JPH0442585A - Photoresponsive excitable synthetic membrane and its manufacture - Google Patents

Abstract

PURPOSE:To control the oscillating phenomenon of the title synthetic membrane withlight by adding a lipid and Bacteriorhodopsins which give excitability to the film to a carrier with fine holes. CONSTITUTION:Bacteriorhodopsins 27 is regularly oriented and adsorbed to an excitable synthetic membrane 21b constituted of a carrier with fine holes impregnated with DOPH. When low-concentration salt solution (KCl aqueous solution) 22 and high-concentratin salt solution (KCl aqueous solution) are separated from each other with this photoresponsive excitable synthetic membrane 28 with the surface adsorbing the Bacteriorhodopsins on the solution 22 side, the DOPH readily makes phase transition between oil drops and multiayered films, because the active transportation 29a of protons produced when the membrane 28 is irradiated with light accelerates the flow 29b of K<+>. As a result, the excitability of the synthetic membrane 28 can be controlled readily with light.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an excitable artificial membrane that imitates the optical information processing function of living organisms and a method for producing the same.

(Prior Art) Conventional computers are mainly constructed of inorganic materials such as silicon semiconductors, and perform serial logical operations using the van Neumann method (hereinafter referred to as Neumann type computers). ). However, although this method was able to perform logical operations accurately, it had the disadvantage that it was difficult to process a large number of information in parallel, and it was not good at pattern recognition.

On the other hand, higher organisms easily perform pattern recognition, etc., as is well known. Therefore, if we can elucidate the principles on which pattern recognition and learning/memory functions found in the brain are carried out, and what elements they are carried out by, and then imitate them, we can It is expected that it will become possible to realize computers, such as biocomputers, that have various functions that could not be satisfied with von Neumann type computers.

For example, the visual function among biological functions is expressed as described below.

In the eye, which is the organ responsible for vision, the retina contains cone cells, which distinguish colors, and rod cells, which distinguish light and darkness. FIG. 6 is a diagram schematically showing the structure of this rod-shaped body cell 20 (hereinafter sometimes referred to as rod-shaped body 20).

In Figure 6, 11 is the disc membrane, 12 is the connecting cilia, and 13 is the
is mitochondria, 14 is Golgi apparatus, 15 is myoid,
16 is the nucleus, 17 is the segment, 18 is the interior, 19 is the synaptic junction, and 20 is the rod. Rods arranged in the retina 2
When light enters into the disk film 11 from the outside (right side of the drawing),
This causes a change in rhodopsin, a photoresponsive protein present in
ans) - disk membrane 1 by changing to retinal
Calcium ions contained within 1 are released into the cytoplasm.

The increased calcium ions in the cytoplasm close the sodium channels in the cell membrane of segment 17, causing hyperpolarization of the cell's membrane potential. This becomes a signal to the synaptic junction 19, reducing the rate of release of inhibitory neurotransmitters and causing excitation of the postsynaptic neuron. These signals propagate between neurons one after another and are processed at a high level in the brain.

In order to realize an artificial functional element that imitates such a biological function, research has been carried out on the creation of an artificial Jl, that is, an excitable artificial membrane, which is excited by external stimulation.

Examples include the literature ■ (MEMB day AN
E), 12 (1) (1987) p. 12-21), there was an excitable artificial membrane constructed by adsorbing a special lipid onto a porous membrane. According to this excitable artificial membrane, a self-supporting oscillation phenomenon similar to the excitatory phenomenon seen in nerves is obtained. This self-assisted oscillation phenomenon will be briefly explained below.

An excitable artificial membrane is produced by adsorbing dioleyl phosphate, which is a synthetic lipid (hereinafter sometimes referred to as a biosimilar lipid), to a porous membrane having a pore diameter of about several um. Such an excitable artificial membrane is brought into contact with a high concentration salt solution on one side and a low concentration salt solution on the other side. The excitable artificial membrane placed in such a state generates a predetermined potential difference in the darkness of these salt solutions over a period of several minutes to several tens of minutes, and generates electrical impulses. Self-sustained oscillation with such a period is called long-period oscillation.

