石 油 学 会 誌 Sekiyu Gakkaishi, 34, (5), 399-406 (1991) 399

Application of Ion-sensitive Field Effect to

Jun-ichi ANZAI, Shouryu LEE, and Tetsuo OSA*

Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980

(Received January 11,1991)

The principle and operation of an ion-sensitive field effect transistor (ISFET) are summarized briefly, and the application of the device to development of is described. Several kinds of techniques are discussed to immobilize ionophores and enzymes on the ISFET.

for/solution interface. The surface potential at 1. Introduction the gate surface is often described reasonably, based The concept of an ion-sensitive fieldeffect tran- on the site-binding model, which explains the sistor(ISFET) was derived from the structure of the interaction between an inorganic insulator and an metal semiconductor field effect transistor adjacent solution, with the assumption (MOSFET), which is a purely electronic semicon- that the surface contains a discrete number of ductor device1),2). In Fig. 1 are schematically surface sites, which can bind or liberate H+ ion3). illustrated the similarities and dissimilarities In the case of ISFET with Si3N4 gate insulator, the between MOSFET and ISFET, together with the gate surface contains silanol groups which can electrical equivalent circuit common to both bind or liberate H+ ions, depending on the pH of devices. The variance between them is that the the measuring solution, as shown in Fig. 2. The of the MOSFET is replaced by the ISFET is often applied in a feedback circuit to for ISFET, while the sample obtain an electrically stable operation. For this solution ensures the electrical contact with the reason, the drain-source voltage has a constant original gate insulator. The pH sensitivityof the preset value and the Id value is also constant, which ISFET is ascribed to the fact that the drain current, affords a gate-source voltage equal to the value of Id,depends on the surface potential at the insula- surface potential at the gate surface. The present paper describes the application of ISFET to the fabrication of microbiosensors. The advantages of ISFET in biosensor application are: (1) easy fabrication of miniature devices through semiconductor manufacturing process, (2) feasibility to accumulate multiple on single tip, (3) applicability of non-conductive materials for sensitive layer ascribed to low output impedance, and (4) suitability for mass production. 2. ISFET Ion Sensors The standard type of pH-sensitive ISFET has been used to detect ionic species other than H+ ion by coating the gate surface with ion-sensitive membranes. A polyvinyl chloride (PVC) membrane containing valinomycin has been used

Fig. 1 Schematic Representation of MOSFET (A) and ISFET (B) and the Equivalent Circuit (C)

* To whom correspondence should be addressed. Fig. 2 pH Response of Si3N4 Gate Surface

石 油 学 会 誌 Sekiyu Gakkaishi, Vol. 34, No. 5, 1991 400 to prepare K+-ion sensor4). The PVC membrane- based ISEFT ion shows some advantages over conventional type of membrane electrode sensors by its small size and low output impedance. The disadvantages, however, such as slow response, short life, etc. remain to be improved. The polymer materials such as polyacrylates and polysiloxanes have been used to improve the potentiometric response of ISFET ion sensors5). We have recently prepared two types of ISFET K+-ion sensors with ion-sensitive layers of mono- molecular level: chemically modified gate and LB membrane-coated gate ISFETs. The fabrication and performance characteristics of these sensors are described below. 2.1 Chemically Modified ISFET Sensors The chemical modification of electrode surface is one of the most promising techniques in devel- oping chemically functional electrodes6). There are some advantages in applying the chemical modification method to the preparation of ISFET ion sensors: owing to the thinness of the ion sensitive layer, minute devices (even in micron Fig. 3 The Proceduree for Surface Modification with order) can easily be prepared, and very rapid Crown Ether response can be expected. Additionally, the covalent bonding between ionophore and gate surface ensures a long life for the sensors without deterioration in sensitivity and selectivity. A procedure for chemical modification of ISFET is shown in Fig. 37). The probe-type Si3N4 gate ISFET coated with poly (p-xylylene) (parylene) film was used as an underlying device. The base insulator material for chemical modification should have less binding site for ions to suppress non-specific noise response. From this view- point, parylene is an excellent material8). First, the carboxylic groups were introduced at the surface of parylene by annealing. The carboxylic Fig. 4 Potentiometric Response of K+-ion Sensitive groups were treated with thionyl chloride to be ISFET Chemically Modified with Crown Ether activated to acid chloride form. The ISFET probe thus activated was immersed in toluene solution of 4'-aminobenzo-18-crown-69), at room temperature, sensor were improved as compared with those of for one day. It was ascertained that the crown PVC membrane-based sensors. The response ether residues are introduced to the parylene time was within 10 sec. After the initial decrease surface through amide linkage, by FTIR in sensitivity, the sensor maintained full response measurement: signals at 1,665cm-1 (amide I) and even after one month. There are some problems 1,569cm-1 (amide II). to be overcome for the chemically modified ISFET The potentiometric response of the K+-ion ion sensors: ca. 20mV/pH of pH response, ionic sensor is shown in Fig. 47). The sensitivity to K+ intensity response, and initial drift of potential. ion was ca. 35mV/log[K+], whereas the Since these problems stem from the chemical conventional type PVC/crown ether membrane- properties of modified parylene film, measure- coated device exhibited ca. 55mV/log[K+]. The ments in different mode with an unmodified difference in sensitivity between the two devices parylene film ISFET may solve the problems. seems to originate from the difference in the 2.2 LB Membrane-modified ISFET Sensors density of crown ether at the gate surface. On the A thin membrane with K+ ion sensitivity was other hand, the response time and the life of the deposited on the parylene gate ISFET by Lang-

