# Patent

## Title
Method of two-dimensionally arraying ferritin on substrate

## Abstract

A novel method for two-dimensionally arraying ferritin on a substrate is provided which obviates the need for a metal ion that permits linking between adjacent two ferritin particles. In a method of two-dimensionally arraying ferritin on a substrate, the surface of the substrate is hydrophilic, and the method includes the steps of: developing a solution containing a solvent and the ferritin on the substrate; and removing the solvent from the solution developed on the substrate, while the ferritin has an amino acid sequence set out in SEQ ID NO: 1 modified at its N-terminus.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of two-dimensionally arraying ferritin on a substrate.
2. Description of Related Art
Ferritin is a spherical protein that includes a metal compound therein which is typified by iron oxide. When any metal compound is not included therein but has a hollow space inside, ferritin is referred to as “apoferritin”.
Quantum dots of a metal that is two-dimensionally arrayed on a substrate can be readily obtained by two-dimensionally arraying ferritin on the substrate followed by removing the ferritin by heating, and reducing metal oxide if necessary. Therefore, two-dimensionally arraying ferritin on a substrate was reported as shown in FIG. 1 (for example, see Pamphlet of International Publication No. 03/040025 and Langmuir, Vol. 23, pp. 1615-1618, (2007)).
SUMMARY OF THE INVENTION
According to the method disclosed in Pamphlet of International Publication No. 03/040025, as shown in FIG. 87, a bivalent metal ion (in FIG. 87, cadmium ion) serves in crosslinkage between adjacent two particles of ferritin 15. After ferritin is removed by heating, this bivalent metal ion remains on the substrate as an impurity. The impurity is supposed to migrate on the substrate in the form of an ion, therefore, an unexpected interface state may be generated due to such an impurity in the quantum dots composed of a two-dimensional array of a metal on a substrate. As a consequence, the impurity adversely affects the quantum dots.
According to the method of Pamphlet of International Publication No. 03/040025, a two-dimensional crystal is formed without using a bivalent metal ion; however, it is necessary to allow a particular peptide (carbon nano material-recognizing peptide, SEQ ID NO: 5) to be presented to the external surface of ferritin, as shown in FIG. 88.
The present invention provides a method of two-dimensionally arraying ferritin on a substrate without such adverse influences.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a schematic view illustrating a state which multiple particles of ferritin form a two-dimensional array on a substrate (FIG. 8 in Pamphlet of International Publication No. 03/040025).
FIG. 2 shows a cross-sectional view of a three-dimensional array.
FIG. 3 shows a schematic view illustrating a Fourier transformation image obtained by two-dimensional Fourier transformation.
FIG. 4 shows a schematic view illustrating a Fourier transformation image obtained by two-dimensional Fourier transformation.
FIG. 5 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Example 1.
FIG. 6 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Example 1.
FIG. 7 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Example 2.
FIG. 8 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Example 2.
FIG. 9 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Example 3.
FIG. 10 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Example 3.
FIG. 11 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Example 4.
FIG. 12 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Example 4.
FIG. 13 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 1.
FIG. 14 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 1.
FIG. 15 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 2.
FIG. 16 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 2.
FIG. 17 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 3.
FIG. 18 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 3.
FIG. 19 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 4.
FIG. 20 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 4.
FIG. 21 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 5.
FIG. 22 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 5.
FIG. 23 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 6.
FIG. 24 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 6.
FIG. 25 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 7.
FIG. 26 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 7.
FIG. 27 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 8.
FIG. 28 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 8.
FIG. 29 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 9.
FIG. 30 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 9.
FIG. 31 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 10.
FIG. 32 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 10.
FIG. 33 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 11.
FIG. 34 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 11.
FIG. 35 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 12.
FIG. 36 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 12.
FIG. 37 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 13.
FIG. 38 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 13.
FIG. 39 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 14.
FIG. 40 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 14.
FIG. 41 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 15.
FIG. 42 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 15.
FIG. 43 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 16.
FIG. 44 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 16.
FIG. 45 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 17.
FIG. 46 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 17.
FIG. 47 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 18.
FIG. 48 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 18.
FIG. 49 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 19.
FIG. 50 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 19.
FIG. 51 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 20.
FIG. 52 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 20.
FIG. 53 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 21.
FIG. 54 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 21.
FIG. 55 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 22.
FIG. 56 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 22.
FIG. 57 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 23.
FIG. 58 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 23.
FIG. 59 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 24.
FIG. 60 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 24.
FIG. 61 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 25.
FIG. 62 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 25.
FIG. 63 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 26.
FIG. 64 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 26.
FIG. 65 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 27.
FIG. 66 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 27.
FIG. 67 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 28.
FIG. 68 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 28.
FIG. 69 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 29.
FIG. 70 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 29.
FIG. 71 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 30.
FIG. 72 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 30.
FIG. 73 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 31.
FIG. 74 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 31.
FIG. 75 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 32.
FIG. 76 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 32.
FIG. 77 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 33.
FIG. 78 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 33.
FIG. 79 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 34.
FIG. 80 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 34.
FIG. 81 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 35.
FIG. 82 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 35.
FIG. 83 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 36.
FIG. 84 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 36.
FIG. 85 shows a photograph illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 37.
FIG. 86 shows a photograph of Fourier transformation image illustrating the appearance of a two-dimensional array of ferritin obtained in Comparative Example 37.
FIG. 87 shows a schematic view illustrating a state in which crosslinking between two adjacent particles of ferritin is effected via a bivalent metal ion (cadmium ion in FIG. 87) which had been shown in FIG. 8 in Pamphlet of International Publication No. 03/040025.
FIG. 88 shows a schematic view illustrating modified ferritin (N1-LF) which had been described in Langmuir, Vol. 23, pp. 1615-1618, (2007), SEQ ID 5 AND SEQ ID 6 with methionine.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In order to solve the aforementioned problems, the present invention provides a method of two-dimensionally arraying ferritin on a substrate, wherein the surface of the substrate is hydrophilic,
