A Simple, Valveless Microfluidic Sample Preparation Device for

Anal. Chem. 2006, 78, 1444-1451

A Simple, Valveless Microfluidic Sample Preparation Device for Extraction and Amplification of DNA from Nanoliter-Volume Samples Lindsay A. Legendre,†,‡ Joan M. Bienvenue,†,‡ Michael G. Roper,† Jerome P. Ferrance,† and James P. Landers*,†,§

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, and Department of Pathology, University of Virginia Health Science Center, Charlottesville, Virginia 22908

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (µSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The µSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min. The goal of developing a micro total analysis system capable of comprehensive genetic analysis requires miniaturization and integration of multiple sample-processing steps. Much progress has been made in the area of electrophoretic separations, which are now readily accomplished in microdevices for both sequencing and size-based genetic analysis.1,2 Other efforts have focused on * To whom correspondence should be addressed. Phone: (434)-243-8658. Fax: (434)-924-3048. E-mail: [email protected]. † University of Virginia. ‡ These authors contributed equally to this work. § University of Virginia Health Science Center. (1) Doherty, E. A.; Kan, C. W.; Paegel, B. M.; Yeung, S. H.; Cao, S.; Mathies, R. A.; Barron, A. E. Anal. Chem. 2004, 76, 5249-5256. (2) Schmalzing, D.; Belenky, A.; Novotny, M. A.; Koutny, L.; Salas-Solano, O.; El-Difrawy, S.; Adourian, A.; Matsudaira, P.; Ehrlich, D. Nucleic Acids Res. 2000, 28, E43.

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the development of polymerase chain reaction (PCR) microchips, either as stand-alone devices3-5 or integrated with separations.6-8 These achievements represent a first and major step toward a totally integrated device for complete genetic analysis. While these developments have advanced microchip technology closer to the goal of a totally integrated system, it is important to note that purified DNA, at least free of PCR inhibitors, is a standard prerequisite for analysis. Consequently, samples that would be considered commonplace in either a clinical, biomedical, or forensic setting still would require time and reagent consuming of large-scale DNA purification techniques to be performed prior to analysis. This exacerbates the importance of miniaturizing and integrating sample preparatory steps (DNA extraction and PCR amplification) in a manner that enhances genetic analysis but also makes it accessible to those outside the microchip development community. While there have been no reports of integrated DNA purification and amplification in the literature, recent reports have demonstrated several devices capable of integrating other sample processing steps. A nanoliter-scale processor that performs cell isolation, cell lysis, and nucleic acid purification on a single platform has been demonstrated.9 In this work, genomic DNA purification was performed from less than 28 Escherichia coli cells; but the amplification step was not performed on-line with DNA purification. In other work, a microchip module for blood cell separation with on-line DNA amplification was reported wherein DNA from white blood cells was isolated and amplified, without incorporating a purification step.10 However, this method utilized (3) Hashimoto, M.; Chen, P. C.; Mitchell, M. W.; Nikitopoulos, D. E.; Soper, S. A.; Murphy, M. C. Lab Chip 2004, 4, 638-645. (4) Lou, X. J.; Panaro, N. J.; Wilding, P.; Fortina, P.; Kricka, L. J. Biotechniques 2004, 36, 248-252. (5) Liu, J.; Enzelberger, M.; Quake, S. Electrophoresis 2002, 23, 1531-1536. (6) Lagally, E. T.; Scherer, J. R.; Blazej, R. G.; Toriello, N. M.; Diep, B. A.; Ramchandani, M.; Sensabaugh, G. F.; Riley, L. W.; Mathies, R. A. Anal. Chem. 2004, 76, 3162-3170. (7) Ferrance, J. P.; Wu, Q.; Giordano, B.; Hernandez, C.; Kwok, Y.; Snow, K.; Thibodeau, S.; Landers, J. P. Anal. Chim. Acta 2003, 500, 223-236. (8) Rodriguez, I. L., M.; Tie, Y.; Zou, Q.; Yu, C.; Singh, J.; Meng, L. T.; Uppili, S.; Li, S. F. Y.; Gopalakrishnakone, P.; Selvanayagam, Z. E. Electrophoresis 2003, 24, 172-178. (9) Hong, J. W. S., V.; Hang, G.; Anderson, F.; Quake, S. R. Nat. Biotechnol. 2004, 22, 435-439. 10.1021/ac0516988 CCC: $33.50

