Addressable Electric Fields for Size-Fractioned Sample Extraction in Microfluidic Devices Rongsheng Lin,†,‡ David T. Burke,§ and Mark A. Burns*,†,| Department of Chemical Engineering, Department of Human Genetics, Department of Biomedical Engineering, and Department of Electrical Engineering and Computer Science, The University of Michigan, Ann Arbor, Michigan 48109-2136 Fraction collection following electrophoresis is of major importance for a variety of biological analyses. These assays typically need to identify specific fractions in the separated sample for further processing and require extraction of one or a group of fragments. In this paper, we have developed and characterized a technique to generate addressable electric fields for improved extraction during electrophoresis in microfluidic devices. The addressable electric field is achieved by applying a low bias voltage (1-2 V) to microelectrode pairs within the electrophoresis microchannel. Theoretical analysis shows the purity of the extracted sample can be improved as much as 30% over extraction without the shaped electric fields, and nearly 100% predicted yield can be achieved. We also describe the theoretical design of shaped electric fields by characterizing the optimal electrode geometry, field strength, channel configuration, and electrophoretic migration behavior needed for efficient band extraction. Electric field-mediated separation is of major importance for analyzing biological samples, such as DNA and protein.1,2 Electrophoresing sample molecules through a sieving matrix allows separation of species on the basis of size and charge and thus provides a means to perform efficient separations on a routine basis. Electrophoresis has been widely used for the sequencing of genomes, DNA fingerprinting, identification of pathogens, and numerous genetic assays used to identify diseases.3 Integrating electrophoresis with fraction collection further extends the power of the separation technique. Commonly, fractionated DNA populations are isolated from a slab gel by cutting out pieces containing the bands or by redirecting the eluting fractions into individual vials in a capillary electrophoresis system.4 The isolated samples then can be recovered for further * To whom correspondence should be addressed. Phone: (734) 764 4315. Fax: (734) 763 0459. E-mail: [email protected]. † Department of Chemical Engineering. ‡ Department of Electrical Engineering and Computer Science. § Department of Human Genetics. | Department of Biomedical Engineering. (1) GaaI, O.; Vereczkey, L.; Medgyesi, G. Electrophoresis in the separation of biological macromolecules; John Wiley & Sons: New York, 1980; pp 11-18. (2) Westermeier, R. Electrophoresis in practice, 3rd ed.; Wiley-VCH: New York, 2000; pp 1-32. (3) Andrews, A. T. Electrophoresis theory, techniques, and biochemical and clinical applications, 2nd ed.; Oxford University Press: New York, 1986. (4) Richwood, D.; Hames, B. D. Gel Electrophoresis of Nucleic Acids: A Practical Approach, 2nd ed.; Oxford University Press: New York, 1990. 10.1021/ac048132o CCC: $30.25 Published on Web 00/00/0000 analysis, such as dot-blot assays.5 Capillary electrophoresis has also been used to collect multiple fractions corresponding to denatured DNA from mutated polymerase chain reaction (PCR) products, and the collected fractions were reamplified by PCR and reanalyzed by electrophoresis.6 More recently, a capillary array electrophoresis system with multiple fraction collections was reported for analysis of the yeast genomic DNA.7 Several other examples of capillary-based fraction collector for DNA fragments,8-10 peptides,11 and protein12 have also been reported. By allowing the electrophoretic separation integrated with fraction collections on a single and compact format, microfludic devices are poised to offer an alternative to perform biological assays with performance comparable to the macroscale counterparts.13-15 Microfabricated systems offer more controllable transport of samples through manipulating the electric field employed in the separation and fraction collection. Electrokinetic techniques in which samples are introduced into the separation matrix using an applied electric field in the order of several hundred volts per centimeter have been widely used in microfabricated electrophoresis devices.16-27 The separated samples can be further (5) Cohen, A. S.; Najarian, D. R.; Paulus, A.; Guttman, A.; Smith, J. A.; Karger, B. L. Proc. Natl Acad. Sci. U. S. A. 1988, 85, 9660-9663. (6) Kuypers, A. W. H. M.; Willems, P. M. W.; Van der Schans, M. J. J. Chromatogr., A 1993, 621, 149-156. (7) Berka, J.; Rultz-Martinez, M. C.; Hammond, R.; Minarik, M.; Foret, F.; Sosic, Z.; Kleparnik, K.; Karger, B. L. Electrophoresis 2003, 24, 639-647. (8) Ekstrom, P. O.; Wasserkort, R.; Minarik, M.; Foret, F.; Thilly, W. G. BioTechniques 2000, 29, 582-589. (9) Irie, T.; Oshida, T.; Hasegawa, H.; Matsuoka, Y.; Li, T.; Oya, Y.; Tanaka, T.; Tsujimoto, G.; Kambara, H. Electrophoresis 2000, 21, 367-374. (10) Magnusdottir, S.; Heller, C.; Sergot, P.; Viovy, J. L. Electrophoresis 1997, 18, 1990-1993. (11) Boss, H. J.; Rohde, M. F.; Rush, R. S. Anal. Biochem. 