石 油 学 会 誌 Sekiyu Gakkaishi, Vol. 34, No. 5, 1991 401

3. ISFET Enzyme Sensors

It is envisaged that the ISFET enzyme sensors can be prepared by modification of gate surface with enzymes which catalyze the reaction con- suming or producing H+ion. Based on this idea, many kinds of ISFET enzyme sensors have been developed so far13)-17). In general, enzymes can be immobilized to the gate surface of ISFET by the use muir-Blodgett (LB) technique10). A mixed of organic thin membranes. The organic mem- monolayer composed of stearic acid and crown branes where enzyme is immobilized should be a ether 1 (9: 1 in weight) was spread with benzene on crucially important factor in determining the water subphase at ca. pH 7 in Langmuir trough. performance of the sensors, because the signal, The surface of the parylene gate ISFET was usually H+ion, is produced as a result of an enzyme covered with the Y-type mixed monolayer. The catalyzed chemical reaction of analyte on and/or in senosor showed a response to K+ ion, over the the membrane. Additionally, the enzyme activity concentration range of 0.01-100mM, with a slope and the diffusion rates of analytes and reaction of ca. 35mV/log[K+]. This sensor, however, products should depend on the chemical and suffers from a drawback in that, in spite of the low physical properties of the membrane. The pH response (5-10mV/pH) of the parylene gate usefulness of direct covalent bonding of enzyme to ISFET without LB layer, the gate potential is ISFET gate has also been discussed very recently18). sensitive to pH, presumably ascribed to the ion- Bearing these in mind, we have examined the exchange sites resulting from the carboxylic performance of ISFET enzyme sensors with two groups in the LB layer. In Table 1 are shown pH different types of organic thin membranes: cross- response of ISFET sensors with and without stearic linked albumin membrane and LB membrane. acid LB membrane11). The use of non-ionic 3.1 Albumin Membrane-modified ISFET En- amphiphiles may be useful for the removal of pH zyme Sensors response. The long-term stability of this sensor is Bovine serum albumin (BSA) has been widely not satisfactory. The improvement of perfor- used to immobilize enzyme in its cross-linked gel mance characteristics of the LB membrane- matrix, because of the inertness of BSA and modified ISFET ion sensors may be possible by the relatively good surface adhesion to solid support. use of LB membranes with higher content of crown We have prepared enzyme sensors by covering the ether, polymerized LB layers, etc. ISFET gate with the enzyme/BSA cross-linked It is possible to miniaturize body of the sensor by membranes19),20). The enzymes used were urease, the use of ISFET device described above. At the trypsin, α-chymotrypsin, penicillinase, and present stage, however, the use of ISFET sensors is oxidase. In this paper, the results of premature in that no reference electrode of urease-modified sensor are described. comparable size is available. Reinhoudt et al. About 10% urease and BSA solutions were mixed have demonstrated that polyacrylate-modified reference FET shows superior pH insensitivity (within 1mV/pH), as compared with previously reported reference FETs coated with ion-blocking thin films by plasma deposition method12).

