JPH0558490B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a bioelement that utilizes the information processing function between cells of living organisms and a method for manufacturing the same.

(Prior Art) The information processing function of a conventional computer is composed of silicon semiconductor elements, etc., and performs serial logical operations using the von Neumann method (hereinafter referred to as "Neumann"). (referred to as a model computer). Although this method can perform quick logical operations, it has the drawback that it is essentially difficult to process a large number of information in parallel.

On the other hand, one of the biological functions for which research is relatively advanced is the visual function. The eye, which is the organ responsible for vision, is made up of photoreceptor cells.
These photoreceptor cells, especially those arranged on the retina,
They can be roughly divided into cone cells, which distinguish colors, and rod cells, which detect light and darkness.

FIG. 5 is a schematic diagram of a rod cell (hereinafter sometimes referred to as a rod). 11 is the disc membrane,
13 is a connecting cilia, 15 is a mitochondria, 17 is a Golgi apparatus, 19 is a myoid, 21 is a nucleus, 21 is an outer segment, 25 is an inner segment, 27 is a synaptic junction, and 28 is a rod. When light enters the rod-shaped bodies 28 arranged in the retina from the outside (left side of the drawing), the disc membrane 11
The disc membrane 11 The calcium ions contained within are pumped out. As these calcium ions are pumped out, an ion concentration gradient is generated within the rod-shaped body 28, and this concentration gradient becomes a stimulus (signal) that is transmitted to the synaptic junction 27, which then successively passes between neurons. It is known to spread. The disc membrane 11 of the rod has been studied by many researchers as a highly differentiated organelle, and the photoresponsive protein rhodobusin accounts for most of its constituent proteins.

In recent years, based on the knowledge of biology and cerebral physiology mentioned above, many researchers have attempted to realize various functions that could not be satisfied with Neumann type computers. It is expected that non-Neumann type computers, such as biocomputers, will be realized by applying information processing functions and information processing elements possessed by living things such as neurons.

(Problems to be Solved by the Invention) Photoresponsive elements of conventional Von-Neumann type computers using semiconductors made of inorganic and organic materials convert incident photons as information from the outside into electrons. The output medium is the current generated by the In this conventional photoresponsive element, output is transmitted only by electric current, which is the flow of electrons. There was a problem in that it had its own limitations when used as a device and an output device.

Conventionally, when patterning a biological material on a substrate, for example, after coating a photoresist on the substrate, a biological material sample is attached by spin-coating, and then subjected to ultrasonic treatment in an organic solvent such as acetone. A patterning method using so-called lift-off, in which the photoresist is peeled off by applying a photoresist, is known. However, these methods have problems in that the manufacturing process is complicated and biological materials constituting the biodevice, especially unstable proteins, may be denatured.

The object of the first invention of this application is to provide a bioelement for converting information from external light into various ions.

Further, the second invention of this application is to provide a method for easily manufacturing a versatile bioelement with high reliability.

(Means for Solving the Problems) In order to achieve this objective, the biodevice of the present invention comprises a substrate and a plurality of proteoliposomes immobilized on the substrate as a two-dimensional array in a single layer. This proteoliposome is composed of a complex containing at least an artificial vesicle made of phospholipid and a photoresponsive membrane protein, and converts the light input into the proteoliposome mentioned above into movement of ions inside and outside the proteoliposome. It is characterized by having the property of

In carrying out this invention, preferably, the plurality of proteoliposomes described above are immobilized on this substrate in a single-layer two-dimensional array by either antigens or antibodies immobilized on this substrate in a two-dimensional array. The binding is performed by an antigen-antibody reaction between one binding factor and the other binding factor inserted into the aforementioned proteoliposome.

According to the method for manufacturing a biodevice of the present invention, a plurality of proteoliposomes each consisting of a complex containing at least an artificial vesicle made of a phospholipid and a photoresponsive membrane protein are arranged in a single-layer two-dimensional array on a substrate. In manufacturing a biodevice that converts light incident on proteoliposomes into movement of ions inside and outside the proteoliposomes, a single layer of binding factors for either antigens or antibodies is placed on the aforementioned substrate. immobilized as a two-dimensional array pattern, inserting the other binding factor of the aforementioned antigen or antibody into the aforementioned proteoliposome, and binding both the aforementioned one and the other binding factors to each other by an antigen-antibody reaction. As a result, the aforementioned proteoliposomes are immobilized on this substrate as a two-dimensional array in a single layer.

