- Email: [email protected]
Biosensors and Micromachining
BIOSENSORS AND MICROMACHINING
lsao Karube, Kenji Yokoyama, Yuji Murakami, and Masayuki Suda
I. INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 11. ELECTROCHEMICAL FLOW CELL . . . . . . . . . . . . . . . . . . . . . 376 111. INTEGRATION OF ENZYME IMMOBILIZED COLUMN AND ELECTROCHEMICAL FLOW CELL . . . . . . . . . . . . . . . . . . 376 IV. INTEGRATION OF ENZYMATIC REACTOR AND CHEMILUMINESCENCE DETECTOR . . . . . . . . . . . . . . . . . . . . 377 V. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
1. INTRODUCTION In recent years, microfabrication techniquesbased on integrated circuit technology, such as photolithography and etching, have been applied to other fields. These techniques, which are used to make some small and efficient three-dimensional devices, are called micromachining (Borky and Wise, 1979; Roylance and Angell, Advances in Molecular and Cell Biology Volume 15B, pages 375-379. Copyright 0 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0114-7
375
376
I . KARUBE, K. YOKOYAMA, Y. MURAKAMI, and M. SUDA A'
PYREX
A
A'
A
figure 1. Structure of the electrochemical flow cell. From Suda et al., 1990.
1979; Fan et al., 1989; Shoji et al., 1990). Furthermore, some studies to make miniaturized chemical analysis systems were already reported (Terry et al., 1979; Manz et al., 1990). These analysis systems have many advantages such as fast response, small amount of sample, and low consumption of reagents as compared With the conventional one. We applied the micromachining technique to make a miniaturized enzyme-based sensor system.
II. ELECTROCHEMICAL FLOW CELL An electrochemical flow cell that has a very small inner volume of about 20 nl was
fabricated (Suda et al., 1990). Figure 1 shows a structure of the micro electrochemical flow cell. This flow cell can be used as an electrochemical detector for liquid chromatography or flow injection analysis (FIA). The enzyme-immobilized flow cell can be employed as an electrochemical biosensor. Glucose oxidase was immobilized onto the sample inlet hole of the cell using glutaraldehydeand bovine serum albumin. Glucose was calibrated in the range of 30 to 1000 mg/dl when 0.2 pl of the sample was injected.
111. INTEGRATION OF ENZYME IMMOBILIZED COLUMN A N D ELECTROCHEMICAL FLOW CELL A long open-tubular column was fabricated on the silicon substrate (Murakami et al., 1993). A glucose sensor was integrated with both an enzyme immobilized column and an electrochemical flow cell (Figure 2). The column was made by anisotropic silicon etching to be 100 pm wide, 70 pm deep, 1 m long, and a total volume of 5 yl. Four gold electrodes were formed on the glass substrate. Both of the two substrates were anodically bonded. Connecting unions to the pump and
Biosensors and Micromachining
377 A
PYREX A
5
Slllcon
I
Inlet
,
,I E
I
R
L
I
P
I
E
Column
#
-7
Outl&
/
A'
Electrodes
A'
figure 2. Electrochemical detector integratedwith enzyme column. From Murakami et al., 1993.
sample injector were glued on the inlet and outlet holes with epoxy resin. Glucose oxidase (GOD) was immobilized on the inner wall of the column using 3-aminopropyltriethoxysilane and glutaraldehyde. This device was applied to a conventional FIA system.
IV. INTEGRATION OF ENZYMATIC REACTOR AND CHEMILUMINESCENCE DETECTOR An enzymatic reactor and a chemiluminescence detector were integrated on the
same chip (Figure 3) (Suda et al., 1992). The reactor consists of a silicon and glass substrate. On the silicon substrate, an enzymatic reaction column, a mixing chamber, a spiral flow cell were added by anisotropic etching. The total size of the measuring unit was 15 mm x 20 mm, and the total internal volume of the device was about 15 yl. Enzyme-immobilized glass beads were packed into the column, and a photodiode was placed onto the spiral flow cell. Using GOD-immobilized glass beads, determinationof glucose concentration was carried out in the range of 10 to 300 mg/dl. Glucose in human serum and urine was measured by a chemiluminescence detector. The correlation coefficient between this chemiluminescence
3 78
I. KARUBE, K. YOKOYAMA, Y. MURAKAMI, and M. SUDA Splral flow cell
T E
E
z
I-
I
\
/
A'
Mlxlng chamber
ErKyWWk mctor
Enzyme lmmobillzed beads
, , PYREX
/,'
J
A
\******.**.,~*J-
.
/
v
A
Figure 3. Chemiluminescence detector integrated with enzyme reactor. From Suda et at., 1992.
method and the conventional method was 0.99. Lactic acid contained in human serum was quantitated using the same procedure as the glucose determination. Samples containing L-lactic acid at concentrations from 4 to 50 mg/dl could be measured. The correlation coefficient between this chemiluminescencemethod and the conventional method was 0.98.
