Inline Injection Microdevice for Attomole-Scale Sanger DNA Sequencing

The inline-injection DNA sequencer channel layout is shown schematically in Figure 1A. Channel patterns are photolithographically transferred to a ...
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Anal. Chem. 2007, 79, 4499-4506

Inline Injection Microdevice for Attomole-Scale Sanger DNA Sequencing Robert G. Blazej,† Palani Kumaresan,‡ Samantha A. Cronier,† and Richard A. Mathies*,†,§

UCSF/UC Berkeley Joint Bioengineering Graduate Group, Department of Mechanical Engineering, and Department of Chemistry, University of California, Berkeley, California 94720

A new affinity-capture-based inline purification, concentration, and injection method is developed for microchip capillary electrophoresis (CE) and used to perform efficient attomole-scale Sanger DNA sequencing separations. The microdevice comprises three axial domains for nanoliter-scale sequencing sample containment, sample plug formation, and high-resolution capillary gel electrophoresis. Purified and concentrated inline sample plugs are formed by electrophoretically driving Sanger sequencing extension fragments into an affinity-capture polymer network positioned within a CE separation channel. Extension fragments selectively hybridize and concentrate at the polymer interface while residual primer, nucleotides, and salts electrophorese out of the system. The plug is thermally released and injected into the CE channel by direct application of the separation voltage. To evaluate this system, 30 nL of sequencing sample prepared from 100 amol (60 million molecules) of human mitochondrial hypervariable region II amplicon was introduced into the microchip, purified, concentrated, and injected, generating a read length of 365 bases with 99% accuracy. This efficient inline injection system obviates the need for the excess sample that is required by cross-injection techniques, thereby enabling Sanger sequencing and other high-performance genetic analysis using DNA quantities approaching theoretical detection limits. The cross-injector was a central enabling concept in the development of microfabricated capillary electrophoresis (µCE) systems.1,2 Nearly all academic and commercial µCE applications utilize cross-injection to form the small, well-defined sample plugs that are required for rapid CE analysis. Most CE microdevices use microliter-scale, off-chip-prepared reactions to provide the excess sample required to establish analyte equilibrium in the cross-injection region. In advanced integrated systems that seek to miniaturize not only CE but all processing steps, the wasteful cross-injector becomes a barrier to reaching theoretical miniatur* Corresponding author. Phone: (510) 642-4192. Fax: (510) 642-3599. E-mail: [email protected]. † UCSF/UC Berkeley Joint Bioengineering Graduate Group. ‡ Department of Mechanical Engineering. § Department of Chemistry. (1) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z. H.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (2) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Ludi, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-258. 10.1021/ac070126f CCC: $37.00 Published on Web 05/12/2007

