JP3155854B2 - Protein molecular membrane - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein molecular film used for electronic devices, for example.

[0002]

2. Description of the Related Art Conventionally, as a molecular film used for an electronic element, for example, a casting method and a Langmuir-Blodgett method (hereinafter, referred to as L) described on page 166, edited by Akio Taniguchi, published by Science Forum, "Organic Electronics Materials".
There is a thin film formed by an electrolytic polymerization method, a vacuum evaporation method, a sputtering method, or the like. FIG.
(A)-(d) is schematic explanatory drawing which shows LB method in order of a process. In the figure, 11, 11a and 11b are functional groups, 1
2, 12a and 12b are alkyl chains, 13 is a compression bar, 1
4 is water, 15 is an electrode, and 16 is the formed organic electronic element.

[0003] The LB method will be described below. The material used is a low molecule (hereinafter, referred to as LB molecule) in which an alkyl chain 12 is added to the functional group 11. This is dissolved in an organic solvent such as chloroform, and this is dropped on the water surface. When the organic solvent evaporates and disappears, a monomolecular film composed of only LB molecules remains on the water surface (FIG. 11A). next,
When the monolayer is compressed by the compression bar 13 in the direction of the arrow, the LB molecules in the monolayer are aligned in a certain direction (FIG. 11B).
Next, when a glass electrode 15 on which a metal such as aluminum is deposited is dipped perpendicularly to the water surface and then pulled up, the monomolecular film is transferred onto the electrode 15, and a monomolecular film of LB molecules is deposited on the electrode 15. It is formed (FIG. 11C). This is called a Langmuir-Blodgett film (LB film).
The junction between the two types of functional groups 11a and 11b can be formed by accumulating a monolayer of the second type molecule on the monolayer of the first molecule (FIG. 11D).

[0004]

The molecular film used in a conventional electronic device is characterized in that the functional groups 11a and 11b are in direct contact with each other in a film of the same kind of molecule, such as the above-mentioned LB film. Was close. For this reason, the direction of electron transfer cannot be controlled at the molecular level, and it is impossible to form one element at the molecular level, and it has been difficult to integrate electronic elements at high density.

[0005] The same applies to a molecular film produced by a casting method, an electrolytic polymerization method, a vacuum evaporation method, a sputtering method, or the like. In addition, these molecular films have problems that they greatly contribute to electron transfer, and that it is very difficult to control the distance and orientation between functional groups.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has a high and stable electron transfer speed between a functional group in a molecule and an electrode, and can be integrated at a very high density. It is an object of the present invention to provide a protein molecular film capable of forming an electronic device such as one ultra-fine, molecular-level rectifying device, switching device, optical switching device, and storage device.

[0007]

Means for Solving the Problems] protein molecular film according to the invention, pro hydrogen bacterial cytochrome c552
It is two-dimensionally aligned on a motor-modified electrode.

[0008]

In the present invention, a hydrogen bacterium-derived cytochrome c552, which is very small as a protein having an electron transfer function in the molecule, has a fast electron transfer with the electrode, and has excellent heat stability, is used. Therefore, the electron transfer speed with the electrodes, other electron transfer proteins, and other redox substances can be performed at high speed and in a stable manner, and an organic electronic device having desired electric characteristics can be formed. A molecular film of the protein thus obtained can be obtained.

[0009]

[Embodiment 1] The protein molecular membrane of the present invention forms a protein complex by combining hydrogen bacterium-derived cytochrome c552, alone or in combination with another electron transfer protein, or combines with another redox substance, and two-dimensionally aligns them on an electrode. It was made.

