JPH0735691A - Chemical sensor, manufacture thereof and chemical sensor unit - Google Patents

Abstract

PURPOSE:To regenerate a chemical sensor by cleaning after end of sensing and to simplify handling by integrating optical waveguide means with a liposome for enclosing film potential sensitive dye. CONSTITUTION:In a chemical sensor 10 for measuring odor, a plurality of liposomes 13a, 13b for enclosing film potential sensitive dyes are fixed to an optical fiber 11 of optical waveguide means and its one end face. Then, an exciting light having a wavelength responsive the dye is input from the fiber 11 to excite the dye in the liposome. When chemical substance is added to sensing liquid, the film potential of the liposome 13 is varied, and it is detected as fluorescence intensity change of the dye. This change is externally output via the fiber 11, and measured by a photoreceiver. After sensing is finished, the sensor 11 is moved to cleanser side such pure water, etc., and cleaned to wash OFF the substance from the liposome 13. Accordingly, it can be repeatedly used for many times of sensings.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemical sensor that imitates the taste and olfactory functions of living organisms, a method for producing the same, and a chemical sensor unit using the chemical sensor.

[0002]

2. Description of the Related Art Enzyme sensors and immunosensors have been conventionally studied as sensors for chemical substances. These selectively detect a specific chemical substance.
On the other hand, the taste and smell of a living organism can be said to be a chemical sensor that simultaneously recognizes a wide variety of chemical substances, and it also recognizes tastes and odors derived from a complex and comprehensive combination of a wide variety of chemical substances. Therefore, if a chemical sensor that mimics taste and smell can be realized, various advantages can be obtained in a wide range of fields such as the food industry, the cosmetics industry, and environmental measurement, which has been attracting attention in recent years. However, the mechanism by which taste and smell recognize taste and odor is often unclear. At present, it is said that the brain processes the responses obtained from many non-specific receptors and recognizes them as odor and taste patterns.

As a specific means for imitating a receptor when imitating taste and smell, for example, reference I (Biochemistry, 26, 6141-6145) is used.
(1987)), liposomes are considered. That is, in a liposome composed of a lipid bilayer membrane, when various odorants are adsorbed on the liposome, the structure of the bilayer membrane changes, and thus the membrane potential changes. This change in membrane potential can be detected by a membrane potential sensitive dye. The mechanism tries to sense odorants (chemical substances) by such a mechanism.

The inventor of the present application also discloses a chemical sensor using a liposome and a membrane potential sensitive dye in, for example, Japanese Patent Application Laid-Open No. 3-245044 (Japanese Patent Application No. 2-41132). Specifically, a suspension containing liposomes in which membrane potential-sensitive dyes having different fluorescence wavelengths are included in at least two types of liposomes having different compositions is used, and the change in the membrane potential of the liposomes is determined by the fluorescence intensity of the dyes. The chemical sensor which detects as a change was disclosed. Since this chemical sensor can obtain multiple information at the same time, it is considered to be advantageous for recognition of chemical substances.

[0005]

However, in the chemical sensor disclosed in Japanese Patent Laid-Open No. 3-245044, a suspension of liposomes is used. Therefore, the suspension is a chemical substance to be measured for each measurement of the chemical substance. Contaminated with material. For this reason,
There is a problem that the handling is complicated to use it as a sensor, for example, it is necessary to prepare a new liposome suspension for each measurement.

Further, in order to distinguish information obtained from a plurality of types of liposomes, it is necessary to prepare as many membrane potential-sensitive dyes as there are types of liposomes, and further, excitation light of a predetermined wavelength for each of these dyes. There is a problem that it is necessary to prepare the above, and it is necessary to switch the measurement wavelength according to the fluorescence wavelength from each dye.

This application has been made in view of the above points, and therefore, an object of the first invention of this application is to provide a chemical sensor using liposomes, which is easier to handle than conventional chemical sensors. It is in. The second object of this application is a chemical sensor using liposomes, which is easier to handle than before, and reduces the number of types of membrane potential sensitive dyes to be used, and a single excitation wavelength or measurement wavelength. It is to provide a chemical sensor capable of achieving wavelength conversion.
Another object of the third invention of this application is to provide a specific method for producing the chemical sensor of the first invention and the second invention. The purpose of the fourth invention of this application is
It is to provide a chemical sensor unit for effectively using the chemical sensor of the first invention and the second invention.

[0008]

In order to achieve the above-mentioned object of the first invention, according to the first invention of this application, an optical waveguide means and a membrane potential sensitive dye fixed to an end of the optical waveguide means. And a liposome encapsulating.

In order to achieve the above-mentioned object of the second invention, the second invention of this application is characterized by comprising a plurality of chemical sensors of the first invention. However, in the plurality of chemical sensors in the chemical sensor of the second invention, the lipid composition of the liposome is different for each chemical sensor (this is referred to as “individual chemical sensor”), or The lipid composition of the liposome is different for each individual chemical sensor group.

