An Integrated Microfluidic Chip For Dna-rna

3297

Electrophoresis 2006, 27, 3297–3305

Fu-Chun Huang1 Chia-Sheng Liao2 Gwo-Bin Lee1, 2 1

Department of Engineering Science, National Cheng Kung University, Tainan, Taiwan 2 Institute of Micro-electromechanical-system Engineering, National Cheng Kung University, Tainan, Taiwan

Received January 23, 2006 Revised March 2, 2006 Accepted March 2, 2006

Research Article

An integrated microfluidic chip for DNA/RNA amplification, electrophoresis separation and on-line optical detection This study presents an integrated microfluidic chip capable of performing DNA/RNA (deoxyribonucleic acid/ribonucleic acid) amplification, electrokinetic sample injection and separation, and on-line optical detection of nucleic acid products in an automatic mode. In the proposed device, DNA/RNA samples are first replicated using a micromachine-based PCR module or reverse transcription PCR (RT-PCR) module and then transported by a pneumatic micropump to a sample reservoir. The samples are subsequently driven electrokinetically into a microchannel, where they are separated electrophoretically and then detected optically by a buried optical fiber. The various modules of the integrated microfluidic chip are fabricated from cheap bio-compatible materials, such as PDMS, polymethylmethacrylate, and soda-lime glass. The functionality of the proposed device is demonstrated through its successful application to the DNA-based bacterial detection of Streptococcus pneumoniae and the RNA-based detection of Dengue-2 virus. It is shown that the low thermal inertia of the PCR/RT-PCR modules reduces the sample and reagent consumption and shortens the reaction time. With less human intervention, the subsequent DNA separation and detection could be performed in an automatic mode. The integrated microfluidic device proposed in this study represents a crucial contribution to the fields of molecular biology, genetic analysis, infectious disease detection, and other biomedical applications. Keywords: Capillary electrophoresis / Laser induced fluorescence / Microfluidics / PCR / RT-PCR DOI 10.1002/elps.200600458

1 Introduction The microfabrication of miniature fluidic devices has attracted considerable interest over the past decade. MEMS (microelectromechanical systems) has proven to be a fundamental enabling technology and has revolutionized much of the existing analytical instrumentation used in the fields of molecular biology, genetic analysis, disease diagnosis, and biomedicine. Due to their ability to conduct the parallel processing of minute amounts of biosamples, the development of microchip

Correspondence: Professor Gwo-Bin Lee, Engineering Science, National Cheng Kung University, 1, University Road, Tainan, 701, Taiwan, China E-mail: [email protected] Fax: 1886-6-2761687 Abbreviations: cDNA, complementary DNA; DNA, deoxyribonucleic acid; MEMS, microelectromechanical system; PMMA, polymethylmethacrylate; RT-PCR, reverse transcription PCR; SEM, scanning electron microscope

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

devices has facilitated the evolution of a wide range of rapid and efficient analytical techniques. Furthermore, the concept of micro total analysis systems (m-TASs), in which sample pretreatment, transportation, mixing, reaction, separation, and detection functions are integrated on a single miniature chip, can now be realized by combining functional microfluidic components manufactured using appropriate micromachining technologies [1]. Compared to the use of conventional analytical instrumentation, amplifying and analyzing deoxyribonucleic acid (DNA) samples in a microchip format has significant advantages. PCR is a well-developed nucleotide amplification method for genetic identification and diagnosis, and is recognized as one of the most essential procedures currently performed in life-science laboratories. Briefly, the PCR process involves amplifying the concentration of a certain segment of dsDNA by thermal cycling. Recent advances in MEMS techniques have enabled the fabrication of micro PCR chips. These chips, which are generally fabricated on either silicon [2–5] or www.electrophoresis-journal.com

3298

F.-C. Huang et al.