The hydrogen bacterium Hydronobacter thermophilus (Hydrogenobacter thermo)
The cytochrome c552 of S. philus is described in the Journal of Bacteriology, Sanbongi et al. (J. Bacteriol., 171, 171).
65 (1989)), Asn-Glu-Gl
u-Leu-Ala-Lys-Gln-Lys-Gly
-Cys-Met-Ala-Cys-His-Asp-
Leu-Lys-Ala-Lys-Lys-Val-G
ly-Pro-Ala-Tyr-Ala-Asp-Va
l-Ala-Lys-Lys-Tys-Ala-Gly
-Arg-Lys-Asp-Ala-Val-Asp-
Tyr-Leu-Ala-Gly-Lys-Ile-L
ys-Lys-Gly-Gly-Ser-Gly-Va
l-Trp-Gly-Ser-Val-Pro-Met
-Pro-Pro-Gln-Asn-Val-Thr-
Asp-Ala-Gln-Als-Lys-Gln-L
eu-Ala-Gln-Trp-Ile-Leu-Se
It is an electron transfer protein consisting of the amino acid sequence of r-Ile-Lys. It has a molecular weight of 7,600 and is about 2/3 of horse cytochrome c, which is very small. Furthermore, it has very high heat resistance, such as being not denatured in an autoclave at 120 ° C. for 10 minutes.

As a method for two-dimensionally aligning proteins, it is advisable to adsorb a monomolecular film on the water surface.
For example, Fromhertz aligned by adsorbing ferritin to a lipid monolayer and performing two-dimensional crystallization.
It is also widely known that 4-mercaptopyridine or 4-mercaptopropionic acid is adsorbed and bonded to the surface of a noble metal such as gold, and the protein is adsorbed by the interaction between the pyridine group or carboxylic acid group and the protein. Although it is not known that proteins are arranged two-dimensionally by this method, proteins can be arranged two-dimensionally by controlling the ionic concentration, hydrogen ion concentration, protein concentration, and temperature of a solution. Was.

As a method for forming a complex of two kinds of proteins, there is a method utilizing ionic bonding by a positive charge and a negative charge. For example, Weber and Trin (PC Weber, G. Tolli)
n) is a magazine: Journal of Biological Chemistry (J. Biological Chemist)
ry, 260, p. 5568 (1985)) shows that horse cytochrome c and cytochrome c551 and flavodoxin form a protein complex.

Embodiment 2 FIG. Hereinafter, an embodiment of the present invention will be specifically described.
First, it is shown that the hydrogen bacterium-derived cytochrome c552 is structurally stable on the electrode and that electrons are rapidly transferred to and from the electrode. Hydrogen bacterium-derived cytochrome c552 was obtained from Sanbongi et al., European Journal of Biochemistry (Eur. J. Bio).
chem. , 198, 7 (1991)). The horse cytochrome c was from Sigma. Both hydrogen bacterium-derived cytochrome c552 and horse cytochrome c were purified by gel chromatography and ion exchange chromatography. Hydrogen bacteria-derived cytochrome c552 or horse cytochrome c was dissolved in phosphate buffer (20 mM, pH 7.0, containing 100 mM sodium perchlorate) to a concentration of 0.1 mM. As an electrode, chromium was first vapor-deposited on glass, and a gold electrode on which gold was vapor-deposited, and this gold electrode was immersed in concentrated nitric acid for 10 hours, washed with water, and immersed in a 1 mM aqueous solution of 4-mercaptopyridine for 5 minutes. The obtained promoter-modified electrode was used. After the hydrogen bacterium-derived aqueous solution of cytochrome c552 or the horse cytochrome c aqueous solution was deoxygenated by aeration with argon, cyclic voltammetry was performed using a gold electrode and a promoter-modified electrode. The obtained cyclic voltammogram (hereinafter abbreviated as CV) was analyzed.

