Anal. Chem. 1998, 70, 4309-4313
Neutral Carrier-Type Ion Sensors Based on Sol-Gel-Derived Membranes Incorporating a Bis(crown ether) Derivative by Covalent Bonding Keiichi Kimura,*,† Takenobu Sunagawa,‡ Setsuko Yajima,† Saori Miyake,† and Masaaki Yokoyama‡
Department of Applied Chemistry, Faculty of Systems Engineering, Wakayama University, Sakae-dani, Wakayama 640-8510, Japan, and Chemical Process Engineering, Faculty of Engineering, Osaka University, Yamada-oka, Suita, Osaka 565-0871, Japan
Sol-gel-derived membranes incorporating crown ether neutral carriers by covalent bonding have been designed for durable, nontoxic neutral carrier-type ion sensors by sol-gel processing using tetraethoxysilane, diethoxydimethylsilane, and their corresponding alkoxysilylated neutral carriers. A tetraphenylborate anion was also bound chemically to the neutral carrier-type sol-gelderived membranes for suppressing anion interference and membrane impedance. Ion-sensitive field effect transistors (ISFETs) based on the sol-gel-derived membranes modified chemically by crown ether derivatives, such as 16-crown-5 and bis(12-crown-4) derivatives, showed high performance, high sensitivity in wide cation activity ranges, short response time, and high electrode durability. Specifically, high ion selectivities of practical use in biological systems were attained in the sodium ISFETs based on the chemically modified bis(12-crown4), which has been successfully applied to sodium assay in blood sera. A variety of materials have been attempted for use as membrane support for neutral carrier-type potentiometric sensors. Although plasticized poly(vinyl chloride) (PVC) is an excellent membrane material, it is not perfect, mainly due to problems with respect to plasticizer exudation1 and protein adsorption in blood.2,3 Silicon-containing materials, whether organic or inorganic, may be promising candidates for the membrane material for the ion sensors, offering the disuse of large amounts of plasticizers and inactivity to biological systems. Cross-linked poly(dimethyl siloxane), also called silicone rubber, is an excellent membrane material for neutral carrier-type ion sensors, allowing some difficulty in the solubility or dispersibility of neutral carriers to be solved.1,4,5 †
Wakayama University. Osaka University. (1) Kimura, K.; Matsuba, T.; Tsujimura, Y.; Yokoyama, M. Anal. Chem. 1992, 64, 2508-2511. (2) Kimura, K.; Tsujimura, Y.; Yokoyama, M. Pure Appl. Chem. 1995, 67, 10851089. (3) Espadas-Torre, C.; Meyerhoff, M. E. Anal. Chem. 1995, 67, 31083114. (4) Pick, J.; To´th, K.; Pungor, E. Anal. Chim. Acta 1973, 64, 477-480. (5) Jenny, H.-B.; Riess, C.; Ammann, D.; Magyar, B.; Asper, R.; Simon, W. Mikrochim. Acta 1980, 1880, 309. ‡
S0003-2700(98)00570-8 CCC: $15.00 Published on Web 09/17/1998
© 1998 American Chemical Society
Sol-gel-derived material, which is useful for hybridization of inorganic and organic compounds,6 can also be silicon-containing for ion-sensing membranes.7-10 Appropriate fabrication of solgel-derived membranes encapsulating neutral carriers such as valinomycin can afford an excellent type of neutral carrier-type ion-sensing membranes for ion-selective field effect transistors (ISFETs).7,8 The simple encapsulation of neutral carriers in solgel-derived membranes, however, has a drawback: the encapsulated neutral carriers are still apt to exude out from the membranes to aqueous sample solutions, which makes the resulting ion sensors less durable and more toxic. This prompted us to incorporate neutral carriers into sol-gel-derived membranes by covalent bonding. We already reported sol-gel-derived ionsensing membranes modified chemically by a bis(crown ether) derivative.9 Here we report in detail the fabrication of sol-gelderived membranes incorporating 16-crown-5 and bis(12-crown4) derivatives, 1 and 2, as the neutral carrier and also tetraphenylborate salts, 3, as the anionic site, by covalent bonding and their applications to Na+-ISFETs. Their durability is compared with that of their corresponding sol-gel-derived membranes simply encapsulating neutral carriers. The practical applicability of Na+-ISFETs based on sol-gel-derived membranes containing a chemically bonded bis(12-crown-4) derivative to sodium assay in blood sera is also described. EXPERIMENTAL SECTION Syntheses. 