© 2007 American Chemical Society

ization limits. Additionally, cross-injection timing requirements can hinder optimization of array CE microdevices and integrated bioprocessor systems operating on limiting amounts of template.3-5 The direct injection method utilized in conventional CE separations eliminates these cross-injection complications. However, in the absence of sample purification and concentration, direct injection yields low-resolution, low-sensitivity separations that are not suitable for DNA sequencing.6-11 Swerdlow’s group has developed on-column sample concentration techniques in fused-silica capillaries based on flow-stream and base-stacking effects.12,13 These methods achieve the signal intensity and peak resolution necessary for DNA sequencing directly from unpurified reactions. Similarly, Yeung’s group has used a fused-silica capillary assembly and freeze-thaw valves in conjunction with capillary zone electrophoresis for purification and direct injection of sequencing samples.14,15 Ueberfeld et al. presented methods for solid-support sequencing sample purification and direct loading in which a micromanipulator is used to position a magnetized electrode carrying paramagnetic microspheres above the sample port of a microfabricated capillary.5 These efforts demonstrate the value of direct sample injection but utilize methods that are not readily incorporated into integrated microchip systems. Although microchip solid-support sample purification methods have been developed, they provide insufficient binding density to achieve (3) Aborn, J. H.; El-Difrawy, S. A.; Novotny, M.; Gismondi, E. A.; Lam, R.; Matsudaira, P.; McKenna, B. K.; O’Neil, T.; Streechon, P.; Ehrlich, D. J. Lab Chip 2005, 5, 669-674. (4) Blazej, R. G.; Kumaresan, P.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7240-7245. (5) Ueberfeld, J.; El-Difrawy, S. A.; Ramdhanie, K.; Ehrlich, D. J. Anal. Chem. 2006, 78, 3632-3637. (6) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484-487. (7) Khandurina, J.; McKnight, T. E.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 2995-3000. (8) Lagally, E. T.; Scherer, J. R.; Blazej, R. G.; Toriello, N. M.; Diep, B. A.; Ramchandani, M.; Sensabaugh, G. F.; Riley, L. W.; Mathies, R. A. Anal. Chem. 2004, 76, 3162-3170. (9) Ruiz-Martinez, M. C.; Salas-Solano, O.; Carrilho, E.; Kotler, L.; Karger, B. L. Anal. Chem. 1998, 70, 1516-1527. (10) Salas-Solano, O.; Ruiz-Martinez, M. C.; Carrilho, E.; Kotler, L.; Karger, B. L. Anal. Chem. 1998, 70, 1528-1535. (11) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (12) Park, S. R.; Swerdlow, H. Anal. Chem. 2003, 75, 4467-4474. (13) Xiong, Y.; Park, S. R.; Swerdlow, H. Anal. Chem. 1998, 70, 3605-3611. (14) Hashimoto, M.; He, Y.; Yeung, E. S. Nucleic Acids Res. 2003, 31. (15) Xue, G.; Pang, H. M.; Yeung, E. S. J. Chromatogr., A 2001, 914, 245-256.

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the required sample preconcentration needed for direct on-column injection.16,17 Bioaffinity purification techniques based on DNA hybridization are well-suited to microchip integration and have unique advantages over alternative purification techniques. A commercially available methacrylate-modified phosphoramidite coupled to a synthetic oligonucleotide enables acrylamide copolymerization to create a matrix with user-definable sequence specificity, hybridization stringency, and fluidic transport properties.18 The matrix network provides the advantages of solid-phase support and positioning while exhibiting three-dimensional liquid-phase reaction kinetics in a replaceable reagent that is easily loaded into microfluidic channels. Pioneering work by Muscate et al. demonstrated two-step capillary affinity gel electrophoresis of short match and mismatch oligonucleotides in a copolymer of acrylamide and an in-house-synthesized acrylamidooligonucleotide.19 More recently, DNA-based affinity capture has been used in a microfluidic system for multianalyte detection and in integrated bioprocessors for sample cleanup prior to Sanger sequencing analysis.4,20,21 To traverse the miniaturization barrier imposed by inefficient cross-injection, here we utilize the high activity of matriximmobilized oligonucleotides to create an efficient inline-injection system capable of both sequencing sample cleanup and narrow sample plug definition. Optimal conditions for sample plug formation and CE analysis are investigated within a microfluidic device consisting of sequential domains for sample containment, affinity-capture, and high-performance CE using an exemplary mitochondrial hypervariable region II amplicon. The ability to modulate hybridization affinity internally through probe design and concentration as well as externally through applied thermal and electric gradients is presented. Elimination of the excess sample previously required for cross-injected CE separations now permits microchip-based Sanger sequencing utilizing a 30-nL reaction containing only 100 amol of template. EXPERIMENTAL SECTION Microdevice Fabrication and Design. The inline-injection DNA sequencer channel layout is shown schematically in Figure 1A. Channel patterns are photolithographically transferred to a 100-mm-diameter glass wafer (Borofloat, Schott, Duryea, PA) and etched to a depth of 30 µm in 49% HF by using a 2,000-Å-thick amorphous silicon hard mask.22 Holes (1.1-mm diameter) are drilled to create cathode, sample, waste, and anode access ports. Enclosed microfluidic channels are formed through thermal bonding with a blank glass wafer in an atmospheric furnace at 668 °C for 8 h. Prior to operation, the channel walls are passivated (16) Wang, H.; Chen, J. F.; Zhu, L.; Shadpour, H.; Hupert, M. L.; Soper, S. A. Anal. Chem. 2006, 78, 6223-6231. (17) Xu, Y. C.; Vaidya, B.; Patel, A. B.; Ford, S. M.; McCarley, R. L.; Soper, S. A. Anal. Chem. 2003, 75, 2975-2984. (18) Rehman, F. N.; Audeh, M.; Abrams, E. S.; Hammond, P. W.; Kenney, M.; Boles, T. C. Nucleic Acids Res. 1999, 27, 649-655. (19) Muscate, A.; Natt, F.; Paulus, A.; Ehrat, M. Anal. Chem. 1998, 70, 14191424. (20) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (21) Paegel, B. M.; Yeung, S. H. I.; Mathies, R. A. Anal. Chem. 2002, 74, 50925098. (22) 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.