V. CONCLUSION Detection units for enzyme-based FIA were fabricated using micromachining techniques. Since these micromachined devices are batch-processed, they can be made at low cost and with good reproducibility. The signal from the detector decreases as the detector size decreases. However, the electrochemical and chemiluminescence methods are more sensitive than the spectroscopic method. Hence, the measurable range of the micromachined detector is almost the same as for the conventional method. Additionally,the measurement could be carried out at a flow rate of less than 50 pl/min and yet the pressure drop over the column was less than 0.1 atm. This suggests the possibility of applying a micromachined pump. Thus, the conventional plunger pump and sample injector used in the experiments described above will be replaced by micromachined devices in the near future.
Biosensors and Micromachining
3 79
REFERENCES Borky, J.M. & Wise, K.D. (1979). Integrated signal conditioning for silicon pressure sensors. IEEE Trans. Electron Devices ED-26, 1906-191 1. Fan, L.S., Tai, Y.C., & Muller, R.S. (1989). IC-processed electrostatic micromotors. Sensors and Actuators 2 0 , 4 1 4 8 . Manz, A,, Graber, N., & Widmer, H.M. (1990). Miniaturized total chemical analysis system: a novel concept for chemical sensing. Sensors and Actuators B1,244-248. Murakami, Y., Suda, M., Takeuchi, T., Yokoyama, K.. Tamiya, E.. & Karube, I. (1993). Integration of enzyme-immobilized column with electrochemical flow cell using micromachining techniques for a glucose detection system. Anal. Chem. 65,273 1-2735. Roylance, L.M. & Angell, J.B. (1979). A batch-fabricated silicon accelerometer. IEEE Trans. Electron Devices ED-26, 1911-1917. Suda, M., Muramatsu, H., Sakuhara, T., Ataka, T., Uchida, T., Murakami. Y.. Suzuki, M.. Tamiya, E.. Masuda, Y., & Karube, I. (1990). Glucose sensor processed with IC technology for microanalysis. Proc. Electrochem. Conf. Sept. 2%30,33. Chiba, Japan. Suda, M., Sakuhara, T., Suzuki, M., Tamiya, E., & Karube, I. (1992). Micromachined biosensors. Proc. 2nd World Congr. Biosensors, 4 0 M 0 6 . Shoji, S., Nakagawa, S., & Esashi, M. (1990). Micropump and sample-injector for integral chemical analyzing systems. Sensors and Actuators A2 1-A23, 18W92. Teny, S.C., Jerman, J.H., & Angell, J.B. (1979). Gaschromatographicairanalyzer fabricatedon a silicon wafer. IEEE Trans. Electron Devices ED-26, 1880-1 886.
lsao Karube, Kenji Yokoyama, Yuji Murakami, and Masayuki Suda
I. INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 11. ELECTROCHEMICAL FLOW CELL . . . . . . . . . . . . . . . . . . . . . 376 111. INTEGRATION OF ENZYME IMMOBILIZED COLUMN AND ELECTROCHEMICAL FLOW CELL . . . . . . . . . . . . . . . . . . 376 IV. INTEGRATION OF ENZYMATIC REACTOR AND CHEMILUMINESCENCE DETECTOR . . . . . . . . . . . . . . . . . . . . 377 V. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
1. INTRODUCTION In recent years, microfabrication techniquesbased on integrated circuit technology, such as photolithography and etching, have been applied to other fields. These techniques, which are used to make some small and efficient three-dimensional devices, are called micromachining (Borky and Wise, 1979; Roylance and Angell, Advances in Molecular and Cell Biology Volume 15B, pages 375-379. Copyright 0 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0114-7
375
376
I . KARUBE, K. YOKOYAMA, Y. MURAKAMI, and M. SUDA A'
PYREX
A
A'
A
figure 1. Structure of the electrochemical flow cell. From Suda et al., 1990.
1979; Fan et al., 1989; Shoji et al., 1990). Furthermore, some studies to make miniaturized chemical analysis systems were already reported (Terry et al., 1979; Manz et al., 1990). These analysis systems have many advantages such as fast response, small amount of sample, and low consumption of reagents as compared With the conventional one. We applied the micromachining technique to make a miniaturized enzyme-based sensor system.
II. ELECTROCHEMICAL FLOW CELL An electrochemical flow cell that has a very small inner volume of about 20 nl was
fabricated (Suda et al., 1990). Figure 1 shows a structure of the micro electrochemical flow cell. This flow cell can be used as an electrochemical detector for liquid chromatography or flow injection analysis (FIA). The enzyme-immobilized flow cell can be employed as an electrochemical biosensor. Glucose oxidase was immobilized onto the sample inlet hole of the cell using glutaraldehydeand bovine serum albumin. Glucose was calibrated in the range of 30 to 1000 mg/dl when 0.2 pl of the sample was injected.