15-Triethoxysilylmethyl-16-crown-5 (1). 15-Methylene-16-crown-5 was prepared according to a procedure in the literature.11 Trichlorosilane (1.48 mmol) and 15-methylene-16crown-5 (1.2 mmol) were dissolved in dry benzene (30 mL), and a tetrahydrofuran (THF) solution (2 mL) of H2PtCl6‚H2O (10 mg) was then added. The mixture was refluxed for 10 h with stirring in an argon atmosphere. After the benzene was evaporated, dry (6) Hench, L. L.; West, J. K. Chemical Processing of Advanced Materials; Wiley: New York, 1992. (7) Kimura, K.; Sunagawa, T.; Yokoyama, M. Chem. Lett. 1995, 967. (8) Kimura, K.; Sunagawa, T.; Yokoyama, M. Anal. Chem. 1997, 69, 23792383. (9) Kimura, K.; Sunagawa, T.; Yokoyama, M. Chem. Commun. 1996, 745746. (10) Kim., W.; Chung, S.; Park, S. B.; Lee, S. C.; Kim, C.; Sung, D. D. Anal. Chem. 1977, 69, 95-98. (11) Tomoi, M.; Abe, O.; Ikeda, M.; Kihara, K.; Kakiuchi, H. Tetrahedron Lett. 1978, 33, 3032-3034.
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ethanol (50 mL) was added to the residue, and then the mixture was stirred in an ice bath. After the catalyst was removed by 30-min centrifugation, evaporation of the excess ethanol and trichlorosilane gave an oily product of 15-triethoxysilylmethyl-16crown-5 (1). The product was used for the subsequent sol-gel processing without further purification. 1 (viscous liquid, yield 91%): 1H NMR (270 MHz, CDCl3) δ 0.8 (2H, t, J ) 7.1 Hz, SiCH2), 1.2 (9H, t, J ) 7.1 Hz, CH2CH3), 3.4-3.8 (27H, m, SiOCH2, OCH2, CH); IR (neat) 2950, 2865, and 1260 cm-1. Bis(2,5,8,11-tetraoxacyclododecylmethyl) 2-Allyl-2-methylmalonate or Bis(12-crown-4-methyl) 2-Allyl-2-methylmalonate. 2-Hydroxyethyl-12-crown-4 was obtained by a modification of a published method.12 Silver cyanide (20 g)13 was suspended in a dry benzene solution (600 mL) of 2-hydroxyethyl-12-crown-4 (90 mmol) and 2-allyl-2-methylmalonyl chloride (40 mmol). The mixture was refluxed for 7 days with stirring. After the reaction, the AgCN was filtered off, and the filtrate was passed through a 2-cm Celite column. Evaporation of the benzene afforded a crude product, which was purified by gel permeation chromatography (CHCl3) to yield an oily product of bis[(12-crown-4-)ylmethyl] 2-allyl-2-methylmalonate (viscous liquid, yield 30%): 1H NMR (270 MHz, CDCl3) δ 1.40 (3H, s, CH3), 2.60 (2H, d, J ) 7.6 Hz, CH2C), 3.5-3.8 (30H, m, CH2OCH2, OCH), 4.1-4.3 (4H, m, COOCH2), 5.0-5.1 (2H, m, CH2dCH), 5.6-5.8 (1H, m, CH2dCH); IR (neat) 2910, 2860, 1730, 1650, and 1240 cm-1; m/z (relative intensity) 535 (M+, 100). Anal. Calcd for C25H42O12: C, 56.17; H, 7.92; O, 35.91. Found: C, 56.26; H, 7.77. Bis[(12-crown-4-)ylmethyl] 2-Triethoxysilylpropyl-2-methylmalonate (2). This compound was synthesized in a way similar to that for 1, using bis[(12-crown-4-)ylmethyl] 2-allyl-2-methylmalonate as the starting ω-vinyl derivative. 2 (viscous liquid, yield 83%): 1H NMR (270 MHz, CDCl3) δ 0.10 (2H, t, J ) 7.6 Hz, SiCH2), 1.21 (9H, t, J ) 6.5 Hz, CH2CH3), 1.42 (3H, s, CH2CCH3), 1.6-1.9 (4H, m, SiCH2(CH2)2C), 3.4-3.9 (36H, m, SiOCH2, CH2OCH2, OCH), 4.1-4.3 (4H, m, COOCH2); IR (neat) 2960, 2870, 1730, 1260, 1100, and 805 cm-1. (12) Miyazaki, T.; Yanagida, S.; Itoh, A.; Okahara, M. Bull. Chem. Soc. Jpn. 1982, 55, 2005-2009. (13) Takimoto, S.; Inanaga, J.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn. 1976, 49, 2335-2336.
