JPH03171686A - Bioelement - Google Patents

Abstract

PURPOSE:To obtain a fluorescence intensity change field corresponding to the potential field of a visual cell wall so as to obtain a required bio-element with out inducing the deterioration of ribosome by a method wherein ribosomes containing Ca<2+> ion sensitive fluorescence dye are two-dimensionally arranged on a substrate, where the ribosome formed using either or both of biomaterial and analogous biomaterial converts incident light into the concentration change of Ca<2+> ion. CONSTITUTION:The surface of a substrate is previously made hydrophobic by dipping the substrate into octadecyltrichlorosilane. Antibody gamma-globulin obtained to phosphorus lipide antigen 35 is spread over a sub-phase water solu tion in a Langmuir-Blodgett film forming device tank, and an antibody monomo lecular film is formed on the surface of the water solution by compressing the surface of the water solution. The monomolecular film is transferred onto the surface of a substrate through a horizontal adhesion method to form an LB film. A substrate 47 provided with an LB film 49 is dipped into a ribosome suspended solution, whereby a antigen-antibody reaction is made to take place to form a bioelement.

Description

[Detailed Description of the Invention] (Field of Industrial Application) This invention is directed to bio-elements that imitate the optical information processing function of living organisms. (Prior Art) Conventional computers are composed of silicon semiconductor elements, etc.
mann) method to perform serial logical operations (
Hereinafter, it will be referred to as a von Neumann computer. ). However, although this method was able to perform logical operation X quickly, it had the main drawback that it was difficult to process a large number of information in parallel. Therefore, based on knowledge of biology and cerebral physiology, many researchers have attempted to create a so-called biocomputer, a computer with various functions that could not be satisfied with a Neumann type computer. There is. One example is a biocomputer that attempts to process visual information by imitating the visual function, which is a relatively well-researched biological function. Here, the visual function in living organisms is as explained below. First, as is well known, the eye, which is the organ responsible for vision, is composed of photoreceptor cells. These photoreceptor cells, especially the cells arranged in a row on the retina, can be broadly divided into cone cells, which detect color, and rod cells, which detect brightness and darkness. Figure 6 is a schematic diagram of a rod cell (hereinafter sometimes referred to as rod ε). 11 is the disc membrane, 13 is the connecting cilia, 15 is the mitochondria, 17 is the Golgi apparatus, ■9 is the myoid, 21 is the nucleus, 23 is the outer segment, 25 is the inner segment, 27 is the synaptic junction, and 28 is the rod. ．． When light enters from the outside of the rod V bodies 281 arranged in the retina (left side of the figure), a change occurs in rhodobusin, a light-responsive protein present in the disc membrane 11, and covalent bonds to this rhodobusin as a prosthetic group. The cis (cis)-retinal is trans (
trans) - Calcium ions contained within the disc membrane 11 by changing into retinal
! released into the body.

The increased calcium ions in the cell membrane close the sodium channels in the cell membrane of the outer segment 23, causing hyperpolarization of the cell's membrane potential. This becomes a signal to the synaptic junction 27, which reduces the release rate of inhibitory neurotransmitters and causes excitation of post-synabular neurons. This signal 1 propagates between neurons one after another and undergoes advanced information processing in the brain. In this way, in vision, the field of changes in membrane potential of photoreceptor cells on the retina caused by light is processed in the brain and recognized as a pattern. As a conventional example of an artificial biodevice imitating a photoreceptor cell in order to construct a biocomputer that processes visual information, for example, the present applicant 1 discloses Japanese Patent Application Laid-open No. 111428/1983 C. There was something that had not been disclosed. This biodevice consists of a two-dimensional array of broteoliposomes, which convert light incident on the biodevice into ions or chemicals, on a substrate. The biodevice disclosed in 1982-111428 was able to convert incident light into ions or chemical substances, but some kind of information conversion process is required to obtain positional information of the incident light on the biodevice. Met.