Table 1 Effect of LB Membranea) Deposition on the pH Response of ISFET

A: ISFET without urease/albumin membrane. B: ISFET coated with 2μm membrane. C: ISFET coated with 5μm membrane.

a) Stearic acid was used as material. Fig. 5 Effect of Urease/Albumin Membrane on the pH b) 4-layer LB membrane. c) 40-layer LB membrane. Response of ISFET

石 油 学 会 誌 Sekiyu Gakkaishi, Vol. 34, No. 5, 1991 402 with 8% glutaraldehyde solution, and an membrane. The detectable range of the sensor appropriate amount of the mixture was applied to was not influenced significantly by the membrane the ISFET gate before its gelation. The probe was thickness. The authors applied the sensor to the dried at ambient temperature for ca. 1h, and was determination of urea in serum. In Table 3 rinsed with working buffer before use. In Fig. 5 is are listed typical results, together with those data shown pH response of ISFETs with and without based on the official method (urease-indophenol enzyme membrane. The potential change was method). The agreement of values of urea level measured by putting the probe in one standard measured by both methods is acceptable for all solution into another solution having different pH samples. Both serum and plasma could be value. The urease membrane-coated ISFET analysed with the urea sensor. Further, required longer time than the probe without urease hemolysis did not cause any disturbance in the membrane for attaining stationary values of potentiometric determination of urea with the output voltage. This can be explained in terms of sensor. the time required for H+ and/or OH- ions to 3.2 LB Membrane-modified ISFET Enzyme diffuse from the membrane/solution interface to Sensors the gate surface or in the opposite direction As summarized in Table 1, the LB membrane according to the concentration gradient in the coated on the ISFET provides a barrier, to some membrane. These data suggest that one should extent, to the transport of H+ ion in the membrane. use thinner membrane to prepare sensors having However, the LB membrane can not block the rapid response. penetration of the ion completely. Matsuo et al. In Table 2 are shown summaries of the per- also examined the chemical and electrical formance characteristics of ISFET urea sensors properties of LB membrane deposited on ISFET. with enzyme membranes of different thicknesses (2, They found that the electrical properties of the LB 5,and 10μm). The mechanism of urea sensors membrane strongly depends on the chemical responding to urea is as follows: the local pH composition of the gate insulator materials of change arising from the decomposition reaction of ISFET11). These results suggest the possible use urea as in Eq. (1) can be detected as a potential of LB membrane-coated ISFET in the preparation of enzyme sensors, by immobilization of enzymes (NH2)2CO+2H2O+H+→2NH4++HCO3- on the surface of the LB membrane. (1) We have prepared apenicillin G sensor by this technique using steari cacid LB membrane21) change at the gate surface. The response time The procedure for the preparation of thesensor is increased proportionally with the membrane as follows: the stearic acid multilayer was thickness. Though the numerical solution of the deposited on Si3N4 gate ISFET. After drying the reaction and diffusion kinetics in the membrane, LB layer on the ISFET, the probe was immersed in which is a function of the diffusion rates of ca. 0.5% penicillinase solution (1mM phosphate participating species and urease activity, is buffer, pH7) at 0-5℃ for ca. 15h to immobilize somewhat complicated, the qualitative explana- the enzyme on the LB membrane. The penicillin tion for this dependency may be given as follows: the diffusion rate of H+ or OH- ion seems to be a main factor in governing the response time of the Table 3 Determination of Urea in Blood Serum and sensors. The slope of the calibration graph was Plasmaa) also affected by the membrane thickness. The probe with thicker membrane resulted in a steeper slope, probably because the pH changes at the gate surface are cancelled in part by the buffer, which effect is thought to be weakened in the thicker

Table 2 Effect of Membrane Thickness on the Poten- tiometric Response of Urease-membrane Coated ISFETs

a) Tris-HCl buffer (pH 7.3, 10mM) was used. b) Blood serum without lipid. c) Blood serum. d) Hemolyzed serum. e) Plasma. f) Urease-indophenol method.

