Anal. Chem. 1999, 71, 1544-1548
pH-Insensitive Ion Selective Optode: A Coextraction-Based Sensor for Potassium Ions Christian Krause, Tobias Werner,* Christian Huber, and Otto S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany Marc J. P. Leiner
Biomedical Division, AVL GmbH, Hans-List-Platz 1, A-8020 Graz, Austria
A novel type of optical potassium sensor is presented whose response mechanism is virtually insensitive to pH and ionic strength. It is based on a coextraction mechanism using a lipophilic ion carrier and a fluorescent anion, the two contained in a solid oil-in-water emulsion. The latter is composed of lipophilic plasticizer droplets (containing valinomycin) and a hydrogel containing the highly solvatochromic anionic dye merocyanine 540. On exposure to a solution of potassium ions, these are extracted, along with the anionic fluorophore, into the plasticizer phase wherein the dye is much more fluorescent than in hydrogel. The dynamic range of sensor response can be adjusted by variation of the membrane composition. Membranes were optimized for measurements in the physiological range. The dynamic range is from 0.1 to 50 mmol/L K+. Response times are 40 12 4 5 10 3.5 3.5 3.5 3.5 3.5 3.5
200 150 200 20 120 220 250 10 25 270 250
a F, fluorescence intensity; F , fluorescence intensity at 0 mM KCl; F , fluorescence intensity at 50 mM KCl; K 0 50 1/2, point of inflection of calibration curve using a Boltzmann fit: (F - F0)/F0 ) (Z - Y)/(1 + exp(log(c/mM) - X/W)) + Y, W, X, Y, and Z are empirical parameters describing the initial value (Z), final value (Y), center (X), and width (W) of the fitting curve. All measurements were performed at constant ionic strength of 140 mM and pH 7.4.
Figure 5. Response time, relative signal change, and reversibility of sensor membrane M1 at constant ionic strength of 140 mM and pH 7.4 (excitation/emission at 575/598 nm).
to eq 2, an increase in the concentration of ion carrier results in lower analyte concentrations needed for the same signal change to occur. This can be seen in the variation of sensor characteristics due to different valinomycin concentrations. The other membrane parameters remain unchanged. The amount of plasticizer is another way to alter sensor characteristics. The total signal change increases with the fraction of plasticizer. A third way to influence response characteristics is the concentration of MC 540 in the soaking solution. As can be seen in Table 2, there is a optimum dye concentration of ∼2 µM MC 540 in the soaking solution of the membrane for detection of K+. Other dye concentrations lead to smaller signal changes while the point of inflection remains unchanged. K1/2 values, calculated from the point of inflection of a sigmoidal fit of calibration curves and signal change on going from 0 to 50 mM K+ of the various membranes, are summarized in Table 2. Cross-Sensitivity to pH. Because the sensing mechanism is not associated with the transport of protons (see eq 2), the response to potassium is supposed to be nearly independent of pH. To prove this, samples of different pH and constant ionic strength of 140 mM were examined. Each calibration curve was measured at least three times. The results are summarized in Figure 6 and Table 3. They show that there are only small deviations for pH variations in the physiological range. Changes of 0.1 in pH at a potassium concentration of 5 mM will not result in a detectable signal change. Signal change on going from 4 to 5 mM K+ is ∼6 ( 0.5% for all examined pH.
Figure 6. Calibration curves for membrane M1 at constant ionic strength of 140 mM but different pH. Table 3. Influence of pH on the Calibration Curve of Membrane M1 at Constant Ionic Strength of 140 mM pH
K1/2a (mM)
total signal change (%)
5.9 7.4 8.3
2.8 3.1 3.3
180 210 160
a K 1/2, point of inflection of calibration curve ((F - F0)/F0) using a Boltzmann fit. The total signal change was calculated from the end point of the Boltzmann fit.
