an immobilized enzyme membrane fabrication method using an ink jet nozzle
TRANSCRIPT
Biosensors 4 (1988) 41-52
An Immobilized Enzyme Membrane Fabrication Method using an Ink Jet Nozzle
J. Kimura, Y. Kawana & T. Kuriyama
NEC Corporation, l-l, Miyazaki 4-Chome, Miyamae-ku, Kawasaki, 213, Japan
(Received 22 March 1988; revised version received and accepted 18 May 1988)
ABSTRACT
A novel enzyme immobilization method, which realized the efficient use of enzymes for biosensors, was developed. An ink jet nozzle, originally developed for printing equipment, was used as a tool for precise enzyme deposition onto an ISFETdevice. Two sorts of immobilization method were attempted: one is liquid phase immobilization, in which an enzyme solution was emitted at first onto an ISFETsensor region. After drying it under room temperature, glutaraldehyde solution was emitted into the enzyme mem- brane for immobilization. In this method, the membrane became very thin (e.g. less than 0.1 pm) in a region around the center. In addition, membrane thickness control was rather difJicult because the enzyme solution redis- solved into the glutaraldehyde solution, and membrane localization at the peripheral region occurred. Next, the method was improved so as to main- tain the shape of the original enzyme drops: gas phase immobilization. At first, enzyme solution drops were emitted onto an ISFET device whose position was precisely determined using an X-Y stage controlled by a personal computer. Enzyme immobilization was realized by keeping the device in a chamber filled with glutaraldehyde vapor. In this case, the resulting membrane shape maintains the original enzyme shape. These methods will have great merit in very expensive enzyme immobilization and in the fabrication of a multi-biosensor, which has various kinds of immobilized enzyme membranes on a sensor chip.
Key words: ISFET, multi-biosensor, ink jet nozzle, gas phase immobiliza- tion , glucose, urease , GOD, biosensor , urea.
41 Biosensors 0265-928X/88/$03.50 @ 1988 Elsevier Science Publishers Ltd, England. Printed in Great Britain
42 J. Kimura, Y. Kawana, T. Kuriyama
INTRODUCTION
After the first work reported by Caras and Janata (1980), many biosensors based on ISFETs (ion sensitive field effect transistors) have been reported (Hanazato & Shiono, 1983; Miyahara et al., 1983; Caras & Janata, 1985a,b; Caras et al., 1985; Kuriyama et al., 1985; Miyahara & Moriizumi, 1985). The authors developed a biosensor fabrication technology by using an SOS (silicon on sapphire)/ISF’ET as a biosensor transducer (Kuriyama et al., 1985). An important advantage of using an SOS/ISFET is its inherent dielectric property, so that the problem of ISFET insulation in an aqueous environment was completely solved. In the work, to realize an enzyme- membrane pattern, the authors introduced a unique method in which partial enzyme inactivation by ultra-violet irradiation was carried out. The biggest problem with the method was that it could not be extended to production of a multi-biosensor. This therefore lacks one of the merits inherent in a semiconductor biosensor, wherein an ISFET is applicable to a multi- biosensor transducer. As a first attempt to realize a multi-biosensor, a manual enzyme membrane deposition method was developed (Kimura et al., 1986). In this method, special skill was needed to deposit an immobilized enzyme membrane on a small sensing region. As a result, the resulting yield rate was low. The next attempt carried out by the authors was a lift-off method (Nakamoto et al., 1987). This method, employing a lift-off process to fabricate small size enzyme membranes, was a first attempt at using a semiconductor manufacturing process in the fabrication of a biosensor. The only disadvantage in the process was that most of the enzyme solution was consumed without contributing to membrane fabrication in the process, so that the application was limited to use for sensors which use rather cheap enzymes. To complement the lift-off process, recently, the authors developed a novel enzyme deposition method, employing an ink jet nozzle as an enzyme deposition tool, which uses enzyme very efficiently and also makes multi-biosensor fabrication easy. In this paper, the enzyme immobi- lization method using an ink jet nozzle is described.
EXPERIMENTAL
Reagents
The reagents used for all experiments are special grade, if no other descrip- tion is given. Glucose oxidase (GOD: from Aspergifh niger, Grade III, 221 U/mg, Lot no. 10306526~06), and urease from jack beans (Urease S, 5 1 U/mg , Lot no. 1046292460) were purchased from Boehringer Mannheim
Immobilized enzyme membrane fabrication using an ink jet nozzle 43
COllNllOl drain
2nwl
4 MOSFET
Source
ISFET
i
Fig. 1. ISFETdevice.
Co. Bovine serum albumin (BSA: Fraction V, Lot No. STN2231) was purchased from Wako Pure Chemical Industries, Ltd. Glutaraldehyde was purchased from Kanto Chemical Co., Inc. as 25% aqueous solution. HEPES (N-2-hydroxylpiperazine-N’-ethanesulfonic acid) was purchased from Dojindo Laboratories, Ltd.
