Glucose sensor based on a field-effect transistor with a photolithographically patterned glucose oxidase membrane

Analytica Chimica Acta, 193 (1987) 87-96 Elsevier Science Publishers B .V ., Amsterdam -Printed in The Netherlands

GLUCOSE SENSOR BASED ON A FIELD-EFFECT TRANSISTOR WITH A PHOTOLITHOGRAPHICALLY PATTERNED GLUCOSE OXIDASE MEMBRANE

Y. HANAZATO*, M . NAKAKO, M . MAEDA and S . SHIONO Central Research Laboratory, Mitsubishi Electric Corporation 1-1, Tsukaguchi-Honmachi 8-Chome, Amagasaki, Hyogo 661 (Japan) (Received 27th August 1986)

SUMMARY A photopolymer solution consisting of polyvinylpyrrolidone and 2,5-bis(4'-azido-2'sulfobenzal)cyclopentanone is used to make a patterned glucose oxidase membrane for a FET-glucose sensor by photolithography . A small patterned glucose oxidase membrane, 0 .2 mm wide and 1 mm long, is made on the gate surface of an ISFET by developing a photocross-linked glucose oxidase membrane with aqueous 1-3% glutaraldehyde solution . The optimum composition of the enzyme/photopolymer solution is described . The sensor with the patterned membrane showed linear response to glucose concentration from 0 .3 to 2 .2 mM and useful response up to 5 mM . Increasing interest has been shown in biosensors based on semiconductor technology, because of the possibility of producing cheap, small and multifunctional sensors . A hydrogen ion-sensitive field-effect transistor (FET) is most widely used as the electronic device for signal transduction for this type of sensor . In general, an enzyme membrane is placed over the gate surface of an ion-selective FET (ISFET) which senses a change in hydrogen ion concentration caused by an enzyme-catalyzed reaction . Enzyme-modified ISFETs have been proposed for the determination of some organic compounds, e .g., penicillin [1], urea [2-4] and glucose [5-7] . It is only recently that multifunctional FET sensors, made by depositing different enzyme membranes on an integrated ISFET having several hydrogen ion-sensitive FET elements, have been shown to provide simultaneous determinations of several compounds . Advances in semiconductor technology have made it possible to fabricate a miniature and/or integrated ISFET, but the methods for depositing an immobilized enzyme membrane so far developed still have problems : the procedures are expensive and timeconsuming, and there is considerable practical difficulty in forming a small membrane on a definite area of an integrated ISFET . Miyahara et al . [8] and Kimura et al . [9] formed micropools with photoresist over ISFET gates by using a photolithographic technique, and injected enzyme-immobilizing solutions into these pools from microsyringes . 0003-2670/87/$03 .50

© 1987 Elsevier Science Publishers B .V.

88 Wen et al. [10] proposed a new method for making a small patterned ion-sensitive membrane on a chemically-sensitive semiconductor device in order to produce an ion sensor . They used a commercially available negative photoresist to deposit an ion-conducting polymer membrane doped with an organic ion-carrier (valinomycin) by this photolithographic technique . This photodefinability was thought to be suitable for producing a miniaturized, multifunctional ion-sensing device. Attempts have been made to apply this photodefinability to immobilized enzyme membranes . Because enzymes may be denatured by organic solvents, a water-soluble photopolymer is preferable for making patterned immobilized enzyme membranes . In previous work [4], photosensitive poly(vinyl alcohol) (PVA) bearing stilbazolium groups (negative photopolymer) [11-13] was used to form immobilized glucose oxidase and urease membranes over the gate surface of an ISFET ; these membranes were made without using the photolithographic technique. In this report, a water-soluble photocross-linkable polymer was used to make a patterned glucose oxidase membrane on the ISFET by photolithography. This photopolymer is more promising than the photosensitive PVA because the preparation of a photopolymer solution and optimization of the composition of the photopolymer mixture are easier. The optimum conditions for making a patterned glucose oxidase membrane and its performance as a glucose sensor are described . EXPERIMENTAL Materials

