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PDMS-based microfluidic devices for biomedical applications
Microelectronic Engineering 61–62 (2002) 907–914 www.elsevier.com / locate / mee
PDMS-based microfluidic devices for biomedical applications Teruo Fujii Institute of Industrial Science, University of Tokyo 4 -6 -1 Komaba, Meguro-ku, Tokyo 153 -8505, Japan
Abstract Microfluidic devices provide a number of advantageous features for microscale biochemical systems for analysis and / or synthesis. A PDMS (polydimethylsiloxane) microchip, for instance, which has microchannels for electrophoretic separation can be easily fabricated through a molding process. Sealing of those channels does not need any elaborate bonding processes which are usually required for glass chips. We have been working on PDMS-based microfluidic devices for biomedical applications, where microreactors, microchips for capillary gel electrophoresis, and hydrophobic vent valves are successfully fabricated and operated. Fundamentals of PDMS-based microfluidic devices and their functions are described as well as the experimental results. 2002 Elsevier Science B.V. All rights reserved. Keywords: Microfluidics; Micro-TAS; Lab-on-a-chip; PDMS
1. Introduction Microfluidic devices provide a number of advantageous features for microscale biochemical systems for analysis and / or synthesis. A PDMS microchip, for instance, which has microchannels for electrophoretic separation can be easily fabricated through a molding process. Sealing of those channels does not need any elaborate bonding processes which are usually required for glass chips. After several preceding works on the PDMS-based microstructure for MCP (microcontact printing [1]), mFN (microfluidic network [2]), and CE (capillary electrophoresis [3,4]), multilayered structures with valving capability have been developed recently [5,6]. We have also been working on PDMS-based microfluidic devices for biomedical applications, where microreactors, microchips for capillary gel electrophoresis, and hydrophobic vent valves are successfully fabricated and operated [7–9]. Unlike the recent reports [5,6], we chose another way of integration in the form of hybrid structures, i.e. a PDMS chip on various rigid substrates on which access ports for liquid, air, and electrodes are integrated. In this hybrid structure, microstrctures on the PDMS chip are used as liquid / air conduits and reaction chambers which could be fabricated easily E-mail address: [email protected] (T. Fujii). 0167-9317 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0167-9317( 02 )00494-X
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T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
and cost effectively. But thanks to the reversible feature of spontaneous bonding of the chip to the substrate, the chip could be a single use device. The substrate on which some of the devices are integrated can be used repeatedly by replacing the PDMS chip. This paper introduces some example of the PDMS-based hybrid structures for microscale biochemical systems following the concepts described above.
2. PDMS microchips Fig. 1 shows a typical fabrication process of PDMS microchips. PDMS microchips can be fabricated through microscale molding processes. For laboratory use, a silicon wafer with patterned photoresist can be used as a mold master. To have a relatively thick structure of microchannels and microchambers for transportation and / or incubation of the reagents and samples, we adopt an ultrathick photoresist, SU-8. After the patterning, prepolymer of PDMS is poured into the mold master. And then cured PDMS is peeled off from the master to be pasted on a flat plate, i.e. PMMA (polymethylmethacrylate), glass, etc., on which access ports for introduction of the reagents and samples should be drilled in advance. PDMS is known to be one of the most attractive materials for microfluidic devices, because of its advantageous features. Fig. 2 shows SEM micrographs of the cross-shaped microchannel fabricated with PDMS. PDMS can replicate fine structures down to a submicron feature size [10]. We can easily get microstructures with smooth surfaces through the simple molding process described above. In most biochemical analysis, fluorescent dyes are widely used for detection and quantification of molecules. PDMS has favorable optical properties for a fluorescence-based detection scheme as shown in Fig. 3. It has almost no absorbance in the range of visible wavelength. Another attractive feature of PDMS as a material of microchip is its spontaneous
Fig. 1. Fabrication process of PDMS microchip.