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Figure 1. Inline injection system for attomole-scale Sanger DNA sequencing. (A) Microfluidic design of the inline injection system showing channels containing sequencing sample (green), affinitycapture matrix (yellow), and denaturing separation matrix (red). Channels are etched 30 µm deep in 100-mm-diameter glass wafers with 1.1-mm-diameter drilled cathode (C), sample (S), waste (W), and anode access ports. Dashed region is shown diagrammatically in (B)(E), where blue arrows indicate the direction of applied electrophoretic potentials. (B) Unpurified sequencing sample (30 nL) is electrophoretically driven toward the affinity-capture matrix at 45 °C. (C) Complementary extension fragments selectively hybridize to the affinity-capture matrix (1) forming a well-defined inline sample plug. Residual fluorescent reagents, salts, and unincorporated nucleotides (2) migrate toward the waste port. (D) Excess sample is flushed from the sample arm (3), and residual reagents are electrophoretically washed out of the inline injection system (4). (E) At 72 °C, the purified sample plug releases from the affinity-capture matrix and is injected toward the separation channel (5).

with a modified Hjerten coating (0.4% 3-(trimethoxysilyl)propyl methacrylate in a 1:1 methanol/water solution, Sigma-Aldrich M6514) to retard DNA adsorption and electroosmotic flow.23 Sample, capture, and CE channels are 200 µm wide, coupling arms connecting the sample and waste ports to the main channels are 70 µm wide, and the tapered turns that fold the CE channel are 65 µm wide.24 The 5.2-mm-long sample region (green, main channel) defines a 30-nL volume. The channel connecting the (23) Hjerten, S. J. Chromatogr. 1985, 347, 191-198. (24) Paegel, B. M.; Hutt, L. D.; Simpson, P. C.; Mathies, R. A. Anal. Chem. 2000, 72, 3030-3037.

