Anal. Chem. 1997, 69, 2379-2383
Applications of Sol-Gel-Derived Membranes to Neutral Carrier-Type Ion-Sensitive Field-Effect Transistors Keiichi Kimura,* Takenobu Sunagawa, and Masaaki Yokoyama
Chemical Process Engineering, Faculty of Engineering, Osaka University, Yamada-oka, Suita, Osaka 565, Japan
Sol-gel-derived membranes encapsulating neutral carriers, such as valinomycin and a bis(crown ether) derivative, were fabricated as novel ion-sensing membranes for neutral carrier-type ion-sensitive field-effect transistors (ISFETs). The neutral carrier-type sol-gel-derived membranes obtained with an initial diethoxydimethylsilane/ tetraethoxysilane ratio of 3 gave excellent results for membrane processibility and sensitivity, response time, and selectivity in the resulting ion sensors. The sol-gelderived ion-sensing membranes containing neutral carriers are superior in the thrombogenic property to their corresponding plasticized poly(vinyl chloride) membranes. The potassium and sodium ISFETs thus obtained were successfully applied to the ion assay in blood sera. A typical membrane material for potentiometric ion sensors based on neutral carriers is plasticized poly(vinyl chloride) (PVC), in which various neutral carriers are soluble. There are, however, some drawbacks in platicized PVC-based ion-sensing membranes. The ion-sensing membranes require a large quantity of a special plasticizer, and PVC itself, without any plasticizer, cannot afford high performance as the membrane material. Comparatively easy exudation of the plasticizers from the membranes to aqueous sample solutions, therefore, causes serious deterioration in the ion-sensing membrane and thereby in the ion sensors.1 Also, some problems induced by adsorption of organic components such as proteins on the plasticized PVC-based ion-sensing membranes cannot be neglected in the cation assay on blood sera.2-5 Sol-gel chemistry enables us to design hybrid inorganicorganic materials, giving an access to the fabrication of glasses and films with novel properties.6 The low reaction temperature on sol-gel processing facilitated incorporation of functional organic compounds into inorganic materials. Sol-gel-derived membranes are therefore promising candidates for sensing membrane materials for ion sensors mainly due to their physical stability and chemical inactivity. Although sol-gel-derived membranes encapsulating enzymes have been applied to biosensors,7 (1) Kimura, K.; Matsuba, T.; Tsujimura, Y.; Yokoyama, M. Anal. Chem. 1992, 64, 2508-2511. (2) Jenny, H.-B.; Riess, C.; Ammann, D.; Magyar, B.; Asper, R.; Simon, W. Mikrochim. Acta 1980, 1880 II, 309-315. (3) Kimura, K.; Tsujimura, Y.; Yokoyama, M. Pure Appl. Chem. 1995, 67, 10851089. (4) Tsujimura, Y.; Yokoyama, M.; Kimura, K. Anal. Chem. 1995, 67, 24012404. (5) Espadas-Torre, C.; Meyerhoff, M. E. Anal. Chem. 1995, 67, 3108-3114. (6) Hench, L. L.; West, J. K. Chemical Processing of Advanced Materials; Wiley: New York, 1992. S0003-2700(96)01106-7 CCC: $14.00
© 1997 American Chemical Society
no work has been reported concerning applications of sol-gelderived membranes to neutral carrier-based potentiometric ion sensors. Recently, we communicated a possibility of their applications to a neutral carrier-based ion sensor.8 Here we describe the applications of sol-gel-derived membranes containing valinomycin and a bis(12-crown-4) derivative to ion-sensing membranes for K+ and Na+ ISFETs, respectively. EXPERIMENTAL SECTION Materials. The starting chemicals for the sol-gel processing, diethoxydimethylsilane (DEDMS) and tetraethoxysilane (TEOS), were purchased from Shin-Etsu Silicon Chemicals. Bis(12-crown4-ylmethyl) 2-dodecyl-2-methylmalonate and valinomycin were employed as received from Dojindo and Aldrich, respectively. Sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB) was also available from Dojindo. The ISFET