JPH04340444A - Detection element and detection method of bitter substance - Google Patents

Abstract

PURPOSE:To enable an amount of a bitter substance to be detected by allowing an excitation light and a fluorescent light to be transmitted through a thin film which consists of a fluorescent substance emitting fluorescence when the excitation light enters and at the same time a fluorescence with a same wavelength as the fluorescence which is emitted from the fluorescent substance to be formed on a substrate which is not excited by the excitation light. CONSTITUTION:A thin film 2 which consists of a fluorescent substance emitting a fluorescence when an excitation light enters allows the excitation light and the fluorescent light which is emitted from the fluorescent substance to be transmitted and at the same time a fluorescence with a same wavelength as the fluorescence which is emitted from the fluorescent substance is formed on a substrate 1 which consists of a material which does not emit light with the excitation light. A fluorescent substance is allowed to contact a bitter substance and the excitation light is emitted to the fluorescent substance thus enabling the fluorescence which is generated by the excitation light to be detected. The fluorescent intensity is changed according to a concentration of the bitter substance, thus enabling the degree of bitterness to be measured quantitatively.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a detecting element and method for detecting bitter substances, and more particularly to a detecting element and method for detecting bitter substances that can quantitatively express the human sense of bitterness.

0002

[Prior art] As a method for detecting bitter substances, Japanese Patent Application Laid-open No. 6
As disclosed in Publication No. 3-222248, a method of adsorbing a bitter substance to a film of an immobilized bilayer membrane and detecting the amount of the bitter substance from changes in membrane potential and/or membrane resistance due to adsorption, or A method has been proposed in which a bilayer membrane is immobilized on a crystal oscillator and the amount of bitter substances adsorbed to the bilayer membrane is determined from changes in the frequency of the crystal oscillator.

[0003] In the method of detecting bitter substances from changes in membrane potential, as shown in FIG. 1 of JP-A No. 63-222248, a measurement sample solution and a reference solution are separated by a bilayer membrane; Since it is necessary to measure the membrane potential by inserting electrodes such as silver-silver chloride into both solutions, it is difficult to solidify the sensing element. The same problem exists when detecting changes in membrane resistance, and it has not been confirmed whether the magnitude of changes in membrane resistance correlates with the intensity of bitterness perceived by humans.

On the other hand, in the method using a crystal oscillator, the amount of adsorption is determined from the amount of change in the oscillation frequency due to changes in the weight of the crystal oscillator. The problem is that the correlation with the strength of

Under these circumstances, there is a demand for a solid-state bitter substance sensing element that has sensitivity characteristics similar to those for bitterness felt by humans.

[0006]

[Problems to be Solved by the Invention] In view of the current situation, the present invention aims to provide a solid-type bitter substance detection element and detection method that can detect the amount of bitter substances as the intensity of bitterness felt by humans. purpose.

[0007]

[Means for Solving the Problems] The first gist of the present invention is as follows:
A thin film made of a fluorescent material that emits fluorescence when excitation light is incident on it transmits the excitation light and the fluorescence emitted from the fluorescent material, and the fluorescence with the same wavelength as the fluorescence emitted from the fluorescent material is transmitted depending on the excitation light. It exists in a bitter substance sensing element characterized in that it is formed on a substrate made of a material that does not emit bitter substances.

[0008] A second aspect resides in the bitter substance sensing element according to the first aspect, characterized in that a thin film made of lipid is formed on the thin film made of the fluorescent substance.

A third aspect resides in the bitter substance sensing element according to the first or second aspect, characterized in that the fluorescent substance is comprised of a cyanine dye represented by the following formula and a salt thereof.

0010

[Chemical formula 2] (However, X=O, S or C(CH3)2, n=0-3
It is. ) The fourth aspect is that the fluorescent substance is brought into contact with a bitter substance or after it is brought into contact with the fluorescent substance, excitation light is irradiated onto the fluorescent substance, and fluorescence generated by the excitation light is detected. It exists in the detection method of bitter substances.

[0011] The fifth aspect is that a bitter substance is brought into contact with the surface on which the fluorescent substance of the bitter substance detection element of the first or second aspect is formed; A method for detecting a bitter substance is characterized in that excitation light is incident on a thin film made of the fluorescent substance from a surface and fluorescence generated from the thin film made of the fluorescent substance is detected.

