Microchip Bioprocessor for Integrated Nanovolume Sample

A microfabricated electrophoretic bioprocessor for inte- grated DNA sequencing sample desalting, template re- moval, preconcentration, and CE analysis...
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Anal. Chem. 2002, 74, 5092-5098

Microchip Bioprocessor for Integrated Nanovolume Sample Purification and DNA Sequencing Brian M. Paegel,† Stephanie H. I. Yeung,‡ and Richard A. Mathies*,†

Department of Chemistry and UCB/UCSF Joint Bioengineering Program, University of California, Berkeley, California 94720

A microfabricated electrophoretic bioprocessor for integrated DNA sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide-copolymerized oligonucleotide complementary to the 20-base sequence directly 3′ of the M13-40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50 °C, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gelpurified duplex at 67 °C and directly injecting onto a 15.9cm effective length CE microchannel. Electrophoretic resolution of the sequencing products is complete in 32 min, producing a total of 560 bp with phred quality q g 20 (accuracy g99%). This fully integrated nanoliter process decreases the purification time ∼10-fold and the process volume ∼100-fold while providing state-of-theart sequencing results. The rapid sequencing of the human genome was made possible in part through innovations in analytical technology.1-3 Although capillary array electrophoresis (CAE)-based instrumentation provided the analytical foundation for current production sequencing efforts,4-7 it is clear that additional improvements in the integration and cost-effectiveness of DNA sequencers are needed to advance the goals of high-throughput biology. The currently deployed CAE instruments do not meet these needs * To whom correspondence should be addressed. Phone: (510) 642-4192. Fax: (510) 642-3599. E-mail: [email protected]. † Department of Chemistry. ‡ UCB/UCSF Joint Bioengineering Program. (1) Collins, F. S.; Patrinos, A.; Jordan, E.; Chakravarti, A.; Gesteland, R.; Walters, L.; Fearon, E.; Hartwelt, L.; et al. Science 1998, 282, 682-689. (2) Lander, E. S.; Linton, L. M.; Birren, B.; Nusbaum, C.; Zody, M. C.; Baldwin, J.; Devon, K.; Dewar, K.; et al. Nature 2001, 409, 860-921. (3) Dovichi, N. J. Electrophoresis 1997, 18, 2393-2399. (4) Mathies, R. A.; Huang, X. C. Nature 1992, 359, 167-169. (5) Kambara, H.; Takahashi, S. Nature 1993, 361, 565-566. (6) Kheterpal, I.; Scherer, J. R.; Clark, S. M.; Radhakrishnan, A.; Ju, J. Y.; Ginther, C. L.; Sensabaugh, G. F.; Mathies, R. A. Electrophoresis 1996, 17, 18521859. (7) Crabtree, H. J.; Bay, S. J.; Lewis, D. F.; Zhang, J. Z.; Coulson, L. D.; Fitzpatrick, G. A.; Delinger, S. L.; Harrison, D. J.; Dovichi, N. J. Electrophoresis 2000, 21, 1329-1335.