tips, pH-sensing devices, were supplied by Shindengen Electric Inc. The gate size is 10 µm wide and 370 µm long. Poly(3-octylthiophene) was prepared by galvanostatic electropolymerization using the working (platinum plate 2 × 2 cm), counter (platinum wire), and reference (Ag-AgCl/3 M KCl) electrodes.9 The resulting polymer on the platinum plate was dissolved in chloroform by ultrasonication, and the evaporation of the solvent then afforded a yellow film of the polymer. PVC with an average polymerization degree of 1020 was purified by reprecipitation from tetrahydrofuran (THF) in methanol. 2-Ethylhexyl sebacate (DOS) was purified by vacuum distillation. Alkali- and alkaline-earth metal chlorides and ammonium chloride were of analytical reagent grade. Water was deionized. Control blood sera (Wako, control serums I and II) were commercially available. The concentrations for Na+ and K+ are as follows: 143 mM Na+ and 4.3 mM K+ for serum I, 124 mM Na+ and 6.8 mM K+ for serum II. 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 general procedure for casting ion-sensing membranes on the ISFET gate surface is as follows. TEOS (22 µL, 1.2 × 10-4 mol), DEDMS (62 µL, 3.5 × 10-4 mol), ethanol (7) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 22, 1120A-1127A. (8) Kimura, K.; Sunagawa, T.; Yokoyama, M. Chem. Lett. 1995, 967. (9) Bobacka, J.; McCarrick, M.; Lewenstam, A.; Ivaska, A. Analyst (London) 1994, 119, 1985-1991.
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(69 µL), 0.1 M HCl aqueous solution (21 µL), and 1 mg of valinomycin or bis(12-crown-4) were mixed in a sample tube. For the bis(12-crown-4) system, the starting solution also contained 0.2 mg of NaTFPB. The mixture was then allowed to stand for about 36 h to afford a viscous sol-gel solution. Unless otherwise stated, an aliquot (1 µL) of the sol-gel solution was placed on the gate surface of a commercially available pH ISFET tip. The device was then heated at 50 °C for 2 days to yield a sol-gelderived membrane of about 150-200 µm thickness. Prior to the sol-gel processing, a poly(3-octylthiophene) interlayer 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. Conditioning of the resultant K+ and Na+ ISFETs was made by soaking in KCl and NaCl solutions of 1 × 10-3 M, respectively, for 12 h. Measurements. Potential measurements were made at 25 °C using an ISFET pH/mV meter (Shindengen Electric Inc.). 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 measured metal ion concentrations were changed by injection of high-concentration solutions to the testing solutions while stirring with a magnetic stir bar. 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).10 Selectivity coefficients for Na+ or K+ with respect to interfering cations were determined by a mixed solution method (fixed interference method). The background cation concentrations were 0.1 M for K+, H+, and NH4+, 0.5 M for Mg2+, Ca2+, and L+, and 1 M for Na+. The sodium assay was carried out by using Gran’s plot method,11 using 5 and 0.1 mL (four times) as the volumes of the sample and added solutions, respectively. Platelet Adhesion Study. Sol-gel-derived membranes with the initial DEDMS/TEOS ratio of 3 were fabricated on a glass substrate (1.5 × 1.5 cm) in a similar manner to the membranes for the ISFET. PVC (about 32 wt %) membranes plasticized by DOS (65 wt %) were similarly cast. The glass-cast membrane were immersed in real human blood serum for 2 h and then washed by water. After drying under vacuum for 3 h, gold was evaporated on the membrane samples. Scanning electron micrography (SEM) of the ion-sensing membranes was undertaken with about 1000 magnifications at an acceleration voltage of 20 kV.
Figure 1. Potential response of K+ ISFETs based on valinomycinencapsulated sol-gel-derived membranes which were fabricated with initial DEDMS/TEOS ratios of 1 (4), 2 (9), and 3 (O).
