JPH0719927B2 - Photoelectric conversion device using avidin-biotin system and manufacturing method thereof - 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, use of cells having photosynthetic ability for photoelectric conversion devices and the like is expected.

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, when measuring
The indirect measurement method using fluorescence or the like has mainly studied the photoelectric conversion mechanism in a solution state (for example, Methods in Enzymology 69, 409-715 (1980).
Year) 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 potential difference or a current.

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] Avidin is bonded onto a substrate provided with an electrode. On the other hand, a specific site of a lipid membrane containing a protein having a photoelectric conversion function is selectively biotinylated.

By immobilizing a lipid membrane containing a biotin-converted protein having a photoelectric conversion function on the avidin-ized substrate surface by avidin-biotin interaction, the lipid membrane is directionally immobilized.

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 crushing cells such as Bacteria sphaeroides (ATCC17023), is a suitable material.

Indium-tin oxide (ITO), tin oxide (S
nO ₂ ), 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 avidin molecule can be bound to the electrode substrate by, for example, chemical binding or adsorption.

When chemically modifying a protein having a photoelectric conversion function with various biotinylation reagents, a specific site can be selectively biotinylated by selecting conditions.

The biotinylated lipid membrane is attached to the avidinized electrode substrate.

[Action] Avidin and biotin have a strong specific interaction.

When a specific site of a protein complex of a lipid membrane containing a protein having a photoelectric conversion function is biotinylated, the lipid membrane containing a protein having a photoelectric conversion function is given directionality with respect to avidin molecule.

Since biotin binds to the avidinized substrate surface,
A lipid film containing a protein having a photoelectric conversion function with directionality can be immobilized on a substrate.

[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, chromatophore membrane vesicles obtained by crushing cells such as photosynthetic bacteria Rhodopseudomonas viridis (ATCC19567) and Rhodobacter sphaeroides (ATCC17023) are suitable materials. In these chromatophore membranes, the subunits of the protein complex at the reaction center for photosynthesis include subunits such as L, M, and H. When the reagent having a biotin group is reacted with the chromatophore membrane suspension to chemically modify the protein, the H subunit can be selectively biotinylated. The chromatophore thus biotinylated can be immobilized on the substrate with directivity via avidin. That is, an avidin molecule is bound to the surface of the substrate by chemical bonding, adsorption or the like, and further a biotinylated chromatophore membrane is made to act. Due to the specific biotin-avidin interaction, the chromatophore membrane is directionally bound to the substrate.

In this way, the photoelectric conversion device having the chromatophore membrane immobilized and the electrodes is used to detect and utilize photoelectric response such as photo-potential response or photo-current response.

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, a membrane fraction was prepared by a centrifugal fractionation method such as a sucrose density gradient centrifugation, and dialyzed against a 50 mM sodium carbonate NaHCO ₃ (pH 8.9) aqueous solution (Arch.Biochem.Biophys ., 223, 282-290 (1979)). 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.

[Biotinylation of chromatophore membrane] 8 mg / ml sulfosuccinimidyl 6- (biotinamide) hexanoate was added to the suspension (A1020 = 10) of the chromatophore membrane, and treated at 30 ° C for 30 minutes to biotin the membrane protein. Chemical modification. As shown in FIG. 1 (B), the biotinylated chromatophore membrane is provided with a biotin group 3 in a directional manner. The chromatophore membrane after biotinylation is suspended in physiological saline (PBS) containing phosphate buffer (pH 7.4).

[Immobilization of Avidin on Substrate Surface] As a substrate for immobilization, tin oxide (Sn
O ₂ ), ITO, gold or other conductive material is deposited to form a thin film electrode 5
As shown in FIG. 1 (C), a thin film 6 is further formed on the surface of the substance such as nitrocellulose having a high protein adsorption capacity, and avidin 7 is added as shown in FIG. 1 (D). The method of adsorption is used. For the preparation of such a thin film, a method in which 50 microliters of 0.002% amyl acetate solution (colodion solution) of nitrocellulose is placed on a 2 cm 2 electrode surface and dried and solidified,
Alternatively, a method may be used in which 20 microliters of a 2% collodion solution is spread on the water surface and the thin film obtained by evaporating the solvent and solidifying is pressed onto the electrode surface. These electrodes are treated with PBS containing 20 μg / ml of avidin at 30 ° C. for 20 minutes to adsorb avidin molecule 7. The immobilization method using the nitrocellulose thin film 6 described above can be widely used for immobilizing avidin on a substrate made of various materials.

Depending on the electrode material, it is also possible to chemically bond the avidin molecule to the electrode plane using a crosslinking reagent such as formaldehyde or glutaraldehyde.

It is considered that the nitrocellulose thin film adsorbs an avidin molecule by a non-covalent bond, and when a residue of a cross-linking reagent is attached, it covalently bonds with an avidin molecule.

[Immobilization of biotinylated chromatophore membrane on the surface of avidinylated substrate]
020 = 5.0) at 30 ° C for 20 minutes, and after the reaction, wash with PBS to remove the unbound chromatophore membrane. By the above operation, as shown in FIG. 1 (E), the chromatophore membrane 8 can be immobilized. After this P containing 1.7% formaldehyde
After treatment with BS at 30 ° C. for 30 minutes, washing with PBS, and returning to the stage of treatment with PBS containing avidin as shown in FIG. 1 (F), the chromatophore membrane 10 is immobilized. By carrying out immobilization a plurality of times in this way, it is possible to carry out multilayer lamination of chromatophore membranes. FIG. 1 (E) shows a case where the nitrocellulose thin film 6 is formed on the surface of the SnO ₂ electrode 5 on the glass substrate 4, and the avidin 7 is adsorbed on the thin film to immobilize the chromatophore film 8 once. Figure 1 (F) shows 2
The case where the layers are fixed once and laminated is shown.