A pH 7.5, 10 mM HEPES buffer solution (O-15 ionic strength), prepared by mixing HEPES, NaOH and NaCl, was used as a buffer solution in all experiments. BSA solution (30%) was prepared by dissolving 300 mg BSA in 700 ~1 HEPES buffer, and storing it in a freezer at less than -20°C for subsequent use as stock solution. The BSA solution of required concentra- tion was prepared by diluting it with a buffer solution.
ISFET device used for experiments
Figure 1 shows the top view of an ISFET device. The device has four ISFET and MOSFET pairs, each pair of which forms a source follower circuit. It is useful as a biosensor transducer, because it needs no peripheral amplifiers (Kimura et al., 1986).
Ink jet nozzle
Figure 2 shows the ink jet nozzle construction. The ink jet nozzle activation principle is based on piezo-electric element contraction induced by an
44 J. Kimura, Y. Kawana, T. Kuriyama
Nozzle 6Qmlal
Pulse 3Opsec -9.W
0
3x10-qlvdrop
Fig. 2. Ink jet nozzle construction.
electric pulse. On the top of the nozzle, a 50 pm diameter hole was created. When chamber contraction is induced, by increasing pressure from the pressure chamber, the enzyme solution filling the pressure chamber is pushed into the air and creates an enzyme solution drop. After an enzyme drop leaves the chamber, the chamber pressure returns to its initial value. To provide the chamber with enzyme solution, an ink inlet is connected to a reservoir. A solution flow resistance element solution flow rate and appropriate drop size.
Experiment 1 (immobiliiation under liquid phase)
Experimental set-up
maintains the constant
Figure 3 shows the experimental set-up for enzyme patterning by an ink jet nozzle. An ink jet nozzle piezoelectric element was stimulated by pulses generated by a pulse generator and an amplifier. An ISFET wafer was mounted on the X-Y stage. The enzyme droplet falling position was con- trolled with the aid of a microscope.
Ink jet nozzle
Fii. 3. Experimental set-up for Experiment 1.
immobilized enzyme membrane fabrication using an ink jet nozzle 45
Fig. 4. Immobilized enzyme membrane fabrication processes for Experiment 1.
Fig. 5. An ISFET device coated by a film-resist photopolymer (hexagonal shading).
Enzyme membrane fabrication process Figure 4 shows an enzyme membrane fabrication process. (1) An ISFET device was previously covered by a film-resist photopolymer, except for sensing regions and a half area which included bonding pads, as shown in Fig. 5. The photopolymer was used for creating enzyme solution pools, and to protect the ISFET surface from mechanical damage. (2) Enzyme solution was emitted by the ink jet nozzle into pools made by the film-resist photo- polymer. The pool was patterned with rounded edges, as shown in Fig. 5. The reason for this shape is that, if the pool shape were to include a sharp edge, most of the enzyme solution would gather in the edge area while
J. Kimura, Y. Kawana, T. Kuriyama
TABLE 1 Solution Contents for Experiment 1
Urease membrane
GOD membrane
Albumin membrane
1% Urease 5% BSA 0.5% GA
4% GOD 5% BSA 0.5% GA
5% BSA 0.5% GA
0@06 /.Ll
o-006 jd
O-006 I*1
0036 /_LI
0.006 /_Ll 0*006 /_&I
drying. The solution contents are given in table 1. About 100 drops were emitted into each pool. (3) The solution was dried at room temperature. (4) Glutaraldehyde solution (O-5%, approx. 100 drops) was formed into a pool on the dried enzyme by the ink jet nozzle. (5) After the crosslinking reaction and drying, an enzyme or protein membrane was obtained.
Experiment 2 (gas phase immobilization)
The typical drop size for emitted enzyme or protein was approximately 50pm in diameter, 60 pl in volume. Although precise patterning was realized by the method described in Experiment 1, redissolving to glutar- aldehyde solution resulted in nonuniformity of membrane thickness: local- ization at the edge of the film-resist photopolymer. To overcome this problem, a technique was developed to control emission of enzyme drops so that they could be placed onto different positions of a small ion-sensitive area. As a result, when drops were emitted and dried, it was possible to realize immobilization while retaining the dried solution shape.
Experimental set-up The main part of the experimental set-up is the same as that used in the previous experiment, as shown in Fig. 3. Enzyme emission position control was realized by employing an X-Y stage controlled by a personal computer, as shown in Fig. 6.
Enzyme membrane fabrication process The process is as follows.
(1) Ten enzyme drops were emitted onto different positions on one ISFET sensing area. These positions are shown in Fig. 7.
(2) Preliminary immobilization was carried out by placing a sensor device in a vapor chamber containing 25% glutaraldehyde aqueous solution for approximately 5 min.
immobilized enzyme membrane fabrication using an ink jet nozzle 47
Microscope
Fig. 6. Experimental set up for Experiment 2.
Fig. 7.