Glucose oxidase (type IX, from Aspergillus niger, 29 .3 U mg -1 ) and bovine serum albumin (BSA) were obtained from Sigma Chemical Co. Glutaraldehyde was used as a 25% solution in water (Ishizu Pharmaceutical Co ., Osaka, Japan) . Polyvinylpyrrolidone (PVP) ; m.w . ca. 360 000 ; extrapure reagent grade) was purchased from Nakarai Chemicals (Kyoto, Japan), and 2,5-bis(4'-azido-2'-sulfobenzal)cyclopentanone (BASC) was obtained from Tokyo Ohka Kogyo Corp. (Kanagawa, Japan) . All other reagents were of analytical grade and were used as received . Distilled/deionized water was used throughout for the preparation of samples, buffer and other solutions . Sensor construction

An integrated ISFET with a chip size of 5 .0 X 6 .5 mm was fabricated on epitaxially grown silicon wafers [10-mm diameter ; p-type epitaxial layer, 8-12 ohm cm, ca . 10 gm thick, n-type substrate, 5-7 ohm cm with (100) orientation] by using a conventional n-channel MOS process . The gate insulator was composed of a thermally grown silicon oxide layer, 50 nm thick, and a silicon nitride layer deposited by low-pressure chemical-vapour deposition . The silicon nitride layer, which also acted as the proton-sensitive gate material, was 70 nm thick . The ISFET had a lightly-doped n-channel

89 (0 .04 X 1 mm) produced by a phosphorus ion-implant before the growth of the gate oxide layer to provide depletion-mode operation . The sensitivity of this ISFET was ca . 50 mV per decade . The structure of the FET glucose sensor is illustrated in Fig . 1 . An integrated ISFET chip was mounted on an epoxy laminate board with gold-plated copper tracks . After electrical connections had been made between the contact pads on the chip and the gold-plated copper tracks with an ultrasonic wire bonder (West Bond, Anaheim, CA ; type 7400), the chip was encapsulated with an epoxy resin to provide electrical insulation for the bonding wires and the exposed-side silicon region of the ISFET chip . The composition of the epoxy resin was 100 parts of DER-332 (Dow Chemical Japan Corp ., Tokyo), 88 parts of an acid anhydride HN-5500 hardener (Hitachi Chemical, Tokyo), 0 .4 parts of 2,4,6-tris(dimethylaminoethyl)phenol as catalyst (Wako Pure Chemicals, Osaka), and 10 parts of fine silicon oxide powder (Japan Aerosol, Tokyo) . A gold electrode as a pseudo-reference electrode was formed on the reverse of the epoxy laminate board . The ISFET surface was silanized with 3-aminopropyltriethoxysilane to improve the adhesion between the surface and the enzyme-containing membrane by the aqueous silanization technique [141 .

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Fig. 1 . Structure of the FET-glucose sensor : (1) epoxy laminate board ; (2) epoxy resin ; (3) aluminium wire ; (4) integrated ISFET chip ; (5) patterned glucose oxidase membrane ; (6) gold electrode .

90 Glucose oxidase membrane Glucose oxidase (10 mg) and 10 mg of BSA were dissolved in 0 .2 ml of a photopolymer solution (100 parts by volume of water, 10 parts of PVP and 1 part of BASC) . This solution was placed dropwise over the exposed gate area of the ISFET electrode illustrated in Fig . 1 . The solution-covered area of the ISFET was placed 2 cm away from the rotational centre of the spinner (Kyowa-Riken, Tokyo ; type K359SD-270) . The whole ISFET shown in Fig . 1 was then spun at 2000 rpm for 2 min to make a thin membrane over the gate surface . The membrane was exposed to ultraviolet irradiation (250 W) on a limited area (0 .2 X 1 mm) around the gate surface through a Hoya UV-34 filter with exposure equipment (Union Optical Co ., Tokyo ; Type ST) for 5 s . The treated ISFET was immersed in aqueous 3% (w/w) glutaraldehyde solution for 5 min at room temperature to form the glucose oxidase membrane . After the electrode had been washed with water, it was immersed in 0 .1 M glycine for 15 min to terminate the cross-linking reaction by glutaraldehyde .