1995, 231, 123-129. (12) Guttman, A.; Cohen, A. S.; Paulus, A.; Karger, B. L.; Rodriguez, H.; Hancock, W. S. In Electrophoresis ′88; Shafer-Nielsen, C., Ed.; VCH Publishers: New York, 1998; p 51. (13) Ugaz, V. M.; Elms, R. D.; Lo, R. C.; Shaikh, F. A.; Burns, M. A. Philos. Trans. R. Soc. London Ser. A 2004, 362 (1818), 1105-1129. (14) Kan, C.; Fredlake, C. P.; Doherty, E. A. S.; Barron, A. E. Electrophoresis 2004, 25, 3564-3588. (15) Paegel, B. M.; Blazej, R. G.; Mathies, R. A. Curr. Opin. Biotechnol. 2003, 14 (1), 42-50. (16) Emrich, C. A.; Tian, H. J., Medintz I. L., Mathies, R. A. Anal. Chem. 2002, 74, 5076-5083. (17) Vazquez, M.; McKinley, G.; Mitnik, L.; Desmarais, S.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 2002, 74, 1952-1961. (18) Mitnik, L.; Carey, L.; Burger, R.; Desmarais, S.; Koutny, L.; Wernet, O.; Matsudaira, P.; Ehrlich, D. Electrophoresis 2002, 23, 719-726. (19) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 66, 11107-1113. (20) Fu, L. M.; Yang, R. J.; Lee, G. B. Anal. Chem. 2003, 75, 1905-1910. (21) Harrison, D. J.; Manz, A.; Fan, Z.; Luedi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. Analytical Chemistry A © xxxx American Chemical Society PAGE EST: 9.8 collected by directing the desired sample zones to the corresponding wells. The addition of a cross-channel allowed the potentials to be reconfigured so that a precise collection of spatially close consecutive bands could be facilitated.28 More controllable transport of analyte was achieved in a three-dimensional fluidic network by employing nuclear track-etched polycarbonate membranes.29 More recently, on-chip electrodes have been employed in the microchannels to extract one or a group of DNA fragments of interest during the separation.30 In addition to performing on-chip fraction collection by electrokinetic manipulation, a new concept with quantitative wholecolumn fraction collection has been developed.31 The CD-like platform uses centrifugal force to move the liquid in the microchannels. The serpentine shape of the separation channel can create segments for fraction collection at each turn. The centrifugal force then can drive the separated fractions simultaneously into individual turns. By allowing whole-column fraction collection, this concept offers a new method to increase the collection efficiency in a parallel manner. We present a technique for generating addressable electric fields to improve extraction performance. Applying finite potentials onto the shaping electrodes modulates the electric field in the vicinity of the extraction region. Using experiments and computational simulations, we show that localized addressable electric fields with tunable control of the shape can be easily constructed. Next, we demonstrate the theoretical study of utilizing shaped electric fields to achieve high-purity extraction. The electric field can be easily and precisely manipulated by adjusting the magnitude of the applied voltage, the electrode positions, or both. This technique is evaluated in terms of the purity of the extracted band, and details on designing such a system are discussed. MATERIALS AND METHODS Modeling and Simulation. The time-dependent concentration field is governed by32-34 ∂C + (µ ‚E B ‚∇)C ) D∇2C ∂t (1) Here C is the concentration of the sample and D is the dispersion coefficient. The use of dispersion coefficient is more accurate in predicting band migration since diffusional band broadening alone (22) Zhang, C. X.; Manz, A. Anal. Chem. 2001, 73, 2656-2662. (23) Jacobson, S. C.; Hergenroder, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132. (24) Effenhauser, C. S. Anal. Methods Instrum. 1993, 1, 172-176. (25) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18-22. (26) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642. (27) Fu, L. M.; Yang, R. J.; Lee, G. B.; Liu, H. H. Anal. Chem. 2002, 74, 50845091. (28) Khandurina, J.; Chovan, T.; Guttman, A. Anal. Chem. 2002, 74, 1737-1740. (29) Kuo, T.-C.; Cannon, D. M.; Chen, Y.; Tulock, J. J.; Shannon, M. A.; Sweedler, J. V.; Bohn, P. W. Anal. Chem. 2003, 75, 1861-1867. (30) Lin, R. S.; Burke, D. T.; Burns M. A. J. Chromatogr., A 2003, 1010, 255268. (31) Speˇsˇny´, M.; Foret, F. Electrophoresis 2003, 24, 3745-3747. (32) Fu, L. M., Yang, R. J.; Lee, G. B. Anal. Chem. 2003, 75, 1905-1910. (33) Tsai, C. H.; Yang, R. J.; Tai., C. H.; Fu, L. M. Electrophoresis 2005, 26, 674-686. (34) Radko, S. P.; Weiss, G. H.; Chrambach, A. J. Chromatogr., A 1997, 781, 277-286. B Analytical Chemistry B is the electric underestimates the observed band broadening.35 E field and µ is the apparent mobility of the band, a combination of electrophoretic and electroosmostic mobility. The externally applied electric field is given by E B ) -∇φ (2) where φ is the electrical potential. For electrophoresis in microchannels, we assume steady electric field, uniform fluid density, and uniformly charged solid surface. For typical conditions (1.0× Tris-Borate-EDTA buffer, TBE) used in the electrophoresis, the Debye length is estimated on the order of 10 nm and therefore is much smaller compared to any channel dimension (several tens of micrometers). Under these restrictions, the electric field potential outside the Debye layer is governed by the Laplace equation:36 ∇2φ ) 0 (3) The es 1-3 were solved simultaneously in a geometry of crosschannel configuration in order to obtain the band migration and electric field distribution. Two-dimensional simulations were performed using Femlab software, a commercial finite element