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Integration of Enzyme-Immobilized Column with Electrochemical Flow Cell Using Micromachining Techniques for a Glucose Detection System Yuji Murakami, Toshifumi Takeuchi, Kenji Yokoyama, Eiichi Tamiya, and Isao Karube. Research Center for Advanced Science and Technology, University of Tokyo 4-6-1 Komaba, Meguro-ku, Tokyo 153, Japan
Masayuki Suda Research Laboratory for Advanced Technology, Seiko Instruments Inc., 563 Takatsuka-shinden, Matsudo-shi, Chiba 271, Japan
A miniaturized enzyme column integrated with a microelectrochemicalflow cell was developed for the flow injection detectionof glucose. The device consistedof a glucose oxidase-immobilizedcolumn and a flow cell with a microelectrode, which were fabricated and integrated on a silicon wafer by micromachining techniques such as photolithography, anisotropic etching, thermal oxidation, vacuum evaporation, and anodic bonding. A silicon wafer was photolithographically developed and anisotropically etched to form a V-shaped groove 1 m long, 100 Hm wide, and 70 Hm deep. A glass plate with thin-film electrodes was anodically bonded to the silicon before enzyme immobilization. The flow injection system gives a linear relationship between peak current and glucose concentration in the range of 1-25 mM.
analysis (FIA) system6 and a high-performance capillary electrophoresis (HPCE) system.' However, no work has been reported on flow-type biosensing systems. Manz et al. have discussed the advantages of miniaturized analysis systems.8 Miniaturized biosensing systems show great promise for medical, biochemical, and environmental applications. In these fields, it is desirable for many different substances to be analyzed rapidly using minimum amounts of sample and of expensive reagents, miniaturization offers obvious benefits in this regard. In addition, miniaturization allowsanalytical systems to be portable, enhancing their value for field use in environmental monitoring. In this paper, we describe a flow-type biosensing system fabricated by micromachining techniques. In this study, we fabricated an enzyme-immobilized column integrated with an electrochemical flow cell on a silicon wafer and demonstrated the functionality of this device for the determination of glucose by connecting it to a conventional flow injection system.
INTRODUCT10N
EXPERIMENTAL SECTION
Recently, advanced IC fabrication techniques, such as photolithography, thin-film deposition, and anisotropic etching, have allowed researchers to fabricate mechanical devices with complicated silicon structures, including pressure sensors,l accelerometers,2micropumps,3 and micro motor^.^ The development of these techniques, called micromachining, has permitted the fabrication of miniaturized gas chromatographic systems for use in space laboratories. Subsequent efforts have extended the technique to the field of chemical measurement, allowing plate-type sensors, such as ISFET and thin-layer electrodes, which handle a sample outside of the device, to be produced by photolithographic techniques. In contrast, there has been limited progress in liquid-flowtype systems, which handle a sample inside of the device, because they require three-dimensional structures with higher pressure resistance than that in gas flow systems,5 and they require micropumping mechanisms as well. Some attempts have been made to fabricate flow systemsfor chemical analysis by micromachining techniques, including a flow injection
Structure and Geometry of Enzyme Column and Electrochemical Flow Cell. Figure 1shows a schematic overview of the enzyme-immobilized column integrated with an electrochemical flow cell. This device consiste of an anisotropically etched silicon chip and a glass plate with thin-filmgold electrodes. The silicon chip has a V-shaped groove (100pm in width, 70 Hm in depth, 962 mm in length), folded 66 time@to fit into an area 28 mm X 19 mm, and penetrated holes (1mm X 1 mm) at both ends of the groove. All patterns for the inner corners were compensatedlOJ1with squares of 120pm to prevent undercutting. The surface of the silicon chip was oxidized to insulate the electrodes. The electrochemical flow cell is constructed with four thin-film gold electrodes (0.5,1, 1.5, and 1 mm in width, 0.05,0.10.15 and 0.1 mm2in effective area, respectively, 18 mm in length, spaced 0.5 mm from each other) at the end of the column. The second electrode of 1mm in width and the third of 1.5 mm were used as working and counter electrodes, respectively. The fist (0.5 mm) and the fourth (1mm) electrodea were designed into the chip for other applicationsand were not used in the research reported in this paper. Glucose oxidase is covalently immobilized on the inner surface of the column.