In addition, the difference in salt concentration between the two sides of the excitable artificial membrane (
In addition to applying a direct current (ion concentration gradient) to the excitable artificial membrane, when a predetermined pressure is applied to the excitable artificial membrane, self-excited oscillation of about a few seconds, called short-period oscillation, occurs. The period of this short-period oscillation can be controlled by changing the above-mentioned DC current.

The mechanism of automatic oscillation described above is thought to be accompanied by a change in the lipid aggregate structure from an oil droplet state to a multilayer film state. This mechanism will be explained in more detail by citing the above-mentioned literature.

Figures 7 (A) and (B) are diagrams for explaining the minute through-holes 21a! Micropores 21 of support body 21 having
FIG. 3 is a diagram showing a state in which a low concentration salt solution 22 and a high concentration salt solution 23 are partitioned off by an excitable artificial membrane formed by impregnating lipid or biosimilar lipid in a. In particular, the area around one microhole is shown.

Dioleyl phosphate (hereinafter referred to as
It is sometimes abbreviated as DOPH. ), the oil droplet state is stable in low-concentration salt solutions and the multilayer film state is stable in high-concentration salt solutions, after a certain salt concentration. Therefore,
As shown in FIG. 7(A), the DOPH has a micropore 21a.
At the outlet on the low concentration salt solution side, it becomes an oil droplet 24 and forms a micropore 2.
1a18: It is blocked, and the cations 25 flow into the micropores 2ia from the highly concentrated salt solution side, so that the oily substance changes into a multilayer film 26. In this state, more cations than anions in the salt solution selectively flow into the micropores 2Ia, so anions (not shown) are left on the support 21, and the membrane potential is lowered. arise. However, as shown in FIG. 7(B), as the change of the oil liquid 24 into the multilayer film 26 progresses, the oil droplets 24 that have formed in the micropores 21a become smaller and the outlet opens, and the micropores 21a penetrate. , the high-concentration cations in the micropores 21a flow out to the low-concentration salt solution side by diffusion, and the cation concentration in the micropores 21a decreases. From this point on, the anions remaining on the support 21 disappear after a certain relaxation time due to diffusion of the anions themselves and recombination with the cations that have diffused.6 After that, the concentration of cations in the micropores 21a becomes low. Since this shows that DOPH is in a stable state when soaked in oil, it returns to the state shown in FIG. 7(A) again. In this way, this excitable artificial membrane repeats self-excited oscillation. Table 1 summarizes the properties of the excitable artificial membrane explained in FIGS. 7(A) and 7(B) using IFr.

Table 1 When using the excitatory artificial H described above, for example, the literature ■ (
Membrane CMMB Sun ANE), 12 (4).

pI), 231-237 (1987)), it is known that oscillation waveforms such as oscillation frequency change in response to various taste substances, and it is known that oscillation waveforms such as oscillation frequency change in response to various taste substances. It becomes possible to construct sensors and construct bio-elements.

(Problems to be Solved by the Invention) However, since the conventional excitable artificial membrane as explained using FIG. 7 responds to various taste substances, it has been possible to construct chemical sensors, etc. However, since it is not possible to control the stopping of self-excited oscillation, the progression of oscillation, the modulation of oscillation, etc. by optical stimulation, it has not been possible to construct biodevices that mimic photoreceptor cells.

This invention was made in view of these points,
Therefore, an object of the present invention is to provide an excitable artificial membrane whose oscillation phenomenon can be controlled by light, a method for manufacturing the same, and 1Fr.

(Means for Solving the Problem) In order to achieve this objective, the inventor of this application has conducted various studies. As a result, the literature ■ (Thin 5
olid Films, 99 (1983) pp, 1
33-138), and attempted to achieve the object of the present invention by combining the technology disclosed in this document (1) and the technology disclosed in the above-mentioned document (2). The technology in the literature @ is a membrane made of a cellulose ester filter adsorbed with 5oya (soybean) lecithin and bacteriorhodopsin, and exhibits proton (H+) transportability in response to light, but has no excitability. It concerned a membrane not shown.

Therefore, the photoresponsive excitable artificial membrane of the first invention of this application is
It is characterized in that a lipid that imparts excitability to the artificial membrane and bacteriorhodopsin are adsorbed onto a support having micropores.