© 2006 American Chemical Society Published on Web 01/31/2006

large PCR volumes (8-9 µL), which can potentially lead to long amplification times. In addition, it was shown that an overabundance of white blood cells present in the amplification step inhibited the reaction, demonstrating that this type of purification will not always produce amplifiable DNA. There are additional benefits to performing DNA purification (as opposed to cell isolation) prior to amplification. Cellular constituents (either proteins or lipids) in cell lysates have long been determined to inhibit or reduce the efficiency of PCR, such as hemoglobin11 and an unidentified factor in eosinophils that specifically inhibits reverse transcription-PCR.12 Also, because several of the benefits associated with performing PCR in microdevices are achieved only when the volume of the PCR chamber is reduced, the use of a concentrating method for DNA isolation is not only advantageous, but necessary, especially for samples that contain a low number of DNA template starting copies. While well-established DNA extraction procedures, such as those involving phenol/chloroform and Chelex methods, have demonstrated efficacy for removal of interfering species, these methods require large reagent volumes that are not easily transferable to the microdevice platform. More recently developed silica-based methods, employed in many commercial extraction kits, are not only effective at extracting highly purified DNA for subsequent analysis, but also readily translatable to microchip formats. This solid-phase extraction (SPE) approach, which relies on DNA adsorption to silica surfaces in the presence of a chaotropic agent, is a well-characterized technology and has been successfully adapted to microdevices to accomplish reducedvolume, high-efficiency extractions, from human, bacterial, and viral sources.13-15 In addition, SPE concentrates the isolated DNA, which is beneficial to interfacing with downstream, small-volume microchip PCR methods. Small-volume microchip PCR allows for a drastic reduction in the volume of reagents used, reduces the volume of sample required, and presents the possibility for invoking faster thermocycling methods. One route to microchip PCR entails the use of integrated heaters and electronics for thermocycling-induced PCR to achieve the aforementioned benefits of microchip-based assays; however, facile transfer of these devices to the average end user needing genetic analysis capabilities is limited until mass production of systems and microdevices makes them commercially available. Tremendous value lies in a device that provides the advantages of microchip miniaturization, but can be interfaced with readily available instrumentation so that researchers can enhance their capabilities for interrogating samples. For example, several reports describe microchip-based PCR systems that performed temperature cycling by placing the microdevice within a benchtop thermocycler,16 a common instrument found in many (10) Yuen, P. K.; Kricka, L. J.; Fortina, P.; Panaro, N. J.; Sakazume, T.; Wilding, P. Genome Res. 2001, 11, 405-412. (11) Rolfs, A.; Schuller, I.; Finckh, U.; Weber-Rolfs, I. PCR: Clinical Diagnostics and Research; Springer-Verlag: Berlin, 1992. (12) Hamalainen, M. M.; Eskola, J. U.; Hellman, J.; Pulkki, K. Clin. Chem. 1999, 45, 465-471. (13) Wolfe, K. A.; Breadmore, M. C.; Ferrance, J. P.; Power, M. E.; Conroy, J. F.; Norris, P. M.; Landers, J. P. Electrophoresis 2002, 23, 727-733. (14) Breadmore, M. C.; Wolfe, K. A.; Arcibal, I. G.; Leung, W. K.; Dickson, D.; Giordano, B. C.; Power, M. E.; Ferrance, J. P.; Feldman, S. H.; Norris, P. M.; Landers, J. P. Anal. Chem. 2003, 75, 1880-1886. (15) Tian, H.; Huhmer, A. F.; Landers, J. P. Anal. Biochem. 2000, 283, 175191.

Figure 1. Design of microfluidic chip for the µSPD. (a) The mask design for the µSPD (dimensions given in the text). The weir in the SPE bed was used to hold the silica beads in place, while the weir in the side channel was used to match flow resistances. (b) A picture of the µSPD accompanied by a picture of the silica beads packed against a weir (bottom right) and a flow diagram illustrating mixing of the reagents necessary for PCR (bottom left).

biochemical, forensic, and clinical laboratories. However, although accessible to many scientists, an important facet overlooked in the methodology was the lack of integration with sample processing prior to PCR amplification. In this report, integration of DNA extraction and PCR amplification on a microdevice for genetic analysis is detailed with application of this device to a variety of sample matrixes relevant to both clinical and forensic testing. A syringe pump, a conventional PCR thermocycler, and a means of analyzing the amplified products are all that are required for the microfluidic sample preparation device (µSPD) to be readily incorporated into standard laboratories. In addition, to further prove the robustness of the method and to achieve a more rapid sample processing, SPE is shown to be interfaceable with noncontact IR-mediated heating for PCR amplification. The enhanced thermocycling speed offered by IR heating, combined with upstream SPE, allows for application of the device to time-sensitive diagnostics, and this is demonstrated by processing a sample for biowarfare agent detection. EXPERIMENTAL SECTION µSPD Preparation. Borofloat glass (Telic Co., Valencia, CA) plates for the microdevices were fabricated using standard photolithographic techniques as described previously.17 The design used for the µSPD is shown in Figure 1, and all microchips were etched 200 µm deep. The photomask contained a SPE channel 10 × 0.14 mm (all dimensions given as length × width) with a weir 2 mm from the exit of the channel, a 15.3 × 0.08 mm side channel, used for introduction of PCR reagents, and a 22.5 × 0.02 mm PCR channel with a 4.0 × 0.8 mm ellipse placed in the middle of the PCR channel to serve as the amplification chamber. A chamber identical to the PCR domain was placed adjacent to the PCR chamber, to serve as a positive control for the integrated analysis with conventional thermocycling and as a reference chamber in the infrared (IR)-heating experiments. Following etching, the etched bottom plates were thermally bonded to borofloat glass top plates; following bonding, the gap between the top of the weir and cover plate was ∼15 µm. For the microchips used in the IR-heating experiments, the dimensions (16) Krishnan, M.; Burke, D. T.; Burns, M. A. Anal. Chem. 2004, 76, 65886593. (17) Roper, M. G.; Shackman, J. G.; Dahlgren, G. M.; Kennedy, R. T. Anal. Chem. 2003, 75, 4711-4717.

