Anal. Chem. 1998, 70, 3983-3985
Luminescence Decay Time-Based Determination of Potassium Ions Christian Krause, Tobias Werner,* Christian Huber, Ingo Klimant, and Otto S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany
A luminescence decay time-based potassium ion sensor is presented which applies the ion-exchange principle. The bulk membrane consists of valinomycin, plasticized PVC, and the ruthenium(II) tris-4,4′-diphenyl-2,2′-bipyridyl bromothymol blue ion pair [Ru(dibipy)3](BTB)2 as a proton donor. The efficacy of radiationless energy transfer from the donor (the ruthenium complex) to the acceptor (BTB) is mediated by the potassium ion concentration. The concentration of potassium ions can be calculated from either luminescence intensity or decay time. At pH 8.7, the working function ranges from 1 to 100 mM KCl. Potassium ion determination is of tremendous interest in the medical science area. It is the major intracellular cation in the blood. Along with sodium, it helps to maintain the osmotic and acid-base balances. Due to its need for proper nerve and muscle action, fast and reliable tests for potassium levels in blood are highly essential to evaluate nerve, muscle, and endocrine disorders. Conventional analytical methods such as flame spectroscopy are time-consuming and do not lend themselves to continuous monitoring.1 The use of potassium electrodes based on plasticized PVC membranes containing valinomycin is most common; however, potassium optical sensors (“optodes”) gained growing interest due to the ease of handling and the specific advantages of disposable optical tests.2 In fact, all commercially available test strips or sensors for potassium are based on ion exchange. The ion-exchange scheme is based on a lipophilic polymer/plasticizer bulk containing a neutral potassium carrier (C; e.g., valinomycin) and a pH indicator (HInd). This indicator becomes deprotonated on potassium ion extraction, followed by the release of protons and a detectable change of its optical properties. + + K+ aq + Corg + HIndorg a ([KC] org + Indorg) + Haq (1)
K)
+ ([[KC] + org][Indorg])[Haq]
[K+ aq][HIndorg][Corg]
(2)
The pH indicator in this approach allows either absorbance or fluorescence intensity measurements to measure potassium ions at a defined pH.3,4 (1) Gibb, I. J. Clin. Pathol. 1987, 40, 298-301. (2) Ng, R. H.; Sparks, K. M.; Statland, B. E. Clin. Chem. 1992, 38, 13711372. S0003-2700(98)00222-4 CCC: $15.00 Published on Web 08/08/1998
© 1998 American Chemical Society
This kind of optical potassium sensor suffers from cross sensitivity to pH and from problems originating in the sample. Real samples are often turbid, colored, or even fluorescent. Optical isolation of the sensing membrane from the sample can minimize this problem but often leads to lower sensitivity and prolonged response times. Furthermore, fluctuations in the optical arrangementssuch as light source, detectors, and optical fiberssare crucial for an application. Photobleaching and leaching of the dye as well as inhomogeneities within the sensing layer add to the problems of intensity-based optical sensors, resulting in frequent recalibration and the need for internal referencing of the system in use. One way to overcome at least some of these problems is to measure luminescence decay time instead of its intensity. Examples of luminescence decay time-based sensors have been published so far.5,6 Up to now, the lack of pH indicators of sufficiently long excitation wavelength and excited-state lifetimes has limited the development of appropriate decay time-based optodes. As previously shown,7 fluorescence resonance energy transfer can be applied to decay time measurements of sensor membranes. A recently developed pH-sensitive absorption dye/ Ru fluorophore ion pair was used for a pH-sensing membrane.7 This application includes a pH-sensitive indicator dye and a ruthenium complex in the form of the [Ru(dibipy)3](BTB)2 ion pair (BTB ) bromothymol blue), which undergoes a change in its luminescence decay time on deprotonation. The change is due to the differences in the absorption maxima of the BTB in the deprotonated and protonated forms. The absorption spectrum of the phenolate (blue) form overlaps with the emission band of the pH-insensitive luminescent ruthenium complex so that radiationless energy transfer can occur.8 The absorption of the phenol (yellow) form, in contrast, does not fit the emission wavelength, so energy transfer is supressed. Radiationless energy transfer affects both the decay time and the intensity of the emission. The apparent pKa of a plasticized PVC membrane containing a pH indicator and valinomycin depends on the concentration of the potassium ion. Therefore, such membranes can be used for potassium detection under controlled pH conditions. (3) Suzuki, K.; Ohzora, H.; Thoda, K.; Miyazaki, K.; Watanabe, K.; Inoue, H.; Shirai, T. Anal. Chim. Acta 1990, 237, 155-164. (4) Wang, K.; Seiler, K.; Morf, W. E.; Spichiger, U. E.; Simon, W.; Lindner, E.; Pungor, E. Anal. Sci. 1990, 6, 715-720. (5) Bacon, J. R, Demas, J. N. Anal. Chem. 1987, 59, 2780-2785. (6) Lakowicz, J. R.; Szmacinski, H. Sens. Actuators B 1993, 11, 133-143. (7) Kosch, U.; Klimant, I.; Werner, T.; Wolfbeis, O. S. Anal. Chem., in press. (8) Gabor, G.; Walt, D. R. Anal. Chem. 1991, 63, 793-797.