The constituent material of the substrate used in the present invention is a material that transmits the excitation light for exciting the fluorescent substance and the fluorescence emitted from the fluorescent substance, and does not emit fluorescence in the same wavelength range as the fluorescent substance due to the excitation light. If so, an appropriate material can be used, such as quartz glass, borosilicate glass, synthetic sapphire, mica, polycarbonate (PC), polymethacrylic acid methacrylate (PMMA), polyethylene terephthalate (PET), and the like. Although the shape of the base is not particularly limited, a typical example is a substrate.

Examples of fluorescent substances include fluorescent dyes such as cyanine dyes, merocyanine dyes, rhodamine dyes, oxonol dyes, and styryl dyes, and long chains of fused aromatic rings such as anthracene, pyrene, and perylene. Hydrocarbon derivatives or long chain hydrocarbon derivatives such as organometallic complexes such as oxine metal complexes can be used.

Among these, preferred examples of cyanine dyes include compounds represented by formula (I) and salts thereof.

Examples of merocyanine dyes include 3
-carboxymethyl-5-[2-(3-octadecyl-
Examples of rhodamine dyes include [9-(o-carboxyphenyl)-6-(N-ethyl-N-octadecylamino)-3H- xanthene-3
-ylidene]-N-ethyl-N-octadecylammonium and the like.

In the present invention, the above-mentioned fluorescent substance may be used alone or in a mixture with, for example, a carboxylic acid, a carboxylic acid ester, a phosphoric acid ester, or the like. Furthermore, two or more of the above fluorescent substances may be used.

The thickness of the thin film made of fluorescent material is 1
~20 molecular layers are preferred, 1 to 10 molecular layers are more preferred, and 2 to 3 molecular layers are most preferred.

In the present invention, it is preferable that a thin film made of lipid is further formed on the thin film made of fluorescent material (claim 2). In this way, by forming the device structure into a two-layer structure consisting of a thin film layer made of lipid and a thin film layer made of fluorescent material, the sensitivity and accuracy of the device to bitter substances are further improved.

The lipid used in the present invention has two or three long-chain saturated or unsaturated hydrocarbon groups with 12 to 20 carbon atoms in the molecule, and has an ester bond with a carboxyl group or a carboxylic acid or It is a compound that has an ester bond with phosphoric acid.

That is, for example, esters of phosphatidylcholine, phosphatidylserine, etc. and long-chain carboxylic acids, esters of long-chain alcohols and phosphoric acid, dicarboxylic acids, or tricarboxylic acids, etc. can be used.
Dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine; dipalmitoylphosphatidylserine, distearoylphosphatidylserine, dioleoylphosphatidylserine, di-n-hexadecyl phosphate, di-n-
Examples include octadecyl phosphate, dioleyl phosphate, and palmityl esters, stearyl esters, and oleyl esters of malonic acid, succinic acid, and the like.

In the present invention, the above lipids may be used alone or in a mixture of two or more. It may also be a mixture of the above lipids and, for example, cholesterol.

The bitter substance sensing element of the present invention can be obtained by forming a thin film of fluorescent substance on the above substrate, or by further forming a thin film of lipid on the thin film of fluorescent substance. For example, vapor deposition, casting, Langmuir-Blodgett (LB) method, etc. are used to form thin films made of fluorescent substances and thin films made of lipids, but from the viewpoint of detection sensitivity and accuracy, it is preferable to use the LB method. It is preferable to do so. In this case, it is preferable that the lipid thin film has two or one layer so that the surface is hydrophilic, and the fluorescent substance thin film has two or three layers and has a hydrophilic surface.

[0023] Examples of bitter substances to be detected in the present invention include quinine, strychnine, nicotine, phenylthiourea, papaverine, caffeine, naringin, and octaacetyl sucrose.

[0024]

[Effect] After bringing a bitter substance into contact with the bitter substance detection element described above, the fluorescent substance is irradiated with light of a specific wavelength and the intensity of the fluorescence emitted from the fluorescent substance is measured. The fluorescence intensity changes. Moreover, this rate of change is proportional to the logarithm of the concentration of the bitter substance, and the bitter substance detection element of the present invention makes it possible to quantitatively measure the intensity of bitterness felt by humans for various bitter substances. It becomes possible.