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Sodium (4-Allyloxyphenyl)triphenylborate. (4-Allyloxyphenyl)magnesium bromide was prepared by the conventional Grignard reaction in THF (30 mL) using allyl 4-bromophenyl ether (40 mmol) and magnesium ribbon (40 mmol). To the resulting (4allyloxyphenyl)magnesium bromide THF solution was added a dichloromethane solution (30 mL) of triphenylboron (40 mmol). The mixture was stirred for 3 h at room temperature. The solvent was evaporated off, and a saturated NaCl aqueous solution (50 mL) was added to the residue. The aqueous solution was extracted with dichloromethane (3 × 30 mL), and the combined dichloromethane solution was subjected to vacuum evaporation. The crude product was purified by recrystallization from xylene to yield a white crystal of sodium (4-allyloxyphenyl)triphenylborate: mp 260 °C dec; 1H NMR (270 MHz, CDCl3) δ 6.0-6.2 (1H, m, CH2dCH), 4.59 (2H, d, J ) 5.1 Hz, CHCH2O), 5.3-5.5 (2H, m, CH2dCH), 6.6-6.7 (19H, m, aromatic H); IR (neat) 3060, 3000, 1640, 1580, 1480, 1430, 1185, and 745 cm-1; m/z (relative intensity) 398 (M+, 10), 153 (100). Anal. Calcd for C27H24OBNa: C, 81.41; H, 6.07. Found: C, 81.60; H, 6.13. Sodium [4-(Triethoxysilylpropyloxy)phenyl]triphenylborate (3). The hydrosilylation of sodium (4-allyloxyphenyl)triphenylborate with trichlorohydrosilane, followed by treatment with ethanol, was performed in a way similar to that of the triethoxysilylpropylsubstituted neutral carriers 1 and 2. The crude product of 3 was subjected to subsequent sol-gel processing. 3 (viscous liquid, yield 91%): 1H NMR (270 MHz, CDCl3) δ 0.81 (2H, t, J ) 7.6 Hz, SiCH2), 1.24 (9H, t, J ) 7.3 Hz, CH2CH3), 3.73 (6H, q, J ) 6.8 Hz, CH2CH3), 4.3-4.5 (4H, m, SiCH2(CH2)2), 7.4-7.8 (19H, m, aromatic H); IR (neat) 2930, 2860, 1585, 1480, 1415, 1190, 1075, and 740 cm-1. Other Materials. Tetraethoxysilane (TEOS) and diethoxydimethylsilane (DEDMS) were used as received from Shin-Etsu Silicon Chemicals. Poly(3-octylthiophene) was prepared by galvanostatic electropolymerization as reported previously.14 Bis[(12crown-4-)ylmethyl] 2-dodecyl-2-methylmalonate and sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate [Na(TFPB)] were purchased from Dojindo Laboratories. Alkali and alkaline earth metal chlorides and ammonium chloride were of analytical reagent grade. Water was deionized. Human blood sera were obtained by centrifugation of whole blood samples in the presence of a small quantity of heparin sodium. The sera were subjected to the serum cation assay and the thrombogenic test as soon as possible. Fabrication. The ISFET tips, pH-sensing devices, were supplied by Shindengen Electric Inc. The gate is 10 µm wide and 370 µm long. A poly(3-octylthiophene) underlayer of about 10 µm thickness was made by casting 1 µL of its chloroform solution (1 mg of polythiophene in 100 µL of chloroform) on the gate surface, followed by vacuum-drying, prior to the sol-gel processing. TEOS (22 µL, 1.2 × 10-3 mol), DEDMS (62 µL, 3.5 × 10-3 mol), ethanol (69 µL), 0.1 M HCl aqueous solution (21 µL), a neutral carrier (1 mg), and a sodium tetraphenylborate derivative (0.2 mg) were mixed in a sample tube, and the mixture was then allowed to stand for 36 h to afford a viscous sol-gel solution. An aliquot (1 µL) of the sol-gel solution was placed on the gate surface of a commercially available pH-ISFET tip. The (14) Bobacka, J.; McCarrick, M.; Lewenstam, A.; Ivaska, A. Analyst 1994, 119, 1985-1991.