glass substrates [6, 7], have shown considerable potential for rapid DNA amplification [2–8]. For example, Northrup et al. [2, 3] presented a microfabricated, silicon-based PCR chamber heated by boron-doped polysilicon resistors positioned outside the PCR chamber. The low thermal inertia of the developed microfluidic device enabled successful amplification of DNA samples with reduced sample and reagent consumption and a shorter reaction time. The use of MEMS-based micro CE chips for DNA sample analysis has been extensively investigated [9]. Compared to their conventional large-scale counterparts, micro CE chips have the fundamental advantages of compactness, low sample/reagent consumption, low cost, and high detection limits. As a consequence, microchip-based electrophoresis is currently employed in an increasing number of DNA analysis procedures. Micro-scale electrophoretic devices have typically been fabricated using glass [10–12], fused-silica substrates [13], and plastics [14–16]. Integrating PCR and electrophoresis devices provides the potential for rapid amplification, separation, and identification of DNA samples [5, 6, 17, 18]. For example, Woolley et al. [5] presented a silicon-based PCR reactor integrated with a glass-based CE chip and showed that the PCR-CE analysis of a b-globin target could be successfully completed within 20 min. Waters et al. [19] demonstrated a microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing analysis. Lagally et al. [6, 7] developed an integrated PCR-CE system with platinum temperature sensors located inside the reaction chamber and heaters arranged outside the chamber. Various researchers have reported success in developing a two-stage DNA amplification/electrophoresis analysis process. For example, Koh et al. [20] presented an integrated plastic microfluidic device capable of PCR, valving, and electrophoretic separation to perform bacterial detection and identification procedures. In their design, screen-printed graphite ink resistors were used to conduct the thermal cycling required in the PCR. Following the PCR operation, the PCR products were injected electrokinetically through a gel valve and then separated electrophoretically. The detection of Escherichia coli O157 and Salmonella typhimurium was successfully demonstrated using the proposed design. Similarly, Rodriguez et al. [21] developed an integrated PCR-CE system for genetic analysis. In their study, the PCR chamber was fabricated using silicon and it incorporated aluminum heaters and temperature sensors. Following PCR, a large-scale external pumping device was used to transport the amplified samples to a glass-based CE chip, where DNA fragments differing in size by 18 bps © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2006, 27, 3297–3305

were successfully resolved. Although the pioneering devices presented above have a proven ability to amplify and analyze DNA samples, their application is limited in practice since they generally involve some form of largescale optical detection system. Moreover, these devices are unable to perform a fully automatic PCR/CE/detection procedure, which is a pressing requirement in many biomedical and chemical analysis applications. A technique commonly associated with the use of CE chips is that of LIF detection. Briefly, in this technique, the samples are labeled with a particular fluorescein, and the fluorescence signals induced by a laser source are then detected as the samples flow through the downstream region of the separation channel. However, the conventional LIF technique utilizes a bulky optical detection apparatus comprising a microscope and various delicate light coupling components. Therefore, the advantages afforded by the miniaturization of the CE device are somewhat lessened. Hence, the integration of miniature optical detection systems with micro CE chips has enormous appeal and provides the means of realizing the online detection of bio-samples. Several approaches toward integrating micro CE chips with optical detection devices have been reported, including liquid core waveguides [22, 23], optic fibers [24], leaky waveguides [25, 26], and optical waveguides [27]. Recently, we developed a novel design in which CE microfluidic devices were fabricated with integrated buried optical waveguides for DNA/protein analysis applications [28]. In the developed chip, optical detection was achieved by means of buried solidcore optical waveguides formed by filling SU-8/SOG (spin-on-glass) double layers within pre-etched waveguide channels. Alternatively, it has been reported that CE detection can be achieved through the use of etched optical fibers inserted directly into embedded channels, orientated perpendicularly to the separation channel [29]. The diameter of etched optical fibers is 100 mm. This arrangement was applied successfully to the separation and detection of DNA samples. The chip fabrication process was both cheap and effective. Hence, the proposed design was a suitable candidate for the mass production of disposable micro-CE chips with integrated on-line optical detection mechanisms. Ro et al. [30] reported integrated optical fibers for CE chip detection. In their study, an additional slit channel between an optical fiber and a fluid channel is used for highly efficient and sensitive optical detection. Experimental data show that the sensitivity greatly improved. The present study develops an integrated microfluidic chip capable of performing DNA/RNA (ribonucleic acid) amplification, electrophoretic separation, and on-line optical detection of DNA samples. In the proposed dewww.electrophoresis-journal.com

Electrophoresis 2006, 27, 3297–3305

Miniaturization

3299

vice, the DNA/RNA samples are replicated using a micro PCR (or reverse transcription PCR (RT-PCR)) module. It is notable that the integrated microfluidic chip is capable of RT-PCR for detection of RNA-based virus. The resulting products are transported by a pneumatic micropump to the sample buffer channel of a CE chip. Using an electrokinetic driving scheme, the samples are injected into the CE chip and separated electrophoretically in the separation channel. Finally, the samples are detected optically by an embedded optical fiber located in the downstream region of the separation channel. The novel combination of pneumatically driven and electrokinetically driven transportation mechanisms ensures the injection of precise amounts of DNA/RNA samples into the CE chip and the segregation of PCR reagents and CE buffer solutions. To the best of our knowledge, this study represents the first reported attempt to utilize such a hybrid driving scheme in integrated microfluidic systems designed for DNA- and RNA-based applications. With less human intervention, the DNA/RNA amplification, separation, and detection could be performed in an automatic mode.