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because they depend on discrete, expensive robotic systems for sample transport and manipulation and do not capitalize on the low-volume analysis capabilities uniquely provided by CE. Microfabricated DNA sequencing systems are capable of providing a solution to this problem because capillary arrays are completely monolithic and analysis is >5-fold faster than conventional instrumentation.8-12 However, the key and largely unexplored advantage of microdevice analysis is the ability to integrate onchip sample handling and preparatory steps on the nanoliter scale. Sample preparation issues are of particular importance in the DNA sequencing process. Challenging steps include processing insert DNA from clones to template for DNA sequencing and preparing extension reactions that are suitable for electrokinetic injection on a CAE instrument.13 In 1998, Ruiz-Martinez and SalasSolano published critical investigations of the effects of DNA sequencing reaction matrix components on read length. Thorough desalting and template removal were found to be essential for maintaining read lengths of 500+ bases.14,15 These operations, referred to collectively as “sample cleanup”, require costly spincolumn purification and multiple sample transfer and centrifugation steps, all of which are deterrents for a production sequencing line. An alternative sample cleanup methodology, utilizing magnetic bead reagents and microliter-scale volumes, requires ∼40 min to complete.16 If high-speed, low-volume cleanup chemistry could be integrated into microfabricated CAE (µCAE) systems, the DNA sequencing paradigm would be dramatically improved. Previous integrated sample purification methods have centered largely on an orthogonal separation dimension such as size exclusion chromatography and capillary zone electrophoresis.17-19 (8) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680. (9) Schmalzing, D.; Adourian, A.; Koutny, L.; Ziagura, L.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 1998, 70, 2303-2310. (10) Liu, S. R.; Shi, Y. N.; Ja, W. W.; Mathies, R. A. Anal. Chem. 1999, 71, 566573. (11) Liu, S. R.; Ren, H. J.; Gao, Q. F.; Roach, D. J.; Loder, R. T.; Armstrong, T. M.; Mao, Q. L.; Blaga, I.; Barker, D. L.; Jovanovich, S. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5369-5374. (12) Paegel, B. M.; Emrich, C. A.; Wedemayer, G. J.; Scherer, J. R.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 574-579. (13) Elkin, C. J.; Richardson, P. M.; Fourcade, H. M.; Hammon, N. M.; Pollard, M. J.; Predki, P. F.; Glavina, T.; Hawkins, T. L. Genome Res. 2001, 11, 1269-1274. (14) Ruiz-Martinez, M. C.; Salas-Solano, O.; Carrilho, E.; Kotler, L.; Karger, B. L. Anal. Chem. 1998, 70, 1516-1527. (15) Salas-Solano, O.; Ruiz-Martinez, M. C.; Carrilho, E.; Kotler, L.; Karger, B. L. Anal. Chem. 1998, 70, 1528-1535. (16) Elkin, C.; Kapur, H.; Smith, T.; Humphries, D.; Pollard, M.; Hammon, N.; Hawkins, T. Biotechniques 2002, 32, 1296-1302. (17) Tan, H. D.; Yeung, E. S. Anal. Chem. 1997, 69, 664-674. (18) Tan, H. D.; Yeung, E. S. Anal. Chem. 1998, 70, 4044-4053. 10.1021/ac0203645 CCC: $22.00

© 2002 American Chemical Society Published on Web 08/31/2002

In each case, the plug containing sequencing fragments is isolated from reaction contaminants in the first separation dimension. The isolated fragments are then injected electrokinetically onto the gel column for electrophoretic resolution. The main disadvantage of these systems is their innate complexity. Coupling multiple capillary separation steps with conventional capillaries requires a complicated network of plumbing including intersections, freezethaw valves, etc. Microfabrication is ideally suited to overcome these systems integration challenges. A small number of microfabricated sample cleanup experiments have already been described in the literature. Oleschuk and co-workers recently implemented bead-based reagents for microdevice solid-phase extraction of a hydrophobic dye from aqueous solution.20 Bead-based reagents are useful for sample extraction because of their high surface area and are attractive for use in microdevices because they are potentially removable. Macroporous monoliths polymerized in situ for use in solid-phase extraction of dyes and peptides have also been successfully developed.21 Recently, researchers at NIST demonstrated two sample preconcentration methods uniquely suited to microfabricated devices: DNA-hydrogel immobilization and temperature gradient concentration.22,23 The former seems particularly promising for the purification of DNA sequencing products. Furthermore, Tian and Landers have explored a variety of silica resins for nonspecific DNA isolation and concentration.24 A thorough review of reagents, methods, and reactors for bioaffinity capture was recently presented by Guzman.25 Gel immobilization of oligonucleotides is a bioaffinity capturebased purification method for nucleic acids that couples the size selectivity of a polyacrylamide matrix with the sequence specificity of hybridization.26 We have chosen this method for sequencing sample purification as it is easily adapted to microfluidic devices and because DNA sequencing products from a given cloning vector and restriction enzyme combination all share a common intervening vector sequence. We present here a microfluidic circuit that uses an oligonucleotide capture matrix to immobilize and preconcentrate with high selectivity only the extension products of a DNA sequencing reaction. The concentrated and desalted products are then released thermally and injected onto a CE column for high-speed electrophoretic resolution. This new method capitalizes on the key advantages of microfabricated electrophoresis devices for DNA sequencing and should be useful in the development of a wide variety of integrated microchip bioprocessors. EXPERIMENTAL SECTION DNA Sequencing Sample Preparation. Four-color DNA sequencing reactions were performed using a dye primer cycle sequencing kit (Amersham Biosciences, Piscataway, NJ) and in(19) He, Y.; Pang, H. M.; Yeung, E. S. J. Chromatogr., A 2000, 894, 179-190. (20) Oleschuk, R. D.; Shultz-Lockyear, L. L.; Ning, Y. B.; Harrison, D. J. Anal. Chem. 2000, 72, 585-590. (21) Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2001, 73, 5088-5096. (22) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (23) Ross, D.; Locascio, L. E. Anal. Chem. 2002, 74, 2556-2564. (24) Tian, H. J.; Huhmer, A. F. R.; Landers, J. P. Anal. Biochem. 2000, 283, 175-191. (25) Guzman, N. A.; Stubbs, R. J. Electrophoresis 2001, 22, 3602-3628. (26) Baba, Y.; Tsuhako, M.; Sawa, T.; Akashi, M.; Yashima, E. Anal. Chem. 1992, 64, 1920-1925.

