free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Genomic DNA has been reproducibly fragmented with 2-10 kbp fragment lengths by applying hydraulic pressure ΔP across micromachined constrictions in the microfluidic channels. The utilization of a series of constrictions reduces the variance of the fragmented DNA length distribution; and parallel microfluidic channels design eliminates the device clogging

Berg, A. van den

Bomer, J.G.

Carlen, E.T.

Jin, M.

Shui, L.

English

Shui, Lingling

Jin, Mingliang

Bomer, Johan G.

Carlen, Edwin

van den Berg, Albert

University of Twente Research Information

MME2010 Workshop 1Proceedings of the 21st Micromechanics and Micro systems Europe Workshop,Enschede, The Netherlands, 26-29 September 2010Edited by Leon Abelmann, Hans Groenland, Joost van Honschoten, and Hein VerputtenISBN 978 90 816737 1 6MME2010 Workshop 2Back to the ProgrammeHIGH-THROUGHPUT ON-CHIP DNA FRAGMENTATION   L. Shui, M. Jin, J.G. Bomer, Edwin T. Carlen, and A. van den Berg  BIOS/Lab-on-Chip Group, MESA+ Institute for Nanotechnology, University of Twente, The Netherlands  Abstract — In this paper we report a high throughput clog-free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Genomic DNA has been reproducibly fragmented with 2-10 kbp fragment lengths by applying hydraulic pressure ΔP across micromachined constrictions in the microfluidic channels. The utilization of a series of constrictions reduces the variance of the fragmented DNA length distribution; and parallel microfluidic channels design eliminates the device clogging.   Keywords: Microfluidics, DNA fragmentation, Hy-drodynamic shearing  I - Introduction  Sample pre-treatment is one of the most important steps in applying lab-on-a-chip (LOC) technology to real world applications. DNA fragmentation is a sample pretreatment step for nucleic acid analysis, which is important for rapid hybridization reaction kinetics and the production of genomic DNA libraries and is often a critical and sometimes rate-limiting step in the DNA sequencing pipeline. Four methods are commonly used for genomic DNA fragmentation including enzymatic digestion, sonica-tion, nebulization and hydrodynamic shearing [1-10]. Although all of these methods are currently used for DNA fragment generation, each has advantages (+) and disadvantages (-), which are listed in Table 1. Table 1: Various DNA fragmentation methods with advan-tages (+) and disadvantages (-). Method + - Restriction enzyme Fragmentation in specific location Denaturation; Fragment bias; Fragmentation non-uniformity Nebulization Cheap and fast Large volumes; Concentrated NA; Difficult LOC integration Sonication Fast and simple Not size specific; NA damage; Difficult LOC integration Mechanical shearing Size specific; Narrow size range; Low NA damage (compared to other methods); Amenable to LOC integration Slow loading in conventional apparatus; conven-tional systems easily clog Enzyme digestion can lead to fragment bias and induces a relatively large number of single-chain breaks as opposed to double-chain scissions. Sonication can result in damage to the fragmentated DNA and is difficult to integrate into a LOC platform. Nebulization typically results in a large fragment length distribution and is also not amenable for LOC integration. Fragmentation based on hydrodynamic shearing is reported to be one of the most robust techniques for the generation of small fragment length and low sheared length variation [10, 11], and the fragments result from double-chain scission [12]. However, conventional single orifice hydrody-namic shearing are systems are expensive and are prone to clogging. The hydrodynamic shearing method is widely used for shearing genomic DNA [8,10]. The DNA molecules are fragmented by hydrodynamic shear stress when they are forced through a small orifice at high speed. Ge-nomic DNA can be efficiently sheared with high quality fragments [8,10]. The length of the fragments is deter-mined by the flow speed of the sample solution and the diameter of the orifice and has been reported to be independent of the initial DNA length, concentration, and salt concentration of the fragmentation buffer [10]. Although DNA fragmentation instruments based on shearing forces are commercially available [13], they require high pressures, complex interconnections, and are prone to clogging. Implementation of DNA frag-mentation in a LOC platform has several advantages: small sample requirements (at least 10× reduction), low dead volume, generating high shear rates at compara-tively low flow rates. In this paper, we present a low cost LOC platform for genomic DNA fragmentation based on the hydrody-namic shearing. The LOC consists of inlet and outlet ports that connect a microfluidic channel with a pre-cisely defined series of constrictions located at the center of the microchannel. Genomic DNA samples are introduced to the inlet port and transported through the constrictions with hydraulic pressure. Assuming a constant volumetric flow rate, as the DNA approaches the constriction, the flow speed increases due to the reduced cross-sectional area of the constriction. Drag forces stretch the DNA molecule until it fragments. The clogging problem has been eliminated by introducing many identical constrictions in parallel, which increases the fragment generation rate without affecting the quality or length of the fragments.  