U.S. patent application number 09/979438 was filed with the patent office on 2003-01-16 for nucleotide detector, process for producing the same and process for morming forming fine particle membrane.
Invention is credited to Yamashita, Ichiro.
Application Number | 20030013096 09/979438 |
Document ID | / |
Family ID | 26587665 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030013096 |
Kind Code |
A1 |
Yamashita, Ichiro |
January 16, 2003 |
Nucleotide detector, process for producing the same and process for
morming forming fine particle membrane
Abstract
A nucleotide detector 10 includes: metal particles 12 having a
size of the order of nanometers (diameter: about 6 nm) placed on a
surface of a substrate 11 at high density with high precision (with
spaces of about 12 nm between adjacent particles); and
single-stranded DNAs (thiol DNAs) 13 having sulfur atoms at ends
bonded to the gold particles 12. The thiol DNAs 13 are placed
uniformly over the entire substrate 11 at high density with high
precision. Therefore, once a fluorescence-labeled single-stranded
DNA is hybridized with any of the thiol DNAs 13, high fluorescence
intensity is stably obtained. This detector is therefore usable as
a high-performance DNA sensor with a high SN ratio.
Inventors: |
Yamashita, Ichiro; (Nara,
JP) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O. BOX 828
BLOOMFIELD HILLS
MI
48303
US
|
Family ID: |
26587665 |
Appl. No.: |
09/979438 |
Filed: |
November 16, 2001 |
PCT Filed: |
March 15, 2001 |
PCT NO: |
PCT/JP01/02083 |
Current U.S.
Class: |
435/6.19 ;
435/287.2 |
Current CPC
Class: |
B01J 2219/00648
20130101; C12Q 1/6837 20130101; B01J 2219/00722 20130101; C12Q
1/6825 20130101; C12Q 1/6837 20130101; C40B 40/06 20130101; C12Q
2565/507 20130101; C12Q 2565/507 20130101; C12Q 1/6825
20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 16, 2000 |
JP |
2000-73805 |
Mar 27, 2000 |
JP |
2000-86116 |
Claims
1. (Deleted)
2. A method for manufacturing a nucleotide detector comprising the
steps of: (a) arranging complex particles each including a metal
particle and a protein molecule holding the metal particle on a
substrate; (b) removing the protein molecules; and (c) bonding one
of a pair of nucleotide molecules capable of conjugating with each
other to each of the metal particles left on the substrate in the
step (b).
3. The method for manufacturing a nucleotide detector of claim 2,
wherein the protein molecules are Dps protein or apoferritin.
4. The method for manufacturing a nucleotide detector of claim 2 or
3, wherein the nucleotide molecules comprise a plurality of types
of nucleotide molecules having different base sequences.
5. (Amended) A method for producing a particulate film comprising
the steps of: (a) placing a substrate in a container so that a
surface of the substrate is vertical to the liquid level of a
liquid containing particulates filled in the container; and (b)
forming a wet film made of the particulates dispersed
two-dimensionally on the surface of the substrate by raising or
lowering the liquid level of the liquid.
6. The method for producing a particulate film of claim 5, wherein
the particulates have a diameter of 50 nm or less.
7. The method for producing a particulate film of claim 5 or 6,
wherein the particulates are protein.
8. The method for producing a particulate film of claim 7, wherein
the protein contains an inorganic material inside.
9. The method for producing a particulate film of claim 7 or 8,
wherein the concentration of the protein in the liquid is 10
.mu.g/ml to 500 mg/ml.
10. The method for producing a particulate film of any of claims 7
to 9, wherein the liquid contains an electrolyte.
11. The method for producing a particulate film of any of claims 7
or 10, wherein a liquid level raising or lowering rate of the
liquid is substantially constant, and it is 10 mm/min. or less.
12. The method for producing a particulate film of any of claims 7
to 11, wherein the liquid is allowed to flow out by gravity.
13. The method for producing a particulate film of any of claims 7
to 12, wherein the substrate has a convex and concave pattern on a
surface.
Description
TECHNICAL FIELD
[0001] The present invention relates to a technology for using a
film made of particulates placed with high precision, and more
particularly, to a technology used suitably for manufacture of a
nucleotide detector and the like.
BACKGROUND ART
[0002] First prior art
[0003] Presently, thanks to the international human genome project
and efforts of researchers involved in the project, it is definite
that the genome DNA sequence of the human species will be
completely clarified in coming several years. However, the genome
DNA sequence (base sequence (gene arrangement)) concerned is a
genome DMA sequence of a specific person, and not that of an
individual. The genome DNA sequence of an individual is slightly
different from that of the specific person, where substitution,
deletion, insertion, and the like of a base may have occurred in a
gene. Normally, such a slight difference is not critical and does
not cause any trouble in the life of the individual.
[0004] However, it has also been clarified that the difference in
genome DNA sequence as described above determines the
predisposition of an individual. For example, this difference
causes predispositions of individuals such as those who are
tolerant to alcohol, those who do not mind the heat, and those who
have a low body temperature.
[0005] In particular, it is recognized that reaction of the body
against a drug differs among individuals, and for this reason, the
difference in genome DNA sequence as described above is considered
as significantly important information from the standpoint of
medical treatment. Therefore, it is strongly desired that the
difference in DNA sequence among individuals as described above be
detected after the coming determination of the DNA sequences of all
the human genes by the human genome project. If genetic information
on an individual is made available, it is possible to provide
medical treatment optimal for the individual.
[0006] To detect the slight difference in genome DNA as described
above, a conventional DNA , base sequence determination method by
use of electrophorasis may be employed. However, this method
requires an exceedingly long time and therefore is not practical as
a method for detecting genetic information on many subjects.
[0007] In addition, it has been discovered that for predispositions
prone to genetic diseases and cancers, for example, only a slight
difference in base sequence (difference of one base pair, for
example) has a critical indication. For example, it has been
discovered that sickle cell anemia, which is a lethal genetic
disease, is caused by mutation of only one base pair. From this
point, also, it is clear that the conventional determination method
is not practical.
[0008] The basics in detection of the DNA sequence of a gene of an
individual are that the DNA sequence of a target gene has been
determined and that how the gene of the individual is different
from the so-called human gene DNA sequence is sought, as in the
instance of sickle cell anemia described above.
[0009] As a method capable of detecting the above difference in a
short time, a technique using a DNA chip has been proposed, and the
effectiveness thereof has been presented.
[0010] For example, first, 1000 types of single-stranded DNAs
slightly different from the human gene DNA sequence (base sequence)
are synthesized in advance, and placed on a substrate. One type of
DNA is placed on one section of the substrate, and the position is
recorded.
[0011] Next, DNA of the subject is taken, and the double helix
structure of the DNA is released into single-stranded DNAs. The DNA
is then cut into pieces of an appropriate length, and the DNA
pieces are fluorescence-labeled.
[0012] Subsequently, the fluorescence-labeled DNAs are allowed to
hybridize (conjugate) with the single-stranded DNAs placed in
advance on the substrate.
[0013] After excess DNA and fluorescent dye are washed away, any
position/section of the substrate that emits fluorescence is
detected. The DNA placed in advance in the section that emits
fluorescence is determined to be the DNA sequence that has
hybridized with the DNA of the subject. In other words, by
detecting the position emitting fluorescence, it is clarified in a
short time how the DNA sequence of the subject has mutated from the
human gene DNA sequence.
[0014] In the technique described above, it is comparatively easy
to increase the number of types of single-stranded DNAs placed in
advance on the substrate to more than 1000. However, in this case,
to attain precise testing, 1000 types or more of single-stranded
DNAs must be placed on extremely fine sections of a chip allocated
for the respective types of DNAs at high and uniform density so
that each section has a uniform amount of DNAs. In particular, in
the case that the area of the section allocated for each type of
single-stranded DNA becomes finer with increase of the number of
types of single-stranded DNAs to be placed, it will become
necessary to realize the requirement described above by
manipulating a trace amount of single-stranded DNAs.
[0015] Second prior art
[0016] Particulates have a large ratio of the surface area to the
volume, and therefore exhibit behaviors generally different from
materials that are small in this ratio. For example, particulates
of an inorganic material such as titanium oxide and zinc oxide have
ultraviolet removal function, antimicrobial function, catalytic
function, and the like. Among particulates of an inorganic
material, those having a diameter of the order of nanometers
(ultra-fine particles) are expected to provide a quantum
effect.
[0017] Such particulates having the above functions have received
attention for their use in the industrial field. In particular, as
for ultra-fine particles having a diameter of the order of
nanometers, it is urgently required to develop a technique for
manufacturing devices using the quantum effect in the industrial
scale.
[0018] Particulates of protein having a diameter of about 10 to 20
nm have received attention for their use for biosensors and the
like. In particular, among a variety of protein particles, there
exist particles capable of containing an inorganic material inside.
