JP2001153805A - Method of optimizing color variation of optode by digitization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method of optimizing optode, which can precisely quantify and simply measure even by a visual observation, and an optimized optode by the method. SOLUTION: A color variation is optimized by digitizing a color variation of optode and plotting on a chromaticity coordinate system. The color variation of optode is selected so that a gray point may be included in a triangle formed by three points, which are a one end, an opposite end and a corresponding coordinate position of a shielding indicator when the color variation of optode is expressed as a chromaticity coordinate QxQy of complementary colors, or a position of the shielding indicator on the chromaticity coordinate QxQy of the complementary colors may lie on a line formed by two points, which are a position of a chemical species to measure optode at a detection point and the gray point on the chromaticity coordinate QxQy of the complementary colors, or the color variation of optode by a change in a concentration of the chemical species to measure it may move in circles about the gray points of each coordinate on a (xy) chromaticity coordinate or a (a*) (b*) chromaticity coordinate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a digital color
Novel design method of optode based on analysis (DCA)
Law, an optode designed in that way. More details
Specifically, the present invention numerically converts the color change of an optode.
The change is represented by the xy chromaticity coordinate system of the color, a^*b ^*Chromaticity coordinates
System or complementary color QxQy chromaticity coordinate system
How to optimize toads with numerical information
Composition and measurement material obtained by
You. More specifically, the present invention relates to the determination of a particular concentration of a measured species.
Design optodes that are colorless and gray in the range of degrees
Method and the color in a wide concentration range of the measured species.
How to design optodes that can change quickly, and how
For the todo compositions and measurement materials designed by these methods
Do

[0002]

2. Description of the Related Art Generally, a sensor captures a physical or chemical quantity of the outside world and converts it into an electrochemical signal for detection. And optical conversion. Both electrochemical and optical sensors are based on the same concept of recognizing an analyte and converting it to a signal for detection. As for the electrochemical conversion, there are many reports on the use of a membrane potential or an oxidation-reduction current represented by an ion-selective electrode (ISE) or an enzyme electrode. Optical sensors are sensors that detect using the interaction of various electromagnetic waves with substances, such as absorption, fluorescence, and chemiluminescence.

Many chemical sensors that have been developed so far have been electrochemical sensors (electrode) using an electrochemical measuring element. In 1978, Seitz et al.
The hydrogen peroxide sensor, which detects the chemiluminescence produced by the reaction of hydrogen peroxide and luminol by the catalytic action of peroxidase with an optical fiber and detects it, was announced as the first optical sensor. Later, the use of the new term opt (r) ode for electrode began to be used, and research progressed rapidly. In 1980 and later, the principle of ion-selective electrodes based on ion extraction of ionophore was applied to optode. Research has flourished. Simons,
Koji Suzuki et al. Included ionophore and proton-selective chromophore (dye) in a plasticized membrane and used the optical signal change caused by the ion exchange reaction between the target ion in the aqueous phase and the proton desorbed to the dye in the membrane. We have developed optodes for many ions. It has been reported that the ionophore used in this case is in principle applicable to the ionophore developed in the ion-selective electrode.

An optical sensor (optode) is
Although it is pointed out that there are problems in response time, durability, running cost, and the like, these are likely to be improved in the future, and optical sensors are very promising as next-generation sensing systems. Especially in the clinical and biomedical fields, from the point of reliability, accuracy and simplicity,
The use of a large number of optodes is expected. Thus, although the optical sensor still has some problems, it is superior to the electrochemical sensor in the following points. 1. Unlike a potential measurement, a reference is not required in principle, so miniaturization is easy. 2. The use of a high-performance photodetector enables more sensitive measurement. 3. Since various types of luminescent reagents and fluorescent reagents can be used directly or in an improved manner, there are many chemical species to be measured. 4. Wide response range. 5. No electrical or magnetic noise is generated or affected. 6. Since the optical material such as optical fiber is glass or plastic, it has excellent electrical insulation and corrosion resistance. 7. Since optical information is transmitted at high speed and with low loss by an optical fiber or the like, remote sensing over a long distance is possible.

[0005] As described above, an optical sensor (optode) has many advantages over a conventional electrochemical sensor, and its development is expected in many application fields. The principle of optode detection that has been developed so far has been to measure a change in color of a detection element according to a change in activity of a measurement object by using a spectroscope. However, the measurement using a spectroscope requires a large-scale apparatus, and when the spectrum data is complicated, it is difficult to directly quantify the data. Therefore, there is a demand for the development of an optode that can perform accurate measurement by visual observation without using a spectroscopic instrument and that can perform simpler measurement.

[0006]

The optode is visual, but in order to reliably function as a sensor visually, the color tone must clearly change in response to a change in the state of the target substance. In addition, it is required that the color tone can be visually and reliably recognized with high reliability. The present inventors have studied a method of quantifying the change in the color of the optode by digital color analysis (DCA), quantifying the change in the color of the optode, and designing the change in the color as digital information. A method for optimizing the change of the value based on the quantified information has been found. For example, the present inventors have found that a method of selecting a shielding indicator can be established so that a change in the color of an optode can be clearly confirmed by visualization by digitizing the chromaticity coordinates QxQy of complementary colors. Therefore, the present invention
The present invention provides an optode that can be accurately measured even by visual observation and that can be measured more easily. More specifically, it provides a new optode optimization method and an optode optimized by the method. It is.

[0007]

SUMMARY OF THE INVENTION The present invention relates to a method for quantifying the color change of an optode, plotting the color change of the optode in a chromaticity coordinate system, and optimizing the color change in the coordinate system. The present invention relates to a novel method of optimizing a change in optode color by digitizing a color using a chromaticity coordinate system. The present invention also provides a more detailed approach to optimization in the chromaticity coordinate system. The present invention
The present invention relates to a method for quantifying a change in the color of an optode, plotting the change in the color of the optode in a chromaticity coordinate system, and optimizing the change in the color of the optode in the coordinate system. More specifically, the present invention relates to a triangle formed by three points of one end and the opposite end when the color change of the optode is represented by the complementary chromaticity coordinates QxQy, and the position of the coordinates of the shielding indicator. The method of selecting an optode shielding indicator so that a gray point falls inside the, and the position of the complementary color of the shielding indicator at the chromaticity coordinate QxQy is the position of the chromaticity coordinate QxQy of the complementary color at the detection point of the measurement chemical species of the optode. The present invention relates to a method for selecting a shielding indicator, which is to select a shielding indicator on a straight line formed from two gray points. Further, the present invention is arranged such that the change in the color of the optode due to the change in the concentration of the measured chemical species draws a circle centered on the gray point of each coordinate in the xy chromaticity coordinates or the a ^* b ^* chromaticity coordinates. It relates to a method of selecting a color change of an optode.

[0008] The present invention also relates to an optode, an indicator composition, a shielding indicator, a measuring material, and a measuring method using these obtained by these methods. The present invention
The present invention relates to a novel method for designing an optode based on digital color analysis (DCA), and an optode designed by the method. The novel optode optimization method of the present invention will be described in detail with reference to specific examples based on the following experimental results.

In conventional optical analysis, optical signals such as an optical spectrum and absorbance are generally measured with a spectrometer and used for highly accurate quantification. Such an analysis method is called spectrophotometry. In Spectrophotometry, since the amount of change in a specific value (such as absorbance) indicated by a spectrum is measured, it is difficult to quantify when analysis is difficult in terms of spectrum. However, in reality, when the spectrum changes, it is first perceived as a "color" change by our eyes. For this reason, it can be said that measurement quantifying “color” itself is a natural method based on perception.

In the field of analytical chemistry, the first use of quantified color information rather than spectra was in 1961.
Reilly, CN, et al., Anal. Chem.,
32, 1218-1232 (1960)). Thereafter, various acid / base indicators and metal indicators have been evaluated. These are mainly used to represent the rate and speed of color change at the end point of the routine as numerical values, and to judge and evaluate the performance as an indicator. However, quantifying the color has involved the complicated task of measuring the light spectrum with a conventional spectroscope and calculating the data using a computer.
Digital color analyzer (COLORTRO)
^NTM ) can easily measure color and can quickly perform calculations for quantification. Therefore, the present inventors
We have considered a new optical analysis method that uses COLORTRON ^™ to treat colors as digital information and digitize and use optical analysis.

In the first place, light is a mixture of all wavelengths in the visible region, and various colors are produced depending on the mixing ratio. When expressing a color, it is often expressed by a numerical value that is a set of three values. These are called three attributes of color, and the three constituent values are called tristimulus values. Based on these tristimulus values, light that can be visually recognized can be synthesized,
Attempts have been made to coordinate.

For example, as the tristimulus values, the spectrum red,
The combination of the three primary colors of green and blue is represented by RGB (Red,
Green, Blue). In this method, when red, green, and blue color lights are added to a state without light, any color can be expressed by the ratio, and this color mixing theory is called additive color mixing. Also, CMY (Cyan, M
Agent, Yellow) is also known. In this method, when subtracting red, green, and blue light from white light, all colors can be expressed by the ratio, and this color mixing theory is called subtractive color mixing.

As a method of expressing a color as a position in a coordinate system based on tristimulus values, a hue (H) is set in xy coordinates, a lightness (B) is set in a z-axis direction, and a saturation (S) is calculated from an origin. HSB (Hue hue, Saturation) expressed as distance
Chroma, Brightness). In this way, all colors can be represented as points in a double cone solid in the xyz coordinate system. In this case, H indicating the color is arranged on the circumference according to the order of the spectrum. S, which indicates vividness, is represented by the length of the radius, and the further away from the center, the more vivid. B, which indicates brightness, has an axis in the vertical direction, and becomes brighter as going upward. The axis passing through the center is an achromatic axis, indicating that the percentage of white increases as going upward. Thus, since a color is represented by three values, it can be represented by one point in three-dimensional rectangular coordinates. The space created by these coordinates is called a color space.

However, in a color space created by RBG, CMY, HSB, or the like, there is a problem that tristimulus values depend on device characteristics. To remedy this, the International Commission on Illumination (CIE) has developed an X, Y and Z tristimulus value calculated based on the standard observer's visual characteristic data, the light source spectral data and the sample spectral data. CI
An "EXYZ" coordinate system and a "CIE xyY" coordinate system, which is a two-dimensional plane coordinate system, are defined. According to this coordinate system, there is no difference between the devices, but the calculation method is difficult and this coordinate system is rarely used directly, but as a relay point when converting from a spectrum to another color space. is important.

The “CIE xyY” coordinate system is based on “CIE xyY”.
Since one point in the three dimensions of the "XYZ" coordinate system is all represented by two-dimensional positions, it is easy to understand perceptually, but has the disadvantage that the color difference does not correspond to the distance on the graph. In this coordinate system, color differences could not be quantified. In order to improve this, CIE stated that "CIE 197
6L ^* u ^* v ^* "coordinate system was defined. In addition, CIE
A “CIE 1976 L ^* a ^* b ^* ” coordinate system based on a red-green axis (a ^* ), a yellow-blue axis (b ^* ) and a value (L ^* ) representing brightness was defined.

Using this coordinate system, for example, an attempt was made to show the color change of a universal pH indicator in a coordinate system. Universal pH indicator can change color and pK
This is a kind of a mixed dye whose pH range that can be measured is widened by mixing several kinds of pH indicators having different a. Specifically, it is a mixture of Methyl Red, Bromoyhtmol B1ue (BTB), and Phenolphthalein at an appropriate ratio, and the color of each indicator is combined at each pH. It gives a very vivid color change at each pH.
Therefore, it is generally used for simple quantitative determination of hydrogen ion concentration by visual observation.
It was examined whether a new quantification could be made by measuring with OLORTRON ^™ .

First, the universal pH measured by a spectrophotometer
Although the absorption spectrum of the indicator was measured, the absorption spectrum at each pH was complex, for which the maximum absorption wavelength and the isosbestic point could not be determined, and it was found that it was difficult to read an accurate pH value by spectrum analysis. . On the other hand, in the measurement by COLORTRON ^™ , the color is recognized as a numerical value, and the numerical value is converted into a numerical value by several methods to determine the position of the coordinate system. For example, CIExy color space,
FIGS. 1 and 2 show the results of plotting the chromaticity coordinates of the CIEL ^* a ^* b ^* color space on the xy chromaticity diagram and the a ^* b ^* plane, respectively.
Shown in In any case, since the observed color change is represented by one calibration curve, it can be seen that accurate quantification can be performed without spectral analysis. In other words, the colors were arranged as digital information, and various calibration curves were obtained by selecting the coordinate system at that time, and it was found that more accurate quantification was possible by selecting the most appropriate one from among them. .

As described above, the CIExy color space and the CIEL
By using the chromaticity coordinates of the ^* a ^* b ^* color space, it was found that the color could be digitized and shown. However, the calibration curve obtained was not a straight line, but a curve of various shapes. It was very difficult to handle. this is,
Such tristimulus values XYZ for light are very convenient values when considering the additive color mixture of light by digitizing the information amount of light, but are digitized when considering subtractive color mixture of light. This indicates that the calculated amount cannot be simply calculated. To this end, Reilly et al. Describe the chromaticity coordinates Qx, Qy of the tristimulus values of the complementary colors.
(Reilly, CN, et al., Anal. Chem.,
32, 1218-1232 (1960); Reilly, CN, et al., Ana
l. Chem., 32, 1233-1240 (1960)). The process of calculating Qx and Qy is almost the same as that of x and y described above, but instead of calculating the tristimulus value XYZ using the transmittance T, the tristimulus value UVW of the complementary color is calculated using the absorbance A. The points are different. The following equation (1) shows a formula for calculating the tristimulus value UVW of the complementary color. FIG. 3 shows the xy coordinate system of the color, and FIG. 4 shows the Qx and Qy coordinate systems of the complementary colors. Although it is shown in black and white in the drawing, it is originally shown in color.

[0019]

(Equation 1)

Where A (λ) is the absorbance of the transmitting object, P
(Λ) is the spectral distribution of the illumination light. Where k in the expression
Is

[0021]

(Equation 2)

## EQU1 ## From these values, the values of Qx and Qy can be obtained by the following equations (2) and (3). Qx = U / (U + V + W) (2) Qy = V / (U + V + W) (3) Since the chromaticity coordinates of Qx and Qy derived from the tristimulus values of the complementary colors focus on the light absorbed by the material, The xy chromaticity coordinates indicating the same value indicate the complementary color of the measured color. Therefore, comparing the color distributions on the xy chromaticity diagram and the QxQy chromaticity diagram, complementary colors are shown in the same region as shown in FIGS. The QyQy chromaticity coordinate has the advantage that it is represented by a point unique to the dye, regardless of the dye concentration in the solution or film. If the colors of solutions and films having different dye concentrations are plotted on the xy chromaticity coordinates, the color shifts from the center of the chromaticity diagram to green as the density increases. And like the additive color mixture on the xy chromaticity diagram,
The color point obtained by subtractive color mixing of the arbitrary two points C and D on the QxQy chromaticity diagram is always on the line connecting C and D, and the position is determined by the mixing ratio of C and D (see FIG. 4).

Based on the above equations (1) to (3), Qx,
Qy can be calculated. Computer software Math
An example using ematica is shown in FIG.

As described above, cyan, magenta, and yellow are three subtractive primary colors. By mixing these at various ratios, all colors can be reproduced. For this reason, it is also used for the three basic colors of ink for color printing. By examining the characteristics of the three primary colors, it is believed that any color can be created by mixing. Therefore, Acid Blue 1 and Acid Red 1 used as dyes for ink jet printers
4 (Acid Red 14), Acid Yellow 23
(Acid Yellow 23) was used to examine the display in the coordinate system. These dyes have the maximum absorption wavelength and the width of the absorption peak so that hue and color purity are improved in order to support full-color printing. When actually commercialized, additives are added to aqueous solutions of these dyes,
It is prepared so as to satisfy the chemical properties and various properties of the ink.

Acid Blue 1 used as a monochromatic dye
(Cyan), Acid Red 14 (Magenta), and Acid Yellow 23 (Yellow) must be invariant in the pH range to be measured. The maximum absorption wavelength, the molar extinction coefficient, and the concentration at the time of measurement of each of Acid Blue 1, Acid Red 14, and Acid Yellow 23 are shown in Table 1 below.

[0026]

[Table 1]

Here, each dye concentration was determined such that the absorbance at the maximum absorption wavelength at pH 7 was all 2. That is, (molar extinction coefficient) × (molar concentration) is constant. Each of the dyes was adjusted to pH 1, 5, 6,
The solutions dissolved in the buffer solutions of 7, 8, 9, 10 and 13 so as to have the concentrations shown in Table 1 were mixed with a spectrophotometer and COLO.
It was measured by the transmission mode of RTRON ^™ . When measuring in transmission mode, use a halogen lamp (PH
L-150; Mejiro precision, Japan) was used. COLO
The measurement of RTRON was performed three times in succession, and the average value was adopted. The measuring apparatus and measuring conditions by COLORTRON were the same as those in the previous experiment.

The absorption spectra of these dyes obtained by a spectrophotometer are shown in FIGS. 6 shows the case of cyan, FIG. 7 shows the case of magenta, and FIG. 8 shows the case of yellow. FIG. 9 shows characteristics on a ^* b ^* coordinates obtained from COLORTRON and observed color changes. FIG. 9 shows a change in color, which is originally a color, but a color diagram is shown in black and white as a drawing. As a result, Acid Blue 1 (cyan), Acid Red 14 (magenta), and Acid Yellow 23 (yellow) in the pH range of 5 to 10 at which the indicator is measured.
Of each color change is 2.90.
7, 26.656 and 17.832. pH indicator BTB (2 × 10 ⁻⁵ M) and PhenoIRed
(6 × 10 ⁻⁵ M) at pH ^{5 to} 10 ΔE is 72.905 and 165.3, respectively. It can be considered that Acid Blue 1 (cyan) does not change color, but Acid Red 14 (Magenta) and Acid Yellow 23 (Yellow) show a color change of 10% or more with respect to ΔE of the indicator. However, in the a ^* b ^* chromaticity diagram, since the yellow region is stretched compared to the other regions, the ΔE of Acid Yellow 23 (yellow) is apparently largely estimated, and the apparent color is almost the same. Since they cannot be distinguished, the color change of Acid Yellow 23 (yellow) is considered to be within an allowable range. Regarding Acid Red 14 (magenta), it is necessary to consider the effect when added to the indicator.

The density of six colors of cyan (C), magenta (M), yellow (Y), cyan + yellow (C + Y), magenta + yellow (M + Y), and cyan + magenta (C + M) is changed in ten steps. Then, the density characteristics were measured with a spectrophotometer and COLORTRON. For cyan (C), magenta (M), and yellow (Y), the density shown in Table 1 was set to the highest density, which was 1/1.
0, 2/10,..., 9/10. Cyan + yellow (C + Y), magenta + yellow (M +
For the two-color mixed system of Y) and cyan + magenta (C + M), a solution obtained by mixing the solutions having the concentrations shown in Table 1 in equal volumes was used as the highest concentration and diluted similarly. The pH was 7.

FIG. 10 shows the results of the density characteristics on a ^* b ^* coordinates. Thus, the higher the density on the a ^* b ^* coordinates, the higher the origin (gray point)
The result of getting away from was obtained. This was a result supporting that the higher the density, the more the vividness increased. The calibration curve for yellow is long and the calibration curve for cyan + magenta (C + M) is short, which is also a ^* b.
^* Consistent with the distortion of this chroma curve in the chromaticity diagram.

Next, Bromoyhtmol Blue (Bromoyht)
mol B1ue (BTB)) was dissolved in a buffer solution having a pH of 5 to 10 and mixed with three kinds of dyes so as to obtain the concentrations shown in Table 2, and the measurement was performed using a spectrophotometer and COLORTRON. The absorbances of the cyan (C), magenta (M), and yellow (Y) dyes at the maximum absorption wavelength are as follows.
In Nos. 1-3, 2; 7 to 9, 1; 10-12
Then, it was set to 0.5.

[0032]

[Table 2]

FIG. 11 shows the resulting color change. FIG.
Indicates a change in color and is originally a color, but a color diagram is shown in black and white as a drawing. BTB
When a monochromatic dye is added to an indicator that changes color near the origin on the a ^* b ^* coordinates from yellow to blue, as in (2 × 10 ⁻⁵ M), all the curves representing the color change move almost in parallel. The direction of the movement coincides with the direction connecting the point of the indicator and the point of the monochromatic dye. Considering that the moving distance may be proportional to the distance between the point of the indicator and the point of the monochromatic dye, the color difference ΔE between the point of the BTB alone and the point of the monochromatic dye at pH 7, and the same The correlation of the color difference ΔE up to the point of the mixed system of BTB + monochrome dye｝ was examined. The result is shown in FIG.
Shown in As a result, the correlation coefficient between the two was r = 0.970, and it was confirmed that the moving distance of the curve was almost proportional to the distance between the BTB point and the monochromatic dye point. Therefore a ^*
It can be said that an indicator whose color changes near the origin in b ^* coordinates can roughly predict the direction and distance of movement when a monochromatic dye is added.

Next, the difference when the amount of dye to be added was changed was examined. Mixed solution No. BTB and cyan, magenta, and yellow dyes 1 to No. Out of 12, N
o. 1 to No. 6 has an absorbance at λmax of 2 (N
o. 4-No. For 6, the two dyes are mixed with a relatively high-concentration monochromatic dye, each having an absorbance of 1).
No. 7-No. No. 12 is No. 1 to No. The dye density is half that of 6. Regarding the magnitude of color change ΔE at pH 5 to 10, no. 1 to No. 6 and no. 7 ~
No. 12 were compared. Table 3 shows the results.

[0035]

[Table 3]

From Table 3, it can be seen that the lower the concentration of any dye, the greater the color change. This is presumably because, when a single-color dye is mixed at a high concentration, the effect of the dye is large and the color change of the BTB itself is hidden. As can be seen from FIG. 7-No. 12
The color change is easier to understand visually.

A ^* b such as BTB (2 × 10 ⁻⁵ M)
^* We examined an indicator that changes color near the origin of the coordinates. To investigate how the color change curve moves when a monochromatic dye is added to the indicator located outside the a ^* b ^* coordinates, Red (Phenol Red) (6 × 1
0 ^-5 M) was subjected to the same measurements for. Phenol Red is dissolved in a buffer solution having a pH of 5 to 10 and mixed with three kinds of dyes so as to have a concentration as shown in Table 4, and a spectrophotometer and a COLOR are used in the same manner as in the case of BTB.
The measurement was performed by TRON. The absorbances of the cyan (C), magenta (M), and yellow (Y) dyes at the maximum absorption wavelength are as follows. 13 to 15; 16 ~
In 18 it was set to 1.

[0038]

[Table 4]

FIG. 13 shows the resulting color change. FIG.
Indicates a change in color and is originally a color, but a color diagram is shown in black and white as a drawing. Phenol Red changes its color relatively from yellow to red on the a ^* b ^* coordinates relatively outside, but further 6 × 10 ⁻⁵ M
By setting a higher concentration (absorbance at λmax 3), it was positioned further outside. In this case, a simple result as in the case of BTB was not obtained. In the same manner as in the case of BTB described above, it was examined whether or not there was a correlation between the moving distance of the curve and the distance from the phenol red point to the monochromatic dye point, and the result was as shown in FIG. The correlation coefficient was r = 0.658, indicating no correlation. Therefore, in the case of such an indicator whose color changes outside on the a ^* b ^* coordinates, it can be said that it is difficult to graphically predict the moving direction and the distance of the curve on the coordinates. Next, the pH of a mixed solution of phenol red and cyan (C), magenta (M), and yellow (Y) dyes is 5 to 10%.
The magnitude ΔE of the color change was determined. Figure 1 shows the results.
It is shown in FIG. From this figure, it can be seen that there is a tendency that ΔE increases as the position moves inward on the a ^* b ^* coordinates, and ΔE decreases as the position moves outward. This trend is
When BTB is mixed with cyan (C), magenta (M), and yellow (Y) dyes, ΔE is smaller when a high-density dye is added (moves outward). I have.

From the above results, generally, a^*b^*Chromaticity coordinates
Above, the color obtained as a result of mixing two dye solutions is quantitatively determined.
I found out that I couldn't do it. This is L^*a^*
b^*The color space is derived from the tristimulus values XYZ,
Because it is a color space where light mixture, that is, additive color mixture
It is. In the case of additive color mixing of light, tristimulus values XYZ, L ^*
a^*b^*By adding values such as
It may be possible to determine the extreme value quantitatively, but the color
In the case of color mixing in a system where subtractive color mixing is possible, such as mixing elemental solutions
This is not possible.

To solve this problem, the present inventors
By using the chromaticity coordinates QxQy of the complementary colors, it was thought that the result could be quantitatively obtained even in the subtractive color mixture. Therefore, QxQy in the case where BTB or phenol red was mixed with cyan, magenta, and yellow dyes was calculated by the calculation software Mathematicala based on the above-described equations (1) to (3). In addition, cyan, magenta, and yellow dyes have λm
concentration at which the absorbance at ax is 0.6 (each of which is 0.
6 × 10 ⁻⁵ M, 4.2 × 10 ⁻⁵ M, 2.4 × 10
^-5 M). 16 to 21 show the results of mixing BTB and cyan, magenta, and yellow dyes using the calculated QxQy, and FIGS. 22 to 27 show the results of mixing phenol red and cyan, magenta, and yellow dyes.

FIG. 16 shows the color change at pH 5 to 10 when BTB is mixed with cyan by QxQy coordinates. FIG. 17 shows the color change at pH 5 to 10 when magenta is mixed with BTB by QxQy coordinates. FIG. 18 shows the color change at pH 5 to 10 when BTB is mixed with yellow, using QxQy coordinates. FIG. 19 shows the color change at pH 5 to 10 when BTB is mixed with cyan + magenta by QxQy coordinates. FIG. 20 shows the color change at pH 5 to 10 when BTB is mixed with cyan + yellow.
This is indicated by the y coordinate. FIG. 21 shows color changes at pH 5 to 10 when BTB is mixed with magenta + yellow by QxQy coordinates. FIG. 22 shows a case where phenol red is mixed with cyan at pH 5-1.
The color change at 0 is shown by QxQy coordinates.
FIG. 23 shows the color change at pH 5 to 10 when magenta is mixed with phenol red on QxQy coordinates. FIG. 24 shows the color change at pH 5 to 10 when phenol red and yellow are mixed by QxQy coordinates. FIG. 25 shows the color change at pH 5 to 10 when phenol red and cyan + magenta are mixed by QxQy coordinates. FIG. 26 shows the pH when phenol red is mixed with cyan + yellow.
The color change in 5 to 10 is shown by QxQy coordinates. FIG. 27 shows the color change at pH 5 to 10 when phenol red and magenta + yellow are mixed, as QxQ.
This is indicated by the y coordinate.

First, a pH indicator such as BTB or phenol red exhibits a color by mixing two chemical species of a broton addition type and a debroton type at various ratios depending on the pH. Changes in the chemical structure and the absorption spectrum at that time are shown in FIGS. 28 and 29. For this reason, the discoloration process of the pH indicator is considered to be a subtractive color mixture of the two colors, and it is considered that the result was represented by one straight line on the QxQy chromaticity coordinates. Regarding the mixture of BTB and cyan (see FIG. 15),
The points resulting from mixing at each pH are located at the same ratio on a straight line connecting the BTB point and the cyan point, and the color change resulting from the mixing is also represented by a straight line. From this, the color created by mixing BTB and cyan dye is
It can be seen that the BTB exists inside a triangle having three vertices of two points at both ends of the BTB and a point of Cyan. The same can be said for the mixture of BTB and yellow (see FIG. 17) and the mixture of BTB and cyan + yellow (see FIG. 19).

However, mixing of BTB and magenta (FIG. 16)
), The color change as a result of mixing is not represented by a straight line. This is because the magenta dye had a ΔE of 26.
This is because the color changes as large as 656, and the point of the magenta dye cannot be represented by a single point on the QxQy chromaticity coordinates. Due to this effect, a mixture of BTB and cyan + magenta (FIG. 1)
8), a mixture of BTB and magenta + yellow (FIG. 20)
), The color change as a result of mixing is a curve.

It was expected that the same tendency as in the case of BTB would be observed with respect to the mixture of phenol red, cyan, magenta, and yellow dyes. . Rather, in any of the dyes, results deviated from the theory in the high pH range. This is because the prepared phenol red solution has a high concentration and has a pH of 8 to 10 as can be seen from FIG.
In, the absorbance at λmax is a very large value of 2 to 3, which is considered to be because the absorbance (approximately 0.5 to 1.0) exceeds the appropriate absorbance measured by the spectrophotometer. . In other words, rather than the influence of the cyan, magenta, and yellow dyes, there was a large error in the measurement of the absorption spectrum of phenol red at a high pH, and it is thought that accurate QxQy could not be calculated.

From the above, cyan, magenta, and yellow dyes suitable for being selected as mixed monochromatic dyes do not change color under the measurement environment, and the absorption spectrum data for calculating QxQy is appropriate. It was also found that it was necessary to measure at a concentration showing the absorbance. Considering what kind of monochromatic dyes can be mixed to achieve a color change passing through the gray point, in all cases in FIGS.
If there is a gray point between the ends of the straight line of the color change of TB or phenol red and the triangle formed by the points of the monochromatic dye, it can be realized by mixing them in an appropriate ratio. In other words, it is only necessary to select a single color pigment having chromaticity coordinates such that such a triangle can be drawn. That is, it was found that by expressing the color change using the QxQy coordinate system, the behavior of the color change when a monochromatic dye was added to the indicator can be systematically grasped. Based on these results, we applied the theory of shielding indicators in analytical chemistry to optode.

In the field of analytical chemistry, shielding indicators have been developed focusing on the quality of visible color change.
This is a kind of a mixed indicator in which a monochromatic dye is mixed with the indicator, and exhibits a gray in which the color disappears at one point of the discoloration process, and the color changes very sharply and sharply before and after that. For this reason, it is mainly used to accurately determine the equivalent point of acid-base titration or metal titration. The shielding indicator is a reference indicator (p
H indicator, metal indicator, etc.) and one or more monochromatic dyes. The monochromatic dye to be mixed is selected to have a color complementary to a specific color of the reference indicator. As a result, at one point in the discoloration process, the color changes to gray, in which the color disappears, and the color changes very sharply and sharply before and after the change, so that it is possible to realize a color change that is easy to see visually.

From the experimental results described above, in order for the optode to be gray at one target point, the gray point in the QxQy coordinate system should be Qx =
Since 0.3101 and Qy = 0.3163, the change in the QxQy coordinate system of the indicator and the QxQy of the monochromatic dye
So that the value of QxQy of the mixed indicator system calculated from the value of is the gray point value in the QxQy coordinate system at one target point, that is, the value of Qx = 0.3101 and the value of Qy = 0.3163 or a value close thereto. You just have to design the optode. Further, when selecting a shielding indicator for the indicator, it is only necessary to select a shielding indicator having a value of the QxQy coordinate system that can obtain a gray point value or a value close to the gray point at a target point. To confirm our novel concept in optodes, we experimented with optodes for Na ⁺ quantification and optodes for Li ⁺ quantification.

An ion sensor is a device that selectively responds to a specific ion and measures the concentration and activity of the ion. Ion exchangers (charged ionoph
ore) and a method using a charge-dipole action by a neutral ionophore. These ionophores are placed in an oil phase such as a plastic PVC (poly vinyl chloride) membrane, and recognition is performed at the interface with the sample solution based on the above-described interaction. The response of the ion-selective electrode is governed by the contribution of the membrane potential due to charge separation at the oil-water interface. When a neutral ionophore is applied to an optode based on this principle, it is necessary that a detection element that changes color dramatically during ion extraction of the ionophore be present in the oil phase. Simon and colleagues have developed an optode that uses a lipid-soluble cationic dye that becomes positively charged and changes color when protonated as a detector. On the other hand, the present inventors have developed an optode using a fat-soluble anionic dye which takes on a negative charge and changes color by being deprotonated.

In this experiment, an optode based on a fat-soluble anionic dye was prepared as a sensing plate using a plasticized PVC membrane as an oil phase, and an optode in which Na ⁺ formed a 1: 1 anchor with an ionophore was used. . When the ionophore in the oil phase partitions into the aqueous phase and forms a complex with Na ⁺ , the lipophilic anionic dye in the oil phase also partitions into the aqueous phase, deprotonates, and changes color. Since the ion-ionophore complex and the deprotonated dye associate with each other to form an ion pair and are extracted into the oil phase, the color change according to the concentration change of the cation is measured at a constant pH. As an ionophore,
DD16C5 represented by the following formula (I), which is a 16-crown-5 derivative developed by the present inventors, was used.

[0051]

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The crown ether easily forms a complex with an alkali metal ion. Particularly, in the case of 16 crown, the ionic radius of Na ⁺ matches the vacancy size. DD16C5 small ionic diameter than primarily Na ⁺ by this as a basic skeleton Li ⁺
With sterically hindered subunits to prevent complex formation with ions larger than the crown ring vacancy such as K ⁺ and Rb ⁺ (ion: ionophore = 1: 2). A certain decalino group is introduced. Lipid-soluble anionic dyes, α- synthesized by the present inventors,
KD-A5 represented by the following formula (II), in which an alkyl side chain was introduced into naphtholphthalein to increase fat solubility, was used.

[0053]

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Further, the following formula (II)
KD-S1 represented by I) is a dye having an increased lipid solubility by introducing an alkyl side chain into Disperse Red 1 used in a solution system.

[0055]

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First, a preliminary experiment was carried out in a solution system in order to realize a color change based on the concept of a shielding indicator with an optode film. BTB was used as a reference indicator, and DR1 was used as a monochromatic dye acting as a shielding dye. BTB is yellow (430 nm) to blue (660 n) depending on the change in pH.
m) and shows green in the middle of the discoloration. DR1 (500
nm) is a red monochromatic pigment, and has a complementary color relationship with BTB green, so that an achromatic gray can be created by mixing at an appropriate ratio. Therefore, the mixing ratio of BTB and DR1 was changed variously, and it was measured whether a color change passing through the gray point could be realized. FIG. 30 shows the result of blotting the color change at that time on a ^* b ^* coordinates.
Shown in From this figure, it was possible to produce a mixed dye system which became gray at pH 8 when mixed at BTB: DRI = 2: 1.

When this result is plotted on the chromaticity coordinates QxQy of the complementary color, the result is as shown in FIG. Using the complementary color QxQy coordinate system of FIG. 31, it can be seen that the position of DR1 in the QxQy coordinate system is clearly located on a straight line connecting the point of pH 8 of BTB and the point of the gray point. Therefore, if BTB is desired to be shielded at pH 8, DR1
Qx can act as an effective shielding dye
I understand Qy. This result clearly demonstrates that the concept of the present invention can be applied to optodes. Based on this result, when an optimum mixing ratio is obtained by calculation using calculation software Mathematicala, BTB: DR1 = 2: 0.
It was calculated to be a gray point at pH 8 when mixed at 829. In the experiment, BTB: DR1 = 2: 1
At this time, the gray point was realized at pH 8, and it can be said that the experimental value and the calculated value almost coincided. It is likely that the slight difference in the numbers is due largely to errors resulting from instability in COLORTRON measurements.