Apparatus The flow-through system described in detail earlier [4] was used for evaluating the performance of the glucose sensor . A sensor was set up at the end of the water jacket thermostated at 34° C for all experiments . Washing solutions of 10 mM acetate buffer (pH 5 .5) and sample solutions prepared by dissolving the required amounts of glucose in 10 mM acetate buffer were pumped alternately past the sensor by a peristaltic pump at intervals of several minutes. The flow rate was 6 ml min -1 . The drain-source voltage of each ISFET element was set at 3 .0 V and the gate voltage at -1 .0 V . The substrate silicon was biased at 4 .0 V . Each differential output voltage between the ISFETs without and with the enzyme membrane was measured by the source-follower mode circuit (constant drain-source voltage and constant drain-current mode) as depicted in Fig . 2 . The enzyme-membrane thickness was measured with a stylus instrument (Tencor Instruments, Mountain View, CA ; type Alpha-Step 100) . RESULTS AND DISCUSSION

Glucose oxidase/photopolymer composition The PVP/BASC photopolymer was insoluble in water after it had been exposed to u .v. irradiation . The photopolymerized membrane was transparent and showed good adhesion to the surface of the silicon oxide layer of the silicon wafer . However, when glucose oxidase was added to this polymer solution, the dried membrane over the silicon wafer obtained after spin-coating was found to have an uneven surface and was not transparent . Additionally, the photopolymerized membrane spontaneously peeled away from the silicon oxide surface ; this might be caused by the swelling of the photocross-linked membrane in aqueous solution . Thus it

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Fig . 2 . Measuring circuit for the FET-glucose sensor, the output voltage is obtained as the differential output voltage of each ISFET element .

was found to be impossible to make a photocross-linked glucose oxidase membrane on a silicon oxide layer by the sole use of this photopolymer . As in the case of photosensitive PVA, glucose oxidase was immobilized by use of a PVP-BASC photopolymer which showed good adhesion to the silicon oxide layer when the chemical cross-linking reaction of glucose oxidase and BSA with glutaraldehyde was used in addition to the photocross-linking reaction by BASC. A glucose sensor with two discrete ISFET chips, as illustrated in Fig . 3, was used in preliminary experiments to establish the optimum composition of the enzyme solution, without photolithographic patterning of the enzyme membranes . A glucose oxidase membrane was formed over the whole surface of one ISFET by several methods . In the first, enzyme solution was coated on the ISFET surface by the method described in the Experimental section . In the second, the coated membrane was photopolymerized by u .v . irradiation over the whole surface of one ISFET . In the third, the membrane was immersed in 25% glutaraldehyde solution for chemical cross-linking of glucose oxidase and BSA with glutaraldehyde . In the fourth, after the membrane had been washed by water, it was immersed in 0 .1 M glycine to terminate the cross-linking reaction. It has been shown [4] that at least 10 mg of BSA in 0 .2 ml of photopoly-



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INNOFig . 3 . Structure of the discrete type of glucose sensor used in preliminary experiments to establish the optimum composition of enzyme/photopolymer solution : (1) epoxy laminate board ; (2) epoxy resin ; (3) aluminium wire ; (4) ISFET chip ; (5) glucose oxidase membrane ; (6) platinum electrode . mer solution is necessary to make an immobilized glucose oxidase membrane . The concentration of BSA was therefore held at 10 mg in 0 .2 ml of the PVP/BASC photopolymer solution in all experiments . The response of the glucose sensor increased with increase in the content of glucose oxidase . Solutions containing <10 mg of glucose oxidase in 0 .2 ml of photopolymer solution provided thin uniform membranes on spin-coating, but solutions containing >10 mg of glucose oxidase were too viscous to be coated uniformly over the ISFET surface . The upper limit of glucose oxidase was concluded to be 10 mg in 0 .2 ml of photopolymer solution . The effect of the concentration of BASC on the response of the glucose sensor was also investigated . For solutions consisting of 100 parts of water, 10 parts of PVP and x parts of BASC (with 10 mg each of glucose oxidase and BSA per 0 .2 ml), the responses when 1 .7 mM glucose was passed through the cell for 5 min at 34°C were 1 .94, 5 .35 and 10 .4 mV for x = 0 .2, 0 .5 and 1 .0, respectively . For x > 1 .5, the enzyme/photopolymer solution separated into two phases . Thus the photopolymer solution containing 1 part of BASC was best for making the glucose oxidase membrane . The enzyme/photopolymer solution described in the Experimental section was used to prepare a discrete glucose sensor, and its performance was evaluated. The calibration graph is shown in Fig . 4 . The sensor is suitable for determining glucose between 0 .3 and 3 mM . The long-term stability of the sensor is depicted in Fig . 5 . The sensor was stored in 10 mM acetate