In the embodiments of the first and second inventions, a concrete example of the optical waveguide means is an optical fiber, or a glass substrate, a polymer thin film or a semiconductor substrate on which a waveguide is formed. Various things, such as a waveguide structure, can be mentioned. Among them, the optical fiber is suitable as the optical waveguide means in consideration of the ease of movement of the chemical sensor during sensing. The optical waveguide means here may be a waveguide structure in which a part of the optical waveguide means is formed of an optical fiber and the other portion is formed on a substrate.

In order to achieve the above-mentioned object of the third invention, according to the third invention of this application, as a method for producing the chemical sensor of the first invention and the second invention, the following (a),
Two aspects of (b) are claimed.

(A). The step of introducing an amino group into the end of the optical waveguide means to be fixed with liposomes, the step of forming a liposome in which a protein is embedded in a lipid bilayer membrane as the liposome, and the liposome in which the protein is embedded are Immobilizing on the end of the amino wave guide-introduced optical waveguide means by utilizing the bond of protein-glutaraldehyde-amino group.

(B). A step of introducing an amino group into the end of the optical waveguide means to be fixed to the liposome, a step of introducing biotin into the introduced amino group, and forming a liposome in which a protein is embedded in a lipid bilayer membrane as the liposome. The step of introducing biotin into the embedded protein, the biotin-introduced liposomes at the end of the biotin-introduced optical waveguide means,
Immobilizing using a biotin-avidin bond or a biotin-streptavidin bond.

Further, in order to achieve the above-mentioned object of the fourth invention, according to the fourth invention of this application, a chemical sensor selected from the chemical sensors of the first and second inventions, and a light sensor provided in the chemical sensor. A light source, which is connected to the end of the wave means opposite to the end to which the liposome is fixed, for supplying excitation light to the membrane potential sensitive fluorescent dye in the liposome,
A light receiving element for measuring fluorescence from the membrane potential sensitive fluorescent dye, the light receiving element being connected to the opposite end portion.

[0015]

According to the structure of the first aspect of the present invention, the sensing state of the chemical substance can be formed by immersing the end of the optical waveguide means on the side where the liposome is fixed in a predetermined liquid (for example, buffer). When a chemical substance to be sensed is added to this liquid, the membrane potential of the liposome changes. This change in membrane potential can be sensed in the same manner as before by the membrane potential-sensitive dye contained in the liposome. However, in this first invention,
The excitation light for exciting the membrane potential sensitive dye can be introduced from the outside by the optical waveguide means, and the change in the fluorescence intensity of the membrane potential sensitive dye caused by the change in the membrane potential can also be extracted to the outside by the optical waveguide means. Furthermore, since the optical waveguide means and the liposome are integrated, after the completion of sensing, this chemical sensor can be moved from a liquid in which an object of chemical sensing is mixed into a suitable washing liquid such as pure water, Then, by cleaning the chemical sensor (particularly liposomes) with this cleaning liquid, the chemical substance at the time of the previous sensing can be washed off, so that the chemical sensor can be regenerated. Therefore, it is not necessary to newly prepare a liposome suspension for each measurement.

Further, according to the structure of the second invention, a plurality of types of individual chemical sensors are constituted by making the lipid composition of the liposome different, and the chemical sensor is constituted by a bundle of these plurality of types of individual chemical sensors. Since individual chemical sensors with liposomes having different lipid compositions show different membrane potential changes with respect to one chemical substance, the membrane potential-sensitive dyes contained in the liposomes of these individual chemical sensors are of the same type. Even so, different fluorescence intensity changes occur in each of these individual chemical sensors. This is
This means that even if the excitation wavelength for exciting the membrane potential sensitive dye is unified, multiple information can be obtained at the same time. On the other hand, if the membrane potential sensitive dyes are unified, the wavelength of the excitation light output from the individual chemical sensor will be the same, but a suitable light receiving element such as a photodiode is individually provided for each optical waveguide means of the individual chemical sensor. Since they can be connected, each excitation light output from the individual chemical sensor can be easily separated and measured. Therefore, the measurement wavelengths can be standardized. Further, in the chemical sensor of the second invention, it is possible to register the excitation light output from each individual chemical sensor for a certain odorant as a pattern, so that the chemical substance can be sensed in a form closer to a biological function. it is conceivable that.

Further, in each method of manufacturing the chemical sensor of the third invention of this application, a predetermined liposome is used as the optical waveguide means, and in the method of the first aspect, the bond of amino group-glutaraldehyde-protein is used. Then, in the method of the second aspect, the biotin-avidin bond or the biotin-streptavidin bond is used for immobilization. Therefore, a predetermined liposome can be fixed to the optical waveguide means with desired and practical strength.

In the chemical sensor unit of the fourth invention of this application, sensing by the chemical sensors of the first invention and the second invention can be easily performed.

[0019]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The first invention of this application will be described below with reference to the drawings.
Each example of the fourth invention will be described. However, the drawings used in the description merely show the dimensions, shapes, and positional relationships of the respective constituents to the extent that the present invention can be understood. Further, in each drawing, the same components are denoted by the same reference numerals, and duplicate description thereof will be omitted in some cases. Further, it should be understood that the conditions such as the material used, the amount used, the processing method, the processing temperature, and the processing time described in the following description are merely examples within the scope of these inventions.

