Fluorescent indicator that measures activity of alkaline metal ion in sample solution

Description: TECHNICAL FIELD [Detailed description of the invention]

【0001】

[Industrial application field]

The present invention relates to a fluorescence-optical indicator for measuring the activity of alkali metal ions in a sample solution that is at least indirectly in contact with the indicator, and a sensor device comprising the indicator.

【0002】

[Conventional technology]

The measurement of alkali metal ion activity is particularly relevant in the medical field. For example, electrolyte imbalance in blood is substantially measured by sodium and potassium in the electrolyte, and thyrium plays a major role in cerebral function.

【0003】

Ion concentration or ionic activity is measured in many ways, for example by potentiometric titration or optical measurements using so-called optodes. Potential difference electrodes have been used in recent years to measure cations in sample solutions. During the measurement process, the ion-selective membrane of the electrode is exposed to a liquid sample. On the surface of the membrane, the ion-selective ionohore contained in the membrane reacts with the ions to be measured in the sample solution. In this method, a membrane potential difference occurs in which the size depends on the concentration of ions present in the sample solution. Despite the very small degree of complex formation, the potentiometric titration that occurs can be reliably measured by the potentiometric method.

【0004】

The major drawback of ion-selective electrodes is that of --Needing a control element -It is difficult to miniaturize --Sensitivity to electrochemical potential and electromagnetic interference --Points that need to be individually tested before measurement.

【0005】

Photodiodes, on the other hand, do not require a control element. The optical signal is independent of external potential difference and current. All of the conventionally known photodiodies for measuring ion concentration are based on measuring the light absorption of chromophor, which depends on the emission intensity or ionic activity of the chromophore. However, given the fact that the optical signal is affected by changes in indicator concentration, intensity of individual light sources and detector sensitivity, calibration with a suitable calibration medium is required prior to measurement.

【0006】

Photodiodes enable the development of miniaturized measuring elements. This is especially important for very small volume samples and for designing catheters for invasive measuring techniques in clinical science and medicine.

【0007】

Recently, many optical sensors have been discussed to measure the ion concentration of alkali metals. These are based on changes in light absorption or rather changes in the fluorescence intensity of the appropriate indicator affected by ions (or, in many general terms, changes in the intensity of photoluminescence of the indicator; simplification. For this reason, the term fluorescence, which is said to include all kinds of photoluminescence, is used below).

【0008】

AT-B384,677 discloses an optical potassium-sensor containing potassium-selective valinomycin-ionophore and optically sensitive fluorophore in a thin polymer membrane. As with the electrodes described at the beginning, the potassium-ion binding by ionohore creates a potential difference in the interface between the membrane and the sample solution. The potential, which depends on the concentration of potassium ions, changes the fluorescence intensity of the potential-sensitive fluorophore. However, the degree of potassium-complexation affected by ionohore is very small in such devices. Therefore, the sensitivity of such a device is low and the cation concentration in the sample cannot be measured accurately and quantitatively.

【0009】

As described in European Patent Application Publication No. 0,358,991, the above drawbacks can be eliminated by adding chromophore to the supporting material containing the cation-selective ionohore. If the cation of the sample to be measured is complexed by the cation-selective ionohore in the membrane, other cations may be transferred to the sample solution. The degree of complexation and changes in the optical signal are significantly greater in this case than in the apparatus described in AT-B384,677. However, it is known that the degree of change in the optical signal depends not only on the activity of carion to be measured in the sample solution but also on the activity of the cation to be transferred from the support film to the sample solution. It is known from the relevant literature that the chromophores of European Patent Application Publication No. 0,358,991 may be exchanged for fluorophore (H.He and OS Wolfbeis; SPIE vol 1368, 1990). For practical purposes, where the cation to be transferred is generally a proton, this implies that the measurement of alkali metal ions by such a device also depends on the pH value of the sample. There is.

【0010】

Sousa and Larson (Journal of the American Chemical Society 99, 307, 1977) explain that the inherent fluorescence intensity of crown-ethers is altered as a result of complexation with alkali metal ions.