Such protein particles are provided with natures of both the
inorganic material and the protein particles.
[0019] The particulates described above are normally available in
the form of a colloid solution. However, the form of a colloid
solution is disadvantageous when the functions of the particulates
are to be effectively used. Therefore, search has been made for a
technique that permits effective use of the functions of the
particulates in the industrial field using the colloid solution as
a raw material.
[0020] At present, as such a technique permitting effective use in
the industrial field, placing the particulates on a substrate is
considered most effective. Therefore, desired is establishment of a
technique in which an idealistic two-dimensional film made of
particulates placed regularly at high density can be easily formed
on a substrate.
[0021] Various techniques have been proposed so far for placing
particulates on a substrate. Some of such techniques handling
comparatively large particles have even been commercialized.
[0022] For example, Nagayama et al. have disclosed the following
method in "Formation of Holoferritin Hexagonal Arrays in Secondary
Films Due To Alder-Type Transition", Lanbgmuir 1996, vol. 12, pp.
1836-1839. That is, as shown in FIG. 18, a substrate 11 is put in a
solution 18 containing particulates 15 (polystyrene spheres having
a diameter of about 1 to 2 .mu.m) dispersed therein, and then
gradually lifted in the position vertical to the liquid level,
forming a wet film 19 on both surfaces of the substrate 11. In this
way, a film made of polystyrene spheres having a diameter of about
1 to 2 .mu.m is formed on the surfaces of the substrate 11.
[0023] However, when it is intended to apply the above method to
ultra-fine particles having a diameter of about 10 nm, the
substrate 11 must be lifted at a very low rate. It is difficult to
keep the lifting rate constant when the rate is low. In addition,
the array of ultra-fine particles of the film may possibly lose
uniformity due to vibration and the like that may be generated
during lifting of the substrate 11. For these reasons, it is
difficult to apply the above method to ultra-fine particles. To
solve this problem, Nagayama et al. disclose a method for forming a
two-dimensional crystal film made of protein (ferritin, diameter:
about 12 nm). This method will be described with reference to FIG.
16.
[0024] FIG. 16 is a view illustrating the method for forming a
two-dimensional crystal film made of ferritin. Referring to FIG.
16, first, a platinum blade 21 is placed in the position vertical
to the surface of a substrate 11 that is mounted on a base 20. A
liquid 16 containing ferritin dispersed therein is then dropped
into a small space between the substrate 11 and the blade 21, so
that the liquid 16 is held in and around the space (hatched portion
in FIG. 16) due to the surface tension of the liquid 16.
Thereafter, while the blade 21 is kept fixed, the base 20 (that is,
the substrate 11) is moved in the direction shown by the arrow at a
constant speed (2 .mu.m/sec. in this case). This results in the
liquid 16 being applied to the substrate 11. The water content of
the liquid 16 is evaporated gradually as the liquid 16 is
sequentially applied to the substrate 11, allowing formation of a
thin film 22 made of ferritin. The thin film 22 has a thickness of
about 10 layers of ferritin particles.
[0025] Problems to be solved
[0026] If the above requirement described in relation with the
first prior art fails to be realized, the DNA chip causes
various-problems. To state specifically, if the density of DNAs
placed in a certain section is too low, the intensity of
fluorescence emitted from hybridized DNAs decreases, deteriorating
the signal to noise (SN) ratio. In other words, the fluorescence
from hybridized DNAs may possibly be buried in background
fluorescence inevitably generated.
[0027] Moreover, if the absolute amount of DNAs placed varies with
the sections, a plurality of sections may emit fluorescence at
different intensities when the DNA of the subject hybridizes in two
or more sections. In this event, it is unknown why the fluorescence
intensity is low in one section compared with that in another
section. Specifically, it is difficult to determine whether the
fluorescence intensity is low because the absolute amount of DNA
placed is small or because the absolute amount DNA placed is so
large that emission of fluorescence is allowed despite of weak
non-specific adsorption. This may results in mistake of the
determination. Furthermore, the variation in the absolute amount of
DNAs placed among chips indicates that the reproducibility of the
chip quality is poor. This may results in generation of defective
DNA chips.
[0028] To overcome the above problems, it is necessary to place
types of DNAs on the substrate at high density (about 10.sup.12
pcs./cm.sup.2) by a uniform amount for each type.
[0029] As for the second prior art, in order to realize
uniform-quality particulate films with high reproducibility by use
of the technique disclosed by Nagayama et al., it is necessary to
ensure the movement of the base 20 (that is, the substrate 11)
while maintaining a constant ultra-low speed of 2 .mu.m/sec.
However, in this movement maintaining a constant ultra-low speed,
the speed tends to be greatly influenced by a subtle variation in
the environment. For example, the moving speed changes with a
slight vibration in the environment. A fluctuation in an atmosphere
(such as wind and operator's respiration) changes the amount of
evaporation of the ferritin solution. By these changes, the
reproducibility of the quality of the particulate films
deteriorates. It is therefore necessary to provide a means for
ensuring the movement of the base 20 while maintaining a constant
ultra-low speed and a means for keeping the surrounding atmosphere
constant. However, it is not easy to actually provide these means.
Therefore, the method disclosed by Nagayama et al. finds difficulty
in providing particulate films with a uniform quality, and thus is
not suitable for applications to formation of a particulate film
over a large-area substrate and to mass production of particulate
films.
[0030] In addition, using a large-size blade 21 made of platinum
costs high. However, if the blade 21 is made of a material other
than platinum, it may possibly be corroded. Moreover, it is very
difficult to produce a large-size rigid blade having
nanometer-order surface precision.
[0031] Other methods have also been proposed, including a method in
which a substrate surface is treated in various ways and a
particulate film prepared in advance is transferred to the
substrate surface (Japanese Laid-Open Patent Publication No.
8-155379), a method in which amphiphilic molecules such as casein
molecules are used as a binder and a particulate thin film is
automatically formed on the binder (Japanese Laid-Open Patent
Publication No. 8-229474), and a lithographic method using a
particulate film as a substrate (Japanese Laid-Open Patent
Publication No. 8-234450). However, all of these methods are not
suitable for mass production.
DISCLOSURE OF THE INVENTION
[0032] An object of the present invention is to provide a
nucleotide detector capable of detecting target nucleotide (DNA,
RNA, or the like) with high precision.
[0033] Another object of the present invention is to provide a
method for easily producing a two-dimensional crystal film made of
particulates of protein or the like having a diameter of the order
of nanometers arranged at high density and at desired positions
with high precision.
[0034] The nucleotide detector of the present invention includes: a
substrate; metal particles placed regularly on the substrate; and
one of a pair of nucleotide molecules capable of conjugating with
each other, the one nucleotide molecule being bonded to each of the
metal particles.
[0035] Either one of a pair of nucleotide molecules capable of
conjugating with each other is bonded to each of the metal
particles placed regularly on the substrate. Therefore, the
conjugation between the one nucleotide molecule and the other
nucleotide molecule capable of conjugating with the former can be
established in uniform over the substrate. Thus, when the other
nucleotide molecule is made detectable with a fluorescent label or
the like, for example, a stable detection signal can be
obtained.
[0036] The method for manufacturing a nucleotide detector of the
present invention includes the steps of: (a) arranging complex
particles each including a metal particle and a protein molecule
holding the metal particle on a substrate; (b) removing the protein
molecules; and (c) bonding one of a pair of nucleotide molecules
capable of conjugating with each other to each of the metal
particles left on the substrate in the step (b).
[0037] By placing the complex particles on the substrate and
removing the protein molecules, only metal particles regularly
placed are left on the substrate. To each of these metal particles,
bonded is one of a pair of nucleotide molecules capable of
conjugating with each other. In this way, attained is a nucleotide
detector in which nucleotide molecules constituting one of a pair
of nucleotide molecules capable of conjugating with each other are
regularly placed on the substrate.
[0038] The protein molecules may be Dps protein or apoferritin.
[0039] The nucleotide molecules may be a plurality of types of
nucleotide molecules having different base sequences.
[0040] The method for producing a particulate film of the present
invention includes the steps of: (a) placing a substrate in a
container so that a surface of the substrate is vertical to the
liquid level of a liquid containing particulates filled in the
container; and (b) raising or lowering the liquid level of the
liquid.
[0041] According to the method for producing a particulate film of
the present invention, by gradually raising or lowering the level
of the liquid, the liquid level slightly rises along the substrate
at the interface between the liquid and the substrate, forming a
meniscus portion. Since the meniscus portion has a large surface
area, a dispersion medium of the liquid evaporates, resulting in
reduction of the amount of the dispersion medium in the meniscus
portion. This causes an effect similar to the micro-capillary
effect in the meniscus portion, where the liquid flows toward this
portion. As a result, the particles exist in the meniscus portion
of the liquid in a significantly high concentration, and thus are
arranged on the surface of the substrate at high density with high
precision. In other words, a film of particulates arranged at high
density with high precision is formed on the surface of the
substrate.
