Anal. Chem. 2001, 73, 1053-1056
Fluoro Reactands and Dual Luminophore Referencing: A Technique To Optically Measure Amines Gerhard J. Mohr,*,† Ingo Klimant,‡ Ursula E. Spichiger-Keller,† and Otto S. Wolfbeis‡
Centre for Chemical Sensors, ETH Technopark, Technopark St. 1, CH-8005 Zurich, Switzerland, and Institute of Analytical Chemistry, Chemo-, and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
An optical sensor for aqueous 1-butylamine is presented which combines two novel techniques: A fluorescent indicator dye (fluoro reactand) embedded in a thin polymer layer performs a reversible chemical reaction with the analyte, causing changes in luminescence intensity. At the same time, inert phosphorescent beads dispersed within the polymer layer provide luminescence signals that act as an internal reference for the indicator dye. As a consequence, the optical sensor is independent of light source fluctuations, ambient light, drifts in optoelectronic setup, or optical fiber bending. The determination of electrically neutral analytes is a difficult task for chemical sensors. In the case of electrochemical or optical sensors for ions, the analyte usually provides changes in membrane potential or affects the electron delocalization of dye molecules.1,2 Neutral analytes have a less significant effect because their interaction with ligand molecules is generally rather weak (hydrogen bonds, van der Waals interactions, etc.), especially in an aqueous environment. Thus, we have developed indicator dyes that interact with neutral analytes by performing a reversible chemical reaction. The formation of a covalent bond between dye and analyte causes a strong change in the optical properties. Several new fluorescence-based indicator dyes (termed “fluoro reactands”) have been presented for analytes such as alcohols, amines, and aldehydes. 3-7 Sensors based on fluorescence mostly use changes in intensity as the analytical information. However, changes in fluorescence intensity can also be caused by fluctuations in the intensity of the light source, bending of optical fibers, exposure to ambient light, and changes in the electronic setup. Consequently, ratiometric methods are required to compensate for these effects. Often, dyes †
ETH Technopark. www.chemsens.ethz.ch. University of Regensburg. www.presens.de. (1) Wolfbeis, O. S., Ed. Fiber Optic Chemical Sensors and Biosensors; CRC Press: Boca Raton, Florida, 1991. (2) Spichiger-Keller, U. E. Chemical Sensors and Biosensors for Medical and Biological Applications, Wiley VCH: Weinheim, Germany, 1997. (3) Mohr, G. J.; Lehmann, F.; Grummt, U. W.; Spichiger-Keller, U. E. Anal. Chim. Acta, 1997, 344, 215. (4) Mohr, G. J.; Spichiger, U. E. Anal. Chim. Acta 1997, 351, 189. (5) Mohr, G. J.; Demuth, C.; Spichiger, U. E. Anal. Chem. 1998, 70, 3868. (6) Mohr, G. J.; Tirelli, N.; Spichiger, U. E. Anal. Chem. 1999, 71, 1534. (7) Mohr, G. J.; Langhals, H.; Jona, W.; Spichiger, U. E. Anal. Chem. 2000, 72, 1084. ‡
10.1021/ac000945z CCC: $20.00 Published on Web 01/27/2001
© 2001 American Chemical Society
with two different fluorescence spectra corresponding to the complexed and uncomplexed forms are used, but they either have to be excited at different wavelengths, or the emission at different wavelengths has to be measured.8 Thus, drifts of light sources or detectors compromise the ratiometric signal. The measurement of luminescence lifetime is also independent of the overall signal intensity but most indicator dyes have lifetimes in the nanosecond range, which requires expensive instrumental setup.9 The present approach combines an inert phosphorescent dye with long luminescence lifetime and an amine-sensitive fluoro reactand with short lifetime. When using modulated excitation light, the phosphorescent dye provides a constant phase angle that is modified by analyte-dependent changes in fluorescence signal of the reactand. Consequently, a phase shift of modulated light caused by the analyte rather than the overall change in luminescence signal is measured. Because measurements are performed at low modulation frequencies adjusted to the longlived reference dye, a cheap and small experimental sensor setup can be developed. EXPERIMENTAL SECTION Reagents. For