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for the SPE domain are the same, but the side channel was 7.0 × 0.02 mm and the PCR channel was 25.0 × 0.02 mm. Hybrid silica bead/sol-gel SPE phases were created using a modified version of the protocol described by Breadmore et al.14 Once the SPE bed had been prepared, the side channel and PCR chamber were dried and a glass syringe filled with SigmaCote (Sigma-Aldrich, St. Louis, MO) was attached to the side channel and flowed through the channel to the PCR chamber outlet. Simultaneously, a syringe filled with air was attached to the inlet of the SPE bed to preclude the passivation agent from entering the SPE bed. This passivation technique was performed prior to every amplification reaction. Sample Preparation. Samples for the elution profiles were prepared by addition of 600 ng of purified human genomic DNA to 500 µL of 6 M guanidine hydrochloride (GuHCl), which was vortexed and used immediately for extraction. To prepare a mock sample for epithelial cell analysis, a cotton swab was rubbed on the inside of a human cheek for 30-60 s, and immediately after, placed in a 1.5-mL tube containing 200 µL of ddH2O to elute the cells. Released cells were allowed to settle to the bottom of the tube for 2-5 min, and 10 µL of settled cells was added to 20 µL of proteinase K solution (Qiagen, Valencia, CA) and 500 µL of 6 M GuHCl. The sample was vortexed for 15 s to lyse the cells and was then ready for introduction into the microchip. Blood samples for the µSPD analysis were prepared by addition of 4 µL of blood to 10 µL of proteinase K solution and 486 µL of 6 M GuHCl and the resultant mixture was then vortexed for 15 s. To lyse sperm cells, a solution of 6 M GuHCl and 40 mM dithiothreitol was prepared immediately before each use.18 The desired amount of semen (5 µL) was diluted to 500 µL using this lysis buffer, vortexed, and loaded into the µSPD. For experiments using IR heating, a swab, rubbed on the inside of a human nostril for 3060 s, was infused with 10 µL of solution containing Sterne strain anthrax spores, 6.6 × 106 cfu/mL. Next, 10 µL of proteinase K was added to the swab and the swab was then placed in a vial containing 180 µL of 6 M GuHCl to elute the sample. µSPD Procedure. Newly filled microdevices were conditioned with 6 M GuHCl, pH 6.1, for 30-45 min at a flow rate of 4.2 µL/ min, to prepare the bed for extraction. Prior to subsequent extractions, the bed was conditioned with 6 M GuHCl for 5 min. The extraction protocol consisted of three pressure-driven steps, each accomplished at a flow rate of 4.2 µL/min using a Harvard Apparatus model 22 dual-syringe pump (Harvard Apparatus, Holliston, MA) and 250-µL Hamilton gastight syringes (Hamilton, Las Vegas, NV). Syringes were connected to the inlet reservoir and the side channel of the microdevice (see Figure 1) using PEEK tubing and NanoPort reservoirs (Upchurch, Scientific, Oak Harbor, WA). All solid-phase extractions were performed as follows, unless otherwise noted in text. First, the lysed sample in 6 M GuHCl was loaded onto the bed for 15 min. Proteins and PCR inhibitors were removed from the SPE bed by passing wash solution (2-propanol/water, 80/20 (v/v)) through the SPE domain for 15 min. During loading and wash steps, water was rinsed through the side channel of the microdevice. Nanopure water was used to elute the DNA from the SPE domain as water flowed simultaneously through the side channel. These initial experi(18) Bienvenue, J. M.; Duncalf, N.; Marchiarullo, D.; Ferrance, J. P.; Landers, J. P. J. Forensic Sci. In press.