Analytical Chemistry, Vol. 70, No. 18, September 15, 1998 3983
EXPERIMENTAL SECTION Materials. Buffers and inorganic salts were of analytical grade (Merck, Darmstadt, Germany). Water was doubly distilled. All buffer compositions were adjusted to constant ionic strength with NaCl according to ref 9. Analytical grade solvents poly(vinyl chloride) (PVC, high molecular weight) and valinomycin were from Fluka (Buchs, Switzerland). Polyester foil (Mylar, 125 µm) was purchased from Goodfellow (Cambridge Ltd., UK). The synthesis of [Ru(dibipy)3](BTB)2 was described elsewhere.7 The plasticizer 2-cyanophenyl dodecyl ether (CPDDE) was synthesized according to ref 10 by reaction of 8.0 g of 2-hydroxybenzonitrile (67 mmol) and 13.8 g of K2CO3 (100 mmol) in 100 mL of diethyl ketone. Dodecyl bromide (12.5 g; 50 mmol) in 20 mL of diethyl ketone was added at room temperature. After refluxing for 24 h, the mixture was filtered to separate KBr and unreacted K2CO3. After addition of 50 mL of water, the filtrate was extracted three times with 30 mL of ether each. The combined ether extracts were washed with 50 mL of water, three times with 50 mL of 5% NaOH, and again with water and dried. After removal of solvents in a vacuum, the remaining yellow oil was distilled at 0.13 hPa (0.1 Torr). The crude product (yield 8.1 g; 88%) was purified by column chromatography on silica gel (eluent, petroether 40-60/ chloroform 1:1). 1H NMR (CDCl3): 7.5 (t, 2 H); 7.0 (t, 2 H); 4.05 (t, 2 H); 1.85 (q, 2 H); 1.3 (s, 18 H); 0.85 ppm (t, 3H). Membrane Composition. A cocktail was prepared by dissolving 120 mg of PVC, 240 mg of CPDDE, 2 mg of valinomycin (corresponding to bvalinomycin ) 5.4 mmol/kg of matrix material), and 1 mg of [Ru(dibipy)3](BTB)2 in 1.5 mL of THF. The membrane was prepared by spreading this cocktail onto the polyester foil using a homemade knife coating device. The resulting layer thickness after solvent evaporation was calculated to be about 10 µm. Measurements. All measurements were performed with airsaturated solutions. The membranes were placed in a Teflon flowthrough cell (volume 900 µL) and measured as shown in Figure 1. A Gilson Minipuls 3 peristaltic pump (Gilson, Villiers-le-Bel, France) was used with a typical flow rate of 1 mL/min. All tubes consisted of silicon. The sample pH was controlled by a WTW 638 pH meter (WTW GmbH, Weilheim, Germany). All membranes were conditioned first in water for 1 h and finally in potassium-free buffer solution for another hour. After changing of the sample in the cell, the membrane was allowed to condition for 10 min. The luminescence decay time measurements were performed using a blue LED light source (λmax ) 470 nm NSPB 500, from Nichia, Nu¨rnberg, Germany), combined with a blue glass filter (BG12, Schott, Mainz, Germany) for excitation. A bifurcated glass fiber bundle (2 mm diameter) was used for light transmission. The LED was modulated at a frequency of 90 kHz via a dualphase lock-in amplifier. Emission light was filtered with a 600nm high-pass filter and detected with a red-sensitive photomultiplier tube (PMT, H5701-02, Hamamatsu, Hersching, Germany). The phase shift was measured by the lock-in amplifier. The average decay time τ was calculated according to eq 3, where f is (9) Perrin, D. D.; Dempsey, B. Buffers for pH and Metal Ion Control, Chapman and Hall Laboratory Manuals; Chapman and Hall: London, 1974. (10) Papkovsky, D. B.; Mohr, G.; Wolfbeis, O. S. Anal. Chim. Acta 1997, 337, 201-205.