RESULTS AND DISCUSSION Optimization of Initial Silane Ratios for Sol-Gel Processing. Sol-gel-derived glasses are often fabricated using tetraethoxysilane (TEOS) as a sole starting alkoxysilane, by its hydrolysis followed by condensation of its resultant silanols. However, the sol-gel processing of TEOS alone gives quite brittle, glassy products which are not suitable for ion-sensing membranes due to the mechanical-strength problems. We, therefore, decided to use mixtures of TEOS and diethoxydimethylsilane (DEDMS) as starting alkoxysilanes for the fabrication of neutral carrier-based ion-sensing membranes. Solutions containing DEDMS and TEOS mixtures with DEDMS/TEOS ratios of 1, 2, and 3 and also a small quantity of neutral carriers were, therefore, tested for the sol-
gel-derived membranes for neutral carrier-type ISFETs. In higher DEDMS/TEOS ratios than 3, the solidification of the resulted membranes was quite difficult or took too long. Figure 1 shows the potential response to K+ activity changes for the K+ ISFETs based on valinomycin-encapsulated sol-gel-derived membranes which were obtained with DEDMS/TEOS ratios of 1, 2, and 3. Only poor sensitivity was found in the ISFETs of sol-gel-derived membranes fabricated with the lower DEDMS/TEOS ratios, 1 and 2. On the other hand, the K+ ISFETs of the membrane with a DEDMS/TEOS ratio of 3 can afford Nernstian response in the wide K+ activity range. The poor sensitivity in the membrane systems with the lower DEDMS/TEOS ratios is probably due to the high degree of cross-linking in their resulted sol-gel-derived membrane, which leads to the low mobility of the encapsulated neutral carrier. Since any of the sol-gel-derived membranes encapsulating valinomycin are quite glassy, one might anticipate tedious potential response in the resultant ISFETs. The potential-time response profiles are demonstrated in Figure 2, showing marked dependence of the response time on the initial DEDMS/TEOS ratios for sol-gel processing. The ISFETs with the initial DEDMS/ TEOS ratios of 1 exhibited time-consuming potential response, the response time (t90) being in the order of 1 min. The higher DEDMS/TEOS ratios allowed rapid potential responses, the response time being about 15 s for the membrane system with the ratio of 2 and only several seconds for that with the ratio of 3. The slow potential response with the lower DEDMS/TEOS ratios can also be attributed to the low mobility of the neutral carrier in the sol-gel-derived membranes, which in turn causes slow ionexchange at the interface between the membrane and aqueous phases. However, the response times for the lower DEDMS/ TEOS ratios, especially with the ratio of 3 (several seconds), are acceptable for practical use of ion sensors. Thus, the initial DEDMS/TEOS ratio of 3 is suitable for the fabrication of solgel-derived membranes encapsulating valinomycin, from the standpoint of the membrane mechanical strength, sensor sensitivity, and response time. Similar sol-gel-derived membranes encapsulating a bis(crown ether) derivative, namely bis(12-crown-4-ylmethyl) 2-dodecyl-2methylmalonate, which is one of the best Na+ neutral carriers,12
(10) Davies, C. W. Ion Association, Butterworth: London, 1962. (11) Gran, G. Analyst (London) 1952, 77, 661-671.
(12) Kimura, K.; Yoshinaga, M.; Funaki, K.; Shibutani, Y.; Yakabe, K.; Shono, T.; Kasai, M.; Mizufune, H.; Tanaka, M. Anal. Sci. 1996, 12, 67-70.
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Figure 4. Dependence of thickness of sol-gel-derived membrane upon slope of calibration graph for K+ ISFETs. The sensor fabrication was made under otherwise identical conditions with those in Figure 3.
Figure 2. Time-course changes of potential response for K+ ISFETs based on valinomycin-encapsulated sol-gel-derived membranes which were fabricated with initial DEDMS/TEOS ratios of 1 (a), 2 (b), and 3 (c) on changing the KCl concentration from 1 × 10-3 to 3 × 10-3 M.