package (COMSOL Inc. Burlington, MA). As the channel width and length is much larger than the depth, 2-D simulations offer sufficient information to probe the feature of the transport process without requiring considerable amounts of computation time. The voltages at the two reservoirs were chosen to match the electric field strength in the experiments, and the two cross-channel potentials were allowed to float. The externally applied electric field was subject to insulating boundary conditions on dielectic microchannel walls; i.e., ∂φ/∂n ) 0. A condition of zero mass flux was applied to the microchannel walls. Other parameters used in the simulations were based on the values measured in the experiment: D ) 10-11 m2/s, µ ) 10-9 m2 s-1 V-1. The initial concentration of the band (C (t ) 0)) was Guassian distribution with σ2 ) 500 µm2 unless specified individually. Device Construction. Detailed fabrication protocols for hybrid glass-silicon devices have been published elsewhere.30 Briefly, the silicon wafer was spin-coated with a positive photoresist (Microposit SC 1827, Marlborough, MA) and patterned with the microelectrodes. After the pattern was developed (MF-319 developer; Shipley Co., Marlborough, MA), a 300-Å thick titanium metal layer followed by a 1000-Å platinum layer was deposited on this wafer by electron beam evaporation. The photoresist and the overlying metal layers were lifted off using acetone, leaving only the microelectrodes. Fabrication of channels in the glass wafer used a wet etching process. First, metal layers of 600-Å chromium followed by 4000-Å gold were deposited on a borofloat glass wafer (500 µm thick, 100-mm diameter). A positive photoresist (Microposit SC 1827; Shipley) was spin-coated, patterned, and developed. The metal layers were etched in a commercial gold etchant (Gold Etchant TFA, Transene Co.) and chromium etchant (CR-14, Cyantek Corp., Fremont, CA), respectively, leaving glass exposed in the locations where the channel network was to be etched. The (35) Ugaz, V. M.; Burke, D. T.; Mastrangelo, C. H.; Burns, M. A. Electrophoresis 2002, 23, 2777-2787. (36) Griffiths, S. K.; Nilson, R. H. Anal. Chem. 2000, 72, 5473-5482. Figure 1. (a) Schematic drawings (top view) depicting the microchannel layout for separation and extraction. X denotes the location of the cross-channel. (b) Simulated image showing band expansion under the normal electric field. (c) Simulated image showing the carryover as the target band is extracted under the normal electric field: main channel 1500 µm × 300 µm; side channel 300 µm × 1500 µm. Vseparation ) Vextraction ) 4 V. accessible glass was then etched in a freshly prepared solution of hydrofluoric acid (49% HF, CMOS grade; J.T. Baker). The rate of etching was 7.0 µm/min, and the etch depth was measured using a stylus surface profilometer. After etching to the desired depth, the metal layers were removed using the corresponding etchants, and the wafer was rinsed in DI water, air-dried, and ovendried at 100 °C for 20 min. The finished microchannel is 30-40 µm deep, and the width is on the order of 300 µm, depending on the design layout. After individual devices on the substrate wafers were diced, holes (300-µm diameter) were drilled in the glass substrate to access the microchannels using an in-house electrochemical discharge apparatus. The glass channel was then bonded to the silicon substrate using a UV-cured optical adhesive (SK-9, Summers Laboratories, Fort Washington, PA). Electrical connections were made by wire bonding the assembled devices to printed circuit boards. Electrophoresis Procedure. Double-stranded DNA separations were performed using 100-bp standard ladders (Bio-Rad Laboratories, Hercule, CA) labeled with YOYO-1 intercalating dye (Molecular Probes, Eugene, OR) at a ratio of 5:1 bp/dye. A 1× TBE solution (Bio-Rad) was used as running buffer. β-Mercaptoethanol (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 10% to reduce photobleaching. Separations were performed using ReproGel (Amersham Pharmacia, Kalamazoo, MI), a commercially available photopolymerized cross-linked polyacrylamide gel, which allows the gel interface to be precisely positioned inside the electrophoresis channel.37 An initial polymerization period of 3 min was used to set the gel interface, after which the interface mask and accompanying unpolyermized solution were removed. The masked region and reservoirs were then refilled with electrophoresis buffer, and UV polymerization was allowed to continue for an additional 5-10 min. The sample (S), sample waste (SW), and buffer (B) reservoirs were filled with the fluorescent sample (Figure 1a). The waste reservoir (W) was filled with the TBE running buffer. Samples were loaded by applying a +70-V potential at the W reservoir and grounding the B reservoir for ∼10 s (E ∼70 V/cm). After a sample plug was formed near the gel interface, the rest of the sample was removed and replaced with TBE running buffer. To perform the separation, +30 V was applied at the W reservoir while grounding the B reservoir. Both the S and SW reservoirs were allowed to electrically float during separation. SC1 and SC2 reservoirs were used to collect the target sample. The shaping voltage is applied on the shaping electrodes using a floating voltage source. Care has to be taken to avoid bubble formation by limiting the