- Email: [email protected]
Microfluidic systems for extracting nucleic acids for DNA and RNA analysis
Sensors and Actuators A 133 (2007) 335–339
Microfluidic systems for extracting nucleic acids for DNA and RNA analysis Wing C. Hui a,∗ , Levent Yobas a , Victor D. Samper a,1 , Chew-Kiat Heng b , Saxon Liw a , Hongmiao Ji a , Yu Chen a , Lin Cong b , Jing Li a , Tit Meng Lim b a
Institute of Microelectronics, 11 Science Park Road, Singapore Science Park II, Singapore b National University of Singapore, 10 Kent Ridge Crescent, Singapore Received 8 July 2005; received in revised form 14 April 2006; accepted 24 April 2006 Available online 9 August 2006
Abstract This paper is to review the differences in the developments of microfluidic chips for extracting genomic deoxyribonucleic acid (DNA) and viral ribonucleic acid (RNA) from blood by the Biosensor Focus Interest Group (BFIG) in Singapore. DNA was extracted in a multi-step process by isolating and lysing white blood cells (WBC), typically ∼10 m in diameter. Viral RNA was extracted directly from the submicron viruses in the blood. In terms of basic microfluidic components required, both DNA and RNA extractions used similar mixers for mixing reagents, filters for capturing or separating the blood cells, and a binder for capturing and purifying the DNA/RNA molecules. The designs of the filters were adapted to either capture WBC for DNA isolation or capture all virus particles for RNA isolation. The designs of these two kinds of filters had to be different. Besides the differences in the sizes of WBC and viruses, the concentration of the virus particles is usually much lower than WBC. Thus, a much higher volume of blood for filtering would be required for extracting viral RNA, especially for the intention to detect the viruses at early onset of infection. With proper modifications of the protocols, it has been demonstrated that both genomics DNA and viral RNA could be extracted successfully in these microfluidic chips. The quality of the extracted samples was verified by polymerase chain reaction (PCR) and gel-electrophoresis after the extractions. © 2006 Elsevier B.V. All rights reserved. Keywords: Nucleic acid; DNA; RNA; Extraction; Microfluidic; Virus
1. Introduction Numerous independent microfluidic components, such as microchannels, microfilters, cell sorters, mixers, valves, binders, etc., have been developed for various biomedical applications [1–4]. In this work microfluidic components have been integrated together to isolate: (1) deoxyribonucleic acid (DNA) from white blood cells and (2) ribonucleic acid (RNA) from viruses in the blood. In general, these two applications use different protocols and require different combinations of microfluidic components for efficient operation. For DNA extraction, the strategy is to capture the white blood cells (WBC) and deplete the concentration of the red blood cells (RBC). The hemoglobin from RBC is a contaminant that can ∗ 1
Corresponding author. Tel.: +65 6770 5900; fax: +65 6774 5747. E-mail address: [email protected] (W.C. Hui). Present address: GE Global Research-Europe, Munich, Germany.
0924-4247/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.sna.2006.06.031
inhibit the downstream process of DNA amplification by polymerase chain reaction (PCR). For viral RNA extraction, it is necessary to remove both WBC and RBC, with a minimal reduction in the concentration of the viruses in the plasma (less than 1 m in size). This demands a submicron filter. RNA is a more fragile molecule than DNA and thus poses additional challenges in the hardware and protocol development when compared to DNA extraction [5]. 2. Development of microfluidic chips 2.1. DNA extraction To extract DNA from WBC, the first step was to isolate the WBC using size- and compliance-based discrimination. The system then had to lyse the WBC using a high salt solution, to release the DNA. A binder was used to reversibly capture the DNA from the lysed cells. The DNA was bound to the surface
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W.C. Hui et al. / Sensors and Actuators A 133 (2007) 335–339
Fig. 1. The DNA extraction chip.