1. Description of Embodiment of Chemical Sensor of First Invention FIGS. 1 (A), 1 (B) and FIGS. 2 (A), (B) are diagrams for explaining a chemical sensor 10 of an embodiment of the first invention. In particular, FIG. 1 (A) is a diagram showing the whole of the chemical sensor 10, and FIG. 1 (B) is a portion of the optical waveguide means 11 provided in the chemical sensor 10 on the end side (FIG. 1) where the liposome 13 is fixed. FIG. 2 is an enlarged view of P part of FIG. 1 (A).
(A) And (B) is explanatory drawing of a liposome.

The chemical sensor 10 of the first embodiment of the present invention is fixed to one end (one end surface) of the optical fiber 11 as the optical waveguide means 11 and the membrane potential sensitive dye ( 2 (A) and 2 (B), which are shown by 21a and 21b). However, in this example, as the liposome 13,
First liposome 13a, which is two kinds of liposomes having different lipid compositions and encapsulating different membrane potential sensitive dyes
2A and 2B show an example in which the second liposome 13b and the second liposome 13b are respectively fixed to the tip surface of the optical fiber 11 (FIG. 1 (B), FIG.
See (A) and (B). ). Of course, the type of liposome is not limited to this, and can be arbitrary depending on the design of the chemical sensor,
It may be one type or three or more types. In FIG. 1 (B), reference numeral 15 indicates a means for fixing the liposome 13 to the tip of the optical fiber 11. The fixing means 15 is not particularly limited, but two suitable examples are shown in the section of the manufacturing method described later.

Here, the optical fiber 11 may be any suitable one depending on the design of the chemical sensor. It is also possible to use an optical fiber capable of wavelength division multiplexing. Also, liposome 1
3a, 13b lipid bilayer 13x (FIG. 2 (A),
What is used as the lipid or the membrane potential-sensitive dyes 21a and 21b to be included in the liposome to form (B)) can be arbitrarily determined according to the design of the chemical sensor. In this example, the lipid of the first liposome 13a is egg phosphatidylcholine (hereinafter, PC), and the membrane potential-sensitive dye is bis- (1,3-dibutylbarbituric acid) -pentamethine oxonol (hereinafter, DiBaC ₄ (5)). ), The lipid of the second liposome 13b is a mixed lipid of the above-mentioned PC and egg phosphatidylethanolamine (hereinafter, PE), and the membrane potential-sensitive dye is bis- (1,3-dibutylbarbituric acid) -trimethyl. It is referred to as tinoxonol (hereinafter, DiBaC ₄ (3)) (details will be described in the section of the production method described later).

The chemical sensor 10 of the first invention will be described in detail in the section of the chemical sensor unit described later.
The chemical substance can be sensed by immersing the tip (the side on which the liposome 13 is fixed) in a suitable liquid (for example, buffer). The membrane potential sensitive fluorescent dye in the liposome 13 is excited by inputting excitation light having a wavelength corresponding to the membrane potential sensitive dye from the other end of the optical fiber 11. When a chemical substance is added to the liquid used for sensing, the membrane potential of the liposome 13 changes. This change in membrane potential can be detected as a change in fluorescence intensity of the dye sensitive to membrane potential. This change in fluorescence intensity can be taken out by the optical fiber 11. After the completion of sensing, the chemical sensor 10 is moved to the side of the cleaning liquid such as pure water and the cleaning liquid causes the chemical sensor 10 to move.
Since the chemical substance can be washed off from the liposome 13, it can be repeatedly used for many times of sensing.

2. Description of Embodiment of Chemical Sensor of Second Invention FIGS. 3A and 3B show a chemical sensor 3 of embodiment of the second invention.
It is a figure with which explanation of 0 is offered. In particular, FIG. 3 (A) is a diagram showing the entire chemical sensor 30, and FIG. 3 (B) is an enlarged diagram showing the tip portion (Q portion of FIG. 3 (A)) of the chemical sensor 30. is there.

The chemical sensor according to the second embodiment of the present invention includes a plurality (seven in this example, as shown in FIG. 3B) of the chemical sensor of the first invention (hereinafter, each is also referred to as an "individual chemical sensor"). 10a to 10g are bundled. However, in the seven individual chemical sensors 10a to 10g, the membrane potential-sensitive dyes encapsulated in the liposomes are all of the same type. In addition, in the plurality of individual chemical sensors 10a to 10g, the lipid composition of the liposome is different for each individual chemical sensor, or the lipid composition of the liposome is different for some individual chemical sensor groups. . In this embodiment, of the seven individual chemical sensors 10a to 10g, a first group composed of four individual chemical sensors 10a to 10d and 10
The lipid composition of the liposome is different from that of the second group composed of three individual chemical sensors represented by e to 10 g.
That is, each liposome of the first group is composed of the first lipid, and each liposome of the second group is composed of the second lipid. In FIG.
Two types of liposomes with different lipid compositions are
Shown as z. The materials used as the first lipid, the second lipid for forming the liposomes 13y and 13z, and the membrane potential sensitive dye to be encapsulated in the liposomes can be arbitrarily selected according to the design of the chemical sensor. In this example, the lipid of each liposome 13y of the individual chemical sensor 10a to 10d is PC, and the lipid of each liposome 13z of the individual chemical sensor 10e to 10g is a mixed lipid of PC and PE. DiBaC which is a membrane potential sensitive dye encapsulated in liposome of individual chemical sensor
₄ (5) (Details will be explained in the section of the manufacturing method described later).