voltage to ∼2 V.38 Introduction of wider shaping electrodes allows slightly higher voltages (∼3 V) to be safely applied for several tens of seconds without bubbling. Fluorescence from the migrating bands was detected using an Olympus SZX 12 fluorescence stereoscope with a mercury arc illumination source and imaged using a Hamamatsu C2400-08 SIT camera (Hamamatsu Corp., Bridgewater, NJ). (37) Brahamasandra, S. N.; Ugaz, V. M.; Burke, D. T.; Mastrangelo, C. H.; Burns, M. A. Electrophoresis 2001, 22, 300-311. (38) Petrucci, R. P. General Chemistry; MacMillan: New York, 1982. Analytical Chemistry C Figure 2. (a) Schematic drawings depicting the separation phase and the generation of the shaped electric field for reduction of band expansion. Vshaping is the voltage bias applied on the shaping electrodes. Lg is the gap between the symmetric electrodes, and Lp is the distance the shaping electrode away from the separation channel. Migration direction is from left to right. Cross-hatch area denotes the target, and 9 denotes the neighboring bands. (b) Simulated image showing the reduction of the band expansion under a single shaped electric field. Main channel 1500 µm × 300 µm; side channel 300 µm × 1500 µm. Vseparation ) 4V. Lp ) 50 µm. Lg ) 200µm. V+⊥ ) 2.5 V, V-⊥ ) 1.5 V. (c) Schematic drawings depicting the extraction phase and the generation of the shaped electric field to reduce the carryover from the neighboring bands. WB denotes the full width of the target band. WCC and WMC denote the width of the cross-channel and the separation channel. Migration direction is from down to up. (d) Simulated image showing the carryover reduction through a second shaped electric field: main channel 1500 µm × 300 µm; side channel 300 µm × 1500 µm. The shaping electrodes (V+II and V-II) are placed 50 µm away from the extraction channel and are 200 µm away from each other. Vextraction ) 4 V, V+II ) 2.5 V, and V-II ) 1.5 V. Particle Imaging Setup. The flow field was observed using 1-µm Fluoresbrite plain YG microspheres (Polyscience Inc.). These particles have an excitation peak at 441 nm and emission peak at 486 nm. The original concentration of fluorescent microspheres was 4.55 × 1012 particles/mL and was diluted to 4.55 × 1010 particles/mL. These particles were introduced into the microchannel by capillary action, and the experiment began when the flow stabilized. The imaging system consists of a Zeiss epifluorescent microscope with illumination provided by a mercury lamp. A Nikon 10× objective lens is used for magnification of the images so that the field of view covered the whole cross-channel. The images were recorded using a Hamamastu C2400 CCD camera with 512 × 512 pixels and 12-bit readout resolution and a DVD recorder. The correlated particle images obtained were analyzed with a PIV interrogation code.39 In the PIV image process, the interrogation cell overlay was given as 50%. Ensemble (39) http://sauron.civil.eng.osaka-cu.ac.jp/∼mori/softwares/mpiv/index.php. D Analytical Chemistry averaging of up to 10 images consecutively captured at a rate of 30 fps was used to obtain the velocity measurements. RESULTS AND DISCUSSION Band Extraction and Shaped Electric Fields. The addition of an electric field perpendicular to the separation direction is the technique used to extract migrating bands from a microchannel. A cross-channel is positioned perpendicular to the separation channel in order to generate the electric field for extraction (Figure 1a). The extraction procedure includes two stages. The first stage involves electrophoresis of the size-fractioned sample through a sieving matrix in the separation channel. After the target fragment is identified from the band pattern, the separation field is stopped and the second stage begins. The capture electrodes in the cross-channel are energized to extract the target band out and finally release it into the buffer reservoir. The simulation results indicate that the impurity inherent in a simple cross-channel configuration is unlikely to be completely eliminated because the band expands laterally into the crosschannel as it reaches the intersection (Figure 1b). A second band can easily expand and come into contact with the end of the first band. In addition, the carryover from the adjacent bands occurs as the target band in the intersection is extracted into the crosschannel (Figure 1c). Both the expansion and carryover are the contributing sources to the impurity in this process. Addition of electrodes in the intersection offers the ability to adjust the electric field shape in favor of reducing the lateral expansion (Figure 2a). By energizing the shaping electrodes with a bias voltage (Vshaping), the bands can be confined in the separation channel and reduce band expansion (Figure 2b). While the lateral expansion can be minimized using shaped electric fields, there is still carryover from the adjacent bands as the target band in the intersection is captured into the extraction channel. The shaped electric field can also be applied in the course of extraction to isolate the target band from its neighbors (Figure 2c). By activating the shaping electrodes in the separation channel with a voltage bias (Vshaping), the shape of the electric field in the vicinity of the intersection can be tuned to reduce the carryover. Isolation between the target and the