house synthesized energy-transfer (ET) primers.27 The 10-µL sequencing reaction for a single color consisted of 800 fmol of ET primer, 0.5 µg of single-stranded M13mp18 template, and 1× of the manufacturer’s premix containing polymerase, nucleotides, terminators, Mg2+, and buffering components. The reaction mixtures were thermally cycled (95 °C 15 s, 55 °C 30 s, 72 °C 60 s, 25 cycles) using a Mastercycler Gradient (Eppendorf, Westbury, NY), and the four separate primer reactions were pooled to make a four-color sample. Pooled reactions were either used as is for on-chip purification or precipitated according to kit protocols. Precipitated reaction products were resuspended in 40 µL of 50% formamide solution in DI H2O. Oligonucleotide Capture Matrix Synthesis. The replaceable, linear polyacrylamide matrix was synthesized by following previously published protocols.28 All reagents were electrophoresis grade. A 2-mL solution of 5% w/v acrylamide, 1× TTE (50 mM Tris, 50 mM TAPS free acid, 1 mM EDTA, pH 8.4), and 20 nmol of the methacrylate-modified oligo (5′-Acrydite-ACTGGCCGTCGTTTTACAA-3′, TM ) 60.4 °C; Operon Technologies, Emeryville, CA) was prepared in a 4-mL autosampler vial with Teflon closure. The solution was sparged with argon for 2 h prior to adding 0.015% w/v APS and TEMED to initiate polymerization. Upon addition of the catalyst, the sparging line was removed from the polymerizing solution and argon was flowed over the reaction mixture for an additional 2 h. All sparging and polymerization steps were carried out at 4 °C. Microdevice Fabrication and Design. The glass wafer sandwich structure was fabricated as described previously.29 All features were isotropically etched to a depth of 25 µm and all channel widths discussed here are post etch. The capture chamber design as well as the microchannel layout of the prototype integrated DNA sequencing bioprocessor is shown in Figure 1. The capture chamber is a simple flow cell with an inlet and outlet well. Each 2-µL well interfaces with a narrow arm 1 mm in length and 100 µm in width. The arm tapers out over 1 mm with an average width of 500 µm. The body of the chamber between the two tapers is 1 mm in width and length. The chamber is flared to lower the electric field strength in the capture region and to accommodate a larger volume per unit channel length. The total chamber volume is 60 nL. The identical chamber design was incorporated into a doublet injector structure based on the radial design of our first-generation µCAE DNA sequencer.12 The inlet side of the chamber is split between a capture inlet well and a coupling channel to the injection intersection (both 75 µm in width). Each doublet of chambers is fluidically addressed by common CE cathode and CE waste wells located in the center of the injector. The microchannel arms leading to the waste are 280 µm in width, and the main CE channels are 200 µm wide. The serpentines are composed of four hyperturns that maintain separation efficiency and extend the effective separation length to 15.9 cm.30 A common anode well is located at the bottom end (27) Ju, J. Y.; Ruan, C. C.; Fuller, C. W.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4347-4351. (28) Ruiz-Martinez, M. C.; Berka, J.; Belenkii, A.; Foret, F.; Miller, A. W.; Karger, B. L. Anal. Chem. 1993, 65, 2851-2858. (29) Simpson, P. C.; Roach, D.; Woolley, A. T.; Thorsen, T.; Johnston, R.; Sensabaugh, G. F.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2256-2261. (30) Paegel, B. M.; Hutt, L. D.; Simpson, P. C.; Mathies, R. A. Anal. Chem. 2000, 72, 3030-3037.