the method comprising the steps of:
developing a solution containing a solvent and the ferritin on the substrate, and removing the solvent from the solution developed on the substrate, wherein the ferritin has an amino acid sequence set out in SEQ ID NO: 1 modified at its N-terminus.
The surface of the substrate is preferably covered by SiO2.
The solution preferably includes at least one of PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), ammonium sulfate, and ammonium acetate.
When the solution includes PIPES, the concentration is preferably not lower than 5 mM and not higher than 50 mM.
In addition, when the solution includes ammonium sulfate, the concentration is preferably not lower than 6.5 mM and not higher than 52 mM.
Moreover, when the solution includes ammonium acetate, the concentration is preferably not lower than 2 mM and not higher than 100 mM.
The solution is preferably adjusted to have a pH falling within the range of not lower than 6.0 and not higher than 8.0.
According to the present invention, there exists no metal ion for binding between adjacent two ferritin particles. Therefore, adverse influences typified by appearance of unexpected interface state on the quantum dot provided by two-dimensionally arraying a metal on a substrate can be suppressed.
Hereinafter, the present invention is described in more detail.
The ferritin which may be used in the present invention has an amino acid sequence represented by: DYYSSSYYEYYS (hereinafter, SEQ ID NO: 1) on the outer peripheral surface. As one example, ferritin used in the present invention is a protein set out in SEQ ID NO: 2. This protein has 187 residues, in which an amino acid sequence of 13 residues that include methionine corresponding to an initiation codon and the amino acid sequence of SEQ ID NO: 1 was added to an amino acid sequence (SEQ ID NO: 6) of ferritin derived from horse having 174 residues at its amino terminal.
In Experimental Examples described later, ferritin used in the present invention is designated as “Y6S4DE-Fer0”. In the case of apoferritin, the designation is made as “apoY6S4DE-Fer0”. In Experimental Examples described later, other apoferritin is also designated with the name of ferritin preceded by a representation “apo”.
Conventional ferritin does not have the amino acid sequence set out in SEQ ID NO: 1. In a conventional example of the ferritin (see, Langmuir, Vol. 23, pp. 1615-1618, (2007)), an amino acid sequence of 13 residues that include methionine corresponding to an initiation codon and a peptide sequence: DYFSSPYYEQLF (SEQ ID NO: 5) is added to an amino acid sequence (SEQ ID NO: 6) of ferritin derived from horse having 174 residues at its amino terminal. As will be also comprehended from Comparative Example described later, the two-dimensional array on a substrate cannot be effected even though conventional ferritin modified at random in the amino acid sequence corresponding to SEQ ID NO: 5 is used. In Experimental Example described later, the conventional ferritin is designated as “N1-LF”.
The term “two-dimensional array” as used herein means an array as shown in the schematic view of FIG. 1, in which multiple particles of ferritin 15 are arranged regularly on substrate 11 viewed in a plane, and a single-layered ferritin film is formed by the multiple particles of ferritin 15 viewed in a cross section.
The array in which a ferritin film including two or more layers as shown in the cross-sectional view of FIG. 2 is not involved in the term “two-dimensional array”. Such an array is referred to as “three-dimensional array” as needed, which is herein distinguished from the term “two-dimensional array”. However, to exclude also the case of the single layered ferritin film that includes a three-dimensional array just in part (i.e., locally) from the term “two-dimensional array” is not intended.
In the method of evaluating “two-dimensional array” herein, an SEM observation image (300 nm×300 nm) including ferritin is subjected to two-dimensional Fourier transformation, and the resulting Fourier transformation image is used. The two-dimensional array in the state of hexagonal closest packing as shown in the schematic view of FIG. 1 has been known to be capable of approximating to overlapping three sinusoidal waves oriented differently by 60 degrees, respectively. In the Fourier transformation image of the two-dimensional array described above, six points corresponding to the three sinusoidal waves appear as shown in FIG. 3. Each point is located at a position provided in rotating 60 degrees each around the point at which the wave number is zero. When the aforementioned approximation is not established (i.e., no two-dimensional array being present), the six points do not appear in the Fourier transformation image.
In the case in which the periodicity of the two-dimensional array shown in the schematic view of FIG. 1 was disorganized (i.e., the case in which the two-dimensional array was deteriorated), or in the case of polycrystalline in which the two-dimensional array was constituted with not monocrystals composed of single crystal alone but multiple crystal grain aggregates, the distinct six points would not appear in the Fourier transformation image, but the points corresponding just to the number of differently oriented polycrystallines appear along the concentric circle, or otherwise a circular pattern may appear as shown in FIG. 4 in an extreme case. Therefore, the case in which distinct six points appear as shown in the schematic view of FIG. 3 is evaluated as “favorable two-dimensional array”; the case in which a “two-dimensional array” is involved in an original SEM observation image but distinct six points do not appear therein as shown in the schematic view of FIG. 4 is evaluated as “inferior two-dimensional array”; and other cases are evaluated as “two-dimensional array not formed”.
The surface of the substrate is hydrophilic. An Si substrate can be used as the substrate.
The hydrophilicity can be imparted to the surface by oxidizing the surface of the Si substrate to give SiO2. In this case, the surface of the substrate has a slightly negative potential.
The method of two-dimensionally arraying ferritin on a substrate according to the present invention has a development step and a removal step. The development step is explained first.
(1) Development Step
In the development step, a solution that contains a solvent, the ferritin, and 12.5 mM PIPES-NaOH (pH 7.0), 12.5 mM PIPES-Tris (pH 7.0), 13 mM ammonium sulfate ((NH4)2SO4), or 20 mM ammonium acetate (CH3CO2NH4) is developed on the substrate. When PIPES (weakly acidic) is to be used as the buffer, the pH is adjusted with NaOH (thus prepared buffer is referred to as “PIPES-NaOH”). Since a metal ion can be an impurity in the present invention, to employ a weakly alkaline buffer, the pH of which is adjusted with an acid such as HCl is more desired.
Specific examples of the process for the development also include the following processes in addition to the process of dropwise addition of the solution on the substrate. More specifically, the solution is added dropwise on a thin film typified by Parafilm, and then the substrate is calmly placed on the solution with the hydrophilic face down. In other words, the solution is sandwiched between the thin film typified by Parafilm, and the substrate with the hydrophilic face down.
(2) Removal Step
Next, the removal step is explained. In the removal step, the solvent is removed from the solution developed on the substrate. Because the solution is typically a buffer, the solvent will be almost water accounting for a major portion of the buffer. Hence, the process for removing water from the substrate is explained in this section.
Specific examples of the process for removing the solvent include a process in which the substrate is subjected to centrifugal separation, as well as a process in which the solvent is evaporated from the substrate, and the like. In light of rapid removal of the solvent, the process in which the substrate is subjected to centrifugal separation is preferred. In any case, the process is acceptable as long as water is removed from the substrate in the removal step, which may include drying and concentration, irrespective of the procedure.
In the manner described above, ferritin can be two-dimensionally arrayed on the substrate. When a quantum dot is to be obtained, in general, thus two-dimensionally arrayed ferritin is heated to eliminate the protein on the external side, and the metal oxide is further reduced as needed, whereby the quantum dot formed by two-dimensionally arraying the metal on the substrate can be readily obtained.
Also, it should be noted that the metal can be substituted with a compound semiconductor (see, pamphlet of International Publication No. 03/099008).
EXAMPLES
Hereinafter, the present invention will be explained in more detail with reference to Experimental Examples.
In the present Experimental Example, the reagents presented in Table 1 below were used.