sample port to the main channel is shortened in Figure 1A for illustrative purposes; on the microdevice it extends 5.5 cm along the separation channel to provide greater fluidic resistance against hydrodynamic sample movement. The inline affinity-capture matrix region (yellow, main channel) is 9.9 mm long, and the separation length of the CE channel is 18.1 cm. DNA Template Preparation. Amplification primers specific to the human mitochondrial hypervariable region II (Forward: 5′AAG CCT AAA TAG CCC ACA CGT TCC-3′. Reverse: 5′-TGG TTA GGC TGG TGT TAG GGT TCT-3′) were selected using the PrimerQuest program (Integrated DNA Technologies, Coralville, IA). The -40 M13 sequence (5′-GTT TTC CCA GTC ACG ACG3′) was added to the 5′-end of the forward primer prior to synthesis (Integrated DNA Technologies) to generate an amplicon compatible with standard sequencing primers. Hypervariable region II DNA is amplified from 20 ng of genomic DNA (NA-13116; Centre d’etude Polymorphisme Humane, Paris, France) in a 50-µL PCR reaction (0.2 mM dNTPs, 1.5 mM MgCl2, 0.2 µM primers, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and 1.5 units of platinum Taq DNA polymerase; Invitrogen, Carlsbad, CA). Following thermal cycling, unincorporated primers, nucleotides, and salts are removed in preparation for cycle sequencing (QIAquick PCR Purification Kit 28104; Qiagen, Valencia, CA) and PCR yield is quantitated by using the dsDNA intercalating dye PicoGreen (Quant-iT PicoGreen dsDNA Assay Kit P-7589; Invitrogen) and a FP-750 spectrofluorometer (Jasco, Great Dunmow, Essex, UK) according to manufacturer protocols. Template DNA is diluted in 1× TE (10 mM Tris, pH 8.0, 0.1 mM EDTA) to a working concentration of 50 fmol/µL. Sequencing Sample Preparation. Dye primer sequencing reactions are performed by using a cycle sequencing kit (79260; USB Corp., Cleveland, OH) and four energy-transfer (ET) -40 M13 forward primers.25 Each 10-µL reaction consists of 75 nM ET primer and 3.3 nM template DNA in a standard sequencing reaction (15 mM Tris-HCL pH 9.5, 3.5 mM MgCl2, 60 µM dNTP, 600 nM ddNTP, 10 units of Thermo Sequenase DNA polymerase, 0.015 unit of Thermoplasma acidophilum inorganic pyrophosphatase). Reactions are thermally cycled (95 °C 30 s, 50 °C 30 s, 72 °C 60 s, 35 cycles) by using a Mastercycler Gradient and then pooled to make a four-color sequencing sample. Unpurified sequencing sample is stored frozen at -20 °C until use. Matrix Design and Synthesis. The affinity-capture oligonucleotide (5′-AGA CCT GTG ATC CAT CGT GA-3′, TM ) 62 °C; 50 mM monovalent salt, 20 µM probe) is selected from the human mitochondrial hypervariable region II DNA sequence 3′ of the forward PCR primer. It is designed to be complementary only to the cycle sequencing extension products and exhibit minimal selfcomplementarity (max ) 4 base pairs). A 5′-Acrydite modification (Integrated DNA Technologies) is incorporated to enable polyacrylamide copolymerization. The affinity-capture matrix is synthesized at 4 °C by sparging a 2-mL solution containing 5% w/v acrylamide, 1× TTE, and 40 nmol of the Acrydite-modified oligonucleotide for 2 h with argon, followed by the addition of 0.015% w/v APS and TEMED. The solution is allowed to polymerize for 24 h prior to loading into a 1-mL syringe. Similarly, linear polyacrylamide sequencing separation matrix is synthesized at 4 (25) 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.