during the clean-up process and subsequently eluted as purified DNA. A silicon/glass microfluidic chip was designed and fabricated for DNA extraction. It consisted of an integrated mixer [6], two paraffin valves, a filter with 3-m gap [7], and a binder. The DNA extraction chip is shown in Fig. 1. 2.2. Viral RNA extraction The basic steps for RNA isolation were similar to DNA extraction. However, some of the detailed requirements were different. In general, the viruses are smaller than WBC and RBC, and the concentration of virus in the blood is much lower than the WBC. Hence, a submicron filter had to be developed for handling the high volume and flow rate of blood necessary to obtain sufficient copies of the virus. The modified filter was able to separate the WBC and the RBC from the viruses in the plasma. A new process technology was developed for making deep submicron filters [8,9]. A filter with 3-m trenches was fabricated by deep reactive ion etching (DRIE). Subsequently, the trench gaps were narrowed down to 0.8 m or less by deposition
Fig. 3. The three submicron filters developed for separating viruses in serum from the WBC and RBC: (A) Submicron filter 1, (B) submicron filter 2, and (C) submicron filter 3.
of silicon dioxide. Fig. 2 shows the SEM pictures of the filter trench gaps before and after the oxide deposition. Based on this fabrication technology, three high flow-rate filters that could handle 40–400 l of blood were designed and fabricated. Fig. 3 shows optical microscope images of the filters. All of these three filters showed >90% efficiencies of filtration
Fig. 2. The SEM photos of the filter trench gaps before (A) and after (B) the deposition of silicon dioxide: (A) 3 m trench gaps fabricated by DRIE and (B) 0.8 m trench gaps after oxide deposition.
W.C. Hui et al. / Sensors and Actuators A 133 (2007) 335–339
337
Table 1 Testing results of the three submicron filters Blood:PBS = 50:50 RBC count in the blood 865 pumped (million) Volume of the diluted blood 400 pumped (l) Submicron filter 1 (5 mm × 10 mm) % EF 90.37 Plasma collected (l) 212
Blood:PBS = 25:75 433 400
99.56 181
Submicron filter 2 (5 mm × 10 mm) % EF 95.08 Plasma collected (l) 224
99.47 139
Submicron filter 3 (10 mm × 10 mm) % EF 91.30 Plasma collected (l) 198
99.81 61
Fig. 5. The basic bench top experimental setup for both the DNA and viral RNA extractions.
was used to drive each syringe to deliver the sample or reagent in the microliter range. The microlinear motion actuators were controlled by a calibrated computer system. A microscope with a CCD video camera was used to monitor the operation. Both experimental parameters and video images could be captured by the computer. Fig. 5 shows the basic experimental setup for DNA and viral RNA extractions. One of the main goals of our efforts was to leverage on the portability offered by microfluidics. Thus, two portable fluidic delivery systems were also developed for the DNA and the viral RNA extractions. 3.1. Submicron filters Fig. 4. The viral RNA extraction test block with the submicron filter and the micro-DNA extraction chip.
(EF) and fulfilled the requirement for the viral RNA extraction process. A summary of their EF can be found in Table 1 [8]. Filter 1 was chosen for the task of viral RNA extraction because experiments showed it to be less prone to unwanted rupture of RBC. The filter was combined with the micro-DNA extraction chip for the viral RNA extraction task. After separation of the viruses by the submicron filter, the micro-DNA extraction chip was used for the lysing of virus, binding and cleaning of the viral RNA, and finally, elution of the RNA. Fig. 4 shows the test kit with both the submicron filter 1 and the original DNA extraction chip. 3. Experimental results and discussions The experimental setups were the same for the initial testing of both the DNA extraction and the viral RNA extraction. They included a test block to connect the microfluidic chips and the external microfluidic delivery system. The microfluidic delivery systems consisted of several syringes containing the sample and the various reagents required. A microlinear motion actuator
Table 1 shows a summary of the filter efficiency of filtration based on the mixture of the phosphate buffered saline (PBS) solutions. The three filters showed >90% efficiencies of filtration and fulfilled the requirement for the viral RNA extraction process [8]. 3.2. DNA extraction On average, approximately 1 ng DNA was obtained per microliter of blood. The results depend strongly on the protocols. PCR results were used to demonstrate the purity of the DNA extracted. Amplified products were put through gelelectrophoresis and visualized by staining the nucleic acids with ethidium bromide, which emitted an orange fluorescence under UV light. If the starting sample was not clean enough, it would inhibit the PCR. Fig. 6 shows the positive PCR result for an extracted DNA sample. In order to make the DNA extraction operation portable, an automatically controlled system was built within a briefcase. It took some of the basic elements from the initial experimental setup, including the microfluidic delivery system with the microlinear motion actuators and the syringes. The system was controlled by a laptop computer. Fig. 7 shows the portable system for the DNA extraction system.