TABLE 1





Abbreviated





name
Trade name
Cat. No.
Lot No.



















Nde I
Nde I
R0111L

New England






Biolabs


Sac I
Sac I
R0156L

New England






Biolabs


x 10 NEB 4
x 10 NEB 4 buffer


New England


buffer



Biolabs


Tris
Trizma base
76066-500G
025K5432
SIGMA-ALDRICH


HEPES
HEPES
342-01375
SF076
Dojindo






Laboratories


AIS
Ammonium iron (II)
091-00855
CEK7339
Wako Pure



sulfate


Chemical



hexahydrate


Industries,






Ltd.


Indium
Indium (III)
20020-32
408C2100
Wako Pure


sulfate
sulfate


Chemical






Industries,






Ltd.


NaH2PO4
Disodium
197-09705
CEJ1855
Wako Pure



hydrogenphosphate


Chemical



(anhydride)


Industries,






Ltd.


NH3
1 mol/L Aqueous
01793-08

KANTO



ammonia (1N)


CHEMICAL CO.,






INC.


Ammonium
Ammonium Sulfate
204501-50G
06810PB
SIGMA-ALDRICH


sulfate
99.999%


Ammonium
Ammonium acetate
019-02835

Wako Pure


acetate



Chemical






Industries,






Ltd.


PIPES
PIPES BioUltra,
80635-250G

Fluka



for molecular



biology, ≧99.5%



(T)









In the present Experimental Example, ferritin having an amino acid sequence including a peptide sequence consisting of the amino acid sequence shown in Table 2 below and methionine corresponding to an initiation codon introduced to an amino acid sequence (SEQ ID NO: 6) of ferritin derived from horse having residues at its amino terminal was used. The DNA sequence corresponding to each amino acid sequence is as listed in Table 2. In the present Experimental Example, each ferritin is designated as “peptide name-Fer0”.










TABLE 2





Peptide
Amino acid




name
sequence
DNA sequence (5′ → 3′)







Y6S4DE
DYYSSSYYEYYS
GAT TAT TAT TCG AGC TCG




(SEQ ID NO: 1)
TAT TAT GAA TAT TAT TCT




(SEQ ID NO: 19)


 


E10S
DYFSSPYYSQLF
GAT TAT TTC TCG AGC CCG



(SEQ ID NO: 7)
TAT TAT TCA CAG CTG TTT




(SEQ ID NO: 20)


 


D2N
NYFSSPYYEQLF
AAT TAT TTC TCG AGC CCG



(SEQ ID NO: 8)
TAT TAT GAA CAG CTG TTT




(SEQ ID NO: 21)


 


E10Q
DYFSSPYYQQLF
GAT TAT TTC TCG AGC CCG



(SEQ ID NO: 9)
TAT TAT CAG CAG CTG TTT




(SEQ ID NO: 22)


 


P7S
DYFSSSYYEQLF
GAT TAT TTC TCG AGC TCG



(SEQ ID NO: 10)
TAT TAT GAA CAG CTG TTT




(SEQ ID NO: 23)


 


Shuffle
FQYLYSYPFESD
TTC CAG TAT CTG TAT TCG



(SEQ ID NO: 11)
TAT CCG TTT GAA AGC GAT




(SEQ ID NO: 24)


 


S5T-S6T
DYFTTPYYEQLF
GAT TAT TTC ACT ACT CCG



(SEQ ID NO: 12)
TAT TAT GAA CAG CTG TTT




(SEQ ID NO: 25)


 


ΔAR
DSASSPSSEQLA
GAT TCG GCT TCG AGC CCG



(SEQ ID NO: 13)
TCG TCG GAA CAG CTG GCT




(SEQ ID NO: 26)


 


Y8F-Y9F
DYFSSPFFEQLF
GAT TAT TTC TCG AGC CCG



(SEQ ID NO: 14)
TTT TTT GAA CAG CTG TTT




(SEQ ID NO: 27)


 


ΔHY
DFFAAPFFEQLF
GAT TTT TTC GCC GCC CCG



(SEQ ID NO: 15)
TTT TTT GAA CAG CTG TTT




(SEQ ID NO: 28)


 


1st half
DYFSSP
GAT TAT TTC TCG AGC CCG



(SEQ ID NO: 16)
(SEQ ID NO: 29)


 


2nd half
YYEQLF
TAT TAT GAA CAG CTG TTT



(SEQ ID NO: 17)
(SEQ ID NO: 30)


 


5AA
DYSSY
GAT TAT TCG AGC TAT



(SEQ ID NO: 18)
(SEQ ID NO: 31)