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ments were performed to determine when DNA eluted from the SPE bed. Aliquots of the eluate were removed from the chip and either assayed for total DNA concentration using the quantitative Picogreen assay19 or amplified by PCR. After these initial experiments were performed, 2× PCR master mix solution was perfused through the side channel while DNA was eluted in water from the SPE bed. When the predetermined time for elution of DNA had passed, the syringe pump was stopped, the syringes and PEEK tubing were removed from the microdevice, and mineral oil was placed over the PCR reservoirs to prevent evaporation during thermocycling. The entire chip was then placed on a conventional thermocycler heating block (GeneAmp 2400 PCR System, Perkin-Elmer, Wellesley, MA) or over a tungsten lamp for IR-mediated temperature cycling according to the procedure dictated below. Following thermocycling, the PCR products were removed from the device for subsequent analysis by capillary electrophoresis. The bed was flushed with ethanol and water at 4.2 µL/min for 15 min each between extractions. Polymerase Chain Reaction. The PCR mixture for analysis of human genomic and anthrax DNA amplifications on the µSPD contained 100 mM KCl, 20 mM Tris-HCl, pH 9.0 at room temperature, 6 mM MgCl2, 0.8 µM concentration of each primer for anthrax or 1.6 µM of each primer for p16 and gelsolin, 0.4 mM dNTP, and 0.5 units/µL Taq polymerase. The conventional thermocycling protocol included a 2-min initial denaturation at 95 °C, followed by 35 cycles of 64 °C “for both the gelsolin and p16 primers”,20 72 °C and 94 °C, with each temperature held for 15 s, followed by a final extension at 72 °C for 2 min. All PCR products were analyzed by capillary electrophoresis as described later in text. The amplification of amelogenin was performed using the Promega Geneprint Fluorescent Sex Determination kit (Madison, WI), and the thermocycling consisted of an initial denaturation at 96 °C for 2 min, 10 cycles of 94 °C, 60 °C, and 70 °C for 15 s each, 20 cycles of 90 °C, 60 °C, and 70 °C for 15 s each, and then a final extension step at 60 °C for 30 min. Thermocycling conditions for IR-mediated cycling for the anthrax amplification were an initial hold at 95 °C for 15 s, followed by 25 cycles of 62 °C for 3 s, 72 °C for 5 s, and 95 °C for 1 s, and a final extension for 30 s at 72 °C. The elution profile incorporating PCR products utilized amplification of a 380-bp fragment of the β-globin gene. A 2-µL aliquot of the PCR products was removed and microchip gel electrophoresis was carried out in an Agilent Bioanalyzer 2100 (Palo Alto, CA) using the commercially available DNA 1000 kit according to the manufacturer’s instructions, to generate the elution profile (Figure 2). Instrumentation for IR-Mediated PCR. The noncontact thermocycling PCR system was constructed in-house as previously described.21 Briefly, this system consisted of a laptop connected to a 50-W tungsten lamp (CXR/CXL, General Electric, Fairfield, CT) and cooling fan through a DAQ-6024 E-series card (National Instruments, Austin, TX). The microchip was aligned above the lamp and a gold-coated mirror was placed above the PCR chamber to enhance heating. The lamp and fan were both controlled using (19) Ahn, S. J.; Costa, J.; Emanuel, J. R. Nucleic Acids Res. 1996, 24, 26232625. (20) Cerilli, L. A. S. J. R.; Saadut, R.; Marshall, C. E.; Rumpel, C. A.; Moskaluk, C. A.; Frierson, H. F. Hum. Pathol. 1999, 30, 1242-1246. (21) Easley, C. J.; Legendre, L. A.; Roper, M. G.; Wavering, T.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2005, 77, 1038-1045.

Figure 2. Elution profiles for timing DNA elution from the SPE bed. The black squares are the amount of DNA in each fraction as determined by PicoGreen (left y-axis), while the gray diamonds are the DNA concentration after PCR amplification of each fraction (right y-axis, DNA quantified after PCR using BioAnalyzer 2100).

a LabVIEW application through solid-state relays and a proportionalintegral-derivative feedback control algorithm written in-house. A miniature type-T copper-constantan thermocouple (model T-240C), obtained from Physitemp Instruments, Inc. (Clifton, NJ), was inserted into the reference channel for temperature detection. The majority of these thermocouples required sanding with fine sandpaper at the sensing end before insertion into the microchannel. A model TAC-386-T thermocouple-to-analog converter (Omega Engineering, Stamford, CT) powered by a 9-V alkaline battery amplified the thermocouple signal 25-fold (amplified to 1.0 mV/°C), which was collected by the DAQ card of the laptop allowing the LabVIEW application to turn on either the lamp or fan to provide heating or cooling of the solution as needed. Capillary Electrophoresis. Amplified DNA was detected using a Beckman MDQ (Fullerton, CA) using hydroxypropyl cellulose for DNA separations, as dictated by Sanders et al.22 At the beginning of each day, a 30-min rinse with 1 M HNO3 was performed, followed by a 15-min rinse with separation polymer consisting of 80 mM MES and 40 mM Tris at pH 6.1 with 3.5% (w/v) hydroxypropyl cellulose and 0.1% (v/v) YO-PRO (Molecular Probes, Eugene, OR).22 For analysis of amplified product from the chip, the solution in the side channel and PCR channel was removed by pipet and diluted to 25 µL with water for capillary electrophoresis injection. Electrophoresis was performed in a 50µm-inner diameter, 30-cm (10-cm effective length) capillary and separation performed with 8 kV applied voltage. The samples were electrokinetically injected for 5 s at 10 kV toward the anode; detection was accomplished using laser-induced fluorescence with excitation by the 488-nm line from an argon ion laser and fluorescence emission recorded at 520 nm (520 ( 10 nm bandpass filter) with a photomultiplier tube. The amplified amelogenin required a single-stranded DNA separation; therefore, electrophoresis was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) per manufacturer’s instructions. RESULTS AND DISCUSSION As stated earlier, a review of the literature showed that microchip-based PCR using conventional thermocyclers has been (22) Sanders, J. C.; Breadmore, M. C.; Kwok, Y. C.; Horsman, K. M.; Landers, J. P. Anal. Chem. 2003, 75, 986-994.