ISFET tip was then heated at 50 °C for about 2 days to yield a sol-gel-derived membrane of about 150-200 µm thickness. Conditioning of the resulting Na+-ISFETs was done by soaking in NaCl solutions of 1 × 10-3 M for 12 h. Measurements. Potential measurements were made at 25 °C using an ISFET pH/mV meter (Shindengen Electric). The source drain voltage (Vds) and current (Ids) were adjusted to 5 V and 100 µA, respectively. The reference electrode was a doublejunction-type Ag/AgCl electrode with 3 M KCl internal solution and 1 M CH3CO2Li external solution. The cation concentrations were changed by injection of high-concentration solutions to the testing solutions while stirring with a magnetic stir bar. For the durability test, the ISFETs were immersed continuously in solutions containing 140 mM NaCl and 50 mM KCl. Selectivity coefficients for Na+ or K+ with respect to other cations were determined by a mixed solution method (FIM). The background cation concentrations were 0.1 M for K+, H+, and NH4+, 0.5 M for Mg2+, Ca2+, and Li+, and 1 M for Na+. For Gran’s plot method15 on sodium assay, the volumes for sample and adding solutions were 10 and 0.1 mL, respectively. The activity coefficients (γ) were calculated according to the Davies equation: log γ ) -0.511(I)1/2/(1 + 0.33R(I)1/2) - 0.10I, using the values of ionic strength (I) and ion size parameter (R).16 The IR spectra were measured by using KBr pellets containing the neutral carrier-based sol-gel membranes. Soxhlet extraction of the sol-gel samples was performed with methanol for 3 days. Scanning electron micrography (SEM) of the ion-sensing membranes was undertaken with about 1000 magnifications at an acceleration voltage of 20 kV. RESULTS AND DISCUSSION Design of Neutral Carriers and Tetraphenylborate Salts Possessing an Alkoxysilyl Moiety. Since sol-gel-derived membranes are generally made by hydrolysis of an alkoxysilane such as TEOS, followed by condensation of the resulting silanol, the easiest way to incorporate a neutral carrier to sol-gel-derived membranes by covalent bonding is by sol-gel processing of the alkoxysilane, together with an alkoxysilylated neutral carrier. So, we incorporated a triethoxysilyl group into crown ether Na+ neutral carriers. As the Na+-selective neutral carriers, 16-crown-5 and bis(12-crown-4) derivatives17 were selected. Thus, triethoxysilylated 16-crown-5 and bis(12-crown-4) derivatives 1 and 2 were synthesized. The triethoxysilylated derivatives were obtained by hydrosilylation of their corresponding ω-vinylsubstituted derivatives with trichlorosilane, followed by treatment with ethanol. The methylene-16-crown-5 was prepared by the cyclization reaction of vinylidene dichloride and tetraethylene glycol in the presence of sodium hydride.11 Since 2,2-disubstituted bis[(12-crown-4-)ylmethyl] malonates are highly Na+-selective neutral carriers, we decided to use as the ω-vinyl-substituted bis(12-crown-4) derivative, its corresponding 2-allyl-2-methylmalonate, which was in turn accessible from 2-allyl-2-methylmalonyl chloride and hydroxymethyl-12-crown-4.12 An anion excluder, sodium tetraphenylborate, should also be immobilized onto sol-gel-derived membranes by covalent bond(15) Gran, G. Analyst 1952, 77, 661-671. (16) Davies, C. W. Ion Association; Butterworth: London, 1962. (17) Kimura, K.; Yoshinaga, M.; Funaki, K.; Shibutani, Y.; Yakabe, K.; Shono, T.; Kasai, M.; Mizufune, H.; Tanaka, M. Anal. Sci. 1996, 12, 67-70.