Here, the lipids that impart excitability to the artificial membrane include:
Examples include dioleyl phosphate as mentioned above, which imparts excitability to the membrane by blocking or penetrating micropores, and lipids, such as triolein and monoolein, which impart excitability by other mechanisms.

Further, in implementing the first invention, it is preferable that the photoresponsive excitable artificial membrane has a structure in which bacteriorhodopsin is adsorbed to an excitable artificial membrane made by adsorbing lipids to a support having micropores. It is. Here, examples of such excitable artificial membranes include those disclosed in the above-mentioned document (2).

Further, according to the second invention of this application, in producing a photoresponsive excitable artificial membrane, the lipid that imparts excitability to the artificial membrane and bacteriorhodopsin are adsorbed onto a support having micropores. , is characterized in that bacteriorhodopsin is adsorbed onto the support by Langmuir-Blodgett method.

According to the third invention of this application, a photoresponsive excitable artificial membrane is produced by adsorbing a lipid that imparts excitability to the artificial membrane and bacteriorhodopsin onto a support having micropores. The method is characterized in that the adsorption of bacteriorhodopsin to the support is carried out by membrane fusion of ribosomes containing bacteriorhodopsin.

(Function) According to the configuration of the first invention, when the membrane receives light, a photoisomerization reaction of bacteriorhodopsin in the membrane occurs. When the photoisomerization reaction of bacteriorhodopsin occurs, proton (H+) transfer occurs, so by regularly aligning bacteriorhodopsin in the membrane, active transport of protons is performed. Therefore, as shown in FIG. 1, for example, bacteriorhodopsin 27 is regularly applied to an excitable artificial membrane 21b which is made up of a support having minute through-holes (not shown) impregnated with DOPH. The photo-responsive excitable artificial membrane 28, which is oriented and adsorbed, creates a gap between the low-concentration salt solution (KCβ aqueous solution) 22 and the high-concentration salt solution (KCβ aqueous solution) 23 on the side on which bacteriorhodopsin has been adsorbed. If the artificial 1!28 is partitioned so that it is on the low concentration salt solution 22 side, the main active transport 29a of protons as a result of irradiating light will promote the flow 29b of K÷, so D 'Facilitates oil immersion in OPH and phase transition between multilayer films. As a result, the excitability of the artificial membrane 28 is easily controlled by light.

Furthermore, according to the configurations of the second and third inventions of this application, it is easy to control the orientation of bacteriorhodopsin, and therefore the photoresponsive excitable artificial membrane of the first invention can be easily manufactured.

(Example) Hereinafter, examples of the photoresponsive excitable artificial membrane and examples of the photoresponsive excitable artificial membrane of the present invention will be described with reference to the drawings.

In the following description, specific conditions will be exemplified and explained to the extent that the present invention can be understood, but it should be understood that the present invention is not limited only to these conditions.

Further, although some sources of chemicals used in the following examples may be omitted, all chemicals were easily available and chemically sufficiently pure. Also,
It should be understood that the drawings used in the explanation only schematically show the dimensions, shapes, and M% of each component to the extent that the present invention can be understood.

・' At the beginning of Step 6, the microporous support was a cellulose ester porous membrane (Millipore filter, manufactured by Millipore) that had many through holes with a pore diameter of 8 um, and the lipid had the following structural formula. Dioley
Sometimes abbreviated as l Phosphate, DOPH. ), and bacteriorhodopsin (hereinafter sometimes abbreviated as bR) is manufactured by Sigma.
The procedure for producing the photoresponsive excitation artificial membrane of the example (hereinafter sometimes simply referred to as the artificial membrane of the example) will be explained.

First, in this example, DOPH was synthesized and purified as follows.

As a starting material, oleyl alcohol (manufactured by Kanto Kagaku ■)
and phosphorus oxychloride (POCl2) (manufactured by Kaidonghua) by well-known synthetic means, and then the resulting synthetic material is hydrolyzed. The DOPH thus obtained is purified by chromatography.