2 Materials and methods 2.1 Design Miniature devices for the rapid PCR-based analysis of DNA samples are crucial for genetic applications. Integrated PCR and electrophoresis devices are ideal candidates for rapid DNA analysis. These devices are not only capable of rapid amplification of DNA samples, but can also conduct the subsequent DNA separation and sizing analysis tasks. This study develops a novel integrated microfluidic device capable of performing DNA/RNA amplification, separation, and on-line detection in an automatic mode. Figure 1a shows a conventional PCR/ CE integrated chip [31]. One of the CE reservoirs was used as a PCR chamber. Thus PCR reagents may be mixed with CE buffers. EDTA in CE buffer solutions is known to be one of the PCR inhibitors [32]. As a result, PCR efficiency could be decreased. Besides, high temperature near the PCR chamber could cause the drying of the CE buffers, thus affecting the CE performance. Figure 1b presents a schematic illustration of the proposed microfluidic chip. The device comprises three major modules, namely a micro PCR (or RT-PCR) chip with three chambers and micropumps, a cross-shaped CE channel for the electrokinetic injection and separation of the amplified DNA samples, and a buried optical fiber to carry out on-line DNA detection. As shown in Fig. 1c, the integrated microfluidic chip is fabricated by bonding together the three separate glass-, polymethylmethacrylate (PMMA)-, and PDMS-based chips. In most of the report© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Schematic illustration of (a) a conventional PCR/CE integrated chip, (b) a proposed microfluidic chip capable of DNA/RNA amplification, electrophoretic injection, separation, and on-line detection of DNA samples, and (c) cross-sectional view of the proposed microfluidic chip comprising glass-, PMMA-, and PDMS-based modules.

ed micro PCR devices, the sensors and heaters required for the PCR process are located outside the PCR chamber. This leads to an inaccurate temperature measurement of the DNA sample and a low heating/cooling rate during thermal cycling. Accordingly, in the proposed design, two microheaters and one temperature sensor are deposited on a glass substrate and are located within the PCR chamber. It has been shown previously that this arrangement improves the precision of the temperature measurement and delivers a more efficient heating/cooling performance [33]. The developed microchip employs electrokinetic forces to carry out sample injection and separation in a crossshaped CE channel. In fabricating this channel, a simple and reliable method involving phowww.electrophoresis-journal.com

3300

F.-C. Huang et al.

toresist as an etch mask is used to fabricate a glass template of the various microchannels [12]. A hot-embossing method is then used to transfer the inverse microstructures onto a PMMA substrate. A second PMMA cover plate with predrilled via holes is thermally bonded to this substrate to seal the microfluidic channels [16]. An etched optical fiber is inserted into an embedded channel perpendicular to the separation channel to detect the LIF signals [29]. Finally, the pneumatic micropumps used to control the flow of the reagents and samples in the PCR process, and to transport the amplified products to the CE sample buffer, each comprise three individual PDMS membranes, which are driven sequentially by external compressed air in order to generate a peristaltic pumping action [34]. The PDMS membranes can be deflected by compressed air to such an extent that they block the microchannels completely. Furthermore, EDTA in CE buffer solutions is known to be a PCR inhibitor. In Fig. 1b, three micropumps consisting of three PDMS membranes were designed. When one pump is activated, the other two serve as valves simultaneously. Therefore, the PDMS membranes can provide a valving function to ensure a proper isolation of the PCR reagents and the CE buffers. Besides, the distance between the PCR chambers and CE channels provides a good thermal isolation, such that drying of the CE buffers could be alleviated. The proposed microfluidic chip design has a number of key advantages. First, the RT-PCR chamber allows the reverse transcription reaction of messenger RNA (mRNA) to be performed if required. In the reverse transcription process, precise amounts of RNA reagents are transported by the pneumatic micropumps to the PCR chamber from the RT-PCR reagent chamber. Synthesized complementary DNA (cDNA) samples are then further amplified after pumping PCR reagents from the PCR reagent chamber. This two-step RT-PCR amplification process provides a reliable operation for RNA samples. If it is only necessary to amplify DNA samples, the reverse transcription process is simply omitted. Second, the pneumatic micropumps also provide a micro-valving function such that the PCR chambers and the CE buffer reservoir can be effectively isolated. It has been shown experimentally that mixing of the reagents and buffer has a detrimental effect on the DNA amplification process [32]. Additionally, the high-temperature field (.957C) in the PCR chamber can cause a drying of the CE buffer, and hence can affect the injection of the amplified DNA samples into the CE channel. Third, the novel combination of pneumatic and electrokinetic pumping mechanisms proposed in the current design represents a promising approach for PCR/CE applications. Micro-CE chips are typically driven by electrokinetic forces. However, when amplified DNA samples and reagents are transported to © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2006, 27, 3297–3305

