U.S. patent application number 11/385006 was filed with the patent office on 2008-06-26 for method of selective arrangement of ferritin.
This patent application is currently assigned to MATSUSHITA ELECTRIC INDUSTRIAL CO., LTD.. Invention is credited to Hiroya Kirimura, Ichiro Yamashita.
Application Number | 20080154024 11/385006 |
Document ID | / |
Family ID | 36587701 |
Filed Date | 2008-06-26 |
United States Patent
Application |
20080154024 |
Kind Code |
A1 |
Kirimura; Hiroya ; et
al. |
June 26, 2008 |
METHOD OF SELECTIVE ARRANGEMENT OF FERRITIN
Abstract
A method for selectively arranging ferritin in a specified
inorganic material part formed on a substrate is provided. The
method for arranging ferritin of the present invention is
characterized in that ferritin is selectively arranged on a part
including titanium or silicon nitride (SiN) in an efficient manner
by adding a nonionic surface active agent. Also, selective
arrangement capability of ferritin can be markedly improved by
modifying the N-terminus of ferritin with a certain peptide.
Inventors: |
Kirimura; Hiroya;
(Kyoto-shi, JP) ; Yamashita; Ichiro; (Nara-shi,
JP) |
Correspondence
Address: |
McDERMOTT WILL & EMERY LLP
600 13th Street, N.W.
Washington
DC
20005-3096
US
|
Assignee: |
MATSUSHITA ELECTRIC INDUSTRIAL CO.,
LTD.
Osaka
JP
|
Family ID: |
36587701 |
Appl. No.: |
11/385006 |
Filed: |
March 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP05/21511 |
Nov 24, 2005 |
|
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11385006 |
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Current U.S.
Class: |
530/400 |
Current CPC
Class: |
C07K 14/47 20130101;
C07K 2319/00 20130101 |
Class at
Publication: |
530/400 |
International
Class: |
C07K 14/79 20060101
C07K014/79 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 2004 |
JP |
2004-361939 |
Feb 10, 2005 |
JP |
2005-034311 |
Claims
1. A method for selectively arranging ferritin on a substrate
comprising: (a) an arrangement step wherein a first solution
containing ferritin and a nonionic surface active agent is exposed
to a portion of said substrate consisting of a first inorganic
material and a portion of said substrate consisting of a second
inorganic material that is different from said first inorganic
material, thereby arranging the ferritin selectively on said
substrate portion consisting of the first inorganic material,
wherein the ferritin is in direct-contact with said portion of
substrate consisting of said first inorganic material, wherein said
first inorganic material is titanium or silicon nitride, and said
second inorganic material is platinum or silicon oxide.
2. The method for selectively arranging ferritin according to claim
1 wherein said first inorganic material and said second inorganic
material are both selected from the combinations consisting of:
titanium and platinum, titanium and silicon oxide, and silicon
nitride and silicon oxide.
3. The method for selectively arranging ferritin according to claim
2 wherein said first inorganic material is titanium and said second
inorganic material is platinum.
4. The method for selectively arranging ferritin according to claim
2 wherein said first inorganic material is titanium part and said
second inorganic material is silicon oxide.
5. The method for selectively arranging ferritin according to claim
2 wherein said first inorganic material is silicon nitride and said
second inorganic material is silicon oxide.
6. The method for selectively arranging ferritin according to claim
1 wherein the concentration of said nonionic surface active agent
is between 0.01 v/v % and 10 v/v %.
7. The method for selectively arranging ferritin according to claim
I wherein the subunit N-terminus part of said first ferritin is set
forth in SEQ ID NO: 4.
8. The method for selectively arranging ferritin according to claim
1 wherein said ferritin in said first solution includes an
inorganic particle therein.
9. A method for selectively arranging ferritin on a substrate
comprising: (a) an arrangement step wherein a first solution
containing ferritin and a nonionic surface active agent is exposed
to a portion of said substrate consisting of a first inorganic
material and a portion of said substrate consisting of a second
inorganic material that is different from said first inorganic
material, thereby arranging the ferritin selectively on said
substrate portion consisting of the first inorganic material,
wherein the ferritin on said substrate portion consisting of said
first inorganic material is in direct-contact with said portion of
substrate consisting of said first inorganic material, wherein said
first inorganic material is titanium or silicon nitride, and said
second inorganic material is platinum or silicon oxide and, (b)
wherein a second solution containing ferritin but not containing
any nonionic surface active agent is exposed to said substrate,
thereby selectively arranging ferritin on said portion of substrate
consisting of said second inorganic material.
10. The method for selectively arranging ferritin according to
claim 9 wherein said ferritin in said second solution includes an
inorganic particle therein.
11. The method for selectively arranging ferritin according to
claim 10 wherein said ferritin in said first solution does not
include an inorganic particle therein.
Description
[0001] This is a continuation application under U.S.C 111(a) of
pending prior International application No.PCT/JP2005/021511, filed
on Nov. 24, 2005.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method for selectively
arranging ferritin.
[0004] 2. Related Art
[0005] Fine particles (inorganic particles) which include a protein
and an inorganic substance and which are arranged on a base
material have attracted attention in industrial fields of
catalysts, sensors, biochips, transistors, semiconductors lasers,
magnetic discs, displays and the like. In particular, patterning
techniques have been desired in which inorganic particles are
selectively arranged in a specified region, or they are regularly
arranged in a fine region of nano-size, when the inorganic
particles are industrially applied. Furthermore, in recent years,
aiming at miniaturization of total analysis systems including
biosensors, applications to fine chemical substance analysis
systems (Micro Total Analysis System (.mu.TAS)) have also attracted
attention. Behind such a situation, advantages such as improvement
of biocompatibility, enablement of lowering of the cost by mass
productivity and measurement in the place (portable) and the like
are involved.
[0006] Techniques for selectively arranging proteins or inorganic
particles on a solid surface involve extraordinary difficulty
because it is very difficult to allow the surface of the protein
and the inorganic substance to have a self-recognizing function.
Known methods for forming a fine pattern using a protein that is a
biomolecule include a method in which photolithography is utilized
(see, A. S. Blawas, W. M. Reichert, Biomaterials, 19, 595 (1998)),
microcontact printing (see, A. Bernard, J. P. Renault, B. Michel,
H. R. Bosshard, E. Delamarche, Adv. Mater., 12, 1067), dip-pen
nanolithography (see, K. B. Lee, S. J. Park, C. A. Mirkin, J. C.
Smith, M. Mrksick, Science, 295, 1702 (2002)), and the like.
However, in light of mass productivity and costs, techniques for
carrying out patterning of fine particles in a nano-size region
have been demanded.