3984 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
Figure 1. Schematic view of the optical arrangement.
the light modulation frequency and φ represents the phase shift.
τ)
tan φ 2πf
(3)
To minimize photobleaching of BTB, the excitation light was switched off during conditioning between measurements. K1/2 values, representing the point of inflection of the analytical calibration curve, as well as pKa values, were calculated using a Boltzmann fit. RESULTS AND DISCUSSION Choice of Material. The sensing membrane was prepared from plasticized PVC. This material is most common for ionexchange-based optodes.4 Valinomycin is a highly selective and easily accessible potassium carrier and, therefore, the preferred ionophore. The [Ru(dibipy)3](BTB)2 ion pair was chosen because it is excitable with a commercially available LED and has a luminescence decay time of up to 1 µs. On deprotonation of BTB, it changes its decay time from typically 1 to 0.6 µs. Due to the perfect match of the absorption band of the deprotonated form with the emission of the pH-insensitive luminophore part of the ion pair, efficient radiationless energy transfer becomes possible. Notwithstanding, the luminophor-indicator ion pair [Ru(dibipy)3](BTB)2 is shown to belong to a versatile class of fluorescent complexes consisting of an absorber pH indicator and a donating luminophor with the advantages of simple preparation and immobilization, availability of a wide range of appropriate indicators and/or other cationic complexes, and easy access to the decay time parameter. A potassium-sensitive membrane was obtained by incorporating the luminophor-indicator ion pair [Ru(dibipy)3](BTB)2 along with the potassium ion carrier molecule valinomycin into a plasticized PVC membrane. As a typical ion-exchange system, the membrane can detect potassium ion concentrations as well
Figure 2. Calibration plot of the potassium sensor membrane in the presence of (A) 100 mM potassium ions and (B) 100 mM sodium ions.
as pH changes of sample solution because the ion-exchange process is directly coupled to a proton transfer. Due to energy transfer from the cationic ruthenium complex (donor) to the deprotonated acceptor BTB (acceptor), both the luminescence intensity and the decay time are affected. As shown by eq 2, the apparent pKa of such membranes and their total signal change are determined by the potassium levels also, so that defined pH conditions are required. Response to pH. pH titration curves were examined in the presence and in the absence of potassium ions (Figure 2). BTB becomes deprotonated (blue) on increasing the pH. Due to better energy transfer from Ru to BTB, the decay time and fluorescence intensity of the Ru complex decrease with pH. pKa values decrease with increasing potassium ion concentration as predicted by eq 2. Furthermore, the total signal change increases at higher potassium levels. Response to Potassium Ion. The response to potassium was examined at different pH values. The dynamic range is from 1 to 100 mM. K1/2 decreases with increasing pH, and the total signal change increases with pH. It was noted that BTB undergoes rapid photodecomposition at pH >9. The results in Figure 3 show that both the dynamic range and the total signal change can be adjusted by proper choice of the sample pH. Because increasing proton concentrations will shift the K1/2 values, all samples must be buffered before measurement. This main limitation makes the membrane applicable only after sample pretreatment. (11) Huber, C.; Werner, T.; Krause, C.; Klimant, I.; Wolfbeis, O. S. Anal. Chim. Acta 1998, 364, 143-151.
Figure 3. Decay time potassium ion titration curve of the sensor membrane at pH 7.9 and 8.7, demonstrating the strong pH dependence of the response. Table 1. pH Dependence of K1/2 Value and [K+] Dependence of the pKa, as Well as Total Signal Change (tsca) of the Sensor Membrane [K+]/mM 0 100 pH 7.9 8.7 a
pKa
tsc/%
9.6 8.6
8 30
K1/2/mM
tsc/%
30 12
7 12
tsc defined as (τmin/τmax) × 100.
This work demonstrates the possibility to adapt the ionexchange sensing scheme to luminescence decay time measurements. If carriers other than valinomycin are incorporated into the plasticized polymer membranes, other ions can be detected as well.11 ACKNOWLEDGMENT The authors thank Hannelore Brunner for technical assistance and Ute Kosch for ion pair preparation. C.K. and C.H. thank the AVL-List GmbH for financial support.
Received for review February 25, 1998. Accepted July 1, 1998. AC9802224
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