[0042] The method for producing a particulate film of the present
invention is particularly preferable when the particulates have a
diameter of 50 nm or less.
[0043] The particulates may be protein.
[0044] The protein may contain an inorganic material inside.
[0045] The concentration of the protein in the liquid is preferably
10 .mu.g/ml to 500 mg/ml.
[0046] The liquid may contain an electrolyte.
[0047] Preferably, a liquid level raising or lowering rate of the
liquid is substantially constant, and it is 10 mm/min. or less.
[0048] The liquid may be allowed to flow out by gravity.
[0049] The substrate may have a convex and concave pattern on a
surface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 is a diagrammatic illustration of a nucleotide
detector of the present invention.
[0051] FIG. 2 diagrammatically illustrates a method for
manufacturing the nucleotide detector of the present invention.
[0052] FIG. 3 is a diagrammatic illustration of a structure of a
complex particle.
[0053] FIG. 4 illustrates a method for arranging and immobilizing
complex particles two-dimensionally on a substrate.
[0054] FIG. 5 illustrates another method for arranging and
immobilizing complex particles two-dimensionally on a
substrate.
[0055] FIG. 6 illustrates yet another method for arranging and
immobilizing complex particles two-dimensionally on a
substrate.
[0056] FIG. 7 illustrates a method for fabricating a nucleotide
detector capable of detecting various types of DNAs.
[0057] FIG. 8 illustrates another method for fabricating a
nucleotide detector capable of detecting various types of DNAs.
[0058] FIG. 9 is an enlarged view of a circled portion C in FIG.
6.
[0059] FIG. 10 is a photomicrograph of a particulate film of
protein ferritin formed on a substrate surface.
[0060] FIG. 11 illustrates examples of the shape and structure of
an open end of a tube used in the present invention.
[0061] FIG. 12 illustrates examples of holes formed through the
bottom of a container used in the present invention.
[0062] FIG. 13 is a view of a substrate having protrusions on a
surface used in the present invention.
[0063] FIG. 14 illustrates a function of the protrusions formed on
the substrate.
[0064] FIG. 15 is an illustration of a conventional method for
placing particles on a substrate.
[0065] FIG. 16 is an illustration of another conventional method
for placing particles on a substrate.
BEST MODE FOR CARRYING OUT THE INVENTION
[0066] Hereinafter, embodiments of the present invention will be
described with reference to the relevant drawings. Note that
nucleotides such as DNAs and RNAs as used herein are
single-stranded unless otherwise specified.
[0067] Embodiment 1
[0068] First, the construction of a nucleotide detector of this
embodiment will be described.
[0069] As shown in FIG. 1, a nucleotide detector 10 of this
embodiment is a DNA sensor, which includes a substrate 11, gold
particles 12 having a size of the order of nanometers (diameter of
about 6 nm) placed on a surface of the substrate 11 at high density
with high precision (with spaces of about 12 nm between adjacent
particles), and single-stranded DNAs (thiol DNAs) 13 each having a
sulfur atom at an end. The thiol DNAs 13 are bonded to the gold
particles 12.
[0070] A method for manufacturing the nucleotide detector 10 of
this embodiment will be described with reference to the relevant
drawings. For fabrication of the nucleotide detector 10 of this
embodiment, it is necessary to place the gold particles 12 having a
diameter of about 6 nm on a surface of the substrate 11 at high
density with high precision. In other words, it is necessary to
arrange and immobilize the gold particles 12 two-dimensionally on a
surface of the substrate 11.
[0071] First, in a step shown in FIG. 2(a), complex particles 15
each composed of a protein molecule 14 holding the gold particle 12
are prepared and placed on the surface of the substrate 11, to
thereby form a complex film in which the complex particles 15 are
placed on the surface of the substrate 11 at high density with high
precision.
[0072] In a step shown in FIG. 2(b), the protein molecules 14 are
removed from the complex particles 15, to leave only the gold
particles 12 behind.
[0073] In a step shown in FIG. 2(c), the thiol DNAs 13 are bonded
to the gold particles 12.
[0074] The step shown in FIG. 2(a) will be described in more
detail.
[0075] As shown in FIG. 3, the complex particle 15 used in this
embodiment is a gold-protein complex where the protein molecules 14
surround the gold particle 12 to hold the gold particle 12 inside.
As the protein molecules 14 of the complex particle 14, used is
apoferritin derived from ferritin extracted from organs such as
spleens and livers of horses, cows, and other animals. The protein
molecules 14 are not limited to this, but other proteins capable of
holding metal particles, such as Dps protein, can also be used
suitably.
[0076] Apoferritin used in this embodiment is a protein of 24
subunits of molecular weight of about 20,000 having an outer
diameter of the entire 24 subunits of about 12 nm, which generally
exists as ferritin in an organ. Ferritin is a complex between the
apoferritin and about 3000 molecules of ferric oxide
(Fe.sub.2O.sub.3).
[0077] Apoferritin has a nature of holding metal particles, and
therefore can be made to hold the gold particles 12 by use of a
solution of KAuCl.sub.4 or HAuCl.sub.4 (concentration: about 1 to 5
mM), for example. Hereinafter, a method for making apoferritin hold
the gold particles 12 using this solution will be described.
[0078] AuCl.sub.4.sup.- is present in the KAuCl.sub.4 or
HAuCl.sub.4 solution. By reducing AuCl.sub.4.sup.-, gold particles
are formed. Using this nature, the gold particles 12 can be
produced by putting a protein that generally has reducing nature in
the HAuCl.sub.4 solution.
[0079] However, in the case of using apoferritin as the protein,
AuCl .sub.4.sup.- fails to enter apoferritin the inside of which is
negatively charged. To overcome this problem, amino acid residues
located inside the apoferritin are genetically changed by
substitution so that the inside of apoferritin is positively
charged. By this operation, AuCl.sub.4.sup.- is allowed to enter
the apoferritin and is reduced inside to produce the gold particle
12. Gold particles are also produced outside the apoferritin. Such
gold particles produced outside can be separated by
centrifugation.
[0080] As a result of the above operation, apoferritin particles
containing the gold particles 12 inside are attained.
[0081] Hereinafter, discussed is a method for placing the complex
particles 15 on a surface of the substrate 11 at high density with
high precision, in other words, a method for arranging and
immobilizing the complex particles 15 two-dimensionally on a
surface of the substrate 11. In this embodiment, either of methods
1 to 5 described below may be employed. Note that all of methods 1
to 5 use apoferritin particles containing the gold particles 12
inside as the complex particles 15.
[0082] Method 1
[0083] As method 1, a method disclosed in Japanese Laid-Open Patent
Publication No. 11-45990 will be described with reference to FIG.
4.
[0084] First, referring to FIG. 4(a), prepared is a liquid 16 with
the complex particles 15 dispersed therein (in this embodiment, a
mixture of a phosphoric acid buffer solution, pH 5.3, having a
concentration of 40 mM and a sodium chloride aqueous solution
having a concentration of 40 mM in equal proportions, with
apoferritin particles containing the gold particles 12 inside
dispersed therein).
[0085] Referring to FIG. 4(b), poly-1-benzil-1-histidine (PBLH) is
gently injected to float on the surface of the liquid 16 with a
syringe or the like, to thereby form a polypeptide film 17 made of
PBLH on the surface of the liquid 16. The pH of the liquid 16 is
then adjusted.
[0086] Referring to FIG. 4(c), an increasing amount of the complex
particles 15 come to attach to the polypeptide film 17 with the
lapse of time, to finally form two-dimensional crystal of the
complex particles 15. This is because while the polypeptide film 17
is positively charged, the complex particles 15 are negatively
charged.
[0087] Referring to FIG. 4(d), the substrate 11 is mounted
(floated) on the polypeptide film 17, to allow the polypeptide film
17 to attach to the substrate 11.
[0088] Referring to FIG. 4(e), the substrate 11 is taken out, to
thereby obtain the substrate 11 with two-dimensional crystal of the
complex particles 15 attaching thereto via the polypeptide film
17.
[0089] Method 2
[0090] Method 2 is the same as the technique by Nagayama et al.
disclosed in "Formation of Holoferritin Hexagonal Arrays in
Secondary Films Due To Alder-Type Transition", Lanbgmuir 1996, vol.
12, pp. 1836-1839, described above in the second prior art (see
FIG. 15).
[0091] First, the complex particles 15 are dispersed in a solution
18 (pure water, pure water with an electrolytic substance such as
sodium chloride added thereto, or the like). Thereafter, as shown
in FIG. 15, the substrate 11 is put in the solution 18. The
substrate 11 is then gradually lifted while the surface of the
substrate 11 is held vertical to the liquid level. This forms a wet
film 19 containing the complex particles 15 dispersed
two-dimensionally on both surfaces of the substrate 11. Once the
wet film 19 is dried, obtained is the substrate 11 with
two-dimensional crystal of the complex particles 15 attaching to
both surfaces thereof.