(1) Canali, C.; Ferla, G.; Morten, B.;Taroni, A. J. Phys. D. 1974,19731983. (2) Roylance, L. M.; Angell, J. B. ZEEE Tram. Electron Devices 1979, ED-26,1911-1917. (3) Shoji, S.; Nakagawa, S.; Esashi, M. Sem. Actuators 1990, A21A23,189-192. (4) Fan, L. S.;Tai, Y. C.; MiiUer,R. S. Sem.Actuators 1989,20,41-48. (5) Terry, S. C.; Jerman, J. H.; Angell, J. B. ZEEE Tram. Electron Devices 1979, ED-26, 1880-1886. 0003-2700/93/0365-2731$04.00/0
(6) Shoji, S.; Esashi, M. Sem. Actuators B 1992,8,205-208.
(7) Harrison, D. J.; Manz, A.; Fan, Z.; Ladi, H.; Widmer, H. M. Anal. Chem. 1992,64, 1926-1932. (8) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators B 1990, I, 244-248. (9) Guvenc, M. G. Micromachining and Micropackaging of Tramdocers; Elsevier: Amsterdam, 1986; pp 215-223. (10) Wu, X. P.; KO,W. H. Sem. Actuators 1989, 18,207-215. (11) Puers,B.; Sansen, W. Sem.Actuators 1990,A21-A23,1036-1041.
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the capillary. (3-Aminopropy1)triethoxisilane[y-APTES;10pL of 10% (v/v)] in toluene was passed through the capillary at room temperature, leaving behind a thin layer of y-APTES on the inner surface of the capillary, followed by heating overnight at 115"C. After a 1-htreatment with 2.5% (v/v)glutaraldehyde in phosphate buffer (pH 7.1), the capillary was filled with 10% (w/v) GOx (type 11, Sigma) in phosphate buffer (pH 7.1) and kept overnightto immobilizedGOx. Finally, flange-typefemale unions made of polyether ether ketone (PEEK) for fingertight fitting were divided into two pieces and bonded onto the inlet and outlet of the capillary with cement (Araldite, Ciba-Geigy). FIA System. The FIA system constructed consisted of the enzyme-immobilizedcolumn integrated with gold electrodes on a silicon chip, a Model 307 pump, (minimum flow rate 0.1 pL/ min, Gilson, France), a Model 8125 injector (sample size 0.2 or 0.5 pL, Rheodyne) a potentiostat (handmade), and a data processor (Model D-2500, Hitachi). Commercially available flange-typefittingswere used for the connection of the biosensing device with other components of the FIA system.
RESULTS AND DISCUSSION Fabrication of V-Shaped Groove. Because etching rates Flgure 1. Schematic overview of the enzyme-immobilized capillary integrated with electrochemical flow cell: (a) V-shaped groove: (b) inlet hole; (c) glass plate: (d) silicon chip; (e) outlet hole; (f) electrodes, f2 working electrode, f3 counter electrode, f l and f4 reserved.
Fabricationof the IntegratedSiliconChip. A siliconwafer [n-type, 2 in., (100) surface, 0.5 mm thick, optically polished] was washed sequentially with water, trichloroethane,a mixture of concentrated sulfuric acid and hydrogen peroxide (2:l) at boiling temperature, and a mixture of ammonia water, hydrogen peroxide, and water (1:1:6) at boiling temperature. The wafer was thermally oxidized in pyrogenicwater at 1000 "C for 200 min with an oven (Model Cobra, Yamato Semiconductor, Japan). The thicknessof the oxidized layer at this stagewas approximately 0.8 pm. Spin-coatingwith negative-type photoresist (OMR-83, Tokyo Ohka Kougyou) was then carried out by a spin coater (Model 1H-D2,Mikasa, Japan). After prebaking at 80 "C for 30 min, the photoresist layer was photolithographically developed with a photomask to make the holes. Postbaking was carried out at 135 "C for 30 min, and the oxidized layer was etched in a hydrogen fluoride-bufferedsolution (hydrogen fluoride:ammonium fluoride = 1:6). The photoresist was then removed completely by washing with a mixture of sulfuric acid and hydrogen peroxide (2:l). The anisotropicetchingwas performed in a mixture of ethylenediamine, pyrocatechol and water (1000 mL:180 g:480 mL) at 110 "C for 8 h to make holes for inlet and outlet of a carrier solution. The reversed side was also etched in the samemanner with a photomask to make a V-shapedgroove. Finally, after stripping of the oxidized layer with the hydrogen fluoride-bufferedsolution, the wafer was thermally oxidized at lo00 "C for 50 min to give an oxidized layer 400 nm thick. In order to form gold electrodes on Pyrex glass plate for electrochemical