JPH0719926B2 - Photoelectric conversion device using antibody and method for manufacturing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a photoelectric conversion device using a lipid membrane containing a protein having a photoelectric conversion function and a method for producing the same, and particularly to a lipid containing a protein having a photoelectric conversion function. The present invention relates to a photoelectric conversion device having a film immobilized on a substrate and a method for manufacturing the photoelectric conversion device.

[Prior Art] As a lipid membrane containing a protein having a photoelectric conversion function, for example, a thylakoid membrane is known. The thylakoid membrane contained in cells having photosynthetic ability such as plants, cyanobacteria, and photosynthetic bacteria is a biological membrane containing proteins and lipids related to photosynthetic function. This kind of membrane has a photosynthetic reaction center protein complex arranged in a directional manner, and has the ability to generate a potential difference across the membrane when absorbing light. Therefore, the thylakoid film is expected to be used in a photoelectric conversion device such as a photoelectric conversion element. According to conventional preparation methods, thylakoid membranes are often obtained as fine membrane fragments or membrane vesicles after disrupting cells. It was difficult to directly take out the potential difference generated by sandwiching such a film. Therefore, also in the case of measurement, research on the photoelectric conversion function in a solution state has mainly been conducted by an indirect measurement method using fluorescence (for example, Methods in Enzymology 69, 409-715 (1980). Academic Press).

[Problems to be Solved by the Invention] In order to use a lipid membrane containing a protein having a photoelectric conversion function as a photoelectric conversion element, a technique is used in which the front and back surfaces of the membrane are aligned and fixed, and an electrical signal generated by sandwiching the membrane is taken out. Development is desired. The electrical signal is, for example, a current or a potential difference.

An object of the present invention is to provide a photoelectric conversion device in which a lipid film containing a protein having a photoelectric conversion function is directionally immobilized on a substrate provided with electrodes.

Another object of the present invention is to provide a method for producing a photoelectric conversion device by directionally immobilizing a lipid film containing a protein having a photoelectric conversion function on a substrate.

[Means for Solving Problems] An antibody against a specific site of a lipid membrane containing a protein having a photoelectric conversion function is prepared, immobilized on a substrate provided with an electrode, and a protein having a photoelectric conversion function is provided on the substrate. Immobilize the lipid membrane containing directionally.

As a lipid membrane containing a protein having a photoelectric conversion function, for example, a thylakoid membrane contained in cells of plant chloroplasts, cyanobacteria, photosynthetic bacteria, etc. is used. A chromatophore membrane, which is a standard product obtained by disrupting cells such as Spheroides (ATCC17023), is a suitable material.

Indium-tin oxide (ITO), tin oxide (S
nO2), metal vapor deposition film, semiconductor, etc. can be used.
The electrode substrate can be prepared by forming a conductive film on the substrate by a method such as vacuum deposition.

The antibody can be immobilized on the electrode substrate by, for example, chemical bonding, adsorption or the like.

The lipid membrane and the antibody can be bound by the antigen-antibody reaction.

In this way, the lipid membrane containing the protein having the photoelectric conversion function can be immobilized on the substrate in a certain direction.

Further, by repeating such an operation, stacking can be performed.

[Action] The antibody recognizes the antigen, causes an antigen-antibody reaction, and binds to the antigen. When an antibody against a specific part of a lipid membrane containing a protein having a photoelectric conversion function is immobilized on a substrate, a lipid membrane (including an antigen) containing a protein having a photoelectric conversion function is directionally immobilized on the substrate. can do.

[Example] The material for the lipid membrane containing a protein having a photoelectric conversion function is, for example, a thylakoid membrane, which can be prepared from cells such as plants, cyanobacteria, and photosynthetic bacteria. Among them, a chromatophore membrane obtained by crushing cells such as photosynthetic bacteria Rhodopseudomonas viridis (ATCC19567) and Rhodobacter sphaeroides (ATCC17023) is a preferable material. In these chromatophore membranes, the protein complex at the photosynthetic reaction center that performs photosynthesis is an integral membrane protein. Among the subunits, there are subunits that have a large portion embedded in the lipid membrane, such as both L and M subunits, and subunits that have a relatively large portion outside the lipid membrane, such as the C subunit and H subunit. To do.

When a foreign body enters the body of a living body, in particular a higher animal, the living body tries to protect the body by repelling this foreign body. One of the mechanisms of this immunity is the function of phagocytic cells that eat foreign substances and decompose them. In that case, it is the antibody that recognizes and binds to the foreign substance as soon as possible, and helps the foreign substance to be eaten by the phagocytes. The binding between a foreign substance serving as an antigen and an antibody is called an antigen-antibody reaction. Antigens stimulate the production of antibodies, but proteins and lipids become antigens along with polysaccharides, certain abiotic polymers, and the like. As a chemical entity of an antibody, a group of protein molecules called γ-globulin are known. There are several types of γ-globulin such as IgG, EgE, and IgM. For example, IgG is a protein with a molecular weight of about 150,000.