[0007] Furthermore, a method for regularly arranging nano-size fine
particles surrounded by protein molecules is disclosed in Japanese
Patent Provisional Publication No. H11-204774. In these methods,
procedures of: subjecting the surface of a SAM membrane
(self-assembled monomolecular membrane), an LB membrane
(monomolecule accumulating membrane) or the like to a processing
for selectively arranging the fine particles; executing patterning
of the fine particles through further conducting photolithography
in combination; forming a region in which the inorganic particles
are selectively arranged on the surface of a base material by
direct drawing or the like of a pattern on the base material with a
nanoprobe such as AFM (Atomic Force Microscope) or the like; and
thereafter arranging the inorganic particles.
[0008] Hereinafter, a method for arranging inorganic particles
using an LB membrane (PBLH membrane) according to the conventional
method (Japanese Patent Provisional Publication No. H11-204774) is
illustrated in FIGS. 1A to 1H.
[0009] First, in the step shown in FIG. 1A, a buffer 11 is reserved
in a water bath 10 made of Teflon (registered trade name), and
native ferritin 21 including an inorganic particle 20 therein is
dispersed in this buffer.
[0010] Next, in the step shown in FIG. 1B, a PBLH membrane 30 is
overlaid on the liquid surface of the solution. Then, the pH is
adjusted with an appropriate acid alkaline solution. Because the
PBLH membrane surface is positively charged, the native ferritin 21
which is negatively charged is attached on the PBLH membrane.
[0011] Next, in the step shown in FIG. 1C, a base material (silicon
substrate) 40 which had been subjected to a hydrophobic surface
treatment is floated on the liquid surface on which the PBLH
membrane 30 was overlaid, thereby allowing the PBLH membrane 30 on
which the native ferritin 21 is attached to be adhered on the base
material.
[0012] Next, in the step shown in FIG. 1D, the silicon substrate 40
having the adhered PBLH membrane 30 on which the native ferritin 21
is attached is removed from the water bath.
[0013] Next, in the step shown in FIG. 1E, after covering the
surface on which the native ferritin 21 is attached with a buffer
solution 11, ultraviolet irradiation is performed using a mask
pattern 50. The native ferritin in the region on which ultraviolet
ray was irradiated is decomposed, and dispersed in the
solution.
[0014] Next, in the step shown in FIG. 1F, the silicon substrate 40
after executing the patterning shown in FIG. 1E is washed with
water.
[0015] Next, in the step shown in FIG. 1G, the silicon substrate 40
is dried to obtain the pattern arrangement of the native ferritin
including the inorganic particle therein.
[0016] Thereafter, in the step shown in FIG. 1H, a heat treatment
at 500.degree. C is carried out in an inert gas 60 (for example, in
nitrogen) to bake the native ferritin 21 including the inorganic
particle therein and the PBLH membrane 30, thereby providing
secondary pattern arrangement of the inorganic particles on the
base material surface. This structure is further processed to give
a structure required for the device as described above.
[0017] However, the SAM membrane is formed on the base material
side, and patterning is executed on the SAM membrane using
ultraviolet ray, or an LB membrane that is an adsorption membrane
of the inorganic particle is utilized as the intermediate layer
with respect to the a base material in the aforementioned
conventional method. Therefore, there are possibilities that the
steps may be complicated, or that impurities included in the
constituents of the SAM membrane or the LB membrane, or in the
solution remain on the arranged surface of the inorganic particles
whereby causing adverse influences on the device.
[0018] Accordingly, an object of the present invention is to
provide a technique for selectively and regularly arranging
inorganic particles, in particular, those having a diameter of
several to several ten nanometers in a necessary region and in a
necessary amount with high mass productivity at low costs by
allowing the inorganic particle to have a base material recognizing
ability.
SUMMARY OF THE INVENTION
[0019] In order to accomplish the object described above, the first
aspect of the present invention is characterized in that binding
force between ferritin and the inorganic material on the substrate
is controlled by a nonionic surface active agent. The nonionic
surface active agent fundamentally has a function to attenuate the
binding force between a protein and the base material on the
substrate that is an inorganic substance through acting on the
interface between them. Thus, this action enables control of the
binding force between ferritin and the base material.
[0020] In other words, this procedure enables control of the
proportion of the ferritin adsorbed on the part where arrangement
is required and on the part where arrangement is not required
(selectivity), and control of the amount of adsorption of the
ferritin on the part where arrangement is required. This term
"control" means rendering the ferritin itself to have an ability of
augmenting inherent binding force between the substrate and the
ferritin, or of lowering the force to the contrary
(self-recognizing ability).
[0021] This self-recognizing ability makes it possible to arrange
the ferritin in a specified inorganic material part on the
substrate where arrangement of the inorganic particles is required,
and to arrange the inorganic particles included in the
ferritin.
[0022] On the other hand, when the inorganic particle is not
included in ferritin, the inorganic particles are not arranged in
the inorganic material part on the substrate, thereby enabling
protection of the specified part with the ferritin.
[0023] The structure of conventional ferritin (native or
recombinant ferritin (basket-shaped protein)) is illustrated in
FIG. 2. Ferritin is a spherical particle having a diameter of about
12 nm and having a cavity (diameter: about 7 nm) inside thereof
formed through binding of 24 subunits. Various inorganic material
particles (core) can be incorporated in this cavity. One subunit
has a specific tertiary structure as shown in the center of FIG. 2,
which was analyzed in detail with X-ray analyses and the like
revealing that it includes a combination of secondary structures of
the .alpha.-helix and .beta.-sheet.
[0024] Amino acid side chains are protruded from the skeleton
(folded polypeptide main chain) of this protein in various
directions, and the sequence of the amino acid residues allows each
protein to have unique chemical characteristics. The ferritin
surface reflects the features of the protruded amino acid residues,
thereby determining the chemical characteristics of the entire
protein (interaction with the base material, interaction among the
proteins and the like).
[0025] The second aspect of the present invention is characterized
in that the chemical characteristic of ferritin is altered by
modifying this ferritin at the N-terminal part with a peptide
(N-terminal modification peptide) thereby allowing the peptide to
protrude from the ferritin surface. Furthermore, binding force
between the ferritin and the inorganic base material is controlled
by the modification with this N-terminal modification peptide.
[0026] The term "modification of N-terminal part of ferritin with a
peptide" referred to herein involves both of: addition of an
N-terminal modification peptide at the N-terminus of ferritin, and
insertion of an N-terminal modification peptide subsequent to the
N-terminal amino acid residue of ferritin (methionine residue).
[0027] Moreover, the third aspect of the present invention is
characterized in that the peptide for modifying the N-terminus of
the ferritin (N-terminal modification peptide) has one or more
polar charged amino group(s). Examples of the polar charged amino
group include lysine (K), arginine (R), histidine (H), aspartic
acid (D) and glutamic acid (E). FIG. 3 illustrates the structure of
the arginine (R) residue as the polar charged amino group. The
peptide which modifies the N-terminal part has a flexible main
chain with a structure that may be variable. Because of the
structure having this flexible main chain protruded from the
surface of the ferritin (FIG. 3, middle figure and right figure),
the charged amino group shall be a factor of action to control the
adsorbing force through recognizing the charge localized on the
inorganic material surface.