detection, chromium and gold layers were deposited on the plate by vacuum evaporator (EBH-6, Ulvac, Japan) and photolithographically etched using positive-type photoresist (OFPR-800, TokyoOhkaKougyou)to give band shape film electrodes. The silicon wafer with the V-shaped groove was anodically bonded to the gold electrode-formed glass plate to give a capillary. In anodicbonding,a ceramicplate (an insulator), a stainless plate (an anode),the siliconwafer, the glass plate, and a stainless plate (a cathode) were put on a hot plate (Model HP46824, Barnstead). The thin-layer gold electrodes were locatedacrossthe end part of the capillary. Direct current voltage was appliedbetween two stainlessplates with a high-voltage power supplyfor electrophoresis (ModelSJ-1065,Atto, Japan). Anodic bonding was performed at 450 "C and lo00 V for 1 h. Auger Electron Spectroscopy of Gold Layer. Gold electrodes of 0.2-pm thickness with a chromium layer of 0.02-pm thickness were prepared in the same manner on glass plate, and heated at 200,400, or 800 "C for 30 min. Immobilization of GOx. For the immobilization of glucose oxidase (GOx), amino groups were covalently attached inside
strongly depend on the temperature and concentration of etchant, it is difficult to reproducibly manufacture silicon structures through control of etching time alone. Because anisotropic etching can be stopped by V-shaped (111)walls, thereby enhancing reproducibility? we chose a V-shaped groove for this study. The V-groove was folded to minimize the area occupied on the silicon chip; as a result, the design features convex corners in series. Although the V-groove could be fabricated with high reproducibility by anisotropic etching, undercutting appeared at every corner, as shown in Figure 2a, if the line on the mask pattern was drawn with the same width from the start to the end. Such undercutting is undesirable because it results in an increase in dead volume at the corners. In order to compensate for undercutting, we used a mask pattern, as shown in Figure 2b. The size of the additional squares at the corners for the compensation was experimentally determined. When the convex corners compensated were etched for 1h, rectangular corners were successfully formed and no undercutting was observed (Figure 2c). Unfortunately, this compensation required control of the etching time period. However, we compensated everycorner of the V-shaped groove for maximum reduction of undesirable dead volume. Adhesion of Glass Plate to Silicon Chip by Anodic Bonding. To form a capillary from the V-groove fabricated on a silicon chip, a glass plate with micro-thin-layer gold electrodes was placed on the silicon chip and attached by anodic bonding. As we applied high pressure to the capillary, a uniform bonding was required to prevent leakage. Because one of the key innovations in this work is the fabrication of electrode-incorporated silicon capillaries, we discuss this process in some detail. Although there are other methods, such as fusion bonding,12 available for bonding glass plate to silicon chip, in this study we employed anodic bonding for two reasons: (1)For the information of thin-layer electrodes on a glass plate, a chromium layer was deposited on the glass plate as a glue layer before the deposition of gold. Depth profiles of the gold electrodes were examined with Auger electron spectroscopy, and it was found that chromium diffused easily into the gold layer and oxidized at the surface when the plate was heated at 200-800 "C (Figure 3). This phenomenon may reduce the adhesion force of the glue layer, so the bonding process should be performed at as low a temperature as possible. In general, anodic bonding allows the use of lower (12) Hanneborg, A.R o c . Micro Electro Mech. Syst. Workshop,Nara, Japan, 1991; pp 92-98.
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Figure 3. AES depth profiles. Chromium (20 nm thick) and gold (200 nm thick) were deposited to glass plate and stored at (a) room temperature, (b) 200 OC, (c) 400 OC, and (d) 800 OC for 30 min. (0) Au; (B) Cr; (A)0; (+) Si; (0)C; (0)N.
