DIGITAL MICROFLUIDIC HUB FOR AUTOMATED NUCLEIC ACID SAMPLE PREPARATION Hanyoup Kim, Michael S. Bartsch, Ronald F. Renzi, Genevieve L. Pezzola, Erin M. Remillard, Eric A. Kittlaus, Jim He, and Kamlesh D. Patel* Sandia National Laboratories, United States of America ABSTRACT We have designed, fabricated, and characterized a digital microfluidic (DMF) platform to function as a central hub for interfacing multiple lab-on-a-chip sample processing modules towards automating the preparation of clinicallyderived DNA samples for ultrahigh throughput sequencing (UHTS). The platform enables plug-and-play installation of a two-plate DMF device with consistent spacing, offers flexible connectivity for transferring samples between modules, and uses an intuitive programmable interface to control droplet/electrode actuations. Additionally, the hub platform uses transparent indium-tin oxide (ITO) electrodes to allow complete top and bottom optical access to the droplets on the DMF array, providing additional flexibility for various detection schemes. KEYWORDS: Digital microfluidic (DMF), Sample preparation, DNA Sequencing, Microdroplet INTRODUCTION While DNA sequencing technology is advancing at an unprecedented rate, sample preparation technology still relies primarily on manual bench-top processes, which are slow, labor-intensive, inefficient and often inconsistent [1]. Automation of sample preparation using microfluidic techniques is well-suited to address these limitations. However, fabricating a monolithic microfluidic device that replicates all the relevant bench top processes can be prohibitively complicated and does not allow the flexibility to execute diverse protocols for processing different samples and sample volumes. Our approach uses a central DMF platform which transports surface-bound microdroplets to interface between multiple sample processing modules in a flexible manner as shown in Figure 1. Ultimately, the platform will interface to downstream ultrahigh throughput sequencing systems with a goal of detecting unknown pathogens by enriching informative nucleic acids sequences (from the pathogen) and suppressing background DNA (from the host).
Figure 1: Schematic representation of an automated molecular biology platform based on a DMF device that functions as a central hub interfacing multiple lab-on-a-chip format modules. Droplets carry sample between modules. DEVICE DESIGN AND EXPERIMENTAL METHODS The platform consists of an engineered polymer frame with opposed recesses to accommodate the 50 x 75 mm DMF and ground plane substrates. Substrates fixed in these recesses by metal compression frames as shown in Figure 2(a) are automatically registered relative to each other with a fixed spacing of either 185 or 400 µm, depending on which platform frame is used. This spacing enables the use of either 150 or 360 µm outer diameter capillary tubes, respectively, which can be positioned in the interstitial space between the DMF and ground plane and fixed in place using CapTiteTM capillary fittings (Sandia National Labs, Livermore, CA) as shown in Figure 2(b). These hydrophobically-coated fused-silica capillaries enable the transfer of sample droplets between the central DMF device and processing modules. Replacing one of the metal compression frames, a special manifold frame (not shown) can also be installed which allows fluidic connections to be made to through-holes drilled in the DMF or ground plane substrates. An array of spring-loaded pogo pins provides electrical connections to the contact pads of the DMF device and to the ground plane.
U.S. Government work not protected by U.S. copyright
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14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands
Figure 2: (a) Schematic and (b) photo of the automatic molecular biology platform (acrylic) with an ITO-based DMF device. Capillary tubes interface with the DMF device to transport sample to and from the surface-bound droplet. Printed circuit boards with spring-loaded pogo pins make electrical connections to the ground plane and DMF device. DMF devices were fabricated by patterning an electrode array onto a 50 x 75 mm ITO-coated glass slide (Delta Technologies, Stillwater, MN), coating the electrodes with dielectric material (SU-8 or parylene C), and coating the dielectric with Teflon AF to yield a hydrophobic surface. A Teflon-coated ITO glass slide was also used as the ground plane. Droplets were actuated by applying optimized AC voltage pulse (typically ~50-100 Vrms at 15 kHz) to the electrodes. A computer-controlled electronic interface was also implemented to activate individual pad(s) either by manual keystrokes or predetermined script sequences. Droplet actuation was monitored with MVX10 (Olympus, Center Valley, PA) microscope with a high speed digital camera (QIClick, Qimaging, Surrey, Canada). The microscope had fluorescence and dark field microscopy capabilities. RESULTS AND DISCUSSION We have successfully actuated 5 different liquids including DI water, TE buffer, and 10, 160, and 330 mM sodium phosphate buffers on DMF devices integrated to the automatic biology platform. Figure 3 shows a preset sequence for manipulating two water droplets from two separate reservoirs.
Figure 3: Two water droplets of ~1.2 µl are split off from two reservoirs on both sides in (a) and (c), actively mixed by circling on four pads in (d) and (e) and then transported to the outlet pad on the bottom right side in (f). The ITO pattern on DMF device is visible. The capillary fluidic interface was tested to transport liquid samples into and out of the DMF device by applying either positive or negative pressure. Figure 4 shows that water was loaded onto the target pad through a 50 µm inner diameter capillary tube by applying pressure created by a manually controlled syringe.
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Figure 4: Sequence of sample loading to a DMF device using a capillary tube integrated to the platform. The target pad was activated while the sample was loaded by applying positive pressure to the syringe. The droplet was actuated away from the capillary once the target volume of 2.5 µl was reached. Finally the droplet was split into two by activating two pads simultaneously. We were also able to perform a DNA labeling assay by mixing two droplets of pico-green dye and DNA (2 µg/ml lambda DNA) solution shown in Figure 5. The transparent ITO DMF device allowed droplet visualization using dark field microscopy without compromising the accuracy of a simultaneous quantitative fluorescence assay. Active mixing by transporting the fused droplet over four pads was used to overcome the slow diffusion-limited mixing [2].
Figure 5: Pico-green assay with lambda DNA. Two droplets of ~600 nl split off from each reservoirs were fused and actively mixed. The concentration of double-stranded DNA can be quantified by the fluorescence intensity as shown in (d) (a) and (b): Dark field only, (c) and (d):Dark field and fluorescence with a GFP filter set. CONCLUSION & FUTURE WORK We have demonstrated and characterized an ITO-based DMF hub with in-plane capillaries to integrate modules for automating sample preparation for host background suppression by normalization, and enzyme-based DNA fragmenting and ligation for platform-specific template formatting. Our goal is to apply this platform to rapidly detect and characterize unknown pathogens through genomic analysis, not possible through probe based molecular techniques such as microarray, polymerase chain reaction or antibody assays. ACKNOLWEDGEMENTS The authors would like to thank Jim Van De Vreugde and Mark Claudnic for their help with the initial platform design. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94Al85000. REFERENCES [1] P. Coupland, Microfluidics for the upstream pipeline of DNA sequencing--a worthy application? Lab on a Chip, 10, pp. 544-7.(2010) [2] P. Paik, V. K. Pamula, M. G. Pollack and R. B. Fair, Electrowetting-based droplet mixers for microfluidic systems. Lab on a Chip, 3, pp. 28-33.(2003) CONTACT *Kamlesh D. Patel, tel: +1-925-2943737;
[email protected]
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