石 油 学 会 誌 Sekiyu Gakkaishi, Vol. 34, No. 5, 1991 403

sensor was rinsed thoroughly with the working 7). For 0.1-10mM penicillin G solutions, the

buffer before use. The structure of the sensor was gate potential always shifted in the positive schematically shown in Fig. 6. The potentiomet- direction to the extent depending on the con- ric response of the penicillin G sensor is based on centration of penicillin G, and reached steady-state the pH change resulting from the enzymatic values in 4-5min. This shows that penicilli- reaction (2) on the surface of LB membrane. nase can be adsorbed to the stearic acid LB membrane without deactivation. Unfortunately, the response time of the sensor was not so rapid as to be expected from the fact that the thickness of the LB layer was 500Å or less. The reproducibility of the response was satisfactory. It is clear that the LB membrane and penicillinase remain on the

gate surface without denaturation, even after repeated use. The long-term stability of the sensor was also checked by measuring the output voltage for 3mM and 10mM penicillin G at pH 8 every day. The

probe was stored in buffer at 4℃ when not in use. In Fig. 8 are shown the results which show that the The potentiometric response of the sensor for sensor is fairly stable under these experimental penicillin G was tested in 5mM buffer at pH 8 (Fig conditions. This sensor could be stored also in dry state (e. g., in air)for a long time without any loss of the enzyme activity, which property is convenient for practical use of the sensor. It is not always possible to immobilize enzyme on the LB membrane by electrostatic or hydro- phobic force of attraction. For this reason, reactive LB membranes which can bind enzyme via covalent linkage have been developed for biosensor applications. Watanabe et al. have immobilized glucose oxidase to the amino groups, via glutaraldehyde, of stearylamine LB membrane which was deposited on SnO2 electrode22). The sensor can be used for detection of D-glucose in the concentration ranging from 1 to 100mM. The response of the sensor was rather slow, probably due to the limited permeation rate of hydrogen Fig. 6 Schematic Illustration of LB Membrane-coated peroxide through the underlying LB membrane. ISFET Enzyme Sensor We have used a reactive polymer as a spacer which links LB membrane and enzyme (Fig, 9)23). The device was prepared in the following manner.

The Egs values were measured for 10 (■) and 3mM (□)

2mM buffer (pH 8.0) was used. penicillin G in 2mM buffer at pH 8.0. The probe was stored in the buffer at 4℃ when not in use. Fig. 7 Potentiometric Response of LB Membrane- coated ISFET Penicillin Sensor Fig. 8 Reusability of the Penicillin Sensor

石 油 学 会 誌 Sekiyu Gakkaishi, Vol. 34, No. 5, 1991 404

Succinimidyl behenoate 2

5-(Octadecyldithio)-2-nitrobenzoic acid 3 Fig. 9 Schematic Representation of LB Membrane Surface Bearing Polyethyleneimine and Fig. 12 Reactive Amphiphiles for LB Membrane Enzyme Preparation

chymotrypsin are shown in Figs. 10 and 11. The

probe exhibited a potentiometric response to N- acetyltyrosine ethyl ester (ATEE)(a model substrate of protein for α-chymotrypsin), showing that the enzyme was immobilized without deactivation and catalyzed hydrolysis reaction of ATEE to change pH value at the gate surface of the ISFET as follows:

Sample: 2mM ATEE in 2mM buffer at pH 8.0.

