JPH0324984B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is a device for determining the CO ₂ content in a sample, such as blood, comprising an indicator embedded in an aqueous solution within a polymer membrane and at least partially in contact with the sample. The present invention relates to a measuring device comprising a PH-dependent fluorescent dye and configured to measure the fluorescent light emitted by an indicator after excitation.

As is well known, the measurement of the partial pressure of CO ₂ (PCO ₂ ) in fluids such as blood or gases is based on PH
Can be changed to measurement. For this purpose, a reaction chamber is generally required, in which the PH- value determined by the respective PCO ₂ value of the measured object is detected: PH = pKs + logHCO ₃ /α·PCO ₂ . The objects to be measured and the reaction chamber are arranged in such a way that the PH value of the reaction chamber is not influenced by the actual PH value of the medium to be measured, and moreover, the CO ₂ is balanced between the object to be measured and the reaction chamber. It must be ensured that CO ₂ can be exchanged between the two.

Usually, this requirement is met in that the object to be measured and the reaction chamber are separated from each other by gas-permeable and proton-impermeable membranes (Teflon, silicone).

PCO ₂ -measuring systems of this kind, which are used for PH measurements in reaction chambers with glass electrodes, are already known as "Severinghaus electrodes" and are used in many fields, for example also for blood gas analysis. There is.

As is further known, this kind of PCO ₂ −
Detection of the PH value in the measuring system can also be carried out in a visual manner using PH-dependent fluorescent dyes. (PCO ₂ -Optical Test Material) In principle, any fluorescent molecule having a dissociable group can be used as an indicator. Such indicators are, for example: quinine, halcone, harmine, harmol, pyronine G, thiochrome, luminol, 1-naphthol-2-sulfonate, 1-naphthol-4-sulfonate, 2-naphthol-
6-sulfonate, umbelliferone, 4-methyl-umbelliferone, fluorescein, 1.
3-dihydroxypyrene-6,8-trisulfonate, 7-hydroxyflavone, 7-hydroxyisoflavone, orthocumaric acid, salicylaldehyde semicarbazone, 3-hydroxyxanthone, 3,6-dihydroxyxanthone, 7-hydroxyrapidone, 3 - Hydrocyacridone, methyl harmol iodide, 7-hydroxy-2-methylchromone In such PCO ₂ -optical specimens, the reaction chamber simultaneously becomes the indicator chamber. The flat reaction chamber, or indicator chamber, is bounded on one side by a gas-permeable, proton-impermeable membrane that borders the object to be measured, and on the other side by a light-transparent surface. I'm wearing it. Through its translucent surface, the intensity of the fluorescence can be measured by well-known methods (light source, monochromator, receiver). In this case, the intensity of the fluorescence is determined by the pH in the reaction chamber determined by the PCO ₂ in the object to be measured.
Becomes a function of value. Furthermore, as is also known, the indicator chamber can preferably be constructed of foil, in which the indicator is leaktightly enclosed. Such containment allows the foil itself to act as a separating membrane that simultaneously blocks the fluid.
and serves as an instruction chamber through which the light rays pass. The advantage of this configuration is the simple and robust construction of the entire measuring device.

The following is recognized as a decisive drawback of this configuration. Foil materials with conventional properties are polymerized. The following shows the difference between hydrophilic and hydrophobic polymers; if the foil is made from a hydrophilic support, CO ₂
- has no separability; the foil material allows protons to pass through; Furthermore, water-soluble indicator molecules may be washed away.

If the foil is made of a hydrophobic polymer, it is not possible to measure the PH value due to the absence of water within the foil.

Since the construction of a simple and durable PCO ₂ -measuring device remained a problem, a system in which the indicator chamber consists of an aqueous indicator solution separated from the analyte by a hydrophobic membrane was proposed. It existed warmly. The disadvantages of this system lie, on the one hand, in the difficulty of reproducibility of its manufacture, and on the other hand, in its mechanical stability. Variations in the layer thickness and non-uniform layer thickness distribution affect the optical properties and consequently the sensitivity of the measurement system. Changes in this property over time may occur if the system is exposed to mechanical or thermal loads, e.g. such loads are necessarily accompanied by use in automated analyzers (cleaning processes, suction pressure). However, this is particularly likely to occur in this case.