Figure 2. SEM photograph of corners of V-groove: (a, top) noncompensated;(b, middle) compensated before etching; (c, bottom) compensated after etching.
temperatures than fusion bonding. (2) It was difficult to achieve control of temperature and pressure adequate for uniform bonding of glass and chip using fusion bonding. The substrates did not bond to each other at low temperature, and the glass bonded to the holder at high temperature. At intermediate temperature, bonding was only partial. With anodic bonding, on the other hand, successful results were achieved with less precise control of the relevant parameters including temperature, pressure, applied voltage, and bonding time. Anodic bonding, well-known as a method for the adhesion of Pyrex glass and silicon structures, is usually carried out at 300-500 "C. The bonding temperature is lower than the softening point of the glass for fusion bonding. In some hypotheticalexplanationsof anodic bonding,the electric field transforms the glass surface to fill the gaps at the interface.13914 However, in our case, the glass plate has thin-layer gold electrodes of thickness much greater than the gaps normally
observed,and the silicon wafer has a thermally oxidized layer, which reduces the strength of the electric field. Thus, the voltage and temperature to be applied are crucial for the anodic bonding of silicon and glass plate with gold electrodes. When the thickness of the oxidized layer, the gold layer, and the chromium layer were approximately 400,100and 10 nm, respectively, the bonding was successfully achieved under conditions of 1000 V applied at 450 "C for 1 h. If the applied voltage, temperature, or reaction time was less than the above, bonding did not succeed. Evidently, these conditions are necessary for the transformation of the glass plate in order to fill and seal the large gaps. Immobilization of GOx on the Inside of the V-Shaped Capillary. GOx is one of the most suitable enzymes for evaluating our device because it is stable and can be easily applied for clinical or industrial use. The immobilizationof enzymes to the surface of glass is commonly done using y-APTES and glutaraldehyde. In addition, it has been reported that y-APTES can be attached on the surface of oxidized silicon15 as well. Therefore, we employed the y-APTES-glutaraldehyde method in this study. (13) Wallis, G.; Pomerantz, D. I. J. Appl. Phys. 1969,40,3946-3949. (14) Kanda,Y.; Matsuda,K.; Murayama,C.; Sugaya,J. Sem.Actuators 1990, A21-A23,939-943. (15) Haller, I. J . Am. Chem. SOC.1978, 100, 8050-8055.
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The -pAPTES treatment requires two steps: In the first step the carrier is exposed to the r-APTES solution. In the second step it is baked at 115 OC to complete the reaction. The first step is usually performed at the reflux temperature of toluene, but in this experiment, r-APTES solution was passed through the capillary at room temperature. We believe that this approach should yield results comparable to those achieved by dipping the carrier. Although no color change, such as to orange or magenta, was observed in the reaction of glutaraldehyde, the sensing system responded to glucose for more than 2 months after glucose oxidase immobilization, implying adequate adhesion of enzyme to surface. When additional immobilization was carried out with a higher concentration of yAPTES or glutaraldehyde, the capillary became clogged due to polymerization by the excess reagents. The entire capillary, including the section downstream of the electrodes, was modified with this method. Thus, there is no dead volume between the enzyme reactor and the electrochemical flow cell. At present, the sample inlet has a large dead space, but we anticipate that this can be reduced through further refinement of the pumping and injection mechanisms. The techniques used for fabrication of long capillaries and the techniques used for modification of its inner surface can be applied to HPLC systems, though an injection mechanism capable of handling nanoliter-order samples would be required. Pressure Drop. The pressure drop of the capillary was about 0.8 MPa when the carrier solution was fed a t 20 pL/ min. The pressure drop of the capillary P m a y be calculated by Poiseuille’s equation as follows: AP = ~ V U L / T ~ ~ (1) where 1 is the viscosity of the carrier solution, u is the flow rate, and L is the length and r is the diameter of the capillary. When 7) is 1.0 X 10-3 PB.s,u is 20 pL/min (3.3 x 10-10 m3/s), r is 3.5 X 1Wm (from the cross-sectionalareaof the capillary), L is 0.96 m, and AP is approximately 0.5 MPa. This value is consistent with the experimental result. The flow rate u is derived by multiplying the mean flow velocity U and the cross-sectional area 1rr2 as per eq 2
u = rr2U Equation 1 can then be written as
a Figure 4. Schematic diagram of glucose detection system: (a) carrier reservoir; (b) pump; (c) inJector; (d) enzyme-immobilized caplliary; (e) electrochemicalflow cell; (f) potentiostatand recorder; (9) waste. The dotted llne shows the area fabricated In this experiment. 2min
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Figure5. Response curve of glucose detectbnsystem. Measurement was carried out with carrier (0.1 M phosphate buffer, pH 7.1); sample volume is 0.2 pL; and flow rate is 10 pL/min. 1400 h
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If we assume Uand L constant to obain a constant response time, the pressure drop is proportional to r2.Thus, although it is easy to fabricate a capillary in which the V-shaped groove’s width is less than 100 pm, such a fine capillary would require a high-pressure pump and an improved connection system. In this experiment, no leak was observed up to 5 MPa. It has been reported that a mechanical pump fabricated by micromachining techniques had a power of less than 0.1 MPa,16 and because it made a parabolic flow profile with laminar flow in the capillary, it may accelerate sample diffusion. We propose the use of electroosmotic flow, providing a plug flow profile and allowing pumping and nanoliter-order injection mechanisms to be made without mobile parts, and allowing control of very small amounts of solution. A glass HPCE system fabricated by micromachining techniques, in which electroosmotic flow moves carrier solution and sample, has been demonstrated.’ Our silicon capillary should be also applicable to a system using electroosmotic flow, because its inner surface is fully insulated with a thermal oxidized layer. Such application may require ~~
~
(16) Shoji, S.;Esashi,M.; Schoot, B.;Rooij, N.Sens. Actuators A 1992,
32, 335-339.