T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
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Fig. 2. SEM micrograph of PDMS structure.
adhesion onto flat surfaces. Generally, microfluidic devices have a confined microchannels and microchambers, which are realized by elaborate bonding processes, for instance, fusion bonding method for glass substrates and anodic bonding method for silicon–glass bonding. But in the case of PDMS, the microstructure can be sealed by just pasting the chip onto a flat substrate. Since the bonding is reversible, you can easily replace the PDMS chip by other chips to avoid cross contamination with an easy washing process of the substrate. We can put some functional devices such as heaters, temperature sensors, etc. on the top of the substrate, and the microfluidic part could be provided in the PDMS microchip. Thus, we are proposing a hybrid structure of PDMS and glass or PDMS and PMMA as a promising format for microfluidic devices for biomedical applications.
3. PDMS microchip for capillary gel electrophoresis Fig. 4 shows a set-up for capillary gel electrophoresis of DNA with a PDMS microchip. An I-shaped microchannel for separation formed on a PDMS chip is pasted on a PMMA plate as shown in Fig. 5. Since PDMS microchips can be replaced repeatedly as a single use device, we can introduce
Fig. 3. Optical properties of PDMS.
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Fig. 4. Set-up for CGE.
agarose gel as a sieving matrix for separation. After loading an initial sample plug into the agarose gel by applying electric field for just 1 s and washing out excessive amount of sample with buffer, sample plug is electrophoresed in the separation channel. Images of the separated band are recorded by a CCD camera through a simple optics for fluorescent detection. We could successfully get an electropherogram of the separation of 100-bp DNA ladder as shown in Fig. 6. Here, 100-bp to 1-kbp DNA fragments can be separated in just 2 min. This is over ten times faster than the conventional slab gel electrophoresis, which usually takes 20–30 min for the same operation. In order to facilitate the advantage of the PDMS microchip as a single use diagnosis device, we have tried to realize sequential operation of gene amplification and detection of the amplified fragment
Fig. 5. Layout of PDMS microchip.
T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
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Fig. 6. Result of DNA separation (2.0% agarose in 1 3 TBE, 100-bp DNA ladder, 71.4 V/ cm).
on a same chip. This operation is basically for checking of the existence of a specific gene on the target DNA. As shown in Fig. 7, a reaction chamber for PCR (polymerase chain reaction) and a separation channel are formed on a PDMS chip, which is pasted on a glass substrate. To avoid the intrusion of the reaction mixture into the separation channel, both structures are connected through a narrowed channel, i.e. so-called ‘capillary break’. Since the surface of PDMS chip is hydrophobic, the narrowed channel works as a simple stop valve up to a certain pressure difference by capillary effect.
Fig. 7. Design of PDMS microchip for PCR and CGE.
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Fig. 8. Results of the DNA amplification and separation: (A) electropherogram of the PCR product (500 bp fragment on lDNA), and (B) electropherogram for sizing.
A whole chip structure is put on a thermoelectric device to apply thermal cycle for the gene amplification reaction. A 500-base fragment on lDNA is amplified and the reaction mixture is introduced into the separation channel by pressurizing the reaction chamber. Consequently we could get a 500-bp band on the electropherogram as shown in Fig. 8.
4. PDMS–glass hybrid microreactor In PDMS–glass hybrid structures, we can integrate functional devices with metal and / or oxide materials on a glass substrate. Fig. 9 shows a PDMS–glass hybrid structure for cell-free protein synthesis. For incubation of the reaction chamber, ITO (indium tin oxide) transparent electrodes are integrated in a multilayer glass substrate on which the PDMS chip with an array of reaction chamber is mounted as shown in Fig. 10. Activity of cell-free protein synthesis is largely dependent on temperature condition. Usually the temperature in the reaction chamber should be kept at 37 8C. The two layers of integrated ITO electrodes could work as heater and temperature sensing devices. Here, upper layer is used as a resistive for temperature sensing and lower layer is used as a heating device. Using the integrated temperature control devices, GFP (green fluorescent protein) is successfully synthesized according to the GFP gene injected into the reactor chamber. Fluorescence in the reaction chamber increases as the coupled transcription–translation reaction takes place as shown in Fig. 11. Currently, the heating pattern for the arrayed reactors are being optimized, and the parallel operation of the reactors with different conditions will be expected [11].