[0025]

EXAMPLES The present invention will be explained in detail below using Examples, but it goes without saying that the present invention is not limited to these Examples.

(Example 1) Perchlorate of 3,3'-dioctadecyl-2,2'-thiacyanine (molecular weight: 887.
80) 3.0 mg was dissolved in 10 ml of chloroform, and 120 μl of this solution was spread on the water surface of the LB film forming apparatus. Maintaining the surface pressure at 30 mN/m, on the transparent quartz substrate 1,
The first molecular layer pulls up the substrate so that the hydrophilic groups are on the surface side, and the second molecular layer pushes down the substrate as shown in Figure 1 (a).
A bilayer film 2 having the structure shown in ) was formed.

Bitter taste was detected using the bitter substance detection element prepared as described above.

As shown in FIG. 2, the container 7 of the measurement cell 6
Fix the bitter taste detection element 4 with the surface on which the bimolecular film (2 in Figure 1) is formed facing the inside of the container 7. First, water containing no bitter substances is placed in the container and the excitation light is emitted as shown in the figure. The intensity of the fluorescence emitted from the fluorescent substance 2 was measured. Next, quinine, a typical bitter substance, was added at various concentrations and the fluorescence intensity was measured.

[0029] The fluorescence intensity was measured using a spectrofluorometer (Spectrofluorometer FP-777 manufactured by JASCO Corporation).
), the excitation light was set to 410 nm, and the intensity of fluorescence at 495 nm was measured.

Based on the results obtained from the above measurements, the relationship between the rate of change in fluorescence intensity and the quinine concentration, expressed by the following equation, is plotted in FIG. Fluorescence intensity change rate = (FQ
- FO ) x 100 (%) FQ
: Fluorescence intensity of quinine aqueous solution FO : Fluorescence intensity of quinine-free water As shown in FIG. 3, it was found that the rate of change in fluorescence intensity was approximately proportional to the logarithm of the quinine concentration. Since the strength of human bitterness sensation is said to be approximately proportional to the logarithm of the concentration of a bitter substance, it was shown that the bitter substance detection element of Example 1 can replace humans and quantitatively represent bitterness.

(Example 2) A solution prepared by dissolving 5.6 mg of 3,3'-dioctadecyl-2,2'-oxacarbocyanine perchlorate (molecular weight 881.72) in 10 ml of chloroform was poured onto the water surface. It was developed to form a bimolecular film in the same manner as in Example 1 with a surface pressure of 40 mN/m. 45 excitation light
0 nm, the fluorescence intensity at 516 nm was measured in the same manner as in Example 1, and the rate of change in fluorescence intensity with respect to quinine was determined. The results are shown in Figure 4.

As is clear from FIG. 4, the rate of decrease in fluorescence intensity of the bitter substance sensing element of Example 2 was also shown to be approximately proportional to the logarithm of the quinine concentration.

(Example 3) 1,1'-Dioctadecyl-3,3,3',3'-tetramethyl-2,2'-indotricarbocyanine perchlorate (molecular weight 985.95
) 3.2 mg dissolved in 10 ml of chloroform was spread on the water surface to form a bimolecular film at a surface pressure of 35 mN/m.

[0034] Using an excitation light of 780 nm, the fluorescence intensity at 825 nm was measured in the same manner as in Example 1 to determine the rate of change in fluorescence intensity with respect to quinine. The results are shown in Figure 5.

As is clear from FIG. 5, the bitter substance detection element of Example 3 has a higher detection limit concentration and inferior sensitivity than the elements of Examples 1 and 2, but the rate of change in fluorescence intensity is similar to that of the quinine concentration. It was shown that there is a nearly proportional relationship with the logarithm value, and that it has sensitivity characteristics similar to the human sense of bitterness.

(Example 4) 3,3'-dioctadecyl-
5.4 mg of 2,2'-oxacarbocyanine perchlorate (molecular weight 881.72) was dissolved in 10 ml of chloroform, and 60 μl of this solution was spread on the water surface of the LB film forming apparatus. While keeping the surface pressure at 40 mN/m, a bimolecular film of the fluorescent substance was formed on the transparent quartz substrate while the first molecular film was pulling up the substrate and the second molecular film was pushing down the substrate.

