogen Corporation) so as
to assemble a calmodulin expression vector (pBAD/gIII/calmodulin).
The vector was introduced into a Escherichia coli LMG194. Thus, a
calmodulin expression clone was obtained. This clone was planted in
an LB/Ampicilin medium of 2 ml, and cultured for one night.
[0157] 5 ml of this culture solution was inoculated into an
LB/Ampicilin medium, and subjected to shaking culture at 37.degree.
C. till OD600 reached 0.5. After that, L-arabinose was added so
that the final concentration was 0.02%. Shaking culture was further
performed at 37.degree. C. for 4 hours. The cultured cells were
collected by centrifugal collection, suspended by Native Binding
Buffer (made by Invitrogen Corporation), crushed ultrasonically,
partially refined by use of Probond.TM. Purification System (made
by Invitrogen Corporation), and then refined uniformly like
SDS/polyacrylamide electrophoresis by use of Hi Load 26/60 Superdex
75 pg (made by Amersham Bioscience Corp.). Thus, calmodulin was
obtained.
[0158] NTA was bonded to the SiO2 film back gate electrode of the
CNT biosensor. After that, the HA was fixed to NTA on the SiO2 film
back gate electrode by His tag attached in advance. HA antibodies
having dilution ratios ranging from 10-8 to 10-2 with respect to a
stock solution were applied, and I-V characteristic curves were
obtained. The results are shown in FIG. 35. Incidentally, the gate
voltage was set to be -20 V.
[0159] In FIG. 35, a curve (i) designates an I-V characteristic
curve when cleaning was performed after NTA was bonded, a curve
(ii) designates an I-V characteristic curve when CaM was bonded to
NTA by His tag attached in advance, a curve (iii) designates an I-V
characteristic curve when the CaM antibody having a dilution ratio
of 10-8 with respect to a stock solution was applied, a curve (iv)
designates an I-V characteristic curve when the CaM antibody having
a dilution ratio of 10-7 with respect to a stock solution was
applied, a curve (v) designates an I-V characteristic curve when
the CaM antibody having a dilution ratio of 10-6 with respect to a
stock solution was applied, a curve (vi) designates an I-V
characteristic curve when the CaM antibody having a dilution ratio
of 10-4 with respect to a stock solution was applied, and a curve
(vii) designates an I-V characteristic curve when the CaM antibody
having a dilution ratio of 10-2 with respect to a stock solution
was applied.
[0160] As is apparent from FIG. 35, the current value changed in
accordance with each concentration when the voltage between the
source electrode and the drain electrode was varied from 0 V to 0.5
V. From this fact, it is understood that the CMA antibody can be
detected even in an area of very high dilution ratio with respect
to the stock solution in the same manner as the HA antibody.
[0161] The results of detection of CaM antibodies and HA antibodies
using ELISA are shown in the following table. Incidentally, in this
measuring procedure, a primary antibody was diluted at the
following dilution ratio and made to stand still for one hour. A
secondary antibody (antimouse HRPO standard antibody) was diluted
5,000 times and made to stand still for one hour again. A substrate
having an absorption wavelength of 450 nm was produced by a TMB
color former, and the absorbance was measured.
TABLE-US-00001 TABLE (CaM antibody) (HA antibody) PBS Neg. Con
0.034 0.030 2.5 .times. 10-2 2.000 1.722 6.3 .times. 10-3 2.439
2.725 1.6 .times. 10-3 2.899 3.378 3.9 .times. 10-4 2.300 3.132
0.98 .times. 10-4 0.650 2.839 2.4 .times. 10-5 0.177 1.413 6.1
.times. 10-6 0.051 0.290
[0162] It is proved that detection becomes difficult in the
dilution ratio of 6.1.times.10-6 by ELISA. On the other hand, in
the aforementioned Examples 3 and 4, the sol-gel method shows the
sensitivity as high as ELISA, while detection is successful in the
dilution ratio of about 10-8 in the other methods.
[0163] CNTs were grown on an Si substrate, and electrodes were
formed on the opposite end portions thereof. The back surface of
the aforementioned Si substrate on the opposite side to the surface
where the CNTs were grown was activated by acid (sulfuric acid).
After that, NTA was fixed by reaction with a silanizing reagent
(3-mercaptopropyltrimethoxysilane) at 180.degree. C. Next, Ni ions
were added to fix an antigen (CaM, HA) histidine was introduced
into. The fixed antigen was made to react with a diluted antibody.
After that, the substrate was cleaned, and negative bias was
applied to the back surface of the substrate. Thus, an I-V
characteristic was measured.
[0164] FIG. 36 is a characteristic graph showing a change in
current value when the CaM fixed as described above was made to
react with the diluted CaM antibody and a voltage of 1.5 V was then
applied between the CNT electrodes. As is apparent from FIG. 36,
little change in current value was observed when the antigen was
not fixed. However, the current value increased with the increase
of the antibody concentration when the antigen was fixed. In
addition, it was proved that the antibody could be detected in a
range of dilution from about 10-10 to 10-8 with respect to the
antibody stock solution.
[0165] When a detection limit of the same antibody by use of FLISA
was examined, it was proved that there was a detection limit in the
dilution of about 10-6 with respect to the antibody stock solution.
It was also proved that there was a difference in detection limit
between CaM and HA, and the detection limit depended on the antigen
and the antibody.