Another possibility for stabilizing PCO ₂ -optical test specimens is, in principle, to manufacture tiny capsules filled with an aqueous indicator solution and to place these capsules uniformly in a membrane material through which the light beam can pass. It may be distributed and embedded.

A method for manufacturing microcapsules in the nanometer (10 ^-9 m) range is described, for example, in German Publication DE-
Described in OS2360384. Aqueous solutions of active substances (eg indicators) and polymerizable molecules are dispersed in the hydrophobic fluid by stirring with the aid of surfactants. In this case, tiny drops of active substance and polymerizable molecules occur in the hydrophobic phase, the polymerization of which forms capsules in the nanometer range. So far, it has been attempted to produce separable (i.e. impermeable to protons) capsule wall materials for PCO ₂ -capsules (hereinafter capsules shall be understood as those filled with the corresponding indicator substance). This situation has been taken into account by embedding the capsule in a foil material that acts as a separating agent.

Further disadvantages arising in this connection are: The production of PCO ₂ -capsules of this type is subject to small changes during testing. During the manufacturing process and during subsequent processing of the capsules, irreversible agglomeration of the capsules is often observed, which makes uniform distribution in the support impossible.

Optimization of this last known method (preparation and embedding of PCO ₂ -capsules in polymer foils) does produce PCO ₂ -optical specimens that are robust in principle, but is a waste of money and time. However, there are disadvantages such as this and difficulties in reproducibility of the manufacturing method.

The problem of the present invention is to solve the problem of PCO ₂ − of the first mentioned type.
Improved measuring device, which eliminates the above-mentioned drawbacks of the known device and in particular allows optical test bodies (i.e. supports containing indicator) to be manufactured simply, at reasonable cost and reproducibly. It is to be.

This means that, according to the invention, an aqueous indicator solution in the form of a droplet with a diameter in the range from 0.1 to 100 μm is incorporated in a uniform distribution within a polymer membrane that allows gases to pass through but not protons. This is achieved by:

The configuration of the mechanically stabilized PCO ₂ -measuring device is such that micelles of an aqueous indicator solution (hereinafter the above-mentioned infusion will be referred to as such, following the nomenclature used in other contexts) are placed in a suitable proton cell. was successfully incorporated in a uniform distribution into the membrane material that encapsulates the

In the case of this type of production of PCO ₂ -optical specimens, certain demands had to be made on the polymers used and the initial mixture of the polymers.

a Requirements for the polymer: - It must be transparent in the spectral region of excitation and emission of the indicator.

- Must have separation properties, - Particularly permeable to gases such as CO ₂ and ions such as protons, oxonium ions,
It must be impermeable to hydronium ions or salts, and further impermeable to macromolecular components of the object to be measured.

b. Requirements for the initial mixture of polymers (an initial mixture can be understood to be one of two things, one of which is a reaction mixture, from which polymerization, polycondensation, polyaddition or network bonding or also vulcanization) The other is a polymer solution, in which case the polymer end product is produced by evaporating this solvent.All methods of converting an initial mixture into a polymer end product (hereinafter referred to as polymerization) -During the production it must be possible to form a stable emulsion of water in the polymer starting material. (The emulsion must be stable until the end of polymerization.) -The presence of water must not cause polymerization to be inhibited or delayed.

- The conditions required to produce the emulsion (e.g. stirring and associated heat generation) must not inhibit or cause polymerization.

- It must be possible to form the emulsion into a suitable form (foil, membrane) before polymerization. In principle, it is possible to block-polymerize an emulsion and then cut out flat optical specimens using a suitable method. Both methods suffer from the following disadvantages: 1. Some micelles are destroyed, resulting in an uneven surface which is disadvantageous for optical measurements.

2 The adhesion of the polymer foil to the transparent support, which can preferably be carried out for mechanical stabilization, represents an additional work step.