CE reservoirs by using electrokinetic forces, they will move at different speeds due to different charge-to-mass ratios. It may affect the following injection and separation process. The proposed design overcomes this problem by using a series of pneumatic micropumps to manipulate the DNA/RNA samples prior to the electrophoretic separation process, and then using conventional electrokinetic forces to conduct sample injection and separation once the DNA/RNA samples have been amplified. Finally, the proposed design incorporates an optical fiber buried at a downstream location in the CE separation channel to detect the induced fluorescence signals. The intensity of the optical signals is enhanced in the current design by means of a side channel filled with index-matching oil [35].

2.2 Fabrication Micro PCR chips are fabricated on glass substrate. Reaction chambers and micropumps are fabricated by using PDMS. Micro CE chips are made up of PMMA. The reasons for using these materials are low cost, bio-compatible and mature manufacturing methods. Figure 2 presents an overview of the fabrication process employed for the integrated microfluidic chip. Briefly, the micro CE channels and optical fiber channels were replicated on a PMMA substrate from a glass template using hotembossing methods (Fig. 2a). A cover PMMA plate with predrilled via holes was then thermally bonded to the substrate to form the sealed micro-CE chip (Fig. 2b). The microheaters and temperature sensor were fabricated on a soda-lime glass substrate (Central Glass Taiwan Trading, Hsin Chu, Taiwan). Initially, a thin layer of titanium (0.02 mm) was deposited on the glass substrate to form an adhesion layer for the subsequent deposition of a 0.1 mm platinum layer for the electron-beam evaporator. The platinum layer was then patterned as a resistor using standard lift-off processes (Fig. 2c). Note that platinum resistors were used for both the temperature sensor and the two heating elements in order to simplify the fabrication process. The resistances of the sensor and heaters were measured to be 400 and 30 O, respectively. A conventional gold metallization (0.4 mm) process was then employed to form electrical leads. A cover glass slide (100 mm thick) was attached to the glass substrate to form an electrical isolation layer (Fig. 2d). Finally, via-holes were drilled (Fig. 2e). The pneumatic micropumps in the integrated microfluidic chip comprise two PDMS structures replicated on silicon substrates from SU-8 templates (Figs. 2f, 3g). The upper PDMS structure incorporates air chambers for the delivery of compressed air to deform the PDMS membranes, www.electrophoresis-journal.com

Electrophoresis 2006, 27, 3297–3305

Miniaturization

3301

Figure 2. Simplified fabrication process of the integrated microfluidic chip. (a) Hot embossing of PMMA plates using glass template with inverse images of CE channels, (b) drilling of via-holes in the second PMMA plate and bonding, (c) deposition/patterning of platinum temperature sensor and heaters on glass substrate for micro PCR device, followed by deposition/patterning of gold as electrical leads, (d) bonding of cover slide for electric insulation, (e) drilling of via-holes, (f) replication of PDMS membranes using SU-8 template on silicon substrate, (g) replication of PDMS fluid channels using SU-8 template on silicon substrate, and (h) peeling of PDMS structures and bonding of two PDMS layers to form micropumps.

while the lower PDMS structure contains the fluidic channels. In fabricating the micropumps, PDMS elastomers and curing agents (Sylgard 184 Silicone Elastomer Kit, Dow Corning, USA) were mixed in the ratio of 10:1 and then cured at 757C for 180 min. The two PDMS structures were then peeled off mechanically and bonded in an oxygen plasma treatment (Fig. 2h). The final integrated microfluidic chip was formed by bonding the glass PCR chip to the PMMA CE chip using UV-sensitive glue. The PDMS micropumps chip was then bonded on top of the glass PCR chip using an oxygen plasma treatment. The assembly process was completed by inserting an etched optical fiber into the buried channel through a coupling device under a microscope [35]. Then, the optical fiber was fixed in position by UV glue.