Preparation 1
Construction of Protein Expression Vector
First, a procedure for constructing a protein expression vector is illustrated in the following.
1. A solution of a plasmid vector pKIS9 (SEQ ID NO: 4) for protein expression was prepared. If necessary, the plasmid vector was isolated and purified from Escherichia coli carrying the plasmid vector (see, also QIAprep_Miniprep Protocol and Trouble Shooting available from QIAGEN).
2. An absorbance of the plasmid solution at a wavelength involving 260 nm was measured with a UV/VIS spectrometer (ND-1000, NanoDrop Technologies, LLC) to determine the concentration of DNA.
3. To 1.5 ml Eppendorf tube which had been subjected to autoclave sterilization were added 2 μL of ×10 NEB 4 buffer, 15 μL of a 148 ng/μL pKIS9 plasmid DNA solution (TE Buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), 1 μL of a Sac I solution, and 1 μL of a Nde I solution. Thus resulting solution was mixed by pipetting, and allowed to react at 37° C. for 1 hour.
4. The reaction mixture was electrophoresed on 2% (w/v) agarose gel, and the target plasmid DNA fragment was separated and extracted from the agarose gel (see, also MinElute Gel Extraction Kit Protocol and Trouble Shooting available from QIAGEN).
5. An absorbance of the DNA fragment solution at a wavelength involving 260 nm was measured with a UV/VIS spectrometer (ND-1000, NanoDrop Technologies, LLC) to determine the concentration of DNA.
6. A single strand oligo DNA was synthesized in by adding an Nde I restriction enzyme cleavage site the 5′ end and a SaI restriction enzyme cleavage site was the 3′ end of a DNA sequence encoding a desired amino acid sequence. Concomitantly, an oligo DNA complementary to the aforementioned single strand oligo DNA was synthesized. The single strand oligo DNA was obtained from Sigma Genosys or Takara Bio Inc.
7. Each same volume of 10 μL of 5 μM single strand oligo DNA solution to be inserted and 5 μM complementary DNA chain solution were mixed, and annealed at 95° C. for 3 min. Thereafter, the mixture was cooled at room temperature to produce a double strand oligo DNA having an Nde I/Sac I site at the end.
8. A pKIS9 plasmid DNA fragment and a double strand oligo DNA were joined to produce a plasmid vector for protein expression (each for expression of ferritin) (see, Manual of DNA Ligation System DNA Ligation Kit Ver. 2.1, available from TaKaRa).
Preparation 2-1
Large Scale Expression and Purification of Y6S4DE-Fer0
First, synthesis and purification procedures of apo Y6S4DE-Fer0 are shown below. The synthesis and purification procedures of Shuffle-Fer0, D2N-Fer0, E10Q-Fer0, E10S-Fer0, P7S-Fer0, HY-Fer0, 2nd half-Fer0, Y8F-Y9F-Fer0, S5T-S6T-Fer0, and NF-LF are also similar except that the N-terminal region of the used plasmid vectors for protein expression has each base sequence shown in Table 2.
1. A plasmid vector for protein expression (SEQ ID NO: 3) was introduced into Escherichia coli XL1-blue (NOVAGENE) to perfect the transformation (see, also ECOS TM Competent E. coli DH5α, JM109, XL1-Blue, BL21 (DE3) Manual (ver. 6) available from NIPPON GENE CO., LTD.).
2. A colony of the transformed Escherichia coli was subjected to shaking culture (apparatus: TAITEC Bio Shaker BR-40LF, preset temperature: 37° C., culture period: 5 to 7 hrs, shaking speed: 120 rpm) in 1 ml of an LB medium (containing 50 μg/ml ampicillin) charged in a 15-ml sterilized Corning tube.
3. The aforementioned culture solution (0.1 to 0.5 ml) was subjected to shaking culture in 50 ml of an LB medium (containing 50 μg/ml ampicillin) in a 500-ml Erlenmeyer flask at 37° C. for 16 to 20 hrs.
4. The turbidity of the medium was measured with a spectrophotometer (Ultrospec 3100 pro, GE Healthcare Bioscience). At a time point when it reached to OD600: 0.1 to 0.5, 50 ml of the aforementioned culture solution was subjected to a spinner culture (apparatus: ABLE BMS-10/05, preset temperature: 37° C., shaking speed: 200 rpm, air flow rate: 4 L/min, culture period: 18 to 20 hrs) in 6 L of an LB medium (containing 100 μg/ml ampicillin).
5. The turbidity of the medium was measured, and after OD600 of 4.0 to 5.0 was ascertained, the bacterial body was harvested with a low-speed centrifuge (Model: Avanti HP-25, rotorModel: JA-10, BECKMAN, preset temperature: 4° C., preset number of revolutions: 8,000 rpm, time: 10 min) in a centrifuge tube for JA-10.
6. Thus harvested bacterial body was suspended in 50 mM Tris-HCl (200 ml to 300 ml), and collected in a centrifuge tube for JA-10 low-speed centrifuge (similarly to the above section 5).
7. The harvested bacterial body was suspended in 50 mM Tris-HCl (120 ml), placed in ice, and the cells were disrupted with an ultrasonic disintegrator (apparatus: Branson Digital Sonifier 450, preset output value: 140 W, pulse setting: on/off 1 sec, disintegration time: 2 min×3).
8. The mixture was centrifuged with a low-speed centrifuge (Model: Avanti HP-25, rotor Model: JA-20, BECKMAN, preset temperature: 4° C., preset centrifugal force: 6,000×g, time: 10 min), and the supernatant was collected.
9. The collected supernatant was subjected to a heat treatment (75° C., 20 min), and was left to stand at room temperature following the heat treatment until the temperature dropped to an ordinary temperature (approximately for 1 hour).
10. The supernatant was centrifuged with a low-speed centrifuge (similar to the above section 8), and the supernatant was collected.
11. To thus collected supernatant was added 5 M NaCl to give a final concentration of 0.5 M NaCl, and then suspended.
12. The suspension was centrifuged with a low-speed centrifuge (similar to the above section 8), and the precipitate was collected.
13. The collected precipitate was suspended in 50 mM Tris-HCl (120 ml), and thereto was added 10.54 ml of 5 M NaCl to give a final concentration of 0.4 M NaCl. Thus resulting mixture was suspended.
14. The suspension was centrifuged with a low-speed centrifuge (similar to the above section 8), and the precipitate was collected.
15. After the precipitate was collected, the operations of 13 to 14 were repeated again.
16. The precipitate was suspended in 50 mM Tris-HCl (60 ml), and passed through a 0.22-μm syringe filter to complete the purification.
Preparation 2-2
Large Scale Expression and Purification of apoΔAR-Fer0
First, synthesis and purification procedures of apoΔAR-Fer0 are shown below. The synthesis and purification procedures of 1st half-Fer0, and 5AA-Fer0 are also similar except that the N-terminal region of the used plasmid vectors for protein expression has each base sequence shown in Table 2.
1. A plasmid vector for protein expression was introduced into Escherichia coli XL1-blue (NOVAGENE) to perfect the transformation (see, also ECOS TM Competent E. coli DH5α, JM109, XL1-Blue, BL21 (DE3) Manual (ver. 6) available from NIPPON GENE CO., LTD.).
