Anal. Chem. 1998, 70, 3452-3457
Reduced Dicyanovinyl Dyes: A Chromophore To Be Used for Optical Sensing in the Red and Near-Infrared Spectral Range Daniel Citterio, Luzi Jenny, and Ursula E. Spichiger*
Center for Chemical Sensors/Biosensors and bioAnalytical Chemistry, Swiss Federal Institute of Technology (ETH), Technoparkstrasse 1, CH-8005 Zu¨ rich, Switzerland
Reduced dicyanovinyl dyes, a new type of pH-sensitive chromophores with absorption bands reaching into the near-infrared (near-IR) spectral range, are presented. Side-chain residues with different properties were attached to the chromophore backbone to influence the pKa values of the indicator dyes. The resulting dyes were suitable to be incorporated into lipophilic optical sensing bulk membranes (optodes) made from plasticized poly(vinyl chloride). The characteristics of the indicators were studied in pH-sensitive membranes. Full signal reversibility for protonation and deprotonation and high chromophore stability were observed. As an example of application for ion-sensing, a calcium-selective optode is demonstrated. The dynamic measuring range for calcium was 10-6-10-1 M in aqueous solutions, buffered at pH 2.7. The response behavior, short-time reproducibility, and stability of the optodes are discussed. New measuring techniques such as integrated optics allow the miniaturization of optical sensors (optodes) and therefore widen the range of application of these devices. Freiner et al. have demonstrated the feasibility of the combination of an optode based on a plasticized poly(vinyl chloride) (PVC) sensing membrane with integrated optics.1 Different approaches for realizing totally integrated optical sensors (TIOSs), where both the light source and the detector are fabricated from the same material, have been presented.2,3 These modules eliminate the necessity of external components and lead to small, lightweight, rugged measuring platforms. They are suitable for fabricating sensor arrays on a single chip which can be mass produced. The materials of choice for manufacturing semiconductor light waveguides are most often III-V semiconductors. A material with very suitable performance in monolithically integrated sensors is the ternary compound AlxGa1-xAs, which emits light in the wavelength range between 750 and 870 nm, depending on the composition.4 For this reason,
the near-infrared (wavelength λ > 780 nm) spectral range is preferred as the working range in integrated optical techniques. A further important advantage of the near-IR spectral range is the high efficiency of light coupling into a dielectric waveguide, which is a prerequisite for realizing integrated optical sensors with high sensitivity.5 In addition, spectral interference caused by unspecific absorption of the sample itself is generally lower in the near-IR range than in the visible.6 In optodes based on an ion-exchange mechanism, as developed by Simon and co-workers,7-11 the chemical recognition step is transduced into an optical signal by a pH-sensitive chromogenic compound, called a chromoionophore. To adapt established optode systems to recent developments in optical technology, new chromophores working in the near-IR spectral range have to be applied. We therefore were looking for dyes fulfilling the following requirements in order to be useful in optical sensors based on plasticized PVC membranes: (i) pH-dependent optical properties; (ii) long-wavelength absorption band; (iii) appropriate pKa values to guarantee sensor reversibility and to cover a wide dynamic range; (iv) high selectivity for protons compared to metal cations; (v) sufficient solubility in plasticized PVC membranes or a chemically reactive site for covalent immobilization onto a different membrane matrix; (vi) high chemical stability and photostability; and (vii) appropriate mobility in the lipophilic membrane phase in order to keep the sensor response time as short as possible. Recently, we have reported on an attempt to incorporate charged cyanine and streptocyanine dyes into optical sensing membranes.12 Charged polymer support materials had to be applied to overcome solubility problems. As a consequence, the combination with conventional, highly lipophilic ligands as used
* Corresponding author. E-mail:
[email protected]. Fax: +41 1 445 12 33. (1) Freiner, D.; Kunz, R. E.; Citterio, D.; Spichiger, U. E.; Gale, M. T. Sensors and Actuators B 1995, 29, 277-285. (2) Kunz, R. E. Proc. SPIEsInt. Soc. Opt. Eng. 1991, 1587, 98-113. (3) Zappe, H. P.; Arnot, H. E. G.; Kunz, R. E. Sens. Actuators A 1994, 41-43, 141-144. (4) Saleh, B. E. A.; Teich, M. C. Fundamentals of Photonics; John Wiley & Sons: New York, 1991.