2.3 Experimental The process of DNA/RNA amplification, electrophoresis separation, and on-line optical detection conducted in the present study can be briefly described as follows (Fig. 1a). Initially, RNA or DNA templates were placed in reservoir 2 (PCR chamber) and the crossshaped CE channel was filled with CE buffer (a mixture of 1.5% HPMC (hydroxypropyl methyl cellulose) in TBE (trisborate-EDTA) and 1% YO-PRO®-1 fluorescence dyes (Molecular Probes, USA)) [36]. RT-PCR and PCR reagents were then loaded in reservoirs 1 and 3, respectively. Note that in this study, the 10 mL RT-PCR reagents contained 1 mg of RNA, 0.5 mL of 10 mM dNTP (deoxynucleoside triphosphates (Yeastern Biotech, Taiwan)), 2 mL of 56 reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, and 15 mM MgCl2), 0.5 mL of 10 mM primer © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

(Merck Taiwan, Taiwan), 1 mL of 0.1 M DTT (Promega, USA), and 0.5 mL of moloney murine leukemia virus RT (200 U/mL, Gibco BRL, MD, USA). Similarly, the 10 mL PCR reagents contained 0.2 mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP) (Yeastern Biotech), 1 mL of 106PCR buffer (15 mM MgCl2, 500 nM KCl, 1.5 M Tris-HCl, pH 8.7), 200 nM of the appropriate paired primers, and 1 U Taq DNA polymerase (Amersham, UK) [37]. In the RT-PCR process, the RT-PCR reagents were pumped from reservoir 1 to reservoir 2 by the pneumatic micropumps. The RNA template was then synthesized to cDNA at a temperature of 437C and maintained for 30 min. Process conditions of 657C for 10 min were then implemented to prevent nonspecific binding prior to the Taq DNA polymerase addition. Following the synthesis of cDNA, 2 mL of cDNA was left in reservoir 2 for the subsequent PCR process. PCR reagents were then transported from reservoir 3 to reservoir 2 to conduct the amplification of cDNA over 20 thermal cycles of 947C for 10 s, 527C for 20 s, and 727C for 20 s. Note that the final cycle included an additional thermal stage of 727C for 1 min. Following cDNA amplification, the DNA samples were transported to reservoir 4, i.e., the CE sample reservoir. The amplified DNA samples were then pumped electrokinetically into the separation channel, where they were separated electrophoretically and then detected by the buried optical fiber. Details about the experimental setup employed for the electrokinetic injection/separation and LIF detection could be found in our previous work [38]. Briefly, a conventional single crossedchannel injection method was utilized to perform sample injection via the appropriate www.electrophoresis-journal.com

3302

F.-C. Huang et al.

switching of a high-voltage power supply. Prior to injection of the amplified DNA samples, the CE channel was filled with CE buffer. When the DNA samples were driven into the CE channel, they were dyed simultaneously. In addition to the integrated microfluidic chip, the whole system needs an additional temperature controller, an air compressor and micropump controller. These peripheral components were installed inside a box with a dimension of 2161268.5 cm3.

3 Results and discussion Figure 3a presents a photograph of the completed microfluidic chip. The chip measures 65645 mm2, and the width and depth of the micro CE channels are 100 and 30 mm, respectively. The separation channel has a total length (as measured from the intersection) of 50 mm and the buried optical fiber is located at a distance of 5 mm

Electrophoresis 2006, 27, 3297–3305

from the downstream end of the channel. The volumes of PCR and reagent chambers are 11.25 mL. Figure 3b provides a close-up view of the micro CE channel and the optical fiber channel. In practical applications, air exists between optical fibers and CE channels, which could affect the optical coupling of the excitation and detection of light. Thus index-matching oil was used to fill a side channel, thus enhancing the optical detection [35]. Figure 4 shows a scanning electron microscope (SEM) image of the temperature sensor and the two heaters. As discussed previously, these resistors are located within the PCR chamber in order to improve the precision of the temperature measurement and to provide a more efficient heating/cooling performance. In this study, a PWM (pulse width modulation) controller and an ASIC (application specific integrated circuit) were used to control the temperature field inside the PCR chamber [8]. During operation, the micro temperature sensor provided real-time measurements of the temperature field, while the micro heaters regulated the temperature of the sample inside the PCR chamber during thermal cycling. The capability of the micro PCR module was demonstrated by conducting a typical PCR cycle. The PCR module is capable of performing typical temperature cycles. The heating and cooling rates of the system were measured to be approximately 207C/s and 107C/s, respectively, with a variation of 0.27C. Such accuracy is important for most experimental protocols and is critical to the scientific communication of reproducible results. Prior to conducting DNA amplification, the performance of the pneumatic micropumps was characterized. Fluid pumping was achieved via the sequential activation of the