5. Summary PDMS-based microfluidic devices enable us to realize microchips for biomedical applications in a relatively easy and cost effective way. By adopting the proposed hybrid structure, we could take another direction toward integrated microfluidic devices in which functional devices, such as
T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
Fig. 9. PDMS–glass hybrid structure with integrated heating and sensing devices.
Fig. 10. Layout of the hybrid structure for a single reactor in the array.
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Fig. 11. Time history of fluorescence in microreactor.
electrodes, heaters, sensors, etc., fabricated on substrates, can easily be interfaced with PDMS-made microstructures. Continuous effort for integrating every single functions into a same microchip leads to one-time use multifunction devices for future biomedical diagnosis.
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
A. Kumar, H.A. Biebuyck, G.M. Whitesides, Langmuir 10 (2000) 1498–1511. E. Delamarche et al., Science 276 (1997) 779–781. C.S. Effenhauser, G.J.M. Bruin, A. Paulus, Electrophoresis 18 (1997) 2203. D.C. Duffy et al., Anal. Chem. 70 (1998) 4974–4984. D.J. Beebe et al., Nature 404 (2000) 588–590. M.A. Unger et al., Science 288 (2000) 113–116. J.W. Hong, K. Hosokawa, T. Fujii, M. Seki, I. Endo, Prof. Transducers ’99, (1999) 760–763. K. Hosokawa, T. Fujii, I. Endo, Anal. Chem. 71 (1999) 4781–4785. S. Kaneda, T. Fujii, J.W. Hong, Y. Kunii, M. Seki, I. Endo, in: Proc. of JSME Annual Conference on Robotics and Mechatronics 2000, 1A1-63-090, 2000, (in Japanese). [10] L. Libioulle et al., Langmuir 15 (1999) 300–304. [11] T. Yamamoto, T. Nojima, T. Fujii, in: Proc. mTAS2001, Monterey, USA, 2001, pp. 69–71.
PDMS-based microfluidic devices for biomedical applications Teruo Fujii Institute of Industrial Science, University of Tokyo 4 -6 -1 Komaba, Meguro-ku, Tokyo 153 -8505, Japan
Abstract Microfluidic devices provide a number of advantageous features for microscale biochemical systems for analysis and / or synthesis. A PDMS (polydimethylsiloxane) microchip, for instance, which has microchannels for electrophoretic separation can be easily fabricated through a molding process. Sealing of those channels does not need any elaborate bonding processes which are usually required for glass chips. We have been working on PDMS-based microfluidic devices for biomedical applications, where microreactors, microchips for capillary gel electrophoresis, and hydrophobic vent valves are successfully fabricated and operated. Fundamentals of PDMS-based microfluidic devices and their functions are described as well as the experimental results. 2002 Elsevier Science B.V. All rights reserved. Keywords: Microfluidics; Micro-TAS; Lab-on-a-chip; PDMS
1. Introduction Microfluidic devices provide a number of advantageous features for microscale biochemical systems for analysis and / or synthesis. A PDMS microchip, for instance, which has microchannels for electrophoretic separation can be easily fabricated through a molding process. Sealing of those channels does not need any elaborate bonding processes which are usually required for glass chips. After several preceding works on the PDMS-based microstructure for MCP (microcontact printing [1]), mFN (microfluidic network [2]), and CE (capillary electrophoresis [3,4]), multilayered structures with valving capability have been developed recently [5,6]. We have also been working on PDMS-based microfluidic devices for biomedical applications, where microreactors, microchips for capillary gel electrophoresis, and hydrophobic vent valves are successfully fabricated and operated [7–9]. Unlike the recent reports [5,6], we chose another way of integration in the form of hybrid structures, i.e. a PDMS chip on various rigid substrates on which access ports for liquid, air, and electrodes are integrated. In this hybrid structure, microstrctures on the PDMS chip are used as liquid / air conduits and reaction chambers which could be fabricated easily E-mail address: [email protected] (T. Fujii). 0167-9317 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0167-9317( 02 )00494-X
908
T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
and cost effectively. But thanks to the reversible feature of spontaneous bonding of the chip to the substrate, the chip could be a single use device. The substrate on which some of the devices are integrated can be used repeatedly by replacing the PDMS chip. This paper introduces some example of the PDMS-based hybrid structures for microscale biochemical systems following the concepts described above.