- The volatile polymer products that arise in any case must not affect the properties of the micelles containing the indicator. For example, the pH value of the micelles must not be changed.

Because of the requirements mentioned above, for example hydrophilic or opaque polymers cannot be used. Similarly, the polymer solution and polymer are separated and they are polymerized at high temperature or pressure.

Surprisingly, from among the group of silicon-based compounds, we were able to find an alternative that meets the above requirements and allows the indicator solution to be placed inside in the form of an emulsion and with uniform distribution.

Accordingly, according to a preferred embodiment of the invention, the polymer membrane is composed of silicone elastic rubber, in particular non-stick silicone.

Although silicone possesses desired properties such as proton impermeability and gas permeability transparency, applications for this polymer were not immediately noticed. Generally, vulcanization of silicones is either initiated or inhibited by water.

As a reactant used for example for the production of silicone types of this kind, products of the company Wurtzker Hemie GmBH of Berghausen, West Germany,
The product number is SLM40060, SLM40061,
The prepolymer, catalyst, and curing agent for SLM40062 are handled. The end product of these is a two-component elastic rubber crosslinked by an addition reaction, and the vulcanization reaction is 1
On the one hand, it is not stopped by the presence of water, and on the other hand, it proceeds under the influence of light energy of a certain wavelength. Under ideal conditions, the vulcanization is completed within seconds.

The production of a durable PCO ₂ -optical specimen or also a measuring device is determined by the properties of the selected polymeric material and is carried out in a simple and reproducible manner.

First of all, the required ingredients for the polymeric material are mixed. An emulsion of the indicator solution is then created within the initial mixture of polymers by adding an aqueous indicator solution and stirring. The emulsion is applied in a thin layer onto a smooth surface by painting, pouring, or any other suitable method.

In a preferred embodiment, at least approximately The membrane is made of a silicone polymer having an evenly distributed O ₂ -fluorescent indicator substance additionally attached in dissolved form to a PH-dependent fluorescent indicator drop; and that both indicators have different emission wavelengths. This very simple method allows you to
A device is created that allows the determination of O ₂ components as well as CO ₂ components. The reaction due to the presence of CO ₂ is then completely independent of the reaction due to the presence of O ₂ since both indicators are separated both spatially and chemically.

The prerequisite for the virtual independence of the sensing functions of O ₂ or also CO ₂ is that the emission wavelengths of the two used indicators have a sufficiently large difference;
However, the fluorescent lights can be separated individually by ordinary optical filters.

Regarding the additional incorporation of an indicator substance in dissolved form that fluoresces in response to O ₂ , the incorporation of the indicator substance in question into the silicone polymer can be achieved by adding an indicator substance which can be incorporated by customary methods. It must be mentioned that the concentration of the substance presented the difficulty of being too small and insufficient in terms of the strength of the fluorescent signal to be of sufficient use for the measuring technique. However, it has surprisingly been found that the indicator substance in question can be contained in silicone in solution in sufficiently high concentrations if the indicator substance in question is chemically denatured or solubilized.

By solubilization we mean here that the solubility of a substance in a solvent (including polymeric solvents) is increased by modification (chemical modification) of that substance.

The modification to the indicator substance is basically achieved following the Friedel-Crafts Alkyl substitution for aromatics, which is well known per se.

At the same time, it has become clear that the absorption properties are maintained over a wide range despite the increased solubility of the fluorophore if the following per se well-known procedures are carried out.

1 The indicator and tertiary butyl chloride are dissolved in a suitable solvent (CS ₂ ) and reacted under the catalysis of aluminum chloride.

2. The extraction step is followed by a washing and drying step; - Excess organic solvent is removed by rotary evaporation, resulting in an oily residue that can be used directly as a “solubilized indicator,” or; as described above, the indicator is Dissolved in excess tertiary butyl chloride without addition of other solvents.