Next, purified DOPH! It is dissolved in benzene, and then the above-mentioned porous membrane made of cellulose ester is immersed in this solution. Thereafter, this porous membrane is removed from the solution and benzene is evaporated to obtain a porous membrane on which DOPH has been adsorbed. In this example, the amount of DOPH adsorbed is 3 to 6 m9/cm2. The reason why such an adsorption amount is set is that if the adsorption amount is too small, it will not be possible to disturb the through-holes of the artificial membrane, and if it is too large, the through-holes will not be penetrated even by the phase transition explained using Figure 7. This is to avoid. However, it should be understood that this amount of adsorption varies depending on the diameter of the through-holes of the filter used, the density of the through-holes, the type of lipid used, etc.

On the other hand, after dispersing bR in a 25% DMF (dimethylformamide) aqueous solution, this bR-dispersed DMF was spread in a water tank containing pure water, and the DMF was roughly evaporated to form a spread film of bR on the water surface. do.

Figure 2 shows the surface pressure-area (TI-A) of this bR development membrane.
The surface pressure (dyne/cm) is plotted on the vertical axis based on the result of measuring the curve.
The area (λ2) is plotted on the horizontal axis.

Next, in order to adsorb bR to the porous membrane that has already adsorbed DOPH, the following treatment is performed in this example.

First, it is fixed on the porous S18 glass substrate to which DOPH has been adsorbed. Next, this glass substrate is immersed in the above-mentioned water tank in which the bR development film is formed so that the angle between the substrate surface and the water surface is approximately perpendicular, and then pulled up.

That is, the Langmuir-Blodgett (LB) vertical immersion method is applied. This dipping and pulling operation is repeated to accumulate 20 layers of the bR film only on the opposite side of the DOPH absorbing porous membrane from the glass substrate. Incidentally, the surface pressure of the bR film when the bR film is accumulated is 25 dyne/cm. The LB film thus formed has a cumulative ratio of approximately 1 when the glass substrate is pulled up, and a cumulative ratio of approximately 0 when immersed in Z.
It was a type.

Next, the DOPH adsorption porous tribute film on which the bR film has been accumulated is removed from the glass substrate. In this way, the artificial membrane of the example is obtained.

In addition, apart from the preparation of the artificial membrane of the example, DOPH
is adsorbed onto porous material 11 (Millipore filter) to produce an artificial S as a comparative example.

Next, we will explain the device used to confirm the self-erecting fi of the artificial membranes of Examples and Comparative Examples (hereinafter referred to as the device for self-assisted oscillation).
FIG. 3 is an explanatory diagram schematically showing the configuration of the self-help oscillation device.

As shown in FIG. 3, the artificial membrane of Example or Comparative Example (representatively indicated by 31 in the figure) was coated with a 100mM KCρ solution 3 contained in the first electrolytic cell 33a on one side.
5a, and the other side is supported by the self-oscillation device in a state in which the other side is in contact with the 5mM KCβ aqueous solution 35b housed in the second electrolytic cell 33b.

A facility (not shown) for circulating constant temperature water is provided around the first and second electrolytic tanks 33a and 33b, so that the temperature of the hot water in the tank can be controlled to an arbitrary value. In this embodiment, the temperature of the KCβ wood solution 35a, 35b is set to 20'C±1°C. Further, a light transmission window 37Ca for irradiating light onto the artificial membrane 31 is provided in a part of the second electrolytic cell 33b.

A standard electrode 37a or 37El made of silver-silver chloride (A9-A9Cl) is immersed in two types of KCρ aqueous solutions 35a and 35b, respectively.

And 100mMKC which is on the high concentration side! The standard electrode 37a immersed in the solution 35a is connected to the anode side of the DC power supply 39, and the standard electrode 37b in the low concentration 5mMK (l aqueous solution 35b) is connected to the cathode side of the DC power supply 39 mentioned above. , a current is applied to the artificial membrane 31.

In general, this self-oscillation device uses a measuring device 411 consisting of a high impedance electrometer and an X-Y recorder in order to measure and record time changes in the potential difference applied to the artificial membrane 31.
It is equipped with Fr. Then, the standard electrode 43a or 43b connected to this measuring device 41 is connected to the above-mentioned K(l aqueous solution 35).
A and 35b were immersed once each.

The self-oscillating device here includes a manometer 45 connected to the first electrolytic cell 33a, and only through this manometer 458 is the external pressure indicated by the arrow a in FIG. can be applied to the artificial membrane 311. In this embodiment, the pressure applied by the above-mentioned manometer 45 will be referred to as external pressure.