(5) Kunz, R. E.; Kempen, L. U. Proc. SPIEsInt. Soc. Opt. Eng. 1994, 2068, 69-86. (6) Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 63, 321A-327A. (7) Seiler, K.; Simon, W. Analytica Chimica Acta 1992, 266, 73-87. (8) Morf, W. E.; Seiler, K.; Lehmann, B.; Behringer, C.; Hartman, K.; Simon, W. Pure Appl. Chem. 1989, 61, 1613-1618. (9) Bakker, E.; Simon, W. Anal. Chem. 1992, 64, 1805-1812. (10) Wang, K.; Seiler, K.; Morf, W. E.; Spichiger, U. E.; Simon, W.; Lindner, E.; Pungor, E. Anal. Sci. 1990, 6, 715-720. (11) Spichiger, U. E. Chemical sensors and biosensors for medical and biological applications; Wiley-VCH: Weinheim, Germany, 1998. (12) Citterio, D.; Ra´sonyi, S.; Spichiger, U. E. Fresenius’ J. Anal. Chem. 1996, 354, 836-840.
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for optical ion sensing13 was impossible; however, pH-sensitive optode membranes have been realized. In a second paper, we sucessfully applied electrically neutral merocyanines as chromophores in pH-sensitive, lipophilic PVC membranes.14 The basicity of the indicators was not high enough to realize metal ion-selective optical sensors. Reversibility would not have been achieved after contact of the sensing membrane with the metal ion, because the complex binding forces are too high with respect to the dye basicity. None of these dyes was able to meet all the requirements listed before. One type of earlier developed indicator dyes,15 which was used in ion-exchange or -coextraction type optical sensors, complies with most of the requirements stated above. Specially, this class of pH indicators covers a wide range of pKa values, leading to a wide dynamic range for the respective sensors. The variation in basicity was achieved without changing the chromophore backbone structure, but with varying the chemical properties of side chains attached to it. The benzophenoxazine type dye Nile blue was used as the template structure. As a result, the optical properties remained almost unchanged over the whole series of indicators. The most intensive absorption band, however, lies in the visible spectral range, and the Nile blue derivatives are, therefore, not fulfilling the most important prerequisite to be usable in available TIOS type devices. One of our goals was to find a new template structure leading to red or near-IR absorbing dyes and allowing the pKa values to be changed by variations of side-chain residues, without affecting the optical properties to a large extent. In this paper, we report on reduced dicyanovinyl dyes, a new type of chromophore with pH-dependent absorption spectra and with characteristics promising for application in optical sensing membranes. EXPERIMENTAL SECTION Materials. The chemical structures of the pH-sensitive reduced dicyanovinyl dyes ETHT 5003-5009 are depicted in Figure 1. The synthesis as well as the structure elucidation is described elsewhere.16 Poly(vinyl chloride) (PVC, high molecular grade), methyltridodecylammonium chloride (MTDDACl), 2-nitrophenyloctyl ether (o-NPOE), bis(2-ethylhexyl) sebacate (DOS), calcium ionophore I (ETH 1001), and tetrahydrofuran (THF) were obtained from Fluka AG (Buchs, Switzerland). All buffer components (boric acid, formic acid, KH2PO4, KCl, KOH, HCl), as well as tetrabutylammonium hydroxide solution, calcium chloride, and methanol for spectrophotometric measurements (puriss p.a.), were purchased from Fluka AG. Aqueous universal buffers were 0.1 M each in boric acid, KH2PO4, and KCl, and the pH was adjusted with 1 M KOH to the appropriate value. Solutions used for calcium measurements were buffered at pH 2.7 with 0.1 M formic acid, adjusted with 1 M KOH. All solutions were prepared from doubly quartz-distilled water. Instruments. Absorption measurements were performed at room temperature, using an UVIKON 930 double-beam spectrophotometer. Herasil quartz cuvettes (d ) 1 cm) were used for (13) Selectophore: Ionophores, membranes, mini-ISE; Catalogue Fluka Chemie AG: Buchs, Switzerland, 1996. (14) Citterio, D.; Jenny, L.; Ra´sonyi, S.; Spichiger, U. E. Sens. and Actuators B 1997, 38-39, 202-206. (15) Bakker, E.; Lerchi, M.; Rosatzin, T.; Rusterholz, B.; Simon, W. Anal. Chim. Acta 1993, 278, 211-225. (16) Jenny, L.; Citterio, D.; Spichiger, U. E. Manuscript in preparation.