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Figure 1. (A) Capture chamber design for binding optimization. The chamber has two 2-µL wells each connected to the body of the chamber by arms that are 100 µm in width and 1 mm in length. The arms are connected to the body via a taper 1 mm in length with average width of 500 µm. The body of the chamber is 1 mm in length and width, thus providing a high-volume and low-field capture region. (B) Design of the CE DNA sequencing channel integrated with a capture chamber. The injector has been modified to include the capture chamber. The chamber contains inlet and outlet wells for oligonucleotide capture operations. The CE waste and cathode provide voltage access points for electrokinetic injection and CE analysis. Four tapered turns, or hyperturns, allow an elongated serpentine separation channel geometry (effective length 15.9 cm) that minimizes turn-induced dispersion of the sequencing bands. The high-voltage anode well is located at the bottom.

of the serpentines. Prior to all electrophoretic operations, the channel walls are charge-neutralized following a modified Hjerten coating.31 Oligonucleotide Capture Optimization. Temperature control on a single capture chamber is achieved by attaching an 8-Ω, 1-cmdiameter polyimide foil heater (Minco, Minneapolis, MN) to the bottom side of the microdevice directly below the chamber of interest. A T-type thermocouple (Omega, Stamford, CT) is attached to the backside of the foil heater and used as the input for a PID temperature controller (Omega). The capture matrix is loaded with a 1-mL syringe from the capture outlet well. Buffer (1× TTE) is placed in the capture outlet well, and 2 µL of (31) Hjerten, S. J. Chromatogr. 1985, 347, 191-198.

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unpurified DNA sequencing sample is placed in the capture inlet well. The chamber is brought to process temperature and equilibrated for 30 s, and sample is electrophoretically driven through the capture matrix using from 50 to 500 V. Progress of the capture process is monitored by epifluorescence microscopy. Laser excitation at 488 nm (2017, Spectra Physics, Mountain View, CA) introduced through a 230-µm multimode fiber optic is directed on a 1-cm-diameter area at ∼3 mW/mm2. Fluorescence is collected through a 520-nm long-pass glass filter attached to a Nikon stereo microscope (SMZ 1500, Nikon) equipped with a 12-bit CoolSnap FX thermoelectrically cooled CCD camera (6-s exposure, Roper Scientific, Tucson, AZ). Images were saved as 16-bit TIFFs and analyzed using NIH Image. Chamber profiles are an average 16 pixels in width starting from the sample well-chamber interface. The 16-pixel width exactly corresponds to the width of the arms of the chamber. Resultant profiles were smoothed with a 10-point moving average to ameliorate chip surface defect-induced noise. Integrated DNA Sequencing Microdevice Operation. The linear polyacrylamide DNA sequencing matrix (CEQ, Beckman Corp., Fullerton, CA) is loaded first from the anode well with a 1-mL syringe until the gel fills the cathode and waste well arms as well as the capture chamber coupling arm. The fluidic resistance of each arm, controlled by modulating channel width, is chosen to ensure that the cathode, waste, and capture chamber coupling arms are completely filled without filling the chamber itself. Capture matrix is then loaded from the capture outlet well sweeping the plume of sequencing matrix into the capture inlet. Unpurified DNA sequencing sample is loaded into the capture inlet well, and the CE cathode, CE waste, anode, and capture outlet wells are filled with 1× TTE. The loaded device is moved to the heated stage of the Berkeley four-color rotary scanner.32 The stage is brought to 50 °C, and the chip is equilibrated for 30 s. Oligonucleotide capture is initiated by applying 250 V to the capture outlet well while the capture inlet well remains grounded. During this procedure, purification progress is monitored by the electrophoretic current. Typically, the current rapidly ramps from a background of 20 to 50 µA in 30 s. As purification progresses, this current slowly falls back to the background level. On average, this process requires only 90 s to complete. When oligonucleotide capture is complete, the capture inlet well is evacuated, washed out, and replaced with fresh 1× TTE. The bound product is electrophoretically washed for 30 s with clean buffer to remove excess primer and other contaminants still present in the capture chamber. After electrophoretic washing, the capture inlet and outlet wells are evacuated, washed out, and replaced with fresh 1× TTE. The stage is ramped to 67 °C and equilibrated for 60 s to allow full denaturation of the product-matrix duplex. Electrokinetic injection is initiated by grounding the capture inlet well, applying 200 V to both the CE cathode and anode, and applying 500 V to the CE waste for 15 s. Separation is accomplished by grounding the CE cathode, applying 100 V to the waste and capture outlet, and applying 2500 V to the anode. This voltage scheme results in an electric field of 150 V/cm in the main separation channel. The entire CE analysis step from injection to coelution required on average 32 ( 1 min to complete for sequencing M13 vector DNA. Buffer wells were periodically (32) Shi, Y.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361.