[0166] Although the aforementioned Examples were described in the
case where the gate voltage was -20 V, it has been proved that
detection is successful in spite of a small change in current value
even when the gate voltage is about 0 V or positive.
[0167] When the CNT biosensor is applied to a solution, there may
occur a problem in reliability of data due to observation of noise.
Therefore, after a sample solution (solution to be tested) is
dropped onto the sensor, the solvent (moisture) may be evaporated
by a blower, a heater, a thermoelectric conversion device (Peltier
device), or the like. Thus, the noise can be reduced extremely.
This countermeasure against the noise was applied to the
aforementioned specific Examples to which a solution was applied.
When the sample solution (solution to be tested) is cooled by a
thermoelectric conversion device (Peltier device), liquid nitrogen
or the like, the influence of the solvent such as water can be
reduced. Particularly when water is frozen and insulated, the noise
can be reduced on a large scale.
[0168] ELISA and Western blotting belong to the background-art
methods. These methods have a limit of sensitivity in the dilution
ratio of about 10-5 with respect to the stock solution. On the
other hand, the sensitivity of the sensor according to the present
invention is about 103 of that by ELISA as to detection of an HA
antibody.
[0169] In addition, due to use of an electric signal, there are not
many chemical reaction processes. Accordingly, the time required
for detection is extremely short. According to examination of the
current characteristic based on an I-V curve, the I-V curve can be
acquired in several seconds by a parameter analyzer.
[0170] PCR or the like known in the background art is accompanied
with a change in temperature. It is therefore necessary to control
the temperature. However, the sensor according to the present
invention can be used in an environment where the temperature is
constant. Thus, temperature control is not required, but the
configuration can be simplified and miniaturized. For example, an
RT-PCR method, an ICAN method, a LAMP method, etc. can be used in
an environment where the temperature is constant. However, any
method has a problem that it takes long time for detection.
[0171] The sensor according to the present invention is applicable
not only to detection of a single kind but also to concurrent
sensing on a large number of kinds in one sample to thereby detect
a plurality of kinds concurrently. Further, detection can be
performed on a plurality of samples in parallel by use of a
plurality of sensors.
[0172] The sensor using nanotube-like structures as a channel
according to the present invention has strength enough to be used
repeatedly. However, the sensor is so inexpensive that the sensor
may be made disposable for detection of a dangerous virus or the
like.
[0173] Although description was made in the aforementioned
embodiment about the case where CNTs were used, ultra fine fibers
which do not have tube-like shapes can be used.
[0174] Although description was made in the aforementioned
embodiment about one kind of biosensor having a DNA probe formed
therein, for example, three CNT biosensors having SiO2 films
respectively may be provided together on a substrate, while a DNA
probe, a protein probe and a glycolipid probe are formed on the
SiO2 films individually. Thus, different biopolymers (DNA, protein
and glycolipid) can be measured concurrently.
[0175] Although description was made in the aforementioned
embodiment about the case where the surface charge distribution
characteristic in DNA was evaluated, the present invention is also
applicable to detection of other biopolymers such as sugar chain,
RNA, amino acid, sugar, virus, etc. Further, the present invention
is also applicable to detection of a change in electron state in
the process where protein such as rhodopsin releases protons in
response to light, a change in electron state in the structural
change of pigment, and so on.
[0176] Although an example in which nanotube-like structures were
connected to the channel portion of SET was shown in the
aforementioned embodiment, nanotube-like structures may be used in
the channel portion of FET.
INDUSTRIAL APPLICABILITY
[0177] When a microorganism such as a virus enters a human body or
another living thing, an antibody against the microorganism begins
to interact therewith. Accordingly, any virus an antibody
correspondingly thereto is present can be detected from body fluid
by the sensor according to the present invention. For example, HA
shown in the aforementioned specific Examples is a protein called a
spike protein covering the surface of an influenza virus. It is
therefore possible to detect the HA by the sensor according to the
present invention. Thus, infectious diseases such as influenza,
SARS, BSE, etc. can be detected sensitively and fast.
[0178] The detection portion of the sensor according to the present
invention is small, and an electric signal is used. Accordingly,
the detection circuit can be formed into a chip. Thus, the sensor
can be used as a portable and inexpensive detector. Accordingly,
the sensor can perform testing in the field, and can be provided
for any medical institution. Thus, the sensor serves as defense
measures of early detection, and also serves as countermeasures
against bio-terrorism.
[0179] Also in the field of basic science, the sensor according to
the present invention realizes detection of bonding strength of
intermolecular interaction on the level of one molecule, or
classification of viruses or proteins based on current
characteristics. Accordingly, a molecule similar to an antibody can
be searched, or designed to make a fundamental experiment for drug
discovery. In addition, one molecule can be detected with time.
Further, the sensor can be used as a basic circuit of a
spectroscopic antigen-antibody reaction detection unit.
[0180] When the gate electrode or the CNTs of the sensor according
to the present invention are modified directly by DNA,
complementary DNA can be electrically detected supersensitively. In
addition, microorganisms such as infectious viruses, bacteria, etc.
can be identified through supersensitive and fast measurement of
DNA.
[0181] Further, environmental hormones, toxins and inorganic
substances can be detected by the sensor according to the present
invention. In addition, since the influence of vapor of a sample
can be detected, the sensor can be applied to not only fluid but
also gas. Accordingly, the sensor can measure concentration of a
specific substance such as a harmful substance in the atmosphere or
other gases.
* * * * *