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Fig. 8. Result of the gel-electrophoresis after reverse transcriptase PCR (RTPCR) of viral RNA extracted by the viral RNA extraction chip.
3.3. Viral RNA extraction
Fig. 6. Result of gel-electrophoresis after PCR on DNA sample extracted by the DNA extraction chip.
Fig. 7. The portable system for DNA extraction.
In terms of handling the nucleic acids, RNA is more unstable and poses greater challenges in preserving their integrity during the extraction process in comparing to DNA. New techniques were developed for capturing and eluting RNA without compromising its integrity. The protocol was first developed for total RNA from WBC and then with viral RNA from a harmless plant virus, such as the Orchid Virus. Fig. 8 shows the results from reverse transcriptase polymerase chain reaction (RT-PCR) that confirm that good quality viral RNA was actually extracted by the viral RNA extraction chip set. The results also depended strongly on the protocols. For the viral RNA extraction, a further minimized version of the automatic microfluidic delivery system was developed (Fig. 9). The size was 31 cm × 23 cm × 11 cm. It contained mechanical micropumps and microvalves to control the fluidics
Fig. 9. The portable system for viral RNA extraction.
W.C. Hui et al. / Sensors and Actuators A 133 (2007) 335–339
down to the microliter level. It could be controlled either: (1) by a PC or laptop computer for flexibility in R&D or (2) by sequential programming with a fixed protocol for routine testing. 4. Conclusions We have successfully demonstrated that both the DNA and viral RNA extraction chips were capable of yielding pure DNA and viral RNA accordingly. The results were confirmed by PCR and RT-PCR, respectively. The performance could vary according to the protocols and run parameters such as flow rates and the duration of incubation times. In addition to the microfluidic extraction chips, portable automatic systems were also developed so that the process of nucleic acid extraction was no longer confined to a laboratory setting. Acknowledgement The authors would like to thank the Agency for Science, Technology and Research (A*STAR) in Singapore for funding the projects under the Biosensor Focus Interest Group (BFIG). References [1] D.R. Meldrum, M.R. Holl, Microscale bioanalytical systems, Science 297 (2002) 1197–1198. [2] J.H. Kim, B.G. Kim, H. Nam, D.E. Park, K.S. Yun, J.B. Yoon, J. You, E. Yoon, A disposable DNA sample preparation microfluidic chip for nucleic acid probe assay, in: Proceedings of the IEEE Micro Electro Mechanical Systems (MEMS), 2002, pp. 133–136. [3] K.A. Melzak, C.S. Shervood, R.F.B. Turner, C.A. Haynes, Driving forces for DNA adsorption to silica in perchlorate solutions, J. Colloid Interface Sci. 181 (1996) 635–644. [4] R. Pal, M. Yang, B.N. Johnson, D.T. Burke, M.A. Burns, Phase change microvalve for integrated devices, Anal. Chem. 76 (2004) 3740–3748. [5] T.W. Smalling, S.E. Sefers, H.J. Li, Y.W. Tang, Molecular approaches to detecting herpes simplex virus and enteroviruses in the central nervous system, J. Clin. Microbiol. 40 (7) (2002) 2317–2322. [6] W.L. Goh, W.H. Ma, W.C. Hui, H.M. Ji, Evaluation of microfluidic mixer designs for RNA extraction, in: International Conference on Materials for Advanced Technologies (ICMAT 2005), Singapore, July 3–8, 2005. [7] Y. Chen, Y. Miao, V. Samper, F. Bte Mustafa, Q.X. Zhang, C.K. Heng, H. Lye, T.M. Lim, Microfabrication of a Si mesh structure depth filter, in: Proceedings of TAS 2002, 6th International Conference on Miniaturized Systems for Chemistry and Life Sciences, vol. 2, 2002, pp. 739–741. [8] L. Yobas, E.L. Gui, H.M. Ji, J. Li, Y. Chen, W.C. Hui, S. Rafeah, S. Swarup, S.M. Wong, T.M. Lim, C.K. Heng, A flow-through shear-type microfilter chip for separating plasma and virus particles from whole blood, in: Proceedings of TAS 2004, 8th International Conference on Miniaturized Systems in Chemistry and Life Sciences, vol. 2, Malmo, Sweden, September 26–30, 2004, pp. 7–9. [9] J. Li, Y. Chen, L. Yobas, H.M. Ji, W.C. Hui, et al., Fabricating submicron microfluidic filter without X-ray lithography, in: Proceedings of International Conference on Materials for Advanced Technologies 2004 (ICMAT 2004), July 4–8, 2004, pp. 409–412.