[0028] Specifically, the present invention relates to a method for
selectively arranging ferritin,
[0029] the method comprising a first arrangement step in which a
solution containing first ferritin and a nonionic surface active
agent is added dropwise to a substrate having on the surface
thereof a first part comprising a first inorganic material and a
second part comprising a second inorganic material that is
different from the first inorganic material, thereby arranging the
first ferritin selectively in the first part,
[0030] the first part comprising titanium or silicon nitride,
and
[0031] the second part comprising platinum or silicon oxide.
[0032] Although ferritin is nonselectively adsorbed to the
inorganic material of the substrate, the present inventor first
found that it is selectively adsorbed to a certain inorganic
material by allowing a nonionic surface active agent to
coexist.
[0033] It is preferred that the first part and the second part
comprise any one combination selected from three combinations
composed of: titanium and platinum, titanium and silicon oxide, and
silicon nitride and silicon oxide, respectively.
[0034] It is more preferred that the first part and the second part
comprise a combination of titanium and platinum, respectively.
[0035] It is more preferred that the first part and the second part
comprise a combination of titanium and silicon oxide,
respectively.
[0036] It is more preferred that the first part and the second part
comprise a combination of silicon nitride and silicon oxide,
respectively.
[0037] It is preferred that the concentration of the nonionic
surface active agent is 0.01 v/v % or greater and 10 v/v % or
less.
[0038] It is preferred that the subunit N-terminal part of the
first ferritin is modified with a peptide set out in SEQ ID NO:
4.
[0039] The first ferritin may include an inorganic particle
therein.
[0040] It is preferred that the second arrangement step in which a
solution containing second ferritin but not containing any nonionic
surface active agent is added dropwise to the substrate, thereby
arranging the second ferritin in the second part is further
included following the first arrangement step.
[0041] In this instance, the second ferritin may include an
inorganic particle therein. Also, the first ferritin may not
include an inorganic particle therein.
[0042] Using the method for arranging ferritin, a device can be
produced by selectively arranging ferritin or the inorganic
particles in the first part or the second part on the
substrate.
[0043] According to the method for arranging ferritin of the
present invention, when ferritin and the inorganic particle
included therein are arranged and fixed on the base material
surface, the physical adsorbing force between the ferritin and the
inorganic material part formed on the base material surface can be
controlled by adding a nonionic surface active agent, and secondary
regular arrangement of the ferritin on the substrate is also
enabled. According to the method for arranging ferritin of the
present invention, the inorganic particles can be arranged in the
region where required in a necessary amount, or the inorganic
particles can be arranged on the substrate with high accuracy in a
regular manner, with high mass productivity and favorable cost
performances.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIGS. 1A to 1H show an explanatory view illustrating the
steps of a conventional method for arranging inorganic
particles.
[0045] FIG. 2 shows an explanatory view illustrating the structure
and the like of conventional ferritin.
[0046] FIG. 3 shows an explanatory view illustrating the structure
and the like of ferritin modified at the N-terminus with a
peptide.
[0047] FIG. 4 shows an explanatory view illustrating the principles
of the method for arranging ferritin of the present invention.
[0048] FIG. 5 shows a schematic view illustrating the principal
construction of a plasmid of L type ferritin subunit, and
incorporation of the plasmid into Escherichia coli.
[0049] FIGS. 6A and 6B show an explanatory view illustrating the
principles of the method for arranging inorganic particles of the
present invention.
[0050] FIG. 7A shows a scanning transmission electron micrograph of
the substrate surface according to Comparative Example 2; and FIG.
7B shows a scanning transmission electron micrograph of the
substrate surface according to Example 1.
[0051] FIG. 8A shows a scanning transmission electron micrograph of
the substrate surface according to Example 2; and FIG. 8B shows a
scanning transmission electron micrograph of the substrate surface
according to Example 3.
[0052] FIG. 9A shows a scanning transmission electron micrograph of
the substrate surface according to Example 3; and FIG. 9B shows a
scanning transmission electron micrograph of the substrate surface
according to Example 3 when Tween 80 in an amount of 0.5 v/v % was
added.
[0053] FIG. 10A shows a schematic explanatory view with respect to
Example 7; and FIG. 10B shows a scanning transmission electron
micrograph of the substrate surface according to Example 7.
[0054] FIG. 11A shows a scanning transmission electron micrograph
of the Ti membrane surface according to Comparative Example 3; and
FIG. 11B shows a scanning transmission electron micrograph of the
SiO.sub.2 substrate surface according to Comparative Example 3.
[0055] FIG. 12A shows a scanning transmission electron micrograph
of the SiO.sub.2 substrate surface according to Example 8; and
[0056] FIG. 12B shows a scanning transmission electron micrograph
of the SiN membrane surface according to Example 8.
[0057] FIG. 13A shows a scanning transmission electron micrograph
of the SiO.sub.2 substrate surface according to Comparative Example
4; and FIG. 13B shows a scanning transmission electron micrograph
of the SiN membrane surface according to Comparative Example 4.
[0058] FIGS. 14A to 14E show an explanatory view illustrating the
method for reverse-selectively arranging inorganic particles
according to Embodiment 3 of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The foregoing objects, other objects, features and
advantages of the present invention will be apparent from the
following detailed description of preferred embodiments with
reference to attached drawings.
[0060] Modes for carrying out the present invention will be
explained below with appropriate reference to the drawings.
However, the present invention is not limited thereto.
PRINCIPLES OF THE PRESENT INVENTION
[0061] Principles of the present invention will be first explained.
In this section, a method for arranging ferritin and the inorganic
particle included in the ferritin on a substrate will be
explained.
[0062] [Method for Arranging Ferritin]
[0063] FIG. 4 shows a flow chart conceptually illustrating the
method for arranging ferritin of the present invention.
[0064] As shown in FIG. 4, the method for arranging ferritin of the
present invention includes three steps, i.e., steps S1 to S3.
[0065] First, in the step S1, a solution containing ferritin is
prepared.
[0066] Next, in the step S2, a nonionic surface active agent is
added to the solution prepared in the step S1.
[0067] Next, in the step S3, the solution prepared in the step S2
is added dropwise on the substrate having two or more inorganic
material parts formed on the surface thereof. Accordingly, binding
force between the ferritin and the inorganic material part on the
substrate is altered by the addition of the nonionic surface active
agent. Consequently, selective arrangement of the ferritin is
enabled in either one of the inorganic material parts.
[0068] As the ferritin which may be used in the step S1,
peptide-modified ferritin and conventional recombinant ferritin
were used in the Embodiments of the present invention described
later.
[0069] The step S1 and the step S2 are explained herein as each
independent step, however, the step S1 and the step S2 can be also
carried out at the same time as single step. Hereinafter, the
method for manufacturing such ferritin will be explained.