Figure 1. Structures and abbreviations of the reduced dicyanovinyl chromophores discussed.
optical characterization of the dyes in methanolic solutions. pH measurements were performed with a combined glass electrode (Orion 8103SC). Membrane Preparation. For the composition of the sensing membranes, refer to Table 1. The membrane components were dissolved in 1 mL of freshly distilled THF. Two hundred microliters of the resulting solution was cast on a rotating quartz disk (35 mm diameter, 2 mm thickness) in a dust-free, THFsaturated environment, using a spin-on device.7 After evaporation of the solvent, homogeneous membranes of approximately 1-3µm thickness on the quartz support were obtained. Experimental Procedure. Tetrabutylammonium hydroxide solution was used for deprotonation of the dyes in methanolic solutions. Continuous measurements with optical sensors were done by pumping the sample solutions at a flow rate of 1.5 mL‚min-1 through a flow-through cell17 containing two sensing membranes. All experiments were performed with the cell mounted in the spectrophotometer in the transmittance mode. Calculations. Molal single-ion activity coefficients were calculated according to the Pitzer formalism.18 The apparent pKa (17) Dinten, O.; Spichiger, U. E.; Chaniotakis, N.; Gehrig, P.; Rusterholz, B.; Morf, W. E.; Simon, W. Anal. Chem. 1991, 63, 596-603. (18) Pitzer, K. S. In Activity coefficients in electrolyte solutions; Pitzer, K. S., Ed.; CRC Press: Boca Raton, FL, 1991; pp 75-153.
Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
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Table 1. Composition of the Optode Membranes membranea
dye, concn (mmol/kg)b
additive, concn (mmol/kg)
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11c
ETHT 5003, 20 ETHT 5004, 20 ETHT 5004, 20 ETHT 5004, 20 ETHT 5004, 20 ETHT 5004, 20 ETHT 5006, 13 ETHT 5007, 20 ETHT 5008, 20 ETHT 5009, 20 ETHT 5009, 10
MTDDACl, 20 MTDDACl, 2 MTDDACl, 10 MTDDACl, 20 MTDDACl, 30 MTDDACl, 40 MTDDACl, 20 MTDDACl, 20
a The compounds were dissolved in 1 mL of freshly distilled THF together with 51 mg of PVC and 102 mg of the plasticizer DOS. b The concentrations refer to the membrane total mass (ionophore, dye, additive, PVC, plasticizer). The total weight of all membrane compounds (excluding the solvent THF) was 167 mg. c Membrane M11 additionally contained 100 mmol/kg of the calcium ionophore ETH 1001.