In the above example, the number of individual chemical sensors is 7, the lipid composition of the liposome is 2 types, and the 7 individual chemical sensors are divided into 2 groups at a ratio of 4: 3. However, the number of individual chemical sensors, the number of types of lipid composition of liposomes, and the method of configuring the group of individual chemical sensors can be changed according to the design of the chemical sensor. For example, if the lipid composition of the liposome is n types (n is an integer of 2 or more), in principle, the number of individual chemical sensors may be n. However, considering the reliability of the sensor, a configuration using a plurality of n kinds of individual sensors is also preferable.

The chemical sensor 30 of the second invention is also capable of sensing chemical substances on the same principle as the chemical sensor of the first invention. However, the individual chemical sensors 10a to 10
Since the lipid composition of the liposomes is made different between the group d) and the groups of the individual chemical sensors 10e to 10g, the individual chemical sensors of each group show unique responsiveness to chemical substances. Therefore, in the case of this example, two types of fluorescence intensity changes, which are a fluorescence intensity change from the first group of individual chemical sensors and a fluorescence intensity change from the second group of individual chemical sensors, can be observed simultaneously. . Moreover, since the membrane potential sensitive dyes to be encapsulated in the liposomes of the individual chemical sensors 10a to 10f are of the same type, the fluorescence intensity change is measured by the excitation light common to the seven individual chemical sensors and the common measurement wavelength. Can be done by That is, from the other end (indicated by 11x in FIG. 3A) of the optical fibers of the individual chemical sensors 10a to 10f, the excitation light of the same wavelength can be put into these individual chemical sensors to perform measurement. Moreover, since the excitation light extracted from the other end 11x of the optical fiber of the individual chemical sensor 10a to 10f can be measured by, for example, a photodiode for each individual chemical sensor 10a to 10f, the measurement wavelengths can be unified.

3. Description of Example of Third Invention (Chemical Sensor Manufacturing Method) 3-1. Description of the first aspect of the third invention As a first method of immobilizing liposomes on the tip of an optical fiber when manufacturing the chemical sensor of the first and second inventions,
A method utilizing the bond of amino group-glutaraldehyde-protein will be described. However, here, in the case of manufacturing the chemical sensor 10 shown in the embodiment of the first invention, that is,
An example of manufacturing a chemical sensor in which the first liposome 13a and the second liposome 13b are immobilized on the tip of the optical fiber 11 will be described. FIG. 4 is a process chart for the explanation.

First, in this case, an optical fiber 11 made of quartz is prepared as an optical waveguide means (FIG. 4A). An amino group is introduced into the tip of this optical fiber. As this method, it is preferable to treat the tip of the optical fiber with a silane coupler having an amino group. Therefore, here, the optical fiber 11 is immersed only for 2 minutes in the 1% aminopropyltriethoxysilane (model number LS-3150: manufactured by Shin-Etsu Silicone) aqueous solution for 2 minutes. This is taken out, washed with pure water, and then heated in a constant temperature bath at a temperature of 110 ° C. for 10 minutes.
An amino group is introduced into the tip of the optical fiber by this series of processes (FIG. 4 (B)).

Then, the tip of the amino group-introduced optical fiber is immersed in a 1% glutaraldehyde aqueous solution for 1 hour to introduce an aldehyde group into the tip of the optical fiber (FIG. 4 (C)).

On the other hand, liposomes are prepared as follows.
First, a solution prepared by dissolving 100 mg of egg phosphatidylcholine (hereinafter, PC), which is one of phospholipids, in 5 ml of chloroform, and a solution of 1 mg of alamethitin (manufactured by Sigma) as a membrane protein in 1 ml of ethanol, having a volume of 50 ml Place in an eggplant-shaped flask, mix, and then evaporate the solvent using a rotary evaporator.
As a result, a thin lipid film remains on the inner wall of the flask. The flask is then charged with bis- (1,3-dibutylbarbituric acid) -pentamethine oxonol (DiBaC ₄ (5)) in this case as a membrane potential sensitive dye.
Add 0 ml bicarbonate buffer (pH 8.5),
Then, the lipid membrane is dispersed by stirring with a vortex mixer at 37 ° C. Ultrasonic waves were applied to the lipid suspension thus prepared to form liposomes, which were then centrifuged (100,00
0 g) and collect the supernatant. Further, the liposome suspension is put into an extruder (made by NOF Liposome Co., Ltd.) so that the diameter of the liposome is adjusted to 200 nm or less. Further, a PC liposome suspension (first liposome suspension) was prepared by diluting 10 times with bicarbonate buffer (pH 8.5). This PC liposome is in the state of FIG. 4 (D) when schematically shown. In FIG. 4 (D), 13 is a liposome encapsulating a membrane potential sensitive dye (not shown), and 15x
Is a protein incorporated in the lipid bilayer of the liposome 13. This PC liposome is shown in detail in FIG. 2 (A).
It corresponds to the one in the state of.