The shape of CV of cytochrome c552 derived from hydrogen bacterium at the promoter-modified electrode was almost equal to that of horse cytochrome c. The oxidation-reduction potential is 27.3 mV (Ag), which is about 30 mV lower than that of horse cytochrome c.
/ AgCl). On the other hand, a stable CV was obtained with the hydrogen bacterium-derived cytochrome c552 on the gold electrode,
No results were obtained with equine cytochrome c. The evaluation of the electrode reaction rate constant k is performed by Nicholson (Nicholso).
The method of n) was used. Oxidation and reduction peak potential difference (m
FIG. 1 shows the relationship between V) and the sweep speed (mV / s). In the figure, a triangle mark (）) shows the result of hydrogen bacterium-derived cytochrome c552 on the gold electrode, a white circle mark (○) shows the result of horse cytochrome c, and a black circle mark (●) shows the result of hydrogen bacterium-derived cytochrome c552 on the promoter-modified electrode. . from this result,
The electrode reaction rate constant k was 1.3 × 10 −2 cm / s for hydrogen bacterium-derived cytochrome c552 on the promoter-modified electrode, 2 × 10 −4 cm / s on the gold electrode, and on the promoter-modified electrode. 4.9 for horse cytochrome c
× 10 −3 cm / s. That is, it was found that the hydrogen bacterium-derived cytochrome c552 can cause electron transfer on the promoter-modified electrode and the gold electrode much faster than the equine cytochrome c. In addition, since stable CV was exhibited on the gold electrode, it was found that cytochrome c552 derived from hydrogen bacteria was structurally very stable.

Embodiment 3 FIG. Next, a method for two-dimensionally aligning hydrogen bacterium-derived cytochrome c552 (hereinafter, referred to as cytochrome c552) on an electrode will be described. Phosphatidylcholine is a lipid,
This chloroform solution (concentration: 1 mg / mL) is spread on the water surface. This water is converted into cytochrome c derived from hydrogen bacteria.
Slowly replace with a 0.1 mM aqueous solution of 552 with a pump.
After another 30 minutes, the cytochrome c552 was two-dimensionally aligned by another replacement with water.

The two-dimensionally arranged film is placed on a gold single crystal electrode by the Langmuir-Blodget method (Langmuir-Bl).
transfer method (odgett method (LB method)). FIG. 2 shows a configuration diagram of the thus prepared cytochrome c552 on the gold electrode. 1 is phosphatidylcholine, 2 is cytochrome c552, and 3 is a gold single crystal electrode. Also,
Similarly, FIG. 3 shows a configuration diagram in which 4-mercaptopyridine was transferred onto a promoter-modified gold single crystal electrode modified with 4-mercaptopyridine. 4 is mercaptopyridine.

FIG. 4 (a) shows cytochrome c552-2.
Using a dimensional array as a storage element, writing information
This shows a state where the probe 5 of the TM performs the operation. 5 for the probe
By applying a voltage pulse of V, any molecule can be denatured. FIG. 4B is a schematic view of a part of the storage element after the writing is completed. 8 × 8 =
A 64-bit element is shown. 6 is non-denatured cytochrome c552, and 7 is denatured cytochrome c552.
Undenatured represents '1', and denatured represents '0'. Therefore, the information in the first horizontal row of the storage element is '11110000'. The voltage of the pulse is not strict.

In the above embodiment, phosphatidylcholine 1 was used as a lipid, but any lipid that forms an LB film can be used. For example, fatty acids such as arachidic acid and stearic acid. Further, those having a π-electron system such as a benzene ring, a double bond, and a triple bond are preferable because a large current value can be obtained.

Embodiment 4 FIG. By directly adsorbing the protein to the gold electrode, the hydrogen bacterium-derived cytochrome c552 can be two-dimensionally arranged. First, after washing the gold single crystal electrode with nitric acid and water, 0.1 mg / m 2 of mercaptopropionic acid was used.
After being immersed in 1 l of an aqueous solution for 1 minute, it is washed with water. this,
After immersion in a 0.1 mM aqueous solution of cytochrome c552 (phosphate buffer 1 mM, pH 7.0) for 5 minutes, washing may be performed with water. FIG. 5 shows a configuration diagram of the two-dimensional array thus manufactured. In the figure, 8 is mercaptopropionic acid. As in Example 2, it was confirmed that the element functions as an ultra-high-density storage element. The concentration and time are not strict. In addition to the mercaptopropionic acid, a molecule having a thiol group and a charged substituent such as a carboxylic acid group, a sulfonic acid group, or an amino group may be used. When the charge is negative, cytochrome c55
It is considered that heme No. 2 is oriented near the electrode and cytochrome c552 is adsorbed on the electrode, and when the charge is positive, the heme separates from the electrode and is adsorbed on the electrode. Further, the substance used for promoter modification shown in Example 1 may be used.