Fig. 10 Typical Response of α-Chymotrypsin-im- mobilized ISFET to ATEE

The disadvantage of this technique is that, in spite of the ordered structure of LB layer, the regulated arrangement of enzyme molecules cannot be expected. Alternative method to immobilize enzyme covalently on the surface of LB membrane is to use reactive amphiphiles as membrane-forming The Egs values were measured for 2mM ATEE in 2mM

buffer at pH 8.0. The probe was stored in the buffer at material. we have prepared two kinds of reactive 4℃ when notin use. amphiphiles: succinimidyl behenoate 224) and 5- octadecyldithio-2-nitrobenzoic acid 325) (Fig. 12). Fig. 11 The Long-term Stability of α-Chymotrypsin The mixed LB membrane of 2 and stearyl alcohol Immobilized with Polyethyleneimine on could be deposited on ISFET gate with deposition ISFET ratio of ca.1.0. The mixed LB membrane-coated ISFET was treated with 0.5% α-chymotrypsin A Y-type LB membrane composed of stearic acid solution to bind the enzyme through amide was deposited on the ISFET gate so as to protrude linkage, by the reaction of active ester with amino carboxyl groups to the uppermost surface. The residues in enzyme. In Fig. 13 is shown a typical LB membrane-coated ISFET was immersed in 0.3% response of the α-chymotrypsin-immobilized polyethyleneimine (PEI) soluion (pH 7) for 2h, by ISFET which was prepared by the use of mixed LB which PEI could be adsorbed to the LB membrane membrane containing 90% active ester. The through electrostatic interaction. After being sensor showed a response to ATEE over a con- treated with 3% glutaraldehyde, the probe was centration range of 0.1-2.0mM. It is rationally dipped in 0.5% enzyme solution to bind enzyme. understood that amino residues in the enzyme

A typical response and the long-term stability of attacked the active ester of the LB layer to form the ISFET protein sensor prepared by the use of α- amide linkage between them. The LB membrane

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3) Yate, D. E., Levine, S., Healy, T. W., J. Chem. Soc., Faraday Trans. I, 70, 1807 (1974). 4) Moss, S. D., Janata, J., Johnson, C. C., Anal. Chem., 47, 2238 (1975). 5) Van der Wal, P. D., Skowronska-Ptasinska, M., Berg, A. V. D., Bergveld, P., Shdholter, E. J. R., Reinhaudt, D. N., Anal. Chim. Acta, 231, 41 (1990). 6) Osa, T., Kagaku, 34, 534 (1979). 7) Matsuo, T., Nakajima, H., Osa, T., Anzai, J., Sens. Actuators, 9, 115 (1986). 8) Fujihira, M., Fukui, M., Osa, T., J. Electroanal. Chem., 106, 413 (1980). 9) Ungaro, R., E1 Haj, B., Smid, J., J. Am. Chem. Soc., 98, 5198 (1976). 2mM buffer (pH 8.0) was used. 10) Anzai, J., Osa, T., Hashimoto, J., Matsuo, T., Denshi Joho Tsushin Gihou, OME87-21, 5 (1987). Fig. 13 Typical Calibration Graph of ATEE Sensor 11) Suzuki, Y., Nakajima, H., Matsuo, T., Iyodenshi to Prepared with ISFET Coated with Active Ester Seitaikogaku, 24, 356 (1986). LB Membrane 12) Skowronska-Ptasinska, M., Van der Wal, P. D. V., Berg, A. V. D., Bergveld, P., Sudholter, F. J. R., Reinhoudt, D. N., Anal. Chim. Acta, 230, 67 (1990). 3 was also deposited on the ISFET gate, and further 13) Caras, S., Janata, J., Anal. Chem., 52, 1935 (1980). modified with urease through thiol/disulfide 14) Miyahara, Y., Moriizumi, T., Shiokawa, S., Matsuoka, H., exchange reaction. Karube, I., Suzuki, S., Nippon Kagaku Kaishi, 1983, 823. Recently Sakai et al. have developed an enzyme- 15) Caras, S. D., Janata, J., Anal. Chem., 57, 1924 (1985). linked immuno FET sensitive to human 16) Nakako, N., Hanazato, Y., Maeda, M., Shiono, S., Anal. Chim. Acta, 185, 179 (1986). immunogloblin G26). They immobilized urease- 17) Van der Schoot, B., Ergveld, P., Anal. Chim. Acta, 199, 157 labelled anti-human immunogloblin G on the (1987). ISFET gate in order to use the urease-catalyzed 18) Gardies, F., Jaffrezic-Renault, N., Martelet, D. N., Perrot, reaction as a signal . This approach H., Valleton, J. M., Alegret, S., Anal. Chim. Acta, 231, 305 will overcome the limitation of direct detection of (1990). 19) Anzai, J., Kusano, T., Osa, T., Nakajima, H., Matsuo, T., protein charges by immuno FET27). Bunseki Kagaku, 33, E131 (1984). 20) Anzai, J., Tezuka, S., Osa, T., Nakajima, H., Matsuo, T., 4. Conclusions Chem. Pharm. Bull., 35, 693 (1987). The ISFET has many advantages which 21) Anzai, J., Hashimoto, J., Osa, T., Matsuo, T., Anal. Sci., 4, contribute significantly to the preparation of high 247 (1988). 22) Tuzuki, T., Watanabe, T., Okawa, Y., Yoshida, S., Yano, performance biosensors. The most significant S., Komoto, K., Komiyama, M., Nihei, N., Chem. Lett., features of ISFET in biosensor application involve 1988, 1265. the miniaturization and multi-sensor capability, 23) Anzai, J., Lee, S., Osa, T., Chem. Pharm. Bull., 37, 3320 based on semiconductor manufacturing process. (1989). The proper design of bio-functional membranes 24) Anzai, J., Lee, S., Osa, T., Bull. Chem. Soc. Jpn., 62, 3018 on the ISFET gate is also important process in (1989). 25) Lee, S., Anzai, J., Osa, T., Bull. Chem. Soc. Jpn., 64, 2019 developing ISFET biosensors. (1991). 26) Sakai, H., Kaneki, N., Hara, H., Ito, K., Anal. Chim. Acta, References 230, 189 (1990). 27) Schasfoort, R. B. M., Bergveld, P., Kooymen, R. P. H., 1) Bergveld, P., IEEE Trans. Biomed. Eng., BME-17, 70 (1970). Greve, J., Anal. Chim. Acta, 238, 323 (1990). 2) Matsuo, T., Wise, K. D., IEEE Trans. Biomed. Eng., BME- 21, 485 (1974).