【0011】

De Silva and co-authors describe an ion-sensor in which ionic bonds suppress the movement of electrons between ionohores and phosphors, thereby increasing fluorescence intensity (eg, Journal of the Chemical Society Perkin Transactions). II 1989, 1559 ~ 1564, 1989). Aoki et al. (Journal of the Chemical Society Chemical Communications 1992, 730-732, 1992) show that the fluorescence intensity is increased by ionic bonding in a system consisting of pyrene, calixarene and nitrobenzene. He et al. (Analytical Chemistry 65, 123-127, 1993) describe a sensor in which the fluorescence intensity of an ion-independent fluorophore is changed by the ion-dependent absorption of ionohore.

【0012】

U.S. Pat. No. 1,154,890 discloses a simple principle. In this case, the potassium-sensitive fluoroionophore is immobilized in an ion-permeable polymer gel whose fluorescence intensity selectively depends on the potassium concentration of the sample.

【0013】

All of the above devices are based on fluorescence altitude as an information carrier. However, such devices have many drawbacks: the measurement signal is almost uncorrectable due to the unavoidable changes in the intensity of the light source, the passage of light through the optics, the sensitivity of the detector and especially the concentration of the indicator. Is affected by This interferes with the actual use of these devices. As a result, each signal sensor element must be individually calibrated at the time of manufacture and a second calibration must be performed prior to actual use of them (or period calibration if the test period is extended). Must be done at the time of use).

【0014】

The above drawbacks can be avoided by using the fluorescence decay time instead of the fluorescence intensity as a measurement variable. Fluorescence decay time is the unique property of each indicator and its molecular environment. This is independent of the thickness of the sensitive layer, the concentration of the indicator, the intensity of the light source, the sensitivity of the indicator and the optical properties of the measurement system. For this reason, changes between individual sensor elements due to the manufacturing process do not produce different sensor characteristics, and the calibration function determined for one element is related to all other elements. , The need for individual calibration is eliminated. Similarly, any changes in the sensor elements due to aging do not change the sensor characteristics, so there is no need to recalibrate prior to actual measurements.

【0015】

Bacon and Demas (US Pat. No. 5,030,420) and Lippitsch et al. (Analytica Chimica Acta 205, 1-6, 1988) have proposed such decay time sensors for oxygen measurement. pH decay time sensor (WO 92/10739), calcium (Lakowicz et al., Cell Calcium 13, 131-147, 1992), aluminum, gallium and indium decay time sensors (Caroll et al., Analytical Chemistry 61, 1768-1772, 1989) ) Has been disclosed conventionally. However, the decay time sensor of alkali metal ions has not been known conventionally.

【0016】

AT-PS393,035 discloses a method for quantitative measurement of chemical parameters of a sample, in which the indicators are composed of two substances that are close to each other, namely chromophore and chromophor. Become. The fluorophore does not respond to the parameter to be measured, but the chromophore is exposed to changes in its absorption spectrum. Due to the partial overlap between the fluorophore emission spectrum and the chromophore absorption spectrum, energy transfer occurs between the two substances, which causes the fluorophore fluorescence decay time to depend on chemical parameters. And decrease.

【0017】

[Problems to be Solved by the Invention]

Therefore, an object of the present invention is to provide a sensor device including a fluorescent indicator and such an indicator based on the measurement of decay time for measuring the activity of alkali metal ions.

【0018】

[Means for solving problems]

In the present invention, the subject is at least three components in which the indicators are chemically bound to each other: a fluorophore, a functional group that can affect the fluorescence decay time of the fluorophore, and an alkali metal to be measured. It has an ionohore, which selectively and reversibly binds to the ion, so that the effect of the functional group on the decay time of the fluorophore can be resolved depending on the activity of the alkali metal ion. Fluorescent.

【0019】

Furthermore, it is also an object of the present invention that the maximum absorption of the fluorophore is within the wavelength of 400-1,200 nm and the fluorescence decay time of the fluorophore is greater than 10 ns. In summary, the indicator of the present invention is At least one type of fluorophore that has absorption, especially in the visible or near-infrared spectrum (400-1,200 nm) and exhibits a favorable fluorescence decay time of> 10 ns. --At least one functional group that affects the fluorescence decay time of the fluorophore by interacting with the fluorophore -Forming a unique reversible bond with a special type of alkali metal ion, thereby altering the interaction between the functional group and the fluorophore, and thus the fluorescence decay time of the fluorophore individually. At least one type of ionohore that is a function of the concentration of alkali metal ions.