°C by sparging a 10-mL solution containing 3.8% w/v acrylamide, 1× TTE, and 6 M urea for 2 h with argon, followed by the addition of 0.015% w/v APS and TEMED. The solution is allowed to polymerize for 24 h and then transferred to a high-pressure matrixloading chuck.26 Inline-Injection DNA Sequencer Operation. The location of reagents in the microdevice is presented in Figure 1A and capture, purification, and inline injection process steps are shown in (B)-(E). The system is first prepared by loading denaturing DNA sequencing matrix from the anode port to the waste arm intersection at 400 psi using the high-pressure loader. Affinitycapture matrix is then loaded with a 1-mL syringe through the cathode port until the capture channel and waste arm are completely filled. The high fluidic resistance of the viscous sequencing matrix prevents the affinity-capture matrix from entering the separation channel. Excess affinity-capture matrix in the sample region is removed by flushing 1× TTE from the sample port to the cathode port. All ports are filled with 5× TTE to provide electrophoresis buffering. The microdevice is transferred to the temperature-controlled stage of the Berkeley four-color rotary scanner where the temperature is ramped at 14.5 °C/min from room temperature to the 72 °C CE separation temperature. The 1× TTE is then flushed through the sample port. The microdevice is cooled at 2.2 °C/ min to the 45 °C capture temperature while an 800-V electrophoresis prerun potential is applied between the cathode and anode. Unpurified sequencing sample is loaded into the 30-nL sample region by pipetting 1 µL of the unpurified sequencing sample into the cathode port and applying 58 kPa vacuum to the sample port for 2 s. The cathode port is then washed 3× with 10 µL of 5× TTE to remove remaining sequencing sample. Equal 10-µL volumes of 5× TTE are placed on the cathode and sample ports to prevent hydrodynamic flow. Inline sample plug formation is initiated by grounding the cathode and applying 25 V to the waste. After extension product capture is complete in 240 s, 1× TTE is flushed through the sample port to remove sequencing sample remaining in the sample arm. The 25-V potential is then reapplied for 15 min to electrophoretically wash interfering residual reagents out of the capture channel. The stage is ramped to 72 °C to release the sample plug from the affinity-capture matrix, and CE separation is accomplished by grounding the cathode and applying 2000 V to the anode. Four-color sequence data were collected 1 cm from the anode in 32 min by the Berkeley inverted four-color fluorescence rotary scanning microscope27 and processed with the Cimarron 3.12 base caller (NNIM, Sandy, UT). Affinity-Capture Optimization. The microdevice is placed on a temperature-controlled stage (Omega, Stamford, CT) and allowed to equilibrate at 35, 40, 45, or 50 °C for 30 s prior to 12-, 25-, or 50-V electrophoretic capture and wash operations. Potentials are applied via platinum wire electrodes connected to a custombuilt, LabVIEW-controlled (National Instruments Corp., Austin, TX) four-channel power supply. Epiillumination from a 488-nm laser (30 mW, Spectra Physics, 2017, Mountain View, CA) is directed onto the microdevice through a 230-µm multimode (26) 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. (27) Shi, Y. N.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361.

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fiber-optic cable aligned at 45° and 1 cm above the stage. Fluorescent images are collected through a 520-nm long-pass filter attached to a SMZ 1500 stereomicroscope (Nikon) equipped with a CoolSNAP FX thermoelectrically cooled CCD camera (Roper Scientific, Tucson, AZ). Pixels are binned 3 × 3 to increase sensitivity and 1-s exposures are stored as 8-bit TIFFs. Images analyzed by the program ImageJ (NIH, Bethesda, MD) are first smoothed and background subtracted to reduce microdevice surface bonding artifacts and to remove background fluorescence from the stage. Fluorescence intensity plot profiles of the captured product are generated starting at the sample arm intersection by averaging 20-pixel segments spanning the channel. Each pixel corresponds to a 10 × 10 µm section. Log-normal peak fits are derived from the intensity plot profiles by using the program GRAMS/AI (Thermo Fisher Corp., Waltham, MA). Captured DNA sequencing extension product quantity is determined by correlating observed fluorescence intensity with an 800 nM ET primer reference sample in 1× TTE. Resolution Measurements. Gaussian peak fits are made to singlet “C” peaks using the program GRAMS/AI (Thermo Fisher Corp.), and resolution is calculated according to the formula RS ) (2 ln2)1/2 ∆t/(w2 + w1)(b2 - b1), where ∆t is the difference in migration time of base numbers b1 and b2 having full-width-athalf-maximum of w1 and w2. RESULTS AND DISCUSSION Microdevice Theory and Design. The microfluidic system presented in Figure 1A is designed to explore the ultimate limits of miniaturized Sanger sequencing sample analysis. Microfabrication provides exquisite control over channel geometry and thus a means to precisely meter the nanoliter volumes needed to probe this sequencing limit. Accordingly, a primary design consideration is the volume of unpurified sequencing sample defined by the sample region dimensions. The minimum necessary volume is dictated by the sensitivity of the detection system and the efficiency of sample processing.4,27 Considering the ideal case of a 1000-base read, a standard cycle-sequencing reaction needs sufficient template to generate 1 billion extension fragments (1 million fluorophores/band × 1000 bands) after 25 cycles. Using a demonstrated linear amplification efficiency of ∼70% per cycle, the required number of starting template molecules is ∼60 million, or 100 amol.4 Typical template concentrations vary from 0.5 to 7.5 nM, the optimal amount being a function of template length and purity as well as cycle number and the desire for uniform band intensity. In practice, the reaction volume is held constant while template concentration and cycle number are varied to achieve optimal results. For this study, we adopted a moderate template concentration of 3.3 nM, thus requiring 30 nL of sample defined by a 5.2-mm-long channel to achieve 100-amol template molecules. Preconcentration and purification of the 1 billion extension fragments contained in this 30-nL sample requires only 83 pL of capture matrix, assuming saturation binding to the 20 µM probe oligonucleotide. It was necessary, however, to increase this volume to 56 nL, defined by a 9.9-mm-long channel, due to two design considerations. First, the capture matrix is in direct contact with the urea-containing separation matrix and must be sufficiently partitioned such that the denaturant does not diffuse into the capture band region. Second, copolymerization with the Acryditemodified oligonucleotide imparts a negative charge on the capture 4502