<Method for Manufacturing Recombinant Ferritin>
[0070] First, a method for manufacturing recombinant ferritin will
be explained without modification of the N-terminus with a
peptide.
[0071] Although native ferritin (derived from equine spleen) is
constructed by assembly of 24 subunits, there are L type and H type
subunits having slightly different structures. Therefore, the
native ferritin does not have a constant structure. In the
following embodiment, recombinant ferritin constructed with only L
type subunits was used.
[0072] First, a DNA encoding L type ferritin (SEQ ID NO: 1, 528
base pairs) was amplified with a PCR method to prepare a large
amount of the L type ferritin DNA. Next, this L type ferritin DNA
was cleaved at sites where restriction enzymes EcoRI and Hind III
will specifically cleave (restriction enzyme sites). By this
cleavage treatment, a solution of L type ferritin DNA fragments
having restriction enzyme sites of EcoRI and Hind III was prepared.
DNA electrophoresis of this solution was performed, and the DNA
fragments encoding the L type ferritin alone were recovered and
purified.
[0073] Thereafter, this L type ferritin DNA fragment and a vector
plasmid (pMK-2) treated with restriction enzymes EcoRI-Hind III
were incubated to perfect ligation. Accordingly, a vector plasmid
pMK-2-fer-0 having the L type ferritin DNA incorporated at the
multicloning site (MSC) of the pMK-2 plasmid was produced. The
vector plasmid pMK-2 employed was selected in light of advantages
in obtaining a large amount of ferritin because it has Tac promoter
as its promoter, and thus is characterized by the large copy number
as a multicopy plasmid. Thus produced plasmid (pMK-2-fer-0) was
introduced (transformed) into E. coli Nova Blue (Novagen), a strain
of Escherichia coli, as a host, thereby yielding a recombinant L
type ferritin strain (fer-0). Schematic view illustrating the
principal construction of the plasmid of the L type ferritin
subunit, and incorporation of the plasmid into Escherichia coli is
shown in FIG. 5.
[0074] Then, in the method for arranging inorganic particles on the
substrate described later, inclusion of inorganic particles
required for a device were executed into thus produced recombinant
ferritin. It was suggested that thermostability of the recombinant
ferritin (fer-0) produced according to the aforementioned method is
improved by the addition of the peptide to the amino terminus.
Although the native ferritin had an allowable temperature limit of
approximately 55.degree. C., in contrast, fer-0 had an allowable
temperature limit of 95.degree. C. By virtue of this heat
resistance, synthesis of nanoparticles utilizing a basket-shaped
protein at a high temperature which had been conventionally
impossible was enabled.
<Method for Manufacturing Ferritin Modified with Peptide>
[0075] Next, a method for manufacturing ferritin including a
peptide having the amino acid sequence set out in SEQ ID NO: 4
inserted subsequent to the N-terminal residue (SEQ ID NO: 2) will
be explained.
[0076] When the amino terminus (N-terminus) of the subunit
constructing ferritin is modified with a peptide, a structure
including the peptide protruded outside of the ferritin particle as
shown in FIG. 3 is provided. Hence, modification of the surface of
the ferritin fine particle with a peptide is enabled by addition or
insertion of an arbitrary peptide at this N-terminal part.
[0077] Hereinafter, a specific method for manufacturing ferritin
having the amino acid sequence set out in SEQ ID NO: 2 will be
demonstrated. A full length gene of the L type subunit of native
ferritin (derived from equine spleen) is set out in SEQ ID NO: 1.
It was reported that 7 residues among amino residues synthesized
from N-terminal 24 bases are processed and deleted in nature. In
other words, ferritin having the amino acid sequence set out in SEQ
ID NO: 2 should be synthesized from the DNA set out in SEQ ID NO:
1, however, ferritin having the amino acid sequence set out in SEQ
ID NO: 3 is yielded in fact because 7 amino acid residues of from
the second to the eighth are deleted from the N-terminus.
[0078] Accordingly, the chemical characteristic of the ferritin was
altered by synthesizing without deletion of the N-terminal 7 amino
acid sequences (SEQ ID NO: 4) to allow a flexible peptide with
variable structure to be protruded outside of the ferritin
particle, and thus, a method for controlling the adsorption of this
ferritin modified with a peptide to the inorganic material in the
presence of a nonionic surface active agent was found.
[0079] A base sequence (DNA) encoding peptide-modified ferritin
having the peptide sequence set out in SEQ ID NO: 4 inserted
subsequent to the N-terminal methionine residue of the subunit was
designed, which was used in a PCR method to prepare a large amount
of the DNA similarly to the aforementioned method for manufacturing
recombinant ferritin. This DNA fragment required for the synthesis
was introduced into a vector plasmid. Thus produced vector plasmid
was introduced into Escherichia coli followed by proliferation
(transformation) to perfect the synthesis of recombinant ferritin
modified with a peptide (peptide-modified ferritin).
[0080] In the present invention, type of the peptide for modifying
the N-terminal part of recombinant ferritin is not particularly
limited, but recombinant ferritin including a peptide having the
amino acid sequence set out in SEQ ID NO: 4 as the N-terminal
modification peptide, inserted subsequent to the N-terminal
methionine residue, i.e., recombinant ferritin having the amino
acid sequence set out in SEQ ID NO: 2 was used in the Embodiments
described below.
[0081] As explained in the foregoings, according to the method for
arranging ferritin of the present invention, the steps are
extremely simplified because binding force between ferritin and the
specified inorganic material part of the substrate surface can be
controlled by merely adding a nonionic surface active agent.
[0082] [Method for Arranging Inorganic Particles on the
Substrate]
[0083] Next, the method for arranging inorganic particles of the
present invention will be explained by way of FIGS. 6A and 6B.
Herein, an example in which ferric oxide (Fe.sub.2O.sub.3) was used
as the inorganic particle will be demonstrated.
[0084] In the step shown in FIG. 6A, after adding dropwise a
solution of conventional ferritin 85 including Fe.sub.2O.sub.3 75
therein to which a nonionic surface active agent 83 was added onto
a substrate 80 having a region 81 where arrangement of inorganic
particles is required, followed by incubation for a given time
period, the substrate was washed with pure water.
[0085] Next, in the step shown in FIG. 6B, because the conventional
ferritin is adsorbed in the specified region 81 on the substrate
80, Fe.sub.2O.sub.3 75 included therein can be arranged also in the
specified region 81. As a consequence, the substrate 82 can be
produced having the fine particles of Fe.sub.2O.sub.3 75
selectively arranged only in the specified region.
[0086] Also, as the alternative example of the method described
above, when the peptide-modified ferritin is used in place of the
conventional ferritin, the amount of adsorption of the inorganic
particles to the certain region 81 is markedly increased, and
adsorption onto the substrate 80 with low interaction can be
suppressed. Accordingly, further highly selective arrangement is
enabled.