Gray point in preliminary experiment in solution system
The color change passing through
Na using C film⁺For color change of optode membrane for quantitative analysis
Tried application. Add red to the pigment that changes color from yellow to green to blue
And shielding were confirmed, but plasticized PVC membrane
Pigments must be highly lipophilic
Therefore, BTB and DR1 cannot be used. There
As a dye that replaces BTB,
Make a similar color change (λmax = 460 nm, 660n
m) and use KD-A5 with high fat solubility.
Was. In addition, as a dye that replaces DR1,
KD-S1 into which a kill side chain was introduced was used. So
By changing the mixing ratio of these two dyes variously,
A toad film is prepared and Na⁺The concentration was measured. This and
Color change^*b ^*Fig. 3 shows the results of blotting on the coordinates.
It is shown in FIG. Looking at the color change, add KD-S1 in excess
The color changes slowly when cut off, so it's perfect for adding
It can be seen that the value exists. And from FIG. 32, KD-
A5: KD-S1 = best when mixed at a ratio of 16: 1.
It can be seen that the color change approaching the ray point is approaching. I
However, this oxide membrane uses FNDPE as the membrane solvent.
Yellow, which is the color of FNDPE,
It appears as a sound, and the curve of color change is
B^*Offset in positive direction of axis, i.e. yellow area
Would. Therefore, the measured Na⁺Not very effective in the concentration range
It cannot be said that the color change is typical.

FIG. 33 shows the result of plotting the color change on the QxQy coordinates. According to the QxQy coordinate system of FIG.
When KD-A5 is used as the detection reagent, the gray point is located at the end of the measured Na ⁺ concentration range of KD-A5, which is very limited for shielding at the target detection position. It must be understood that a shielding indicator must be used. The use of QxQy coordinates reveals that it is difficult for the shielding dye KD-S1 used in this experiment to pass through the gray point at the target detection position. Therefore, when KD-A5 is used as a detection reagent, in order for the optode to show gray at the target detection position, it is necessary to use a shielding indicator having a larger value of Qx and Qy in the QxQy coordinates. The system will show.

Next, as a fat-soluble anionic dye, KD-
A similar experiment was performed using KD-A4 in which the naphthol moiety of KD-A5 was a 3-methyl-4-hydroxyphenyl group instead of A5. In this case, the shielding dye KD-
The sensing plate containing no S1 changed its color from yellow to green to blue as the Na ⁺ concentration increased, while the sensing plate containing the shielding dye KD-S1 changed its color from orange to gray to purple and turned gray without any color change. It exhibited a vivid color change with large contrast. When these responses are compared with the color difference ΔE in digital color analysis, ΔE can be increased (sensitivity is increased) in the sensing plate containing the shielding dye. Therefore, it was found that by adding a shielding dye, a sensing plate capable of more accurate visual quantification could be produced.

Next, an experiment was conducted using TTD14C4 represented by the following formula (IV), which is a 14-crown-4 derivative which forms a 1: 1 complex with Li ⁺ ion as an ionophore.

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Measurement of the lithium ion concentration in blood has become a very important problem for treatment and prevention of various diseases. For example, lithium salts have been used as pharmaceuticals for treating manic depression and suppressing thyroid function, and the lithium ion concentration in blood in treating patients with manic depression is 0.75 to 1.
It has been reported that the concentration is 25 mM, and when it exceeds 1.5 mM, it causes intoxication, and when it exceeds 3.5-4.5 mM, it causes death. As described above, excessive administration of lithium salt is harmful to the human body, and the therapeutic range is narrow. There must be.

Although it is possible to know the lithium ion concentration by analyzing the blood, if the patient himself can easily monitor the medication at home,
It is effective to measure lithium ions in saliva, which is easy to handle. It is said that the concentration of lithium ion in saliva is about twice as high as in a dish. That is, the salium ion concentration in the treated saliva is about 1.5 to 2.5 mM. In order to be able to measure the lithium ion concentration in blood at home, it must be easy to handle and quantitatively quantified to some extent visually. Therefore, a new sensing device (P) with immobilized dye or ionophore
If it is possible to easily determine the lithium ion concentration using a VC film, paper, or the like), the patient himself will be able to carry out treatment with lithium salts in the course of the process. In addition, the quantitative determination of lithium ions by saliva must be such that accurate visual quantification can be ensured as described above. for that purpose,
It is necessary to emphasize the color in the therapeutic density range, and it is effective to apply the concept of color change passing through the gray point.

TTD14C4 used as ionophore
Is a kind of crown ether and easily forms a complex with an alkali metal ion. In particular, in the case of 14 crown, the ion diameter of lithium matches the pore diameter. TTD14C4 has this as a basic skeleton, into which a tetramethyl group, which is a sterically hindered subunit, is introduced so as to prevent complex formation with ions such as Na ⁺ and K ⁺ which are larger than the vacancy of the crown. Examples of the fat-soluble cationic dye include KD described above.
-A5 and KD-M11 represented by the following formula (V) developed by the present inventors were used.

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Since KD-M11 has a negative charge with a brass via a conjugated system in one molecule, it has a normal pH.
Although not only a spectral change depending on the pH but also a spectral change depending on the nature of the solvent (a change in the maximum absorption wavelength: solvatochromism) appears remarkably, here, only a spectral change depending on a pH change is used.

First, measurement conditions of these ionophores and fat-soluble cationic dyes were examined in a system using a PVC membrane in the same manner as in the above-mentioned experiment. First, examination was performed using a fat-soluble anionic dye KD-A5 as a dye. As described above, KD-A5 is yellow (460 nm) to blue (660 n).
m), which shows a green color during the discoloration, but the extraction of lithium ions was not sufficient, and a response with high sensitivity could not be obtained. There is also a method of increasing the pH of the sample solution in order to achieve a larger response at a low concentration,
Considering the application to the measurement of a biological sample (pH 7.4), it is not the best method. Therefore, it was decided to use a dye having a lower pKa to promote broton elimination and raise the response sensitivity. We decided to use M11. Since KD-M11 also changes color from yellow to green to blue, it is considered that KD-S1 can provide an effective shielding effect. The membrane solvent is NPOE (2-nitrophenyl)
Octyl ether) and adjust the pH of the sample solution to 7 and 9
The experiment was performed by changing to. In this case, since the response mechanism is different from the case where the fat-soluble anionic dye is used, TFPB represented by the following formula (VI) was also added as a fat-soluble anion additive.

[0069]

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According to the absorption spectrum and the response curve, K
DM-M11 showed remarkably good response even at a lower concentration than when KD-A5 was used. The color change of KD-M11 at this time was extremely clear, and the results of blotting the color change on the QxQy chromaticity coordinates are shown in FIGS. 34 (at pH 7) and 35 (at pH 9). According to the display using the QxQy coordinate system in FIGS. 34 and 35, KD-
It can be seen that the concentration that can be shielded using S1 is 10 ⁻¹ M and has not reached the therapeutic concentration of 10 ⁻³ M, but it can be shielded at around 10 ⁻³ M when the pH is 9.

Therefore, the calculation software Mathematical
The mixing amount was calculated using a, and shielding was performed. The absorption spectrum at this time was measured. The color change at this time is just 10
Achromatic gray was shown around ^-3 M, and it was found that the color was prominent from the colors before and after. When no shielding indicator is added, green is shown around 10 ⁻³ M,
Since the color change occurred continuously from blue to green, the difference between the color before and after the change was not clear. That is, it is difficult to distinguish ^10-3M green from yellow-green or blue-green. However, gray is completely different from other colors in that it has no color,
It is possible to make a clear distinction even with human eyes. Also,
FIG. 36 shows the result of plotting the change in the case of this mixing amount on the QxQy coordinates. From the QxQy coordinates in FIG.
It can be seen that the plot overlaps the gray point at exactly 10 ⁻³ M. Therefore, it can be seen that if a dye is mixed based on the shielding theory of the present invention, optimization for producing an optode film that can be quantified accurately with human eyes can be performed.

From the above, it has been found that in a PVC film which is a system in which an optode response mechanism is established, the color change can be optimized without changing the response mechanism by mixing a shielding dye. However, considering the application to sensors that can be used at home, PVC films are inconvenient to handle, and reflecting objects can perform more stable measurements on reflective objects than on transmissive objects even with a digital color analyzer measurement system. Therefore, a new optode device was constructed using "paper", which is the most familiar and easy-to-handle reflective object. This is an application to so-called dry chemistry. Dry chemistry is a chemical analysis method in which reagents necessary for a detection reaction are attached to an analytical material incorporated in filter paper or a plastic film, and a small amount of a sample solution is attached thereto, and the amount of generated dye is quantified by reflection-side light. It is used mainly in the clinical testing field. It is thus called from a comparison between a homogeneous reaction in a diluted aqueous solution and wet chemistry that has been developed based on light on the transmission side.

The beginning of dry chemistry is a urine test strip or a blood glucose test strip used in a group test or the like, and dates back to the 1950s. Although the judgment of these test papers was limited to visual semi-quantitative analysis, the performance was improved by the development of reflection type spectrophotometer and the improvement of analytical materials.
Quantification has been possible since 975. Dry chemistry materials are broadly divided into two types. One is a test paper method, which is based on the reaction in a filter paper impregnated with a detection reagent system and dried and the reflection side light. The second is a multilayer analytical film. In a multilayer analysis film, a dye is generated in a uniform and transparent thin layer by the progress of a detection reaction through various functional layers, and this is measured by reflection-side light.

On the other hand, the response of the conventional optode is based on the solvent extraction mechanism between the oil phase as a sensing layer containing various functional molecules and the aqueous phase of the sample solution. Therefore, the substance and shape used for the oil phase matrix are important factors that affect the sensitivity and characteristics of the sensor. Paper has a high hydrophilicity because it contains cellulose as a main component, so that it is not possible to retain ionophore or pigment having high fat solubility as it is, and that the sample solution may permeate. Therefore,
Coated paper was used in which a coating solution was applied to the paper surface to increase the hydrophobicity of the surface.

Although there are various types of coated paper, it is necessary to perform conditioning with an acid due to the response mechanism of the optode, and therefore, paper in which calcium carbonate is affected cannot be used. Moreover, if paper to which aluminum sulfate is added is used, the paper may be affected by aluminum ions. Therefore, in this experiment, a special coated paper made of a coating liquid containing only kaolin 100% without using calcium carbonate as a pigment and adding only 8% of SBR to the kaolin was used. The paper pH of this paper is 6
Met.

The coated layer of the coated paper was used as an oil phase to hold various functional substances. That is, a sensing paper was prepared by spin-casting a THF solution of an ionophore, a dye and a film solvent on the surface. After vacuum drying for about 1 hour, the various substances were very well fixed uniformly on the paper surface, and the state of the paper surface did not change even if touched by hand. When the sample solution was dropped, the water droplets became hemispherical due to surface tension, and water did not permeate into the paper. A color change on the paper surface according to the change in ion concentration was also observed. From this, it can be considered that the paper surface functions as the oil phase and the water droplets of the sample function as the water phase, and the extraction of ions between these two layers based on the solvent extraction mechanism is performed.

From this, it was found that the response mechanism of the conventional optode was established also in the sensing paper manufactured here, and the same measurement conditions as in the case of the PVC film were optimized. The dye is KD-M11,
The ionophore utilized the same molecules as in the TTD14C4 PVC membrane. Since KD-M11 is a fat-soluble cationic dye, the dye must be completely brotonized as an initial state before measurement. However, at the time of film formation, since an ion pair is formed with Na ⁺ which is a counter cation of the fat-soluble anion additive TFPB, it is necessary to perform conditioning with a strong acid or the like before measurement. In the case of a PVC membrane, Na ⁺ and H ⁺ could be easily exchanged by immersing the membrane in a strong acid. However, if conditioning is performed in the same manner when using paper, the calcium carbonate contained in the paper reacts with the acid to release carbon dioxide and change to calcium sulfate. Then, it is considered that the generated Ca ²⁺ affects the measurement of Li ⁺ as an interfering ion.

Therefore, a special coated paper which does not contain calcium carbonate and has a low paper surface pH is used, and at the same time, it is not necessary to perform conditioning with an acid before measurement.
Before spin casting on paper,
It was decided to exchange a ⁺ and H ⁺ . The method is
Equimolar amounts of KD-M11 and TFPB are dissolved in an appropriate organic solvent, and the mixture is separated and extracted with a strong acid. The organic layer is concentrated, and the cation KD-M11-H ^{+ of} brotonized KD-M11 is obtained.
Is that making the ion pair ^- and TFPB.
If spin casting is performed on paper in this state, measurement can be started without conditioning. The following experimental results were all performed by this method.