Figure 3. (a) Photograph of the integrated microfluidic chip. The three layers of the integrated chip are composed of a PMMA CE chip, a glass micro PCR chip and PDMS micropumps (bottom-up). The size of chip is measured to be 6.563.960.8 cm3. The channel is of 100 mm (width)630 mm (depth). The volumes of the PCR and reagent chambers are 11.25 mL. (b) Close-up SEM image of the buried optic fiber channel. Note the side channel filled with index-matching oil. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 4. SEM image of the temperature sensor and heaters. www.electrophoresis-journal.com

Electrophoresis 2006, 27, 3297–3305

Miniaturization

3303

three PDMS membranes in the micropump. A peristaltic pumping action was induced by changing the phase and frequency of the driving pressure and applying a sequential control to the individual membranes. The experimental data confirmed that the pump was capable of successful sample transportation. Moreover, these PDMS membranes could be used as microvalves while they are deflected completely. Therefore, they can provide a valving function to ensure a proper isolation of the PCR reagents and the CE buffers. Experimental data showed that PCR efficiency could be greatly affected if the PCR reagents were mixed with the CE buffers. In practical applications, DNA/RNA could not be successfully amplified without proper isolation of the PCR reagents and the CE buffers. As shown in Fig. 5, the pumping rate can be controlled simply by changing the frequency of the applied driving signal. It is noted that compressed air is supplied at a constant pressure of 20 psi in this figure. It could be clearly seen that an increase in activation frequency significantly increases the pumping rate of the peristaltic micro-pumps. The pumping rate will eventually reach a saturation value limited by the maximum driving frequency of the electromagnetic valves (about 15 Hz). Although not shown here, the experimental data revealed that the pumping rate can be increased by increasing the driving air pressure [34].

Aftercharacterizing the PCR module and the pneumatic pumps, the developed microfluidic chip was used to perform the DNA-based bacterial detection of Streptococcus pneumoniae (S. pneumoniae) and the RNAbased viral detection of Dengue fever (Type 2). In the bacterial detection trial, a gene of length 240 bps is amplified for detection of the resistance of S. pneumoniae to the penicillin antibiotic in 20 thermal cycles. The concentration of the PCR products increases with thermal cycles [8]. Figure 6 presents a slab-gel electrophoregram of the amplified PCR products. The micro PCR operation was completed within 15 min and consumed a total sample volume of just 10 mL. To prevent evaporation of PCR reagents, mineral oil was used to cover the PCR reagents. For comparison purposes, the PCR process was also performed using a conventional large-scale PCR machine. This traditional technique required 2 h to complete and consume 25 mL of sample. In Figure 6, the fluorescence signals in the first lane correspond to the 100 bp DNA ladders. Lane (C) shows the fluorescence signal obtained from the micro PCR chip, and Lane (M) shows the PCR product obtained using the bench-top PCR machine (PCR Sprint HBSP02, Thermo Electron, USA). The results confirm the ability of the developed PCR microchip to perform rapid and accurate PCR operations.

Figure 5. Relationship between the pumping rate of the pneumatic micropump and driving frequency. Note compressed air pressure is 20 psi. An increase in activation frequency significantly increases the pumping rate. The pumping rate will eventually reach a saturation value limited by the maximum driving frequency of the electromagnetic valves (about 15 Hz).

Figure 6. Slab-gel electropherogram showing successful amplification of DNA samples using developed micro PCR module. (Lane L: 100 bp DNA ladders; Lane C: PCR products using micro PCR chip; Lane M: PCR products using conventional PCR machine.)

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.electrophoresis-journal.com

3304

F.-C. Huang et al.