[0087] In Embodiments described below, the ferritin which had been
including Fe.sub.2O.sub.3 therein was baked by subjecting the
substrate to a heat treatment at 500.degree. C. in nitrogen gas
after washing with water, whereby allowing Fe.sub.2O.sub.3 to be
fixed in the specified region 81. In place of nitrogen gas, inert
gas or oxygen gas, hydrogen gas or the like can be also used.
[0088] Next, introduction of the inorganic particle into ferritin
will be explained.
[0089] <Introduction of Inorganic Particle into Ferritin>
[0090] In the present invention, type of the inorganic particle to
be included into the recombinant ferritin is not particularly
limited, but in the foregoing descriptions and Embodiments
described later, ferric oxide (Fe.sub.2O.sub.3) was used as the
inorganic particle. Introduction of the Fe.sub.2O.sub.3 core into
the peptide-modified ferritin was conducted as described below.
[0091] As the reaction solution, 0.5 mg/ml peptide-modified
ferritin/100 mM HEPES-NaOH (pH 7.0) was prepared, and thereto was
added 5 mM ammonium iron acetate. The reaction was allowed at
25.degree. C. overnight, and the peptide-modified ferritin having
the core of Fe.sub.2O.sub.3 formed was recovered from the solution
following the reaction through molecular purification by
centrifugal separation and gel filtration. The centrifugal
separation was conducted under the conditions of 1,600 G for 10
min, and 10,000 G, for 30 min. Thus, unwanted portions other than
the ferritin were eliminated stepwise as the precipitate, and then
the peptide-modified ferritin having a Fe.sub.2O.sub.3 core formed
therein was recovered from the finally remaining supernatant by
ultracentrifugal separation at 230,000 G for 1 hour as the pellet.
Thus resulting peptide-modified ferritin was loaded on gel
filtration using HPLC [column: TSK-GEL G4000SWXL PEEK/flow rate: 1
ml/min/buffer: 50 mM Tris-HCl (pH 8.0)+150 mM NaCl] to fractionate
to give a peak of 24-mer (about 480 kDa). Solution of the
fractionated peptide-modified ferritin was concentrated using an
ultrafilter to obtain the peptide-modified ferritin including
Fe.sub.2O.sub.3 therein.
[0092] In addition, by carrying out a similar operation to that
described above on recombinant ferritin without modification of the
N-terminus with a peptide, recombinant ferritin including
Fe.sub.2O.sub.3 therein was obtained. Also, by carrying out a
similar operation to that described above on the native ferritin
(derived from equine spleen), native ferritin including
Fe.sub.2O.sub.3 therein was obtained.
[0093] Hereinafter, specific embodiments of the present invention
will be explained sequentially.
Embodiment 1
[0094] Embodiment 1 of the present invention demonstrates a method
for arranging ferritin and inorganic particles on a substrate. In
this Embodiment, a Pt part and a Ti part are formed on a substrate
as two kinds of inorganic material parts.
[0095] Specific examples of this Embodiment will be demonstrated by
way of Examples below, and the effect thereof will be explained
with reference to Comparative Examples. In the following
Comparative Examples 1 and 2, and Reference Example, the nonionic
surface active agent was not used.
COMPARATIVE EXAMPLE 1
[0096] In Comparative Example 1, inorganic particles were arranged
on a substrate as described below.
[0097] First, native ferritin (manufactured by Sigma Corporation,
derived from equine spleen) including Fe.sub.2O.sub.3 therein was
adjusted to give the concentration of 2 mg/ml using a buffer
solution (10 mM Tris-HCl, pH 8.0). On a Ti substrate having a
platinum membrane (Pt membrane) formed on a part of its surface was
added the native ferritin solution dropwise. After leaving to stand
at room temperature for 1 hour, the substrate was washed with pure
water. After washing, the substrate was subjected to a heat
treatment according to the method described above, thereby allowing
Fe.sub.2O.sub.3 to be fixed on the substrate.
[0098] Thereafter, a scanning transmission electron micrograph of
the substrate surface was taken, and the number of Fe.sub.2O.sub.3
arranged on the Ti substrate and the Pt membrane was counted in a
square of 200 nm. The number on the Ti substrate was 79, while the
number on the Pt membrane was 76, exhibiting the selective
arrangement ratio of 1.0.
[0099] Herein, the selective arrangement ratio means the ratio of
the number of Fe.sub.2O.sub.3 adsorbed on the Ti substrate,
N.sub.(Ti), to the number of Fe.sub.2O.sub.3 adsorbed on the Pt
membrane, N.sub.(Pt), i.e., N.sub.(Ti)/N.sub.(Pt).
COMPARATIVE EXAMPLE 2
[0100] In Comparative Example 2, a similar operation to that in
Comparative Example 1 was carried out on the recombinant ferritin
including Fe.sub.2O.sub.3 therein.
[0101] FIG. 7A shows a scanning transmission electron micrograph of
the substrate surface in an experiment in which the recombinant
ferritin including Fe.sub.2O.sub.3 therein was arranged on the Ti
substrate including a Pt membrane formed on a part of its
surface.
[0102] In FIG. 7A, when the number of Fe.sub.2O.sub.3 arranged on
the Ti substrate and the Pt membrane was counted in a square of 200
nm, the number on the Ti substrate was 200, and in contrast, the
number on the Pt membrane was 195, exhibiting the selective
arrangement ratio of 1.0. [Reference Example] In Reference Example,
a similar operation to that in Comparative Example 1 was carried
out on the peptide-modified ferritin including Fe.sub.2O.sub.3
therein.
[0103] FIG. 7B shows a scanning transmission electron micrograph of
the substrate surface in an experiment in which the
peptide-modified ferritin including Fe.sub.2O.sub.3 therein was
arranged on the Ti substrate including a Pt membrane formed on a
part of its surface.
[0104] In FIG. 7B, the number of adsorption of Fe.sub.2O.sub.3 in a
square of 200 nm was 116 on the Ti substrate, and was 100 on the Pt
membrane, exhibiting the selective arrangement ratio of 1.2.
Therefore, the selective arrangement ratio was increased by 20%
when the peptide-modified ferritin was used, in comparison with the
case in which the conventional ferritin was used. Accordingly, it
was ascertained that ferritin became more apt to be adsorbed on the
Ti substrate than on the Pt membrane.
EXAMPLE 1
[0105] In Example 1, a similar operation to that in Reference
Example was carried out using a solution to which 0.5 v/v % Tween
20 manufactured by ICI Inc., was added as a nonionic surface active
agent, when native ferritin including Fe.sub.2O.sub.3 therein was
prepared.
[0106] When the number of Fe.sub.2O.sub.3 arranged on the Ti
substrate and the Pt membrane was counted in a square of 200 nm,
the number on the Ti substrate was 79, while the number on the Pt
membrane was 12, exhibiting the selective arrangement ratio of 6.6.