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Glucose concentration (mM) Figure 6. Callbratlon curve for glucose. Measurement was carried out with carrier (0.1 M phosphate buffer, pH 7.1); sample volume is 0.2 pL; and flow rate is 10 pL/min.
further refinement of voltage control in order to avoid electrical breakdown during system operation. Glucose Detection. A glucose detection system was constructed with the enzyme-immobilized capillary integrated with an electrochemical flow cell (Figure 4). In this system, the carrier and sample solution containing glucose come from the inlet hole a t the end of the column. Passing through the column, glucose reacts with dissolved oxygen in the carrier solution in a reaction catalyzed by the GOx on the inside wall of the column to give gluconolactone and hydrogen peroxide. Eventually the electrodes detect oxidation current from the hydrogen peroxide. Figure 5 shows a typical response curve of the glucose detection system using phosphate buffer (pH 7.1) as carrier solution fed at 10pL/min, with 0.2r L of glucose contained sample injected and 700 mV applied between two integrated electrodes. The oxidation current of hydrogen peroxide peaked at 1min after injection. The baseline was
ANALYTICAL CHEMISTRY, VOL. 05, NO. 20, OCTOBER 15, 1993
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Flow rate (pl/min) Flguro 7.- Flow rate dependence of hydrogen peroxide and glucose. Measurement was carried out with carrier (0.1 M phosphate buffer, pH 7.1); sample volume 1s 0.5 pL. (0)Glucose 10 mM; )(. glucose 1 mM; (A)hydrogen peroxide 1 mM.
noisy, and we think one reason was absence of the reference electrode, such as Ag/AgCl, which was difficult to fabricate in the capillary. If 0.2 pL sample zone passes the flow cell without any dispersion, it takes 1.2s at 10pL/min. However, half-value widths were approximately 20 s. The calibration curve for glucose is shown in Figure 6. A linear relationship was observed between current and glucose in the range of 1-25 mM, which includes typical human blood glucose values. The correlation coefficient, r, is 0.993 (n = 23) in the range of 1-25 mM at a flow of 10 pL/min. The
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sensing system gave no response to lOOmM maltose. Glucose solutions in the range below 1 mM were injected, but peak heights were difficult to measure against the baseline noise. The reaction rate was investigated by changing flow rate. Figure 7 shows the flow rate dependence of the response to glucose or hydrogen peroxide. Because the peak current of hydrogen peroxide depends upon its mass transportation rate, a faster flow gave a higher response. The peak current of glucose, however, depends upon reaction time in the reactor, and a slower flow gave a higher response. Comparing the response of hydrogen peroxide and glucose at the same concentration (1 mM), we found that glucose was not completely oxidized about 10% of the glucose was oxidized at 20 pL/min. When the flow rate was 5 pL/min, glucose reacted completely. As noted previously, fabricated sensors remained usable for more than 2 months after glucose oxidase immobilization. In addition, we were unable to detect any decrease in sensor response after 1 month's storage at room temperature.
ACKNOWLEDGMENT The authors thank Seiko I Technoresearch for the AES measurement and also thank M&S Instruments Trading Inc. for providing HPLC supplies.
RECEIVEDfor review March 3, 1993. Accepted July 6, 1993.' e Abstract published in Advance ACS
Abstracts, September 1,1993.