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Fig. 4 . Calibration graph for discrete glucose sensor prepared without use of photolithography (34°C, pH 5 .5). Fig. 5 . Long-term stability of discrete glucose sensor used in Fig . 4 . The output voltage indicates the response to 1 .7 mM glucose (for conditions, see text) . buffer (pH 5 .5) in a refrigerator when not in use . The responses gradually decreased over the first 20 days and then became reasonably constant at ca . 60% of the initial response . The initial decrease is presumably due to enzyme leakage from the membrane . Photolithographicallypatterned glucose oxidase membrane After u.v . irradiation of the limited area of the dried enzyme membrane which was obtained from spin-coating of the enzyme/photopolymer solution described above on the silicon oxide layer of a silicon wafer, water was first tested as the developer to obtain photolithographically-patterned glucose oxidase membranes . A patterned membrane could be formed, but the membrane thus obtained often peeled off, partially or completely from the surface . To overcome this problem, glutaraldehyde solutions (1-25% w/w) in water were examined as the developer, and were found to be suitable . Strong adhesion was achieved by using 1-3% glutaraldehyde solutions as developer . Typical patterned membranes obtained after development in 2% glutaraldehyde solution are shown in Fig . 6. The membranes are observed as black rectangular shapes in the photograph . This improved adhesion was thought to be due to the higher cross-linking density of the membrane given by the chemical cross-linking reaction of proteins with glutaraldehyde . When the membrane was developed by >5% glutaraldehyde solutions, even membranes which had not been exposed to u .v . irradiation became insoluble in water, and a thin membrane was observed under the microscope over the whole silicon oxide layer on the silicon wafer . The development of the membrane



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Fig . 6 . Photograph of typical patterned glucose oxidase membranes . Widths are 0 .1 mm (right side) and 0 .2 mm (left side) . The membranes were developed with 2% glutaraldehyde solution at room temperature . Fig . 7 . Effect of exposure time on membrane thickness .

with 1-3% glutaraldehyde solution was also thought to give rise to a thin layer containing glucose oxidase over the whole surface of the ISFET, but the response of the reference ISFET to 1 .7 mM glucose was found to be negligible . The effect of the exposure time on the membrane thickness is depicted in Fig . 7 . The membrane thickness was measured from the profile of a patterned glucose oxidase membrane with a stylus instrument . This figure indicates that an exposure time longer than 2 s gives a patterned membrane of constant thickness . However, a rectangular-patterned membrane tended to have halation at its side edge after u .v. irradiation for >10 s . The effect of the exposure time on the response of the glucose sensor with a patterned membrane was also examined ; the results indicated that at least a 2-s exposure time is desirable for obtaining a constant response and that glucose oxidase is not inactivated by u .v. irradiation for <10 s . Accordingly, an exposure time of 5 s is recommended for making a patterned glucose oxidase membrane .