Figure 5. Selectivity coefficients for K+ and Na+ ISFETs based on optimized sol-gel-derived membranes of valinomycin (a) and bis(12-crown-4) (b) with a polythiophene interlayer. The membranes were identical with those in Figure 3.
Figure 3. Potential response of K+ and Na+ ISFETs based on optimized sol-gel-derived membranes of valinomycin (b) and bis(12-crown-4) (O) with an interlayer of polythiophene. The initial DEDMS/TEOS ratio for sol-gel processing is 3. The measuring solutions are KCl and NaCl aqueous solutions for the K+ and Na+ ISFETs, respectively.
were fabricated for Na+ ISFETs. In the bis(12-crown-4) membrane system, too, the initial DEDMS/TEOS ratio of 3 gave the best results. The Na+ ISFETs showed Nernstian response to Na+ activity changes in the activity range of 1 to 1 × 10-4 M and a short response time (t90 ) 2 s). Although the sol-gel-derived membranes containing valinomycin and bis(12-crown-4), fabricated with the initial DEDMS/TEOS ratio of 3, can afford highperformance K+ and Na+ ISFETs, respectively, the ion sensors still have some problems regarding instability in the potential and increase in the lower detection limit due to the lack of internal reference electrolyte between the membrane and gate surface.13 We therefore decided to adopt poly(3-octylthiophene) as the solid internal electrolyte.9 Thus the employment of a polythiophene (13) Brunink, J. A. J.; Haak, J. R.; Bomer, J. G.; Reinhoudt, D. N. Anal. Chim. Acta 1991, 254, 75-80.
interlayer improved the potential instability and the lower detection limit in both of the K+ and Na+ ISFETs based on the sol-gelderived membranes with the initial DEDMS/TEOS ratio of 3, as demonstrated in the calibration graphs (Figure 3). Thin ion-sensing membranes may be ideal for fast establishment of ion exchange equilibrium at the membrane interface of ISFETs. Figure 4 depicts the dependence of thickness in the solgel-derived membranes containing valinomycin on the sensor sensitivity for the resultant K+ ISFETs. Only poor sensitivity was found in the ISFET based on sol-gel-derived membranes with 30 µm thickness, while the thickness of 100 and 200 µm afforded a Nernstian response. Thus, a membrane thickness of at least 100 µm is required for attaining high sensitivity with a Nernstian response in the ISFETs of the neutral carrier-based sol-gelderived membranes, as is the case with the other membrane systems for neutral carrier-type ISFETs.1 Ion Selectivities for K+ and Na+ ISFETs with Optimized Ion-Sensing Membranes. Ion selectivity is an important factor for practical applications of ion sensors. Selectivity coefficients values for K+ and Na+ ISFETs with the optimized ion-sensing membranes encapsulating valinomycin and bis(12-crown-4) are summarized in Figure 5. The selectivity coefficient for K+ with respect to Na+ in the K+ ISFET is 2 × 10-4 and that for Na+ with respect to K+ in the Na+ ISFET is 3 × 10-3. The selectivity Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
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Figure 6. SEM photographs for sol-gel-derived and plasticized PVC membranes encapsulating bis(12-crown-4) after the platelet adhesion test.