II - Experimental Details  Figure 1 depicts the concept of hydrodynamic shear-ing-based DNA fragmentation in a microfluidic chan-nel. Long DNA molecules are introduced into left side of the channel (inlet with pressure P1) and transported through the microchannel with a hydraulic pressure MME2010 Workshop 124Back to the Programmedifference (∆P=P1-P2). As the DNA approaches the constriction, the flow speed increases and the coiled DNA molecules are elongated and then fragmented by drag forces when the DNA is located in the constric-tions. The series of constrictions are used to reduce the     Figure 1: Schematic drawing of DNA fragmentation device using hydrodynamic shearing forces. ∆P=P1-P2. variance in the fragment length distribution. Figure 2 shows a top-view of a single constriction with length L and width d, and main channel width a. The angled constriction design reduces bubble entry into the frag-mentation stream [11].  Figure 2: Top-view schematic drawing of fragmentation constriction with length L and width d. Two types of device structures have been designed and fabricated: series constrictions and parallel chan-nels. The fragmentation devices are fabricated entirely from glass (Borofloat) substrates. A single lithography mask is used to define the planar channel and constric-tion structures and etched in the glass substrate using reactive ion etching (Adixen AMS100DE). A second glass capping substrate is then patterned and the inlet and outlet through-wafer holes are created using pow-der-blasting with 10 µm diameter alumina particles. The glass wafers are then thermally bonded and subse-quently diced into 1 × 3 cm2 size chips. Figure 3 shows a scanning electron microscopy (SEM) image of a constriction region. The depth of the channels is 5 µm.  Figure 3: SEM image of a fragmentation constriction region. Figure 4a shows optical microscopy images of com-pleted glass microfluidic chips used for DNA fragmen-tation. The lower image shows an example a single fragmentation channel with multiple constrictions and multiple channels with constrictions. Figure 4b shows an example of various fragmentation chip designs.      Figure 4: (a) Top: A Borofloat glass fragmentation chip (1×3 cm2). Bottom: series and parallel fragmentation channels. (b) Fragmentation chips with varying constriction dimensions. microchannel inlet outlet constrictions DNA motion (a) (b) series parallel MME2010 Workshop 125Back to the ProgrammeThe fragmentation chip was clamped into a custom-made chip-holder and connected to a gas-tight syringe (Hamilton) driven by a syringe pump (Harvard PHD2000) [14]. Sheared DNA samples were collected into a transparent cuvette. A schematic drawing of the DNA fragmentation setup is shown in Figure 5. The samples were then separated and analyzed using a chip-based automated gel electrophoresis system (Experion, Bio-Rad). The fluid flow rate and constriction size determines the final DNA fragment sizes. Passing the DNA through the serial constrictions reduces the vari-ability of the fragmented length and the parallel con-striction design prevents clogging.  Figure 5: Schematic setup for DNA fragmentation.  III - Results and Discussion  A. Effect of constriction channel structure  Microchannel clogging is a common problem for microfluidic devices. A device with a series of constric-tions has been expected to reduce the fragmented DNA size distribution and parallel channels to eliminate clogging [11,15]. In order to study the effect of the constriction channel structure on clogging, we have designed two types: series and parallel channels, as shown in Figure 4a. We have tested the chips with single and serial constriction channels with different channel size and number as show in Figure 4b. All constrictions with single or several serial channels had clogging problems during the experiments. When any constriction in the series channel was clogged, fluid is not able to pass through the channel and experiments have to be terminated. The parallel constriction channels eliminate the clogging problem (Figure 4a). When a dust particle of organic debris enters the main channel into the constric-tions then a single constriction can be clogged, however, fluid could still pass through the unclogged constriction channels to the outlet. In this case it is important to have a large number of parallel constriction channels so that one or two clogged channels does not result in a large change in the pressure drop ∆P across the chip. The chip with parallel channel constriction can be used repeatedly without obvious change in performance, which makes this type of chip very important for reli-able fragmentation performance. Therefore, the experi-mental results from the parallel constriction channel design are presented in this paper.  