Also, egg phosphatidylcholine (PC) 5
0 mg and egg phosphatidylethanolamine (hereinafter,
PE) 50 mg dissolved in chloroform 5 ml, and alamethitin as a membrane protein (manufactured by Sigma)
A solution of 1 mg dissolved in 1 ml of ethanol was added to a volume of 5
The mixture was placed in a 0 ml eggplant-shaped flask and mixed to prepare PC + PE liposome suspension air (second liposome suspension) according to the method for preparing the PC liposome suspension. However, in this case, bis-
(1,3-Dibutylbarbituric acid) -trimethine oxonol (DiBaC ₄ (3)) is used. This PC + PE liposome is also in the state of FIG. 4 (D) when it is schematically shown. In detail, it corresponds to the state of FIG. 2 (B),
The lipid bilayer 13x is composed of PC and PE.

The above-mentioned aldehyde-introduced optical fiber was added to a mixture of the first liposome suspension prepared as described above and the second liposome suspension in a ratio of 1: 1.
Immerse for 2 hours at room temperature. The aldehyde group reacts with and binds to the amino group of the protein alamethicin embedded in the liposome, so that the liposome 13 is fixed to the tip of the optical fiber 11 (FIG. 4 (E)). Unnecessary liposomes not bound to the optical fiber are bicarbonate buffer (p
H8.5) to remove it by washing the end of the optical fiber.

3-2. Description of the second aspect of the third invention As a second method of immobilizing liposomes on the tip of an optical fiber when manufacturing the chemical sensor of the first and second inventions, a method of utilizing biotin-streptavidin binding is described. explain. However, here again, an example of manufacturing the chemical sensor 10 shown in the embodiment of the first invention, that is, the first liposome 13a and the second liposome 1 at the tip of the optical fiber 11 is manufactured.
An example of manufacturing a chemical sensor in which 3b is fixed will be described. 5 and 6 are process diagrams used for the description.

First, an amino group is introduced into the tip of the optical fiber 11 by the procedure described in the first embodiment (FIG. 5).
(A) and (B)). Next, the tip of the amino group-introduced optical fiber was dipped in 10 ml of a bicarbonate buffer (pH 8.5), and 3 parts of the buffer were immersed in the buffer.
mg sulfosuccinimidyl-6- (biotinamide) hexanoate [Sulfosuccinimidyl-6- (biotinamido) Hex
anoate] (trade name NHS-LC-BIOTIN: manufactured by Funakoshi Chemical Co., Ltd.) is added and reacted for 1 hour. As a result, biotin (biotin long arm) is introduced into the tip of the optical fiber 11 via the amino group (FIG. 5).
(C)). Note that, after FIG. 5C, the bonding state of the optical fiber 11 and the biotin 41 is schematically shown without using a chemical structural formula.

Next, the biotin-introduced optical fiber is washed with a phosphate buffer (pH 7.3) to remove unnecessary biotin. Then, the tip of this optical fiber is immersed in a 0.1 mg / ml streptavidin (Funakoshi Chemical Co., Ltd.) bicarbonate buffer (pH 8.5) solution for 2 hours at room temperature. This allows the optical fiber 1
Streptavidin is introduced into the tip of 1 via an amino group and biotin (FIG. 5 (D)).

On the other hand, liposomes are prepared as follows.
First, a solution prepared by dissolving 100 mg of egg phosphatidylcholine (hereinafter, PC), which is one of phospholipids, in 5 ml of chloroform, and a solution of 1 mg of alamethitin (manufactured by Sigma) as a membrane protein in 1 ml of ethanol, having a volume of 50 ml Place in an eggplant-shaped flask, mix, and then evaporate the solvent using a rotary evaporator.
As a result, a thin lipid film remains on the inner wall of the flask. The flask is then charged with bis- (1,3-dibutylbarbituric acid) -pentamethine oxonol (DiBaC ₄ (5)) in this case as a membrane potential sensitive dye.
Add 0 ml bicarbonate buffer (pH 8.5),
Then, the lipid membrane is dispersed by stirring with a vortex mixer at 37 ° C. Ultrasonic waves were applied to the lipid suspension thus prepared to form liposomes, which were then centrifuged (100,00
0 g) and collect the supernatant. Further, the liposome suspension is put into an extruder (made by NOF Liposome Co., Ltd.) so that the diameter of the liposome is adjusted to 200 nm or less. To this suspension 1
1 ml of a bicarbonate buffer solution of NHS-LC-BIOTIN having a concentration of mg / l is added, and the reaction is performed while stirring for 2 hours (FIG. 6 (A)). In addition, bicarbonate buffer (pH
8.5)) to prepare a PC liposome suspension in the second embodiment (first liposome suspension in the second embodiment). This PC liposome is in a state shown in FIG. 6 (B) when schematically shown.