Therefore, it is difficult to directly obtain a field for changes in ion J degree or a field for changes in chemical substance concentration that correspond to the field for changes in membrane potential of photoreceptor cells, so there are naturally limits to how they can play the role of photoreceptor cells. Ta. In addition, the role of photoreceptor cells can be fulfilled by a two-dimensional array of photoresponsive elements such as photodiodes made of inorganic or organic semiconductors, and the field of membrane potential of photoreceptor cells can be improved. It is also possible to obtain the corresponding field of current change, but
Because there are limits to the fine arrangement of photodiodes, etc., it is difficult to obtain a field of precise current changes that corresponds to the membrane potential field of a photoreceptor cell. This invention has been made in view of these points, and therefore, the purpose of this invention is to provide a bioelement that can be used to construct a 1M biocomputer that has an information processing form that imitates the visual sense of living things. (Means for Solving the Problems) In order to achieve this object, the biodevice of the present invention is formed using one or both of a biological material and a biosimilar material, and converts incident light into Ca. ``A liposome that converts into a change in the concentration of ions, and is characterized by a two-dimensional array of liposomes containing Ca2+ ion-sensitive fluorescent balms on a substrate.In carrying out this invention, the above-mentioned liposomes are Preferably, the liposome is constructed by reconfiguring the disc membrane present in photoreceptor cells of living organisms.Furthermore, in carrying out the present invention, the above-mentioned liposome is immobilized on the substrate using an antigen-antibody reaction. (Operation) According to the biodevice of the present invention, incident light is converted into Ca2+
Changes in ion concentration {Since the liposomes are two-dimensionally arranged and can be converted into changes in fluorescence intensity according to changes in Ca2+ ion concentration, the position information of the incident light on the bioelement and the field of the membrane potential of photoreceptor cells are A field of fluorescence intensity changes corresponding to is obtained. In addition, when immobilizing liposomes on a substrate by an antigen-antibody reaction, the procedure is to first form a film from antibodies on the substrate, and then irradiate unnecessary parts of this film with an ion beam such as gallium ions. A desired two-dimensional pattern can be created from the remaining antibody after denaturation and removal, and then liposomes can be immobilized on this antibody. For this reason, the liposome itself is not subjected to M-contact processing using an ion beam or the like, so the desired bioelement can be obtained without causing any deterioration of the liposome. Examples of the biodevice of the invention will be explained in detail. Each figure used in the explanation schematically shows the dimensions, shapes, and arrangement relationships of each component to the extent that this invention can be understood. First, we detached the adapted retina from the cow's eyeball,
To destroy this retinal structure, prepare a photoreceptor suspension by suspending and stirring in erythrodextrin-Ringer's solution. The erythrodextrin-Ringer's solution used at this time contained 0.65% by weight of NaCj7 and 0.65% by weight of K (l).
014% by weight, Q a G R 2 is 0.012% by weight
, Erythrodextrin was added to Ringer's solution adjusted to have a NaHCOs content of 0.012% by weight to give a specific gravity of 1.10. This photoreceptor suspension is subjected to density gradient centrifugation using a centrifuge at a density gradient of the above-mentioned Eryto-O dextrin-Ringer's solution in the range of 1.10 to about 1.07. Collect both precipitated fractions by this centrifugation (this will yield the outer segment fraction. Next, wash both outer segment fractions by suspending them in 1 sucrose solution, then centrifuge to separate them. Through these purification operations described above, an almost single disk membrane fraction can be obtained.
For the operations described in this example, see, for example:
The method described in Biochemistry Experiment Course, Vol. 14, "Biomembranes", p. 431, etc. is used.

Subsequently, this disc membrane fraction @ IIOmM-N a C
(1, 2mM-KCA, l.8mM-C a C II
Suspend in an aqueous solution consisting of 2 and 20 mM sucrose (hereinafter, this aqueous solution will be abbreviated as solution N). A nonionic surfactant (hereinafter sometimes referred to as a solubilizer) Triton X-100 (
polyoxyethylene glycol pt-octylphenyl ether (trade name) at critical micelle concentration (approximately 0.24 m
M) The above addition destroys the fat-containing double layer. The state of the sample solution thus obtained (hereinafter referred to as solubilized extract) is shown in Figure 2 (A). The solubilized extract is actually an insoluble substance (a state in which the above-mentioned surfactant is attached to the photoresponsive protein 29 and phospholipid 31 in the form of micelles (in a solubilized state)). However, for convenience of illustration, in Fig. 2 (A) and Fig. 2 (B) described below,
These insoluble substances are shown as monodisperse. In addition, in this solubilization operation, the solubilizer is
on-100, but the solubilizer is not limited to this. For example, NP-40W nonionic surfactant may have negative effects on proteins (inactivation, deformation, etc.)
It can be used because there are few Broteoliposome Next, the phospholipid antigen represented by the formula (corresponding to the other binding factor when obtaining a biodevice by an antigen-antibody reaction) was added to the solubilized extract obtained in this way. Phosphorus Tribute 3
1 (see Figure 2 (A)) and Ca2+ ion concentration-sensitive fluorescent dye Quin2 (
(trade name, manufactured by Dojindo Kagaku Kenkyusho) to a concentration of 10 uM and stir to make it uniform. The solubilized extract in this state is shown in Figure 2 (B). In Figure 2 (B), 35 is a phospholipid antigen and 37 is a fluorescent dye. The phospholipid antigen represented by formula (■) is obtained by synthesis. CH3 Next, the solubilized extract containing the above-mentioned phospholipid antigen 35 and fluorescent dye 37 is placed in a dialysis membrane, and the solubilizing agent is removed by a modified cholic acid dialysis method using the above-mentioned solution N as the external dialysis liquid. is removed to reconstitute the liposomes.