By preparing and utilizing an antibody against a specific site of a lipid membrane containing a protein having a photoelectric conversion function, the lipid membrane is directionally immobilized. Preferably, an antibody against a site located on one side of the membrane among the subunits having a large portion outside the lipid membrane is prepared, and chemically bound to the surface of the substrate,
Immobilize by adsorption.

When a residue is attached to an antibody-attached substrate by a treatment with a crosslinking reagent such as formaldehyde to bind the antibody, covalent bond is considered to occur. It is considered that antibody molecules are non-covalently adsorbed on the nitrocellulose membrane or the like.

When the chromatophore membrane is brought into contact with the substrate to which the antibody is bound, the chromatophore membrane is directionally and strongly bound by a specific antigen-antibody reaction.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

[Preparation of chromatophore membrane] Rhodopseudomonas viridis (ATCC19) which is a photosynthetic bacterium
567) was cultured in the anaerobic state at 30 ° C. under light irradiation. A chromatophore membrane was prepared from the obtained bacterial cells by the following procedure. That is, the cells were disrupted by a French press, membrane vesicles were prepared by a centrifugal fractionation method such as sucrose density gradient centrifugation, and dialyzed against 10 mM Tris-HCl Tris-HCl (pH 8.2) aqueous solution (Arch.Biochem. Biophys., 223, 282-290 (1
979)). Thus, a chromatophore membrane as shown in FIG. 1 (A) was prepared. It has a structure in which the protein complex 1 of the photosynthetic reaction center is embedded in the lipid membrane 2.

[Preparation of antibody] 20% N, N-dimethyldodecylamine-N-oxide (N, N-dimethyldodecy) was added to the chromatophore membrane suspension (A1020 = 100).
lamine-N-oxide, LDAO) is added for solubilization, and the photosynthetic reaction center protein is purified using Sepharose CL-6B gel. The obtained purified sample was subjected to sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (Nature, Vol. 227, pages 680-685 (1970)), and the H subunit and C subunit bands were respectively cut out to obtain the protein. Extract and collect. Rabbits are immunized with 3-5 mg of the subunit protein thus purified to obtain antisera against each subunit. Γ-globulin is purified from this antiserum. For purification of γ-globulin, a salting-out method, an antigen absorption method, various column chromatography and the like can be used if necessary. In this example, the ammonium sulfate salting-out method was used.

[Immobilization of Antibody on Substrate Surface] As an immobilization substrate, tin oxide (SnO2),
A thin film electrode 5 is formed by depositing a conductive material such as TO and gold. As shown in FIG. 1 (B), a thin film 4 is further formed on the surface of the SnO2 electrode 5 by using a substance such as nitrocellulose having a high protein adsorbing ability to adsorb the antibody.

For making such a thin film,
A method in which 50 microliters of 0.002% amyl acetate solution (colodion solution) is placed on an electrode surface of 2 square centimeters to dry and solidify, and a thin film in which 20 microliters of 2% collodion solution is spread on the water surface and the solvent is evaporated to solidify It is possible to use a method of pressure-bonding to the electrode surface.

These electrodes were washed with physiological saline (PBS) containing phosphate buffer (pH 7.4) and then with PBS containing 20 μg / ml antibody at 30 ° C for 2 days.
Treatment is performed for 0 minutes to adsorb the antibody molecule 3.

The above-mentioned immobilization using a nitrocellulose membrane can be widely used for immobilizing an antibody on a substrate made of various materials.

Depending on the material of the electrode, it is also possible to form a residue using a cross-linking reagent such as formaldehyde or glutaraldehyde to chemically bond the antibody molecule to the electrode plane.

[Immobilization of chromatophore membrane on solid surface] A glass substrate on which an antibody is immobilized is used as a chromatophore membrane (A1020).
= 5.0) in PBS at 30 ℃ for 20 minutes. After the reaction, wash with PBS to remove the unbound chromatophore membrane. By the above operation, the chromatophore membrane 9 can be immobilized as shown in FIG. 1 (C). After this, P containing 1.7% formaldehyde
The bound chromatophore membrane was immobilized by treatment with BS at 30 ° C for 30 minutes. Figure 2 shows the optical absorption spectrum of the immobilized chromatophore membrane measured in PBS. The substrate on which the chromatophore membrane was immobilized in this manner was used as a 1% bovine serum albumin (BS
A) Soak in aqueous solution and dry.

Also, by stacking a collodion thin film on the immobilized chromatophore membrane and immobilizing it again, it is possible to stack a plurality of times as shown in FIG. 1 (E).

As shown in FIG. 1 (D), if the antibody 7 is directed to the opposite side of the chromatophore membrane, the chromatophore membrane 9
Is fixed in the opposite direction to the case of FIG. 1 (C).

By the immobilization method as described above, the thylakoid film can be directionally immobilized. It can also be laminated. It is considered that not only thylakoid membranes but also lipid membranes containing proteins can be immobilized in a directional manner.