【0020】

These three components are located in close proximity to each other, either by chemically bonding to each other or by being anchored together in a suitable matrix. Below are some examples of suitable fluorophore, functional groups and ionohores with individual properties that are essential to the present invention: Abbreviations used: 1_a Long wave end of absorption band t_f Fluorescence decay time K_s Ionophore / cation binding constant Common logarithm of pKS Ks Example of fluorophore Substance name 1_a(nm) t_f(ns) 2-Amino Anthracene 480 31 Rubrene 560 17 Big Cyclone 490 28 Ruthenium-Bipridyl 550 545 Ruthenium-Phenanthroline 447 950 If the fluorescence decay times of polycyclic-and heterocyclic aromatic compounds and metal ion complexes with heterocyclic ligands can be influenced by functional groups, these are also other possible phosphors.

【0021】

Examples of functional groups Substance name t-Type of effect Nitro-, cyano-electron acceptor Amino-, methoxy-electron donor Biologen electron acceptor Halides, pseudohalides electron acceptors Ionohoa example Substance name Cation pKS Calixi (4) Allen-ester Na⁺ 4.3 K⁺ 2.9 1,4,-Benzodioxine-23-Crown K⁺ 3.0 -7- Lariat ether Na⁺ <0.6 2,9-Dibutyl-1,10- Phenanthroline Li⁺ About 4.5 Na⁺ <2 Next, Na⁺-, K⁺-And Li⁺Possible indicators used to measure activity are described. While these examples serve the purpose of demonstrating the invention, they do not limit the subject matter of the invention. 1. Na⁺Indicator for measuring ion activity [0022]

[Chemical 1]

<img file="JPH0772084A_D0001.tif" />In this example, the fluorophore is 2-aminoanthracene, the functional group is nitrophenylmethyl, and the ionohore is calix (4) allene. If the ionohore is not bound to a cation, the ring to which the functional group or fluorophore is bound is free to rotate. Due to thermal motion, the two components often approach each other. At that time, the nitro group deactivates the fluorophore and reduces the fluorescence decay time. Ionohoa is Na⁺Shows high selectivity for binding. When such ions are bound, the ring position is fixed by the interaction of the phenolic oxygen atom and the ion, no longer deactivates, and the decay time reaches the value of the uninactivated fluorophore. Almost reached. 2. K⁺Indicator for measuring ion activity [0023]

[Chemical 2]

<img file="JPH0772084A_D0002.tif" />In example (a), the fluorophore is decacyclene, the functional group is nitrophenylmethyl, and the ionohore is benzodioxin-23-crown-7-lariat ether. In example (b), tris-lutenium-phenanthroline is used as the fluorophore, dinitrobenzene is used as the functional group and benzodioxin-23-crown-7-lariat ether is used as the ionohore. If the ionohore is not attached to the cation, the lateral arm to which the functional group is attached is free to move. Due to thermal motion, this group often approaches the fluorophore, thereby deactivating its fluorescence and reducing the fluorescence decay time. Ionohoa is K⁺Has high selectivity for binding. When such ions are bound, the position of the lateral arm is fixed by the interaction of the ester oxygen atom and the ion, no longer inactivates, and the decay time of the uninactivated fluorophore. Almost reach the value. 3. Na⁺Indicator for measuring ion activity [0024]

[Chemical 3]

<img file="JPH0772084A_D0003.tif" />The fluorophore in this case is rubrene, the functional group is diethylamine, and the ionohore is dibutylphenanthroline. If the ionohore is not attached to a cation, the butyl group to which the functional group is attached is free to rotate. These two components often approach the fluorophore due to thermal motion, in which the amino group acts as a fluorescence inactivating agent and the fluorescence decay time is reduced. Ionohoa is Li⁺Has high selectivity for binding. When such ions are bound, the position of the amino group is fixed by interaction with this ion, and the positive charge of the ion binds the free ion pair of the nitrogen atom. Deactivation no longer occurs, and the decay time almost reaches the value of the uninactivated fluorophore.

【0025】

In the sensor device using the indicator described in the present invention, the indicator must be mixed with the hydrophilic ion-permeable matrix on the carrier substrate that transmits the excitation-and measurement radiation. It is preferably physically dissolved in a hydrophilic ion permeable matrix. However, it is also possible to incorporate the indicator into the matrix electrostatically or by covalent bond.