As a method of stacking, a method of stacking a collodion thin film on an immobilized chromatophore membrane and then immobilizing it again is used.

In this way, the Rhodopseudomonas viridis chromatophore membranes 8 and 10 are formed on the glass substrate 4 having the SnO ₂ electrode 5.
FIG. 2 shows the absorption spectrum of the photoelectric conversion layer obtained by laminating 10 times. It can be seen that characteristic light absorption is performed.

By the above-mentioned immobilization method, the thylakoid film can be immobilized with directionality.

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.

The substrate on which the chromatophore membrane is immobilized is immersed in a 1% bovine serum albumin (BSA) aqueous solution and then dried.

[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 schematically shows a photoelectric response detection system of the photoelectric conversion device.
Nitrocellulose thin film on SnO ₂ electrode 14 on glass substrate 15
The chromatophore membrane layer 12 was immobilized and laminated via 13. The counter electrode 11 was formed on it by a method such as placing a mercury ball.

From the lower side in the figure, a light stimulus from a light source 20 such as a strobe lamp or an LED is applied to the chromatophore membrane layer 12 via the glass substrate 15, SnO ₂ electrode 14, and nitrocellulose thin film 13. The potential change generated between the electrodes 11 and 14 by the optical stimulation was taken out by the conductors 16 and 19, and was observed by the oscilloscope 18 via the differential amplifier 17.

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, 10
The photoelectric conversion layer that was once immobilized was used.

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

Thereby, a photoelectric conversion device can be realized.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing immobilization of a chromatophore membrane. (A) is an untreated chromatophore membrane, (B) is a biotinylated chromatophore membrane, and (C) is a SnO ₂ electrode coated with a nitrocellulose membrane. A glass substrate equipped with (D) a nitrocellulose membrane and a glass substrate provided with an avidin-adsorbed SnO ₂ electrode, (E) a glass substrate coated with a nitrocellulose thin film on the SnO ₂ electrode, and avidin-coated When the adsorbed chromatophore membrane is immobilized, (F) shows the case where immobilization similar to (E) is performed twice and laminated. Figure 2 shows the absorption spectrum measured in PBS after a nitrocellulose thin film was placed on the SnO ₂ electrode and the chromatophore membrane was immobilized 10 times. FIG. 3 is a schematic diagram of a photoelectric response detection system of the photoelectric conversion device. FIG. 4 is a diagram showing the potential response of the photoelectric conversion device to the optical stimulation of strobe lamp light. The measurement was performed using a photoelectric conversion layer in which a chromatophore membrane was immobilized and laminated 10 times. Explanation of symbols 1 ... Photosynthetic reaction center protein complex 2 ... Lipid membrane 3 ... Biotinylation reagent: sulfosuccinimidyl 6- (biotinamide) hexanoate 4 ... Glass substrate 5 ... SnO ₂ electrode 6 ... Nitrocellulose thin film 7 …… Avidin adsorbed on nitrocellulose thin film 8 …… Immobilized first layer lipid membrane 9 …… Avidin bound on immobilized chromatophore membrane 10 …… Immobilized second layer lipid membrane 11 …… … Counter electrode 12 …… Immobilized and laminated chromatophore membrane layer 13 …… Nitrocellulose thin film 14 …… SnO ₂ electrode 15 …… Glass substrate 16 …… Counter conductor 17… Differential amplifier 18 …… Oscilloscope 19 …… SnO ₂ electrode lead wire 20 …… Light source

Front page continuation (72) Inventor Sugio Kawamura 1-3-3 Higashi, Tsukuba-shi, Ibaraki Institute of Industrial Technology, Institute of Microbial Technology (72) Inventor Noboru Totsuka 1-3-1 Higashi, Tsukuba-shi, Ibaraki Industrial Technology Institute of Microbial Science and Technology (72) Inventor Toshikazu Mashima 1-4-1, Umezono, Tsukuba-shi, Ibaraki Inside Institute of Electronics Technology, Institute of Industrial Technology (72) Hideki Toyoda 5-9-5 Tokodai, Tsukuba, Ibaraki No. Stanley Electric Co., Ltd. Tsukuba Research Laboratory (72) Inventor Hiroaki Sugino 5-9-5 Tokodai, Tsukuba City, Ibaraki Prefecture Stanley Electric Co., Ltd. Tsukuba Research Center (72) Inventor Shuichi Aji 5 Tokodai, Tsukuba City, Ibaraki Prefecture 9-5, Stanley Electric Co., Ltd., Tsukuba Research Laboratory (56) References JP-A-63-295600 (JP, A)

Claims (2)

[Claims] 1. A substrate comprising a first electrode portion, avidin 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, the specific site of which is selectively biotinylated, and which is bound by the interaction of the avidin and avidin-biotin, and the photoelectric conversion layer. And a second electrode portion formed on the photoelectric conversion layer so as to face the first electrode portion, and between the avidin and the first electrode to which the avidin is fixed. Is a photoelectric conversion device that does not contain proteins or lipids. 2. A step of treating a substrate with a treatment agent containing avidin to avidinize the surface, and a lipid membrane containing a protein having a photoelectric conversion function is treated with a treatment agent containing biotin to modify the biotinylated protein. The steps to be performed and a lipid membrane containing a biotin-modified protein having a photoelectric conversion function are allowed to act on a substrate whose surface is avidinized, and a lipid membrane containing a protein having a photoelectric conversion function is utilized by utilizing the avidin-biotin interaction. A method of manufacturing a photoelectric conversion device, comprising the step of immobilizing on a substrate.