Biographies Wing C. Hui obtained both his BS and MS degrees in chemical engineering from University of California at Berkeley, in 1978 and 1980, respectively. Wing had
339
worked at Dow Chemicals for 4 years and then Lawrence Livermore National Laboratory in US for 11 years in the areas of semiconductor processing and micro-electro-mechanical systems (MEMS). From 1994 to 1999, he was heading the Application and Marketing Department for GaSonics International in Asia. He joined Institute of Microelectronics (IME) in Singapore in 1999 and has been the senior technical manager for MEMS and senior industry development manager for Bioelectronics and BioMEMS. Levent Yobas received his BSc degree in electrical engineering from Hacettepe University, Turkey, and both his MSc and PhD degrees in biomedical engineering from Case Western Reserve University, Cleveland, OH. Levent has been with the Bioelectronics and BioMEMS (BEBM) program at the Institute of Microelectronics (IME), Singapore. Prior to joining the IME in 2002, he worked in the US in the development of the micro/nanotechnology products for drug discovery and delivery. Victor D. Samper received his PhD degree from Heriot-Watt University (Scotland) in 1997, specializing in LIGA and microactuators for cardiac catheter system. Then he worked for the Institute of Microelectronics in Singapore for 6 years, leading several R&D projects in microrelay and DNA microextraction. Between 2003 and 2005 he worked as a principal research scientist at the Institute of Bioengineering and Nanotechnology (IBN) in Singapore. He is currently a lead scientist at the General Electric Global Research Center in Germany. Chew-Kiat Heng received his PhD in human genetics from National University of Singapore (NUS) in 1996. He is currently a senior research scientist at the Department of Pediatrics in NUS. He is the principal investigator of several projects related to lab-on-a chip and DNA and RNA extractions. His other research interests include genetic epidemiology of coronary artery disease. Saxon Liw received his BEng and MEng degrees in electrical engineering from the National University of Singapore, in 1999 and 2002, respectively. He was working in the Institute of Microelectronics from 2002 to 2005. He is currently with Perlos Asia in Singapore, engaging in antenna design for mobile communications. His research interests include active antenna design, rf circuit design, waveguides, optics, MEMS and sensors. Hongmiao Ji obtained her BEng in mechanical engineering from Xi’an JiaoTong University, PR China, in 1995 and MEng in mechanical engineering from National University of Singapore in 2000. She joined Institute of Microelectronics in 2001. Her research interests are simulation and design in microfluidic biochip. Yu Chen obtained her BEng and MEng in chemical engineering from Tsinghua University, PR China, in 1984 and 1989 and her PhD in chemical engineering from National University of Singapore in 1995. She was a lecturer in Tsinghua University from 1984 to 1991. She joined Institute of Microelectronics in 1997. Currently she is a member of technical staff in the Bioelectronics and BioMEMS program in IME. Her major research interests are electrochemical biochips and biosensors as well as microfluidic device and process integration. Lin Cong obtained his bachelor and master degrees from Huangzhong University of Science and Technology in China in 1996 and 1999, respectively, and PhD degree from Nanyang Technological University in Singapore in 2003, all in electrical engineering. His research interests include advanced control system design, instrumentation design, etc. Jing Li received her PhD degree from NanYang Technological University, Singapore, in 2006 and ME and BE from Xi’an Jiaotong University, China, in 1998 and 1995, respectively. Since 2003, she has been in Institute of Microelectronics, Singapore. Her research interest includes microfabrication, optical MEMS design and characterization. Lim obtained his PhD from the University of Cambridge, UK, in 1987. He is currently an associate professor at the Department of Biological Sciences, National University of Singapore. His research interest is in developmental biology.