(3)
(4)
Enzyme drop emission positions in an ISFET. Ten circles show positions from which enzyme drops are emitted along an ISFET channel.
By repeating processes (1) and (2) five successive times, 50 enzyme drops were finally immobilized on one ISFET sensing area. Immobilization was completed by immersing the sensor device in 1% glutaraldehyde solution for approximately 5 min. Figure 8 shows patterned enzyme membranes on the ISFET sensing areas.
RESULTS AND DISCUSSION
Relationship between solution concentration and patterned shape
Figure 9 shows cross-sectional profiles for enzyme drops after being emitted onto a silicon wafer and dried at 25”C, as detected by a stylus membrane thickness monitor. The dried enzyme membrane has two parts: a rather thick peripheral doughnut-like area, and a thin center area. With a low protein concentration (3%) the center area is particularly thin, e.g.
J. Kimura, Y. Kawana, T. Kukiyama
Albumin membrane
GOD membrane I Urease membrane
Fig. 8. Patterned immobilized membranes on an WET device.
-0.05 pm. With a 6% protein concentration, the center area thickness became about O-2 pm. A higher concentration of 9% gave a thickness of -0.3 pm. Use of a low concentration protein solution was preferable to prevent nozzle fouling by dried enzyme. In Experiment 1, a 5% protein solution was used. In Experiment 2, a 3% solution was used.
Liquid phase immobilization method
Figure 10 shows the sensor top region with patterned enzyme or albumin membranes. As shown in the figure, the membrane center area seemed very thin. This phenomenon is explained by the affinity between enzyme solution and film-resist photopolymer. Figure 11 shows the response to glucose and urea.
In this method, the resulting membrane shape does not reflect the shape of the initial enzyme membrane deposited on the ISFET device. The greatest advantage of the method is precise patterning of a very small amount of enzyme. One problem is that enzyme redissolving into the glutaraldehyde solution destroys its original shape, and introduces localiza- tion of the membrane in boundary areas around the film-resist photo- polymer. To solve the problem, enzyme must be prevented from dissolving into a glutaraldehyde solution.
Immobilized enzyme membrane fabrication using an ink jet nozzle 49
Fig. 9. Emitted enzyme drop shape, after drying.
Albumin membrane
GOD membrane I Ureas8 membrane
Fig. 10. Immobilized enzyme membranes in Experiment 1.
50 J. Kimura, Y. Kawana, T. Kuriyama
1 Glucose lOOmg/dl
S s I 1 min
I-
Urea 1 OOmg/dl
_.I p- Fig. 11. Response curves for a biosensor in Experiment 1.
TABLE 2 Solution Contents for Experiment 2
Urease membrane
GOD membrane
1 %Urease 3% BSA
1% GOD 3% BSA
Albumin membrane 3% BSA
Gas phase immobilization method
In this method, a personal computer controls the shapes of immobilized enzyme membranes by partial emission on the channel area of the ISFET. The immobilized enzyme membrane is needed to cover the channel region; there is no need to cover the other region. In this method, only ten drops of enzyme solution cover a large part of the channel region. It is very important that the part covered by the enzyme membrane be sufficient for a biosensor enzyme membrane. In addition, this method presents a rather uniform membrane.
Figures 12 and 13 show a response curve and a calibration curve, respect- ively. Altnough the whole surface was not covered, good responses were obtained.
The only problem in using this method is the total time taken. In practice,
Immobilized enzyme membrane fabrication using an ink jet nozzle
Fig. 12. Response curves for a biosensor in Experiment 2.
51
Glucose concentration hg/dl) Urea concentration (mg/dl)
Fig. 13. Relationship between substrate concentration and biosensor output.
enzyme emission and immobilization processes were repeated five times. For each emission and immobilization, approximately 1 h is needed, requiring therefore, approximately 5 h in total. A way to reduce this time consumption will be realized by introducing a higher concentration protein solution or simultaneous realization of emission and immobilization.
52 J. Kimura, Y. Kawana, T. Kuriyama
CONCLUSIONS
A new enzyme immobilization method was developed, employing an ink jet nozzle as an enzyme drop emission tool. This method was applied to the fabrication of a multi-biosensor, which detects urea and glucose simul- taneously. Only a very small amount of enzyme is needed in this method, so that expensive enzyme can be used efficiently for a biosensor. This method will find greatest application when a multi-biosensor is realized which uses expensive enzyme. A further merit of this method is that, by employing a personal computer, it is easy to select an appropriate enzyme from among several enzymes, and to determine the enzyme emission position, so that many kinds of multi-biosensors, which have different enzyme combinations, will be realized on one wafer. In the experiment, a biosensor chip was mounted onto a flexible printed circuit board after enzyme membrane deposition. However, using this method it will be easy to deposit an enzyme after mounting a chip onto a flexible printed circuit board, or to reactivate a biosensor enzyme activity by emitting an enzyme onto an inactivated enzyme membrane.
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