2. A colony of the transformed Escherichia coli was subjected to shaking culture (apparatus: TAITEC Bio Shaker BR-40LF, preset temperature: 37° C., culture period: 5 to 7 hrs, shaking speed: 120 rpm) in 1 ml of an LB medium (containing 50 μg/ml ampicillin) charged in a 15-ml sterilized Corning tube.
3. The aforementioned culture solution (0.1 to 0.5 ml) was subjected to shaking culture in 50 ml of an LB medium (containing 50 μg/ml ampicillin) in a 500-ml Erlenmeyer flask at 37° C. for 16 to 20 hrs.
4. The turbidity of the medium was measured with a spectrophotometer (Ultrospec 3100 pro, GE Healthcare Bioscience). At a time point when it reached to OD600: 0.1 to 0.5, 50 ml of the aforementioned culture solution was subjected to a spinner culture (apparatus: ABLE BMS-10/05, preset temperature: 37° C., shaking speed: 200 rpm, air flow rate: 4 L/min, culture period: 18 to 20 hrs) in 6 L of an LB medium (containing 100 μg/ml ampicillin).
5. The turbidity of the medium was measured, and after OD600 of 4.0 to 5.0 was ascertained, the bacterial body was harvested with a low-speed centrifuge (Model: Avanti HP-25, rotorModel: JA-10, BECKMAN, preset temperature: 4° C., preset number of revolutions: 8,000 rpm, time: 10 min) in a centrifuge tube for JA-10.
6. Thus harvested bacterial body was suspended in 50 mM Tris-HCl (200 ml to 300 ml), and collected in a centrifuge tube for JA-10 low-speed centrifuge (similarly to the above section 5).
7. The harvested bacterial body was suspended in 50 mM Tris-HCl (120 ml), placed in ice, and the cells were disrupted with an ultrasonic disintegrator (apparatus: Branson Digital Sonifier 450, preset output value: 140 W, pulse setting: on/off 1 sec, disintegration time: 2 min×3).
8. The mixture was centrifuged with a low-speed centrifuge (Model: Avanti HP-25, rotor Model: JA-20, BECKMAN, preset temperature: 4° C., preset centrifugal force: 6,000×g, time: 10 min), and the supernatant was collected.
9. The collected supernatant was subjected to a heat treatment (75° C., 20 min), and was left to stand at room temperature following the heat treatment until the temperature dropped to an ordinary temperature (approximately for 1 hour).
10. The supernatant was centrifuged with a low-speed centrifuge (similar to the above section 8), and the supernatant was collected.
11. However, when the disrupted product does not precipitate sufficiently, it was centrifuged with a low-speed centrifuge (Model: Avanti HP-25, rotorModel: JA-20, BECKMAN, preset temperature: 4° C., preset centrifugal force: 15,000×g, time: 10 min), and the supernatant was collected.
12. The disruption supernatant was filtrated with a 0.22 μm filter (MilieX-GV Cat. No. SLGV033RS, Millipore) to give a filtrate 1.
13. An anion exchange column HiLoad-26/10 Q Sepharose-HP (GE Healthcare) was connected to an HPLC system for protein purification (AKTA explorer 10S, GE Healthcare).
14. Using 50 mM Tris-HCl buffer, pH 8.0 as a starting mobile phase (buffer A), 400 ml or more of the buffer was fed at a flow rate of 7.0 ml per min beforehand.
15. The aforementioned filtrate 1 was charged in a sample loop, and injected into the column at a flow rate of 7 ml per min.
16. Using 50 mM Tris-HCl buffer, pH 8.0 as a starting mobile phase (buffer A), 400 ml or more of the buffer was fed at a flow rate of 7.0 ml per min to remove the unabsorbed fraction.
17. Using 50 mM Tris-HCl buffer, pH 8.0 containing 1 M NaCl as a gradient mobile phase (buffer B), elution of the protein fraction was performed with gradient from 10 to 60% of the mixing ratio with the buffer A with ten times volume (530.93 ml) of the column bed volume (53.093 ml).
18. The target protein was eluted at around a mixing ratio of approximately 30%.
19. From each fraction involving the mixing ratio of 30%, 10 μL of an aliquot was taken, and SDS-PAGE was performed using an SDS-PAGE gel at an acrylamide concentration of 12.5%.
20. The target protein fraction was determined by SDS-PAGE, and the fraction was transferred to a ultrafiltration apparatus (Centriprep YM30, Millipore).
21. The Centriprep YM30 was placed into a swing rotor of a centrifuge (LC-200 TOMY, preset temperature: 4° C.).
22. The fraction was concentrated by repeating the centrifugal separation at 3,000 rpm until the volume of the solution left in the collection tube was reduced to no greater than 3 ml.
23. The concentrated solution was taken from the collection tube.
24. When the purity of the target protein was more inferior as compared with the purification procedure shown in Preparation 2-1, gel filtration was performed by the following procedure.
25. A Tricorn 10/600 column (GE Healthcare) packed with a TSK-GEL BIOASSIST G4SWXL resin (Tosoh Corporation) was connected to a general-purpose HPLC system (8020 series Tosoh Corporation).
26. Using 50 mM Tris-HCl buffer, pH 8.0 containing 150 mM NaCl as a mobile phase, 100 ml or more of the buffer was fed at a flow rate of 0.5 ml per min beforehand.
27. The concentrated solution 1 of not more than 3 ml was charged in a sample loop, and injected into the column at a flow rate of 0.5 ml per min.
28. Monitoring was carried out with a UV/VIS detector (UV-8020 Tosoh Corporation) at a wavelength of 280 nm, and the results were recorded using a software (LC-8020 Model IITosoh Corporation) installed in PC for regulation.
29. The eluate was collected each 1.0 ml with a fraction collector (FC-8020 Tosoh Corporation), and the fraction including the target protein was collected.
30. The concentration of thus resulting protein solution was unknown.
Preparation 3
Determination of Protein Concentration
The concentration of the protein solution obtained by the aforementioned large-scale expression and purification (for example, solution containing apoY6S4DE0-Fer0) was unknown. Thus, the concentration of the protein solution having an unknown concentration was measured according to the following method.
The determination of the protein concentration was carried out according to a Lowry method using a DC protein assay kit (Cat. No. 500-0112JA, BioRad).
1. As a standard protein, a solution of BSA (Bovine Albumin Serum, Cat. No. 23209 PIERCE) having a known concentration diluted with ultrapure water to give certain concentrations (0.2, 0.4, 0.6, 1.0, 2.0 mg/ml) was used.
2. The reaction mixture was produced by the following procedure. The protein solution (or ultrapure water as a control) in a volume of 25 μl and 125 μl of the reagent A were charged in a microtube and mixed.
3. Subsequently, 1 ml of the reagent B was charged in the same microtube and mixed, and then the reaction was allowed at room temperature, 25 (±1)° C. for not shorter than 15 min.
4. After completing the reaction, the absorbance was measured within one hour at a wavelength of 750 nm with a spectrophotometer (Ultrospec 3100 pro, GE Healthcare Bioscience).
5. The absorbance at 750 nm of the BSA solutions was plotted with respect to the protein concentration, and a formula of:

(Protein concentration of unknown sample)=A (Absorbance of unknown sample at 750 nm)+C 

was derived by a least square method.

6. The protein concentration of an arbitrarily diluted sample solutions was determined according to the aforementioned procedure, and the determined protein concentration was multiplied by the dilution ratio to derive the concentration of the sample stock solution. Thus derived protein concentration (concentration of apoY6S4DE-Fer0 included in the solution) was 2.5 mg/ml.
Preparation 4
Purity Test of Apoferritin
With respect to the purity of the resulting apoferritin (for example, apoY6S4DE-Fer0) whether or not it is suited for core synthesis, a test was carried out by the following procedure.
The purity was determined by the gel filtration as in the following.
1. A general-purpose HPLC system (8020 series Tosoh Corporation) to which a TSK-GEL BIOASSIST G4SWXL column was connected (Tosoh Corporation) was used.
2. Using a 50 mM Tris-HCl buffer, pH 8.0 as a mobile phase, 50 ml or more of the buffer was fed at a flow rate of 1.0 ml per min beforehand.
3. The purified solution having a concentration of 1 mg/ml of 0.1 ml was charged in a sample loop, and injected into the column at a flow rate of 1.0 ml per min.
4. Monitoring was carried out with a UV/VIS detector (UV-8020 Tosoh Corporation) at a wavelength of 280 nm, and the results were recorded using a software (LC-8020 Model IITosoh Corporation) installed in PC for regulation.
5. It was ascertained that the peak corresponding to the ferritin subunit included in the sample (elution time: 13 to 14 min) was not beyond the detection level, and was only the peak derived from apoferritin (monomer: 8.6 min, dimer: 7.8 min).
Preparation 5
Synthesis of Ferritin Including Indium
Indium oxide used for producing a two-dimensional array was synthesized in side apoferritin (for example, apoY6S4DE-Fer0) as follows.
In this Example, 80 ml of a reaction mixture was prepared according to the following procedure to give the final composition of the solution of: 0.2 M disodium hydrogenphosphate, 12 mM ammonia, 40 mM HCl, 0.1 mg/ml apoY6S4DE-Fer0, and 1 mM indium sulfate.
1. To a 125-ml square medium bottle (Nalge Nunc 2019-0125) were added 16 ml of 1 M disodium hydrogenphosphate, 0.96 ml of 1 M ammonia, 3.2 ml of 1 N HCl and 56.64 ml of ultrapure water in this order, and the mixture was stirred with a stirrer.
2. The pH was measured with a pH meter and ascertained that the mixture had a pH of 2.88 (within ±0.02).
3. 3.2 ml of a 2 mM Tris (pH 8.0) solution containing 2.5 mg/ml apoY6S4DE-Fer0 was added thereto, and the mixture was stirred with a stirrer.
4. Thereto was added 41.4 mg of indium sulfate powder, and the powder was dissolved in the reaction mixture.
5. The square medium bottle charged with the reaction mixture was sealed with a cap, and the reaction was allowed while stirring at 25° C. (±1° C.) for 3 hours.
6. After completing the reaction, each 40 ml of the reaction mixture was dispensed in a 50-ml Falcon tube.
7. The Falcon tube was placed in a swing rotor for a centrifuge LC-200 (TOMY), and centrifuged at 3,000 rpm for 10 min. Thus, supernatant 1 was eliminated to collect precipitate 1.
8. To the precipitate 1 was added 5 ml of a 50 mM Tris-HCl buffer (pH 8.0), and suspended with a vortex mixer.
9. The Falcon tube including the precipitate 1 was placed in a swing rotor for the centrifuge LC-200, and centrifuged at 3,000 rpm for 10 min to obtain supernatant 2 and precipitate 2. The supernatant 2 was dispensed in a new Falcon tube.
10. To the precipitate 2 was added 5 ml of the 50 mM Tris-HCl buffer (pH 8.0), and suspended with a vortex mixer.
11. The Falcon tube including the precipitate 2 was placed in a swing rotor for the centrifuge LC-200, and centrifuged at 3,000 rpm for 10 min to obtain supernatant 2 and precipitate 2. The supernatant 2 was dispensed in a new Falcon tube.
12. To the precipitate 2′ was added 5 ml of a 50 mM Tris-HCl buffer (pH 8.0), and suspended with a vortex mixer.
13. The Falcon tube including the precipitate 2′ was placed in a swing rotor for the centrifuge LC-200, and centrifuged at 3,000 rpm for 10 min to obtain supernatant 2′′ and precipitate 2′′. The supernatant 2′′ was dispensed in a new Falcon tube.
14. To each of the supernatant 2 (about 5 ml), the supernatant 2′ (about 5 ml), and the supernatant 2′′ (about 5 ml) was added 0.5 ml of 5M NaCl, and the Falcon tube was inverted to allow for stirring.
15. The Falcon tube was placed in a swing rotor for the centrifuge LC-200, and centrifuged at 3,000 rpm for 10 min. Thus, supernatant 3, supernatant 3′ and supernatant 3′′ were eliminated to collect precipitate 3, precipitate 3′ and precipitate 3′′.
16. To the precipitate 3 was added 10 ml of a 50 mM Tris-HCl buffer (pH 8.0), and suspended with a vortex mixer to obtain suspension 3.
17. To the precipitate 3′ was added the suspension 3, and suspended with a vortex mixer to obtain suspension 3′.
18. To the precipitate 3′′ was added 10 ml of the suspension 3′, and suspended with a vortex mixer to obtain suspension 3′′.
19. To the suspension 3′′ (about 10 ml) was added 0.9 ml of 5 M NaCl, and the Falcon tube was inverted to allow for stirring.
20. The Falcon tube was placed in a swing rotor for the centrifuge LC-200, and centrifuged at 3,000 rpm for 10 min. Thus, supernatant 4 was eliminated to collect precipitate 4.
21. To the precipitate 4 was added 10 ml of the 50 mM Tris-HCl buffer (pH 8.0), and suspended with a vortex mixer to obtain suspension 4.
22. Suspension 5 was transferred to a collection tube of an Apollo 20-ml (QMWL 150 kDa) centrifugal concentrator.
23. The Apollo 20-ml centrifugal concentrator was placed in a swing rotor of the centrifuge LC-200, and the centrifugal separation was repeated at 3,000 rpm to concentrate until the volume of the solution left in the collection tube was reduced to not more than 1 ml.
24. Concentrated solution 1 was taken from the collection tube.
25. In the procedure shown in “Preparation 3: Determination of Protein Concentration” described above, the concentration of Y6S4DE-Fer0 (hereinafter, designated as Y6S4DE-Fer0 (In)) having indium oxide as a core was determined. In the description hereinbelow, ferritin having indium oxide as a core is represented by adding a denotation of (In) to the end of the name of the ferritin.
Preparation 6
High Level of Purification of X-Fer0 (In)
For the two-dimensional regular arraying, highly pure ferritin (monomer purity: not less than 99.5%) having an indium oxide core inside (hereinafter, designated as X-Fer0 (In), wherein X represents a peptide name such as Y6S4DE) is desired.
Accordingly, X-Fer0 (In) for use in the two-dimensional regular arraying was highly purified in this Example as shown below.
1. Tricorn 10/600 column (GE Healthcare) packed with TSK-GEL BIOASSIST G4SWXL resin (Tosoh Corporation) was connected to a general-purpose HPLC system (8020 series Tosoh Corporation).
2. Using a 50 mM Tris-HCl buffer, pH 8.0 as a mobile phase, 100 ml or more of the buffer was fed at a flow rate of 0.5 ml per min beforehand.
3. The concentrated solution 1 of not more than 3 ml was charged in a sample loop, and injected into the column at a flow rate of 0.5 ml per min.
4. Monitoring was carried out with a UV/VIS detector (UV-8020 Tosoh Corporation) at a wavelength of 280 nm, and the results were recorded using a software (LC-8020 Model IITosoh Corporation) installed in PC for regulation.
5. Each 1.0-ml fraction of the eluate was collected with a fraction collector (FC-8020 Tosoh Corporation), thereby collecting the fraction containing the X-Fer0 (In) monomer.
6. In the procedure shown in “Preparation 3: Determination of Protein Concentration” described above, the concentration of X-Fer0 (In) was determined.
Preparation 7
Preparation of Substrate
A substrate having a hydrophilic surface for use in the two-dimensional arraying of the present invention is prepared.
Thermally Oxidized Silicon Substrate
A procedure for removing organic matters on the surface by a UV/ozone treatment (washing with ultraviolet ray/ozone) is demonstrated below.
1. Just before (i.e., immediately before allowing for two-dimensionally arraying of ferritin as described later), a thermally oxidized silicon substrate (SiO2 membrane having a thickness of 3 nm) was cut into a piece having a size of 5×10 mm.
2. Using an apparatus (Model UV-1, SAMCO Inc.), the thermally oxidized silicon substrate was subjected to an UV/ozone treatment under conditions at a substrate temperature of 110° C. and an oxygen flow rate of 0.5 L/min for a washing time of 10 min.
Two-Dimensional Arraying of Ferritin
After completing the foregoing Preparations 1 to 7, ferritin was two-dimensionally arrayed according to the following procedure (hereinafter, may be referred to as “sandwich method”).
1. A protein/2 mM Tris buffer having a concentration twice of the final concentration was prepared. For example, in the case of Y6S4DE-Fer0 (In) in which the final concentration was to be 0.5 mg/ml, 1.0 mg/ml Y6S4DE-Fer0 (In) was prepared.
2. A solution for arraying having a concentration two times of the final concentration was prepared. For example, in the case of ammonium sulfate in which the final concentration was to be 13 mM, a 26 mM ammonium sulfate solution was prepared.
3. Each 5 μl of the protein solution and a crystallization solution was charged in a micro test tube, and mixed by pipetting or with a Vortex mixer.
4. An appropriate size of Parafilm was provided in a plastic dish, and 5 μl of the mixed solution was dropped on the Parafilm.
5. The substrate prepared according to “Preparation 7: Preparation of Substrate” was provided such that the droplet was brought into contact with its surface subjected to a hydrophilizing treatment.
6. The plastic dish was covered by a lid, and left to stand still in an incubator (LTI-2000, Tokyo Rikakikai Co., LTD.) at 20 (±0.5)° C. for 30 min.
7. After the lapse of a predetermined time period, the substrate was peeled off from the Parafilm with vacuum tweezers, and transferred into a 1.5-ml micro test tube.
8. The micro test tube was centrifuged with a centrifuge (5415D, Eppendorf Co., Ltd.) at 1500 G for 10 min, and an excess solution on the substrate was removed.
9. The substrate was removed from the micro test tube, and SEM (JEOL SEM7400F) observation was carried out. The observation condition involved an accelerating voltage of 5 kV, and an emission electric current of 10 μA.
The results are as shown below.
Example 1
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Y6F4DE-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 5, and a Fourier transformation image thereof is shown in FIG. 6.
Example 2
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Y6F4DE-Fer0 (In) and 12.5 mM PIPES-Tris (pH 7.0) is shown in FIG. 7, and a Fourier transformation image thereof is shown in FIG. 8.
Example 3