FIG. 3 is a cross-sectional view schematically showing the structure of broteoliposome 41 obtained by the above-mentioned operation. As can be understood from this figure, rhodobucin 29 is inserted into the lipid bilayer 43 of the broteobinome 4l, and the inside of the lipid bilayer 43 is , a part of the soluble protein matrix 33 and the fluorescent membrane 3 that constituted the disc membrane 11 in a state of being isolated from the outside.
7 is included together with the above-mentioned external dialysis fluid. Roughly speaking, this lipid bilayer 43 is composed of the phospholipid antigen 31 that constituted the disc membrane 11 and the phospholipid antigen 35 that was artificially added.

J:. In addition, as can be understood from the formula
The i group is bulky. Therefore, in the cell bilayer 43, there is a high probability that the phospholipid antigen 35 exists in the outer layer with high curvature. Similarly, if the photoresponsive protein 21 in the proteoliposome 41 is reconstituted after a sufficient period of time as in the method described by Moe, the lipid bilayer 4
3, it appears that the device was inserted in the same direction as in the living body. In this example, the amount of the phospholipid antigen 35 added was approximately 1% of the phospholipid antigen 31, and this added li+
The brodeoliposome 41 used in the biodevice structure is attached to an antibody (described later) attached to a substrate (described later).

), the strain may be less than 1% by volume or more than 1% by weight, as long as it is sufficient to cause an antigen-antibody reaction and be immobilized on the substrate. Next, in order to construct the biodevice of the present invention, a processing operation for forming an antibody monolayer on a substrate will be explained. This large embodiment C is a case where a glass substrate is used as the substrate due to the simplicity of the substrate surface and the simplicity of the processing operation. However, the substrate may be made of boron styrene or any other suitable material.

First, the surface of the substrate is hydrophobized by immersing it in octadecyltrichloride silane in advance.

In addition, the phospholipid antigen 35 (specifically (near the aromatic ring of the phospholipid antigen of this type) (7): The obtained antibody γ-globulin was incubated with Langmuir-Blodgetto (Langmuir-Blodgetto).
A monomolecular film of the antibody is formed on the aqueous solution by spreading the antibody on the subphase aqueous solution in the water tank of the membrane-forming device and compressing the surface of the aqueous solution. Due to the polar distribution of the MJ't5 antibody, it is expressed in a certain direction with the antigen binding region of the MJ't5 antibody facing downward.

Next, the monomolecular film made of this antibody was transferred by a horizontal method to the surface of a substrate whose surface had been made hydrophobic. Figure 4 is a sectional view schematically showing the substrate bubble in this state. In the figure, 45 is an antibody, 47 is a substrate, and 49 is an antibody 45 and indicates a bit. The antibody 45 is transferred so that the binding region with the phospholipid antigen 35 faces the opposite side to the substrate 47 side (in the upward direction of the substrate). In this example, the substrate 4 is formed using the [B film forming apparatus].
However, for simplicity, it is possible to dissolve the antibody 45 in an appropriate solution and directly immerse the hydrophobically treated substrate 47 in this solution. It can be adsorbed onto 47. Ribbon Room 2;-1 The substrate 47 having the antibody membrane 49 described above is immersed in a solution in which liposomes prepared as described above are suspended, thereby causing an antigen-antibody reaction. As a result,
A biodevice of Example is obtained. M1 diagrams (A) and (B) are diagrams for explaining this biodevice, and in particular, FIG. 1 (A) is a diagram schematically showing a state in which proteoliposomes 41 are fixed to a substrate 47, FIG. 1(B) is a diagram schematically showing a part of the biodevice of the example. When arranging the broteoliposomes in a predetermined two-dimensional pattern, first, an unnecessary portion of the 1B film 49 made of antibodies on the substrate 47 is irradiated with an electron beam such as gallium ion or an electron beam while scanning. It is preferable to perform denaturation and removal, create a predetermined two-dimensional pattern using the remaining antibody, and then immobilize the broteoliposomes on this antibody. According to this method, the broteoliposomes themselves are exposed to ion beams or electron beams.
As a result, the broteoliposomes are not directly processed, which prevents deterioration of the broteoliposomes, which is preferable. In order to confirm that the biodevice of the present invention responds to light, the biodevice of the example prepared as described above was irradiated with visible light and then exposed to light at a wavelength of 340 nm. The bioelement is excited by irradiating light, and the fluorescence intensity (F) at a wavelength of 490 nm is measured.