reported, although coupling of DNA purification methods with microchip PCR amplification from biological matrixes has not been demonstrated. The difficulty in achieving this integration can be attributed to a number of issues, including incompatibility of SPE reagents with either the device or the passivation reagents used or possible incompatibility of solid-phase supports with the device. The microfluidic platform described in this report provides a simple, yet effective, method for removing PCR inhibitors before on-chip DNA amplification and should be applicable to research in diverse fields (environmental, forensic, clinical). For these microdevices to be readily implemented into already existing laboratory protocols, ease of use was heavily weighted in their design. The µSPD was fabricated to have valveless fluidic control by incorporating a side channel that allowed for dilution and direct introduction of PCR reagents without their having to traverse the SPE bed. As a result of all external connections to the microchip being reversible, the µSPD could simply be placed on the heating element of a conventional thermocycler for PCR amplification. Device Design and Passivation. The fluidic architecture of the µSPD allowed for connection of the SPE domain to the PCR chamber without the need for valves (Figure 1). Consequently, DNA was eluted from the SPE domain directly into the PCR chamber for subsequent amplification. The use of glass as the microdevice substrate necessitated passivation of the silica surface to avoid adsorption of Taq polymerase prior to and during PCR amplification. In addition, the gastight glass syringes used to contain sample and reagents for extraction and PCR also required passivation. Since passivation had to be carried out prior to DNA extraction, the passivation reagent needed to be stable to the reagents used for SPE, specifically 6 M GuHCl and 80% 2-propanol. While numerous silanizing reagents have been reported in the literature for electrophoretic capillaries, effectiveness with fused silica did not necessarily ensure effectiveness with borofloat glass. Moreover, in line with the overall simplicity of the µSPD, a passivation chemistry was sought that was rapid (one step) and simple. The evaluation of a variety of passivating reagents that met these criteria led to the use of a commercially available silanizing reagent, SigmaCote. This reagent provided a microchip passivation process that was one-step and was complete in less than 5 min. To perform the passivation, a syringe was filled with SigmaCote and flowed through the side channel and PCR chamber. This reagent required only a dry glass surface as a prerequisite, eliminating the often extensive rinsing and cleaning steps associated with other microchip coating procedures. Timing of DNA Elution. The effectiveness and efficiency of a silica bead/sol-gel solid phase for the extraction of DNA has been previously established.13-15 In single-process microdevices containing this particular hybrid solid phase, the majority of the DNA is eluted in a volume greater than 10 µL;14,18 however, the total volume of the PCR channel in the µSPD is only 1.6 µL. As a result of this volume mismatch, it was critical to determine the precise elution of DNA from the solid phase to ensure that the appropriate aliquot of eluted nucleic acids (heart cut of the elution peak) mixed with PCR master mix fed in through the sidearm during the dual-syringe pumping process. To define this, a fluorescence timing study was carried out with prepurified human genomic DNA with aliquots of eluate (2 µL during the initial DNA release phase, 5-µL fractions thereafter) collected for DNA Analytical Chemistry, Vol. 78, No. 5, March 1, 2006

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quantitation using a commercial fluorescence assay. As depicted in Figure 2 (black squares), the majority of DNA was desorbed from the SPE bed in the first 10 µL of the elution phase as expected, while each of the subsequent aliquots had negligible concentrations of DNA (n ) 5). To determine the correlation between the mass of DNA present in each fraction and the PCR amplifiability of that DNA, identical aliquots were collected and conventional PCR was carried out. The results of analysis by microchip electrophoresis are given in Figure 2 (gray diamonds). Fractions 2-4 (6 µL total volume) reproducibly contained amplifiable DNA (n ) 3); however, there was a distinct shift when comparing the maximum amount of DNA detected in the two assays. The first 2-µL fraction amplified poorly or not at all, and while the second fraction was reliably amplifiable, it was the third 2-µL fraction, 45 s after the initiation of elution of DNA from the SPE bed, that amplified optimally. Therefore, for all subsequent analyses performed on the µSPD, the syringe pump was stopped 45 s after initiation of DNA elution to ensure that this fraction was utilized for the PCR amplification. We speculate that the PCR amplification was inhibited in the early fractions (the first 2-4 µL) due to residual 2-propanol in the SPE and PCR domains. To avoid this problem on the µSPD, the longer elution time into the PCR chamber was used to wash the 2-propanol out of the device. There are other possible methods for ensuring the eluted DNA is free from the 2-propanol found in the wash step. Either hydrogel plugs23 or a polymer24 containing capture oligonucleotides can be placed between the SPE bed and the PCR domain to bind the eluted DNA. While these methods are feasible, the use of a second “cleanup” step after the SPE bed seemed redundant and the method described here was reproducible and simple. On-Chip Mixing of Eluted DNA and PCR Components. In preparation for the PCR process, co-mobilization of the eluted DNA (from the SPE domain) and the PCR master mix (from the sidearm) into the PCR domain was required. Ideally, mixing of these would occur as the solutions traverse the microfluidic channel leading to the elliptical PCR chamber. However, as is normally observed in microfluidic chips, low Reynolds numbers allow mixing based only on lateral diffusion which, depending on the diffusion coefficient of the analytes, can involve long times, contradictory to the benefits of fast analysis times associated with microfluidic devices. Numerous papers have described elegant schemes for both active and passive mixing of solutions in microdomains (for a recent review, see Hessel et al.25). However, it was anticipated that the combination of two simplistic modes, diffusion and convection, would inherently provide adequate mixing of the eluted DNA and the PCR master mix as a result of the design of the chip and the processes that it executes. Diffusive mixing occurs during transfer from the SPE bed to the PCR chamber and then in the chamber itself, while convective mixing should result from thermocycling during the PCR process. It was necessary to ascertain that mixing of the eluted DNA and PCR master mix was complete early in the thermocycling as the efficiency of PCR amplification would be compromised in an inadequately mixed sample. A calculation was performed to (23) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (24) Paegel, B. M.; Yeung, S. H. I.; Mathies, R. A. Anal. Chem. 2002, 74, 50925098. (25) Hessel, V.; Lowe, H.; Schonfeld, F. Chem. Eng. Sci. 2005, 60, 2479-2501.