2. PDMS microchips Fig. 1 shows a typical fabrication process of PDMS microchips. PDMS microchips can be fabricated through microscale molding processes. For laboratory use, a silicon wafer with patterned photoresist can be used as a mold master. To have a relatively thick structure of microchannels and microchambers for transportation and / or incubation of the reagents and samples, we adopt an ultrathick photoresist, SU-8. After the patterning, prepolymer of PDMS is poured into the mold master. And then cured PDMS is peeled off from the master to be pasted on a flat plate, i.e. PMMA (polymethylmethacrylate), glass, etc., on which access ports for introduction of the reagents and samples should be drilled in advance. PDMS is known to be one of the most attractive materials for microfluidic devices, because of its advantageous features. Fig. 2 shows SEM micrographs of the cross-shaped microchannel fabricated with PDMS. PDMS can replicate fine structures down to a submicron feature size [10]. We can easily get microstructures with smooth surfaces through the simple molding process described above. In most biochemical analysis, fluorescent dyes are widely used for detection and quantification of molecules. PDMS has favorable optical properties for a fluorescence-based detection scheme as shown in Fig. 3. It has almost no absorbance in the range of visible wavelength. Another attractive feature of PDMS as a material of microchip is its spontaneous
Fig. 1. Fabrication process of PDMS microchip.
T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
909
Fig. 2. SEM micrograph of PDMS structure.
adhesion onto flat surfaces. Generally, microfluidic devices have a confined microchannels and microchambers, which are realized by elaborate bonding processes, for instance, fusion bonding method for glass substrates and anodic bonding method for silicon–glass bonding. But in the case of PDMS, the microstructure can be sealed by just pasting the chip onto a flat substrate. Since the bonding is reversible, you can easily replace the PDMS chip by other chips to avoid cross contamination with an easy washing process of the substrate. We can put some functional devices such as heaters, temperature sensors, etc. on the top of the substrate, and the microfluidic part could be provided in the PDMS microchip. Thus, we are proposing a hybrid structure of PDMS and glass or PDMS and PMMA as a promising format for microfluidic devices for biomedical applications.
3. PDMS microchip for capillary gel electrophoresis Fig. 4 shows a set-up for capillary gel electrophoresis of DNA with a PDMS microchip. An I-shaped microchannel for separation formed on a PDMS chip is pasted on a PMMA plate as shown in Fig. 5. Since PDMS microchips can be replaced repeatedly as a single use device, we can introduce
Fig. 3. Optical properties of PDMS.
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T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
Fig. 4. Set-up for CGE.
agarose gel as a sieving matrix for separation. After loading an initial sample plug into the agarose gel by applying electric field for just 1 s and washing out excessive amount of sample with buffer, sample plug is electrophoresed in the separation channel. Images of the separated band are recorded by a CCD camera through a simple optics for fluorescent detection. We could successfully get an electropherogram of the separation of 100-bp DNA ladder as shown in Fig. 6. Here, 100-bp to 1-kbp DNA fragments can be separated in just 2 min. This is over ten times faster than the conventional slab gel electrophoresis, which usually takes 20–30 min for the same operation. In order to facilitate the advantage of the PDMS microchip as a single use diagnosis device, we have tried to realize sequential operation of gene amplification and detection of the amplified fragment
Fig. 5. Layout of PDMS microchip.