neighboring band is clearly demonstrated in the simulation (Figure 2d). For the purpose of quantification, we define the yield and purity as ∫∫ Ci dA upper cross-channel Yi ) M0 (4) P1 ) ∫∫ C1 dA upper cross-channel ∫∫ upper cross-channel C1 dA + ∫∫ C2 dA upper cross-channel (5) Here Yi is the yield of the band (1 stands for the target and 2 denotes the neighboring band); Ci is the concentration of the band; M0 is the initial band mass and can be calculated by integrating the concentration over the entire channel area. P1 is the purity of the target band: P1 ) Y1/(Y1 + Y2) for the case of the bands (C1, C2) having the same initial band mass. These definitions only consider a simple case with one neighboring band and could be extended to other situations, such as two adjacent fragments. Shaped electric fields greatly improve the band extraction. Figure 3 shows the computed purity (a) and yield (b) of the target band as a function of extraction time. The purity under shaped electric fields can reach 96% in a very short time (40 s) after extraction began, whereas it has a maximum value around 66% under a normal electric field. Figure 3b compares the yield in the case with and without shaped electric field. The yield under shaped electric fields increases almost linearly with time at a rate of 0.0073 s-1 after extraction began and can reach 96% in 150 s, whereas the rate is only 0.0045 s-1 and the yield goes up to 84% only for the case in the absence of the shaped electric field. However, the purity starts to decrease after 40 s due to carryover from the neighboring bands (Figure 3a single electric field). Figure 3. (a) Computed purity as a function of time during extraction under (I) normal electric field, (II) single shaped electric field, and (III) double shaped electric field. (b) Computed yield as a function of time during extraction under (I) normal electric field, (II) single shaped electric field, and (III) double shaped electric field. The channel geometry is the same as the one used in Figure 2. Main channel 1500 µm × 300 µm; side channel 300 µm × 1500 µm. The shaping electrodes (V+⊥ and V-⊥) are placed 50 µm away from the separation channel and are 200 µm away from each other. Vseparation ) 4 V, V+⊥ ) 2.5 V, and V-⊥ ) 1.5 V. The other shaping electrodes (V+II and V-II) are placed 50 µm away from the extraction channel and are 200 µm away from each other. Vextraction ) 4 V, V+II ) 2.5 V, and V-II ) 1.5 V. Formation of another shaped electric field can minimize the carryover. After the shaping, electrodes in the separation channel are energized (Figure 3a double electric field), and the purity remains almost constant once it reaches 96%. Experimental Results. Based on the simulation, we fabricated a silicon-glass hybrid device (1.3 cm × 0.7 cm) as shown in Figure 4a. The device contains an offset double-T for sample injection, separation channel, and cross-channel for extraction. A close-up micrograph of the intersection is shown in Figure 4b. Note that there are some shadows along the edge of the microchannels, resulting from undercut during the glass etching process. The anisotropic nature of the process generates a channel cross section with a trapezoid shape instead of rectangle. Generating an addressable electric field requires the appropriate setting of potentials at each shaping electrodes. The potentials will be redistributed after a bias voltage is applied on the shaping electrodes. By applying Vshaping, on the shaping electrodes, the Analytical Chemistry E Figure 4. (a) Photograph of a microfabricated device used for separation and extraction (b) a close-up of the intersection with the shaping electrodes. potential at each electrode (V+, V-) can be tuned: V+ increases whereas V- decreases linearly with the shaping potential. Electric Field Distribution. The shaped electric field distribution was measured using particle tracking techniques, and the experiments agreed with the simulations. The use of microparticle image velocimetry (PIV) enables spatially resolved measurements of instantaneous velocity fields and, therefore, has been widely used to characterize the pressure-driven flow or electroosmosis flow in microchannels.40-42 In this work, we chose 1-µm negative charged fluorescent latex microspheres to visualize the movement under the shaped electric field. Figure 5 shows a comparison between the flow field from the particle tracking experiment and the corresponding electric field predictions. The velocity is proportional to the electric field (v b ) µ‚E B), and the results between the experiment and simulation are similar. We also note that the field is more curved in the vicinity of the electrodes in the simulation. The motion of trace particles in the third dimension in the experiment may cause the discrepancy. Nevertheless, these results qualitatively verify the formation of shaped electric fields. Band Migration. The band migration without shaped electric fields was first demonstrated experimentally using a single size DNA fragment. A 400-base pair double-stranded DNA sample was loaded into the gel and migrated through the intersection (Figure 6aI). As a comparison, we simulated the initial band using a Gaussian distribution (Figure 6bI). The comparison shows a very good agreement in terms of band shape. The simulation results (bII) show the similar expansion