In these ways it is possible to produce polymer mixtures or also prepolymer mixtures with an oxygen-sensitive indicator substance formed in the membrane, the concentration of indicator in this mixture being adjusted even in thin layers (e.g. 50 μ or less) is high enough to obtain sufficient signal strength in terms of measurement technology. The polymer mixture is further processed to obtain a thin film by conventional methods. These methods include painting, pouring, or other methods used to apply layers to the surface of the polymer. A further advantage of this process is that during the polymerization step thin films can be created on solid support materials with adhesive bonds.

Thin polymer membranes with an O ₂ indicator material of the type described above affixed to a support could be used for additional fluorescence measurements of oxygen in gas. At that time, it became clear that a special advantage of this measurement method was that the detection time from pure nitrogen to pure oxygen was within 0.15 seconds.

Although dissolution of the indicator within the polymeric support material is nearly sufficient to prevent loss of indicator to the surroundings, other methods are used to prevent material migration through the polymer for various purposes. There is an advantage.

This can be achieved, for example, by: a) limiting the migration of the indicator within the polymer by chemical modification of the indicator (alkylation with long-chain alkyl groups); b) using the present invention as an indicator material that emits fluorescence in response to the covalent bond O ₂ between the polymeric material and the indicator. In a preferred embodiment, a molecule of a polycyclic or heavy ring carbocyclic aromatic compound or a heterocyclic compound,
More preferably, a polycyclic or heavy ring aromatic hydrocarbon having a fluorescence extinction time of 5 nanoseconds or more is used.

In further advantageous embodiments of the invention,
Some transparent layered support materials have a polymer film vulcanized or polymerized on the smooth surface. The transparent support material may be, for example, glass or vinyl glass. The advantage is that the membrane sticks to the surface and does not come off. Its adhesion can still be increased using suitable adhesives. The transparent support material confines the optical specimen to the fluorometry apparatus, which serves a dual function, and also serves for the mechanical stability of the membrane.

In a further aspect of the invention, a carrier is provided for the micelles containing the indicator within the polymeric membrane material, the carrier having an indicator solution on its surface or impregnated with the indicator solution. There is. This reduces the possibility of deformation of the micelles under the action of external forces.

Such carriers, which have the same size as drops or even micelles, can have hydrophilic properties due to their composition or their composition (e.g. silica gel or polyacrylamide), which makes it possible to dry membranes and so-called optical specimens. prevent or at least delay. Moreover, rewetting the micelles is easy and quick.

It is a further novel feature of the present invention that the polymeric membrane material be incorporated with a carrier which can be made from spherules of porous material, such as glass spherules;
This can be done in two ways:

1 The carrier carrying the indicator solution is preferably a single layer and is applied to a transparent support which shields the optical specimen on the measuring device side. This layer is loaded with a polymer initial mixture and vulcanized.

2. The carrier carrying the indicator solution is added to the polymer initial mixture and uniformly distributed by stirring. The method of the following working steps corresponds to the method for carrier-free emulsions.

In order to avoid mechanical breakage of the micelles near the surface with or without a carrier, in another embodiment of the invention the side of the polymer membrane facing the sample is preferably provided with a densely polymerized protective layer. There is.

This additional protective layer can be made light-tight for even greater advantages, or it can also be made light-tight in any suitable way (for example by adding pigments or dyes). This protects the actual optical specimen membrane from surface damage and
At the same time, it is optically isolated from the sample.

In another embodiment of the invention, an indicator solution (with or without a carrier) is present within the micelles.
However, some contain additional buffering systems, such as bicarbonate buffers or phosphate buffers. Such an addition is not essential, since the indicator liquid itself acts as a buffering system, and by appropriate selection of the indicator concentration a suitable buffering capacity can be adjusted to the measuring device.

Example of manufacturing a PCO ₂ specimen for the measuring device according to the invention; Example 1 10 g of SLM40060, 40 mg of SLM40061 and 400 mg of SLM40061
Mix SLM40062. 0.01 mol 1 in this mixture
An emulsion is prepared by adding 1 g of an aqueous solution of pyranine and stirring using a high-speed stirrer. Stirring time is approximately 30 seconds. This emulsion is applied to a degreased glass support using an applicator in a layer thickness of approximately 50 μm. This layer is vulcanized by the action of ultraviolet light.