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Figure 3. Microfluidic mixing in the µSPD. (a) Blue dye was flowed through the side channel while yellow food dye was flowed through the SPE bed (flow rate as used in µSPD, 4.2 µL/min). (b) After entrance into the PCR chamber, blue and yellow dyes were only mixed in the middle due to diffusion. (c) Green color was observed in the PCR chamber indicating mixing of the two streams after initial denaturation step of thermocycling.

approximate the time required for diffusive mixing of the DNA and PCR master mix components. Relying solely on diffusion and assuming 50-kbp DNA fragments (due to shearing of genomic DNA strands during pipetting) with a diffusion coefficient of 5 × 10-9 cm2/s,26 DNA fragments would traverse half the width of the bottom of the PCR channel in 4 × 104 s (667 min) and half the ellipse width in 2 × 105 s (2667 min), clearly making diffusive mixing too slow for rapid analysis. To determine this visually, blue and yellow dyes were pumped through the SPE channel and side channel respectively, using syringe-driven flow at the same rates used for the µSPD. Figure 3a shows the laminar flow of the two streams at the “T” where the side channel (blue) and the SPE domain (yellow) streams meet. Evidence of slight mixing is seen in the center of the channel (indicated by the green color), and upon reaching the PCR chamber, only minimal diffusive mixing had occurred (Figure 3b). To demonstrate that adequate heatinduced convective mixing occurred during the initial denaturation step, the flow was stopped and the solution in the PCR chamber heated to the initial denaturation temperature (94 °C) for 120 s. This provided complete mixing of the solutions within the PCR chamber as shown in Figure 3c. A control experiment, allowing the solutions to diffuse without any heating, showed incomplete mixing even after 10 min as expected (data not shown). SPE-PCR for Clinical and Forensic Applications. Ideally, a generic platform and method that exploits the advantages of microminiaturization (low-volume reagent consumption) but uses common laboratory instrumentation (e.g., conventional thermocyclers) would enable a large number of researchers to reap the benefits of microfabricated devices. Consequently, an integrated (26) Smith, D. E. P., T. T.; Chu, S. Macromolecules 1996, 29, 1372-1373.

Figure 4. Capillary electrophoretic separation of amplified product from the µSPD coinjected with standard DNA fragments. DNA from lysed buccal cells was loaded onto the device for 15 min, and integrated PCR was successful as shown by the 80-bp fragment from the p16 gene that was amplified.

method to extract DNA and then carry out target PCR amplification in the same microfluidic device using a common benchtop thermocycler was sought. Having determined the timing for DNA elution from the SPE domain, and that the eluted DNA could be comprehensively mixed with the PCR reagents during the initial heat denaturation, the versatility of the µSPD was tested with a variety of biologically derived samples. To account for the diverse array of sample matrixes encountered when performing DNA analysis with clinical and forensic applications, extraction and amplification of DNA from buccal cells, blood, and semen were evaluated. Each of these samples contained molecular species capable of inhibiting DNA amplification, which therefore requires DNA purification before PCR,27-29 providing a test for sample bandwidth with the µSPD. Coupling of the SPE and PCR processes was first tested with the extraction of DNA from buccal epithelial cells and the subsequent on-chip amplification of a fragment of the p16 gene. This gene codes for a family of cell cycle regulators (cyclindependent kinase inhibitors, CDKI) that binds to “cyclin-CDK” complexes to induce cell cycle arrest in the G1 phase. As such, p16 is a tumor suppressor gene that is suppressed in such cancers as melanoma (familia) and pancreatic adenocarcinoma, squamous cell carcinoma of the head and neck, and carcinoma of the esophagus, to name a few,20,30 and represents a target of interest for molecular diagnosis of gene mutations or deletions. The integrated extraction and amplification successfully amplified an 80-bp fragment of the p16 gene, which can be seen in the electropherogram shown in Figure 4. A DNA ladder was coinjected for sizing the 80-bp PCR product. A plot of DNA fragment size versus migration time established a fragment length of 82 bp for the amplicon, a reasonable estimation knowing that Taq polymerase can add an extra nucleotide to the fragment during amplification.31 Since PCR is not quantitative, success of the method was measured by the repeatability of the results (the (27) Al-Soud, W. A.; Radstrom, P. J. Clin. Microbiol. 2001, 39, 485-493. (28) Rudbeck, L. D., J. Biotechniques 1998, 25, 588-590. (29) Butler, J. M. Forensic DNA Typing, Biology and Technology Behind STR Markers; Academic Press: San Diego, 2001. (30) Liggett, W. H.; Sidransky, D. J. Clin. Oncol. 1998, 16, 1197-1206. (31) Clark, J. M. Nucleic Acids Res. 1988, 16, 9677-9686.