Further, by controlling the concentration, pH, temperature, etc. of the salt of phosphoric acid or the like in the aqueous solution of cytochrome c552, a metal electrode such as gold or platinum, a semiconductor electrode such as graphite, fullerene deposited film, silicon or tin oxide, polypyrrole, etc. Without using a modifier for organometallic electrodes such as
It was also possible to directly sequence cytochrome c552 derived from hydrogen bacteria.

Embodiment 5 FIG. An example is shown in which a complex is formed with hydrogen bacterium-derived cytochrome c552 and another protein and used as an electronic element. Desulfovibrio Bulgaris
rio vulgaris) and 1 ml each of a 0.1 mM aqueous solution of cytochrome c552 (phosphate buffer 1 mM, pH 7.0) were mixed and allowed to stand, and the mixture was allowed to stand. FC). This aqueous solution could be two-dimensionally arrayed using phosphatidylcholine in the same manner as in Example 2.
This cytochrome c552 monomolecular film could be transferred onto the gold single crystal electrode and the promoter-modified electrode. FIG. 6 is a sectional view showing the protein molecular membrane. In the figure, 9 is flavodoxin. FIG. 7 is a characteristic diagram showing a result of measuring current-voltage characteristics of one molecule using STM. The horizontal axis is voltage (V), and the vertical axis is current (nA).
It is. Flavodoxine has a redox potential of about -600 m
V (Ag / AgCl), 6 more than cytochrome c552
It is lower than 00 mV. Therefore, electron transfer occurs only in the direction from flavodoxin to cytochrome c552. As a result, rectification characteristics were observed as shown in FIG. That is,
FC was shown to have the function of one rectifying element with one complex consisting of two molecules.

In the above embodiment, desalphovibrio was used as a protein forming a complex with cytochrome c552.
Although an example using flavodoxin obtained from Bulgaris is shown, flavodoxin, cytochrome c oxidase, cytochrome reductase, and the like obtained from other organisms also form a complex and exhibit a rectifying function. Also, as in Example 3, without using a phosphatidylcholine film,
A molecular film may be formed by directly attaching a complex to an electrode such as a gold single crystal or a modified gold single crystal.

Embodiment 6 FIG. When heme is irradiated with visible light, electrons are excited.
As a result, the electron transfer speed with an electrode such as a metal changes. An example in which this property is used to function as an optical switch element will be described. FIG. 8 is a characteristic diagram illustrating a change in current (nA) with respect to time (second) when light of 410 nm is irradiated. It can be seen that the current value when light is applied is greatly increased as compared with the case where no light is applied. This proved that the optical switch functioned. The absorption of flavodoxin has peaks at around 350 nm and 450 nm. Therefore, even when light in this wavelength range was irradiated, a change in current due to electronic excitation of flavodoxin was observed.
Due to the respective absorption effects of flavodoxin and cytochrome c552, the function of the optical switch was observed by irradiation with light in the wavelength range of 200 to 700 nm.

Embodiment 7 FIG. Next, FIG. 9 shows an example in which a hydrogen bacterium-derived cytochrome c552 monolayer is formed on another redox substance. The riboflavin is heated to 300 ° C. in a vacuum of 1 × 10 −7 torr, and the flavin film 10 is
Deposited with a thickness of nm. To this, cytochrome c552 was two-dimensionally aligned and attached using phosphatidylcholine as in Example 2. Riboflavin has a redox potential of about -400 mV (Ag / AgCl) and is about 400 mV lower than cytochrome c552. Therefore, electron transfer from riboflavin 10 to cytochrome c552
Happens only in the direction of. FIG. 10 shows a current (nA) -voltage (V) characteristic of one molecule using STM. This figure also shows that it has a rectifying function.