[0092] Method 3
[0093] Method 3 is the same as the other technique by Nagayama et
al. described above in the second prior art (see FIG. 16).
[0094] As shown in FIG. 16, the platinum blade 21 is placed
vertical to the surface of the substrate 11 that is mounted on the
base 20. The liquid 16 containing the complex particles 15
dispersed therein is then dropped in a small space between the
substrate 11 and the blade 21, so that the liquid 16 is held in and
around the space due to the surface tension of the liquid 16.
Thereafter, while the blade 21 is kept fixed, the base 20 (that is,
the substrate 11) is gradually moved in the direction indicated by
the arrow at a constant rate (2 .mu.m/sec. in this case). This
results in formation of a thin film 22 of the liquid 16 on the
substrate 11. The thin film 22 includes the complex particles 15
dispersed two-dimensionally. Once the thin film 22 is dried,
obtained is the substrate 11 with two-dimensional crystal of the
complex particles 15 attaching to one surface thereof. This
two-dimensional crystal film of the complex particles 15 has a
thickness of about 10 layers of the complex particles 15.
[0095] Method 4
[0096] As method 4, a method based on a transfer method developed
by Yoshimoto et al. (Adv. Biophys., vol. 34, pp. 99-107 (1997))
will be described with reference to FIG. 5.
[0097] In a step shown in FIG. 5(a), a liquid 24 containing the
complex particles 14 dispersed therein (a suspension containing
ferric oxide-containing apoferritin) are injected into a sucrose
solution 23 having a concentration of 2% with a syringe 25.
[0098] In a step shown in FIG. 5(b), drops of the liquid 24 emerge
on the sucrose solution 23.
[0099] In a step shown in FIG. 5(c), drops of the liquid 24
arriving first at the gas-liquid interface form an amorphous film
26 of apoferritin, and drops of the liquid 24 arriving late attach
to the bottom surface of the amorphous film 26.
[0100] In a step shown in FIG. 5(d), two-dimensional crystal 27
made of the complex particles 15 is formed under the amorphous film
26. Thereafter, the substrate 11 (a silicon wafer, a carbon grid, a
glass substrate, and the like) is mounted on a film 28 composed of
the amorphous film 26 and the two-dimensional crystal 27 of the
complex particles 15. The film 28 made of the complex particles 15
is thus transferred to the surface of the substrate 11.
[0101] The surface of the substrate 11 may be subjected to
hydrophobic treatment before the transfer, to facilitate the
transfer of the film 28 to the surface of the substrate 11. As the
hydrophobic treatment of the substrate 11, usable is treatment of
the surface with hexamethyldisilazane (HMDS,
(CH.sub.3).sub.3SiNHSi(CH.sub.3).sub.3) or the like when the
substrate 11 is a silicon substrate, and coating of the surface
with a fluorocarbon monomolecular film when the substrate 11 is a
glass substrate, for example.
[0102] Method 5
[0103] Method 5 will be described with reference to FIG. 6.
[0104] First, referring to FIG. 6, the substrate 11 is put in a
container 29 containing the liquid 16 used in method 1 so that the
surface of the substrate 11 is substantially vertical to the level
of the liquid 16. The liquid 16 is then gradually drawn out at a
constant rate from the container 29 via a tube 30 or the like.
Alternatively, as will be described later, a hole may be formed in
the lower portion of the container 29 to gradually draw out the
liquid 16 at a constant rate.
[0105] By the above drawing, a wet film is formed on both surfaces
of the substrate 11. This wet film includes the complex particles
15 dispersed two-dimensionally, as the wet film shown in FIG. 2.
Therefore, once the wet film is dried, obtained is the substrate 11
with two-dimensional crystal of the complex particles 15 attaching
thereto.
[0106] Method 5 will be described later in more detail following
description of Embodiment 3.
[0107] Next, the step shown in FIG. 2(b) will be described in more
detail as follows.
[0108] Protein molecules are normally susceptible to heat and thus
can be removed by applying heat. The protein molecules 14 of the
complex particles 15 are therefore removed by heat treatment. For
example, the protein molecules 14, and the polypeptide film 17 if
method 1 was adopted, are consumed by being left to stand in an
atmosphere of inert gas such as nitrogen at 400 to 500.degree. C.
for about one hour. As a result, the gold particles 12 are left
behind on the substrate 11 in the shape of dots arranged regularly
at high density with high precision.
[0109] Thus, the gold particles 12 that had been held inside the
complex particles 15 are uncovered in the state of a
two-dimensional array on the substrate 11 arranged at high density
with high precision.
[0110] Next, the step shown in FIG. 2(c) will be described in more
detail.
[0111] The nucleotide detector 10 of this embodiment is constructed
of thiol DNAs 13 bonded to the gold particles 12 that are placed on
the substrate 11 in the manner described above.
[0112] The gold particles 12 and the thiol DNAs 13 can be bonded
together by putting the substrate 11 with the gold particles 12
placed thereon in contact with an aqueous solution of the thiol
DNAs 13 and leaving to stand for a predetermined period of time.
The reason is that since sulfur easily reacts with gold, the sulfur
at the end of the thiol DNA or RNA easily conjugates with the gold
particle 12.
[0113] Specifically, when the thiol DNA 13 in the aqueous solution
comes into contact with the gold particle 12 on the substrate 11,
the sulfur atom S of the thiol DNA 13 and the gold particle 12
establish one-to-one conjugation as shown in FIG. 2(c). As a
result, the thiol DNAs 13 are placed on the substrate 11 at
significantly high density with significantly high precision. Since
the gold particles 12 are arranged two-dimensionally on the
substrate 11 at high density with high precision, the resultant
nucleotide detector 10 has the thiol DNAs 13 bonded to the gold
particles 12 arranged two-dimensionally on the substrate 11 at high
density with high precision, and also has particles placed with a
uniform number of particles per unit area determined depending on
the size of the particles.
[0114] Note that in this step, nucleotides such as thiol RNAs and
PCR primers with thiol ends may be used in place of the thiol DNAs
13.
[0115] In the above step, the concentration of the thiol DNAs 13 in
the aqueous solution may be determined, theoretically, so that the
number of the thiol DNAs 13 matches with the number of the gold
particles 12 on the substrate 11. Actually, however, it is
preferable to set the number of the thiol DNAs 13 greater than the
number of the gold particles 12. Therefore, in this embodiment, a
high-concentration aqueous solution of thiol DNAs is prepared to
ensure that the solution contains the thiol DNAs 13 greater in
number than the complex particles 15 contained in the
complex-dispersed liquid 16.
[0116] As the temperature of the aqueous solution of the thiol DNAs
13 is higher, the bonding of the sulfur atoms S of the thiol DNAs
13 to the gold particles 12 is more facilitated. However, if the
temperature is excessively high, handling of the aqueous solution
of the thiol DNAs 13 becomes difficult due to intensified
convection and the like. Excessively high temperature is also
disadvantageous from the standpoint of energy consumption. In
normal, therefore, the aqueous solution of the thiol DNAs 13 is
preferably warmed to about 20 to 60.degree. C.
[0117] Thus, the nucleotide detector 10 of this embodiment capable
of easily detecting DNA or RNA of which detection is desired is
attained.
[0118] Next, a DNA detecting method using the nucleotide detector
10 as the DNA sensor will be described.
[0119] First, a solution containing a group of DNAs for detection
(subject DNA group) is prepared. The DNAs in the subject DNA group
are labeled with fluorescence in advance.
[0120] The solution of the fluorescence-labeled subject DNA group
is put in contact with the nucleotide detector 10 with the thiol
DNAs placed thereon and left to stand in this state.
[0121] After the lapse of a certain period of time, if there exists
a DNA in the subject DNA group that hybridizes with the thiol DNA
of the nucleotide detector 10, the thiol DNA of the nucleotide
detector 10 and the DNA in question in the subject DNA group
constitute a double helix, establishing stable bonding.
[0122] The resultant nucleotide detector 10 is washed with a
solution such as water containing no fluorescent substance, to
remove the remaining DNAs in the subject DNA group that have not
bonded to the thiol DNAs of the nucleotide detector, together with
a slight amount of the fluorescent substance left behind on the
nucleotide detector 10.
[0123] Thereafter, the surface of the nucleotide detector 10 is
irradiated with light such as laser light to observe fluorescence.
During this observation, fluorescence is emitted if the subject DNA
group includes a DNA having a sequence that hybridizes with the
thiol DNA of the nucleotide detector 10.
[0124] Thus, as described above, whether or not a DNA having a
predetermined sequence exists in the subject DNA group can be
detected by examining whether or not fluorescence is emitted.