Sensitivity to Ionic Strength. Buffers of different ionic strength and pH 7.4 were examined. Ionic strength was adjusted with NaCl. Each calibration curve was measured at least three times. As can be seen in Table 4, ionic strength does not affect the point of inflection of the calibration curves significantly. Signal change on going from 4 to 5 mM K+ is ∼6 ( 0.5% for all examined ionic strengths. The total signal change is lower for high ionic strength. Cross-Sensitivity to Lipophilic Anions. The effect of different lipophilic anions on sensor response was examined. Samples of constant ionic strength, pH, and potassium concentration but containing various lipophilic anions were pumped through the flow-through cell. Lipophilic anions lower the fluorescence of the membrane in the presence of potassium as can be seen in Figure Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
1547
Table 4. Influence of Ionic Strength on Calibration Curve of M1 at Constant pH of 7.4 ionic strength (mM)
K1/2a (mM)
total signal change (%)
80 140 200
2.8 3.1 3.0
220 210 160
a K 1/2, point of inflection of calibration curve ((F - F0)/F0) using a Boltzmann fit. The total signal change was calculated from the end point of the Boltzmann fit.
Figure 7. Calibration curves for membrane M1 at constant pH of 7.4 but different ionic strengths.
Figure 8. Cross-sensitivity of M1 to different lipophilic anions at constant ionic strength, pH, and 5 mM potassium.
8. While this effect is reversible for most anions, SDS causes an irreversible effect. The more lipophilic the anion, the more the fluorescence is lowered. In the absence of potassium, lipophilic anions do not cause a signal change. It was observed that lipophilic anions accelerate leaching of the dye, which occurs in both the presence and absence of potassium. DISCUSSION Choice of Materials. Solid-state emulsions composed of HN80 and CPDDE proved to be stable without the addition of a emulgator. Stabilization by the hydrogel is preferable over the addition of a surfactant due to the ease of membrane preparation and small droplet size. Furthermore, MC 540 leaches little out of such membranes (in the absence of lipophilic anions). HN80 seems to offer adsorption sites for lipophilic anions, which can be occupied by the anionic dye. Adsorption is strong enough to minimize leaching, but the dye is still mobile enough to be 1548 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
coextracted into the lipophilic droplets with potassium. This assumption is confirmed also by the accelerated leaching of the dye in the presence of lipophilic anions. Membrane Parameters. Increasing the ion carrier concentration led to the expected higher affinity to the analyte (see eq 2). Increasing the fraction of plasticizer increased the relative signal change. This may be due to a increased inner surface. Increasing the plasticizer amount (and therefore increasing the amount of valinomycin) also lowers background fluorescence of the remaining dye in the hydrophilic phase during coextraction. This leads to higher total signal changes. According to eq 2, increasing the dye concentration should have an effect similar to increasing carrier concentration. If a surface-controlled mechanism is involved, the concentration of the dye in the hydrophilic bulk is not important for the sensor characteristics, but only the concentration of the dye near the hydrophobic, carrier-containing plasticizer droplets. The point of inflection is mainly determined by the HLB (eq 1) of the dye and then by the concentration in the hydrophilic bulk. The decrease of total signal change at high dye concentration originates from a self-quenching of the dye. Cross-Sensitivity. The results confirm the expected low pH interference of such sensor membranes. The deviations between different ionic strengths and pH may result from other reasons: (a) swelling properties of the HN80 are pH and ionic strength dependent and therefore the HLB of the dye and (b) deviations between different membranes make it difficult to compare such small changes in response characteristics. The results of cross-sensitivity to a lipophilic anion are in accordance with eq 1. The more lipophilic the interfering anion is, the more likely it will be coextracted instead of a MC 540 anion. The lower cross-sensitivity in the absence of potassium shows that the lowering of fluorescence intensity is not due to a quench effect (well-known for iodide) but to a coextraction of the interfering anion instead of MC 540. The irreversible effect of SDS is explained by the formation of a highly charged SDS layer around the lipophilic droplets. This micelle formation is common for surfactants. The charged layer makes the coextraction process impossible because the anionic dye is rejected by the negatively charged SDS layer. The accelerated leaching of MC540 in the presence of lipophilic anions is explained by a displacement process. SUMMARY The sensors presented here can measure potassium ion concentration in the physiological range with sufficient accuracy and low cross-sensitivity to the examined interfering ions. An attractive feature of this approach is the fact that a variety of ions can be measured with the same wavelength.19 Optical sensors for sodium and calcium are now under development in our laboratory. ACKNOWLEDGMENT The authors thank Hannelore Brunner for technical assistance. C.K. and C.H. thank the AVL-List GmbH for financial support. Received for review September 18, 1998. Accepted February 2, 1999. AC981042X (19) Huber, C.; Werner, T.; Krause, C.; Wolfbeis, O. S.; Leiner, M. J. P. Anal. Chim. Acta, submitted for publication.