The performance o f the FET glucose sensor Figure 8A shows the calibration graph obtained for glucose . The steadystate response was achieved after 2 min . The output voltage shows a linear response to glucose concentration up to 2 .2 mM but it saturates at glucose concentrations exceeding 5 mM . Because the oxidation reaction catalyzed by glucose oxidase requires dissolved oxygen, the concentration of dissolved oxygen influences the output voltage of the glucose sensor . The influence of the dissolved oxygen concentration on the output voltage of the sensor is



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Fig. 8 . Calibration graphs for the FET-glucose sensor prepared with use of photolithography : (A) 0-6 mM glucose ; (B) 1 .7-11 mM glucose . (0) Air-saturated conditions ; (0) pure oxygen-saturated conditions . (34°C, pH 5.5 .)

depicted in Fig. 8B . Almost the same responses are obtained up to 3 mM glucose for air- or oxygen-saturated solutions . However, above 3 mM, the responses under oxygen-saturated conditions are larger . These results suggest that insufficient dissolved oxygen restricts the response at ca . > 5 mM glucose, when this type of sensor is used under air-saturated conditions . Compared with the calibration graph in Fig . 4, the graph for the sensor with a patterned membrane (Fig. 8A) shows lower sensitivity but a higher glucose concentration before the response becomes saturated . These differences between the two calibration graphs are mainly due to the amount of glucose oxidase immobilized in the membranes . The thickness of the photocross-linked membrane decreased during development in the dilute glutaraldehyde solution, therefore the amount of glucose oxidase immobilized in the developed membrane also decreased in comparison with that immobilized in an undeveloped membrane . The long-term stability is shown in Fig. 9 . The FET-glucose sensor was stored in a water jacket thermostated at 34 ° C, with 10 mM acetate buffer (pH 5 .5) flowing through at 6 ml min - ' (under the same conditions as those used to measure the glucose concentration) . The response to 2 .2 mM glucose gradually decreased to 50% of the initial response after 15 days . Peeling of the patterned glucose oxidase membrane from the ISFET surface was not



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Fig . 9 . Long-term stability of the FET-glucose sensor used in Fig . 8 (for conditions, see text) .

observed, so the decrease in sensitivity could have been due to thermal or bacterial degradation of glucose oxidase or leakage from the membrane . The authors are grateful to Mr. Miyazawa for supplying the BASC and for valuable discussions . REFERENCES 1 S . Cares and J. Janata, Anal . Chem., 52 (1980) 1935 . 2 Y . Miyahara, F . Matsu, T . Moriizumi, H . Matsuoka, I. Karube and S . Suzuki, Proc . Int. Meeting Chem . Sensors, Fukuoka, September 19-22, 1983, p . 501 . 3 J . Anzai, Y . Ohki, T . Osa, H . Nakajima and T . Matsuo, Chem . Pharm. Bull ., 33 (1985) 2556. 4 Y . Hanazato, M . Nakako and S . Shiono, IEEE Trans . Electron Devices, (1986) 47 . 5 Y . Hanazato and S . Shiono, Proc . Int. Meeting Chem . Sensors, Fukuoka, September 19-22, 1983, p . 513. 6 M . J. Eddowes, D . G . Pedley and B . C . Webb, Sensors and Actuators 7 (1985) 233 . 7 S. D. Caras, D . Petelenz and J . Janata, Anal . Chem ., 57 (1985) 1920 . 8 Y. Miyahara, T . Moriizumi and K. Ichimura, Sensors and Actuators, 7 (1985) 1 . 9J . Kimura, T . Kuriyama and Y . Kawana, Third Int . Conf. on Solid-State Sensors and Actuators (Transducers '85), Philadelphia, PA, 1985, Digest of Technical Papers, p . 152. 10 C . C. Wen, I . Lauks and J . N. Zemel, Thin Solid Films, 70 (1980) 333 . 11 K . Ichimura and S . Watanabe, J . Polym. Sci . Polym. Chem . Ed ., 18 (1980) 891 . 12 K . Ichimura and S. Watanabe, Polym . Sci. Polym. Chem . Ed ., 20 (1982) 1419 . 13 K . Ichimura, J . Polym. Sci . Polym. Chem. Ed ., 22 (1984) 2817 . 14 S . P. Colowick and N . O . Kaplan, in K . Mosback (Ed .), Methods in Enzymology, Academic Press, New York, Vol . 44, 1976, p . 139 .