Egg phosphatidylcholine (PC) 5
0 mg and egg phosphatidylethanolamine (hereinafter,
PE) 50 mg dissolved in chloroform 5 ml, and alamethitin as a membrane protein (manufactured by Sigma)
A solution of 1 mg dissolved in 1 ml of ethanol was added to a volume of 5
Pour in a 0 ml eggplant-shaped flask and mix to prepare a PC liposome suspension according to the second embodiment.
A C + PE liposome suspension (second liposome suspension in the second embodiment) was prepared. However, in this case,
As a membrane potential sensitive dye, bis- (1,3-dibutylbarbituric acid) -trimethine oxonol (DiBa
C ₄ (3)) is used. This PC + PE liposome is also in the state of FIG. 6 (B) when it is schematically shown.

The above-prepared optical fiber in which streptavidin has been introduced into a mixture of the first liposome suspension and the second liposome suspension in the second embodiment prepared in the above-described manner at a ratio of 1: 1. Is immersed at room temperature for 2 hours (FIG. 6 (C)). The streptavidin introduced at the tip of the optical fiber forms a strong bond with biotin bound to the liposome, so that the liposome 1 is attached to the tip of the optical fiber 11.
3 is fixed (FIG. 6 (D)). Unnecessary liposomes not bound to the optical fiber are bicarbonate buffer (pH
It is removed by washing the end of the optical fiber with that of 8.5).

According to each of the above-mentioned third embodiments of the invention, a liposome containing a membrane potential sensitive dye can be immobilized on the tip of an optical fiber with industrial and practical strength. Also, instead of using the biotin-streptavidin bond, when using the biotin-avidin bond,
The same fixing was possible.

When the chemical sensor 30 of the second embodiment of the invention (see FIG. 3) is manufactured by the method of the third invention, only the first liposome suspension is used and the individual chemistry is performed according to the above procedure. Each of the sensors 10a to 10d is manufactured,
Since the individual chemical sensors 10e to 10g may be produced in accordance with the above procedure using only the second liposome suspension, detailed description thereof will be omitted.

4. Description of Example of Fourth Invention (Chemical Sensor Unit) 4-1. First Embodiment of Fourth Invention An embodiment of a chemical sensor unit (of the fourth invention) suitable for sensing a chemical substance by the chemical sensors of the first and second inventions will be described. Here, an example in which the chemical sensor 10 of the embodiment of the first invention is used as the chemical sensor will be described.
FIG. 7 is a diagram showing the configuration of the chemical sensor unit of the example.

The chemical sensor unit of this embodiment includes the chemical sensor 10 of the first embodiment of the present invention and this chemical sensor 10.
A light source 51 for supplying excitation light to the membrane potential sensitive fluorescent dye in the liposome 13, which is connected to the end 11x of the optical fiber 11 provided in the opposite side to the end to which the liposome 13 is fixed. It is provided with a light receiving means connected to the opposite end 11x for measuring fluorescence from the membrane potential sensitive fluorescent dye. However, in the case of this embodiment,
A photomultiplier tube is used as the light receiving element. Also light source 5
1 is optically connected to the other end 11x of the optical fiber 11 via the interference filter 61, the half mirror 63, and the lens 65. The photomultiplier tube 53 is optically connected to the other end 11x of the optical fiber 11 via the monochromator 67, the lens 69, the half mirror 63, and the lens 65.

Here, the interference filter 61 is used by changing it according to the pumping wavelength in order to change the wavelength of the pumping light. The monochromator 67 is provided to change the measurement wavelength. These are the chemical sensors 10 of the embodiment of the first invention.
Since the first liposome and the second liposome having different membrane potential-sensitive dyes are fixed to the tip of the optical fiber, it is necessary to change the wavelength of the excitation light and the wavelength of the measurement light according to these dyes. Therefore, it is provided. But,
If one kind of liposome is fixed to the tip of the optical fiber, or if there is one kind of membrane potential sensitive dye as in the chemical sensor 30 of the second invention, one kind of interference filter 61 is fixedly used. Better yet, monochromator 6
7 can be omitted (for details, refer to the section of the second embodiment of the fourth invention).

In this chemical sensor unit, the tip of the chemical sensor 10 (liposome fixed side) is dipped in, for example, the bicarbonate buffer 73 in the beaker 71 to enable sensing. In the experiment, a chemical substance (in this case, an odorant is used) 75 was added to the bicarbonate buffer 73 using a microsyringe.

In this chemical sensor unit, from the light source 51 to the interference filter 61, the half mirror 63 and the lens 65.
The excitation light can be guided to the optical fiber 11 through. The excitation light is applied to the liposome 13 through the optical fiber 11. On the other hand, the fluorescence emitted from the membrane potential sensitive dye according to the membrane potential change caused by the adsorption of the chemical substance on the liposome 13 passes through the optical fiber 11 and further passes through the lens 65, the half mirror 63, the lens 69 and the monochromator 67 to generate photoelectrons. It reaches the multiplier 53 and is received there. One of the membrane potential sensitive fluorescent dyes DiBaC used in the experiment
The excitation wavelength of ₄ (5) is 591 nm, and the maximum wavelength of fluorescence is 616 nm. The excitation wavelength of the other membrane potential sensitive fluorescent dye DiBaC ₄ (3) used in the experiment is 494 nm, and the maximum wavelength of fluorescence is 520 nm. Therefore, when the interference filter 61 and the monochromator 67 are set so as to correspond to the membrane potential sensitive fluorescent dye DiBaC ₄ (5), the change in fluorescence intensity in the first liposome 13a can be observed. Further, an interference filter 61 and the monochromator 67 membrane potential sensitive fluorescent dye DiBAC ₄
When set to correspond to (3), the second liposome 1
The change in fluorescence intensity at 3b can be observed.