Accordingly, addition of the nonionic surface active agent in an
amount of 0.5 v/v % could allow the native ferritin to be
selectively arranged on the Ti membrane.
EXAMPLE 2
[0107] In Example 2, a similar operation to that in Reference
Example was carried out using a solution to which 0.5 v/v % Tween
20 manufactured by ICI Inc., was added as a nonionic surface active
agent, when recombinant ferritin including Fe.sub.2O.sub.3 therein
was prepared.
[0108] FIG. 8A shows a scanning transmission electron micrograph of
the substrate surface in an experiment in which the recombinant
ferritin including Fe.sub.2O.sub.3 therein was arranged in the
presence of the nonionic surface active agent, on the Ti substrate
including a Pt membrane formed on a part of its surface.
EXAMPLE 3
[0109] In Example 3, a similar operation to that in Reference
Example was carried out using a solution to which 0.5 v/v % Tween
20 manufactured by ICI Inc., was added as a nonionic surface active
agent, when peptide-modified ferritin including Fe.sub.2O.sub.3
therein was prepared.
[0110] FIG. 8B shows a scanning transmission electron micrograph of
the substrate surface in an experiment in which the
peptide-modified ferritin including Fe.sub.2O.sub.3 therein was
arranged in the presence of the nonionic surface active agent, on
the Ti substrate including a Pt membrane formed on a part of its
surface.
[0111] Referring to FIGS. 8A and 8B, it is revealed that
Fe.sub.2O.sub.3 was arranged on the Ti substrate and the Pt
membrane with greater difference in the number of adsorption
compared with FIGS. 7A and 7B. In FIG. 8A, the number of adsorption
of Fe.sub.2O.sub.3 in a square of 200 nm was 150 on the Ti
substrate, and in contrast, the number on the Pt membrane was 3,
exhibiting the selective arrangement ratio of 50.0. Moreover, in
FIG. 8B, the number of adsorption of Fe.sub.2O.sub.3 in a square of
200 nm was 146 on the Ti substrate, and was 1 on the Pt membrane,
exhibiting the selective arrangement ratio of 146.0 which suggested
remarkable improvement of the selectivity.
[0112] Hence, it was verified that addition of the nonionic surface
active agent improved selective adsorptivity for the Ti substrate
in any case in which the native ferritin, the recombinant ferritin
or the peptide-modified ferritin is used. In particular, adsorbing
force between ferritin and the inorganic base material could be
controlled by modification of the ferritin surface with the
peptide.
[0113] Additionally, because the selective arrangement ratio in
Example 2 was 50.0, which was greater than the selective
arrangement ratio of 6.6 in Example 1, it was indicated that the
effect of improving selectivity for the inorganic base material (Ti
substrate) by addition of the nonionic surface active agent was
greater on the recombinant ferritin than on the native
ferritin.
EXAMPLE 4
[0114] In Example 4, a method of the arrangement was attempted in
which a nonionic surface active agent was not added to the native
ferritin solution but a solution containing the nonionic surface
active agent was added dropwise to the substrate, followed by
adding a native ferritin solution without including a nonionic
surface active agent dropwise to the substrate. Accordingly, it was
verified that the number of adsorption and selectivity that are
almost equivalent to Example 1 in which the nonionic surface active
agent was added to the native ferritin solution could be
achieved.
EXAMPLE 5
[0115] In Example 5, a method of the arrangement was attempted in
which a nonionic surface active agent was not added to the
recombinant ferritin solution but a solution containing the
nonionic surface active agent was added dropwise to the substrate,
followed by adding a recombinant ferritin solution without
including a nonionic surface active agent dropwise to the
substrate. Accordingly, it was verified that the number of
adsorption and selectivity that are almost equivalent to Example 2
in which the nonionic surface active agent was added to the
recombinant ferritin solution could be achieved.
EXAMPLE 6
[0116] In Example 6, a method of the arrangement was also attempted
in which a nonionic surface active agent was not added to the
peptide-modified ferritin solution, but a solution containing the
nonionic surface active agent was added dropwise to the substrate,
followed by adding a peptide-modified ferritin solution without
including a nonionic surface active agent dropwise to the
substrate. Accordingly, it was verified that the number of
adsorption and selectivity that are almost equivalent to Example 3
in which the nonionic surface active agent was added to the
peptide-modified ferritin solution could be achieved.
[0117] Moreover, because the selective arrangement ratio in Example
5 was 50.0, which was greater than the selective arrangement ratio
of 6.0 in Example 4, it was indicated that the effect of improving
selectivity for the inorganic base material (Ti substrate) was
greater on the recombinant ferritin than the native ferritin also
in the case in which the substrate was treated with the nonionic
surface active agent.
[0118] For reference, experimental results of Comparative Examples
1 to 2, Reference Example and Examples 1 to 6 are summarized in
Table 1.
TABLE-US-00001 TABLE 1 Method of Native Recombinant
Peptide-modified arrangement ferritin ferritin ferritin 1. No
treatment of On Selective On Selective On Selective ferritin
solution, Ti: arrangement Ti: arrangement Ti: arrangement substrate
with surface 79 ratio: 1.0 200 ratio: 1.0 116 ratio: activating
agent On [Comparative On [Comparative On 1.2 Pt: Example Pt:
Example Pt: [Reference 76 1] 195 2] 100 Example] 2. 0.5 v/v %
nonionic On Selective On Selective On Selective surface activating
Ti: arrangement Ti: arrangement Ti: arrangement agent added to 79
ratio: 6.6 150 ratio: 50.0 146 ratio: ferritin solution On [Example
1] On [Example 2] On 146.0 Pt: Pt: 3 Pt: 1 [Example 12 3] 3.
Arranged by adding On Selective On Selective On Selective solution
containing 0.5 v/v % Ti: arrangement Ti: arrangement Ti:
arrangement surface activating 77 ratio: 6.0 150 ratio: 50.0 140
ratio: agent dropwise, followed On [Example 4] On [Example 5] On
140 .0 by addition of ferritin Pt: Pt: 3 Pt: 1 [Example solution
dropwise 13 6]
[0119] In addition, also in the cases in which 0.5 v/v % Tween 80
manufactured by ICI Inc., was added as the nonionic surface active
agent, similar results to those in Table 1 were entirely achieved.
Just for reference, scanning transmission electron micrographs of
the substrate surface in the case in which 0.5 v/v % Tween 20 or
Tween 80 was added in Example 3 are shown in FIGS. 9A and 9B,
respectively.
[0120] As in the foregoings, the adsorbing force between the
ferritin, and the Ti substrate and the Pt membrane of the substrate
surface could be controlled by adding a nonionic surface active
agent. In particular, use of the nonionic surface active agent in
combination with the peptide-modified ferritin enabled synergistic
control of the adsorption.