coefficient values obtained here are similar to those for the ISFETs and ion-selective electrodes with the previous membrane materials containing the same neutral carriers. The high sensitivity and selectivity for the neutral carrier-type ISFETs based on sol-gelderived membranes have lasted for at least 3 weeks. Thrombogenic Property of Sol-Gel-Derived Membranes Containing Neutral Carriers. Adsorption of blood platelets on the sol-gel-derived membrane containing a neutral carrier was tested to obtain some information about biocompatibility of the ion-sensing membranes and therefore the resultant ISFETs in the cation assay on blood. For comparison, plasticized PVC membranes containing the same neutral carrier were also investigated for the thrombogenic property. Comparison in the SEM photographs for a sol-gel-derived membrane and its corresponding plasticized PVC membrane indicates a striking difference in the platelet adsorption (Figure 6). The sol-gel-derived membrane hardly adsorbs blood platelets, whereas a large amount of platelets can be seen on the plasticized PVC membrane. This indicates that the sol-gel-derived ion-sensing membranes are superior to the corresponding platicized PVC membranes in the biocompatibility. The adsorption of organic components in blood on plasticized PVC ion-sensing membranes often causes some negative errors in the cation assay on blood by the resulted membrane ion sensors.2,14 Also, due to the serious platelet adhesion, it is difficult to use plasticized PVC membrane ion sensors for cation monitoring in vivo. On the contrary, the markedly alleviated platelet adhesion on the sol-gel-derived membrane suggests a possibility for intraarterial use of the neutral carrier-type ISFETs designed here. Applications of Sol-Gel-Derived Membrane ISFETs to the Cation Assay on Blood Sera. Attempts were made to assay Na+ and K+ in blood sera with the sol-gel-derived membrane ISFETs based on bis(12-crown-4) and valinomycin, by using Gran’s plot method. For a reproducibility check, the cation assay was firstly carried out ten times for each sample, using two different control (14) Tsujimura, Y.; Yokoyama, M.; Kimura, K. Sens. Actuators, B 1994, 22, 195199.
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Table 1. Cation Assay in Control Sera by Na+ and K+ ISFETs Based on Sol-Gel-Derived Membranes Encapsulating Bis(crown ether) and Valinomycin Na+ concn (mM)
sample control serum I control serum II
K+ concn (mM)
coeff of coeff of variation variation actual found (%) actual found (%) 143.0 124.0
143.5 124.4
0.59 0.78
4.30 6.80
4.32 6.82
0.45 0.61
Table 2. Comparison between Actual and Found Values of Na+ and K+ Concentrations on Serum Assay of Human Blood by Using ISFETs with Sol-Gel-Derived Membranes of Bis(12-crown-4) and Valinomycin Na+ concn (mM)
K+ concn (mM)
sample
actual
found
actual
found
1 2 3 4 5 6 7 8 9 10
139.0 149.9 143.3 134.7 151.8 145.5 143.9 142.0 144.5 145.6
140.0 148.9 142.9 135.3 151.4 146.0 142.0 142.3 146.0 143.9
4.71 4.22 4.58 4.58 4.60 4.90 5.18 4.53 4.55 4.49
4.80 4.31 4.76 4.69 4.76 4.87 5.19 4.54 4.52 4.53
sera (Table 1). The ISFETs gave reliable data both in Na+ and K+ assay. The coefficient of variation ranges between 0.45 and 0.78%, thus indicating excellent reproducibility in the serum cation assay with the neutral carrier-type sol-gel-derived ISFETs. Next we employed real blood sera for normal human for the cation assay. Table 2 shows a comparison between the found and actual values in the Na+ and K+ assay. The relative errors are within 2% for the Na+ assay and within 4% for the K+ assay. These data clearly indicate that the found values are in good agreement with their corresponding actual values in both the Na+ and K+ assay. Thus, the Na+ and K+ ISFETs based on sol-gel-derived mem-
branes containing bis(12-crown-4) and valinomycin, respectively, are highly reliable on the serum cation assay. In conclusion, the sol-gel-derived membranes encapsulating neutral carriers, when fabricated with an initial DEDMS/TEOS ratio of 3, can afford excellent K+ and Na+ ISFETs possessing high performance in sensitivity, response time, and ion selectivity, in spite of the high rigidity of the membrane materials. Also, the ion-sensing membranes exhibit alleviated platelet adhesion in the serum cation assay, which suggests high biocompatibility. The neutral carrier-type sol-gel-derived membrane ISFETs are promising for the cation assay in blood sera.
ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture.
Received for review October 29, 1996. Accepted April 11, 1997.X AC9611069 X
Abstract published in Advance ACS Abstracts, June 1, 1997.
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