values of the dyes in the membrane phase were estimated by analyzing the slope of the optode response curve during titration of the sensor membranes. For this purpose, the optode membranes were equilibrated with universal buffer solutions in the flow-through cell. Buffer solutions with identical concentrations but variable pH were used in all experiments in order to account for changing ionic strength. The measured absorbance was plotted against the pH of the respective buffer solutions. The pH value at the point of maximum slope is reported as the apparent pKa. This value coincides with the relative acidity of the chromophores, referred to the specific membrane environment and the specific measuring conditions. RESULTS AND DISCUSSION During synthesis of the dicyanovinyl dye 1,3-bis(dicyanomethylene)-2-[4′-(N,N-dialkylamino)phenylimino]indan,19 the reduced dicyanovinyl dye ETHT 5003, a new type of chromophore system, which has, to our knowledge, not been described before, was isolated as a byproduct (for NMR data and mass spectrum, see ref 16). It is an electrically neutral dye molecule showing pH-dependent color properties. Formally, the structure is derived from the dicyanovinyl indicator by addition of hydrogen to the C-N double bond. The presence of a functional chemical group in the molecule, the free secondary amino group, allows a variety of modifications to be made with the template structure, leading to the series of dyes ETHT 5004-5009. Spectral Behavior and Optical Properties of the Chromophores in Methanolic Solutions. Representative absorption spectra of the pH-sensitive chromophores ETHT 5003 and ETHT 5009 in methanolic solution are shown in Figure 2. The spectroscopic properties of all the reduced dicyanovinyl dyes discussed in this paper are summarized in Table 2. In the protonated electrically neutral state (CH), no absorption bands are observed in the near-IR range. In the negatively charged deprotonated state (C-), however, broad bands extending into the near-IR range appear. Compared with cyanine and streptocyanine dyes, the (19) Bello, K. A.; Cheng, L.; Griffiths, J. J. Chem. Soc., Perkin Trans. 2 1987, 815-818.
3454 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
Figure 2. Representative absorption spectra of two pH-sensitive reduced dicyanovinyl dyes in methanolic solution. Electrically neutral protonated state (- - -) and negatively charged deprotonated state (s). Table 2. Spectral Properties of the Dyes ETHT 5003-5009: λmax, Wavelength of Maximum Absorption, and Emax, Maximum Molar Decadic Absorption Coefficient protonated form
deprotonated form
dye
λmax (nm)
max (L mol-1 cm-1)
λmax (nm)
max (L mol-1 cm-1)
ETHT 5003 ETHT 5004 ETHT 5005 ETHT 5006 ETHT 5007 ETHT 5008 ETHT 5009
588 597 609 604 513 525 500
8000 8000 8000 ndb 3000 3000 3000
717 759 a 752 576 594 637
12000 12000 a ndb 7000 7000 9000
a No acidic proton. b Not determined (the structure of the dye molecule was not confirmed).
extinction coefficients are much lower, and the absorption bands are less sharp. A deprotonation at the carbon atom in position 2 of the five-membered ring of the indan subsystem was assumed to be responsible for the spectral changes observed upon treatment of the dyes with acid or base. This assumption is strongly supported by the fact that the spiro compound ETHT 5005, missing a proton in the C-2 position, did not show any spectral changes upon treatment with base. This excludes the possibility that a deprotonation and protonation at a basic nitrogen center is leading to the spectral changes observed for ETHT 5003. In addition, the working schemes of the membranes, which are based on the mass balances presented later in eqs 1 and 2, can be explained only if the chromophore is deprotonated into a negatively charged species. We assume the structure shown in Figure 3 to represent the main chromophoric system of the dye in its deprotonated state, responsible for the low-energy transition. The two dicyanomethylene residues in the molecule are known to act as highly efficient
Figure 3. Primary chromophoric system of the reduced dicyanovinyl dyes in the anionic, deprotonated state.