Microfluidic systems for extracting nucleic acids for DNA and RNA analysis Wing C. Hui a,∗ , Levent Yobas a , Victor D. Samper a,1 , Chew-Kiat Heng b , Saxon Liw a , Hongmiao Ji a , Yu Chen a , Lin Cong b , Jing Li a , Tit Meng Lim b a
Institute of Microelectronics, 11 Science Park Road, Singapore Science Park II, Singapore b National University of Singapore, 10 Kent Ridge Crescent, Singapore Received 8 July 2005; received in revised form 14 April 2006; accepted 24 April 2006 Available online 9 August 2006
Abstract This paper is to review the differences in the developments of microfluidic chips for extracting genomic deoxyribonucleic acid (DNA) and viral ribonucleic acid (RNA) from blood by the Biosensor Focus Interest Group (BFIG) in Singapore. DNA was extracted in a multi-step process by isolating and lysing white blood cells (WBC), typically ∼10 m in diameter. Viral RNA was extracted directly from the submicron viruses in the blood. In terms of basic microfluidic components required, both DNA and RNA extractions used similar mixers for mixing reagents, filters for capturing or separating the blood cells, and a binder for capturing and purifying the DNA/RNA molecules. The designs of the filters were adapted to either capture WBC for DNA isolation or capture all virus particles for RNA isolation. The designs of these two kinds of filters had to be different. Besides the differences in the sizes of WBC and viruses, the concentration of the virus particles is usually much lower than WBC. Thus, a much higher volume of blood for filtering would be required for extracting viral RNA, especially for the intention to detect the viruses at early onset of infection. With proper modifications of the protocols, it has been demonstrated that both genomics DNA and viral RNA could be extracted successfully in these microfluidic chips. The quality of the extracted samples was verified by polymerase chain reaction (PCR) and gel-electrophoresis after the extractions. © 2006 Elsevier B.V. All rights reserved. Keywords: Nucleic acid; DNA; RNA; Extraction; Microfluidic; Virus
1. Introduction Numerous independent microfluidic components, such as microchannels, microfilters, cell sorters, mixers, valves, binders, etc., have been developed for various biomedical applications [1–4]. In this work microfluidic components have been integrated together to isolate: (1) deoxyribonucleic acid (DNA) from white blood cells and (2) ribonucleic acid (RNA) from viruses in the blood. In general, these two applications use different protocols and require different combinations of microfluidic components for efficient operation. For DNA extraction, the strategy is to capture the white blood cells (WBC) and deplete the concentration of the red blood cells (RBC). The hemoglobin from RBC is a contaminant that can ∗ 1
Corresponding author. Tel.: +65 6770 5900; fax: +65 6774 5747. E-mail address: [email protected] (W.C. Hui). Present address: GE Global Research-Europe, Munich, Germany.
0924-4247/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.sna.2006.06.031
inhibit the downstream process of DNA amplification by polymerase chain reaction (PCR). For viral RNA extraction, it is necessary to remove both WBC and RBC, with a minimal reduction in the concentration of the viruses in the plasma (less than 1 m in size). This demands a submicron filter. RNA is a more fragile molecule than DNA and thus poses additional challenges in the hardware and protocol development when compared to DNA extraction [5]. 2. Development of microfluidic chips 2.1. DNA extraction To extract DNA from WBC, the first step was to isolate the WBC using size- and compliance-based discrimination. The system then had to lyse the WBC using a high salt solution, to release the DNA. A binder was used to reversibly capture the DNA from the lysed cells. The DNA was bound to the surface
336
W.C. Hui et al. / Sensors and Actuators A 133 (2007) 335–339
Fig. 1. The DNA extraction chip.