membrane preparation, poly(vinyl chloride) (PVC, high molecular weight), bis(2-ethylhexyl)sebacate (DOS), and tetrahydrofuran (THF) were obtained from Fluka. Polyacrylonitrile (PAN) was from Hoechst (Kelheim, Germany). The synthesis and the optical properties of 4-N,N-dibutylamino-4′-trifluoroacetylstilbene (ETHT 4003) and copolymer CP2, composed of the monomers hexyl methacrylate and 4-[N,N-bis(11-methacryloxyundecyl)amino]-4′-trifluoroacetyl-stilbene (ETHT 4014), have already been described in detail elsewhere.3,6 The Ru(dpp)/PAN beads composed of ruthenium(II)tris(4,7-diphenyl-1,10-phenanthroline) perchlorate (Ru(dpp)) dissolved in PAN were synthesized according to ref 11. Amine solutions were prepared by dissolving the appropriate amount of 1-butylamine in 0.1 M sodium hydroxide solution. The high pH value is necessary to provide 1-butylamine in the (8) Kopelman, R.; Song, A.; Parus, S. Anal. Chem. 1997, 69, 863. (9) Werner T.; Fahnrich, K.; Huber, C.; Wolfbeis, O. S. Photochem. Photobiol. 1999, 70, 585. (10) Lide, D. R. Handbook of Chemistry and Physics, 74th ed.; CRC Press: Boca Raton, 1993-1994, 8, 43-45). (11) Huber, C.; Klimant, I.; Krause, C.; Werner, T.; Mayr, T.; Wolfbeis, O. S. Fresenius J. Anal. Chem. 2000, 368, 196.
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Figure 1. Luminescence excitation spectra of (a) Ru(dpp)/PAN and (b) ETHT 4003 in PVC/DOS, and (c) emission spectrum of the blue LED.
electrically neutral form and not in the ammonium form. The correct amine concentration was calculated by using the Henderson-Hasselbach equation and the pKa value of 1-butylamine.10 Membrane Preparation. The sensor membrane M1 was obtained by dissolving 40 mg of PVC, 85 mg of DOS, and 0.5 mg of ETHT 4003 in 0.8 mL of THF and adding 60 mg of the Ru(dpp)/PAN beads. M2 was obtained by dissolving 120 mg of copolymer CP2 (containing 0.22 mg of ETHT 4014) in 0.8 mL of THF and adding 30 mg of the Ru(dpp)/PAN beads. The mixtures were spread on a polyester support using a knife at a spacer distance of 100 µm to the polyester support. The resulting polymer membranes containing the dispersed beads were placed in ambient air for drying. Apparatus. The fluorescence intensity measurements were performed on a Perkin-Elmer LS 50 B spectrofluorimeter by placing the membranes in a cuvette and adding the respective analyte solutions. Phase-angle measurements were performed using a dual-phase lock-in amplifier (DSP 830, Stanford Research Inc.) for sine wave modulation of the blue LED at a frequency of 45 kHz and for detection. The optical system consisted of a blue LED (λmax 470 nm, Nichia, Nu¨rnberg, Germany), equipped with a blue band-pass filter (BG 12, Schott, Mainz, Germany), a bifurcated glass fiber bundle (diameter, 2 mm), and a red-sensitive PMT module (H5701-02, Hammamatsu, Hersching, Germany) equipped with a long-pass filter (Schott, Mainz, Germany). The sensor membrane was placed in a flow-through cell and the fiber bundle was in contact with the outside of the sensor membrane. Solutions were pumped through the cell at a flow rate of 1.8 mL min-1 by using a peristaltic pump from Minipuls (Gilson, Villiers, France) and silicone tubings of 1.0 mm i.d. RESULTS AND DISCUSSION Sensor mechanism. Two different fluorescent dyes are present in one sensor layer. The first is the polymer-based fluoro reactand, which changes its fluorescence intensity upon interaction with the analyte. The second is the phosphorescent dye which, for the sake of chemical and physical inertness, is embedded in poly(acrylonitrile) beads. Both dyes exhibit overlapping excitation and emission spectra (Figures 1 and 2). The fluoro reactand exhibits a short fluorescence lifetime. Consequently, there is virtually no phase shift between the modulated light of the LED and the emitted light of the reactand (Figure 3). The ruthenium reference, in contrast, has a lifetime in the range of microseconds, causing a strong phase shift as compared to the light source. If 1054
Analytical Chemistry, Vol. 73, No. 5, March 1, 2001
Figure 2. Luminescence emission spectra of (a) copolymer CP2, (b) ETHT 4003 in PVC/DOS, (c) Ru(dpp)/PAN beads dispersed in PVC/DOS, and (d) cutoff range of the band-pass filter OG 530.