[0125] In particular, the nucleotide detector 10 of this embodiment
has thiol DNAs placed in uniform over the substrate at high density
with high precision. Therefore, this nucleotide detector can be
used as a high-performance DNA sensor that provides fluorescence at
high intensity in uniform with high precision and is significantly
high in SN ratio. Therefore, by using the nucleotide detector 10 of
this embodiment as the DNA sensor, it is possible to determine that
a DNA having a predetermined sequence exists in the subject DNA
group if the detected fluorescence intensity is higher than a
predetermined value. In other words, it is possible to
substantially eliminate the possibility of erroneous determination
on whether or not a DNA having a predetermined sequence exists.
[0126] Moreover, the nucleotide detector 10 of this embodiment has
thiol DNAs placed in uniform over the substrate at high density
with high precision. Therefore, the fluorescence intensity
exhibited after hybridization of a DNA having a predetermined
sequence hardly differs among substrates. This eliminates the
necessity of changing the setting of the threshold value of the
fluorescence intensity for each substrate for determination on
whether or not a hybridized DNA exists, and thus produces a
remarkable effect of widely reducing the trouble for adjusting the
fluorescence detector.
[0127] In this embodiment, the nucleotide detector 10 was used as
the DNA sensor. Alternatively, the nucleotide detector 10 may be
used as a RNA sensor by using an RNA group as the subject for
detection, in place of the DNA group.
[0128] In this embodiment, the gold particles 12 and the thiol DNAs
13 were used. Alternatively, a combination of particles of other
metal and DNAs treated to permit bonding to the metal particles may
be used, in place of the combination of the gold particles 12 and
the thiol DNAs 13.
[0129] Conventionally, nucleotide detectors such as DNA chips are
not reusable. In the nucleotide detector 10 of this embodiment,
DNAs (or RNAs) are fixed to the substrate via sulfur atoms and the
gold particles so firmly that this fixation can be maintained even
at a temperature of 100.degree. C. Thus, the nucleotide detector of
this embodiment can be reused by dissociating the hybridized DNA
from the thiol DNA and washing it away.
[0130] Embodiment 2
[0131] In embodiment 1, detection of one type of DNA was described.
In reality, there is an occasion that many types of DNAs are
detected simultaneously. In this embodiment, therefore, detection
of many types of DNAs simultaneously will be described.
[0132] In detection of many types of DNAs, a nucleotide detector
capable of detecting many types of DNAs is manufactured by a
technique as shown in FIG. 7, for example.
[0133] First, referring to FIG. 7(a), after the two-dimensional
placement of the gold particles 12 on the substrate 11 as in
Embodiment 1, a resin resist film 31 that can be denatured with
light and removed with a developer is formed on the substrate.
Thereafter, a mask 33 having an opening 32 is formed on the resin
resist film 31, and the resultant substrate is irradiated with
light a incident from above the mask 33. This denatures a portion
31 of the resin resist film 31 located in the opening 32.
[0134] Referring to FIG. 7(b), the resultant substrate is treated
with a developer to remove the denatured portion 31 of the resin
resist film 31 and thus expose the corresponding gold particles 12
on the substrate 11.
[0135] The resultant substrate is then treated with the aqueous
solution of the thiol DNAs in a manner as described with reference
to FIGS. 2(b) and 2(c). As a result, the thiol DNAs 13 are bonded
to only the gold particles in the exposed portion.
[0136] The type of the thiol DNAs 13 (in base sequence or the like)
to be bonded to the exposed gold particles 12 is changed one after
another, and the above operation is repeated for each type, to
finally produce a nucleotide detector including DNAs bonded to the
substrate in which the sequences of the DNAs are different among
sections of the substrate.
[0137] By use of the nucleotide detector of this embodiment, it is
possible to detect a plurality of different DNAs simultaneously.
Detection using the nucleotide detector of this embodiment is
performed in the following manner.
[0138] First, as in Embodiment 1, a solution of a subject DNA group
labeled with fluorescence in advance is put in contact with the
nucleotide detector of this embodiment and left to stand in this
state.
[0139] If there exists a DNA in the subject DNA group that
hybridizes with the thiol DNA of the nucleotide detector of this
embodiment, the thiol DNA of the nucleotide detector and the DNA in
question in the subject DNA group constitute a double helix,
providing stable bonding.
[0140] The resultant nucleotide detector is washed with water or
the like, and irradiated with light to observe fluorescence. During
this observation, fluorescence is emitted if a DNA in the subject
DNA group has bonded to the thiol DNA of the nucleotide detector of
this embodiment. By specifying the position from which fluorescence
is emitted, the sequence of the hybridized DNA can be detected.
[0141] The nucleotide detector of this embodiment also has thiol
DNAs placed in uniform over the entire substrate at high density
with high precision. Therefore, this nucleotide detector can be
used as a high-performance DNA sensor that provides fluorescence at
high intensity in uniform with high precision and is high in SN
ratio. In particular, in the nucleotide detector of this
embodiment, the thiol DNAs having different sequences among
sections are fixed to the substrate by a uniform amount for each
section. Therefore, the nucleotide detector of this embodiment can
overcome the problem of the conventional DNA sensor that it is
difficult to determine whether or not a DNA having a predetermined
sequence exists in a section with low fluorescence intensity.
[0142] Moreover, in the nucleotide detector of this embodiment, the
fluorescence intensity hardly differs among substrates. This
eliminates the necessity of changing the setting of the threshold
value of the fluorescence intensity for each substrate for
determination on whether or not a hybridized DNA exists, and thus
produces a remarkable effect of widely reducing the trouble for
adjusting the fluorescence detector.
[0143] Embodiment 3
[0144] In this embodiment, another case of detecting many types of
DNAs simultaneously will be described.
[0145] 1) First, as shown in FIG. 8(a), electrodes 34 are placed in
advance on the substrate 11, and the gold particles 12 are placed
on the electrodes 34 as in Embodiment 1.
[0146] 2) As shown in FIG. 8(b), a positive potential is applied to
an electrode 34" in a region in which DNAs having a specific
sequence are to be placed, and a negative potential is applied to
the other electrodes 34".
[0147] 3) The resultant substrate is put in contact with a solution
of the thiol DNAs 13. The thiol DNAs 13, which are negatively
charged intensively, are kept away from the electrodes 34" to which
a negative potential has been applied, and thus concentrate on the
electrode 34', to which a positive potential has been applied. As a
result, the thiol DNAs 13 establish one-to-one bonding to the gold
particles 12 on the electrode 34'.
[0148] The operations 1) to 3) above are repeated changing the type
of the thiol DNAs 13 (in base sequence or the like) one after
another and also changing the electrode 34' to which the positive
potential is applied. In this way, a nucleotide detector including
many thiol DNAs having different sequences bonded to one substrate
11 (multi-type DNA sensor) can be manufactured.
[0149] The nucleotide detector of this embodiment can be used as a
high-performance multi-type DNA sensor as in Embodiment 2.
[0150] Detailed description of method 5
[0151] Method 5 in Embodiment 1 will be described in more detail
with reference to FIGS. 6 and 9. FIG. 9 is an enlarged view of the
circled portion C in FIG. 6.
[0152] First, the container 29 containing the liquid 16 is
prepared. As described in Embodiment 1, the liquid 16 is a mixture
of a phosphoric acid buffer solution, pH 5.3, having a
concentration of 40 mM and a sodium chloride aqueous solution
having a concentration of 40 mm in equal proportions, with the
complex particles 15 dispersed therein.
[0153] The substrate 11 is then prepared, and, as shown in FIG. 6,
put in the container 29 so that the surface of the substrate 11
stands vertical to the level of the liquid 16.
[0154] The level of the liquid 16 is then lowered or raised. In
Embodiment 1 above, the liquid level was lowered. This process of
lowering the level of the liquid 16 will be described in
detail.
[0155] As the level of the liquid 16 is gradually lowered, the
liquid level slightly rises along the substrate 11 at the interface
between the liquid 16 containing the complex particles 15 and the
substrate 11 as shown in FIG. 9, forming a meniscus portion M. In
the meniscus portion M that has a large surface area, a dispersion
medium (water in this embodiment) of the liquid 16 evaporates,
resulting in reduction of the amount of the dispersion medium. This
causes an effect similar to the micro-capillary effect in the
meniscus portion M, where the liquid 16 flows toward this portion.
As a result, the complex particles 15 exist in a significantly high
concentration in the meniscus portion M of the liquid 16 and thus
are arranged on the surface of the substrate 11 at high density
with high precision. That is, a wet film 35 including the complex
particles 15 arranged at high density with high precision is formed
on the surface of the substrate 11. The meniscus portion M is also
formed when the level of the liquid 16 is raised, and a wet film 35
including the complex particles 15 arranged at high density with
high precision is formed on the surface of the substrate 11.