According to a preferred embodiment of the method invention, one of the aforementioned binding factors is an antibody and the other aforementioned binding element is an antigen.

Also, according to a preferred embodiment of this method invention:
The monolayer two-dimensional array pattern of one of the above-mentioned binding factors is produced by irradiating the above-mentioned one of the binding factors uniformly adhered and fixed on the above-mentioned substrate with an ion beam or an electron beam, and denaturing and removing the irradiated portion. It is better to form it by

Furthermore, according to this method invention, preferably:
Insertion of the other binding factor into the proteoliposome is preferably carried out by adding the other binding factor to the solubilized extract.

Further, in carrying out any of the inventions of the above-mentioned biodevice and its manufacturing method, the above-mentioned antibody may be γ-
Glopulins and the above-mentioned antigens are preferably hapten-conjugated phospholipids.

(Function) The bio-element of the first invention of this application is an element made of biological material or bio-analogous material, and is constructed in imitation of an organ related to vision, which is an information input means in higher organisms. be.

Therefore, this biodevice imitates the information processing mechanism of these living things by placing proteoliposomes, which are complexes containing at least artificial vesicles made of phospholipids and membrane proteins, on a single-layer, two-dimensional structure. It is an element formed as an array.

Hereinafter, the principle of the biodevice of the first invention of this application will be explained with reference to the drawings.

FIG. 6 is a sectional view of the biodevice of the present invention for explaining the principle thereof. 29 is a photoresponsive protein (hereinafter in this specification, the photoresponsive protein may be expressed as, for example, rhodopsin); 31 is a phospholipid that constitutes the disc membrane 11; 33 is a soluble protein; 35 is a phospholipid antigen synthesized by covalently bonding a phospholipid and a hapten. In addition, in FIGS. 1A and B, 37 is an antibody obtained against the phospholipid antigen 35, 39 is a substrate, 41 is a spherical proteoliposome, and 43 is a lipid bilayer. Also, in FIG. 6, arrow a
is light as a signal from the outside, arrow b is arrow a
This is an ion that is released in response to a signal. Incidentally, these antigen 35 and antibody 37 are binding factors for performing an antigen-antibody reaction.

In this biodevice, a proteoliposome 41 composed of a photoresponsive protein 29, a phospholipid 31, and a phospholipid antigen 35 is placed on a substrate 39 by an antibody 3.
It has a fixed configuration via 7. This fixation is achieved by the lipid bilayer 43 of the proteoliposome 41.
This is carried out by the antigen-antibody reaction that occurs between the phospholipid antigen 35 contained in the phospholipid antigen 35 and the antibody 37 obtained against the phospholipid antigen 35.

Furthermore, in this figure, the internal and external environments of the proteoliposome 41 are omitted, but the internal and external environments of the proteoliposome 41 are filled with an aqueous solution for protecting biological materials (hereinafter referred to as Unless otherwise specified, the internal and external environments of the ribosome shall conform to this.) Furthermore, the interior of the proteoliposome 41 contains calcium ions, which are the information transmitting substance of this bioelement. (described later). Therefore,
When this bioelement is exposed to light, calcium ions within the proteoliposome 41 are pumped out by the action of rhodopsin 29 inserted into the proteoliposome 41.

Further, according to the method for manufacturing a biodevice, which is the second invention of this application, a photoresponsive protein 29, which is a target protein, is inserted into a proteoliposome 41. An antibody 37 for immobilizing this proteoliposome 41 on the substrate is formed as a monolayer on the substrate 39. Next, the antibody 37 on the substrate 39 is denatured and removed using an ion beam or the like, and an antigen-antibody reaction is performed with the phospholipid antigen 35 contained in the lipid bilayer 43 of the proteoliposome 41. The proteoliposomes 41 are patterned.

(Example) Hereinafter, examples of the biodevice of the present invention and its manufacturing method will be described in detail with reference to the drawings.

Preparation of solubilized extract First, the dark-adapted retina was detached from the bovine eyeball, and in order to destroy the retinal structure, it was suspended in erythrodextrin-Ringer's solution and stirred to prepare a photoreceptor cell suspension. do. The Ringer's solution used at this time contained 0.65% by weight of NaCl, 0.014% by weight of KCl,
CaCl ₂ and NaHCO ₃ were prepared to be 0.012% by weight and 0.012% by weight, and erythrodextrin was added to this Ringer's solution to give a specific gravity of 1.10, which was used as an erythrodextrin-Ringer's solution. This photoreceptor suspension was centrifuged to obtain 1.10% of the erythrodextrin-Ringer's solution mentioned above.
Centrifuge the density gradient in a range of approximately 1.07 to 1.07. The outer segment fraction is obtained by collecting the fraction that precipitates during this centrifugation.