during the clean-up process and subsequently eluted as purified DNA. A silicon/glass microfluidic chip was designed and fabricated for DNA extraction. It consisted of an integrated mixer [6], two paraffin valves, a filter with 3-m gap [7], and a binder. The DNA extraction chip is shown in Fig. 1. 2.2. Viral RNA extraction The basic steps for RNA isolation were similar to DNA extraction. However, some of the detailed requirements were different. In general, the viruses are smaller than WBC and RBC, and the concentration of virus in the blood is much lower than the WBC. Hence, a submicron filter had to be developed for handling the high volume and flow rate of blood necessary to obtain sufficient copies of the virus. The modified filter was able to separate the WBC and the RBC from the viruses in the plasma. A new process technology was developed for making deep submicron filters [8,9]. A filter with 3-m trenches was fabricated by deep reactive ion etching (DRIE). Subsequently, the trench gaps were narrowed down to 0.8 m or less by deposition
Fig. 3. The three submicron filters developed for separating viruses in serum from the WBC and RBC: (A) Submicron filter 1, (B) submicron filter 2, and (C) submicron filter 3.
of silicon dioxide. Fig. 2 shows the SEM pictures of the filter trench gaps before and after the oxide deposition. Based on this fabrication technology, three high flow-rate filters that could handle 40–400 l of blood were designed and fabricated. Fig. 3 shows optical microscope images of the filters. All of these three filters showed >90% efficiencies of filtration
Fig. 2. The SEM photos of the filter trench gaps before (A) and after (B) the deposition of silicon dioxide: (A) 3 m trench gaps fabricated by DRIE and (B) 0.8 m trench gaps after oxide deposition.
W.C. Hui et al. / Sensors and Actuators A 133 (2007) 335–339
337
Table 1 Testing results of the three submicron filters Blood:PBS = 50:50 RBC count in the blood 865 pumped (million) Volume of the diluted blood 400 pumped (l) Submicron filter 1 (5 mm × 10 mm) % EF 90.37 Plasma collected (l) 212
Blood:PBS = 25:75 433 400
99.56 181
Submicron filter 2 (5 mm × 10 mm) % EF 95.08 Plasma collected (l) 224
99.47 139
Submicron filter 3 (10 mm × 10 mm) % EF 91.30 Plasma collected (l) 198
99.81 61
Fig. 5. The basic bench top experimental setup for both the DNA and viral RNA extractions.
was used to drive each syringe to deliver the sample or reagent in the microliter range. The microlinear motion actuators were controlled by a calibrated computer system. A microscope with a CCD video camera was used to monitor the operation. Both experimental parameters and video images could be captured by the computer. Fig. 5 shows the basic experimental setup for DNA and viral RNA extractions. One of the main goals of our efforts was to leverage on the portability offered by microfluidics. Thus, two portable fluidic delivery systems were also developed for the DNA and the viral RNA extractions. 3.1. Submicron filters Fig. 4. The viral RNA extraction test block with the submicron filter and the micro-DNA extraction chip.
(EF) and fulfilled the requirement for the viral RNA extraction process. A summary of their EF can be found in Table 1 [8]. Filter 1 was chosen for the task of viral RNA extraction because experiments showed it to be less prone to unwanted rupture of RBC. The filter was combined with the micro-DNA extraction chip for the viral RNA extraction task. After separation of the viruses by the submicron filter, the micro-DNA extraction chip was used for the lysing of virus, binding and cleaning of the viral RNA, and finally, elution of the RNA. Fig. 4 shows the test kit with both the submicron filter 1 and the original DNA extraction chip. 3. Experimental results and discussions The experimental setups were the same for the initial testing of both the DNA extraction and the viral RNA extraction. They included a test block to connect the microfluidic chips and the external microfluidic delivery system. The microfluidic delivery systems consisted of several syringes containing the sample and the various reagents required. A microlinear motion actuator
Table 1 shows a summary of the filter efficiency of filtration based on the mixture of the phosphate buffered saline (PBS) solutions. The three filters showed >90% efficiencies of filtration and fulfilled the requirement for the viral RNA extraction process [8]. 3.2. DNA extraction On average, approximately 1 ng DNA was obtained per microliter of blood. The results depend strongly on the protocols. PCR results were used to demonstrate the purity of the DNA extracted. Amplified products were put through gelelectrophoresis and visualized by staining the nucleic acids with ethidium bromide, which emitted an orange fluorescence under UV light. If the starting sample was not clean enough, it would inhibit the PCR. Fig. 6 shows the positive PCR result for an extracted DNA sample. In order to make the DNA extraction operation portable, an automatically controlled system was built within a briefcase. It took some of the basic elements from the initial experimental setup, including the microfluidic delivery system with the microlinear motion actuators and the syringes. The system was controlled by a laptop computer. Fig. 7 shows the portable system for the DNA extraction system.