Figure 5. Representative electropherogram of a gelsolin gene fragment amplified from a blood sample on the µSPD. Presence of the 139-bp amplicon shows successful purification and amplification from a total of 600 nL of blood. To show the reproducibility of the device, electropherograms representing multiple experiments with the µSPD are shown.

detection of amplified product) in multiple attempts with buccal cells. While the integrated buccal epithelial cell DNA purification and amplification demonstrated the µSPD could effectively extract DNA and allow for chip-based integration of the amplification step, extraction and amplification of a fragment of the gelsolin gene from DNA in whole blood would further test the versatility of the device. Gelsolin plays an important role in the “gel” to “sol” transformation in cell motility, through the severing and capping of actin filaments, thereby regulating filament lengthsthese filaments are involved in cell structure, motility, apoptosis, and cancer.32,33 Integrated DNA extraction and amplification of a gelsolin-specific fragment from whole blood using the µSPD yielded the 139-bp amplicon seen in the electropherogram in Figure 5sthe successful amplification confirmed that microchip extraction removed PCR inhibitors that might have been present in the sample. The volume of blood loaded onto the µSPD was 600 nL, and replicate purification of DNA from whole blood and amplification of the gelsolin fragment was performed three times over multiple days on three different microdevices. These results (Figure 5) illustrate that the µSPD was capable of robust, repeatable amplification of DNA purified from a biofluid. Following successful demonstration of integrated purification and amplification of DNA from both simple (buccal cells) and complex (blood) biological samples with clinically relevant amplification targets, the µSPD was challenged with a forensically relevant analysis involving extraction and amplification of DNA from semen. The amplification target chosen for this experiment was the same as that included in routine forensic testing, to illustrate the range of the µSPD. In addition, a commercially available kit was used for the amplification to further evaluate the robustness and utility of the microchip PCR process. For this assay, a total of 630 nL of semen was loaded onto the µSPD. The protocol for the commercial kit amplification was modified for the small volume required in the microchip analysis by reducing the (32) Mielnicki, L. M.; Ying, A. M.; Head, K. L.; Asch, H. L.; Asch, B. B. Exp. Cell Res. 1999, 249, 161-176. (33) Ohtsu, M.; Sakai, N.; Fujita, H.; Kashiwagi, M.; Gasa, S.; Shimizu, S.; Eguchi, Y.; Tsujimoto, Y.; Sakiyama, Y.; Kobayashi, K.; Kuzumaki, N. Embo J. 1997, 16, 4650-4656.


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Figure 6. Evaluation of the µSPD with a semen sample. (a) Two peaks in the electropherogram, labeled X and Y, were observed at the expected size (212 and 218 bp) of the amelogenin gene fragment demonstrating successful amplification.

volumes used in the large-volume PCR reactions (25-50 µL) to those more suitable for the miniaturized analysis. As seen in Figure 6, successful amplification of amelogenin gene fragments resulted in the two peaks at 212 and 218 bp (representing X and Y chromosomes) in the electropherogram (n ) 3). The ability to adapt commercially designed amplification kits to the microchip format is an important step in paving the way for acceptance of microminiaturized analytical systems in research and clinical/ forensic laboratories in a way that is compatible with already established methods. In addition, the amelogenin marker, utilized for human identification, was amplified from a forensically relevant target, important for eventual application of this technique to genetic typing for forensic analysis using short tandem repeat (STR) fragments.29 The results obtained from this evaluation of the µSPD for genetic analysis of buccal cells, blood, and semen represent several significant accomplishments. Through successful coupling of SPE and PCR on a single device, we demonstrate the first example of a valveless µSPD that can be utilized with standard benchtop thermocyclers. The versatility of this device is clear from the successful purification and amplification of DNA from the types of complex biological samples often encountered in both clinical and forensic analyses. Successful amplification of amelogenin demonstrated that commercial kits could be used with this µSPD, maintaining the integrity of the amplification while reducing both the analysis time and cost of the method through the decreased amount of reagents used. This adaptation is especially crucial for application of this device to forensic analysis, as most current PCR amplifications for forensic casework utilize validated and approved commercial amplification kits to generate STR fragments for genetic profiling.29 To integrate into forensic laboratories, new technology must interface with existing standardssdemonstrating that these amplification kits can be adapted to the microchip format indicates that application of these microdevices to casework analysis remains a viable and practical opportunity. Another aspect of interest to the clinical/forensic sector is a µSPD that is singleuse and, following analysis, discarded. In terms of glass microdevices that are commercially produced for one-time analysis, a precedent has been set with the microchip analysis system manufactured by Agilent, where glass electrophoretic microchips 1450