The fluorescence intensity measurement results are plotted on the vertical axis as the rate of change in fluorescence intensity Δ(
%) and time is plotted on the horizontal axis as shown in Figure 5. here,
ΔF is determined by the following formula. However, ■F in the formula. is the fluorescence intensity before visible light irradiation. ΔF=100 CF-Fo )/Fo- This is the end point of irradiation. As can be understood from Figure 5, Ca'' in the internal solution was clearly significant before and after visible light irradiation.
A decrease in Ca2+ and ions was observed, indicating that Ca2+ was pumped out from the liposome of the present invention by the action of the photoresponsive protein. Therefore, the liposome included in the biodevice of this invention converts incident light into Ca2+
It can be seen that the Ca2+ concentration change can be converted into a change in the fluorescence intensity of the fluorescent dye encapsulated in the liposome. Furthermore, according to the biodevice of the present invention, it is clear that if the biodevice is irradiated with light in a certain pattern without irradiating the entire surface with light, a pattern of fluorescence intensity change corresponding to this pattern can be obtained. ．． Although the embodiments of the biodevice of the present invention have been described above, the present invention is not limited to the above-mentioned embodiments; various changes or modifications can be made as described below. ．． The method for producing a bio-element, the numerical conditions used therein, and the materials used for producing the bio-element described in the above-mentioned Examples are preferred examples to facilitate understanding of the present invention, and are examples of the present invention. It is clear that changes in the design are possible within the scope of the purpose. For example, in the above-mentioned example, bio-element production (bovine rhodopsin was used, but other squid-mouth dobuscin,
Children can use aiorodobsin from chickens, porphyrobuscin from crucian carp, bacteriorhodopsin from halophilic bacteria (however, bacteriorhodobuscin has undergone proton membrane permeation), etc.

Further, in the above-described embodiment, the antibody 45 was attached to the substrate 47, and the phospholipid antigen 35 was inserted into the lipid bilayer 43 of the broteoliposome 41 to cause an antigen-antibody reaction to form a biodevice. However, the phospholipid antigen 35 is attached onto the substrate 47 by the method of attaching a monolayer described above, and on the other hand, a crosslinking agent such as glutaraldehyde and maleimide compound is applied to the phospholipid antigen 31 and the antibody 45. The lipid bilayer 4 of the broteoliposome 41 is
3 (By inserting this, it is expected that the same results as in the example will be obtained. In addition, in manufacturing the biodevice of this invention, antibodies and antigens may be used as binding factors to immobilize the broteoliposomes on the substrate. However, by replacing these binding factors with, for example, avidin and biotin contained in egg white, the same biodevice as in the example can be obtained.In addition, in the above example, Ca2+ ion transport Although the liposome was explained as a broteoliposome because the donor for transporting Ca2+ ions was a protein donor, the same effects as in the example can be expected with liposomes in which the transport substance for Ca2+ ions is a substance other than a protein donor. (Effects of the Invention) As mentioned above. As is clear from the above description, the biodevice of the present invention is capable of converting incident light into a field of fluorescence intensity changes corresponding to a field of Ca'◆ ion concentration changes in two-dimensionally arranged liposomes. Therefore, information on the position of the incident light on the bio-element can be obtained. Therefore, it is possible to use the information processing device, input device M or output device ffi!, etc. of a biocomputer that has an information processing form that imitates the vision of living things. You can.

[Brief explanation of the drawing]

Figures 1 (A) and (B) are diagrams used to explain the biodevice of the example, Figures 2 (A) and (B) are diagrams explaining the state of the solubilized extract, and Figure 3 is a diagram used to explain the state of the solubilized extract. FIG. 4 is a diagram schematically showing the structure of the ribbon beam in the example. FIG. 4 is a diagram that provides an explanation of the antibody-attached substrate. FIG. , a diagram schematically showing a rod cell. 29...Photoresponsive protein v4 (rhodobusin) 31...
Phospholipid, 3 3... Soluble protein 35.
...Phospholipid antigen, 37...Fluorescent fluorophore 41...
Proteoliposome 43... Phospholipid bilayer, 47... Substrate, 45... Antibody 4 9-... 4 LBII from antibody. Patent application filed by Oki Electric Industry Co., Ltd. Time (min) Figure 5 showing the photoresponse failure of the biodevice of the example

Claims (3)

[Claims] (1) A liposome that is formed using one or both of a biological material and a biosimilar substance, converts incident light into a change in the concentration of Ca^2^+ ions, and contains a Ca^2^+ ion-sensitive fluorescent dye. A biodevice characterized by a two-dimensional arrangement of liposomes on a substrate. (2) The biodevice according to claim 1, wherein the liposome is composed of a reconstituted disc membrane present in photoreceptor cells of living organisms. (3) A biodevice, wherein the liposome is immobilized on the substrate using an antigen-antibody reaction.