of a migrating band, resulting from cross-channel configuration to the experiment data (aII). As the band migrates forward leaving the junction, the expanded portions are left behind in both the experiment and simulation (aIII,bIII). The similar experiment and simulation was performed to demonstrate band migration under the shaped electric field. After the shaping electrodes are energized, the band migrates in a (40) Meinhart, C. D.; Wereley, S. T.; Santiago, J. G. Exp. Fluids 1999, 27, 414419. (41) Devasenathipathy, S.; Santiago, J. G. Anal. Chem. 2002, 74, 3704-3713. (42) Devasenathipathy, S.; Santiago, J. G. Exp. Fluids 2003, 34, 504-514. F Analytical Chemistry Figure 5. (a) Velocity field extracted from the particle tracking images Vshaping ) 2 V. (b) Simulation of the shaped electric field distribution under the similar condition (Vshaping ) 2V). There is no voltage spanning the separation channel. confined region with limited expansion into the cross-channel (Figure 7(aII,III). The simulation results (Figure 7bII,III) show the similar band migration behavior, indicating that the model can accurately describe experimental behavior. The difference on the level of curvature between experiment and simulation is most Figure 6. (a) Experimental snapshot of (I) the initial status of a migrating band, (II) band migrating in the junction, and (III) band leaving the junction. Eseparation ) 25 V/cm, (b) Simulated image of (I) initial concentration (C(t ) 0) ) Guassian distribution with σ2 ) 500 µm2), (II) band migrating in the junction, and (III) band leaving the junction under the simulation condition similar to the experiment: Vseparation ) 4 V corresponding to Eseparation around 27 V/cm, Migration direction is from left to right. likely due to the nonuniformity of the initial band observed in the experiment. Designing Shaped Electric Fields. We have demonstrated shaped electric fields both theoretically and experimentally. However, challenges still remain in terms of designing such electric fields that are capable of reducing band expansion and carryover. For the purpose of reduction of band expansion, the shaping electrodes arrangement (e.g., size and relative position to the intersection) is of major importance. On the other hand, the channel geometry and band migration are more crucial in reducing carryover during band extraction. Position and Size of the Shaping Electrodes (Lp, Lg). The shaped electric field for reduction of band expansion is affected by several parameters (Figure 2a). These include the distance of the shaping electrodes away from the main separation channel (Lp), the gap between two symmetrical electrodes (Lg), and the applied shaping voltage (Vshaping and Vseparation). To characterize the band expansion, we define the retained band mass as ∫∫ MR separation channel retained band mass ) ) M0 Mo C dA (6) Figure 7. (a) Experimental snapshot of (I) the initial status of a migrating band, (II) band migrating in the junction, and (III) band leaving the junction in the experiment Eseparation ) 25 V/cm, Vshaping ) 1 V. (b) Simulated image of (I) initial concentration (C(t ) 0) ) Guassian distribution with σ2 ) 500 µm2), (II) band migrating in the junction, and (III) band leaving the junction under the condition similar to the experiment: Vseparation ) 4 V corresponding to Eseparation around 27 V/cm, V+⊥ ) 2.5 V, V-⊥ ) 1.5 V. Migration direction is from left to right. over the entire channel area. MR is the band mass retained in the separation channel. The retained band mass defines the percentage of the band mass remaining in the separation channel as the band passes by the cross-channel. The position of the shaping electrodes (Lp) has a signficant impact on the shaping effect, whereas the gap between the electrodes (Lg) is minor. Figure 8 shows the retained band mass as a function of Lp under a set of different electrode gaps (Lg). The closer to the separation channel the shaping electrodes are placed, the less band expansion is obtained. The band migration is not sensitive to Lg and remains essentially constant once the position of the electrodes is fixed. These results imply that the shape of the electrodes has little influence on the band migration as a consequence of the minor discrepancy in the electric field distributions. The electrodes have a minimal distance of approximately 2030 µm away from the separation channel (Figure 8). The minimum distance (Lpc) can be estimated by scale analysis. The time necessary for diffusive transport (tdiff) by a distance of Lp can be estimated using the following relationship.43 tdiff ∼ Lp2/D (7) The convective transport time scale, tconv, is on the order of time Here C is the DNA concentration distribution. M0 is the initial band mass and can be calculated by integrating the concentration (43) Bird, R. B.; Stewart, W. E.; Lightfoot, E. N. Transport Pheomenona, 2nd ed.; J. Wiley: New York, 2002. Analytical Chemistry G Figure 8. Retained band mass (the lowest value) as a function of Lp at a fixed Lg ) 50 (b), 100 (1), 150 (2), and 200 µm (9). The following parameters are used for simulations: main channel 1500 µm × 300 µm; side channel 300 µm × 1500 µm, Vseparation) 4 V, V+⊥ ) 2.5 V, and V-⊥ ) 1.5 V. required to drive the band out of the intersection by Lp distance: tconv ∼ Lp/(µE) (8) As tconv must be less than tdiff, for the band