B. Effect of fluid flow rates (pressure)  The effect of fluid flow rates has been studied by varying the applied fluid flow rates in the same micro-fluidic device. A syringe pump was used to pump fluids through the chip. The chip was first flushed by DI water (dH2O) for 15 minutes. The DNA sample (50 kbp genomic DNA, 40 ng/μL in dH2O) was then pumped through the chip and collected in a transparent cuvette. The DNA sample was then transferred and analyzed using the Experion 12K DNA analysis system. Figure 6 shows representative electropherograms of DNA sam-ples sheared at different flow rates. It indicates a clear shift towards smaller fragment sizes as flow rate is increased.   Figure 6: Electropherograms of sheared DNA samples at different flow rates. The fragment size decreases with increas-ing flow rate. The constriction channel depth, width and length are 3.8, 10 and 1000 µm, respectively. The results shown in Figure 7 were obtained using the same device. In Figure 7, we summarize experimental results of DNA fragment sizes varying with applied pressure using a chip with parallel constrictions (10 parallel constriction channels, channel depth, width and length are h=3.8 μm, d=10 μm and L=1000 μm, respectively) at room temperature. Here, we use pressure instead of fluid flow rates to eliminate the effect of channel di-mensions and directly show the pressure requirement for experimental performance. The pressure difference between a microfluidic channel inlet and outlet is calculated using the Hagen-Poiseuille equation:  4dLQCPη=Δ ,  MME2010 Workshop 126Back to the Programmewhere C is a geometrical constant, η is the fluid dy-namic viscosity (Pa s); L is the channel length (m), Q is the volumetric sample flow rate (m3/s), and d is the characteristic dimension of the microfluidic device (m). The average fragment length was calculated by dividing the sum of the raw intensity values by the sum of the relative population values. The relative population of each fragment length was calculated by dividing the raw intensity values with their corresponding base pair number.  Figure 7: DNA fragment length (FL) as a function of ∆P at room temperature. The pressure difference along fragmenta-tion channels is calculated using Hagen-Poiseuille equation. As seen in Figure 7, the fragmented DNA length de-creases as the pressure is increased. The DNA sample can be fragmented to 10 kbp-6 kbp in a pressure range of 1-7 Bar using the current device, thus demonstrating that this method is useful for integration with other steps for on-chip sample pre-treatment (e.g. DNA extraction) or post-treatment (e.g. DNA separation and hybridiza-tion).  Acknowledgement  The authors gratefully acknowledge the Framework 7 European Union project TheraEDGE for financial support.  References  [1] P. N. Hengen, Trends in Biochemical Sciences, 22(7), 273-274, 1997. [2] S. Anderson, Nucleic Acids Research, 9, 3015-3027, 1981. [3] P. M. Slocombe, Heredity, 36(Apr), 289-289, 1976. [4] P.L. Deininger, Analytical Biochemistry, 135, 247-263, 1983. [5] B. M. Dancis, Biophysical Journal, 24(2), 489-503, 1978. [6] C. M. Okpodu, D. Robertson, W. F. Boss, R. K. Togasaki, and S.J. Surzycki, BioTechniques, 16, 154-159, 1994. [7] M. L. Wu, S. S. Freitas, G. A. Monteiro, D. M. F. Prazeres and J. A. L. Santos, Biotechnology and Applied Biochemistry, 53, 237-246, 2009. [8] P.J. Oefner, S.P. Hunicke-Smith, L. Chiang, F. Dietrich, J. Mulligan, and R.W. Davis. Nucleic Ac-ids Research, 24, 3879-3886, 1996. [9] Y. K. Lentz, L. R. Worden, T. J. Anchordoquy and C. S. Lengsfeld, Journal of Aerosol Science, 36(8), 973-990, 2005. [10] Y. R. Thorstenson, S. P. Hunicke-Smith, P. J. Oefner and R. W. Davis, Genome Research, 8(8), 848-855, 1998. [11] J. Lichtenberg, L. Ceriotti, N. A. Lacher, S. M. Lunte, N. F. D. Rooij, and E. Verpoorte. in mTAS2003, Squaw Valley, California USA, 172-174, 2003. [12] L. F. Cavalieri and B. H. Rosenberg, Journal of the American Chemical Society, 81(19), 5136-5139, 1959. [13] P. F. Davison, Proceedings of the National Acad-emy of Sciences of the United States of America, 45(11), 1560-1568, 1959. [14] L. L. Shui, F. Mugele, A. van den Berg and J. C. T. Eijkel, Applied Physics Letters, 93(15), 153113, 2008. [15] A. Joneja and X. H. Huang, Biotechniques, 46(7), 553-556, 2009.  MME2010 Workshop 127

High-throughput on-chip DNA fragmentation

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High-throughput on-chip DNA fragmentation

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