4-2. Second Embodiment of Fourth Invention As already described with reference to FIG. 3, in the chemical sensor 30 of the second invention, the individual chemical sensors 10a to 10g can be the same kind of membrane potential sensitive dyes used therein. It was
Therefore, the wavelengths for exciting the individual chemical sensors 10a to 10g and the measurement wavelengths can be made common. An example of the chemical sensor unit in that case will be described below as a second embodiment of the fourth invention. FIG. 8 is an explanatory view of the chemical sensor unit of the second embodiment.

In the chemical sensor unit of these two embodiments,
The monochromator used in the first embodiment is not used. Further, the light emitting diode array 81 is used as the light receiving means. The diode array 81 includes a lens 69 and a filter 8
3, it is optically connected to the other end 30a of the chemical sensor of the second invention through the half mirror 63 and the lens 65. The other structure is basically the same as that of the first embodiment.

Here, the chemical sensor 3 according to the second embodiment of the invention.
The excitation wavelength of the sensitive fluorescent dye DiBaC ₄ (5) used in 0 was 591 nm, and the maximum wavelength of fluorescence was 616 n.
Since it is m, the interference filter 61 is supposed to be able to transmit light having a wavelength of 591 nm. Further, the filter 83 is supposed to be capable of cutting light having a wavelength of 600 nm or less.
This is so that only the fluorescence of the liposome can be supplied to the photodiode array 81. Further, the photodiode array 81 has the individual photodiodes in the array arranged so as to correspond to the arrangement of the individual chemical sensors of the chemical sensor 30.

The chemical sensor unit of the second embodiment is also
Basically, the chemical substance can be sensed by the same principle as the chemical sensor unit of the first embodiment. However, sensing can be performed in a state where the wavelengths of the excitation light of the individual chemical sensors are common and the wavelengths of the measurement light are common.

5. Explanation of Sensing Results of Chemical Substances The following four types of chemical sensors are sequentially incorporated in the chemical sensor unit described with reference to FIG. 7 or FIG. 8 and the characteristics of each chemical sensor unit with respect to several kinds of odorants are explained below. Measure as you do.

A chemical sensor 10 according to the first embodiment of the present invention, in which liposomes are immobilized on an optical fiber by utilizing an amino group-glutaraldehyde-protein bond.

The chemical sensor 10 of the first embodiment of the present invention, in which liposomes are immobilized on an optical fiber by utilizing the binding of biotin-streptavidin.

A chemical sensor 30 according to an embodiment of the second invention, wherein liposomes are immobilized on an optical fiber by utilizing the amino group-glutaraldehyde-protein bond in the preparation of the individual chemical sensors 10a to 10g. .

The chemical sensor 30 of the second embodiment of the present invention, wherein liposomes are immobilized on an optical fiber by utilizing the biotin-streptavidin bond in the preparation of the individual chemical sensors 10a to 10g.

With respect to each of the above-mentioned sensors and, the responses to the odorants nonanol, β-ionone, acetic acid-n-amyl and dl-muscon are examined.
In addition, with respect to each of the above sensors and, the response to each of the odorants nonanol, menthone dl-muscon, and citral is examined. Here, the response means that the fluorescence intensity F ₀ of the chemical sensor before the odorant is added to the beaker 71 of the unit of FIG. 7 and the fluorescence intensity F of the chemical sensor after the addition are measured, These are shown by the fluorescence intensity change ΔF obtained by substituting these into the following equation (1).

ΔF = (F−F ₀ ) × 100 / F ₀ (1) FIGS. 9A to 9D show response characteristics of the above chemical sensor to each odorant. 10 (A) to (D)
Shows the response characteristics of the above chemical sensor to each odorant. 11 (A) to (D) show the response characteristics of the above chemical sensor to each odorant. 12
(A)-(D) showed the response characteristic with respect to each odor substance of the said chemical sensor. 9 to 12 (A) to
In the figure (D), A is the response when the liposome lipid is composed of PC, and B is the response when the liposome lipid is composed of a mixed lipid of PC and PE. 9-
From FIG. 12, it can be seen that even when the same odorant is sensed, different response characteristics are exhibited when the lipid composition of the liposome is different.

Although the embodiments of the inventions of this application have been described above, the inventions are not limited to the above-mentioned embodiments.

For example, phosphatidylserine, sphingomyelin, cholesterol and the like can be used as the lipid for forming the liposome. By using these, more various types of liposomes having different lipid compositions, that is, different response characteristics to chemical substances can be constructed, and therefore, more advanced recognition of chemical substances is possible. Although alamethitin was used as the membrane protein to be embedded in the liposome, other proteins can be used.
The property of the liposome membrane is also changed by changing the type of protein, and thus the ability to recognize chemical substances can be changed.