[Detection of photoelectric response] An electrode serving as a counter electrode can be attached on a lipid membrane immobilized on a substrate to apply a light stimulus, and an electric signal between both electrodes due to photoelectric response can be extracted. For example, measure changes in potential and current. The counter electrode can be formed by a method such as placing a mercury ball on the surface of the lipid film which is the photoelectric conversion layer, pressing a metal thin film, or vapor depositing a metal thin film. The light source of the light stimulus may be natural light such as sunlight, strobe lamp, light emitting diode (LED), laser, arc lamp, or the like.

FIG. 3 shows an outline of the photoelectric response detection system of the photoelectric conversion device. The chromatophore membrane layer 9 is fixed and laminated on the SnO2 electrode 11 on the glass substrate 12 via the nitrocellulose thin film 10. The counter electrode 8 is created by a method such as placing a mercury ball on it.

From the lower side in the figure, a light stimulus from a light source 13 such as a strobe lamp or an LED is applied to the chromatophore membrane layer 9 through the glass substrate 12, the SnO2 electrode 11, and the nitrocellulose thin film 10. The change in electric potential generated between the electrodes 8 and 11 by the light stimulation was taken out through the conductors 14 and 15 and observed by the oscilloscope 17 via the differential amplifier 16.

FIG. 4 shows an example of the potential response of the photoelectric conversion device to the optical stimulus. A rapid rise of a millisecond or less was observed in response to the stimulus of strobe lamp light. In this example, anti-H
The photoelectric conversion layer was immobilized once using the subunit antibody.

[Advantages of the Invention] A lipid membrane containing a protein having a photoelectric conversion function can be immobilized in a directional manner.

As a result, a photoelectric conversion device can be realized.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing immobilization of a chromatophore membrane, where (A) is a chromatophore membrane, (B) is the case where an anti-H subunit antibody is adsorbed on an electrode coated with a nitrocellulose thin film, (C) Shows the case where the chromatophore membrane is immobilized on (B), (D) shows the case where the chromatophore membrane is immobilized using the anti-C subunit antibody, and (E) shows the case where the layer is immobilized twice. Figure 2 shows the absorption spectrum measured in PBS after a nitrocellulose thin film was placed on the SnO2 electrode and the chromatophore film was immobilized once. FIG. 3 is a schematic diagram of a photoelectric response detection system of the photoelectric conversion device. FIG. 4 shows the potential response of the photoelectric conversion device to the photostimulation of strobe lamp light, which was detected using the photoelectric conversion layer after the chromatophore membrane was immobilized once using the anti-H subunit antibody. Explanation of symbols 1 …… Photosynthetic reaction center protein complex 2 …… Lipid membrane 3 …… Anti-H subunit antibody adsorbed on nitrocellulose thin film 4 …… Nitrocellulose thin film 5 …… SnO2 electrode 6 …… Glass substrate 7 ・ ・ ・… Anti-C subunit antibody 8 …… An electrode that serves as the counter electrode 9 …… Immobilized and laminated chromatophore membrane layer 10 …… Nitrocellulose thin film 11 …… SnO2 electrode 12 …… Glass substrate 13 …… Light source 14 …… Connected to the counter electrode Conductor 15 …… Conductor connected to SnO2 electrode 16 …… Differential amplifier 17 …… Oscilloscope

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Sugio Kawamura 1-3-1, Higashi Tsukuba, Ibaraki Prefecture Institute of Microbial Technology, Industrial Technology Institute (72) Noboru Tomizuka 1-3-3 East, Tsukuba, Ibaraki (72) Inventor Toshikazu Majima, 1-4 Umezono, Tsukuba-shi, Ibaraki Institute of Electronic Technology Research Institute, Industrial Technology Institute (72) Hideki Toyoda 5-chome, Tokodai, Tsukuba, Ibaraki Prefecture 9-5 Stanley Electric Co., Ltd. Tsukuba Research Center (72) Inventor Hiroaki Sugino 5-9-5 Tokodai, Tsukuba City, Ibaraki Prefecture Stanley Electric Co., Ltd. Tsukuba Research Center (72) Inventor Shuichi Aji, East Tsukuba City, Ibaraki Prefecture 5-9-5 Hikaridai, Stanley Electric Co., Ltd., Tsukuba Research Laboratory (56) References JP-A-63-295600 JP, A)

Claims (2)

[Claims] 1. A substrate having a first electrode portion, an antibody immobilized on the first electrode portion, a lipid prepared from cells having photosynthetic ability, and a protein having a photoelectric conversion function. A photoelectric conversion layer formed of a lipid film, a specific portion of which functions as an antigen and is bound to the antibody by an antigen antireaction, and a first electrode with the photoelectric conversion layer sandwiched therebetween. And a second electrode portion formed on the photoelectric conversion layer so as to face the portion, and a protein or lipid is present between the antibody and the first electrode to which the antibody is immobilized. Not photoelectric conversion device. 2. A step of immobilizing an antibody against a specific portion of a lipid membrane containing a protein having a photoelectric conversion function on a substrate, and a lipid membrane containing a protein having a photoelectric conversion function is immobilized on the substrate on which the antibody is immobilized. A method for manufacturing a photoelectric conversion device, including the step of: