JPH01280242A - Instrument and method of measuring concentration of oxygen - Google Patents

Abstract

PURPOSE:To enhance the affinity to the fluid of a vital system and to enable exact measurement by coating a high-polymer film contg. a specific light emitting material with a silk film. CONSTITUTION:The high-polymer film 3 contg. the light emitting material is coated by the silk film 4, by which the measuring instrument 1 is constituted. The light emitting material is at least one kind selected from the group consisting of tris(2,2'-bipyridine)ruthenium (II) complex, tris(o-phenanthroline) ruthenin (II) complex, pyrene, and pyrene deriv. The optical excitation wavelength may be UV light or visible light and the visible light is more preferable. The silk film 4 is obtd. by spreading an aq. soln. of silk fibroin on, for example, a glass or plastic plate and air drying the same. The high-polymer film is a high-polymer film excluding silk. The concn. of oxygen is obtd. by fixing the measuring instrument 1 to the front end of a conductor of light and measuring the emission intensity of the light emitting material after the instrument is irradiated by light.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention utilizes the phenomenon in which light emission (photoluminescence) generated from the photoexcited state of a light-emitting substance is quenched by oxygen, and is applied to water, blood, and various solutions. Alternatively, the present invention relates to an oxygen concentration measuring device used for optically measuring the oxygen concentration in a medium such as a gas, and an oxygen concentration measuring method using the same.

[Conventional technology]

Conventionally, a so-called electrochemical method using an oxygen electrode has been used to measure oxygen concentration. However, such electrochemical methods have many technical hurdles, such as slow response coupling, being susceptible to external electromagnetic noise, and difficulty in miniaturization. Therefore, an oxygen concentration measurement method was devised that utilizes the phenomenon in which oxygen quenches luminescence such as fluorescence and phosphorescence.

For example, pyrenebutyric acid has been shown to be effective as a luminescent substance for oxygen concentration measurements (W, M, Bogan (V).
aughan), G. Weber,
Biochemistry (Biochem), 9,464
(1970)), research on luminescent materials using pyrene derivatives has been active. However, this method requires the use of ultraviolet light as the excitation light, and is subject to limitations such as the need to use a material that transmits ultraviolet light in the optical system for guiding the excitation light.

Further, PCT/JP87100463 discloses an oxygen concentration measuring device comprising an immobilized polypyridine metal complex. Here, a polypyridine gold R complex is dispersed in a polymer or fixed by being adsorbed on an adsorbent, and a technique for using a polypyridine complex by immobilizing it on a visible light conductor is also disclosed.

[Problem to be solved by the invention]

In this way, various oxygen concentration measuring devices that utilize oxygen quenching have been developed, but when a biological sample such as blood is used as the test liquid, the affinity between the material of the membrane that fixes the luminescent material and the test liquid is It was difficult to make accurate measurements because of the low

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide an oxygen concentration measuring device that is particularly suitable for measuring oxygen concentration in biological fluids and has a high affinity with biological fluids, and an oxygen concentration measuring method using the same. There is a particular thing.

[Means to solve the problem]

The present invention relates to an oxygen concentration measuring device comprising a thin film containing a luminescent substance.

The present invention also relates to an oxygen concentration measuring device formed by covering a polymer film containing a luminescent substance with a thin film.

Further, the present invention provides that the above-mentioned luminescent substance is tris (2゜2''-
The above oxygen concentration measuring device is characterized in that the oxygen concentration measuring device is at least one selected from the group consisting of bipyridine) ruthenium (I[) complex, one tris(o-phenanthroline) ruthenium (II), pyrene, and a pyrene transparent conductor. Regarding.

The present invention also relates to an oxygen concentration measuring device with a light guide, in which the above oxygen concentration measuring device is fixed to the tip of a light guide.

The present invention also relates to an oxygen concentration measuring method characterized in that the above oxygen concentration measuring device or the oxygen concentration measuring device with a photoconductor is irradiated with light to measure the luminous intensity of a luminescent substance.

The present invention will be explained in more detail below.

I> Luminescent substance As the luminescent substance used in the present invention, any luminescent substance that emits luminescence from a photoexcited state upon irradiation with light and whose light is quenched by oxygen can be physically contained in a polymer film.
Chemically bonded substances are used. Here, a luminescent substance can be bonded to a luminescent substance or a polymeric substance pendant, or fine particles (luminescent substance) can be used in a gel. Specifically, PCT/JP871
Polypyridine metal complexes disclosed in No. 00463, such as tris(2,2'-bipyridine)ruthenium (
II) complexes, tris(o-phenanthroline)ruthenium(n) complexes, pyrene and pyrene derivatives, and the like.

The optical excitation wavelength may be ultraviolet light or visible light, but visible light is preferred. For example, tris(2,2°-bipyridine)ruthenium(II) complex has a long excitation wavelength and a maximum absorption wavelength λma
x is 452 nm) and the emission wavelength of luminescence (maximum emission wavelength Emax is 603 nm), both of which are in the visible region, and since luminescence is efficiently quenched by oxygen, it is used as a luminescent substance. Furthermore, although pyrene and its derivatives have an excitation wavelength of around 340 nm, which is in the ultraviolet region, their luminescence is around 400 nm, which is close to the visible region, and is efficiently quenched by oxygen, so they are still excellent as luminescent substances.

11) Silk membrane The main feature of the present invention is that silk, which has excellent biocompatibility, is used as a membrane component on the outer surface of the oxygen concentration measuring device that comes into contact with the test liquid.

Silk, a natural product, is known as an excellent material that is compatible with living organisms, and is used for sutures, cosmetics, etc., and is also being considered for use in contact lenses, etc.

The silk membrane used in the present invention can be prepared from an aqueous solution of silk fibroin by a casting method, for example, by spreading the aqueous silk fibroin solution on a glass or plastic plate and air drying.

The concentration of the silk fibroin aqueous solution used at this time is 0.1~
It is preferably 30% by weight, and the thickness of the silk film is 0.1
The density of ~500 μm1 silk membrane is 1-1.5 g/cu
It is preferable that it is t.

To directly immobilize a luminescent substance into a silk membrane, the luminescent substance may be mixed into an aqueous solution of silk fibroin and then the silk membrane prepared, or the luminescent substance may be physically adsorbed onto the prepared silk membrane.

iii) Polymer film In the present invention, the term "polymer film" refers to any polymer film made of a polymer compound other than silk, which is known to be usable for fixing a luminescent substance in an oxygen concentration measuring device. Contains polymer membranes.

In the present invention, the luminescent substance may be directly immobilized in the silk film, but in that case, the quenching efficiency of the luminescent substance by oxygen is low. Therefore, a luminescent substance is dispersed and fixed in a polymer film, and
The polymer membrane may be covered with a silk membrane to form an oxygen concentration measuring device.

In order to manufacture such an oxygen concentration measuring device, a polymer film on which a luminescent substance is immobilized is bonded to a silk film, or an aqueous solution of silk fibroin is cast onto the polymer film to form a film, or the polymer film is bonded to a silk film. The membrane may be sandwiched between silk membranes so that the surface that comes into contact with the test liquid is the silk membrane.

The polymer may be either a synthetic product or a natural product, but one having a hydrophilic group is preferred. For example, methyl methacrylic acid,
Examples include N-vinylpyrrolidone, acrylic acid, vinyl alcohol, acrylamide, vinylpyridine, vinylbipyridine, hydroxyethyl methacrylate, styrene sulfonate, etc. alone or in copolymers with other monomers. In addition, Nafion, which is a cation conversion membrane having a sulfonate group, an anion conversion membrane having a quaternary ammonium group or a pyridinium group, and natural products such as cellulose, gelatin, and collagen can be used.

Various methods can be considered for immobilizing a luminescent substance onto a polymer membrane. The conditions for immobilization are: (1) the immobilized luminescent substance should not elute into the test solution (if it elutes, the intensity of luminescence will change, making stable measurement difficult);
(2) It must not undergo irreversible changes due to chemical reactions with the test liquid or gas.

First of all, as a first method, the luminescent substance can be most easily immobilized by dissolving or dispersing it in a polymer.

In this case, many polymers can be used. For example, many general-purpose plastics used in boat fishing,
For example, low density polyethylene, polypropylene, polyvinyl chloride, ethylene vinyl acetate copolymer, polystyrene,
Polymethyl methacrylate, silicone resins, polyurethanes, and the like are particularly suitable for use. The specific means for mixing the luminescent substance with these polymers is appropriately selected depending on the luminescent probe and polymer used. For example, when polypyridine metal complexes are used in luminescent probes, thermoplastics with high solvent resistance such as low-density polyethylene may be mixed by heating and melting, and polystyrene may be mixed by solution mixing (polymer in an organic solvent solution). mixture) can be used.

The concentration of the polypyridine metal complex in the mixture with the polymer is I x 10-"mo j1!/dm' to 1
mo j2 /dm' is suitable. IX 10-8
Mo! ! If the concentration is less than 1mo j!/dm, sufficient luminescence cannot be obtained and therefore the sensitivity is insufficient.
At a high concentration exceeding dm3, the intensity of emitted light does not change depending on the oxygen concentration and is not suitable for use.

Next, as a second method, there is a method in which a luminescent substance is chemically or physically adsorbed onto an adsorbent. Examples of the adsorbent include inorganic substances such as silica gel and glass, organic substances such as porous polymers, various ion exchange resins, natural products such as polysaccharides, and proteins. Any adsorbent can be used. In particular, more stable immobilization can be achieved by using a cation exchange resin or a chelate type adsorbent.

A third method is to form an immobilized complex that is physically and chemically more stable by introducing a luminescent substance as a constituent unit of a polymer. This method requires a slightly more complicated operation than the two methods described above, but it is a method in which the most stable immobilized product is selected. For example, when using a polypyridine metal complex, polymerizable functional groups are A method of polymerizing polypyridine or copolymerizing it with a copolymerizable monomer to obtain a polymer or copolymer, and then forming a metal complex, such as styrene, methacrylic acid, acrylic acid, Vinyl compounds such as acrylonitrile and 4-methyl-4'-vinyl-2,2'-
A method of forming a metal complex after copolymerizing polypyridine (ligand) having a vinyl group such as bipyridine,
A method may be adopted in which a monomer having a functional group capable of chemically bonding with a substituent of the polypyridine metal complex is prepolymerized, and then the substituent and the functional group are bonded. Here, the copolymer includes a random copolymer, a block copolymer, a graft copolymer, or a polymer crosslinked with a complex.

An example of a polymer to which a luminescent substance is chemically covalently bonded is tris(2,2'-bipyridineruthenium(II)).
The following polymers have a complex (hereinafter abbreviated as Ru(bpy)s''+) as a pendant substituent.

However, X- is an anion such as Cβ-1Cβ04-, M is 4
-Methyl-4'-vinyl-2,2'-bipyridine, methyl meccrylic acid, acrylic acid, N-vinylpyrrolidone,
Monomer units such as hydroxyethyl methacrylate and styrene, XS7%2 is the molar fraction of repeating units, x+
y+z=1, and xSy is 0 to 0.99.2.
Selected from the range 01-1.

It is also possible to combine a plurality of the above methods.

For example, it is also possible to mix the polymer complex with other polymers.

iv) Light Guide As the light guide, it is preferable to use, for example, a flexible plastic optical fiber.

■) Oxygen Concentration Measuring Device and Oxygen Concentration Measuring Method In the present invention, since the interstitial membrane is highly permeable to media such as water, oxygen dissolved in the test liquid passes through the interstitial membrane and reaches the luminous body. Since it emits light corresponding to the oxygen concentration, the oxygen concentration can be determined from the luminescence intensity. Furthermore, since gas also permeates through the intermembrane, it is also possible to measure the oxygen concentration in the gas phase.

Usually, light having a wavelength at or near the absorption maximum is irradiated to create an excited state of the light emitter, and the intensity of light emitted from this excited state is measured. In order to block the scattered light from the film, it is preferable to use a cutoff filter on the luminescence monitor side that blocks the excitation light wavelength range and transmits the luminescence.

Next, the oxygen concentration measuring device and oxygen concentration measuring method of the present invention will be explained using examples shown in FIGS. 1, 2, and 3.

FIG. 1 is a conceptual diagram showing an oxygen concentration measuring device 1 using a quartz plate as a base and its usage.

The oxygen concentration measuring device 1 has a three-layer structure including a quartz plate 2, a polymer film 3 on which a luminescent substance is fixed, and an interlayer film 4. This oxygen concentration measuring device 1 is immersed in a test liquid or test gas, irradiation light 5 is applied from the outside, and the intensity of the obtained luminescence 6 is measured with a monitor (e.g., Photomaru) 7, and the test liquid or test gas is measured. Measures the oxygen concentration of gas.

FIG. 2 shows an oxygen concentration measuring device with a photoguide of the present invention,
Figure 3 shows its usage.

A polymer film 3 on which a luminescent substance is fixed and a membrane 4 covering it are fixed to the tip of the optical fiber 8, and the silk film portion is immersed in a test liquid 18 or a test gas. The irradiation light emitted from the xenon lamp 9 is passed through an interference filter 10.
The light passes through the dichroic mirror 11 and reaches the polymer film 3 via the optical fiber 8, bringing the luminescent substance into a photoexcited state. Luminescence emitted from the luminescent substance passes through the optical fiber 8 and reaches the optical fiber coupler 12 and the dichroic mirror 11.
There, it is guided to the monitor system, passes through a monochromator 13, a photomultiplier tube 14, a preamplifier 15, and a recorder 1.
6, the luminescence intensity is recorded.

〔Effect of the invention〕

By using the oxygen concentration measuring device and oxygen concentration measuring method of the present invention, it is possible to easily measure the acid sa level in liquids of biological systems, which has been difficult to measure in the past. Further, the acid sa level measuring device of the present invention is suitable for measuring oxygen concentration in a minute site, and can also measure oxygen concentration in gas.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example I In a 1QmM aqueous solution of Ru (bpy) s”CR2
30 × 3 cm Nafion membrane (thickness 173 μm)
After soaking for a minute to adsorb the complex into the membrane, it was washed with water, dried, and fixed on a Teflon plate. A silk fibroin membrane (thickness: approximately 100 μm) was adhered thereon to form a two-layer membrane type oxygen concentration measuring device. This was immersed in water with various dissolved oxygen concentrations, and 4.50 nm was measured at a 45 degree angle to the membrane.
The emission spectrum was measured through a 0-56 cut-off filter, and the intensity of the emission maximum (about 600 nm) was investigated. The relationship between oxygen concentration and luminescence intensity was obtained as shown in . From this, the oxygen concentration can be determined by measuring the luminescence intensity.

Example 2 In structural formula 1, copolymer component M is methyl methacrylate, X is 0.943, y is 0.020.2 is 0.03
7. An acetone solution of a polymer pendant type luminescent probe in which X- is CI- is cast onto a silk fibroin film and dried to form a film. A silk fibroin aqueous solution was cast onto this polymer membrane to form a third membrane, thereby creating an oxygen concentration measuring device having a structure in which the polymer membrane was sandwiched between the silk fibroin membranes.

When this is fixed to a frame and the relationship between the oxygen concentration in water and the luminescence intensity is measured in the same manner as in Example 1, the result is as shown in FIG. 5, and from this, the oxygen concentration can be determined from the luminescence intensity.

Example 3 Same as Example 2, M is N-vinylpyrrolidone, X is 0, 828, y as a polymer pendant type luminescent probe.
The relationship between the oxygen concentration in water and the luminescence intensity was measured in the same manner as in Example 2, except that a compound with 0.150.2 and 0.022 was used, and a linear relationship was obtained.

Example 4 In Example 1, when the relationship between oxygen partial pressure and relative luminescence intensity was determined in oxygen/argon mixed gases having different oxygen partial pressures, a linear relationship was obtained.

Example 5 A double-layer membrane type oxygen concentration measuring device was manufactured in the same manner as in Example 11 except that nihilene butyric acid was used instead of Ru(bpy) 3''Cβ2, and the oxygen concentration in water and the relative luminescence intensity were measured. When finding the relationship, we used 338 nm light as the excitation light, L-39 as the cutoff filter, and 400 nm as the monitor wavelength, and a similar linear relationship was obtained with i, etc.

Performance Example 6 In Example 2, a physiological saline solution (9% sodium chloride aqueous solution) was used to measure the relationship between the oxygen concentration and luminescence intensity in this aqueous solution, and results similar to those obtained in Example 2 were obtained.

[Brief explanation of the drawing]

FIG. 1 is a conceptual diagram of an example of the oxygen concentration measuring device of the present invention and its usage. FIG. 2 is a conceptual diagram of an example of the oxygen concentration measuring device with a photoguide according to the present invention. FIG. 3 is a conceptual diagram of how to use the oxygen concentration measuring device with a photoguide of the present invention shown in FIG. 2. FIG. 4 and FIG. 5 show the correlation between luminescence intensity and oxygen concentration measured using various oxygen concentration measuring devices of the present invention. 1... Oxygen concentration measuring device 2... Quartz plate 3... Polymer membrane 4... Silk film 5... Irradiation light 6. ...Luminescence 7 ...Monitor 8 ...Optical fiber 9 ...Xenon lamp 10 ...Interference filter 11 ...Two colors Mirror 12... Optical fiber coupler 13...
- Monochromator 14...Photomultiplier tube 15...Preamplifier 16...Recorder 17...High voltage power supply 18...Test liquid 1st Figure 2 Figure 6

Claims (5)

[Claims] (1) An oxygen concentration measuring device consisting of a silk film containing a luminescent substance. (2) An oxygen concentration measuring device formed by covering a polymer film containing a luminescent substance with a silk film. (3) A claim in which the luminescent substance is at least one member selected from the group consisting of tris(2,2'-bipyridineruthenium(II) complex, tris(o-phenanthroline)ruthenium(II) complex, pyrene, and pyrene derivatives) The oxygen concentration measuring device according to item (1) or (2). (4) An oxygen concentration measuring device with a light guide, comprising the oxygen concentration measuring device according to any one of claims (1) to (3) fixed to the tip of a light conductor. (5) The oxygen concentration measuring device or the oxygen concentration measuring device with a photoconductor according to any one of claims (1) to (4) is irradiated with light to measure the luminescence intensity of the luminescent substance. Oxygen concentration measurement method.