being able to migrate out of the intersection, the following condition derived using the above equations has to be satisfied: Lp . Lpc. Here Lpc is the minimal distance defined by the relation Lpc ) D/(µE) (9) For typical values of diffusivity and mobility in experiments and simulations (D ∼ 10-11 m2/s, µ ∼ 10-9 m2 s-1 V-1), Lpc is on the order of 10 µm under the electric field strength of 103 V/m. Electric Field Strength (Eshaping and Eseparation). Compared to the voltages applied on the shaping electrodes and cross the separation channel (Vshaping and Vseparation), the shaping electric field strength (Eshaping) is a more intuitive factor influencing the field distribution associated with the separation field (Eseparation) in the vicinity of the cross-channel. The shaping field strength (Eshaping) can be calculated by Vshaping/WCC, where WCC is the width of the cross-channel. Because the band migration is insensitive to the electrode gap (Lg), the use of cross-channel width is more reasonable than the electrode gap. Applied separation voltage divided by the length is used to estimate the field strength for separation (Eseparation). Computational modeling shows that retained band mass increases in a manner that scales with Eshaping/Eseparation (Figure 9). To reduce the band migration laterally into the cross-channel (<5%), a value of the ratio of Eshaping/Eseparation greater than 1 has to be maintained. Since the shaping field strength is proportional to the applied voltage on the electrodes (Vshaping), higher Vshaping generates stronger shaping fields and, therefore, allows less band expansion. This plot also offers a basic guideline for generating a shaped electric field by matching the Eshaping with Eseparation. An Eshaping/Eseparation ratio of ∼1 can be achieved by adjusting the shaping voltage (Vshaping) and the cross-channel width (WCC). The H Analytical Chemistry Figure 9. Retained band mass (the lowest value) as a function of Eshaping/Eseparation. Eshaping is the shaped electric field strength and is calculated by Vshaping divided by the cross-channel width. Eseparation is the electric field strength for separation and is calculated by the voltage spanning the separation channel divided by the channel length. The following parameters are used for simulations: main channel 1500 µm × 300 µm; side channel 300 µm × 1500 µm. Vseparation) 4, 6, 8, and 10 V. Vshaping ) V+⊥ - V-⊥ ) 0.5 (1), 1 (2), 1.5 (b), and 2 V (9). Lp ) 50 µm. Lg ) 200µm. analysis indicates that a shaped electric field is achievable for separation field strengths ranging from 20 to 200 V/cm by using cross-channel widths from 50 to 500 µm (Vshaping ) 1 V). Length of Channel (X). The separation length determines how far two consecutive bands can be separated or resolved. The bandwidth (WB) affects the yield whereas the purity is influenced by both the bandwidth and resolution (R). Discussions centering on these two parameters (WB and R) are essential to determine the position of the cross-channel (X) in order to achieve a certain purity and yield. The bandwidth (WB) can be estimated using the following equation for a Gaussian distribution: WB ) 4‚xσ2inj + 2(D/µE)X (10) Here σ2inj is the peak variance of the injection plug. WB0 ) 4σinj is the full width of the injection plug. D is the dispersion coefficient. By plotting the ratio of the bandwidth (WB) over the migration distance (X) as a function of the migration distance (X) (Figure 10a), two dominant regions can be represented using two series of straight lines corresponding to different initial injection plug width (WB0) and dispersion (2D/(µE)). This plot offers the flexibility of determining the transition region between the two dominant regions by simply finding the intersection of the two lines (e.g.,WB0 ) 100 µm, 2D/(µE) ) 0.1 µm). The procedure is easy to follow and is suitable for any combination of settings of WB0 and 2D/(µE). The bandwidth is inherently related to the resolution (R) by44 R ) 2(xn - X)/(wn + WB) (11) Here xn is the migration distance of the neighboring band and wn Figure 10. (a) Ratio (WB/X) as a function of migration distance (X) under various settings of injection plug width (WB0) and 2D/µE. The dashed line interpolates the curve with a fixed plug width and dispersion (WB0 ) 100 µm, 2D/µE ) 0.1 µm). (b) Resolution (R) as a function of the ratio of bandwidth over the migration distance (WB/ X) under different selectivity settings (selectivity (S) is from 0.2 to 1.6%). is the width of the neighboring peak at the baseline. For two neighboring peaks (wn ) WB) R ) S‚(WB/X)-1 Figure 11. (a) Purity as a function of R(WB/WCC) in various WCC/ WMC settings under normal electric fields. The purity is obtained 140 s after the extraction starts. The main channel is 1500 µm × 300 µm and the cross-channel is 1500 µm long. The width of the crosschannel (WCC) and the bandwidth (WB) varies from 75 to 300 µm simultaneously to maintain the ratio of WB/WCC equal to 1. For the case that the ratio of WCC/WMC maintains 0.5, WB/WCC ) 0.5 (1), 1 (2), and 1.5 (b). (b) Purity as a function of R(WB/WCC) in various WCC/WMC settings under shaped electric fields. The channel geometry is the same as those in (a) except that extra electrodes are placed aligning the cross-channel. The distance between electrodes are 200 µm. Vextraction ) 4 V, V+II ) 2.5 V, and V-II ) 