Figure 3. Phase angle shift of the overall luminescence, φm, the reference, φref, and the fluoro reactand, φfr. Luminescence of the fluoro reactand before (A) and after (B) interaction with 1-butylamine.
the reactand is present in its unreacted trifluoroacetyl form with strong luminescence at around 610 nm, then the amplitude composed of the luminescence signal of reactand and reference luminophore is measured (A). This causes a shift of the phase angle of the overall luminescence, φm, as compared to the phase angle of the reference dye, φref. If the reactand reacts with 1-butylamine and no luminescence of the reactand around 610 nm is present, then the overall amplitude consists only of the reference dye (B), and a phase angle, φm, being equal to the phase angle of the reference, φref, is observed. Therefore, the phase angle, φm, directly reflects the intensity of the fluoro reactand and, consequently, the concentration of 1-butylamine. The modulation frequency is adjusted to the decay time of the reference dye. The calculation of the phase shift and of cot φ is performed according to
cot φm ) cot φref + 1/sin φref‚Afr/Aref
(1)
provided that the phase angle of the fluoro reactand, φfr, is equal to zero, and φref is constant.11,12 Consequently, the measured phase angle, φm, depends on the ratio of amplitudes of fluoro reactand, Afr, and reference beads Aref, because φref is constant.
Optical Properties of the Fluorogenic Reactand ETHT 4003, the Copolymer CP2, and the Ru(dpp)/PAN beads. The fluorogenic reactands ETHT 4003 and 4014 belong to a class of stilbene dyes which exhibit high molar extinction coefficients, large fluorescence quantum yields, short fluorescence lifetimes, and Stokes’ shifts of e200 nm.3,6 In general, they consist of the stilbene moiety and terminal electron-donor and -acceptor groups. In the present case, the alkylamino group serves as an electron donor and the alkyl chains attached to the nitrogen atom render the reactand polymer soluble. In the case of the copolymer CP2 based on ETHT 4014, the alkyl chains were chemically modified by polymerizable methacrylate groups. The electronaccepting trifluoroacetyl group can perform reversible chemical reactions with nucleophilic species such as amines and alcohols to form hemiaminals, zwitterions, Schiff bases, or hemiacetals. This causes the fluorescence of the trifluoroacetyl form to disappear.3-6 The phosphorescent beads based on ruthenium(II)tris(4,7diphenyl-1,10-phenanthroline)perchlorate dissolved in poly(acrylonitrile) exhibit excitation and emission at wavelengths that are typical for organo-ruthenium complexes, namely at 460 and 610 nm, respectively.13 However, by using the oxygen-impermeable poly(acrylonitrile) as the polymer matrix, one prominent feature of these metal complexes widely used for oxygen sensing13 is eliminated; namely, its quenchability by molecular oxygen. Aniline or iodobutane also do not affect the luminescence of Ru(dpp) in the beads. This is of utmost importance because the luminescence of the reference dye must not be affected by ions or electrically neutral molecules. Spectral Compatibility Between Fluoro Reactands in Polymers and Ru(dpp)/PAN Beads. One important requirement for DLR is a significant match between the optical properties of the indicator dye, the reference, and the light source. This is necessary to allow the use of one common light source and one common detector in the experimental setup. Furthermore, a short fluorescence lifetime for the indicator and a long lifetime for the reference are required. Ru(dpp)/PAN beads exhibit luminescence lifetime around 6 µs, whereas the fluorescence lifetime of ETHT 4003 measured in ethyl acetate was found to be 2.1 ns.3 Ru(dpp)/PAN beads have an excitation maximum at around 460 nm, which is matched by the excitation maxima