refreshed with 1× TTE to maintain electrical contact and to compensate for evaporation. Matrix was cleared from the device using a custom-built, high-pressure cleaning apparatus.33 Fourcolor fluorescence DNA sequencing data were processed in BaseFinder using a script written in-house and output as an SCF file.34 The SCF file was used as input for base-calling and quality scoring by phred.35 RESULTS AND DISCUSSION The purification of DNA sequencing extension products from contaminating buffer ions, template DNA, and unused nucleotide triphosphates and primers can be readily accomplished by utilizing the exquisite, sequence-specific binding properties of DNA. With the goal of binding only extension products from the DNA cycle sequencing reaction, we have chosen a probe sequence that is complementary to the 20-base sequence directly 3′ of the M1340 universal forward primer in the M13mp18 phage cloning vector. This sequence is flanked by the priming sequence and the polylinker cloning site. Thus, this matrix should bind only those strands that have been extended past the first 20 bases. Interfering small molecules (e.g., buffer and reaction components) and noncomplementary DNA (e.g., template and primer) will not be retained in the capture matrix. In the experiments presented here, the probe length and content was chosen such that the melting temperature, TM, was several degrees below the sequencing process temperature (67 °C). Thus, release of the captured product, electrokinetic injection, and electrophoretic analysis all occur at the same temperature. DNA hybridization and duplex denaturation are classically modeled as a simple equilibrium between single-stranded reactants and double-stranded products: Keq

S + C y\z S:C

(1)

where S is the free electrophoresing ssDNA sequencing extension fragment, C is the gel-immobilized ssDNA complementary to S, and S:C is the double-stranded duplex immobilized in the gel. In addition to interspecies conversion described by this chemical equilibrium, each component of the system is subject to diffusion described by Fick’s law and electrophoresis governed by the species’ electrophoretic mobility constant and the local electric field. Each of these terms is added to obtain the generalized diffusion equation model of the concentration profile as a function of distance across the chamber, x, and time, t:36, 37

∂2 ∂ C(x,t) ) DC 2C(x,t) - kf[S][C] + kb[S:C] ∂t ∂x µCE(x)

∂ C(x,t) (2) ∂x

∂ ∂2 S(x,t) ) DS 2S(x,t) - kf[S][C] + kb[S:C] ∂t ∂x µSE(x)

∂ S(x,t) (3) ∂x

∂ ∂2 S:C(x,t) ) DS:C 2S:C(x,t) + kf[S][C] - kb[S:C] ∂t ∂x µS:CE(x)

∂ S:C(x,t) (4) ∂x

where C(x, t), S(x, t), and S:C(x, t) are the concentration profiles of each species in time and space, D is the species-specific diffusion coefficient, kf and kb are the forward and backward rate constants related to Keq, µ is the species-specific electrophoretic mobility constant, and E(x) is the time-independent spatial distribution of electric fields (for a chamber of nonuniform width). Although no analytical solution exists for this system of equations, we can simplify the system for an understanding of how key parameters affect the process. For example, the gel-immobilized oligonucleotide and the gel-immobilized duplex both have approximately zero electrophoretic mobility. Furthermore, because the system is in a viscous gel and the species involved have large molecular weights, the diffusion-mediated transport of any species is likely to be negligible on the time scale of the analysis. Under these conditions, the above system of equations is reduced to

∂ ∂ S(x,t) ) - kf[S][C] + kb[S:C] - µSE(x) S(x,t) (5) ∂t ∂x We can immediately identify the key parameters necessary for optimizing the system. Equation 5 can be divided into two parts: the duplex source and sink terms consisting of -kf[S][C] and kb[S:C] and the electromigration term, -µSE(x)(∂/∂x)S(x,t). For optimal saturation binding to occur in a given distance element described by this equation, the analyte should be delivered as rapidly as possible to the sample space but should not be removed so rapidly that binding is incomplete. System parameters that affect this balance are the magnitudes of the association and dissociation rate constants, kf and kb and the magnitude of the local electric field distribution, E(x). Thus, an empirical study of these parameters is required to obtain optimal binding. The progress of a typical oligonucleotide capture operation is shown in Figure 2. The unpurified DNA sequencing sample was placed in the well at the bottom of the frame. At 250 V driving potential and at 40 °C, images were taken at 30-s intervals through 120 s. Images are dark-corrected to the frame at 0 s. As the capture proceeds, the fluorescent primers, which have no specificity for the capture matrix, are not retained and begin to accumulate in the waste well located at the top of the frame. As discussed above, one critical factor in determining the binding of analyte is the distribution of electric fields inside the chamber, E(x). Microfabrication allows very specific control of chamber geometry and, hence, E(x). The utility of chamber geometry can be seen in the final image. The doubly tapered chamber contains two arms with conductivity a factor of 10 lower than the body of the chamber, resulting in a drop of the electric field from ∼600 V/cm in the feeding arm to ∼60 V/cm in the chamber. Equation 5 suggests that an optimal geometry for localizing product formation should feature a high-field sample introduction region sweeping the product into the chamber with minimal binding in the coupling arm, followed by a sudden drop to low field where binding kinetics (33) Scherer, J. R.; Paegel, B. M.; Wedemayer, G. J.; Emrich, C. A.; Lo, J.; Medintz, I. L.; Mathies, R. A. Biotechniques 2001, 31, 1150-1156. (34) Giddings, M. C.; Severin, J.; Westphall, M.; Wu, J. Z.; Smith, L. M. Genome Res. 1998, 8, 644-665. (35) Ewing, B.; Green, P. Genome Res. 1998, 8, 186-194. (36) Atkins, P. Physical Chemistry, 5th ed.; W. H. Freeman and Co.: New York, 1994. (37) Patterson, D. H.; Harmon, B. J.; Regnier, F. E. J. Chromatogr., A 1996, 732, 119-132.