electron acceptors,20 whereas the free electron pair resulting from the deprotonation could act as a donor. The broad absorption bands indicate an intramolecular charge-transfer type of chromophore.21 The exact position of the red or near-IR absorption band is influenced by the donor-acceptor properties of the residue R, attached to the indan subsystem at C-2. Electron donors lead to a bathochromic spectral shift, whereas electron-withdrawing groups result in a hypsochromic shift. During experiments with the chromophore ETHT 5003 in methanolic solution, a very high basicity of the compound was noted. Deprotonation was achievable only under strongly basic conditions (concentrated KOH in methanol). Using tetrabutylammonium hydroxide solution as a base, the deprotonated state was reached after the addition of even a small amount of the reagent, therefore lowering the apparent basicity of the dye in the solution to a great extent. This indicates that the presence of a lipophilic cation is required in order to stabilize the anion resulting after deprotonation. Alkylation at the amine nitrogen of ETHT 5003 leads to the dye ETHT 5004. This compound shows spectral behavior very similar to that of the precursor ETHT 5003, which further supports the assumption of a protonation/deprotonation reaction at the carbon atom C-2. A slightly increased basicity was noted, due to the inductive effect of the alkyl residue. The substitution with electron-withdrawing groups (ETHT 5007, 5008, and 5009) resulted in dyes with lower basicity. pH-Sensitive Optodes. In DOS-plasticized PVC membranes containing only the indicator dye ETHT 5003 and no further additives, no deprotonation of the compound based on cation exchange could be achieved, even in highly basic samples (pH 14). Replacing the plasticizer DOS by the more polar o-NPOE, the situation remained unchanged. However, when lipophilic cationic sites were added in the form of the anion-exchange salt MTDDACl (100 mol % relative to the amount of indicator), reversible deprotonation and protonation of the dye became possible. Further experiments confirmed that ETHT 5003 incorporated into a lipophilic membrane phase can be deprotonated only in the presence of freely available lipophilic cations (R+) in the membrane phase. In analogy to the observations in methanolic solutions, the lipophilic cation is necessary as a counterion for the stabilization of the resulting anionic dye molecule. Figure 4 shows the absorption spectra of an optode with membrane composition M1 incorporating the dye ETHT 5003. The sensor was exposed to a series of universal buffer solutions with varying pH. Due to the high amount of cationic additive R+ (100 mol %), a reversible coextraction mechanism of protons and anions between the aqueous sample solution (aq) and the organic (20) Griffiths, J. In Modern Colorants: Synthesis and Structure; Blackie Academic & Professional: London, 1995; Vol. 3, pp 40-62. (21) Fabian, J.; Nakazumi, H.; Matsuoka, M. Chem. Rev. 1992, 92, 1197-1226.
Figure 4. Absorption spectra of a pH-sensitive optode (membrane M1, DOS/PVC/MTDDACl) with chromophore ETHT 5003 after equilibration with universal buffer solutions (pH 6.0-12.0; H3BO3/KH2PO4/ KCl/KOH) and KOH (pH 13.0-14.0) in a flow-through system. The absorption maxima are at 579 nm for the protonated and 734 nm for the deprotonated forms.
Figure 5. Short-time reproducibility of the absorbance of a pHsensitive optode (membrane M1) with chromophore ETHT 5003, alternately equilibrated with 0.01 M hydrochloric acid and 0.01 M potassium hydroxide solution in continuous-flow operation.
membrane phase (m) is postulated:
CHm + R+X-m h C-m + R+m + H+aq + X-aq
(1)
The lipophilicity of the anions present in the buffer solution directly influences the degree of protonation of the chromophore. At constant pH value, lipophilic anions lead to a higher degree of protonation than the more hydrophilic counterparts. To avoid a sudden change of the anion in the buffer system, a universal buffer solution was chosen for all titration experiments (see Figure 4). In addition, a constant high background ionic strength (0.1 M KCl) was adjusted. Figure 5 illustrates the absorbance response versus time for the optode with membrane M1 (see Figure 4) while it was exposed to 10-2 M hydrochloric acid and 10-2 M potassium hydroxide, alternately. The deprotonation and protonation process of ETHT 5003 incorporated into an optode membrane is obviously fully reversible. Even upon exposure to more concentrated potassium hydroxide solution (1 M), stable and reproducible signals were observed. After the sensor was stored in contact with buffer solution (pH 7.0), but protected from light, for several days, signals identical to those obtained with the unused membranes were Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
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Table 3. Apparent pKa Values of the Dyes ETHT 5003-5009 Embedded in Plasticized PVC Optode Membranes dye (membrane type) ETHT
5003 (M1) ETHT 5004 (M2) ETHT 5006 (M7) ETHT 5007 (M8) ETHT 5008 (M9) ETHT 5009 (M10)
apparent pKaa
composition
8.8 9.5 8.5 4.8