With respect to the lithium ion sensing vapor produced by the above method, the measurement was performed while changing the pH of the sample solution to 7.4 and 8. The reflection spectrum at pH 7.4 and pH 8 was measured by COLORTRON. From these results, it was found that the reflection spectrum was quantitatively changed in accordance with the change in the Li ⁺ concentration, and that there was no problem even if the Optod response theory was applied. Further, the color change at this time was observed. FIG. 37 shows changes on the QxQy coordinates.
(In the case of pH 7.4) and FIG. 38 (in the case of pH 8). From the results of the QxQy coordinates, it can be seen that the concentration that can be shielded by KD-S1 is 10 ⁻² M at pH 7.4 and exactly 10 ⁻³ M at pH 8.

Therefore, as in the case of the PVC film, the pH 8
The ratio of KD-S1 to be mixed in the case of was calculated and shielding was performed. The reflection spectrum at this time was measured in the same manner, and the color change was observed. Again, as in the case of the PVC film, achromatic gray was shown at 10 ⁻³ M, and it was confirmed that the color was prominent from the colors before and after that. Also, QxQ
FIG. 39 shows the result of blotting on the y coordinate. Also from the QxQy coordinates in FIG. 39, it was confirmed that the plot overlapped the gray point at exactly 10 ⁻³ M. From the above, it was confirmed that the theory of the present invention when shielding with a PVC film can be applied to sensing paper as it is.

Up to now, the optimization of the color change focusing on the hue has been performed. Then, next, it was decided to optimize the dye concentration in the film so as to give a larger color difference while maintaining the same hue. There is a color difference ΔE as a parameter representing a color difference felt by our eyes, which is given by the following equation (4).

[0082]

(Equation 3)

Using this value, it is possible to determine which density gives the maximum ΔE when the density of the dye is changed. Specifically, the vertical and horizontal axes are as follows. • Vertical axis: ΔlogC _{Li +} / ΔE (reciprocal of color difference variation ΔE with respect to logarithmic variation ΔlogC _{Li +} of sample concentration) • Horizontal axis: C _dye / C _{dye, 0} (dye density relative to experimental dye density C _{dye, 0)} C
₍ Dye ratio) The value on the horizontal axis that gives the minimum value on the vertical axis gives the optimum dye density.

Therefore, the data of the color change (pH 8) of the sensing paper before shielding is used, and 10 ⁻² M to 1
FIG. 40 shows the result of creating the above graph in the concentration range of 0 ⁻⁴ M Li ⁺ . Here, when theoretically calculating the color difference when the dye concentration is changed, COLOR
After converting the reflection spectrum obtained by the measurement of TRON into an absorption spectrum, assuming that the Lambert-Beer rule is satisfied, the dye concentration is doubled, tripled, and so on.
・ With the change, the absorbance is doubled, tripled, etc.
Was changed. As a result, when C _dye / C _{dye, 0} was 3.34, it was found that ΔlogC _{Li +} / ΔE became the minimum. In other words, the maximum color change is given when the dye concentration is 3.34 times the dye concentration when this data is measured. So, COLORTRO
FIG. 41 shows the result of simulating the color change when N was increased by 3.34 times. Although FIG. 41 is originally color, it is shown here as black and white. From this, it was found that the dye concentration can be optimized based on the theory of the present invention when constructing the sensor.

The most disturbing factors in measuring Li ⁺ in saliva are Na ⁺ and K ⁺ . Therefore, the selectivity for these was examined. The result is shown in FIG. The vertical axis shows the ratio of the color difference to the total color difference of the sensing paper based on the concept of the normalized absorbance when the absorption spectrum is used. As a result, it hardly responded to K ⁺ and showed about 100-fold selectivity for Na ⁺ . Na ⁺ and K ⁺ concentrations in saliva are both about 20 mM
(2 × 10 ⁻² M), it can be considered that this sensing paper is hardly affected by Na ⁺ and K ⁺ . FIG. 43 shows the color change at this time. FIG.
3 is originally color, but is shown here as black and white. This indicates that the sensing paper of the present invention hardly responds to anything other than high-concentration Na ⁺ .

The conventional PVC film has a problem that the response time is long. Therefore, the response time of the sensing paper produced this time was examined. FIG. 44 shows the response profile of the PVC membrane, and FIG. 45 shows the response profile of the sensing paper. It takes 5 to 10 minutes to stabilize at the earliest with PVC membrane, but 9
It can be seen that it has almost reached stability at 0 seconds.

Until now, the use of a shielding dye that turned gray at the target detection point has been studied. In this case, the only point at which the detection and quantification can be clarified visually is that only the target detection point is used. become. This is an extremely effective means when it is determined whether or not the concentration is within a specific range as in the case of the lithium ion concentration described above. An example of such is a universal pH indicator. A universal pH indicator can indicate a wide pH range by a change in color. As described above, the absorption spectrum measured by the spectrophotometer at each pH of the universal pH indicator is a complex one whose maximum absorption wavelength, iso-absorption point, etc. cannot be determined, and accurate pH
Although it was difficult to read the value, the change in color of the universal pH indicator was digitized (digitized),
When plotted on the chromaticity coordinates of the Exy color space and the CIEL ^* a ^* b ^* color space, a curve like a circle was observed as shown in FIGS.

In the xy chromaticity coordinates shown in FIG. 1, since the gray point is such that x is about 0.31 and y is about 0.32,
The curve drawn by the universal pH indicator was found to be circular with the gray point at the center. The same can be seen from the a ^* b ^* chromaticity coordinates in FIG. A ^* b ^{* in} FIG.
Since the point where a ^* and b ^* are 0 in the chromaticity coordinates is the gray point, it can be seen that the point is a circle centered on the gray point as in FIG. In the first place, these coordinate systems are intended to indicate a change in color by coordinates, and a large movement in the coordinate system indicates a large change in color. The results of FIGS. 1 and 2 show that the point that becomes the center of the change is a gray point without color (gray point).

As described above, in the digital color analysis (DCA), colors can be displayed in a three-dimensional or two-dimensional coordinate system, so that intuitive color changes can be accurately grasped from the position of the coordinate system. Will be able to By utilizing this theory, it is possible to develop an optode that shows a visually apparent color change according to the concentration of the target substance. Based on this theory, the present inventors examined a combination of discoloring dyes for producing many hues in the process of color change, based on TMA ⁺ extraction, which can approximate the response characteristics in the extraction of an ion-ionophore complex. .

LCD-2 which is a fat-soluble cationic dye
Changes from purple to yellow when the pH value increases, and KD
-M11 is a pigment that changes color from yellow to blue when the pH value increases. Absorption spectra were measured using LCD-2 and KD-M11, respectively, and their response curves are shown in FIG. Looking at the total extraction coefficient κ having a correlation with pKa from the response curve, the total extraction coefficient κ when LCD-2 is used is 10 ^−1.3 (5.01 × 10 ⁻² ).
Therefore, only a part of the response curve, which should originally show an S-shaped curve, appeared. And a shown in FIG.
^* B ^* As can be seen from the results of the color space coordinates and the color change of the film, in the low pH range, except for pH 1, no purple color was exhibited.
2 indicated colorless. The total extraction coefficient κ when KD-M11 is used is 10 ^−8.8 (1.58 × 10 ⁻⁹ ).
Although the response curve showed a beautiful S-shaped curve,
Looking at the results of the a ^* b ^* color space coordinates and the color change of the film shown in FIG. 47, yellow was observed around pH1 to pH6, and the color change became difficult to see visually.

Looking at the case where these two dyes are mixed, it is expected that the color changes of purple-colorless-yellow-green-blue are shown from the change of each total extraction coefficient κ and the color. Looking at the results of the a ^* b ^* color space coordinates and the color change of the film, the colorless
Shows yellow around pH 3-7, low and medium pH
It was found that the color change was difficult to see in the region.

KD-M9, which is a lipid-soluble cationic dye, changes color from yellow to red when the pH value increases when the polarity of the membrane is high, and KD-M13 changes from yellow to red when the pH value increases.
It is a pigment that changes color to blue. KD-M9 and KD-M1
Absorption spectrum was measured using No. 3 and their response curves are shown in FIG. Looking at the total extraction coefficient κ having a correlation with pKa from the response curve, the total extraction coefficient κ when KD-M9 is used is 10 ^−11.7 (2.00
× 10 ⁻¹² ). As can be seen from the results of a ^* b ^* color space coordinates and film color change shown in FIG.
It showed a yellow color at around pH 10 and did not give an easily understandable color change. Furthermore, in the high pH range, the color was purple instead of red. Therefore, NPOE is used as the membrane solvent.
-OH to increase the polarity of the membrane to increase the overall extraction coefficient κ
Was increased, and an attempt was made to make red appear in a high pH range. As a result, the result was almost the same as the previous experiment using NPOE as the membrane solvent, and the discoloration pH range was pH 11 to 11.
It was as high as 12 and did not show red color in the high pH range.

The total extraction coefficient κ when KD-M13 is used is 10 ^−11.0 (1.00 × 10 ⁻¹¹ ), and the a ^* b ^* color space coordinates and film thickness shown in FIG. Looking at the results of the color change, the color change also showed yellow around pH 1 to pH 10, and a visually apparent color change was not obtained. From the above results, considering the case where these dyes are mixed, the total extraction coefficient κ of KD-M9 and KD-M13 is extremely high, so that discoloration in a low pH range is hardly apparent, and two more values are obtained. It is very close and it is expected that the discoloration of the dye will occur almost simultaneously, and it is considered that many hues did not appear in the process of color change.

KD-C4 which is a fat-soluble cationic dye
Is a pigment that changes color from red to yellow when the pH value increases. The absorption spectrum when KD-C4 was used was measured, and this response curve is shown in FIG. Looking at the response curve, the total extraction coefficient κ waiting for correlation with pKa is 10
^−5.2 (6.31 × 10 ⁻⁶ ). Therefore, considering the case of mixing with KD-M11 having a total extraction coefficient κ of 10 ^−8.8 (1.58 × 10 ⁻⁹ ),
From each of the total extraction coefficient κ and the color change, it is expected to show a color change of red-orange-yellow-green-blue.

First, when the mixing ratio of the dyes of KD-M11 and KD-C4 is 1: 1 (absorbance ratio), the results of a ^* b ^* color space coordinates and film color change shown in FIG. 52 are shown. And began to show yellow near pH 6-10 and green near pH 11, resulting in a color change that was hard to see in the middle pH range. Next, KD-M11 and KD-
In the case where the C4 dye mixture ratio is 3: 2 (absorbance ratio), the results of a ^* b ^* color space coordinates and film color change shown in FIG. The color change was green, and the color change in the middle pH range became visually apparent as compared with the case where the dye mixing ratio was 1: 1 (absorbance ratio). When the mixture ratio of the dyes of KD-M11 and KD-C4 is 2: 1 (absorbance ratio), the color mixture shows green near pH 7 but shows orange instead of red in the low pH range, and the dye mixture ratio becomes 3 : 2 (absorbance ratio), lower p
The color change in the appearance at H Castle became difficult to understand. From the above, it can be seen that the most visually recognizable color change was obtained when the dye mixture ratio of KD-M11 and KD-C4 was 3: 2 (absorbance ratio). The chemical formulas of the dyes used in these experiments are shown below.

[0096]

Embedded image

From the above experimental results, KD-M11 and KD-C4 were the combinations of dyes that exhibited the most hues in the process of color change and showed a color change that was easy to see. The mixing conditions of the dyes that represent many hues in the process of color change are that the pKas of the mixed dyes differ by about 3 to 4. A dye having a low pKa changes from a deep color to a light color. A dye having a high pKa changes from a pale color to a deep color. The hues on the lighter side of the dyes to be mixed are almost the same. The hue on the deep color side of the dye to be mixed is visually different. Turned out to be necessary.