Next, this outer segment fraction is suspended in a sucrose solution, washed, and then separated by centrifugation. Through these purification operations described above, a substantially single disk membrane fraction can be obtained. The various operations described in this example were carried out by the methods described in, for example, the literature: Biochemistry Experimental Course, Volume 14, "Biological Membranes", Page 431.

Subsequently, this disc membrane fraction was treated with 110mM NaCl,
Suspend in an aqueous solution (hereinafter, this aqueous solution is referred to as solution N) consisting of 2mM KCl, 1.8mM CaCl ₂ and 20mM sucrose. The nonionic surfactant Triton
The above addition destroys the lipid bilayer 43. The state of the sample solution thus obtained (hereinafter referred to as solubilized extract) is shown in FIG. 3A. However, in this figure, the same components as those in FIG. 6 are denoted by the same reference numerals. The actual state is such that the above-mentioned surfactant is attached to insoluble substances (i.e., photoresponsive protein 29 and phospholipid 31) in the form of micelles (solubilized state), forming a solubilized extract. However, for convenience of illustration, these insoluble properties are shown in a monodisperse state in FIG. 3A and FIG. 3B described later.

In this solubilization procedure, the solubilizer is
X-100, but other than this, such as NP-40
A nonionic surfactant such as the above is preferable because it has less adverse effects (activation, denaturation, etc.) on proteins.

Reconstitution of proteoliposomes Phospholipid antigen 35 (corresponding to the other binding factor in this example) expressed by the formula (next page) was added to the solubilized extract thus obtained.
Add about 1% by weight of 1 and stir until uniform. The solubilized extract in this state is shown in FIG. 3B.

The above-mentioned solubilized extract containing the phospholipid antigen 35 is placed in a dialysis membrane, the above-mentioned solution N is used as the external dialysis solution, and the solubilizing agent is removed by a modified cholic acid dialysis method to reconstitute proteoliposomes 41. I let it happen.

FIG. 2 is a schematic cross-sectional view of proteoliposome 41 obtained by the above-described operation. As can be understood from this figure, proteoliposome 4
Rhodopsin 29 is inserted into the lipid bilayer 43 of 1, penetrating the lipid bilayer 43, and the disc membrane 11 is formed inside the lipid bilayer 43, isolated from the outside. A part of the soluble protein 33 contained in the dialysis fluid is contained together with the above-mentioned dialysis fluid. Furthermore, this lipid double phase layer 43
The phosphorus that made up 1 Lipid 31 and artificially added phospholipid antigen 35
It is composed of.

Furthermore, as can be understood from the formula, the phospholipid antigen 35 has an aromatic ring in its hydrophilic group, so the head polar group is bulky. Therefore, in the lipid bilayer 43, there is a high probability that it exists in the outer layer with high curvature. Similarly, if the photoresponsive protein 29 in the proteoliposome 41 is reconstituted after a sufficient period of time as in the method described above, it is thought that it will be inserted in the same direction as in vivo.

Further, in this example, the amount of the phospholipid antigen 35 added was approximately % by weight of the phospholipid 31, but this amount was
1 by weight is sufficient to cause an antigen-antibody reaction with the antibody 37 (corresponding to one binding factor in this example) attached to the substrate 39 and to be immobilized on the substrate 39. % or more or less than 1% by weight.

Formation of Antibody-Adhering Substrate Hereinafter, a processing operation for forming an antibody as a monomolecular film on the substrate 39 for constructing the biodevice of the present invention will be described. In this example, we will explain the case where a glass substrate is used due to the flatness of the substrate surface and the ease of processing operations, but polystyrene etc.
The reference may be made of any other suitable material.

First, the substrate surface is hydrophobized by immersing the substrate in octadecyltrichlorosilane in advance.

In addition, the water tank of the Langmuir-Blodgett membrane forming apparatus was filled with the above-mentioned solution N, and the antibody 37 obtained against the phospholipid antigen 35 (particularly near the aromatic ring of the phospholipid antigen of the formula γ-Globulin) was dissolved in the aforementioned Ringer's solution at a concentration of 1 mg/ml, this antibody solution was spread on the water surface of the aforementioned water tank, and by compressing the water surface, the antibody 37 was dissolved on the aforementioned water surface. forms a monolayer of Due to the polar distribution of antibody molecules, this molecular membrane is formed in a certain direction, ie, with the antigen-binding region ( _Fab region) of the antibody facing down.