[0156] In the above process, as the time given for the arrangement
of the complex particles 15 is longer, the complex particles 15 can
be arranged at higher density with higher precision. It is
therefore desirable to control the humidity at and around the
liquid level so that the evaporation rate of the dispersion medium
is low. For example, the process in this embodiment may be
performed in a closed system, and an air conditioner or the like
may be provided to control the humidity inside the closed system so
that the dispersion medium can be gradually evaporated.
[0157] During the gradual lowering or raising of the level of the
liquid 16, any vibration should desirably be eliminated to prevent
the liquid level from being influenced by the vibration. For
example, as a measure against vibration, the method in this
embodiment may be performed on a vibration isolation base.
[0158] By the process described above, the wet film 35 is formed on
both surfaces of the substrate 11. Although the wet film 35
includes the complex particles 15 arranged two-dimensionally at
high density with high precision, it is not completely free from
the dispersion medium. The wet film 35 is therefore completely
dried to obtain the substrate 11 with two-dimensional crystal of
the complex particles 15 formed thereon. FIG. 10 is a
photomicrograph of a two-dimensional crystal film of the complex
particles 15 formed on the surfaces of the substrate 11 of this
embodiment. In this method, the complex particles 15 are
crystallized while being aligned in parallel with the liquid level.
When the resultant two-dimensional crystal film is assumed to be in
the (0001) face of a dense hexagonal lattice, the arrangement
orientation is such that the direction vertical to the start line
of the growth of the two-dimensional crystal film, that is, the
growth direction is in the <1-100> direction. This indicates
that the complex particles 15 can be arranged regularly at high
density with high precision.
[0159] In the two-dimensional crystal film obtained by this method,
since the arrangement orientation of the complex particles 15 is in
order, the number of the complex particles 15 placed on the surface
of the substrate 11 can be easily calculated. In other words, the
number of the complex particles 15 placed on the surface of the
substrate 11 can be easily controlled by changing the area of the
substrate 11.
[0160] The level of the liquid 16 can be gradually lowered by
forming a hole through the bottom of the container 29 to allow the
liquid to drip through this hole, for example. The level of the
liquid 16 may be raised by gradually increasing the liquid 16 in
the container 29 utilizing siphonage, for example. In either case,
the liquid level lowering or raising rate is preferably kept
constant. Also, in general, the liquid level lowering or raising
rate is preferably lower to ensure formation of a good film of the
complex particles on the substrate 11. In particular, the liquid
level lowering or raising rate for the liquid 16 is preferably 10
mm/min. or less. No specific lower limit is defined, but to achieve
good economy industrially, it is appropriate to set the liquid
level lowering or raising rate at about 0.1 mm/min. Thus, the
liquid level lowering or raising rate is preferably about 0.1 to
about 1 mm/min., more preferably about 0.12 to about 0.24
mm/min.
[0161] To lower the liquid level in this method, the liquid 16 may
be drawn out by sucking the liquid 16 from above the container 29
via a tube having one open end positioned inside the container 29
near the bottom thereof and the other open end coupled to a suction
means located outside the container 29, or by allowing the liquid
to drop by gravity via a hole formed through the bottom of the
container 29.
[0162] An arbitrary means may be taken to draw out the liquid 16.
For example, a tube 30 may be used as shown in FIG. 6. One open end
37 of the tube 30 is positioned inside the container 29 near the
bottom thereof and the other open end is coupled to a suction means
(a syringe, an aspirator, a suction pump, or the like) located
outside the container 29. By operating the suction means, the
liquid 16 in the container 29 is sucked at the open end 37 of the
tube positioned near the bottom of the container 29 upward through
the tube to be drawn out from the container 29.
[0163] The open end 37 of the tube 30 positioned near the bottom of
the container 29 is not specifically restricted in shape and
structure. The open end may be a circular shape or an obliquely cut
shape. Alternatively, a rectangular parallelepiped structure of
which the bottom is open may be attached to the open end (see FIG.
11(a)), or the open end may have a funnel-shaped structure widened
downwardly of which the bottom is open (see FIG. 11(b)).
[0164] When the open end 37 of the tube 30 has the rectangular
parallelepiped structure or the funnel-shaped structure as shown in
FIG. 11(a) or 11(b), the tube 30 is preferably positioned so that
the bottom of the rectangular parallelepiped structure or the
funnel-shaped structure is in parallel with the bottom of the
container 29.
[0165] When the liquid is drawn out by suction as described above,
any disturbance of flow of the liquid 16 occurring near the
substrate 11 may influence the formation of the film of the complex
particles 15 (growth of the two-dimensional crystal film) on the
surface of the substrate 11. Therefore, the substrate 11 is
preferably placed at a position sufficiently apart from the open
end of the tube positioned near the bottom of the container 29 (the
opening of the rectangular parallelepiped structure or the
funnel-shaped structure if such a structure is provided at the end
of the tube), that is, at a position free from influence of
disturbance of the flow of the liquid 16 caused by suction of the
liquid 16.
[0166] In this method, the liquid 16 may be drawn out by dropping
the liquid via a hole formed through the bottom of the container 29
by gravity, in place of sucking from above the container 29.
Specifically, using a container 29 provided with a hole that
enables the liquid 16 to flow (drop) therethrough by gravity at a
flowing (dropping) rate equal to the liquid level lowering rate
described above, the liquid 16 is drawn out from the bottom of the
container 29.
[0167] When the liquid is drawn out by gravity as described above,
only one hole 38 may be formed through the bottom of the container
29, or a plurality of holes 38 may be formed, as shown in FIG. 12.
The hole 38 may have a circular shape, or a triangular or other
polygonal shape (hereinafter, collectively called a polygonal
shape). It may also be a slit. Such a slit-shaped hole 38 may be
long or short. When a plurality of holes 38 in a circular,
polygonal, or short-slit shape are formed through the bottom of the
container 29, the plurality of holes 38 are preferably arranged in
a line as shown in FIGS. 12(a) and 12(b), or they may be arranged
in parallel lines as shown in FIGS. 12(c) and 12(d). In the case of
forming a plurality of long slit-shaped holes 38 through the bottom
of the container 29, they may be arranged in parallel with each
other as shown in FIG. 12(f).
[0168] The plurality of holes 38 formed through the bottom of the
container 29 are preferably lined in parallel with the surface of
the substrate 11. In other words, the substrate 11 is preferably
placed so that the surface of the substrate 11 is positioned in
parallel with the plurality of holes 38 formed through the bottom
of the container 29.
[0169] In the case described above where the liquid is drawn out by
gravity, also, any disturbance of flow of the liquid 16 occurring
near the substrate 11 may influence the formation of the film of
the complex particles 15 (growth of the two-dimensional crystal
film) on the surface of the substrate 11. Therefore, the substrate
11 is preferably placed at a position sufficiently apart from the
portion where the holes are formed, that is, at a position free
from influence of disturbance of the flow of the liquid 16 caused
by the drawing of the liquid 16.
[0170] In this method, as shown in FIG. 13, the substrate 11 may
have protrusions 39 on the surface thereof. As shown in FIGS. 14(a)
to 14(c), by forming the protrusions 39 on the surface of the
substrate 11, a larger amount of the liquid 16 is left behind on a
portion 40 surrounding each protrusion 39 by the surface tension of
the liquid 16. Therefore, a comparatively large number of complex
particles 15 tend to gather on the surrounding portion 40. As a
result, a multilayer film of complex particles 15 can be
selectively formed on the portion 40 surrounding the protrusion 39.
In short, by forming the protrusions 39 on the surface of the
substrate 11, it is possible to form a film of complex particles 15
arranged at higher density with higher precision.
[0171] For example, by using a substrate having a number of
protrusions 39 formed on the surface in a matrix pattern, it is
possible to form a film made of complex particles 15 arranged in
layers at high density with high precision.
[0172] In addition, when the protrusions 39 are electrodes, the
complex particles 15 are ferritin particles containing particles of
an inorganic material such as iron inside, and the substrate 11 is
a semiconductor substrate, for example, a ferritin film is formed
on the semiconductor substrate. By removing the protein moiety of
the ferritin by heat treatment or the like, only the particles of
an inorganic material such as gold and iron are left behind on the
semiconductor substrate. By using the thus-produced semiconductor
substrate with a film made of the inorganic particles formed
thereon, super-fine electronic devices such as transistors and
diodes can be produced.
[0173] The formation of the protrusions 39 is not restricted to
that shown in FIG. 13. Only one protrusion may be formed on the
surface of the substrate 11, or a plurality of protrusions may be
formed at random. Alternatively, a plurality of protrusions 39 may
be placed regularly in a pattern other than the matrix pattern
shown in FIG. 13.
[0174] In method 5 in Embodiment 1, the liquid 16 (a mixture of a
phosphoric acid buffer solution, pH 5.3, having a concentration of
40 mM and a sodium chloride aqueous solution having a concentration
of 40 mM in equal proportions, with the complex particles 15
dispersed therein) was used. This method is not restricted to this.
