JP2001305060A - pH VALUE MEASURING ELEMENT AND SENSOR HAVING IT - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pH measuring element and a pH sensor for measuring a pH value over a wide range by combining a proton sensitive phosphor compound with a single polymer bondable to it. SOLUTION: This pH value measuring element can measure a pH value over a wide range by chemically bonding a proton sensitive phosphor substance having a carboxyl group and an organic polymer having an amino group together. This pH sensor is provided with a light source radiating excitation light, a base board transmitting the excitation light from the light source, a fluorescence detection part arranged on the base board and provided with the pH measuring element showing an increase in fluorescence intensity under the excitation light according to a pH value, and a signal processing part processing an output signal from the fluorescence detecting part so as to output a signal indicating the pH value.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pH value measuring element using a proton-sensitive fluorescent substance.
The present invention further provides a p-type device having such a pH-measuring element.
It also relates to the H sensor.

[0002]

2. Description of the Related Art As a method for measuring a pH value, a hydrogen electrode method, a glass electrode method and the like have been established as standard methods. On the other hand, since the late 1980's, with the progress of optical fibers and various new light sources, the development of "optical fiber fluorescent pH sensors" using proton-sensitive fluorescent compounds has been promoted. According to this method, an extremely thin sensor can be manufactured as compared with a hydrogen electrode or a glass electrode, and a p-type sample in a living body or a small amount of a sample can be obtained.
The measurement of the H value becomes possible. In addition, there are many advantages such as measurement from a long distance, no need for replenishment of the electrolytic solution, and susceptibility to electric and magnetic fields. This utilizes the "fluorescence quenching phenomenon" in which the fluorescence emitted by light excitation reduces the fluorescence intensity according to the concentration of existing protons. For example,
Analytical Chemistry 59, 437-439 (19
1987) discloses a method using phenol red as a fluorescent compound. Analytical Chemistry
61, pp. 174-177 (1989) discloses a method using 8-hydroxypyrene-1,3,6-trisulfonic acid as a fluorescent compound. U.S. Pat. No. 5,774,603 discloses a method using Nile Blue Fluorescein Oxazine 1, Bromocresol Green, or the like as a fluorescent compound.

[0003]

However, according to the conventionally proposed method, a combination of one fluorescent compound and one polymer for immobilizing the fluorescent compound can measure only a narrow range of pH value. Its use was very limited. For example, Analytical Chemistry 61 Vol. 174-1
On page 77 (1989), 8-hydroxypyrene-
A method for bonding 1,3,6-trisulfonic acid to an ethylene-vinyl acetate polymer is disclosed. But,
In this method, the measurable pH value was 5.5 to 8.0. In addition, in International Publication WO 92/15962,
Combination of fluorescein and silica glass, pH
A method for measuring the range from 3.5 to 6.5 is disclosed. U.S. Pat. No. 5,774,603 discloses a method in which Nile Blue, Fluorescein, Oxazine 1, Bromocresol Green, or the like is used as a fluorescent compound and immobilized with Zr / Si sol-gel glass. In this case, measurement in the range of pH 12 to 13 is possible.

As a method for obtaining a wide pH range, a combination of a plurality of fluorescent substances can be considered, and there are several literatures on this method. However, according to these methods, the immobilization of a plurality of fluorescent substances itself becomes a technically difficult problem, for example, each fluorescent substance requires a different polymer. Furthermore, since each fluorescent substance is excited by light of a different wavelength, and the fluorescent light emitted from each has a different wavelength, there is a disadvantage that the control unit of the sensor becomes extremely complicated.

Accordingly, an object of the present invention is to provide a pH-measuring element capable of measuring a wider range of pH values by changing the form of chemical bonding between a proton-sensitive fluorescent compound and an immobilized substance, and a pH-measuring element comprising the same. It is to provide a sensor.

[0006]

The pH-measuring element according to the present invention provides a pH-measuring element for a wide range of pH values by chemically bonding at least a part of the carboxyl group of at least one proton-sensitive fluorescent substance to an immobilizing substance. It is characterized in that measurement is enabled.

Hitherto, in proton-sensitive fluorescent substances, it has been considered that carboxyl groups contribute to the fluorescence quenching phenomenon. Therefore, when the fluorescence quenching phenomenon was desired to be used, there was one that was immobilized using an alkyl group other than a carboxyl group, but there was no one that used a carboxyl group. The novel point of the present invention is that the carboxyl group is chemically bonded to the immobilizing substance, focusing on the fluorescence quenching phenomenon caused by the carbonyl group and the hydroxyl group after the carboxyl group is reacted.

In order to immobilize a plurality of types of fluorescent substances, a plurality of types of immobilizing substances are required. Typical proton-sensitive fluorescent substances (for example, uranine and o-coumaric acid) are usually used. According to the present invention, because of having a carboxyl group, a plurality of fluorescent substances can be combined with one immobilizing substance. Therefore, according to the present invention, it is also possible to obtain a substance having both of two fluorescence intensity change characteristics by mixing a plurality of fluorescent materials having different fluorescence intensity change characteristics and binding the same to an immobilization material. It is. For example, a substance having a desired fluorescence intensity change characteristic can be obtained by mixing a fluorescent substance whose fluorescence intensity changes mainly in an acidic state and a fluorescent substance whose fluorescence intensity changes in an alkaline state and adjusting the ratio thereof. For example, a substance whose fluorescence intensity changes over a wide range from acidic to alkaline can be obtained. Alternatively, a substance having high sensitivity on the acidic side or the alkaline side can be obtained.

Further, a pH sensor according to the present invention includes a light source that emits excitation light, a substrate that transmits the excitation light from the light source, and at least one type of proton provided under the excitation light under the excitation light. At least a part of the carboxyl group of the sensitive fluorescent substance is chemically bonded to the immobilization substance, and under the excitation light, a fluorescence detection unit including a pH value measuring element showing a change in fluorescence intensity according to the pH value, A signal processing unit that processes the output signal from the fluorescence detection unit and outputs a signal indicating a pH value.

The light intensity of the fluorescent light is very weak. If light enters from outside, it causes disturbance noise, which causes a measurement error and makes the measurement unstable, so that it is necessary to block external light. Therefore, it is necessary to measure the pH value in a dark room in which external light is blocked, or to provide a layer for blocking external light in the fluorescence detector. However, measurement in a dark room is inconvenient due to its limited use, so the fluorescent detector is provided with a fluorescent substance immobilized thereon and provided on a pH value measuring element that generates fluorescence. It is preferable to provide a fluorescence reflection external light blocking layer that reflects emitted fluorescent light, blocks external light, and transmits a test substance. Further, the fluorescence reflection external light blocking layer also has a function as a protective layer. As the fluorescence reflection external light blocking layer, a material obtained by applying a substance having light-shielding properties and chemical resistance (for example, metal titanium or the like) to a porous polymer having hydrophilicity or the like is suitable.

The upper part of FIG. 1 shows the principle of operation of the pH value measuring element according to the present invention utilizing the fluorescence quenching phenomenon. As shown at the top of FIG. 1, for example, a typical proton-sensitive fluorescent compound, fluorescein sodium (uranin)
When there are many protons, that is, when the pH is low (acid side), the dissociation of the protons of the carboxyl group and the hydroxyl group is small, and the emission of fluorescence is small even when irradiated with excitation light. On the other hand, when the presence of protons is small, that is, when the number of hydroxyl groups is large and the pH is high (alkali side), the hydroxyl groups are attacked and the protons are dissociated in a large amount. Thus, the higher the pH (ie, the more protons are dissociated), the greater the fluorescence intensity. Therefore, as shown in the lower graph of FIG. 1, if a calibration curve of the fluorescence intensity ratio at an arbitrary wavelength with respect to pH is prepared in advance, the unknown proton concentration (ie, pH) can be evaluated from the measurement of the fluorescence intensity. Such a region where the fluorescence intensity changes depends on the pKa of the fluorescent substance. The reaction mechanism described above is a simplification, and the actual reaction mechanism has a more complicated mechanism. The details of this reaction mechanism will be described later.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First, an aqueous uranine solution (concentration of 0.
The fluorescence intensity of 1 mmol / l) was measured at an excitation wavelength of l _ex = 485 nm and a fluorescence emission wavelength of l _em = 510 nm in the range of pH = 2 to pH = 14. FIG. 2 is a graph showing the measurement results of the fluorescence intensity of the uranine aqueous solution, in which the vertical axis represents the fluorescence intensity at each pH as a fluorescence intensity ratio based on the maximum fluorescence intensity, and the horizontal axis represents pH. As shown in FIG. 2, in this uranine aqueous solution, the fluorescence intensity ratio greatly changed between pH = 4 and pH = 7, and became approximately constant before and after these. That is,
As described above, if only one fluorescent compound is used, the response region to pH is as narrow as 4 to 7, and it is difficult to extend this change region to a wide pH region.

A typical example of a method for manufacturing a pH measuring element according to the present invention will be described below. Nylon 6 film is cut into 10 cm x 6 cm and placed in a 100 ml Erlenmeyer flask. This flask is charged with 10 ml of 10 ^-2 mol / l HCl, and further diluted with 50 ml of ion-exchanged water. Nylon 6
1 g of water-soluble carbodiimide is added to the solution containing the film, and 0.083 g of uranine is further added. The solution is reacted for 9 hours with stirring while shielding the solution from light with aluminum foil while maintaining a constant temperature of 25 ° C., so that the carboxyl group of uranine and the amino group of nylon 6 are selectively amide-bonded. Nylon 6 film with uranine bonded to ion-exchanged water 10
Wash 10 times in total with 0 ml for a total of 5 hours. The film thus produced is stored in ion-exchanged water,
An element is cut out to a diameter of 1 cm.

FIG. 3 is a diagram showing the structure of the fluorescence detecting section 10 according to the present invention. As shown in FIG. 3, the fluorescence detecting unit was attached to the pH measuring element 14 at the center of a 3 cm × 4 cm transparent substrate 12 manufactured using an acrylic plate.
The fluorescent detection unit 10 is formed. This substrate 12
It is preferable to use a material that transmits excitation light and generated fluorescence, for example, acrylic, optical glass, or the like.

FIG. 4 is a view showing a modification of the structure of the fluorescence detecting section 10. As shown in FIG. A pH measuring element 14 is provided at the center of the substrate 12.
FIG. 3 is a view showing a configuration in which a fluorescence reflection external light blocking layer 16 is further provided on the pH value measuring element 14 to block external light, thereby forming a fluorescence detection unit 10. In this embodiment, the fluorescence reflection external light blocking layer 16 is formed by applying a material in which metal titanium powder having light-shielding properties and chemical resistance is dispersed in a porous and hydrophilic polymer.

FIG. 5 is a schematic diagram of a pH sensor according to the present invention. The above-described three-layered fluorescence detector 10 is housed at the tip of the probe holder 20. A light guide rod LGR having one end abutting on the back surface of the substrate 12 is provided inside the probe holder 20 having the fluorescence detector 10 housed at the tip.
And a pair of optical fibers 22 and 24 are extended so that one end of the optical fiber 22 abuts on the other end, and the other end (incident side) of one optical fiber 22 is connected to the light source via the incident side band-pass filter 26. And the light emitting diode 30 which generates the light. The other end (outgoing side) of the other optical fiber 24 is opposed to the photodiode 32 via the outgoing side bandpass filter 28. Incident side bandpass filter 2
On the emission side of 6, an excitation light compensation photodiode 34 is provided at a position off the optical axis, and its output is fed back to an arithmetic and control circuit 40 for driving the light emitting diode 30. A reference signal is supplied from the central processing unit (CPU) 50 to the arithmetic / control circuit 40, and the drive current of the light emitting diode 30 is controlled based on the difference between the reference signal and the signal from the photodiode 34, and is always constant. The excitation light having the luminance of is emitted.

After the output signal of the photodiode 32 is amplified by the preamplifier 60, the sample / hold circuit 62
, And obtains the sampled value.
After further amplification by the amplifier 4 and the amplifier 66 on the instrument body side,
The signal is converted into a digital signal by the A / D converter 68 and
Supply 0. The CPU 50 can calculate the pH value by processing the output signal of the photodiode 32 thus supplied.

A temperature sensor 70 is also provided at the tip of the probe holder 20, and its output signal is amplified by an amplifier 72 and sent to the main body of the instrument, converted into a digital signal by an A / D converter 74, and supplied to the CPU 50. I do. The correlation between the pH value and the temperature is determined in advance, and when the pH value is calculated by the CPU 50, the correction based on the temperature detected by the temperature sensor 70 is performed, so that the measurement accuracy can be improved.

The signal representing the pH value obtained as described above is displayed on a liquid crystal display 76 connected to the CPU 50, converted into an analog signal by a D / A converter 78 and output, and output from an RS-232C interface. It is output as a digital signal via 80.

Example 1 The fluorescence detector 10 thus prepared was mounted on a fluorescence spectrophotometer (JASCO FP750) and irradiated with excitation light (λ _ex = 485 nm) to measure the intensity of the emitted fluorescence (λ _em = 530 nm). . FIG. 6 is a graph showing the measurement results of the fluorescence intensity of the element on which uranine is immobilized, in which the vertical axis represents the fluorescence intensity at each pH with respect to the maximum fluorescence intensity as a fluorescence intensity ratio, and the horizontal axis represents pH. It is. At this time, the pH was changed from 1 to 14, but when pH = 14, the maximum fluorescence intensity was obtained. The fluorescence intensity ratio at each pH was determined based on this maximum fluorescence intensity.
As a result, as shown in FIG. 6, it was found that the fluorescence intensity ratio changes continuously between pH = 1 and 14, that is, a wide pH response region can be obtained.

Here, the details of the reaction mechanism of the generation of fluorescence in the device on which uranine is immobilized will be described. In this device, the carboxyl group of uranine and the amino group at the chain end of nylon 6 are amide-bonded as described below. -NH2 + -COOH → -NH-CO-
The groups involved in the + H2O fluorescence quenching phenomenon are considered to be a hydroxyl group (-OH), a carbonyl group of an amide bond (-CO-), and a carboxyl group (-COOOH) remaining unreacted.
The continuous pH change of the fluorescence intensity is considered to be due to the following mechanism. On the acidic side (pH 3.5 or less), there are many protons of the test substance, so the protons hardly dissociate from the OH group of uranine, but the protons are added to the carbonyl group of the amide bond, and this portion is fluorescence quenched. Show the phenomenon. It is considered that such proton addition greatly contributes to a continuous change in fluorescence intensity at pH 3.5 or less. At neutral (pH 3.5 to pH 12), the proton sensitivity of uranine dissociates the proton of the OH group of uranine according to the proton concentration of the test substance, and this is thought to contribute significantly to the change in fluorescence intensity. Can be On the other hand, on the alkaline side, since the amount of protons in the test substance is small, free hydroxyl groups are attacked, protons are easily dissociated from OH groups of uranine, and the fluorescence intensity is increased. Also, under strong alkalinity (pH 12 or more), a hydroxyl group is added to the carbonyl group of the amide bond, and it is considered that the addition of the hydroxyl group greatly contributes to a continuous change in the fluorescence intensity. That is, addition of a proton to a carbonyl group of an amide bond, dissociation of a proton from a hydroxyl group,
And addition of a hydroxyl group to the carbonyl group of the amide bond,
It is considered that the fluorescence intensity changes continuously over a wide range depending on one factor. When an unreacted carboxyl group remains, it is considered that protons are also dissociated from the remaining carboxyl group and contribute to a change in fluorescence intensity.

Embodiment 2 The following is an example in which two kinds of fluorescent substances having different characteristics of the change of the fluorescence intensity are combined with one immobilizing substance. Uranine (response area pH4 ~ 7) and o-coumaric acid (response area pH11 ~ 14)
Was chemically bonded to nylon 6 as an immobilizing substance. Using this, a device was prepared in the same manner as in Example 1, and the pH fluorescence intensity was measured under the same conditions. As shown in FIG. 7, a wide pH response region could be obtained from pH1 to pH14. The wavelength of the excitation light was set at 390 nm (square point) and 485 nm (triangle point) and measured.
At 485 nm (square point), a better pH response could be obtained.

The present invention is not limited to only the above-described embodiment, and many modifications and variations are possible. For example, in the above-described embodiment, uranine and o-coumaric acid are used as the proton-sensitive substances, but other proton-sensitive substances such as eosin can be used. Although nylon 6 is used as the immobilizing substance, other high molecular substances, low molecular substances such as aniline and amino acids, and inorganic substances such as glass can also be used.

[0024]

The advantages of the present invention can be summarized as follows. A pH sensor that utilizes the fluorescence quenching phenomenon,
The value can be measured. 2. The response (measureable on the order of milliseconds) is very good because it is an optical pH measurement. 3. An extremely thin element can be manufactured as compared with a hydrogen electrode or a glass electrode, and the sensor can be downsized. Further, a thin and easily bendable element can be manufactured. 4. By adjusting the residual ratio of carboxyl groups (or hydroxyl groups) or the mixing ratio of two kinds of fluorescent substances, a device having desired pH response characteristics can be manufactured. 5. Telemetry is possible. 6. There is no need to supply an electrolyte, and it is hardly affected by electric and magnetic fields. And the like.

Therefore, the pH value measuring element and its sensor can be applied to a wide range of applications. For example,
It is expected to be used for measuring the pH value of a living body or a small amount of a sample.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating the operating principle of a pH value measuring element using the fluorescence quenching phenomenon according to the present invention.

[Fig. 2] The vertical axis shows the pH values based on the maximum fluorescence intensity.
Is expressed as a fluorescence intensity ratio, and the horizontal axis represents pH.
FIG.

FIG. 3 is a cross-sectional view illustrating a structure of a fluorescence detection unit 10 according to the present invention.

FIG. 4 is a cross-sectional view showing a structure of a modified example of the fluorescence detector 10 according to the present invention.

FIG. 5 is a diagram schematically showing an overall configuration of a pH sensor according to the present invention.

FIG. 6 is a graph showing a measurement result of a fluorescence intensity of a device on which uranine is immobilized.

FIG. 7 is a graph showing the results of measurement of the fluorescence intensity of a device in which a mixture of uranine (response region pH 4 to 7) and o-coumaric acid (response region pH 11 to 14) was immobilized.

[Explanation of symbols]

10 fluorescence detector, 12 substrates, 14 pH measuring element,
16 Fluorescence reflection external light blocking layer, 20 probe holder, 2
2, 24 Optical fiber, 26 Incident-side bandpass filter, 28 Outgoing-side bandpass filter 28, 30 Light emitting diode, 32 photodiode, 34 Excitation light compensation photodiode, 40 Arithmetic / control circuit, 50 C
PU, 60 preamplifier, 62 sample and hold circuit, 64F amplifier, 66 amplifier, 70 temperature sensor,
72 amplifier, 74 A / D converter, 76 liquid crystal display, 78 D / A converter, 80 RS-232C interface

Continued on the front page (72) Inventor Kazuhiro Kobayashi 2-12-1 Ookayama, Meguro-ku, Tokyo Inside Tokyo Institute of Technology (72) Inventor Kozo Inoue 1-10-5 Iwamotocho, Chiyoda-ku, Tokyo Automatic System Co., Ltd. F-term in the research (reference) 2G043 AA03 CA03 EA01 GA08 GB01 GB21 HA01 HA05 JA03 KA05 LA01 MA01 NA11 2G054 AA02 AA06 AB07 CA21 CE02 EA03 FA32 GA04 GB02 JA01 JA04 JA05

Claims (9)

[Claims] 1. A pH value measuring element, wherein at least a part of a carboxyl group of at least one kind of proton-sensitive fluorescent substance is chemically bonded to an immobilizing substance. 2. The pH value measuring element according to claim 1, wherein the proton-sensitive fluorescent substance having a carboxyl group is a fluorescein-based compound. 3. The pH value measuring element according to claim 1, wherein the immobilizing substance is a compound having an amino group. 4. The pH value measuring device according to claim 3, wherein the compound having an amino group is an amide compound or an aramid compound. 5. The method according to claim 1, wherein the amide compound is nylon 6,6.
The pH value measuring element according to claim 4, wherein the pH value is 10 or 66. 6. A light source for emitting excitation light, a substrate transmitting the excitation light from the light source, and at least a part of at least one carboxyl group of at least one proton-sensitive fluorescent substance provided on the substrate. A fluorescent detector having a pH value measuring element showing a change in fluorescence intensity according to the pH value under the excitation light, and processing an output signal from the fluorescent detector under the excitation light to obtain a pH value. And a signal processing unit that outputs a signal indicating the following. 7. The method according to claim 7, wherein the fluorescence detecting section comprises:
And a fluorescent reflection external light blocking layer provided on the element, which reflects fluorescence emitted from the element, blocks external light, and transmits a test substance whose pH value is to be measured. The pH sensor according to claim 6, wherein: 8. An LED that emits an excitation light beam as the light source
The pH sensor according to claim 6 or 7, further comprising: 9. A band-pass filter having a pass band centered on the wavelength of the excitation light of the fluorescent substance, a photodiode for receiving the excitation light transmitted through the band-pass filter, and receiving an output of the photodiode for excitation. 9. The pH sensor according to claim 6, further comprising a light source driving amplifier for maintaining a constant light intensity.