The above-mentioned applied current and external pressure are conditions necessary to cause the artificial membranes of the Examples and Comparative Examples to oscillate, and are set to certain values.

This self-oscillation device is an electrolytic cell 33a.

331), and a light source 49 for irradiating light onto the excitable artificial membrane 31 through the light transmission window 37c. The light source 49 in this embodiment is composed of a 100W halogen lamp equipped with a filter for cutting off heat rays. The reason why the light source 49 is configured as described above is that with this configuration, the absorption wavelength peak (approximately 560 nm) of bacteriorhodopsin for the artificial membrane 31 is achieved! This is because it is possible to effectively irradiate light in a wavelength range including the above.

-1 Next, using the self-sustained oscillation device 1 described above, the self-sustained oscillation of the artificial membranes of the examples and comparative examples was measured as described below.

<Measurement of oscillation characteristics with and without light irradiation> First, the artificial membrane of the example was placed in a 5mM KCA aqueous solution 3 on the side where the bR membrane was accumulated.
5b side (set in the self-excited oscillation device). Then, in a state where no light is irradiated, that is, with the light source 49 turned off, 100mMK (100mMK from the aqueous solution 35a side!!
Direct current and external pressure are applied to the aqueous solution 35t) side. And Tato pressure! When fixed at 20cmH2O,
Changes in the self-supporting oscillation frequency of the artificial membrane of the example with respect to changes in direct current are measured. The characteristic indicated by the square in FIG. 4 is a diagram showing the results, and is a characteristic diagram in which the horizontal axis represents the DC current and the vertical axis represents the launching frequency.

As is clear from FIG. 4, the artificial membrane of the example without light irradiation requires a current of 0.45 uA to cause oscillation.

Next, when the light source 497N is operated to irradiate the artificial membrane with light and the external pressure is fixed at 20 cmH2O, changes in the automatic oscillation frequency of the artificial membrane of the example with respect to changes in the DC power source are measured. The results are indicated by ■ in FIG. As is clear from this figure, in the light irradiation state, the artificial membrane of the example generates oscillation even without the application of current. It can be seen that the oscillation frequency is also higher than that without light irradiation.

On the other hand, when the same experiment as described above was conducted on the artificial membrane of the comparative example, the characteristics shown by I in the M4 diagram were obtained in the same way as in the example. No phenomena such as a decrease in the oscillation starting current or an increase in the oscillation frequency were observed.

Measurement of controllability using oscillating light> Next, the external pressure applied to the artificial membrane of the example was set at 20 cmH.
The artificial film of the example was repeatedly irradiated with light for 20 seconds and then not irradiated for the next 20 seconds M with 2O and a current of 0.2A for 2 days. Then, changes in the oscillation characteristics of the artificial film of the example under such light irradiation conditions are measured. Figure 5 shows this measurement result by 1 on the vertical axis.
The potential of a 00mM Kcβ aqueous solution is plotted and time is plotted on the horizontal axis. Note that the potential on the vertical axis is measured using the potential of a 5 mM KCρ aqueous solution as a reference potential. Further, among the displays of "ON" and rOFFJ alternately shown along the horizontal axis, "ON" indicates the time when the light source 49 is turned on, and rOFFJ indicates the time when the light source 49 is turned off.

As is clear from FIG. 5, the artificial membrane of the example oscillates at a period of about 1 second while being irradiated with light, and does not oscillate at all in the dark when it is not irradiated with light.

Further, the controllability of oscillation by light was measured for the artificial membrane of the comparative example under the same conditions as in the example, but no oscillation was observed regardless of the light irradiation.

4. In addition, DOPH! is used as a membrane filter. After inhaling in the same manner as described above to form excitable artificial N, inhalation of bacteriorhodopsin to this excitable artificial membrane is performed by membrane fusion of ribosomes containing bacteriorhodopsin.

As a specific method, for example, the literature (FEBS
LETTE Japan S (Fence Letters) 76 (197
7) There is a method disclosed in p. 45-50).

In the case of a photoresponsive excitatory artificial membrane obtained by adsorbing bR into an excitable artificial membrane by membrane fusion, does it have the same properties as the light dependence of the oscillation shown above? Admitted.