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Y6F4DE-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 9, and a Fourier transformation image thereof is shown in FIG. 10.
Example 4
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Y6F4DE-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 11, and a Fourier transformation image thereof is shown in FIG. 12.
As shown in FIG. 5 to FIG. 12, it was verified that the ferritin having an amino acid sequence set out in SEQ ID NO: 1 on its outer peripheral surface formed “favorable two-dimensional array” or “inferior two-dimensional array”.
Comparative Example 1
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml N1-LF (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 13, and a Fourier transformation image thereof is shown in FIG. 14.
Comparative Example 2
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml N1-LF (In) and 12.5 mM PIPES-Tris (pH 7.0) is shown in FIG. 15, and a Fourier transformation image thereof is shown in FIG. 16.
Comparative Example 3
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml N1-LF (In) and 13 mM ammonium sulfate is shown in FIG. 17, and a Fourier transformation image thereof is shown in FIG. 18.
Comparative Example 4
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml N1-LF (In) and 20 mM ammonium acetate is shown in FIG. 19, and a Fourier transformation image thereof is shown in FIG. 20.
It could be verified that the ferritin having an amino acid sequence set out in SEQ ID NO: 1 on its outer peripheral surface formed “favorable two-dimensional array” or “inferior two-dimensional array”, although when compared with N1-LF in conventional examples shown in FIG. 13 to FIG. 20, they were equal to or equal to with limitations (for example, domain size of the two-dimensional array being small) to the same.
Comparative Example 5
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml D2N-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 21, and a Fourier transformation image thereof is shown in FIG. 22.
Comparative Example 6
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml D2N-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 23, and a Fourier transformation image thereof is shown in FIG. 24.
Comparative Example 7
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml D2N-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 25, and a Fourier transformation image thereof is shown in FIG. 26.
Comparative Example 8
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml E10Q-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 27, and a Fourier transformation image thereof is shown in FIG. 28.
Comparative Example 9
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml E10Q-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 29, and a Fourier transformation image thereof is shown in FIG. 30.
Comparative Example 10
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml E10Q-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 31, and a Fourier transformation image thereof is shown in FIG. 32.
Comparative Example 11
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using suspended E10S-Fer0 (In) and 2 mM Tris-HCl buffer (pH8.0), and a thermally oxidized silicon substrate is shown in FIG. 33, and a Fourier transformation image thereof is shown in FIG. 34.
Comparative Example 12
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml P7S-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 35, and a Fourier transformation image thereof is shown in FIG. 36.
Comparative Example 13
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml P7S-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 37, and a Fourier transformation image thereof is shown in FIG. 38.
Comparative Example 14
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml P7S-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 39, and a Fourier transformation image thereof is shown in FIG. 40.
It could be verified that the ferritin D2N-Fer0, E10Q-Fer0, E10S-Fer0 and P7S-Fer0 having an amino acid sequence with substitution by a different amino acid residue from N1-LF in conventional example on its outer peripheral surface formed “favorable” or “inferior” two-dimensional array.
Comparative Example 15
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Y8FY9F-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 41, and a Fourier transformation image thereof is shown in FIG. 42.
Comparative Example 16
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Y8FY9F-Fer0 (In) and 12.5 mM PIPES-Tris (pH 7.0) is shown in FIG. 43, and a Fourier transformation image thereof is shown in FIG. 44.
Comparative Example 17
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Y8FY9F-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 45, and a Fourier transformation image thereof is shown in FIG. 46.
Comparative Example 18
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml S5TS6T-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 47, and a Fourier transformation image thereof is shown in FIG. 48.
Comparative Example 19
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml S5TS6T-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 49, and a Fourier transformation image thereof is shown in FIG. 50.
Comparative Example 20
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml S5TS6T-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 51, and a Fourier transformation image thereof is shown in FIG. 52.
It could be verified that the ferritin Y8FY9F-Fer0 and S5TS6T-Fer0 having an amino acid sequence with substitution by two different amino acid residues from N1-LF in conventional example on its outer peripheral surface formed “inferior two-dimensional array”.
Comparative Example 21
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml ΔHY-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 53, and a Fourier transformation image thereof is shown in FIG. 54.
Comparative Example 22
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml ΔHY-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 55, and a Fourier transformation image thereof is shown in FIG. 56.
Comparative Example 23
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml ΔHY-Fer0 (In), and 20 mM ammonium acetate is shown in FIG. 57, and a Fourier transformation image thereof is shown in FIG. 58.
It could be verified that the ferritin ΔHY-Fer0 having an amino acid sequence with substitution by five different amino acid residues from N1-LF in conventional example on its outer peripheral surface did not form a two-dimensional array.
Comparative Example 24
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml ΔR-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 59, and a Fourier transformation image thereof is shown in FIG. 60.
Comparative Example 25
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml ΔAR-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 61, and a Fourier transformation image thereof is shown in FIG. 62.
Comparative Example 26
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml ΔAR-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 63, and a Fourier transformation image thereof is shown in FIG. 64.
It could be verified that the ferritin ΔAR-Fer0 having an amino acid sequence with substitution by five different amino acid residues from N1-LF in conventional example on its outer peripheral surface formed “favorable two-dimensional array” or “inferior two-dimensional array”.
Comparative Example 27
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml Shuffle-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 65, and a Fourier transformation image thereof is shown in FIG. 66.
Comparative Example 28
A photograph illustrating the appearance of a two-dimensional array of ferritin obtained using 0.5 mg/ml shuffle-Fer0 (In), and 20 mM ammonium acetate is shown in FIG. 67, and a Fourier transformation image thereof is shown in FIG. 68.
It could be verified that the ferritin Shuffle-Fer0 having an amino acid sequence with substitution by twelve different amino acid residues from N1-LF in conventional example on its outer peripheral surface formed “inferior two-dimensional array”.
Comparative Example 29
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 1st-half-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 69, and a Fourier transformation image thereof is shown in FIG. 70.
Comparative Example 30
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 1st-half-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 71, and a Fourier transformation image thereof is shown in FIG. 72.
Comparative Example 31
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 1st-half-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 73, and a Fourier transformation image thereof is shown in FIG. 74.
Comparative Example 32
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 2nd-half-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 75, and a Fourier transformation image thereof is shown in FIG. 76.
Comparative Example 33
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 2nd-half-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 77, and a Fourier transformation image thereof is shown in FIG. 78.
Comparative Example 34
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 2nd-half-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 79, and a Fourier transformation image thereof is shown in FIG. 80.
It could be verified that the ferritin 1st-half-Fer0 and 2nd-half-Fer0 having an amino acid sequence with deletion of amino acid residues from N1-LF (having twelve amino acid residues) in conventional example, i.e., the ferritin having a shorter amino acid sequence (having six amino acid residues) on its outer peripheral surface “did not form a two-dimensional array”.
Comparative Example 35
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 5AA-Fer0 (In) and 12.5 mM PIPES-NaOH (pH 7.0) is shown in FIG. 81, and a Fourier transformation image thereof is shown in FIG. 82.
Comparative Example 36
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 5AA-Fer0 (In) and 13 mM ammonium sulfate is shown in FIG. 83, and a Fourier transformation image thereof is shown in FIG. 84.
Comparative Example 37
A photograph illustrating the appearance of attachment of ferritin obtained using 0.5 mg/ml 5AA-Fer0 (In) and 20 mM ammonium acetate is shown in FIG. 85, and a Fourier transformation image thereof is shown in FIG. 86.
It could be verified that the ferritin 5AA-Fer0 having an amino acid sequence with deletion of seven amino acid residues from N1-LF (having twelve amino acid residues) in conventional example, i.e., the ferritin having a shorter amino acid sequence (having five amino acid residues) on its outer peripheral surface “did not form a two-dimensional array”.
The correlation between formation of the two-dimensional array, and the number of modified amino acid residues (the number of modification from N1-LF) is summarized in Table 3 below.