Electrophoresis 2006, 27, 3297–3305

After amplifying the DNA, we pipette DNA markers in reservoir 4. Then, amplified DNA samples were pumped to reservoir 4 and mixed with DNA markers. After the mixing process, the mixture of DNA markers and amplified DNA samples was injected and separated in the CE channels and then detected by the integrated optical fiber. This trial was conducted using a CE buffer of 1.5% HPMC in TBE with 1% YO-PRO-1 fluorescence dye. Sample injection was performed by applying a voltage of 200 kV for 0.5 min, while separation was conducted under a voltage of 1.3 kV applied for 3.5 min. For comparison purposes, Hae III digested fx-174 DNA markers with a concentration of 10 ng/mL were mixed with the PCR products and separated at the same time. DNA fluorescence signals (509 nm wavelength) were induced using a Hg excitation light with a 491 nm wavelength. Fluorescence signals were collected by the buried optical fiber downstream of the separation channel and amplified via a PMT (photomultiplier tube) module (C3830, R928, Hamamatsu, Japan). The amplified analog signals were converted to digital signals by 24-bit ADC (Model 0224-2, SISC, Taipei, Taiwan), and recorded. Figure 7a presents an electrophoregram of the mixture of DNA markers and PCR products obtained during the detection of S. pneumoniae. It can be seen that all the 11 peaks of the DNA markers and the single peak of the PCR product (240 bps) from the S. pneumoniae bacteria were separated successfully within 1.4 min. In addition to fast analysis and efficient separation, chip-base electrophoresis has been known to have a higher detection limit while compared to conventional slab-gel electrophoresis (about one order of magnitude improvement). Figure 7b shows the electrophoregram corresponding to the PCR product (419 bps) obtained from the Dengue-2 RNA virus. Again, all the 11 peaks of the DNA markers and the single peak of the RT-PCR product (419 bps) were separated successfully within 1.4 min. These two tests confirm the capability of the proposed device for DNA/RNA amplification and detection. Typically, LIF detection requires complicated optics for focusing excitation light on the microchannel and collecting induced fluorescence signals back to a sensitive photo-detector. In this study, optical fiber can guide fluorescence light from the chip to a PMT without using any complicated focus lens, pinholes, and alignment stages. It is an improved design and a user-friendly process to acquire optical signals directly.

4 Concluding remarks This study has presented an integrated microfluidic chip capable of performing DNA/RNA amplification, sample transportation, CE separation, and on-line optical detec© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 7. (a) Electropherogram of amplified DNA products associated with detection gene (240 bps) of S. pneumoniae. (b) Electropherogram of amplified RNA products associated with detection gene (419 bps) of Dengue-2 virus.

tion in an automatic mode. The micromachine-based PCR (or RT-PCR) chip was fabricated using established MEMS-based techniques; and consumes minimum reagent and sample volumes, and provides higher heating/cooling rates together with a more precise temperature control. A clever design was used to ensure a proper www.electrophoresis-journal.com

Electrophoresis 2006, 27, 3297–3305

isolation of the PCR reagents and the CE buffers. The micropump can be used to transport the PCR products automatically. The ability of the integrated microfluidic device to perform the successful amplification of the detection genes for S. pneumoniae bacteria and Dengue2 viruses, and the ability to carry out the injection and separation of the amplified PCR products have been demonstrated. The ultimate goal of the study is to use “optical-fiber only” structures for light in/out of the chip and a fully-integrated biochip for detection of pathogens. The authors gratefully acknowledge the financial support provided to this study by the National Science Council, Taiwan (grant number NSC 92-2323-B-006-010) and the MOE Program for Promoting Academic Excellence of Universities (grant number EX-91-E-FA09-5-4). The authors also thank the Center for Micro/Nano Technology Research, National Cheng Kung University for access provided to major fabrication equipments. Finally, the authors would like to extend their thanks to Dr. Che-Hsin Lin, Mr. Tsung-Min Hsieh, and Dr. Ching-Hsing Luo for their valuable input to project discussions, and to Dr. Jiunn-Jong Wu, Dr. Chih-Ching Chang, and Dr. HsiaoSheng Liu for their kind assistance in the preparation of DNA/RNA samples.

5 References [1] Manz, A., Graber, N., Widmer, H. M., Sens. Actuators B 1990, 1, 244–248. [2] Northrup, M. A., Ching, M. T., White, R. M., Wltson, R. T., Transducers 1993, 924–926. [3] Northrup, M. A., Gonzalez, C., Hadley, D., Hills, R. F. et al., Transducers 1995, 764–767. [4] Daniel, J. H., Iqbal, S., Millington, R. B., Moore, D. F. et al., Sens. Actuators A 1998, 71, 81–88. [5] Woolley, A. T., Hadley, D., Landre, P., deMello, A. J. et al., Anal. Chem. 1996, 68, 4081–4086. [6] Lagally, E. T., Simpson, P. C., Mathies, R. A., Sens. Actuators B 2000, 63, 138–146. [7] Lagally, E. T., Emrich, C. A., Mathies, R. A., Lab chip 2001, 1, 102–107. [8] Liao, C.-S., Lee, G.-B., Wu, J.-J., Chang, C.-C. et al., Biosens. Bioelectron. 2005, 20, 1341–1348. [9] Lin, C.-C., Lee, G.-B., Chen, S.-H., Electrophoresis 2002, 23, 3550–3557. [10] Wang, H. Y., Foote, R. S., Jacobson, S. C., Schneibel, J. H., Ramsey, J. M., Sens. Actuators B 1997, 45, 199–207. [11] Harrison, D. J., Fluki, K., Seiler, K., Fan, Z. et al., Science 1993, 261, 895–897.