338
W.C. Hui et al. / Sensors and Actuators A 133 (2007) 335–339
Fig. 8. Result of the gel-electrophoresis after reverse transcriptase PCR (RTPCR) of viral RNA extracted by the viral RNA extraction chip.
3.3. Viral RNA extraction
Fig. 6. Result of gel-electrophoresis after PCR on DNA sample extracted by the DNA extraction chip.
Fig. 7. The portable system for DNA extraction.
In terms of handling the nucleic acids, RNA is more unstable and poses greater challenges in preserving their integrity during the extraction process in comparing to DNA. New techniques were developed for capturing and eluting RNA without compromising its integrity. The protocol was first developed for total RNA from WBC and then with viral RNA from a harmless plant virus, such as the Orchid Virus. Fig. 8 shows the results from reverse transcriptase polymerase chain reaction (RT-PCR) that confirm that good quality viral RNA was actually extracted by the viral RNA extraction chip set. The results also depended strongly on the protocols. For the viral RNA extraction, a further minimized version of the automatic microfluidic delivery system was developed (Fig. 9). The size was 31 cm × 23 cm × 11 cm. It contained mechanical micropumps and microvalves to control the fluidics
Fig. 9. The portable system for viral RNA extraction.
W.C. Hui et al. / Sensors and Actuators A 133 (2007) 335–339
down to the microliter level. It could be controlled either: (1) by a PC or laptop computer for flexibility in R&D or (2) by sequential programming with a fixed protocol for routine testing. 4. Conclusions We have successfully demonstrated that both the DNA and viral RNA extraction chips were capable of yielding pure DNA and viral RNA accordingly. The results were confirmed by PCR and RT-PCR, respectively. The performance could vary according to the protocols and run parameters such as flow rates and the duration of incubation times. In addition to the microfluidic extraction chips, portable automatic systems were also developed so that the process of nucleic acid extraction was no longer confined to a laboratory setting. Acknowledgement The authors would like to thank the Agency for Science, Technology and Research (A*STAR) in Singapore for funding the projects under the Biosensor Focus Interest Group (BFIG). References [1] D.R. Meldrum, M.R. Holl, Microscale bioanalytical systems, Science 297 (2002) 1197–1198. [2] J.H. Kim, B.G. Kim, H. Nam, D.E. Park, K.S. Yun, J.B. Yoon, J. You, E. Yoon, A disposable DNA sample preparation microfluidic chip for nucleic acid probe assay, in: Proceedings of the IEEE Micro Electro Mechanical Systems (MEMS), 2002, pp. 133–136. [3] K.A. Melzak, C.S. Shervood, R.F.B. Turner, C.A. Haynes, Driving forces for DNA adsorption to silica in perchlorate solutions, J. Colloid Interface Sci. 181 (1996) 635–644. [4] R. Pal, M. Yang, B.N. Johnson, D.T. Burke, M.A. Burns, Phase change microvalve for integrated devices, Anal. Chem. 76 (2004) 3740–3748. [5] T.W. Smalling, S.E. Sefers, H.J. Li, Y.W. Tang, Molecular approaches to detecting herpes simplex virus and enteroviruses in the central nervous system, J. Clin. Microbiol. 40 (7) (2002) 2317–2322. [6] W.L. Goh, W.H. Ma, W.C. Hui, H.M. Ji, Evaluation of microfluidic mixer designs for RNA extraction, in: International Conference on Materials for Advanced Technologies (ICMAT 2005), Singapore, July 3–8, 2005. [7] Y. Chen, Y. Miao, V. Samper, F. Bte Mustafa, Q.X. Zhang, C.K. Heng, H. Lye, T.M. Lim, Microfabrication of a Si mesh structure depth filter, in: Proceedings of TAS 2002, 6th International Conference on Miniaturized Systems for Chemistry and Life Sciences, vol. 2, 2002, pp. 739–741. [8] L. Yobas, E.L. Gui, H.M. Ji, J. Li, Y. Chen, W.C. Hui, S. Rafeah, S. Swarup, S.M. Wong, T.M. Lim, C.K. Heng, A flow-through shear-type microfilter chip for separating plasma and virus particles from whole blood, in: Proceedings of TAS 2004, 8th International Conference on Miniaturized Systems in Chemistry and Life Sciences, vol. 2, Malmo, Sweden, September 26–30, 2004, pp. 7–9. [9] J. Li, Y. Chen, L. Yobas, H.M. Ji, W.C. Hui, et al., Fabricating submicron microfluidic filter without X-ray lithography, in: Proceedings of International Conference on Materials for Advanced Technologies 2004 (ICMAT 2004), July 4–8, 2004, pp. 409–412.