In this embodiment, a deposited film of riboflavin is used as the redox substance. However, a deposited film of another flavin derivative such as lumiflavin may be used. In addition, even when riboflavin was dissolved in distilled water at a concentration of 0.1 μM and immersed in a single crystal of gold to adsorb riboflavin, a protein molecule membrane having a rectifying function and a light switching function was obtained.

Also, instead of riboflavin, organometallic complexes such as metalloporphyrin, metallophthalocyanine, ferrocene, porphyrin, phthalocyanine, quinone, pyridine nucleotide, viologen or derivatives thereof,
Organic charge-transfer complexes such as tetracyanoquinodimethane (TCNQ) and redox substances such as iron-sulfur clusters can be formed by depositing thin films of redox substances on electrodes by vapor deposition or adsorption onto electrodes using solutions. ,
A similar molecular film was obtained.

In Examples 1 to 3, the protein expressed by Escherichia coli was used as the cytochrome c552 derived from the hydrogen bacterium. Of course, the cytochrome c5 obtained by culturing the original hydrogenobacter thermophilus was used.
A similar effect was obtained with 52. Similar effects can be obtained by introducing a gene into another microorganism. Furthermore, N
Similar results were obtained with proteins in which one or more than ten amino acids were added to one or both of the C-terminal and C-terminal.

[0028]

As described above, according to the present invention, the electron transfer speed between the functional group in the molecule and the electrode is increased by aligning the hydrogen bacterium-derived cytochrome c552 two-dimensionally on the promoter-modified electrode. A stable, stable, and ultra-high-density integrated, ultra-fine, molecular-level protein molecular film capable of forming one electronic device such as a rectifier, switching, optical switching, or storage device is obtained. Can be

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the relationship between the oxidation and reduction peak potential differences and the sweep rate in cyclic voltammograms of a promoter-modified gold electrode and a hydrogen bacterium-derived cytochrome c552 at a gold electrode and a horse cytochrome c at a promoter-modified electrode.

FIG. 2 is a configuration diagram showing a protein molecular membrane according to Example 2 of the present invention.

FIG. 3 is a configuration diagram showing a modified example of the protein molecular membrane according to the second embodiment of the present invention.

FIGS. 4A and 4B are a configuration diagram illustrating a state in which information is written by STM by applying the protein molecular film according to the second embodiment of the present invention as a storage element, and a schematic view illustrating the storage element in which the information is stored.

FIG. 5 is a configuration diagram showing a protein molecular membrane according to Example 3 of the present invention.

FIG. 6 is a configuration diagram showing a protein molecular membrane according to Example 4 of the present invention.

FIG. 7 is a current-voltage characteristic diagram showing a rectifying function of an element by a protein molecular film of Example 4 of the present invention.

FIG. 8 is a characteristic diagram showing a current change due to light irradiation of an element by the protein molecular film of Example 5 of the present invention.

FIG. 9 is a configuration diagram showing a protein molecular membrane according to Example 6 of the present invention.

FIG. 10 is a current-voltage characteristic diagram showing a rectifying function of an element by a protein molecular film according to Example 6 of the present invention.

FIG. 11 is a schematic explanatory view showing an LB method, which is a conventional method for producing a molecular film used for an electronic element, in the order of steps.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Phosphatidylcholine 2 Hydrogen bacteria-derived cytochrome c552 4 4-mercaptopyridine 5 STM probe 6 Non-denatured cytochrome c552 7 Denatured cytochrome c552 9 Flavodoxin

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Satoshi Ueyama 8-1-1 Tsukaguchi Honcho, Amagasaki City Inside the Central Research Laboratory, Mitsubishi Electric Corporation (72) Inventor Satoru Isoda 8-1-1 Honcho Tsukaguchi, Amagasaki City Mitsubishi (56) References JP 5-75096 (JP, A) JP 5-109263 (JP, A) JP 5-75097 (JP, A) JP 2- 32358 (JP, B2) (58) Field surveyed (Int. Cl. ⁷ , DB name) H01L 51/00-51/40

Claims (1)

(57) [Claims] 1. A flop hydrogen bacterial cytochrome c552
Protein molecular membranes two-dimensionally aligned on a motor-modified electrode.