of ETHT 4003 in PVC/DOS and ETHT 4014 in copolymer CP2 (both at 468 nm) (Figure 1). The maximum of the blue LED is at 470 nm. The overlap of the luminescence emission is, however, less pronounced because the maximum of the reference dye in the beads is around 610 nm, whereas the maximum of ETHT 4003 in PVC/DOS is at 561 nm, and is 522 nm in the unpolar copolymer CP2 (Figure 2). This difference in emission is due to the pronounced positive solvatochromism of both ETHT 4003 and 4014, resulting in optical properties that are strongly affected by the polarity of the polymer matrix.3,6 It is possible to improve the spectral overlap in emission by using more polar plasticizers, such as o-cyanophenyloctyl ether (CPOE),14 which due to the positive solvatochromism of ETHT 4003, shifts its emission (12) Huber, C.; Klimant, I.; Krause, C.; Wolfbeis, O. S. Anal. Chem. 2001, in press. (13) Klimant, I.; Wolfbeis, O. S. Anal. Chem. 1995, 67, 3160. (14) Papkovsky, D. B.; Mohr, G. J.; Wolfbeis, O. S. Anal. Chim. Acta, 1997, 337, 201.
Figure 4. Luminescence spectra of a sensor layer, M1, composed of plasticized PVC, ETHT 4003, and Ru(dpp)/PAN beads in contact with water and aqueous 1-butylamine, all at pH 13.0.
maximum up to 610 nm (as compared to 561 nm in DOS and 522 nm in CP2). Furthermore, recent investigations have shown that a change in polarity by, for example, using the highly polar plasticizer CPOE instead of the relatively unpolar plasticizer DOS does not significantly affect the sensitivity toward amines.15 The situation is slightly different for copolymer membranes. Although they are advantageous over membranes based on plasticized PVC in terms of operational stability and shelf life, their preparation is more sophisticated. They have to be synthesized in a way such that no plasticizers are required, which can be achieved by, for example, using the monomer hexyl methacrylate together with a reactand methacrylate such as it is for the case of CP2. However, the resulting copolymer layers exhibit low polarity, which shifts the emission maximum to shorter wavelengths and decreases the spectral overlap between reactand and reference beads. To improve the optical properties without affecting the sensitivity, the use of more polar methacrylates and acrylates with 2-cyanoethyl, N,N-diethylaminoethyl or 2-alkoxyethyl groups for copolymer preparation is conceivable. To use such copolymers for a wider range of analytes (e.g., alcohols, where the addition of quaternary ammonium catalysts is required4), the copolymerization of methacrylate derivatives of quaternary ammonium ions is conceivable, as well. 16 Performance of 1-Butylamine-Sensitive Membranes M1 and M2. The fluorescence emission maximum of ETHT 4003 in M1 was at 561 nm, whereas the reference beads exhibited luminescence emission at wavelengths >590 nm. Exposure to airsaturated aqueous 1-butylamine resulted in a decrease in fluorescence intensity of the reactand due to the chemical reaction of the trifluoroacetyl group with 1-butylamine, but the emission of the inert reference luminophore remained unchanged (Figure 4). The sensor membrane, M1, exhibited the highest sensitivity to 1-butylamine in the 1-100 mM range. A shift in the phase angle φ from 13.4 to 50.8° was observed (corresponding to a shift of cot φ from 4.02 to 0.82) when changing from 0.1 M sodium hydroxide solution to 0.5 M 1-butylamine (Figure 5). This was due to the changes in the ratio of intensities of the reference dye and the reactand, causing the phase angle to increase (Figure 3). To evaluate the cross-sensitivity to molecular oxygen, M1 was also exposed to 1-butylamine solutions deoxygenated by addition of 0.5% (w/v) sodium sulfite. The corresponding calibration plot is (15) Mohr, G. J.; Nezel, T.; Spichiger-Keller, U. E. Anal. Chim. Acta, 2000, 414, 181.