[0060]

As is apparent from the above description, according to the chemical sensor of the first invention, the optical waveguide means and the liposome are integrated, and therefore, after completion of the sensing, the chemical sensor is, for example, pure water. Can be transferred into a suitable washing liquid of (3), and by cleaning the chemical sensor (particularly liposome) with this washing liquid, the chemical substance at the previous sensing can be washed off. Therefore, it is not necessary to newly prepare a liposome suspension for each measurement, so that a chemical sensor that is easier to handle than before can be provided.

Further, according to the chemical sensor of the second invention, even if the membrane potential-sensitive dyes contained in the liposome of the individual chemical sensor are of the same type, different fluorescence intensity changes occur in each of these individual chemical sensors. Occurs. Therefore,
Even if the excitation wavelength for exciting the membrane potential sensitive dye is unified, multiple information can be obtained at the same time. On the other hand, if the membrane potential sensitive dyes are unified, the wavelength of the excitation light output from the individual chemical sensor will be the same, but a suitable light receiving element such as a photodiode is individually provided for each optical waveguide means of the individual chemical sensor. Since they can be connected, each excitation light output from the individual chemical sensor can be easily separated and measured. Therefore, the measurement wavelengths can be standardized. For this reason,
It is possible to provide a chemical sensor capable of reducing the number of kinds of membrane potential sensitive dyes to be used and making each excitation wavelength and measurement wavelength into a single wavelength.

Further, in each of the methods for manufacturing a chemical sensor of the third invention, a predetermined liposome can be fixed to the optical waveguide means with desired strength and practical strength.

Further, in the chemical sensor unit of the fourth invention, sensing by the chemical sensor of the first invention and the second invention can be easily carried out.

[Brief description of drawings]

1A and 1B are explanatory views of an embodiment of a chemical sensor of the first invention.

FIG. 2 (A) and (B) are explanatory views of a liposome.

3 (A) and 3 (B) are explanatory views of an embodiment of the chemical sensor of the second invention.

4A to 4E are manufacturing process diagrams according to the first aspect of the third invention.

5A to 5D are manufacturing process diagrams according to the second aspect of the third invention.

6A to 6D are manufacturing process diagrams following FIG. 5 according to the second embodiment of the third invention.

FIG. 7 is a diagram for explaining the first embodiment of the fourth invention.

FIG. 8 is a diagram which is used for describing a second embodiment of the fourth invention.

FIG. 9 is an explanatory diagram of characteristics of the sensor.

10 is a characteristic explanatory diagram of the sensor following FIG. 9. FIG.

FIG. 11 is a characteristic explanatory diagram of the sensor following FIG. 10;

12 is a characteristic explanatory diagram of the sensor following FIG. 11. FIG.

[Explanation of symbols]

 10: Chemical Sensor of Example of First Invention 11: Optical Waveguide Means (Optical Fiber) 13: Liposomes Encapsulating Membrane Potential Sensitive Dye 13a: First Liposomes 13b: Second Liposomes 30: Examples of Second Invention Chemical Sensors 10a-10g: Individual Chemical Sensors 13y, 13z: Liposomes with Different Lipid Compositions

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Masakazu Kato 1-7-12 Toranomon, Minato-ku, Tokyo Oki Electric Industry Co., Ltd.

Claims (6)

[Claims] 1. A chemical sensor comprising: an optical waveguide means; and a liposome fixed to an end of the optical waveguide means and encapsulating a membrane potential sensitive dye. 2. A chemical sensor comprising a plurality of chemical sensors according to claim 1 (however, in the plurality of chemical sensors, the lipid composition of the liposome is different for each chemical sensor (individual chemical sensor)). , Or the lipid composition of liposomes is different for each of several individual chemosensor groups.). 3. The chemical sensor according to claim 1, wherein the optical waveguide means is an optical fiber. 4. The method for producing the chemical sensor according to claim 1, wherein the step of introducing an amino group into the end of the optical waveguide means to be fixed with the liposome, and the step of implanting a protein into a lipid bilayer membrane as the liposome. And a step of immobilizing the protein-embedded liposome on the end of the amino group-introduced optical waveguide means using a protein-glutaraldehyde-amino group bond. A method of manufacturing a chemical sensor, comprising: 5. The method for producing the chemical sensor according to claim 1, wherein the step of introducing an amino group into the end of the optical waveguide means to be fixed with the liposome, and the step of introducing biotin into the introduced amino group. A step of forming a liposome in which a protein is embedded in a lipid bilayer membrane as the liposome, a step of introducing biotin into the embedded protein, and a step of introducing the biotin-introduced liposome into the biotin-introduced optical waveguide. A step of immobilizing at the end of the means by utilizing a biotin-avidin bond or a biotin-streptavidin bond. 6. The chemical sensor according to any one of claims 1 to 3, which is connected to an end portion of the optical waveguide means provided in the chemical sensor, which is opposite to the end portion to which the liposome is fixed. A light source for supplying excitation light to the membrane potential sensitive fluorescent dye in the liposome, and a light receiving device connected to the opposite end of the optical waveguide means for measuring fluorescence from the membrane potential sensitive fluorescent dye. And a chemical sensor unit.