are single-use, disposable devices. As a result of the intended single analysis use, sequential run carryover was not thoroughly investigated. Accelerating the SPE-PCR Process for Anthrax Detection. The integrated analyses demonstrated in this report utilized a conventional thermocycler for heating and cooling during PCR amplification to illustrate the ease of use and compatibility with current conventional instrumentation. However, a drawback to the use of the conventional PCR instrumentation was long amplification times (∼75 min) resulting from the slow temperature transitions of heating the metal block in the thermocycler. Reduction of scale to the volumes typically associated with microchip analysis typically reaps decreased analysis time (increased speed)34 that would make these devices applicable in settings that required rapid analysis. These would include pointof-care testing for clinical diagnostic purposes or bioagent detection in forensic or military applications. To demonstrate that rapid analyses could be performed with the µSPD, noncontact temperature cycling was employed for PCR using IR-mediated excitation of the vibrational bands of water.34-36 The size of the PCR chamber was decreased (from 1.6 µL to 330 nL volume) to allow positioning of both the PCR and temperature reference chambers in the focal spot of the tungsten lamp. In addition, the solid-phase extraction process was further refined by decreasing the volume of lysis solution added to sample, which decreased the load time, and decreasing the wash time, to provide faster DNA purification. This rapid analysis µSPD method was challenged with a mock sample created by inoculating a nasal swab with anthrax spores. The modified DNA extraction step for this sample, achieved purification in ∼9 min (3 min for load, 5 min for wash, 45 s for elution). Following the addition of mineral oil and insertion of a thermocouple for temperature sensing, IR-mediated thermocycling was completed in a total amplification time of 16 minsthis is roughly a 5-fold reduction in PCR time and reduces the time required for the entire microchip process to 25 min. To ensure the reproducibility and robustness of this analysis, the entire procedure was repeated on different daysssuccessful amplification was observed in multiple attempts with this chip (n ) 3). The electropherogram in Figure 7 show the presence of a significant amount of unused primer; however, this can be explained by the design of the device. The entire channel from the SPE bed to the PCR outlet reservoir holds a volume of 1.6 µL and the side channel, which contained 2× master mix, holds an additional 0.5 µL. However, only a small fraction of that total volume (330 nL) was actually heated and, therefore, experienced the PCR amplification. Following thermocycling, the entire volume from the side channel to the PCR outlet was removed for capillary electrophoresis analysis, including unused primers from outside the heated region. While the excess primer in the electropherogram was apparent, it did not impact the performance of the device, as the amplified product was clearly observed. It is noteworthy that, when interpreting the results from analysis of samples potentially containing biowarfare agents, time is of critical importance. Moreover, a device capable of fast sample (34) Giordano, B. C.; Ferrance, J.; Swedberg, S.; Huhmer, A. F.; Landers, J. P. Anal. Biochem. 2001, 291, 124-132. (35) Oda, R. P.; Strausbauch, M. A.; Huhmer, A. F.; Borson, N.; Jurrens, S. R.; Craighead, J.; Wettstein, P. J.; Eckloff, B.; Kline, B.; Landers, J. P. Anal. Chem. 1998, 70, 4361-4368. (36) Huhmer, A. F.; Landers, J. P. Anal. Chem. 2000, 72, 5507-5512.

Figure 7. Electropherogram showing anthrax present in the sample analyzed by the µSPD from the swab. Anthrax spores on a nasal swab were eluted in lysis buffer and loaded onto the µSPD for purification of DNA and integrated with IR-mediated PCR. The capillary electropherogram shows successful amplification of the 211bp product peak; the total analysis required only 25 min.

processing, reducing contamination due to minimal sample handling, and compatibility with multiple amplification procedures and sample matrixes is essential. The work presented here represents the first application of the valveless µSPD to biological samples, paving the way for the future extension of integrated sample preparation systems to analysis of bona fide clinical and forensic samples in a diagnostic/ crime laboratory. On-line integration of DNA purification from crude biological samples allows for subsequent, on-line PCR in a (37) Legendre, L. A., Ferrance, J. P., Horseman, K. M., Guillo, C., Bienvenue, J. M., Landers, J. P. Ninth International Conference on Miniaturized Systems for Chemistry and Life Sciences; 2005; Vol. 2, pp 1519-1521.

completely self-contained device, thereby reducing the susceptibility of the system to contamination. The noncontact approach that we have described for heating and cooling during thermocycling simplifies fabrication of the device, by eliminating the need for incorporating heaters or valves. The simplicity of the device design utilized here also allows for facile adaptation to more complicated applications, such as STR genotyping using commercially available amplification kits. For example, by using two or more PCR chambers fluidically connected to a SPE bed, multiple amplifications can be performed on the same sample encompassed in one device. In addition, one can envision that through interfacing of direct fluorescence detection with the system described here, to either a noncontact thermocycling system37 or a conventional thermocycler, real-time PCR, could be performed. The incorporation of an amplicon detection step into the µSPD would allow for a microdevice capable of performing a fully integrated genetic analysis. ACKNOWLEDGMENT The authors acknowledge Agilent Technologies, Inc. for the DNA 1000 kits used in this work and Sandy Feldman for the anthrax spores. The authors also acknowledge Mr. Christopher Easley for his contributions to the IR-mediated thermocycling system and Mr. Sushil Shrinivasan for photographing the microdevice. Funding for this work was provided by the NIH through the U.S. National Human Genome Research Institute award numbers R01 HG002613 and R01 HG001832.

Received for review September 22, 2005. Accepted December 27, 2005. AC0516988


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A Simple, Valveless Microfluidic Sample Preparation Device for

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