In the above, each embodiment of the photoresponsive excitable artificial membrane and the method for manufacturing the same of the present invention has been described, but the invention is not limited to the above-mentioned embodiments only, and various modifications as described below can be made. It is possible to add

For example, in the embodiment, the support having micropores is a Millipore filter, but the support is not limited to this, and can be changed to various types depending on the purpose. Further, in principle, the support may have one micropore.

Alternatively, a support made of an inorganic material such as a silicon substrate with micropores may be used.

In addition, in the examples, DOPH was used as a lipid that imparts excitability.
However, the lipid that can be used is not limited to DOPH, and other lipids, whether natural or synthetic, may be used as long as they can obtain the same effect.

In addition, in the above embodiment, for ease of explanation, the third
Illustrating a specific experimental setup N as explained using the diagram,
The cases where various characteristics were measured were explained. However, it is clear that the effects of the photoresponsive excitable artificial membrane of the present invention are not achieved only by a specific device.

(Effects of the Invention) As is clear from the above description, the photoresponsive excitable artificial membrane of the present invention, in addition to the physical properties of conventional excitable artificial membranes, has the same properties as the excitable artificial membrane when irradiated with light from the outside. We newly demonstrate the physical property that automatic oscillation stops when self-sustained oscillation is started and light irradiation is stopped, and the physical property that the oscillation frequency changes due to light irradiation.

Therefore, the photoresponsive excitable artificial membrane of the present invention is useful in constructing a bioelement that mimics the photoreceptor cells of living organisms.

[Brief explanation of the drawing]

Fig. 1 is a diagram used to explain the present invention, Fig. 2 is a diagram showing a surface pressure-area curve of a bacteriorhodopsin-developed membrane, and Fig. 3 is an explanation of the apparatus used to confirm self-oscillation. Figure 4 is a diagram showing the oscillation patent of the artificial membrane of the example with and without light irradiation; Figure 5 is a diagram showing the controllability of the oscillation of the artificial membrane of the example by light; The figure is a diagram schematically showing rod cells, Figure 7 (A
) and (8) are diagrams for explaining the mechanism of self-assisted oscillation. 1...Support, 21a--Micropore 1b
... Excitable artificial membrane 2 ... Low concentration salt solution (KCβ aqueous solution) 3 ... High concentration salt solution (K (l aqueous solution)) 4 ... Lipid in the form of oil droplets 5 ... Cation 6 ... Lipid in a multilayered membrane state 7 ... Bacteriophore dobuscin 28 - ... Photoresponsive excitatory artificial membrane 29 a - ... Active transport of protons 29 b - + flow 31 - ... Comparative example or example Artificial membrane 33a - first electrolytic cell, 33b -! second electrolytic cell 35a - 100mM K
(l aqueous solution 35b - 5mM K (l aqueous solution 37a, 37b, 43a, 43b - standard electrode 37C
- Light transmission window, 39-... DC power source 41-... Measuring device, 45-... Manometer a-- External pressure, 47... Dark box 49-... Light source. Patent applicant Oki Electric Industry Co., Ltd.

Claims (5)

[Claims] (1) A photoresponsive excitable artificial membrane characterized by adsorbing a lipid that imparts excitability to the artificial membrane and bacteriorhodopsin onto a support having micropores. (2) The photoresponsive excitable artificial membrane according to claim 1, wherein bacteriorhodopsin is adsorbed to an excitable artificial membrane formed by adsorbing lipids to a support having micropores. (3) The photoresponsive excitable artificial membrane according to claim 1 or 2, wherein the lipid is dioleyl phosphate. (4) Adsorption of bacteriorhodopsin to the support in producing a photoresponsive excitable artificial membrane in which a lipid that imparts excitability to the artificial membrane and bacteriorhodopsin are adsorbed onto a support having micropores. A method for producing a photoresponsive excitable artificial membrane, characterized in that the process is carried out by the Langmuir-Prodgett method. (5) Adsorption of bacteriorhodopsin to the support in producing a photoresponsive excitable artificial membrane in which a lipid that imparts excitability to the artificial membrane and bacteriorhodopsin are adsorbed onto a support having micropores. A method for producing a light-responsive excitable artificial membrane, characterized in that this is carried out by membrane fusion of ribosomes containing bacteriorhodopsin.