Based on the above results, application of the PVC film to ammonium ions was examined. TD19C6 for ionophore, KD for fat-soluble cationic dye
Response to ammonium ion was examined using -M11 and KD-C4 mixed dyes. The ammonium ion dissolution at this time was carried out using one having a pH of 6. Looking at the response curve shown in FIG. 54, the response is best in the concentration range of 10 ⁻⁴ M to 10 ⁻² M, but the a ^* shown in FIG.
Looking at the b ^* color space coordinates, the starting point of the calibration curve is near a ^* value = 0, indicating orange. This can be seen from the result of the color change of the film, and the color changed from orange to yellow to green to blue as the concentration of ammonium ion increased.

The above-mentioned fat-soluble cationic dye KD-M1
The color change of 1 and KD-C4 mixed dyes is red-orange-yellow-green-blue, and despite the appearance of red in the process of color change, in the film containing the ionophore TD19C6, the color change in the process of color change Could not be obtained. This is probably due to the structural problem of the ionophore.
Therefore, the response to lithium ions was examined using TTD14C4 having a different structure from TD19C6.

The response to lithium ions was examined using TTD14C4 as an ionophore and a mixed dye of a fat-soluble cationic dye KD-M11 and KD-C4 as a dye. At this time, the lithium ion solution having a pH of 6 or 7 was used. First, an optode film containing each of the dyes KD-M11 and KD-C4 was prepared, and the absorption spectra of the respective dyes were measured.
FIG. 56 shows the result of examining the response curve of the H6 lithium ion solution. Looking at the results of the response curve shown in FIG. 56, the slope of the KD-C4 membrane is largest at low concentrations in the range of 10 ⁻⁶ to 10 ⁻² M, and the absorbance changes.
At this time, the color changes from red to yellow. On the other hand, in the film of KD-M11 having a higher pKa than that of KD-C4, 10 ^{−4 to} 1M
The gradient is greatest at high densities in the range, and the color changes from yellow to green and blue. This indicates that the degree of color change of the film is the largest in this concentration range, and can be seen from the result of the actual color change.

The dye containing each dye obtained as described above
From the measurement results of the putod film, the color change of the mixed dye
I did a simulation. That is, the spectral data of each dye
L when the dye is mixed from the^*a^*b^*Calculate the value of
And change the color^*b ^*Plot on the coordinates,
Simulate the appearance of color changes by changing the mixing ratio of dyes
Was created. In addition, when the lithium ion concentration is 1
0^-6M is the start point of the color change, 1M is the end point
Change of hue angle from start point to end point_abAsk for
When, Mixing ratio of dye Δh _ab  (KD-C4 / KD-M11) 9/2 187.0 ° 3/1 194.7 ° 2/1 182.2 °3/2 166.0 ° And the mixing ratio of KD-C4 and KD-M11 is 3: 1.
It can be seen that the change in hue is largest at the molar ratio.
won.

Next, when the dye is actually mixed, the pH 6
The response of optode films in lithium ion solution was investigated. FIG. 58 shows the result. KD-C4 and KD
The mixture ratio of -M11 is based on the result of the simulation,
The molar ratio was 3: 1. When the color change at this time is plotted on a ^* b ^* coordinates, only KD-M11, KD-C4
The change in hue is wider than in the case of only
The color change is clear and easy to understand. A low concentration range of ¹⁰ -4 M to ^-3 M than the response curve shown in Figure ^{58, 10} - 1 slope in the high concentration range of M~1.0M is large. This indicates that the degree of color change of the film is the largest in this concentration range,
It can be seen from the result of the color change. In addition, the color change of the film is red at low concentrations, and becomes red to red as the ion concentration increases.
Color changes of orange-yellow-yellow-green-green-blue-green-blue were obtained, and it was found that visual quantification could be performed in a wider concentration range. Further, the color difference ΔE from the point where the color change end point, and the change in hue angle Delta] h _ab, and, when comparing the case where the dye mixture of the single dyes for dynamic range, both become greater in the dye mixture I have. In particular, it can be seen that the hue angle was greatly improved.

As described above, sodium ions and potassium ions interfere most when measuring lithium ions in saliva. Therefore, the selectivity for interfering ions in this system was investigated. The response curve of each ion at pH 6 is shown in FIG. 57 (the vertical axis is the color difference ΔE). According to this, it did not respond so strongly to potassium ions, and showed 100-fold or more selectivity to sodium ions. Looking at the color difference ΔE with respect to the color difference from the start point to the end point, ΔE _{total, it} is about 1000 times for Na ⁺ and about 50 times for K ⁺ .
It turned out to show good selectivity of 00 times. The concentration of both sodium and potassium ions in saliva is about 2
Since it is 0 mM (2 · 10 ⁻² M), it is considered that there is almost no interference. From the results of the color change, both sodium ions and potassium ions were 10 ⁻⁵ M or more.
The color change in the concentration range of 1.0 M is from red to orange, and it can be seen that there is almost no response except for the high concentration of sodium ions.

Next, looking at the results of the mixed dye in the lithium ion solution at pH 7, as shown in FIG. 59 (the vertical axis is the color difference ΔE), when the pH changes by 1, the response curve S
It can be seen that the curve also moves equilibrium by about one digit with respect to the lithium ion concentration. Here, this case will be applied to a theoretical formula for the activity a _Co of the measured chemical species Co. Assuming that the target ion is monovalent and the ion-ionophore complex is 1: 1, the following equation (5) is obtained.

[0105]

(Equation 4)

In the equation (5), Κ is a total extraction constant.
(5) the left side of the equation is constant, i.e. a _Siro when absorbance is _constant, also a _{CHR o} becomes constant, also must be constant even a _So. to balance in this case system. Therefore, the ion concentration a corresponding to a certain absorbance a
_{i +} is, _{a H +} 10 ² times the change (pH changes only 2) Then likewise varies 10 ^twice. That is, it is shifted by two digits.
From the above, it can be said that when the pH changes by 1, the S-curve moves equilibrium by about one digit with respect to the lithium ion concentration, indicating a tendency consistent with the theory.

The reason that the low concentration ammonium ion did not show a red color while the low concentration lithium ion showed a red color was that the vacancy of TTD14C4 was smaller than that of TD19C6, It is difficult to form a complex with sodium ion which is an ion pair of TFPB, and the number of oxygen atoms of TTD14C4 is TD
It is considered that the binding constant with sodium ions is also small since it is smaller than that of 19C6. Therefore, it is also important to select an ion recognition element (ionophore) to be used, and it is understood that it is preferable to select and use an element that does not interact with other membrane components.

Thus, with the ion optode of the present invention,
Can be combined with an appropriate dye to
Many hues appear as the density changes,
The change was found to be advantageous in visual quantification. Ma
In addition, the discoloring dyes KD-C4 and KD-M11 were used alone.
In each case, the measurable concentration range is 10^-6-10
^-2M and 10^-4M to 1M, but of the mixed dye
Case 10^-6Visual quantification in a wide range of up to 1M possible
It turned out that it would work. Therefore, digital color
Calibration by color sample is possible by using an analyzer
Not only that, but also accurate
Quantification and optimization of dye mixing
I found out.

In the previous experiments, the digital color
Narizer (for example, COLORTRON^TM)
But how much does this digital color analyzer
We examined whether the density could be determined finely.
Blot to QxQy coordinates based on measured color change
The results obtained are shown in FIG. Seen from the measured color change
The color observed in the eyes can be read 10 times difference
Although 10^-4M, 10^-3M-order fine cousin
Is almost indistinguishable. However, from FIG.
0^-3At concentrations lower than M, data fluctuates
Although 10 ^-3There is a quantitative difference at concentrations above M
Has been measured. Therefore, if you want to know the exact value
If you use a digital color analyzer,
It turned out that the concentration could be measured with a reader. Digital camera
The color analyzer is compact in size and very easy to handle
And it is easy to bring it home.
Conceivable. Therefore, along with visual quantification, digital
Use at home with a color analyzer
Accurate measurement can be realized.

As described above, the present invention provides a new technical idea for optimizing the optode color change by quantifying the change. Therefore, the present invention includes all the optodes that have been optimized according to the purpose by quantifying the color change of the optode based on the technical idea of the present invention. As a means for quantifying color change of the present invention, a method using a digital color analyzer is preferable, but there is no particular limitation as long as color elements can be analyzed and quantified. The coordinate system for digitization can be appropriately selected according to the purpose of the optode, but xy chromaticity coordinates, a ^* b ^* chromaticity coordinates, or chromaticity coordinates QxQy of complementary colors are preferable.

In the above examples, the optode of the present invention mainly includes a combination of an ionophore and a dye. However, the present invention is not limited to this, and the indicator itself may be a dye. Further, it may be composed of more complicated components. The optode of the present invention may be any as long as it can detect, quantify, and / or identify the concentration or state of a measured chemical species by a change in color. The measurement chemical species is preferably, but not limited to, an ion. Preferred measurement species include Na ⁺ , L
metal ions such as i ^+, and non-metal ions such as H ⁺ , OH ⁻ and halogen ions. Further, the chemical species to be measured may be an organic compound, preferably a bioactive organic compound. As the dye used in the optode of the present invention,
The lipid-soluble cationic dyes and lipid-soluble anionic dyes mentioned in the examples are preferable, but not limited thereto. The dyes used may be used alone or in combination of two or more. In addition, the dyes may be used in combination with a substance that assists the dye, not the dye itself.

One end when the color change of the optode in the method of the present invention is represented by the chromaticity coordinate QxQy of the complementary color and the opposite end are usually one end and the other end of the measurement limit of the optode. It does not necessarily have to be the measurement limit. One end and the other end of an arbitrary measurement range including the range to be measured may be used. The "gray point" described in the method of the present invention is strictly one of the coordinate systems.
In this regard, the present invention does not use the gray point in such a strict sense, but uses the term “gray point” including the vicinity of the gray point. The range that can be visually recognized as gray or a color close to gray is the “gray point” in the present invention. Therefore, the straight line formed from the two points of the chromaticity coordinates QxQy of the complementary color and the gray point at the detection point of the measurement chemical species of the optode of the present invention is not a straight line in a strict sense, and the detection point itself is not a straight line. Since it has a certain range and the gray point also has a range including the neighborhood as described above, the gray point is not the only one in the coordinate system and falls within such a range. There will be multiple straight lines.

As the ionophore used in the optode of the present invention, a crown ether derivative has been exemplified, but the ionophore is not limited to this. There is no particular limitation as long as it is a chemical species that can selectively supplement a specific ionic species. Preferred ionophores of the invention include crown ethers or derivatives thereof, with TTD14C4 being particularly preferred for lithium ions. Examples of the support of the optode of the present invention include a PVC film and a coated paper, but are not limited thereto. For example, paper, a polymer film, or the like can be used, and a material that has affinity for components such as an optode component and a dye, is inexpensive, has physical strength as a material, and is hydrophobic is preferable. Like the coated paper described above, a hydrophilic material can be processed and used as the support of the present invention.

The indicator composition of the present invention is used in the sense of comprising an optode and a shielding indicator. In the present invention, the indicator composition may be referred to as an optode in a broad sense. As described above, the optode in the present invention is not limited to a component having a function as an optode, but is simply referred to as an optode, including a so-called optode and a component that assists the function of the optode. The shielding indicator of the present invention may be a single type of dye, or may be a combination of two or more types of dyes. The dye used for the shielding indicator is preferably a monochromatic dye, but is not limited thereto. As the monochromatic dye, those having no structural change that causes discoloration under the measurement environment are preferable. It is pH that contributes to such color change
It is preferable to select a dye having no broton desorption site as the monochromatic dye because proton desorption of the dye is often caused by the change of the dye. As a method for measuring a target chemical species using the optode or the indicator composition of the present invention, a usual measurement method can be applied. Since the optode or indicator composition of the present invention has a large change in color, it can be sufficiently measured visually, but can also be automatically measured using an optical instrument. Examples of the target chemical species include the measurement chemical species described above.