【0026】

An inexpensive method of immobilizing an alkali metal ion-selective fluorescent indicator in an ion-permeable hydrophilic polymer-matrix is to physically dissolve the indicator in this matrix. Unlike measurements of fluorescence intensity or light absorption, measurements of fluorescence decay time depend on changes in indicator concentration and geometric changes in the light passage (because the physicochemical properties of the sample cause the polymer matrix to swell). ing. For this reason, the measuring device is resistant to weak bleaching effects, diffusion of the indicator into the sample and geometric changes in the light passage. This fixation method is particularly useful when the device is used as a one-way measuring device.

【0027】

Another possible method of incorporating an indicator of the invention into a polymer matrix is to chemically immobilize the indicator by covalent bond. This is particularly useful if the device is in contact with the sample for extended periods of time (continuous measurements, long-term measurements) or if the sample should be protected from being contaminated with the indicator substance (in vivo measurements). Chemical fixation of the indicator has already been disclosed in related literature, such as E. Koller and OS Wolfbeis's Fiber Optic Chemical Sensorsand Biosensors, Volume 1, Chapter 7, by OS Wolfbeis, CRC Press, Boca Raton 1991. There are many methods available.

【0028】

The attached drawings show a typical sensor device using the indicator of the present invention. Alkali metal ions in sample solution P (eg Na⁺A fluorescence indicator I for measuring the activity of (ions) is present in the hydrophilic ion permeable matrix M, in which it is (preferably) physically dissolved. The ion-permeable matrix M forms a layer on a support or carrier T that transmits excitation-and measurement radiation. It also has a device A for excitation and a device M'for measurement, which are in optical contact with the indicator I. The signal of the measuring device M'is transmitted to the evaluation device P for determining the decay time or its change and the ionic activity associated therewith.

【0029】

A sensor S containing a carrier T, a matrix M and an indicator I dissolved therein can be stored dry until it is used (once), in which case the polymeric hydrophilic matrix M contacts the aqueous sample P. It swells only when. Hydrophilic polymer support materials that swell upon contact with an aqueous sample for a few seconds have been conventionally known. A major advantage of the present invention is that the measurement system does not need to be calibrated with a calibration solution prior to the actual measurement process. Unlike in conventional systems that optically measure alkali metal ions, the measurement signal is independent of fluctuations in light source intensity, detector sensitivity and indicator concentration. The properties of the measuring device, which are dependent on measurement variability, depend only on the type of indicator and polymer-carrier material used.

【0030】

Suitable substances for the ion-permeable polymer matrix M are hydrogels, polyvinyl alcohols, polyglycols, polysaccharides, polyacrylates, polyacrylamides, polyimides, polyurethanes and derivatives thereof. Suitable carriers T for Matrix M are light conductors, glass slides or plastic films such as polyester, polycarbonate and the like.

【0031】

As is known from the prior art, there are two essentially different methods of measuring fluorescence decay time. That is, by time-divided recording of excitation and generation of fluorescence using short light waves, or by measurement of phase rubbing between excitation and excitation light modulation with high frequency modulated light and fluorescence modulation or By measuring the intensity of fluorescence modulation. Either method can be used in the sensor device of the present invention. In one embodiment of the invention, indicator I is immobilized in, for example, a polymer matrix and contacted with sample solution P. Fluorescence is excited by short-term use of a light source such as a coaxial flash lamp, pulsating laser or pulsating LED. Fluorescence is separated from the excitation light by the cutoff-filter F and fast photodetectors (photoelectron doubling means, pin-photodiodes, avalanche-photodiodes) are used to detect the fluorescence. Attenuation time is measured by suitable electronic means. The ion concentration or activity can be calculated from the measured decay time through a calibration function stored only once.

【0032】

When using a phase-fluorescence meter, the light sources (lamps, lasers, LEDs) are highly frequency-modulated, the fluorescence is separated and detected by a fast photodetector. In this case, the phase rubbing between the modulation of the excitation light and that of the fluorescence is a function of the fluorescence decay time and therefore the function of the ion concentration.

[Simple explanation of drawings]

[Figure 1]

FIG. 1 illustrates a typical sensor device using the indicator of the present invention.

[Explanation of symbols]

I ... Indicator P Liquid sample M Ion permeability matrix T ... Support or carrier M' Measuring device A ... Excitation device P Evaluation device