Figure 1. IR spectra for sol-gel-derived membranes incorporating bis(12-crown-4) and anionic sites by covalent bonding (a) and encapsulation (b) after hot methanol extraction.
ing. The corresponding triethoxysilylated derivative 3 was therefore designed, which can be prepared by a similar hydrosilylation reaction of sodium (4-allyloxyphenyl)triphenylborate. Chemical Modification of Sol-Gel-Derived Membranes. Since sol-gel-derived membranes made from solutions containing only TEOS are very brittle,8 sol-gel-derived ion-sensing membranes were fabricated by using solutions containing TEOS and DEDMS with a 1:3 ratio. An aliquot of a gelified HCl-ethanol solution containing TEOS, DEDMS, an alkoxysilylated neutral carrier (1 and 2), and alkoxysilylated sodium tetraphenylborate 3 was placed on gate surface of a pH-ISFET with a polythiophene underlayer and was then allowed to solidify in an oven. The incorporation of alkoxysilyl-substituted bis(12-crown-4) 2 and anion excluder 3 into sol-gel-derived membranes by covalent bonding was checked by comparison of IR spectra of the membranes before and after extraction by hot methanol, which can easily remove the chemically nonbonded neutral carrier and salt (Figure 1). In the IR spectrum for the sol-gel-derived membrane modified chemically by 2 and 3, strong peaks assigned to νCdO of 2 and νCdC of 3 were found even after the methanol extraction. This means that the neutral carrier and anion excluder can be stably immobilized in the membrane. A similar sol-gel-derived membrane simply encapsulating bis[(12-crown-4-)ylmethyl] 2-dodecyl-2-methylmalonate and Na(TFPB) was also employed for Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
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Figure 2. Potential response to Na+ activity changes of Na+ISFETs based on sol-gel-derived membranes modified chemically by alkoxysilyl-substituted neutral carriers 1 (O) and 2 (b), together with anion excluder 3.
Figure 4. Selectivity comparison between Na+-ISFETs based on sol-gel-derived membranes modified chemically by alkoxysilylsubstituted neutral carriers 1 and 2.
Figure 3. Time course changes of potential response for Na+ISFETs based on sol-gel-derived membranes modified chemically by alkoxysilyl-substituted bis(12-crown-4) 2, on changing Na+ concentration from 1 × 10-3 to 3 × 10-3 M.
comparison. The IR spectrum shows that most of the encapsulated neutral carrier and anion excluder was removed from the membrane by the methanol extraction. Thus, by the present solgel processing using the alkoxysilyl-substituted neutral carrier and anion excluder, the neutral carrier and anion excluder can be chemically bonded to the membranes to a great extent. Cation Response for ISFETs Based on Sol-Gel-Derived Membranes Containing Chemically Bonded Neutral Carriers. Figure 2 presents typical profiles for the potential response of Na+-ISFETs based on the sol-gel-derived membranes modified chemically by alkoxysilylated 16-crown-5 1 and bis(12-crown-4) 2, together with anion excluder 3. Both of the Na+-ISFETs exhibited high sensitivity, with a Nernstian response to Na+ activity changes in a wide activity range from 3 × 10-5 to 1 M. Surprisingly enough, the potential response is quite fast in the Na+-ISFETs, despite the covalent bonding of the neutral carriers to the ion-sensing membranes, as demonstrated in the membrane system of the bis(12-crown-4) 2 (Figure 3). Several attempts has been so far made to bond a neutral carrier to membrane supports such as PVC for neutral carrier-type ion sensors,18,19 but their potential response was generally sluggish due to the poor mobility of the neutral carrier in the membrane because the ion-sensing membranes do not include a special plasticizer. Conceivably, some mobility of the chemically bonded neutral carriers can be maintained in the present sol-gel-derived membranes containing (18) Tietje-Girault, J.; MacInnes, I.; Schroder, M.; Tennant, G.; Girault, H. H. Electrochim. Acta 1990, 35, 777-783. (19) Cross, G. G.; Fyles, T. M.; Suresh, V. V. Talanta 1994, 41, 1589-1595.