For example, this method is greatly usable for production of a
particulate film using particulates other than the complex
particles 15..
[0175] For example, particles of inorganic materials and organic
materials having a diameter of 50 nm or less are usable as the
particulates in this method. Examples of the organic materials
include synthetic polymers and proteins. Examples of the proteins
include (1) virus proteins forming capsids or envelopes of viruses
(for example, adenovirus, rotavirus, poliovirus, HK97, CCMV, and
the like), (2) proteins belonging to the ferritin family such as
ferritin and apoferritin, and (3) Dps proteins and MrgA proteins
(refer to the protein data banks).
[0176] In this method, especially preferable are particles of the
ferritin family such as ferritin and apoferritin having a diameter
of 10 to 12 nm, or viruses, Dps proteins (protein particles one
size smaller than ferritin particles having a diameter of about 9
nm and a core diameter of about 4 nm), and MrgA proteins having a
diameter of 9 nm or less.
[0177] It is preferable to use protein particles obtained by
translating DNAs or RNAs having the same base sequence. A plurality
of protein particles obtained by translating the same DNA or RNA
have completely the same structure and are not likely to have
diameters varying every molecule. Moreover, protein particles have
the self-assembling ability where they can construct a high-order
structure together by recognizing one another. The protein
particles having this ability can be arranged at high density with
high precision.
[0178] Hereinafter, described is a case in which protein particles
obtained by translating the same DNA are used as particulates in
place of the complex particles 15, and a protein suspension with
the protein particles suspended therein is used as the liquid
16.
[0179] In use of the protein suspension described above, if the
concentration of the protein particles in the suspension is
excessively low, the protein particles can exert only insufficient
self-assembling function, failing to provide a good particulate
film (two-dimensional crystal film). On the contrary, if the
concentration is excessively high, the self-assembling function
saturates. This is not only uneconomical, but also may possibly
lead to layered arrangement of protein particles, resulting in
formation of a locally three-dimensional crystal film and thus
failing to provide a film that can be effectively used
industrially. In view of these, in this method, when a suspension
containing ferritin particles is used as the liquid 16, for
example, the protein concentration is in the range of 10 .mu.g/ml
to 500 mg/ml, preferably in the range of 10 .mu.g/ml to 200 mg/ml,
more preferably in the range of 0.5 mg/ml to 100 mg/ml.
[0180] In this method, the protein suspension may contain only
protein in a dispersion medium (generally, pure water), or may
additionally contain an electrolytic substance. The suspension
containing only protein in pure water may generate large
electrostatic repulsion between the substrate 11 and the protein
particles, and this may cause slow adsorption of the protein
particles to the substrate 11. To avoid this occurrence and
accelerate formation of a film made of the protein particles, an
electrolytic substance is added in this method. Examples of the
electrolytic substance include sodium chloride, potassium chloride,
calcium chloride, and magnesium chloride. If the content of the
electrolytic substance is excessively large, the electrolytic
substance is precipitated. In view of this, the content is suitably
300 mM or less, preferably about 150 mM (this is roughly the
concentration of a normal saline solution), more preferably about
50 mM, in the case of sodium chloride.
[0181] Alternatively, the protein suspension may be heated to
accelerate the formation of the particulate film made of the
protein particles. During the heating of the protein suspension,
however, convection is generated in the protein suspension. This
may adversely affect the array of the protein particles and thus
block the formation of the film of particulates arranged
beautifully at high density with high precision. Therefore, when
the protein suspension is heated to accelerate the formation of the
particulate film, it is desirable to heat the suspension, the
substrate, and all of the other components in uniform so that the
particulate film can be formed in the equilibrium state blocking
generation of convection of the protein suspension.
[0182] As another accelerating means, the surface of the substrate
11 may be subjected to hydrophilic treatment to charge the surface.
Examples of the hydrophilic treatment of the surface of the
substrate 11 include active ozone treatment under ultraviolet
irradiation at high temperature (about 110.degree. C.), oxygen
plasma treatment, and amino silane treatment.
[0183] As yet another accelerating means, the pH of the protein
suspension may be adjusted to fall within the range in which the
charge of the substrate 11 immersed in the protein suspension is
the opposite to the charge of the protein particles adsorbed to the
surface of the substrate 11.
[0184] For example, the charge of the substrate subjected to amino
silane hydrophilic treatment is plus when the pH is 11 or less,
while the charge of the ferritin particles is minus when the pH is
5 or more. Therefore, if the pH of the ferritin suspension is
adjusted to fall within the range of 5 to 11, it is possible to
facilitate the adsorption of the ferritin particles to the
substrate subjected to amino silane hydrophilic treatment
(formation of the ferritin particle film on the substrate). In
other words, the adsorption of the ferritin particles to the
substrate subjected to amino silane hydrophilic treatment is
facilitated by use of the attraction between the plus charge and
the minus charge. Only specific regions of the substrate surface
may be subjected to hydrophilic treatment to form the wet film 35
according to the pattern of the regions.
[0185] The substrate 11 is not necessarily subjected to hydrophilic
treatment as described above, but may preferably be subjected to
hydrophobic treatment depending on the type of the liquid 16. That
is, depending on the type of the liquid 16 (or the dispersion
medium), the wet film 35 can be formed only on a hydrophilic
substrate surface or only on a hydrophobic substrate surface. For
example, when a hydrophobic dispersion medium is used, the wet film
35 is formed only on a hydrophobic substrate surface, not a
hydrophilic substrate surface. Exceptionally, in the case of
protein, the substrate 11 is usable even when the surface thereof
is hydrophobic in some cases. This is because protein is denatured
on the surface of the substrate 11 and by this denaturation the
surface of the substrate 11 becomes hydrophilic, to allow the wet
film 35 to be formed on the hydrophilic surface.
EXAMPLES
Example 1
[0186] On a surface of a silicon substrate subjected to hydrophilic
treatment with active ozone under ultraviolet irradiation at
110.degree. C., apoferritin particles containing gold particles
inside were arranged two-dimensionally at high density with high
precision as shown in FIG. 2(a) by method 5 described above. For
this process, used was a liquid containing gold particle-containing
apoferritin in a normal saline solution in a concentration of 50
mg/ml. The liquid was drawn out from a container containing the
liquid with a syringe at a drawing rate (liquid level lowering
rate) of 0.1 mm/min.
[0187] The thus-produced substrate was heat-treated in a nitrogen
gas atmosphere at 450.degree. C. for one hour, to remove the
apoferritin as the protein moiety and thus attain a substrate with
only gold particles placed thereon in the shape of dots
two-dimensionally at high density with high precision.
[0188] The resultant substrate was then put in contact with a thiol
DNA aqueous solution, to attain a DNA sensor having one-to-one
bonding of thiol DNAs to the gold particles. As the thiol DNA
aqueous solution, used was an aqueous solution containing thiol
DNAs in a concentration of 70 mg/ml warmed to 37.degree. C. T4
phage DNAs were used as the DNAs and sulfur atoms were bonded to
ends of the DNAs. The substrate was kept in contact with the thiol
DNA aqueous solution for one hour.
[0189] Using the resultant DNA sensor, detection tests were
conducted for the T4 phage DNA and M13 phage DNA unrelated at all
to the T4 phage DNA. In the detection test for the T4 phage DNA,
the DNA sensor exhibited high fluorescence intensity stably
compared with the conventional DNA sensor, and thus detection was
very easy. On the contrary, in the detection test for the M13 phage
DNA, only background fluorescence intensity was detected,
indicating that no hybridized DNA existed.
[0190] The DNA sensor used for the detection test for the T4 phase
DNA was immersed in 100.degree. C. pure water for 10 minutes, and
then the surface of the DNA sensor having the hybridized DNA was
exposed to flow of 100.degree. C. pure water for 10 minutes. The
resultant surface of the DNA sensor was measured for fluorescence
and found to have the same fluorescence intensity as that of the
background. From this result, it was confirmed that the hybridized
DNA had dissociated from the thiol DNAs of the DNA sensor.
[0191] Using the resultant DNA sensor, a detection test for T4
phage DNA was performed again. As a result, the fluorescence
intensity increased, and therefore, it was confirmed that the
detection of DNA and the dissociation of hybridized DNA could be
repeated a plurality of times.
Example 2
[0192] An RNA sensor was produced in the same manner as that
described in Example 1, except that thiol RNAs (thiol RNAs produced
using mRNAs obtained by transcription of T4 phages with sulfur
atoms bonded to ends of the mRNAs) was used in place of the thiol
DNA.
[0193] Using the resultant RNA sensor, a detection test was
performed for separately synthesized complementary RNA. As a
result, high fluorescence intensity was exhibited stably, and thus
detection was very easy.