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要 旨

イオ ン感応性電界効果 トランジスターのバ イオセンサ ーヘの応用

安斉 順一, 李 昇龍, 長 哲郎

東北大学薬学部, 980 仙台市青葉区荒巻字青葉

イ オ ン感 応 性 電 界 効 果 トラ ン ジ ス ター (ISFET) を小 型 バ イ は従 来 のPVC膜 セ ンサ ー と比 較 して, 応 答 速 度 や 耐 久 性 の 向 オ セ ンサ ー に応 用 す る 試 み が行 わ れ て い る。 そ の 際 のISFET 上 が み られ た。 の特 徴 は, (1) 半 導 体 製造 工 程 で生 産 され, 小 型 化 が 容 易 で あ ま た, ISFETを 応 用 した 小 型 酵 素 セ ンサ ー が 作 製 され た。 る, (2) 単 一 素 子 に複 数 の セ ンサ ー を多 重 化 で きる, (3) 低 出 ISFETを ウ レ アー ゼ ・ア ル ブ ミ ン膜 で 被 覆 した尿 素 セ ンサ ー 力 イ ン ピー ダ ン ス の た め 絶 縁 性 感 応 膜 を使 用 で き る, お よ び は, ヒ ト血 清 ま た は血 し ょう中 の 尿 素 の 測定 に利 用 す る こ とが (4) 大 量 生 産 に適 して い る こ と, な どで あ る. 可 能 で あ った。 さ ら に, 反 応 性LB膜 被 覆ISFETに 酵 素 を固 クラ ウ ンエ ー テ ル類 を用 い て, 化 学 修 飾 法 お よ びLB膜 法 に 定 化 して, ペ ニ シ リンや タ ンパ ク質 測 定 用 の セ ンサ ー が作 製 さ よ りISFETイ オ ン セ ンサ ー が 開 発 され た。 こ れ らの セ ンサ ー れ た。

Keywords Biosensor, Supported catalyst, Ion sensitive field effect transistor, Enzyme sensor, LB membrane

石 油 学 会 誌 Sekiyu Gakkaishi, Vol. 34, No. 5, 1991