1.5 V. (12) Here S ) ∆µ/µ is the selectivity of two neighboring bands. Figure 10b shows the predicted resolution as a function of the ratio of bandwidth over migration distance (WB/X) under different selectivity settings. High resolution requires a small ratio of WB/X, which can be realized either by prolonging the separation length (X) or by narrowing the bandwidth (WB). Figure 10 offers a simple means to estimate the separation distance (X) to achieve a certain resolution and bandwidth needed for high purity/yield extraction. Channel Dimensions (WCC, WMC). The simulation results show that the purity of the isolated band is a function of resolution (R), migrating bandwidth (WB), cross-channel width (WCC), and main channel width (WMC). R and WB are parameters associated with the band migration, whereas WCC and WMC are microchannel (44) Landers, J. P. Handbook of Capillary Electrophoresis, 2nd ed.: CRC Press: New York, 1995. geometry related (Figure 2c). To make the design process scalable in channel geometry, we computed purity as a function of the multiplication of resolution and WB/WCC under various fixed settings of WCC/WMC (Figure 11a). We found that, for a range of WB/WCC (0.5-1.5), all the curves can collapse into one with WB/ WCC equal to 1 (Figure 11a, WCC/WMC ) 0.5). This approximation offers a simple means to capture the essential features of the problem and provides valuable insights into the scaling of the system. A similar model using shaped electric fields can also be generated (Figure 11b). Purity improvement is observed through shaping the electric field to isolate the target band from its neighbors. The ability to scale down offered by these two plots makes them suitable to design the channel geometry in terms of purity and resolution under both normal electric fields and shaped electric fields. For instance, there is little difference in purity between two channel dimensions (I) WB ) 100 µm, WCC ) 100 µm, and WMC ) 100 µm and (II) WB ) 300 µm, WCC ) 300 µm, Analytical Chemistry I relationship can be derived: 1 e (WCC/WB) e 2R - 1 (13) This equation suggests the minimum requirement for imposing a shaped electric field during extraction is that the consecutive bands have to be well resolved (R g 1). In practice, migrating bands are not exactly rectangular but approximate a Guassian distribution, allowing the upper and lower limits of WCC/WB to be slightly different from the derived relationship. This analysis indicates maintaining the ratio of WCC/WB around 1 is important to make full use of shaped electric fields for applications requiring both high yield and purity. Figure 12. (a) Purity as a function of resolution in various WCC/WB settings under the shaped electric field. (b) Yield as a function of WCC/ WB under the shaped electric field. WMC ) 300 µm. WB ) 150 µm. WCC varies from 75 to 300 µm corresponding to the ratio of WCC/WB from 0.5 to 2. The distance between electrodes is 200 µm. Vextraction ) 4 V, V+II ) 2.5 V, and V-II ) 1.5 V. The initial target band is in the center of the intersection. The resolution data are calculated based on the bandwidth and the distance the neighboring band is away from the target band. and WMC ) 300 µm as long as the ratio of WB:WCC:WMC remains constant. However, there is a tradeoff between the purity and yield. As the cross-channel becomes narrower compared to the bandwidth, much purer sample can be extracted. Nevertheless, the yield decreases as a result of the narrower cross-channel (Figure 12(a)(b)). Ideally, the cross-channel width should be less than the distance between two consecutive bands and greater than the bandwidth in order to maintain a high yield. The following J Analytical Chemistry PAGE EST: 9.8 CONCLUSIONS We have developed a technique using addressable electric fields to control and minimize the contamination from the adjacent bands and selectively extract target bands during electrophoresis. The precise control of electrode characteristics, such as position and size, makes microfabrication an ideal tool for the development of such a system. In this work, we theoretically characterized shaping electrode configurations and investigated band migration behavior and electric field distribution under these conditions. The use of shaping electrodes within the cross-channel resulted in improved predicted extraction selectivity and reduced carryover by tuning the localized electric field into the desired shape. The principle of this method is general and versatile and should be applicable to any system that fractionates charged molecules. The described computational models and experiments provide a foundation for designing more complex addressable electric fields using multiple electrodes for on-chip sample extraction. Future work involving fully three-dimensional simulations and rigorous comparison with experimental results would be beneficial to understand the role of localized shaped electric fields in a more quantitative manner. By allowing integration with microfluidic controls, this type of devices could add more functionalities onto the existing µTAS systems. ACKNOWLEDGMENT The authors acknowledge the support of the National Institutes of Health under the Grant NIHGRI P01-HG001984. Received for review December 17, 2004. Accepted April 22, 2005. AC048132O
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