Subsequently, this monomolecular film composed of the antibody 37 is transferred by a horizontal adhesion method onto the surface of the substrate 39 whose surface has been made hydrophobic, thereby forming a single layer LB film on the substrate 38. FIG. 4A is a sectional view schematically showing the substrate 39 in this state. 45 indicates the LB membrane consisting of antibody 37. By the method described above,
A monomolecular film of the antibody 37 is formed on the hydrophobized substrate 39 with the antigen-binding region facing upward (hereinafter, the monomolecular film of the antibody 37 attached to the substrate 39 is referred to as LB film 47).

Furthermore, in this example, the antibody 37 was attached onto the surface of the substrate 39 using an LB film forming apparatus, but for simplicity, the substrate 37 was subjected to hydrophobic treatment.
The antibody 37 can also be adsorbed onto the substrate 39 by directly immersing it in the aforementioned antibody solvent (γ-globulin dissolved at 1 mg/ml).

Patterning process of LB film attached substrate Next, patterning process of LB film attached substrate will be explained.

The substrate 39 on which the LB film 47 is formed is denatured and removed by irradiating the portion where the antibody 37 is unnecessary (the portion where the proteoliposome 41 is not immobilized) with an ion beam such as gallium ions or an electro beam while scanning. By this removal process, antibodies 37 are formed on the surface of the substrate 39 at intervals of 200 nm.
A single-layer two-dimensional pattern was created using the method. FIG. 4B is a cross-sectional view of the substrate schematically showing this state.

Creation of two-dimensional array The substrate 39 on which the above-mentioned LB film 47 has been two-dimensionally patterned is immersed in a solution N in which the above-mentioned reconstituted proteoliposomes 41 are suspended to perform an antigen-antibody reaction. . The state in which a monolayer two-dimensional array of proteoliposomes was thus formed is shown in FIG. 1A.

Figure 1A shows reconstituted proteoliposome 41
is shown as a schematic cross-sectional view of the element substrate in a state where it is fixed to the substrate 39.

Further, FIG. 1B is a perspective view schematically showing a portion of the biodevices, in order to show that the biodevices produced by the above-described method are arranged in a two-dimensional array.

Confirmation of photoresponsiveness In order to confirm that the biodevice of the present invention responds to light, the biodevice prepared by the above method was irradiated with visible light, and then the solution N in which the biodevice was immersed was irradiated with visible light. A partial specimen was collected.

Quantification of calcium ions in the collected solution N is as follows:
After chelating calcium ions using Quin2 (manufactured by Dojindo Laboratories, trade name), the excitation wavelength was
This was done by measuring the fluorescence intensity at 340nm and a fluorescence wavelength of 490nm. According to this measurement, a clearly significant increase in calcium ions in the external solution was observed before and after visible light irradiation (data not shown). It was confirmed that the calcium ions contained in proteoliposome 41 were pumped out.

The above-described embodiments are preferred examples to facilitate understanding of the present invention, and these numerical conditions are as follows:
It is clear that changes in design are possible within the scope of the purpose of this invention. For example, in the above-mentioned example, bovine rhodopsin was used to create the biodevice, but other types of icarhodopsin, chicken iodopsin, crucian carp borphylopsin, and halophilic bacteriorhodopsin (however, bacterial dopsin is a proton membrane Transmission.) etc. can also be used.

Further, in the above-described embodiment, the antibody 37 is attached onto the substrate 39, and the phospholipid antigen 35 is inserted into the lipid bilayer 43 of the proteoliposome 41, so that the antigen-
This is the case where an antibody reaction was performed. however,
The substrate 3 is formed by the above-described method of attaching a monomolecular film.
9, and on the other hand, for example, the phospholipid 31 and antibody 35 are crosslinked by acting on a crosslinking agent such as glutaraldehyde and a maleimide compound, and then inserted into the lipid bilayer 43 of the proteoliposome 41. It is expected that similar results will be obtained by

Furthermore, in the method for manufacturing a biodevice of the present invention,
Although antibodies and antigens have been described as examples of binding factors that immobilize proteoliposomes on a substrate, by replacing these binding factors with avidin and biotin contained in egg white, for example, a method similar to this invention can be obtained. bio-elements can be obtained.