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Figure 2. Epifluorescence images showing the progress of a typical oligonucleotide capture operation in which the product is concentrated exclusively in the inlet (bottom side) taper region. The “wash” time step is taken after the 30-s electrophoretic wash with clean 1× TTE in the capture inlet well. The capture inlet well is at the bottom of the image, and the capture outlet is at the top of the image. The outline of the chamber is shown as a thin, white line superimposed on the fluorescence image.

are dominant over electromigration. Reproducible sample localization is critical for further electrokinetic manipulation on a microfluidic chip. Factors influencing binding according to eq 5 also include the process temperature and the magnitude of the applied field. To explore the effects of temperature on binding, we varied the temperature from room temperature to the TM in increments of 5 °C at a driving voltage of 250 V. The final fluorescence profiles of the bound and washed product in the capture chamber are shown in Figure 3A. The peak concentration of bound product was found to increase with temperature initially. The profiles at increasing temperature suggest that binding is enhanced as more material is able to bind earlier in the chamber. Increasing the temperature from 25 to 50 °C shifts all of the bound material into the first tapering region starting 1 mm away from the capture inlet well and ending 2 mm away from the capture inlet well. The maximum peak height, corresponding to the absolute concentration of analyte in this region, is almost 5-fold higher at 50 °C. At 60 °C, which is approximately equal to TM, relatively little material remains in the column after washing. The inset of Figure 3A shows the maximum concentration for all temperatures examined. This dependence of binding efficiency on temperature is expected because the reaction rate constants exhibit Arrhenius behavior. Equation 5 dictates that optimal binding occurs by increasing temperature such that kf[S][C] is maximized relative to kb[S:C], but not approaching too near TM. While the relative values of field strengths, E(x), are governed by chamber geometry, the absolute magnitude is determined by the voltage applied to the chamber. To explore the effect of field on product binding, the experiment of Figure 3A was repeated at constant optimum temperature (50 °C) while scanning the driving 5096 Analytical Chemistry, Vol. 74, No. 19, October 1, 2002

Figure 3. Fluorescence profiles of the capture chamber with product bound under various conditions. (A) Select binding profiles at room temperature (RT) and 40, 50, and 60 °C are shown at a constant driving potential of 250 V. The inset plots the maximum peak intensity as a function of temperature. (B) Select binding profiles at driving voltages of 150, 250, 350, and 450 V are shown at a constant capture temperature of 50 °C. The inset plots the maximum peak intensity as a function of driving voltage. In both cases, unpurified DNA sequencing sample was used. The capture matrix was 5% linear polyacrylamide copolymerized with 10 µM capture oligonucleotide.

voltage from 100 to 550 V. The resultant profiles from these experiments are presented in Figure 3B. The peak maximums acquired at all measured voltages are presented in the inset to Figure 3B. At and below 100 V driving voltage, product binding was not observed because the sample is not being efficiently delivered to the chamber from the sample well. Starting at 150 V, a modest amount of binding was observed and was centered early in the chamber. Additionally, binding was also observed in the arm leading to the entrance taper, as can be seen in the offset of the blue curve from 0 in the first 1 mm. Maximum concentration was observed at 250 V where the peak maximum value is centered just after the beginning of the entrance taper. At successively higher applied voltages, lower concentration and lower binding were observed, and furthermore, the peak maximum value shifted to later points in the chamber. At voltages higher than 450 V, there was no detectable binding in the chamber. In agreement with theory, binding kinetics cannot successfully compete with the higher electrophoretic flow rate that occurs at high field and no duplex formation is observed despite a thermodynamically favored duplex state and an electric field drop of a factor of 10 in the chamber. Under optimized binding conditions, the DNA sequencing product is isolated from contaminating buffer ions, template DNA, and excess fluorescent sequencing primers in 120 s. This is a 10-