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Figure 5. Calibration function of M1 and M2 on exposure to various concentrations of aqueous air-saturated (o) and deoxygenated (×) 1-butylamine. The solid lines were calculated according to ref 5.
shown in Figure 5 and indicates low quenchability of the beads by oxygen. The relative standard deviation for sensor signals at concentrations of 10, 50, and 100 mM aqueous 1-butylamine (n ) 8) was determined to be 0.4, 0.2, and 0.1%, respectively, where the measurements were performed during a 2-h period. The forward response time, t95 (time needed for 95% of the total signal change to occur), of a PVC/DOS/bead membrane was in the range of 5 min, whereas the time for the reverse response was in the range of 8 min. A reference membrane composed of only PVC, DOS, and beads was prepared in order to investigate any effect of 1-butylamine on the luminescence of the Ru(dpp)/PAN beads. No phase shift upon exposure to 1-butylamine was observed. Because there is no effect of the analyte on the luminescence of the beads, and because the layer’s response is not compromised by the presence of the beads, the sensor’s selectivity behavior for different amines is similar to sensor layers without beads.5,6 The sensitivity of M1 is slightly smaller than that of an optode membrane for 1-butylamine based on a lipophilic calix[6]arene hexaester and a pH indicator dye which exhibits a sensitive range from 1 to 100 mM and a detection limit around 0.1 mM.17 Membrane M2 exhibited comparable behavior to M1 in terms of sensitivity to 1-butylamine and spectral behavior (Figure 6). However, due to the smaller spectral overlap in luminescence emission between reactand and reference beads, the phase shift was significantly smaller. Thus, the shift in the phase angle φ was from 28.4 to 54.2° (corresponding to a shift of cot φ from 1.73 to 0.73) when changing from 0.1 M sodium hydroxide solution to 0.5 M 1-butylamine (Figure 5). Figure 5 shows the corresponding (16) Antonisse, M. M. G.; Lugtenberg, R. J. W.; Egberink, R. J. M.; Engbersen, J. F. J.; Reinhoudt, D. N. Anal. Chim. Acta 1996, 332, 123. (17) Chan, W. H.; Lee, A. W. M.; Wang, K. Analyst 1994, 119, 2809.
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Figure 6. Luminescence spectra of a sensor layer M2 composed of copolymer CP2 and Ru(dpp)/PAN beads in contact with water and aqueous 1-butylamine, all at pH 13.0.
calibration plots of M2 for air-saturated and deoxygenated 1-butylamine solutions. The relative standard deviation for sensor signals at concentrations of 10, 50, and 100 mM 1-butylamine (n ) 8) was determined to be 0.4, 0.3, and 0.4%. The covalent immobilization of ETHT 4014 in the copolymer affected the response time in that the forward response time of M2 was in the range of 10-15 min, whereas the time for the reverse response was in the range of 15-30 min. CONCLUSION Fluoro reactands are new tools for the molecular recognition of neutral analytes. It is possible to use fast reversible chemical reactions for the development of optical sensors to detect aliphatic amines. The presented approach of using fluoro reactands together with phosphorescent beads for referencing purposes is a further step in the development of optical sensors with simplified calibration and enhanced signal stability. With the help of the new referencing mechanism, cheap and small electronic devices can be used to evaluate the sensor signals. ACKNOWLEDGMENT This work was supported in part by the Swiss National Science Foundation within project 20-55588.98 and by the Swiss Federal Institute of Technology project TH-2/99-1 (Reg. No. 03145). This support is gratefully acknowledged. G.J.M. also thanks Frank Lehmann (www.dyomics.com) for financial support and stimulating discussions within this investigation. Received for review August 10, 2000. Accepted November 13, 2000. AC000945Z