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Miniaturization

3305

[12] Lin, C.-H., Lee, G.-B., Lin, Y.-H., Chang, G.-L., J. Micromech. Microeng. 2001, 11, 726–732. [13] Lee, G.-B., Chen, S.-H., Lin, C.-S., Huang, G.-R., Lin, Y.-H., J. Chin. Chem. Soc. 2001, 48, 6B. [14] McCormick, R. M., Nelson, R. J., Alonso-Amigo, M. G., Benvegnu, D. J., Hooper, H. H., Anal. Chem. 1997, 69, 2626–2630. [15] Ogura, M., Agata, Y., Watanabe, K., McCormick, R. M. et al., Clin. Chem. 1998, 44, 2249–2255. [16] Lee, G.-B., Chen, S.-H., Huang, G.-R., Sung, W.-C., Lin, Y.H., Sens. Actuators B 2001, 75, 142–148. [17] Khandurina, J., Mcknight, T. E., Jacobson, S. C., Waters, L. C. et al., Anal. Chem. 2000, 72, 2995–3000. [18] Fujii, T., Microelectron. Eng. 2002, 61–62, 907–914. [19] Waters, L., Jacobson, S. C., Kroutchinina, N., Khandruina, J. et al., Anal. Chem. 1998, 70, 158–162. [20] Koh, C. G., Tan, W., Zhao, M., Ricco, A. J., Hugh, Z. F., Anal. Chem. 2003, 75, 4591–4598. [21] Rodriguez, I., Lesaicherre, M., Tie, Y., Zou, Q. et al., Electrophoresis 2003, 24, 172–178. [22] Hanning, A., Lindberg, P., Westberg, J., Roeraade, J., Anal. Chem. 2000, 72, 3423–3430. [23] Wang, S. L., Huang, X. J., Fang, Z. L., Anal. Chem. 2001, 73, 4545–4549. [24] Liang, Z. H., Chiem, N., Ocvirk, G., Tand, T. et al., Anal. Chem. 1996, 68, 1040–1046. [25] Grewe, M., Gross, A., Fouckhardt, H., Appl. Phys. B 2000, 70, S839–S847. [26] Grosse, A., Grewe, M., Fouckhardt, H., J. Micromech. Microeng. 2001, 11, 257–262. [27] Mogensen, K. B., Pertersen, N. J., Hübner, J., Kutter, J. P., Electrophoresis 2001, 22, 3930–3938. [28] Lin, C.-H., Lee, G.-B., Chen, S.-H., Chang, G.-L., Sens. Actuators A 2003, 107, 125–131. [29] Lin, C.-H., Lee, G.-B., Fu, L.-M., Chen, S.-H., Biosens. Bioelectron. 2004, 20, 83–90. [30] Ro, K. W., Lim, K., Shim, B. C., Hahn, J. H., Anal. Chem. 2005, 77, 5160–5166. [31] Lee, G.-B., Lin, C.-H., Huang, F.-C., Liao, C.-S. et al., IEEE The 16th International Micro Electro Mechanical Systems Conference 2003, Kyoto, Japan, pp. 423–426. [32] Waleed, A. A.-S., Peter, R., J. Clin. Microbiol. 2001, 39, 485– 493. [33] Lee, C.-Y., Lee, G.-B., Liu, H.-H., Huang, F.-C., Int J. Nonlinear Sci. Numer. Simul. 2002, 3(3–4), 177–180. [34] Wang, C.-H., Lee, G.-B., Biosens. Bioelectron. 2005, 21, 419–425. [35] Hsiung, S.-K., Lin, C.-H., Lee, G.-B., Electrophoresis 2005, 26, 1122–1129. [36] Xu, F., Jabasini, M., Baba, Y., Electrophoresis 2002, 23, 3608–3614. [37] Liu, H.-S., Tzeng, H.-C., Chen, C.-C., J. Virol. Meth. 1995, 51, 55–60. [38] Fu, L.-M., Yang, R.-J., Lee, G.-B., Anal. Chem. 2003, 75, 1905–1910.

www.electrophoresis-journal.com