Biographies Wing C. Hui obtained both his BS and MS degrees in chemical engineering from University of California at Berkeley, in 1978 and 1980, respectively. Wing had
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worked at Dow Chemicals for 4 years and then Lawrence Livermore National Laboratory in US for 11 years in the areas of semiconductor processing and micro-electro-mechanical systems (MEMS). From 1994 to 1999, he was heading the Application and Marketing Department for GaSonics International in Asia. He joined Institute of Microelectronics (IME) in Singapore in 1999 and has been the senior technical manager for MEMS and senior industry development manager for Bioelectronics and BioMEMS. Levent Yobas received his BSc degree in electrical engineering from Hacettepe University, Turkey, and both his MSc and PhD degrees in biomedical engineering from Case Western Reserve University, Cleveland, OH. Levent has been with the Bioelectronics and BioMEMS (BEBM) program at the Institute of Microelectronics (IME), Singapore. Prior to joining the IME in 2002, he worked in the US in the development of the micro/nanotechnology products for drug discovery and delivery. Victor D. Samper received his PhD degree from Heriot-Watt University (Scotland) in 1997, specializing in LIGA and microactuators for cardiac catheter system. Then he worked for the Institute of Microelectronics in Singapore for 6 years, leading several R&D projects in microrelay and DNA microextraction. Between 2003 and 2005 he worked as a principal research scientist at the Institute of Bioengineering and Nanotechnology (IBN) in Singapore. He is currently a lead scientist at the General Electric Global Research Center in Germany. Chew-Kiat Heng received his PhD in human genetics from National University of Singapore (NUS) in 1996. He is currently a senior research scientist at the Department of Pediatrics in NUS. He is the principal investigator of several projects related to lab-on-a chip and DNA and RNA extractions. His other research interests include genetic epidemiology of coronary artery disease. Saxon Liw received his BEng and MEng degrees in electrical engineering from the National University of Singapore, in 1999 and 2002, respectively. He was working in the Institute of Microelectronics from 2002 to 2005. He is currently with Perlos Asia in Singapore, engaging in antenna design for mobile communications. His research interests include active antenna design, rf circuit design, waveguides, optics, MEMS and sensors. Hongmiao Ji obtained her BEng in mechanical engineering from Xi’an JiaoTong University, PR China, in 1995 and MEng in mechanical engineering from National University of Singapore in 2000. She joined Institute of Microelectronics in 2001. Her research interests are simulation and design in microfluidic biochip. Yu Chen obtained her BEng and MEng in chemical engineering from Tsinghua University, PR China, in 1984 and 1989 and her PhD in chemical engineering from National University of Singapore in 1995. She was a lecturer in Tsinghua University from 1984 to 1991. She joined Institute of Microelectronics in 1997. Currently she is a member of technical staff in the Bioelectronics and BioMEMS program in IME. Her major research interests are electrochemical biochips and biosensors as well as microfluidic device and process integration. Lin Cong obtained his bachelor and master degrees from Huangzhong University of Science and Technology in China in 1996 and 1999, respectively, and PhD degree from Nanyang Technological University in Singapore in 2003, all in electrical engineering. His research interests include advanced control system design, instrumentation design, etc. Jing Li received her PhD degree from NanYang Technological University, Singapore, in 2006 and ME and BE from Xi’an Jiaotong University, China, in 1998 and 1995, respectively. Since 2003, she has been in Institute of Microelectronics, Singapore. Her research interest includes microfabrication, optical MEMS design and characterization. Lim obtained his PhD from the University of Cambridge, UK, in 1987. He is currently an associate professor at the Department of Biological Sciences, National University of Singapore. His research interest is in developmental biology.