Example 3
[0194] First, as in Example 1, gold particles were placed
two-dimensionally on a silicon substrate. A resin resist film made
of polymthyl methacrylate (PMMA) was then formed on the substrate,
and a photomask having openings was formed on the resin resist
film.
[0195] Subsequently, the substrate was irradiated with light from
above of the photomask, and then treated with a developer to
pattern the resin resist film, to thereby expose part of the gold
particles on the substrate.
[0196] Thereafter, thiol DNAs were bonded to the gold particles on
the substrate.
[0197] The above operation was repeated while the type of the thiol
DNAs (in base sequence or the like) to be bonded to the exposed
gold particles was changed one after another, to attain a
multi-type DNA sensor with DNAs having a number of different
sequences bonded to the substrate. As the thiol DNAs, T4 phage DNAs
were used, and sulfur atoms were bonded to ends of the DNAs. A
solution containing such thiol DNAs in a concentration of 70 mg/ml
and warmed to 37.degree. C. was used. The substrate was kept in
contact with the thiol DNA solution for one hour.
[0198] Using the thus-produced multi-type DNA sensor, detection
tests were performed using a plurality of types of DNAs having
different base sequences derived from T4 phage and a plurality of
types of DNAs having different base sequences derived from M13
phage.
[0199] As a result, fluorescence was observed in expected sections
in the detection test for the plurality of types of DNAs having
different base sequences derived from T4 phage. The multi-type DNA
sensor exhibited high fluorescence intensity stably compared with
the conventional multi-type DNA sensor, and thus detection was very
easy. On the contrary, in the detection test for the plurality of
types of DNAs having different base sequences derived from M13
phage, only the background fluorescence intensity was detected,
indicating that no hybridized DNA existed.
Example 4
[0200] A multi-type RNA sensor was produced in the same manner as
that described in Example 3, except that thiol RNAs having a number
of different sequences (thiol RNAs produced using part of T4 phage
base sequences with sulfur atoms bonded to ends of the sequences)
were used in place of the thiol DNAs having a number of different
sequences.
[0201] Using in the resultant multi-type RNA sensor, detection
tests were performed using a plurality of mRNA sequences derived
from T4 phage and a plurality of mRNA sequences derived from M13
phage. As a result, the multi-type RNA sensor exhibited high
fluorescence intensity stably in expected sections, compared with
the conventional multi-type RNA sensor, in the detection test for
the T4 phage-derived mRNAs, and thus detection was very easy. On
the contrary, in the detection test for the M13 phage-derived
mRNAs, only the background fluorescence intensity was detected,
indicating that no hybridized mRNA existed.
Example 5
[0202] First, a metal mask having openings was placed on a surface
of a silicon substrate, and chromium and gold thin films were
deposited by sputtering to form electrodes on the surface of the
silicon substrate.
[0203] Gold particles were then placed as in Example 1 on the
electrodes.
[0204] A positive potential was applied to an electrode in a region
in which DNAs having a specific sequence were to be placed, and a
negative potential was applied to the other electrodes. While
applying these potentials, the substrate was put in contact with a
thiol DNA solution, to allow thiol DNAs to establish one-to-one
bonding to gold particles an the electrode.
[0205] The above operation was repeated while the type of the thiol
DNAs (in base sequence or the like) was changed one after another
and also the electrode to which the positive potential was applied
was sequentially changed, to attain a multi-type DNA sensor with
thiol DNAs having a number of different sequences bonded to the
substrate.
[0206] As the types of thiol DNAs, those described in Embodiment 3
were used.
[0207] Using the thus-produced multi-type DNA sensor, detection
tests as described in Example 3 were conducted, and substantially
the same results as those described in Example 3 were obtained.
Example 6
[0208] A multi-type RNA sensor was produced in the same manner as
that described in Example 5, except that thiol RNAs having a number
of different sequences (thiol RNAs produced using part of T4 phage
base sequences with sulfur atoms bonded to ends of the sequences)
were used in place of the thiol DNAs having a number of different
sequences.
[0209] As the types of thiol RNAs, those described in Embodiment 4
were used.
[0210] Using the thus-produced multi-type RNA sensor, detection
tests as described in Example 4 were performed, and substantially
the same results as those described in Example 4 were obtained.
[0211] Hereinafter, examples of production of a particulate film
will be described.
Example 7
[0212] First, prepared was a ferritin suspension containing
ferritin as particulates (containing ferritin particles having a
size of 12 nm derived from a horse spleen in a normal saline
solution in a concentration of 100 mg/ml). As the substrate,
prepared was a silicon substrate (with a surface subjected to
hydrophilic treatment with oxygen plasma) having a size of 40 mm
wide.times.50 mm long.times.500 .mu.m thick.
[0213] The silicon substrate was put in a container containing the
ferritin suspension so as to stand vertical to the liquid level of
the ferritin suspension. A tube (diameter: 1 mm) was placed in the
container so that one open end thereof was positioned near the
bottom of the container inside the container and the other open end
was coupled to a syringe. The ferritin suspension was gradually
drawn out from above the container at a liquid level lowering rate
of 0.12 mm/min.
[0214] To prevent the substrate from being influenced by
disturbance of the ferritin suspension generated due to the
suction, the tube was placed so that the open end positioned near
the bottom of the container was 20 mm apart from the silicon
substrate.
[0215] By the above operation, a wet film was formed on both
surfaces of the silicon substrate. By drying the wet film, attained
was a substrate on both surfaces of which a particulate film made
of ferritin particles arranged two-dimensionally at high density
with high precision was formed.
Example 8
[0216] A particulate film was produced in the same manner as that
described in Example 7, except that a container (10 cm.times.10
cm.times.10 cm, capacity: 1 liter) having one circular hole
(diameter: 2 mm) formed through the bottom was used, in place of
the syringe, so that the ferritin suspension in the container was
dropped through this hole by gravity. As a result, in the
particulate film, ferritin particles were arranged
two-dimensionally on both surfaces of the silicon substrate at high
density with high precision.
Example 9
[0217] A ferritin particle film was produced on both surfaces of
the substrate 3 in the same manner as that described in Example 1,
except that a plurality of protrusions (each protrusion has a
lattice shape and part of the lattice has been circularly stamped)
were formed on one surface of the silicon substrate (the size and
surface treatment were the same as those in Example 1). This
example will be described with reference to FIG. 14.
[0218] As the liquid level of the ferritin suspension 16 is lowered
by suction with the syringe, the wet film 36 is formed on the
portion 40 surrounding the protrusion 39 on the surface of the
silicon substrate 11. The wet film 36 has a divergent drop-like
shape widening downwardly toward the silicon substrate as shown in
FIG. 14(b). The ferritin particles of the wet film 36 gather toward
the protrusion 39 as indicated by the arrows due to decrease in the
amount of the dispersion medium of the wet film 36 and the
micro-capillary effect. Therefore, the concentration of the
ferritin particles increases along the gathering path toward the
protrusion 39.
[0219] Thus, as shown in FIG. 14(c), the particulate film produced
in this example with ferritin particles arranged two-dimensionally
at high density with high precision has a thickness that is largest
at a position adjacent to the protrusion 39 and gradually decreases
as the position is farther from the protrusion 39.
Example 10
[0220] A particulate film was produced in the same manner as that
described in Example 9, except that an apoferritin suspension with
zinc oxide-containing apoferritin particles, in place of the
ferritin particles, suspended therein was used.
[0221] The particulate film was heat-treated in a nitrogen
atmosphere at 450.degree. C. for two hours. As a result, the
protein moiety was removed, and super-fine particles of zinc oxide
were formed on the silicon substrate.
[0222] The air containing a trace amount of mercaptan was sprayed
to the resultant substrate. As a result, the amount of mercaptan in
the air was reduced.
[0223] The substrate was then irradiated with an electronic beam to
measure fluorescence, and it was found that the fluorescence
intensity was different between before and after the exposure to
the air containing a trace amount of mercaptan. It was therefore
confirmed that the substrate with the particulate film formed
thereon in this example was effective as a microsensor.
[0224] As described above, according to the method for producing a
particulate film of the present invention, it is possible to easily
produce a two-dimensional crystal film made of particulates having
a diameter of the order of nanometers arranged at high density and
at desired positions with high precision.
[0225] Therefore, according to the method for producing a
particulate film of the present invention, two-dimensional crystal
films made of particulates can be easily mass-produced in the
industrial scale.
INDUSTRIAL APPLICABILITY
[0226] The nucleotide detector of the present invention is usable
for devices utilizing complementarity of nucleotides, such as DNA
sensors and RNA sensors.
[0227] The method for producing a particulate film of the present
invention is usable for fabrication of devices requiring super-fine
patterns, in particular, for fabrication of diffraction gratings,
nucleotide detectors, and super-fine electronic devices such as
transistors and diodes.
* * * * *