Figure 4. C termination sequencing traces for three sample input conditions. (A) On-chip oligonucleotide capture-purified M13mp18 four-color sequencing sample. Capture operations were performed at 250 V applied across the chamber and at 50 °C. Sample was captured for 120 s and then replaced with fresh 1× TTE buffer for a 30-s electrophoretic wash. The captured sample was released and electrophoretically resolved at 67 °C, Beckman CEQ LPA gel, 150 V/cm. B) An aliquot of the same sample was precipitated with ethanol, resuspended in 50% formamide in DI H2O, and run on our 96-lane DNA sequencing chip. (C) The same unpurified sample was directly analyzed in parallel with the precipitated sample. The insets present an enlarged view of the early region of each trace. The oligonucleotide capture-purified sample contains termination events starting at C21 counting from the 3′-end of the M13-40 universal forward sequencing primer. The ethanol-precipitated sample additionally exhibits the excess primer peak and four termination events held within the oligonucleotide capture sequence (C9, C12, C15, C16). The unpurified sequencing sample exhibits poor peak intensity and contains all termination events found in the precipitated sample.

fold improvement in processing time compared to ethanol precipitation. Additionally, sample manipulation is entirely integrated and electrokinetic, requiring no centrifugation and no incubation steps for completion. The 2-µL unpurified sample is concentrated into a ∼10-nL volume in the capture chamber, yielding a volumetric concentration factor of ∼200. Calibration against a FAM standard in the chamber indicates that the bound product has an overall concentration of ∼6 µM fluorophore.38 The optimized binding temperature and electric field were next used for an oligonucleotide capture-based cleanup of unpurified DNA sequencing sample followed by injection and microchip CE analysis. Figure 4 presents a comparison of four-color microchip CE DNA sequencing traces of M13mp18 ssDNA vector showing only the C terminations (FAM-labeled fragments). Trace A shows results from extension fragments of sample that was oligonucleotide capture-purified on the integrated microchip. Traces B and C were run in parallel on our 96-lane DNA sequencing microchip under identical injection and separation conditions.12 Trace B is product that was precipitated with ethanol and resuspended in 50% formamide in DI H2O, and trace C is unpurified DNA (38) Because the sequencing sample is actually a mix of energy-transfer dye pairs with quantum efficiencies slightly differing from FAM, this number is only approximate.

sequencing sample taken directly from the thermal cycler. The capture-purified peaks are much higher in intensity and peak area compared to the unpurified sequencing product in trace C, and the ratio of the average peak area against its corresponding peak in ethanol-purified trace B is 85% over the 10-650-base range. The unpurified sequencing sample exhibits vastly inferior signal strength throughout the run compared to the precipitated sample and oligonucleotide capture-purified fragments. The peak areas for trace C are on average 10% of the corresponding peak area from trace B (10-650 bp). This observation is consistent with previously published studies that cycle sequencing reaction components significantly interfere with electrokinetic injection.15,39 Ethanol precipitation typically removes Cl-, the predominant interference in the sequencing reaction buffer, to a final concentration of 20-200 µM, and excess nucleotide triphosphates to below detectable limits.14 In the case of the integrated bioprocessor, electrophoretic washing of the bound product ensures that it is equilibrated ionically with the electrophoretic run buffer, which contains neither Cl- nor nucleotide triphosphates. Further examination of Figure 4 shows the peak intensity dropped slightly for trace A at longer fragments compared to the ethanol-precipitated products. Increasing the cross-injection time did not increase the signal of larger molecular weight peaks, suggesting that template DNA is partially occluding the capture matrix inlet before larger fragments have achieved steady-state loading into the chamber. The addition of a low concentration of sieving matrix to the sample prior to loading would retard the high molecular weight template and eliminate this effect. The inset and magnified traces in Figure 4 show the first 60 bases off the primer (C terminations only), demonstrating the high sequence specificity of this technique. Not only is the primer missing from trace A but the first four C terminations are also missing. These first four C terminations occur within the capture sequence itself. Because these products do not contain the entire capture sequence, their melting temperatures are intrinsically lower, and as a result, these fragments are not retained during the capture step. The first sequencing peak observed is the first base 3′ of the capture region. Interestingly, the last base of the capture sequence (T) is not readily discernible in the raw data. The processed and base-called data from on-chip purification followed by CE analysis of M13mp18 sequencing extension fragments are shown in Figure 5. Sequencing essentially starts from the first base 3′ of the capture sequence at base position 21, which begins GCCAA. As in most sequencing traces, the first bases called routinely contain errors. A compression at ∼60 bases from the primer (CCCCGGG) was miscalled, but no other errors were observed until after base position 590. The sequence at 614 bases (TTTTTTGGGG) was called correctly as seen in Figure 5. Base-calling accuracy was evaluated using quality scores generated by phred. Phred quality values are logarithmically proportional to the base-calling error rate by the relation P ) 10-q/10, where P is the probability of an erroneous call and q is the phred quality score. A plot of the 10-base window moving average of the phred quality scores for the on-chip-purified DNA sequencing run is shown in Figure 6. A phred quality score of 20, or basecalling accuracy of 99%, is used as the benchmark for the read (39) Kleparnik, K.; Garner, M.; Bocek, P. J. Chromatogr., A 1995, 698, 375383.