TABLE 3





Peptide
Number of modified
Formation of two-dimensional


name
amino acid residues
array







Y6S4DE
5 amino acid
“favorable two-dimensional



residues
array” or “inferior



substitution
two-dimensional array”


D2N
1 amino acid residue
“favorable two-dimensional



substitution
array”


E10Q
1 amino acid residue
“favorable two-dimensional



substitution
array” or “inferior




two-dimensional array”


E10S
1 amino acid residue
“favorable two-dimensional



substitution
array”


P7S
1 amino acid residue
“favorable two-dimensional



substitution
array”


Y8F-Y9F
2 amino acid
“inferior two-dimensional



residues
array”



substitution


S5T-S6T
2 amino acid
“inferior two-dimensional



residues
array”



substitution


ΔHY
5 amino acid
“two-dimensional array not



residues
formed”



substitution


ΔAR
5 amino acid
“inferior two-dimensional



residues
array”



substitution


Shuffle
12 amino acid
“inferior two-dimensional



residues
array”



substitution


1st half
6 amino acid
“two-dimensional array not



residues deletion
formed”


2nd half
6 amino acid
“two-dimensional array not



residues deletion
formed”


5AA
7 amino acid
“two-dimensional array not



residues deletion
formed”









The ferritin used in the present invention does not require a bivalent metal ion in forming a two-dimensional array. Therefore, adverse influences typified by appearance of unexpected interface state on the quantum dot provided by two-dimensionally arraying a metal on a substrate can be suppressed.
The peptide set out in SEQ ID NO: 1 presented on the outer surface of ferritin used in the present invention has a novel sequence in which five amino acid residues among 12 amino acid residues are different as compared with the amino acid sequence of the peptide in conventional example set out in SEQ ID NO: 5, and can form a “favorable two-dimensional array” or an “inferior two-dimensional array” on a substrate.
From the foregoing description, it can be construed that the ferritin used in the present invention can be two-dimensionally arrayed on a substrate without exerting adverse influences typified by appearance of unexpected interface state, and that it presents a novel peptide on the outer surface thereof, the peptide having a sequence distinct from those in conventional examples.
The present invention can be utilized in a variety of devices, semiconductor apparatuses and the like, which make use of nanoparticles arranged on a substrate.

## Claims

1. A method of two-dimensionally arraying ferritin on a substrate, comprising:
(a) developing a solution and the ferritin on the substrate,
wherein, the solution comprises:
a solvent consisting of water, and
at least one from the group consisting of PIPES, ammonium sulfate, and ammonium acetate,

wherein the N-terminal of the ferritin is SEQ ID NO: 1 and the surface of the substrate is hydrophilic and;
(b) removing the solvent from the solution developed on the substrate.

2. The method according to claim 1 wherein the surface of the substrate is covered by SiO2.

3. The method according to claim 1 wherein the concentration of PIPES in the solution is not lower than 5 mM and not higher than 50 mM.

4. The method according to claim 1 wherein the concentration of ammonium sulfate in the solution is not lower than 6.5 mM and not higher than 52 mM.

5. The method according to claim 1 wherein the concentration of ammonium acetate in the solution is not lower than 2 mM and not higher than 100 mM.