111. INTEGRATION OF ENZYME IMMOBILIZED COLUMN A N D ELECTROCHEMICAL FLOW CELL A long open-tubular column was fabricated on the silicon substrate (Murakami et al., 1993). A glucose sensor was integrated with both an enzyme immobilized column and an electrochemical flow cell (Figure 2). The column was made by anisotropic silicon etching to be 100 pm wide, 70 pm deep, 1 m long, and a total volume of 5 yl. Four gold electrodes were formed on the glass substrate. Both of the two substrates were anodically bonded. Connecting unions to the pump and
Biosensors and Micromachining
377 A
PYREX A
5
Slllcon
I
Inlet
,
,I E
I
R
L
I
P
I
E
Column
#
-7
Outl&
/
A'
Electrodes
A'
figure 2. Electrochemical detector integratedwith enzyme column. From Murakami et al., 1993.
sample injector were glued on the inlet and outlet holes with epoxy resin. Glucose oxidase (GOD) was immobilized on the inner wall of the column using 3-aminopropyltriethoxysilane and glutaraldehyde. This device was applied to a conventional FIA system.
IV. INTEGRATION OF ENZYMATIC REACTOR AND CHEMILUMINESCENCE DETECTOR An enzymatic reactor and a chemiluminescence detector were integrated on the
same chip (Figure 3) (Suda et al., 1992). The reactor consists of a silicon and glass substrate. On the silicon substrate, an enzymatic reaction column, a mixing chamber, a spiral flow cell were added by anisotropic etching. The total size of the measuring unit was 15 mm x 20 mm, and the total internal volume of the device was about 15 yl. Enzyme-immobilized glass beads were packed into the column, and a photodiode was placed onto the spiral flow cell. Using GOD-immobilized glass beads, determinationof glucose concentration was carried out in the range of 10 to 300 mg/dl. Glucose in human serum and urine was measured by a chemiluminescence detector. The correlation coefficient between this chemiluminescence
3 78
I. KARUBE, K. YOKOYAMA, Y. MURAKAMI, and M. SUDA Splral flow cell
T E
E
z
I-
I
\
/
A'
Mlxlng chamber
ErKyWWk mctor
Enzyme lmmobillzed beads
, , PYREX
/,'
J
A
\******.**.,~*J-
.
/
v
A
Figure 3. Chemiluminescence detector integrated with enzyme reactor. From Suda et at., 1992.
method and the conventional method was 0.99. Lactic acid contained in human serum was quantitated using the same procedure as the glucose determination. Samples containing L-lactic acid at concentrations from 4 to 50 mg/dl could be measured. The correlation coefficient between this chemiluminescencemethod and the conventional method was 0.98.
V. CONCLUSION Detection units for enzyme-based FIA were fabricated using micromachining techniques. Since these micromachined devices are batch-processed, they can be made at low cost and with good reproducibility. The signal from the detector decreases as the detector size decreases. However, the electrochemical and chemiluminescence methods are more sensitive than the spectroscopic method. Hence, the measurable range of the micromachined detector is almost the same as for the conventional method. Additionally,the measurement could be carried out at a flow rate of less than 50 pl/min and yet the pressure drop over the column was less than 0.1 atm. This suggests the possibility of applying a micromachined pump. Thus, the conventional plunger pump and sample injector used in the experiments described above will be replaced by micromachined devices in the near future.
Biosensors and Micromachining
3 79
REFERENCES Borky, J.M. & Wise, K.D. (1979). Integrated signal conditioning for silicon pressure sensors. IEEE Trans. Electron Devices ED-26, 1906-191 1. Fan, L.S., Tai, Y.C., & Muller, R.S. (1989). IC-processed electrostatic micromotors. Sensors and Actuators 2 0 , 4 1 4 8 . Manz, A,, Graber, N., & Widmer, H.M. (1990). Miniaturized total chemical analysis system: a novel concept for chemical sensing. Sensors and Actuators B1,244-248. Murakami, Y., Suda, M., Takeuchi, T., Yokoyama, K.. Tamiya, E.. & Karube, I. (1993). Integration of enzyme-immobilized column with electrochemical flow cell using micromachining techniques for a glucose detection system. Anal. Chem. 65,273 1-2735. Roylance, L.M. & Angell, J.B. (1979). A batch-fabricated silicon accelerometer. IEEE Trans. Electron Devices ED-26, 1911-1917. Suda, M., Muramatsu, H., Sakuhara, T., Ataka, T., Uchida, T., Murakami. Y.. Suzuki, M.. Tamiya, E.. Masuda, Y., & Karube, I. (1990). Glucose sensor processed with IC technology for microanalysis. Proc. Electrochem. Conf. Sept. 2%30,33. Chiba, Japan. Suda, M., Sakuhara, T., Suzuki, M., Tamiya, E., & Karube, I. (1992). Micromachined biosensors. Proc. 2nd World Congr. Biosensors, 4 0 M 0 6 . Shoji, S., Nakagawa, S., & Esashi, M. (1990). Micropump and sample-injector for integral chemical analyzing systems. Sensors and Actuators A2 1-A23, 18W92. Teny, S.C., Jerman, J.H., & Angell, J.B. (1979). Gaschromatographicairanalyzer fabricatedon a silicon wafer. IEEE Trans. Electron Devices ED-26, 1880-1 886.