[0121] When the concentration of the added nonionic surface active
agent was less than 0.006 v/v %, controllability of adsorption of
the conventional ferritin and the peptide-modified ferritin was
reduced, resulting in to decrease of the selective arrangement
ratio. In contrast, when the concentration of the nonionic surface
active agent was beyond 10 v/v %, the amount of adsorption to the
Ti substrate was lowered. Therefore, judging from the practical
usefulness, the nonionic surface active agent in the solution
containing ferritin according to the present invention may be
preferably in the range of the concentration of 0.006 v/v % or
greater and 10 v/v % or less, and more preferably in the range of
the concentration of 0.01 v/v % or greater and 1 v/v % or less.
[0122] Meanwhile, Tween 20 and Tween 80 used herein as the nonionic
surface active agent are substances characterized by: belonging to
polyoxyethylene sorbitans (polyoxyethylene sorbitan alkyl esters),
being readily dissolved particularly at a low temperature, not
having a group dissociable into an ion in the aqueous solution, and
the hydrophilicity thereof being adjustable. General structural
formulae of Tween 20 and Tween 80 are shown below.
##STR00001##
Embodiment 2
[0123] Embodiment 2 of the present invention demonstrates a method
for arranging ferritin and inorganic particles on a substrate. In
this Embodiment, two kinds of substrates, i.e., a substrate having
a Ti membrane formed in a part on a SiO.sub.2 substrate, and a
substrate having a SiN membrane in a part on a SiO.sub.2 substrate
are used.
[0124] Specific examples of this Embodiment will be demonstrated by
way of Examples below, and the effect thereof will be explained
with reference to Comparative Examples.
EXAMPLE 7
[0125] In connection with Example 7, FIG. 10A shows a schematic
view illustrating results of an experiment in which
peptide-modified ferritin 300 including Fe.sub.2O.sub.3 301 therein
was arranged on a silicon oxide (SiO.sub.2) substrate 100 having a
titanium membrane (Ti membrane) 200 formed in a part of the
surface.
[0126] Peptide-modified ferritin 300 including Fe.sub.2O.sub.3 301
therein was adjusted to give the concentration of 2 mg/ml with a
buffer solution (10 mM Tris-HCl, pH 8.0), and thereto was further
added 0.5 v/v % Tween 20 manufactured by ICI Inc., as a nonionic
surface active agent. On the SiO.sub.2 substrate 100 having a Ti
membrane 200 formed in a part of the surface was added the
peptide-modified ferritin solution dropwise. After leaving to stand
at room temperature for 1 hour, the substrate was washed with pure
water. After washing, the substrate was subjected to a heat
treatment according to the method described above, thereby allowing
Fe.sub.2O.sub.3 301 to be fixed on the substrate.
[0127] FIG. 10B shows a scanning transmission electron micrograph
of the substrate surface after allowing the Fe.sub.2O.sub.3 301 to
be fixed as corresponded to FIG. 10A. Fe.sub.2O.sub.3 301 was
hardly arranged on the SiO.sub.2 substrate 100, but was selectively
arranged on the Ti membrane 200, therefore, it was verified that
the peptide-modified ferritin 300 did not adsorb on the SiO.sub.2
substrate 100 when it is coexistent with the nonionic surface
active agent but specifically adsorbed on the Ti membrane 200.
COMPARATIVE EXAMPLE 3
[0128] In Comparative Example 3, the inorganic particles were
arranged on the substrate through carrying out a similar operation
to that in Example 5 except that Tween 20 was not added.
[0129] FIGS. 11A and 11B show scanning transmission electron
micrographs of the substrate surface in Comparative Example 3. FIG.
11A shows the micrograph of the Ti membrane surface, while FIG. 11B
shows the micrograph of the SiO.sub.2 substrate surface.
Fe.sub.2O.sub.3 was arranged on both the SiO.sub.2 substrate
surface and the Ti membrane surface at almost the same level.
Hence, selectivity for the base material was not found at all. In
other words, under the condition in which the nonionic surface
active agent did not coexist, the peptide-modified ferritin did not
exhibit selective adsorptivity for the Ti membrane and the
SiO.sub.2 substrate.
[0130] Accordingly, the adsorbing force between the
peptide-modified ferritin, and the SiO.sub.2 substrate and the Ti
membrane of the substrate surface could be controlled by adding the
nonionic surface active agent. Although Tween 20 was used as the
nonionic surface active agent herein, the agent should not be
limited thereto. For example, also in the cases in which Tween 80
manufactured by ICI Inc., was added at the same concentration,
similar experimental results were achieved.
EXAMPLE 8
[0131] In Example 8, an experiment in which the peptide-modified
ferritin including Fe.sub.2O.sub.3 therein was arranged on a
silicon oxide (SiO.sub.2) substrate having a silicon nitride
membrane (SiN membrane) formed in a part of the surface was
conducted similarly to Example 7. The used buffer, nonionic surface
active agent and the concentration thereof and the like are the
same as in Example 7.
[0132] FIGS. 12A and 12B show scanning transmission electron
micrographs of the substrate surface in Example 8. FIG. 12A shows
the micrograph of the SiO.sub.2 substrate surface, while FIG. 12B
shows the micrograph of the SiN membrane surface. Because
Fe.sub.2O.sub.3 was not arranged on the SiO.sub.2 substrate but was
selectively arranged only on the SiN membrane, it was verified that
the peptide-modified ferritin did not adsorb on the SiO.sub.2
substrate in the presence of the nonionic surface active agent but
specifically adsorbed on the SiN membrane.
COMPARATIVE EXAMPLE 4
[0133] In Comparative Example 4, the inorganic particles were
arranged on the substrate through carrying out a similar operation
to that in Example 8 except that Tween 20 was not added.
[0134] FIGS. 13A and 13B show scanning transmission electron
micrographs of the substrate surface in Comparative Example 4. FIG.
13A shows the micrograph of the SiO.sub.2 substrate surface, while
FIG. 13B shows the micrograph of the SiN membrane surface.
Fe.sub.2O.sub.3 was arranged on both the SiO.sub.2 substrate
surface and the SiN membrane surface. Hence, selectivity for the
base material was not found at all. In other words, under the
condition in which the nonionic surface active agent was not
present, the peptide-modified ferritin did not exhibit selective
adsorptivity for the SiO.sub.2 substrate and the SiN membrane.
Accordingly, the adsorbing force between the peptide-modified
ferritin, and the SiO.sub.2 membrane and the SiN membrane of the
substrate surface could be controlled by adding the nonionic
surface active agent.
Embodiment 3
[0135] Embodiment 3 of the present invention demonstrates a method
for reverse-selective arrangement of ferritin and inorganic
particles on a substrate.
[0136] <Method for Reverse-Selective Arrangement of Inorganic
Particles Using Apoferritin>
[0137] In Embodiment 1 and 2, the method for arranging ferritin and
inorganic particles in the region where ferritin is specifically
adsorbed was explained. A method for arranging ferritin and
inorganic particles in a region other than the region where the
ferritin is specifically adsorbed in a reverse manner will be
explained with reference to FIGS. 14A to 14E.