The change in the color of the optode due to the change in the concentration of the measured chemical species of the present invention is such that a circle centered on the gray point of each coordinate is drawn in the xy chromaticity coordinates or a ^* b ^* chromaticity coordinates. In the method of choosing the color change of the optode, the circle should not be a strict circle, but should surround the gray point and be distributed over a wide area of the coordinate system instead of being concentrated in a specific part of the coordinate system. Means a simple curve. The fact that the detection line is concentrated at a specific position in the coordinate system means that the color change is concentrated on the specific color part, so it is not so, and it is expressed as a circle in the sense that it is widely distributed It is using. Since the color becomes more vivid as the distance from the gray point increases, a distribution away from the gray point is preferable.

Further, the present invention relates to a material for measuring a lithium ion concentration in a specimen, which contains an optode and a shielding indicator so that the lithium ion concentration in the specimen is gray when the concentration is in an appropriate concentration range. More specifically, a material for measuring a lithium ion concentration in which the optode comprises an ionophore of TTD14C4 and the dye KD-M11 and the shielding indicator is KD-S1 is preferable. further,
A material for measuring lithium ion concentration immobilized on coated paper or the like is preferable. The material for measuring the lithium ion concentration of the present invention can be used to determine the blood concentration of lithium ions from saliva, and has a gray colorless color when the lithium ion concentration is appropriate. Therefore, since the lithium ion concentration can be accurately grasped visually without requiring any special technique, it can be easily used by individuals at home. The ionophore of the present invention is TTD14C4, and the dye is KD-M11.
The optode comprising KD and KD-C4 can display the lithium ion concentration over a wide range by vivid color change. The optode immobilized on a support such as paper or a polymer film is described above. Like the lithium ion concentration measuring material of the present invention, it can be easily used by individuals at home.

In the examples described above, the application of the optode optimization method of the present invention to the measurement of lithium ion concentration has been exemplified. However, the method of the present invention is limited to the measurement of the lithium ion concentration described above. Instead, it can be applied to various types of measurement chemical species, and in the future, it will be applied to the quantification of other ions and molecules in the human body to develop a simple sensing device that can comprehensively check one's own health condition. There is expected.

[0118]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples and specific description.

Example 1 (Quantification of color change of universal pH indicator by COLORTRON ^™ ) 0.2 ml of universal pH indicator (manufactured by Merck) was added to 0.05
M citric acid / NaOH (pH 4, 5, 6), 0.05
M Na ₂ HPO ₄ / NaH ₂ PO ₄ (pH 7) and 0.05 M boric acid / 0.05 M KCl / NaOH
10 m buffer solution of each pH consisting of (pH 8, 9, 10)
and placed in a cell with a lid, and the spectrophotometer and C
It was measured by the transmission mode of OLORTRON ^™ . At the time of measurement in the transmission mode, a halogen lamp (PHL-150; Mejiro Precision, Japan) was used as an external light source. The measurement was performed three times in succession, and the average value was adopted.
The absorption spectrum of the universal pH indicator at each pH measured by a spectrophotometer is a complex one whose maximum absorption wavelength, iso-absorption point, etc. cannot be discriminated.
It turned out to be difficult to read the H value. COL
Color is quantified in several ways in ORTROM ^™ measurements. Among them, the chromaticity coordinates of the commonly used CIExy color space and CIEL ^* a ^* b ^* color space are respectively plotted on an xy chromaticity diagram and a ^* b ^* plane, and the results are shown in FIGS. 1 and 2, respectively. In any case, since the observed color change is represented by one calibration curve, it can be seen that accurate quantification is possible without spectral analysis.

Example 2 (Color change when BTB and dye are mixed) Bromoyhtmol B1ue (BTB)
(Manufactured by Junsei Chemical Co., Ltd.), was dissolved in a buffer solution of pH 5 to 10, mixed with three types of dyes such that the concentration as shown in Table 2, similar to Example 1 by spectrophotometer and Colortron ^TM The measurement was performed by the method. The buffer used was 0.05M KCl / HCl (pH 1), 0.0M
5M Tris / HCl + 0.1M TMACl (pH
5, 6, 7, 8, 9, 10), 0.05M KCl / N
aOH (pH 13). The buffer solution of pH 5 to 10 was added with high concentration of TMACl (tetramethylammonium chloride) in consideration of the influence of the difference in ionic strength. The results are shown in FIG. 11 and FIG.

Example 3 (Color change when phenol red and a dye are mixed) The same procedure as in Example 2 was carried out except that phenol red (manufactured by Junsei Chemical Co., Ltd.) was used instead of BTB. The results are shown in FIG. 13, FIG. 14 and FIG.

Example 4 (Formation of QxQy Coordinates of Color Change when BTB and Dye are Mixed) Based on the data of color change when BTB obtained in Example 2 and each dye are mixed, calculation software Mathematical
The value of the chromaticity coordinates QxQy of the complementary color was calculated by ica.
The results are shown in FIGS.

Example 5 (Formation of QxQy Coordinate of Color Change When Phenol Red and Dye are Mixed) Based on the data obtained when the phenol red and each dye are mixed in Example 3, calculation software Mathema
The value of the chromaticity coordinates QxQy of the complementary color was calculated by tica. The results are shown in FIGS.

Example 6 (BTB and dye D in solution system)
Mixing with R1) In a buffer at each pH, add 2 × 10 ⁻⁵ M BTB and DR1
(Manufactured by Aldrich) at 2 × 10 ⁻⁵ M and 1 × 10 ⁻⁵ M
M dissolved sample solution (buffer: ethanol = 1: 1)
Was prepared and placed in a cell with a lid.
It was measured as in Example 1 by a transmission mode ORTRON ^{T M.} The buffer used here was 0.05 M citric acid / NaOH (pH 4, 5, 6), 0.05 M Na
₂ HPO ₄ / NaH ₂ PO ₄ (pH 7), 0.05M
Boric acid / 0.05M KCl / NaOH (pH 8, 9,
10) and 0.05 M NaH ₂ PO ₄ / NaOH (p
H11). The results are shown in FIGS.

Example 7 (Mixing of ionophore and dye in membrane system) Polyvinyl chloride (PVC) (manufactured by SIGMA) was used as a membrane agent and 2-fluoro-2'-nitrodiphenyl ether (FNDPE) was used as a membrane solvent. Was. Dye KD
-A5, KD-S1 and ionophore DD16C5 were dissolved in THF together with PVC and FNDPE at a mixing ratio as shown in Table 5 below, dropped on a plastic plate (OHP sheet) fixed to a spin coater, and rotated at 4000 rpm for 5 seconds. This produced a thin film.

[0126]

[Table 5]

[0127] After cutting to size into the cells of a spectrophotometer is sequentially immersed in will color change Na ⁺ solution of ¹⁰ -5 M~1M is the absorption spectrum was measured from the stable. Immediately after that, remove the membrane from the cell, wipe off the moisture gently, place a Teflon sheet underneath, and
The color of the film was measured by the reflection mode of ^TM . Fig. 3 shows the results.
2 and FIG.

Example 8 (Measurement of Lithium Ion in PVC Membrane) Film agent PVC: 32.3 wt%, membrane solvent: NPOE (2-nitrophenyl octyl ether (manufactured by Dojindo)) 6
4.5 wt%, and indicator KD-A5 or KD-M11
Is contained in a film composition comprising 3.2 wt% of a shielding dye KD-S.
1 and the ionophore TTD14C4 to the dye
00 mol% was added. Again, KD-M as an indicator
When 11 was used, TFBP was added to the dye at 100
Mole% was further added. Each of the obtained compositions was dissolved in THF, and a plastic plate (OH) fixed to a spin coater was used.
(P sheet) and rotated at 4000 rpm for 5 seconds to produce a thin film. After vacuum drying, this is cut into a size that fits into the cell of the spectrophotometer, and then 10 ⁻⁵ M to 1 M
Was sequentially immersed in a solution of Li ⁺ and after the color change was stabilized, the absorption spectrum was measured. Immediately after that, the film was taken out of the cell, and after wiping off the water lightly, a Teflon sheet was placed under the cell, and the color of the film was measured by a reflection mode of COLORTRON ^™ . The results are shown in FIGS.

Example 9 (Measurement of Lithium Ions in Sensing Paper) First, KD-M11 and TFPB were dissolved in chloroform in an equimolar amount, and this was separated and extracted with a 0.1 M HCl solution to extract TFB in the oil phase. Na ⁺ which is a counter cation
Is replaced with H ^+, and protonated KD-M11 and TF
An ion pair of PB was prepared. This KD-M11 TFP
B ion pair, KD-S1 and TTD14C4
E was dissolved in THF together with E, dropped on coated paper fixed to a spin coater, and spin-cast in the same manner as in Example 8. At this time, the mixing ratio of NPOE was 95.
2 wt%, KD-M11 4.7 wt%, TFPB K
100 mol% based on D-M11, TTD14C4 is K
It was 100 mol% based on D-M11. After vacuum drying, a few drops of a ^10-5 M to 1 M Li ⁺ solution are dropped, and the sample is left for 2 minutes to stabilize the color change, and the sample is wiped off. Immediately, the color of the paper is measured by the reflection mode of COLORTRON ^™. Was measured. The results are shown in FIGS.

Example 10 (Synthesis of Dye KD-C4) About 0.4 g of neutral red was dissolved in 500 ml of water, an appropriate amount of an aqueous NaOH solution and chloroform were added thereto, and the mixture was shaken. And evaporated under reduced pressure. About 0.3 g of triethylamine and chloroform were added thereto, and the mixture was stirred at room temperature. Further, about 0.9 g of stearoyl chloride was added and reacted overnight to obtain the desired KD-C4 into which neutral amino red stearoyl group was introduced.

Example 11 (Ion optode using mixed dye) Lithium ionophore (TTD14C4), fat-soluble cationic dye (KD-M11 and KD-C4), fat-soluble anion (Tetrakis [3,5-bis (trifluoromethyl) ) -Phenyl] b
orate, sodium salt, dihydrate), membrane solvent (2-Nitrophen
yloctylether) was dissolved in tetrahydrofuran (THF) and spin-cast on an OHP sheet to produce an optode film. This optode film was placed in a cell for measuring absorbance, immersed in a 10 ⁻⁶ M to 1.0 M Li ⁺ solution (pH 6), and the change in absorbance with time at the maximum absorption wavelength of the dye was measured with a double beam spectrophotometer. After confirming that the absorbance was constant, the absorbance spectrum was recorded.
Also, after taking out this membrane from the cell and gently removing moisture,
Place it on a white Teflon plate and use a spectrophotometer (COLORT
The color of the film was measured by RON ^™ ).

Example 12 (Preparation of Response Curve) The color change in each sample solution was measured with a digital color analyzer (COLORTRON ^™ ), and 0.1 M
HCl color change is 0, 0.1M NaOH color change is 1
, The degree of color change (color difference ΔE) in each sample solution was determined. This was plotted against the ion concentration of each sample solution.

[0133]

According to the method of the present invention for optimizing the color change of the optode based on the digitized information, it is possible to carry out a simpler measurement, which enables accurate quantification even by visual observation without using a spectroscopic instrument. The optode can be designed purposefully by quantification, not by repeating experiments by mere thought and error. The optode designed by the method of the present invention can be easily and accurately quantified visually, does not require a large-scale device such as a spectroscopic instrument, and is a measurement material that can be easily used by homes and individuals. Is provided. For example, if the optode of the present invention is used to measure lithium ions in blood, it can be simply and accurately performed using saliva without the need for a special device or a special discrimination technique. Therefore, the present invention provides an optode that can be accurately measured even by visual observation and that can be measured more easily, and more specifically, a novel optode optimization method for that purpose, and an optode optimized by the method. Optodes.

[Brief description of the drawings]

FIG. 1 shows the results of plotting the color change at each pH of a universal pH indicator on an xy chromaticity diagram.

FIG. 2 shows the results of plotting the color change at each pH of the universal pH indicator on the a ^* b ^* plane.

FIG. 3 shows an xy coordinate system for colors.