T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
911
Fig. 6. Result of DNA separation (2.0% agarose in 1 3 TBE, 100-bp DNA ladder, 71.4 V/ cm).
on a same chip. This operation is basically for checking of the existence of a specific gene on the target DNA. As shown in Fig. 7, a reaction chamber for PCR (polymerase chain reaction) and a separation channel are formed on a PDMS chip, which is pasted on a glass substrate. To avoid the intrusion of the reaction mixture into the separation channel, both structures are connected through a narrowed channel, i.e. so-called ‘capillary break’. Since the surface of PDMS chip is hydrophobic, the narrowed channel works as a simple stop valve up to a certain pressure difference by capillary effect.
Fig. 7. Design of PDMS microchip for PCR and CGE.
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T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
Fig. 8. Results of the DNA amplification and separation: (A) electropherogram of the PCR product (500 bp fragment on lDNA), and (B) electropherogram for sizing.
A whole chip structure is put on a thermoelectric device to apply thermal cycle for the gene amplification reaction. A 500-base fragment on lDNA is amplified and the reaction mixture is introduced into the separation channel by pressurizing the reaction chamber. Consequently we could get a 500-bp band on the electropherogram as shown in Fig. 8.
4. PDMS–glass hybrid microreactor In PDMS–glass hybrid structures, we can integrate functional devices with metal and / or oxide materials on a glass substrate. Fig. 9 shows a PDMS–glass hybrid structure for cell-free protein synthesis. For incubation of the reaction chamber, ITO (indium tin oxide) transparent electrodes are integrated in a multilayer glass substrate on which the PDMS chip with an array of reaction chamber is mounted as shown in Fig. 10. Activity of cell-free protein synthesis is largely dependent on temperature condition. Usually the temperature in the reaction chamber should be kept at 37 8C. The two layers of integrated ITO electrodes could work as heater and temperature sensing devices. Here, upper layer is used as a resistive for temperature sensing and lower layer is used as a heating device. Using the integrated temperature control devices, GFP (green fluorescent protein) is successfully synthesized according to the GFP gene injected into the reactor chamber. Fluorescence in the reaction chamber increases as the coupled transcription–translation reaction takes place as shown in Fig. 11. Currently, the heating pattern for the arrayed reactors are being optimized, and the parallel operation of the reactors with different conditions will be expected [11].
5. Summary PDMS-based microfluidic devices enable us to realize microchips for biomedical applications in a relatively easy and cost effective way. By adopting the proposed hybrid structure, we could take another direction toward integrated microfluidic devices in which functional devices, such as
T. Fujii / Microelectronic Engineering 61 – 62 (2002) 907 – 914
Fig. 9. PDMS–glass hybrid structure with integrated heating and sensing devices.
Fig. 10. Layout of the hybrid structure for a single reactor in the array.
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Fig. 11. Time history of fluorescence in microreactor.
electrodes, heaters, sensors, etc., fabricated on substrates, can easily be interfaced with PDMS-made microstructures. Continuous effort for integrating every single functions into a same microchip leads to one-time use multifunction devices for future biomedical diagnosis.
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
A. Kumar, H.A. Biebuyck, G.M. Whitesides, Langmuir 10 (2000) 1498–1511. E. Delamarche et al., Science 276 (1997) 779–781. C.S. Effenhauser, G.J.M. Bruin, A. Paulus, Electrophoresis 18 (1997) 2203. D.C. Duffy et al., Anal. Chem. 70 (1998) 4974–4984. D.J. Beebe et al., Nature 404 (2000) 588–590. M.A. Unger et al., Science 288 (2000) 113–116. J.W. Hong, K. Hosokawa, T. Fujii, M. Seki, I. Endo, Prof. Transducers ’99, (1999) 760–763. K. Hosokawa, T. Fujii, I. Endo, Anal. Chem. 71 (1999) 4781–4785. S. Kaneda, T. Fujii, J.W. Hong, Y. Kunii, M. Seki, I. Endo, in: Proc. of JSME Annual Conference on Robotics and Mechatronics 2000, 1A1-63-090, 2000, (in Japanese). [10] L. Libioulle et al., Langmuir 15 (1999) 300–304. [11] T. Yamamoto, T. Nojima, T. Fujii, in: Proc. mTAS2001, Monterey, USA, 2001, pp. 69–71.