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Figure 5. Base-called four-color fluorescence sequencing data from the on-chip oligonucleotide capture-purified sample. Windows of the first 30 of each century of bases are presented. Data were processed in BaseFinder followed by base-calling and quality score analysis using phred.

length of a DNA sequencing run. This run contained a total of 653 called bases, consisting of 560 q g 20 bases, 510 q g 30 bases, and 410 q g 40 bases. The data from the unpurified sample did not produce any phred 20 bases. The precipitated sample yielded 601 q g 20 bases. The longer read length achieved with the precipitated sample is most likely due to the increased signal intensity seen for longer fragments. Gel immobilization of oligonucleotides is attractive as a method for the high-speed purification of DNA sequencing samples; gels are easily introduced into (and removed from) microfluidic systems,33 and electrokinetic transport of analyte through gel is well characterized in the literature. Furthermore, gel-based chemistries combine the advantageous elements of the solid phase as well as the liquid phase. The solid phase provides a support for sample immobilization and geometric control of localization. Liquid-phase processes involving chemical reactions are advantageous because the thermodynamic activity of reagents is neither hindered by bulk-surface diffusion-mediated transport nor limited by the surface area of the chosen solid support. These advantages facilitate the low-volume, electrokinetic manipulation of sample shown here as well as the rapid hybridization of product in 120 s. Current sample purification protocols in genome sequencing centers rely primarily on ethanol precipitation, which is difficult to automate due to multiple centrifugation steps. More sophisticated protocols utilizing magnetic bead reagents circumvent centrifugation at the expense of more costly reagents and added time. Furthermore, both methods still require g10-µL reaction volumes. The integrated on-chip preparation and analysis microdevice presented here reduces both processing time and volume by more than 1 order of magnitude and obviates the need for robotic transfer of sample from purification to CE analysis. The oligonucleotide-capture purification chemistry and associated microfluidic circuit design presented here illustrate the important

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Figure 6. Average phred quality value (q, 10-base moving window) from the experiment in Figure 5. The run produced 560 bases with q g 20 (accuracy g99%).

benefits that microfabricated chemical analysis devices can bring to the field of bioanalytical chemistry: integrated and rapid processing of nanoliter volumes. CONCLUSIONS We have demonstrated a novel chamber geometry, capture process, and affinity capture chemistry for the purification of DNA sequencing products and integrated this process with high-speed microdevice sequencing. Sample immobilization, preconcentration, and desalting are completed in only 120 s, a greater than 10-fold reduction in time and a greater than 100-fold reduction in reagent volume. Binding optimization is understood through the interplay of binding kinetics, binding thermodynamics, and electrophoretic transport. The ability to functionalize microfluidic devices with integrated high-speed, nanovolume affinity capture is a powerful new tool for chemical analysis that will find application in a wide variety of sample purification, concentration, and analysis processes. ACKNOWLEDGMENT The authors gratefully acknowledge discussions on buffering with Dr. Oscar Salas-Solano and colleagues at Amersham Biosciences. Microfabrication was carried out at the University of California Berkeley Microfabrication Laboratory. This research was supported by grants from the National Institutes of Health (HG01399) and from the Director, Office of Science, Office of Biological and Environmental Research of the U.S. Department of Energy under Contract DEFG91ER61125. Additional support was provided by Amersham Biosciences. B.M.P. was supported by a NIH trainee fellowship from the Berkeley Program in Genomics (T32 HG00047) and by a summer fellowship from the American Chemical Society Division of Analytical Chemistry. Received for review June 3, 2002. Accepted July 25, 2002. AC0203645