FIG. 4 shows Qx and Qy coordinate systems of complementary colors in colors.

FIG. 5 shows an example of calculating Qx and Qy using the computer software Mathematica.

FIG. 6 shows an absorption spectrum obtained by a cyan dye spectrophotometer.

FIG. 7 shows an absorption spectrum obtained with a magenta dye spectrophotometer.

FIG. 8 shows an absorption spectrum obtained with a yellow dye spectrophotometer.

FIG. 9 shows the change in color due to Acid Blue 1 (cyan), Acid Red 14 (Magenta), and Acid Yellow 23 (Yellow) on the a ^* b ^* coordinates and the observed color change. . The color change in the figure is originally a color, but the color figure is shown in black and white in the drawing.

FIG. 10 shows the results of density characteristics of cyan, magenta, yellow, and a mixed color thereof on a ^* b ^* coordinates.

FIG. 11 shows cyan (C), magenta (M),
The change in color when the yellow (Y) dye and these mixed dyes are mixed with BTB is shown by the characteristics on the a ^* b ^* coordinates and the observed color change. The color change in the figure is originally a color, but the color figure is shown in black and white in the drawing.

FIG. 12 shows a correlation between a color difference ΔE between a point of BTB alone and a point of a single color dye, and a color difference ΔE between a point of BTB alone and a point of a mixed system of {BTB + single color dye}. The results are shown.

FIG. 13 shows cyan (C), magenta (M),
The change in color when a yellow (Y) dye and a mixed dye thereof are mixed with phenol red is shown as characteristics on a ^* b ^* coordinates and the observed color change. The color change in the figure is originally a color, but the color figure is shown in black and white in the drawing.

FIG. 14 is a correlation between a color difference ΔE between a point of phenol red alone and a point of a single color dye, and a color difference ΔE between a point of phenol red alone and a point of a mixed system of {phenol red + single color dye}. The result of examining the relationship is shown.

FIG. 15 shows a magnitude ΔE of a color change of a mixed solution of phenol red and cyan (C), magenta (M), and yellow (Y) dyes at pH 5 to 10,
The results are shown on a ^* b ^* coordinates.

FIG. 16 shows the result of mixing BTB and cyan (C) dye in QxQy coordinates.

FIG. 17 shows the result of mixing BTB and magenta (M) dye in QxQy coordinates.

FIG. 18 shows a result of mixing BTB and a yellow (Y) dye in QxQy coordinates.

FIG. 19 shows the result of mixing BTB and cyan (C) + magenta (M) dyes in QxQy coordinates.

FIG. 20 shows the result of mixing BTB and cyan (C) + yellow (Y) dyes represented by QxQy coordinates.

FIG. 21 shows the result of mixing BTB and magenta (M) + yellow (Y) dyes in QxQy coordinates.

FIG. 22 shows phenol red and cyan (C).
The result of the mixing of the dye is represented by QxQy coordinates.

FIG. 23 shows a result of mixing phenol red and a magenta (M) dye in QxQy coordinates.

FIG. 24 shows the result of mixing phenol red and yellow (Y) dyes in QxQy coordinates.

FIG. 25 shows phenol red and cyan (C).
The result of mixing + magenta (M) dye is represented by QxQy coordinates.

FIG. 26 shows phenol red and cyan (C).
The result of mixing + yellow (Y) dye is represented by QxQy coordinates.

FIG. 27 shows the result of mixing phenol red and magenta (M) + yellow (Y) dyes in QxQy coordinates.

FIG. 28 shows a change in the chemical structure and a change in the absorption spectrum when two chemical species of a broton addition type and a de-broton type are mixed at various ratios to give a color according to the pH of BTB. It is a thing.

FIG. 29 shows the change in the chemical structure and the change in the absorption spectrum when two species of a broton addition type and a debrotonization type are mixed at various ratios to give a color according to the pH of phenol red. It is shown.

FIG. 30 shows the results of blotting the color change on the a ^* b ^* coordinates when the measurement was performed with variously changing the mixing ratio of BTB and DR1.

FIG. 31 is a graph showing complementary color changes QxQy obtained when measurement is performed with variously changing the mixing ratio of BTB and DR1.
It shows the result of blotting on coordinates.

FIG. 32 shows the results of blotting the color change when measuring the Na ⁺ concentration on the a ^* b ^* coordinates when the mixing ratio of KD-A5 and KD-S1 was variously changed. is there.

FIG. 33 shows the results of blotting the color change when measuring the Na ⁺ concentration on various QxQy coordinates of the complementary colors by changing the mixing ratio of KD-A5 and KD-S1 variously. .

FIG. 34 shows KD- at the time of measuring Li ⁺ concentration blotted on QxQy coordinates at pH7.
FIG. 9 shows the relationship between the calibration curve of M11 and the blot on the QxQy coordinates of KD-S1.

FIG. 35 shows the KD- at the time of measuring the Li ⁺ concentration blotted on the QxQy coordinates at pH9.
FIG. 9 shows the relationship between the calibration curve of M11 and the blot on the QxQy coordinates of KD-S1.

FIG. 36 shows the results of blotting the color change when measuring the Li ⁺ concentration when the mixing ratio of KD-M11 and KD-S1 was optimized at pH 9 on the complementary color QxQy coordinates. It is shown.

FIG. 37 shows pH 7.4 on sensing paper.
, The calibration curve of KD-M11 and the KD-S1 when the Li ⁺ concentration was blotted on the QxQy coordinates.
3 shows the relationship with the blot on the QxQy coordinate of the graph.

FIG. 38 shows the calibration curve of KD-M11 and the Q of KD-S1 when measuring Li ⁺ concentration blotted on QxQy coordinates at pH 8 on sensing paper.
It shows the relationship with the blot on the xQy coordinates.

FIG. 39 is a graph showing the change in color when the Li ⁺ concentration is measured when the mixing ratio of KD-M11 and KD-S1 is optimized at pH 8 on the sensing paper on the QxQy coordinate of the complementary color. It shows the result of blotting.

FIG. 40 is a diagram showing a color change (p
Utilizing the data of the ^{H8), 10 - 2 M~10 -4} M of L
It is a figure which shows the result of having plotted the change of the density | concentration of dye in the density range of i ⁺ .

FIG. 41 shows a result of simulating a color change at a dye density 3.34 times the dye density when the data shown in FIG. 40 was measured. Although the color change in the figure is originally a color, it is shown here as black and white.

FIG. 42 shows the relationship between the respective ion concentrations of Li ⁺ , Na ^+, and K ⁺ (horizontal axis) and the ratio of the color difference to the total color difference of the sensing paper (vertical axis).

FIG. 43 shows a change in color of the sensing paper with respect to each of Li ⁺ , Na ^+, and K ⁺ ions. In addition, although the color piece in the figure is originally a color,
Here, it is shown as black and white.

FIG. 44 shows a response profile of response time in a PVC membrane.

FIG. 45 shows a response profile of response time in sensing paper.

FIG. 46 shows LCD-2 and K in PVC film.
The response curve in the case of using each of D-M11 is shown.

FIG. 47 shows LCD-2 and K in a PVC film.
Shows a plot by each of ^a * ^{b *} color space coordinates of D-M11.

FIG. 48 shows LCD-2 and K in PVC film.
3 shows a plot of a ^* b ^* color space coordinates for the case where D-M11 was mixed.

FIG. 49 shows KD-M9 and K in PVC membrane.
The response curve when each of D-M13 is used is shown.

FIG. 50 shows KD-M9 and K in PVC membrane.
Shows a plot by each of ^a * ^{b *} color space coordinates of D-M13.

FIG. 51 shows a response curve when using KD-C4 in a PVC film.

FIG. 52 shows a ^* for the case where KD-M11 and KD-C4 in a PVC film are mixed (1: 1) ^.
b ^* shows a plot by the color space coordinates.

FIG. 53 shows a ^* for the case where KD-M11 and KD-C4 in a PVC membrane are mixed (3: 2) ^.
b ^* shows a plot by the color space coordinates.

FIG. 54 shows a response curve to ammonium ions at pH 6 in a PVC membrane using TD19C6 as an ionophore and a mixed dye of a fat-soluble cationic dye KD-M11 and KD-C4 as a dye.

FIG. 55 shows a plot of a ^* b ^* color space coordinates for ammonium ions using a mixed dye of KD-M11 and KD-C4 in a PVC film.

FIG. 56 shows a response curve to lithium ions when TTD14C4 is used as the ionophore and fat-soluble cationic dyes KD-M11 and KD-C4 are used as the ionophore at pH 6 in the PVC membrane.

FIG. 57 shows Li ⁺ , Na ^+, and K ^{+ at} pH 6 in a PVC membrane when TTD14C4 is used as the ionophore and a mixed dye of the lipid-soluble cationic dyes KD-M11 and KD-C4 is used as the dye. The response curve is shown.

FIG. 58 shows a plot using a ^* b ^* color space coordinates for lithium ions when using a mixed dye of KD-M11 and KD-C4 in a PVC film.

FIG. 59 shows TTD14C4 as an ionophore and a lipophilic cationic dye KD- as a dye in a PVC membrane.
3 shows response curves at pH 6 and pH 7 for lithium ions when a mixed dye of M11 and KD-C4 is used.

FIG. 60 shows a result of blotting on QxQy coordinates based on a color change measured by a digital color analyzer in a lithium ion sensing paper.

Continued on the front page F term (reference) 2G020 AA08 BA02 BA03 BA14 CD33 CD34 CD36 2G042 AA01 BB02 DA08 FA11 HA10 2G054 CA10 CE01 FA27 JA01 JA04 JA07 JA20

Claims (18)

[Claims] 1. A method of quantifying an optode color change, plotting the optode color change in a chromaticity coordinate system, and optimizing the optode color change in the coordinate system. 2. The optimization method according to claim 1, wherein the change in the color of the optode in the coordinate system is based on a relationship with a gray point in the coordinate system. 3. The chromaticity coordinate system includes xy chromaticity coordinates, a ^* b ^*
3. The method according to claim 1, wherein the chromaticity coordinates are complementary chromaticity coordinates QxQy. 4. A gray color inside a triangle formed by three points, one end and the other end when the change in color of the optode is represented by the chromaticity coordinates QxQy of the complementary color, and the position of the coordinates of the shielding indicator. How to select optode shielding indicators to get points. 5. The position at the chromaticity coordinate QxQy of the complementary color of the shielding indicator is two of the position of the chromaticity coordinate QxQy of the complementary color at the detection point of the measurement chemical species of the optode and the gray point.
A method for selecting a shielding indicator, which comprises selecting the shielding indicator to be on a straight line formed from points. 6. The method according to claim 1, wherein the optode is an indicator of hydrogen ion concentration or metal ion concentration. 7. The method according to claim 1, wherein the optode comprises an ionophore and a dye. 8. An indicator composition comprising an optode and a shielding indicator is fixed to a support.
The method according to any of the above. 9. An indicator composition comprising an optode and a shielding indicator selected by the method of claim 4 or 5. 10. The composition according to claim 9, wherein optode is an indicator of metal ion concentration. 11. The composition according to claim 9, wherein an indicator composition comprising an optode and a shielding indicator is fixed to a support. 12. A shielding indicator for an optode selected by the method according to claim 4. Description: 13. A method for measuring a target chemical species using the indicator composition according to any one of claims 9 to 11. 14. The method according to claim 13, wherein the target chemical species is a metal ion. 15. A material for measuring a lithium ion concentration in a specimen, comprising an optode and a shielding indicator so that the lithium ion concentration in the specimen is gray when the concentration is within an appropriate concentration range. 16. The optode change in the color of the optode due to the change in the concentration of the measured chemical species such that the optode draws a circle centered on the gray point of each coordinate in the xy chromaticity coordinates or a ^* b ^* chromaticity coordinates. How to choose a color change. 17. The method according to claim 16, wherein the selection of the color change of the optode is by the choice of a dye incorporated in the optode. 18. An optode, selected by the method according to claim 16 or 17, which can change color vividly over a wide concentration range of the measured species.