After removing the fluorescent substance remaining on the water surface, di-n-hexadecyl phosphate (molecular weight 546.86)
Solution 10 of 3.5 mg dissolved in 10 ml of chloroform
0 μl was spread on the water surface, and a bilayer film of di-n-hexadecyl phosphate was formed on the bilayer film of the fluorescent material under a surface pressure of 40 mN/m as in the case of the fluorescent material. The structure of the obtained film is as shown in FIG. 1(b).

Using this element, in the same manner as in Example 1,
Fluorescence intensity was measured for various concentrations of quinine. The obtained results are summarized in Figure 6.

As is clear from the comparison with Example 2, by forming the lipid thin film on the fluorescent substance thin film, the amount of change in fluorescence intensity with respect to the concentration of the bitter substance increases, making it possible to measure with higher precision. It turns out that

(Example 5) [9-(o-carboxyphenyl)-6-(N-ethyl-N-octadecylamino)
-3H-xanthene-3-ylidene]-N-ethyl-N
- Using octadecyl ammonium as a fluorescent substance, a bitter substance detection element was prepared in the same manner as in Example 1, and the rate of change in fluorescence intensity with respect to quinine was determined. here,. The excitation light wavelength was 520 nm, and the fluorescence wavelength was 582 nm. The results are shown in FIG.

As shown in FIG. 7, although the sensitivity and accuracy were inferior to the devices of Examples 1 to 3, it was found that the rate of change in fluorescence intensity was proportional to the logarithm of the quinine concentration.

[0042]

According to the present invention, it is possible to provide a bitter substance detection element capable of quantitatively detecting the intensity of bitterness felt by humans.

[Brief explanation of the drawing]

FIG. 1 (a) is a conceptual diagram showing the structure of a bitter substance sensing element of Example 1. (b) A conceptual diagram showing the structure of the bitter substance detection element of Example 4.

FIG. 2: Schematic diagram of fluorescence measurement.

FIG. 3 is a graph showing the relationship between the fluorescence intensity change rate of the bitter substance sensing element of Example 1 and the quinine concentration.

FIG. 4 is a graph showing the relationship between the rate of change in fluorescence intensity of the bitter substance detection element of Example 2 and the quinine concentration.

FIG. 5 is a graph showing the relationship between the fluorescence intensity change rate and quinine concentration of the bitter substance detection element of Example 3.

FIG. 6 is a graph showing the relationship between the fluorescence intensity change rate and quinine concentration of the bitter substance detection element of Example 4.

FIG. 7 is a graph showing the relationship between the rate of change in fluorescence intensity of the bitter substance sensing element of Example 5 and the quinine concentration.

[Explanation of symbols]

1 base (substrate), 2 thin film made of fluorescent substance, 3
A membrane made of lipid, 4 a detection element for a bitter substance, 5 a bitter substance solution, 6 a measurement cell, and 7 a container.

Claims (5)

[Claims] Claim 1: A thin film made of a fluorescent material that emits fluorescence when excitation light is incident thereon, transmits the excitation light and the fluorescence emitted from the fluorescent material, and transmits fluorescence of the same wavelength as the fluorescence emitted from the fluorescent material. . A bitter substance sensing element, characterized in that it is formed on a substrate made of a material that does not emit light depending on the excitation light. 2. The bitter substance sensing element according to claim 1, wherein a thin film made of lipid is formed on the thin film made of the fluorescent substance. 3. The bitter substance detection element according to claim 1, wherein the fluorescent substance is composed of a cyanine dye represented by the following formula and a salt thereof. [Chemical formula 1] (However, X=O, S or C(CH3)2, n=0-3
It is. ) 4. Bitter taste characterized by irradiating the fluorescent substance with excitation light while or after bringing the fluorescent substance into contact with the bitter substance, and detecting the fluorescence generated by the excitation light. How to detect substances. 5. Bringing a bitter substance into contact with the surface on which the fluorescent substance of the bitter substance detection element according to claim 1 or 2 is formed, and the surface opposite to the surface on which the fluorescent substance is formed. 1. A method for detecting a bitter substance, which comprises making excitation light incident on a thin film made of the fluorescent material through the substrate and detecting fluorescence generated from the thin film made of the fluorescent material.