(Effects of the Invention) As is clear from the above description, the biodevice which is the first invention of this application rapidly responds to light as an external signal in a two-dimensional array of proteoliposome monolayers on a substrate. Since it emits ions, it can be used as a new image information input device.

In addition, according to the method for manufacturing a biodevice, which is the second invention of this application, antibodies on a substrate are patterned using an ion beam or the like and then immobilized, so that the antibodies respond to external signals. However, there are few adverse effects such as protein denaturation.
Further, according to this production method, an antigen-antibody in which one binding factor is a phospholipid antigen obtained by binding a hapten to a phospholipid constituting a proteoliposome, and an antibody obtained against the phospholipid antigen is the other binding factor. Since a reaction is utilized, a responsive biological substance can be immobilized on a substrate without adverse effects such as inactivation of physiological activity and precipitation of proteins that occur when an antibody binds to an antigen.

[Brief explanation of drawings]

FIG. 1A is a schematic cross-sectional view of a device substrate for explaining an embodiment of the biodevice of the present invention, and FIG. 1B is a schematic cross-sectional view for explaining the biodevice of the present invention and its manufacturing method. FIG. 2 is a perspective view of the device substrate, and FIG.
Figures A and B are explanatory diagrams of the state of the solubilized extract for explaining the method for producing bioelements of the present invention, and Figures 4A and B are for the attachment of monomolecular films in the method for producing bioelements of the present invention. FIG. 5 is a cross-sectional view of a substrate to explain the state, FIG. 5 is a schematic diagram of a cell to explain the rod-shaped body, and FIG. 6 is a schematic cross-sectional view of a proteoliposome to explain the action of the present invention. FIG. 11... Disc membrane, 13... Connecting cilia, 15... Mitochondria, 17... Golgi apparatus, 19... Myoid, 2
1... Nucleus, 23... Outer segment, 25... Inner segment, 27... Synaptic junction, 28... Rod, 29... Light responsive protein (rhodopsin), 31... Phospholipid, 33... Soluble protein, 35... Phospholipid antigen , 37...antibody, 39...
Substrate, 41... Proteoliposome, 43... Lipid bilayer, 45... LB membrane.

Claims (1)

[Claims] 1. Comprising a substrate and a plurality of proteoliposomes immobilized on the substrate as a two-dimensional array in a single layer, the proteoliposomes comprising artificial vesicles made of phospholipids and a photoresponsive membrane. 1. A biodevice comprising a complex containing at least a protein and having a property of converting light input into the proteoliposome into movement of ions inside and outside the proteoliposome. 2. In the biodevice according to claim 1, the immobilization of the plurality of proteoliposomes to form a two-dimensional array in a single layer on the substrate includes antigens immobilized on the substrate in a two-dimensional array. Alternatively, binding is performed by an antigen-antibody reaction between one of the binding factors of the antibody and the other binding factor inserted into the proteoliposome. A bio element characterized by: 3. The biodevice according to claim 2, wherein the antibody is Y-globulin. 4. The biodevice according to claim 2, wherein the antigen is a phospholipid to which a hapten is bound. 5 A plurality of proteoliposomes each consisting of a complex containing at least an artificial vesicle made of phospholipid and a photoresponsive membrane protein are immobilized on a substrate as a two-dimensional array in a single layer. In manufacturing a biodevice that converts light into movement of ions inside and outside the proteoliposome, a binding factor for either an antigen or an antibody is immobilized on the substrate as a two-dimensional array pattern in a single layer, and the antigen or Inserting the other binding factor of the antibody into the proteoliposome, and binding both the one and the other binding factors to each other by an antigen-antibody reaction, thereby forming the proteoliposome into a monolayer two-dimensional structure on the substrate. A method for manufacturing a biodevice, characterized by fixing it in an array. 6. The method for manufacturing a biodevice according to claim 5, wherein the one binding factor is an antibody, and the other binding device is an antigen. 7. In the method for manufacturing a biodevice according to claim 5, the monolayer two-dimensional array pattern of the one binding factor is formed on the one binding factor uniformly adhered and immobilized on the substrate. 1. A method for producing a bio-element, characterized in that the bio-element is formed by irradiating an ion beam or an electron beam and denaturing and removing the irradiated portion. 8. In the method for producing a biodevice according to claim 5, the other binding factor is inserted into the proteoliposome by adding the other binding factor to the solubilized extract. Characteristic method for manufacturing biodevices. 9. The method for manufacturing a biodevice according to claim 5, wherein the antibody is Y-globulin, and the antigen is a phospholipid to which a hapten is bound.