[0138] First, in the step shown in FIG. 14A, a solution containing
peptide-modified ferritin (apoferritin) 84 without including
Fe.sub.2O.sub.3 therein and a nonionic surface active agent is
added dropwise to a substrate 80 having a specified region 81
configured with a certain inorganic material in a part of the
surface. Then, after incubation for a predetermined time period,
the substrate is washed with pure water.
[0139] Next, in the step shown in FIG. 14B, the apoferritin 84
adsorbs only in the specified region 81, thereby giving the
substrate 80 with selective arrangement.
[0140] Next, in the step shown in FIG. 14C, a solution containing
conventional ferritin including an inorganic particle therein 85 is
added dropwise to the substrate 80, and a similar operation to that
described above is carried out. In this step, any nonionic surface
active agent is not used.
[0141] Next, in the step shown in FIG. 14D, the conventional
ferritin 85 including an inorganic particle therein is adsorbed
only in a region 86 where arrangement of the inorganic particles is
required which is a region other than the specified region 81 where
the apoferritin 84 was already adsorbed.
[0142] Thereafter, in the step shown in FIG. 14E, the substrate 80
is subjected to a heat treatment according to the method described
above, whereby obtaining a substrate 87 having inorganic particle
88 reverse-selectively arranged in the region other than the
specified region 81.
[0143] The protein including an inorganic particle therein is not
limited to ferritin but other type of protein can be used. Also, in
place of the ferritin including an inorganic particle therein, a
protein without including an inorganic particle therein can be also
reverse-selectively arranged. This technique shall be useful in the
cases in which, for example, an enzyme having a certain function is
arranged in a specified region on a substrate to manufacture a
biosensor.
[0144] In addition, Fe.sub.2O.sub.3 is selectively arranged in a
specified region on a substrate using the ferritin including
Fe.sub.2O.sub.3 therein as an inorganic particle in the above
Embodiments, however, just the same results shall be achieved when
ferritin without including the inorganic particle therein is
used.
[0145] From the foregoing description, many modifications and other
embodiments of the present invention will be apparent to persons
skilled in the art. Therefore, the foregoing description should be
construed as merely illustrative exemplification, which was
provided for the purpose of teaching the best embodiment for
carrying out the present invention to persons skilled in the art.
Details of the constitution and/or function of the present
invention can be substantially altered without departing from the
spirit thereof.
[0146] The present invention relates to a method for arranging
ferritin or inorganic particles on a substrate surface with high
mass productivity and favorable cost performances. In particular, a
technique for selectively arranging inorganic particles having a
diameter of several to several ten nm in a region where required,
or for regularly arranging them in a nano-region. According to this
technique, arrangement of fine particles of an inorganic material
on a required base material in a self-selective manner on a
nano-scale is enabled. The technique can be applied in manufacture
steps in industrial fields of catalysts, sensors, biochips,
transistors, semiconductor lasers, magnetic discs, displays and the
like.
Sequence CWU 1
1
41528DNAEquus caballus 1atgagctccc agattcgtca gaattattct actgaagtgg
aggccgccgt caaccgcctg 60gtcaacctgt acctgcgggc ctcctacacc tacctctctc
tgggcttcta tttcgaccgc 120gacgatgtgg ctctggaggg cgtatgccac
ttcttccgcg agttggcgga ggagaagcgc 180gagggtgccg agcgtctctt
gaagatgcaa aaccagcgcg gcggccgcgc tctcttccag 240gacttgcaga
agccgtccca ggatgaatgg ggtacaaccc cagacgccat gaaagccgcc
300attgtcctgg agaagagcct gaaccaggcc cttttggatc tgcatgccct
gggttctgcc 360caggcagacc cccatctctg tagcttcttg tctagccact
tcctagacga ggaggtgaaa 420ctcatcaaga agatgggcga ccatctgacc
aacatccaga ggctcgttgg ctcccaagct 480gggctgggcg agtatctctt
tgaaaggctc actctcaagc acgactaa 5282175PRTArtificialChemically
Synthesized 2Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Glu Val
Glu Ala Ala1 5 10 15Val Asn Arg Leu Val Asn Leu Tyr Leu Arg Ala Ser
Tyr Thr Tyr Leu 20 25 30Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val
Ala Leu Glu Gly Val 35 40 45Cys His Phe Phe Arg Glu Leu Ala Glu Glu
Lys Arg Glu Gly Ala Glu 50 55 60Arg Leu Leu Lys Met Gln Asn Gln Arg
Gly Gly Arg Ala Leu Phe Gln65 70 75 80Asp Leu Gln Lys Pro Ser Gln
Asp Glu Trp Gly Thr Thr Pro Asp Ala 85 90 95Met Lys Ala Ala Ile Val
Leu Glu Lys Ser Leu Asn Gln Ala Leu Leu 100 105 110Asp Leu His Ala
Leu Gly Ser Ala Gln Ala Asp Pro His Leu Cys Ser 115 120 125Phe Leu
Ser Ser His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys 130 135
140Met Gly Asp His Leu Thr Asn Ile Gln Arg Leu Val Gly Ser Gln
Ala145 150 155 160Gly Leu Gly Glu Tyr Leu Phe Glu Arg Leu Thr Leu
Lys His Asp 165 170 1753168PRTEquus caballus 3Met Tyr Ser Thr Glu
Val Glu Ala Ala Val Asn Arg Leu Val Asn Leu1 5 10 15Tyr Leu Arg Ala
Ser Tyr Thr Tyr Leu Ser Leu Gly Phe Tyr Phe Asp 20 25 30Arg Asp Asp
Val Ala Leu Glu Gly Val Cys His Phe Phe Arg Glu Leu 35 40 45Ala Glu
Glu Lys Arg Glu Gly Ala Glu Arg Leu Leu Lys Met Gln Asn 50 55 60Gln
Arg Gly Gly Arg Ala Leu Phe Gln Asp Leu Gln Lys Pro Ser Gln65 70 75
80Asp Glu Trp Gly Thr Thr Pro Asp Ala Met Lys Ala Ala Ile Val Leu
85 90 95Glu Lys Ser Leu Asn Gln Ala Leu Leu Asp Leu His Ala Leu Gly
Ser 100 105 110Ala Gln Ala Asp Pro His Leu Cys Ser Phe Leu Ser Ser
His Phe Leu 115 120 125Asp Glu Glu Val Lys Leu Ile Lys Lys Met Gly
Asp His Leu Thr Asn 130 135 140Ile Gln Arg Leu Val Gly Ser Gln Ala
Gly Leu Gly Glu Tyr Leu Phe145 150 155 160Glu Arg Leu Thr Leu Lys
His Asp 16547PRTArtificialChemically Synthesized 4Ser Ser Gln Ile
Arg Gln Asn1 5
* * * * *