Example 2 Average diameter of 15 ml of pyranine aqueous solution of 0.01 mol 1
Two 20 μm cross-linked polyacrylamide spherules
Add g. The spherules impregnated with the indicator solution and acting as a carrier are filtered and mixed with an aqueous pyranine solution in a weight ratio of 5:1 to make them suitable as a polymer initial mixture. The subsequent steps are the same as in Example 1.

Example of manufacturing a measuring device according to the invention for determining CO ₂ and O ₂ ; 0.1 g of dissolved benzo(ghi) perylene is
SLM40060 (Watskerchemy, West Germany)
dissolve in

2 g of crosslinked polyacrylamide globules with an average diameter of 20 μm are added to 15 ml of a 0.01 mol/ml aqueous pyranine solution. The pellets, which act as carriers impregnated with the indicator solution, are filtered and stirred into the next polymer initial mixture in a weight ratio of 1:5 (1 part pellets, 5 parts polymer initial mixture).
), 10 g...SLM40060 containing dissolved benzo(ghi)perylene 40 mg...SLM40061 400 mg...SLM40062 This suspension was applied to a degreased glass support using an applicator in a layer thickness of approximately 50 μm. It will be done. This layer is vulcanized by the action of ultraviolet light.

Dissolved benzo(ghi)perylene dissolved in a hydrophobic membrane matrix acts as an O ₂ indicator. Pyranine dissolved in the water contained in the suspended globules acts as a _CO2 -indicator.

Claims (1)

[Claims] 1. PH as an indicator for determining the CO ₂ component in a sample, which is embedded in an aqueous solution within a polymer membrane and is at least partially in contact with the sample.
In a measuring device comprising a fluorescent dye dependent on protons and configured to measure the fluorescence emitted by the indicator after excitation, an aqueous indicator solution in the form of a droplet with a diameter in the range of 0.1 to 100 μm is used to detect protons. A device characterized in that it is incorporated in a uniform distribution within a polymeric membrane that allows gases to pass through without being allowed to pass through. 2. The measuring device according to claim 1, wherein the polymer film is made of silicone elastic rubber such as non-adhesive silicone. 3. Said membrane is composed of a silicone polymer additionally bound in dissolved form to an indicator drop that fluoresces in response to PH in at least approximately uniform distribution an indicator substance that fluoresces in response to O ₂ . A measuring device according to claim 1. 4 Selected from the group consisting of polycyclic or heavy ring carbocyclic aromatic compounds and heterocyclic compound molecules 5
4. The measuring device according to claim 3, wherein a polycyclic or heavy ring aromatic hydrocarbon having a fluorescence extinction time t ₀ of nanoseconds or more is used as an indicator substance that emits fluorescence in response to O ₂ . 5 In order to eliminate the mobility of the indicator substance that emits fluorescence in response to O ₂ , it is chemically modified such as alkylation with a long chain branched or unbranched alkyl group having 3 to 20 carbon atoms. The measuring device according to claim 3 or 4, wherein movement of the indicator within the measuring device is restricted. 6. The measuring device according to claims 1 to 5, wherein the polymer film is vulcanized or alternatively polymerized on the smooth surface of a transparent, layered support material. 7. A carrier for an infusion containing an indicator in a polymeric membrane material is provided with the same size as the infusion, the carrier carrying an indicator solution on its surface or being impregnated with an indicator solution. The measuring device according to the ranges 1 to 6. 8. Measuring device according to claim 7, wherein the carrier is made of globules or alternatively microparticles of a hygroscopic material such as silica gel or polyacrylamide. 9. A measuring device according to claim 7, wherein the carrier is made of a porous material, such as a glass pellet. 10. The measuring device according to claims 1 to 9, wherein a densely polymerized protective film is attached to the side of the polymer film facing the sample. 11. The measuring device according to claim 10, wherein the protective film, which is not included in the fluorescence measurement, has a light-shielding property. 12. The measuring device according to claims 1 to 11, wherein the indicator solution contains a buffer system such as a bicarbonate buffer or a phosphate buffer.