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Figure 5. Time course changes of sensitivity (slope of calibration plots) for Na+-ISFETs based on sol-gel-derived membranes incorporating bis(12-crown-4) by covalent bonding using 2 (b) and by encapsulation using bis[(12-crown-4-)ylmethyl] 2-dodecyl-2-methylmalonate (O).
1 and 2. The response time (t90) is several seconds for both of the Na+-ISFETs based on the sol-gel-derived membranes of 1 and 2, being almost comparable to those for similar ISFETs based on sol-gel-derived membranes simply encapsulating bis[(12crown-4-)ylmethyl] 2-dodecyl-2-methylmalonate.8 Ion Selectivity. Selectivity coefficients for Na+ with respect to interfering cations which exist in biological and environmental systems were determined for the Na+-ISFETs based on solgel-derived membranes incorporating neutral carriers 1 and 2 by covalent bonding (Figure 4). In the ISFETs of the 16-crown-5 membrane system, the Na+ selectivities against H+, Li+, NH4+, Mg2+, and Ca2+ are quite good, but the selectivity against K+ is not high enough, especially for Na+ assay in biological systems such as blood. To the contrary, high Na+ selectivity against K+ as well as the other interfering ions was found in the sensor of the bis(crown ether) membrane system, which is of practical use in biological systems due to the ion selectivity. Durability of ISFETs. One of the advantages of ion-sensing membranes modified chemically by a neutral carrier and an anion excluder over those simply encapsulating them is the high durability of the ion-sensing membranes and, therefore, of the
Figure 6. Correlation between actual and found values of Na+ concentrations on serum assay with ISFETs based on sol-gel-derived membranes of bis(12-crown-4) 2.
resulted ion sensors. Durability was checked with Na+-ISFETs based on the sol-gel-derived membranes incorporating the alkoxysilylated bis(12-crown-4) and anion excluder, 2 and 3, and simply encapsulating bis[(12-crown-4-)ylmethyl] 2-dodecyl-2methylmalonate and Na(TFPB) (Figure 5). Changes in the sensor sensitivity, that is, the slope for the calibration graph, were followed with time. In the ISFETs of the membranes simply encapsulating the neutral carrier and anion excluder, an abrupt decrease in the sensitivity was observed after 30 days, probably due to their easy extrusion from the membrane to sample solutions. In the ISFETs of the membranes modified chemically by the neutral carrier and anion excluder, on the other hand, high sensitivity of a Nernstian response was attained even after 100 days. The long lifetime for the ISFETs of the membranes modified chemically by the neutral carrier and anion excluder is useful for practical applications. The high durability of the ion
sensors also means that the active materials for ion-sensing membranes hardly dissolve out from the membrane to aqueous sample solutions during the ion assay, in turn implying low toxicity of the ion sensors for use in biological systems. Application to Serum Sodium Assay. The sol-gel-derived membranes incorporating the alkoxysilylated bis(crown ether) were tested for their thrombogenic property. Any significant adhesion of blood platelets was hardly observed on the membranes, as was the case with sol-gel-derived membranes encapsulating a neutral carrier.8 Such an excellent thrombogenic property of the present sol-gel-derived membranes modified chemically by the bis(crown ether) suggests a high possibility for their application to Na+ assay in blood sera. Serum Na+ assay was carried out with the Na+-ISFETs using normal human blood samples. The results are given in Figure 6, showing that the values found for Na+ concentrations are in good agreement with the corresponding actual values. In conclusion, the present Na+-ISFETs based on the solgel-derived membranes incorporating the bis(12-crown-4) neutral carrier and anion excluder by covalent bonding possess high sensitivity, selectivity, and durability and fast potential response. The ion sensors are, therefore, quite promising for Na+ assay in blood sera and also may be applicable as intraarterial Na+ sensors. ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports. Financial support from the Suzuken Memorial Foundation and the Salt Science Research Foundation is also acknowledged. Received for review May 26, 1998. Accepted August 6, 1998. AC9805706
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