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matrix, resulting in electromigration under applied potentials. Extending the capture region channel increases fluidic resistance and reduces matrix movement. The separation portion of the microdevice is based on previous microfluidic designs for miniaturized Sanger sequencing with the important modification that cross-injector structures are eliminated.21,28,29 The high capture matrix binding capacity enables sample plug definition through hybridization kinetics rather than channel geometry, thereby eliminating injection timing and excess sample requirements inherent to cross-injection. Tapered turns that minimize turn-induced band dispersion are utilized to fold a 18.1-cmlong CE channel onto the 100-mm-diameter glass wafer substrate.24 Inline-Injection DNA Sequencer Operation. Conceptual operation of the inline injection system presented in Figure 1B-E shows the serial positioning of unpurified sequencing sample, affinity-capture matrix, and denaturing capillary electrophoresis matrix within a channel. Unpurified sample is electrophoretically driven into the affinity-capture matrix, which is utilized for sample preconcentration and purification as well as inline sample plug formation. The capture matrix simultaneously performs all of these operations through sequence-specific hybridization of a covalently linked oligonucleotide that is complementary only to the sequencing extension fragments. Matrix affinity is thermally switchable, enabling release and inline injection of the sample plug at elevated temperature. Ideally, the sample plug location remains fixed during all processing steps. However, electromigration and thermal matrix expansion collaborate to shift the sample plug location, necessitating regulation of these physical properties during microdevice operation. Figure 2 presents epifluorescence images of inline capture and injection of sequencing extension products within the microdevice. An elevated temperature flush at 72 °C followed by cooling to 45 °C combined with an electrophoresis prerun are used to move the capture matrix interface ∼1.5 mm away from the sample arm junction. This manipulation ensures that subsequent movement of the sample plug is confined to axial displacement within the main channel. In the absence of this pretreatment, matrix loaded at room temperature thermally expands beyond the sample arm junction, resulting in undesirable orthogonal sample plug movement into the sample arm during capture, release, and injection. Electromigration and thermal expansion effects are clearly seen in Figure 2B and C, respectively. During the 15-min electrophoretic wash operation, the sample plug migrates down the channel due to bulk movement of the capture matrix. When the microdevice is heated from the capture to the separation temperature, matrix expansion shifts the sample plug back toward the cathode. Once released and injected at 72 °C, the extension fragments move independently of the capture matrix and begin to size-fractionate as seen in Figure 2D. Affinity-Capture Optimization. Previous work in a crossinjection affinity-capture/CE microdevice demonstrated that extension fragment binding is described by

∂ ∂ [S(x,t)] ) -kf[S][C] + kb[S:C] - µsE(x) [S(x,t)] ∂t ∂x

(1)

where S is the extension fragment, C is the matrix-immobilized complementary oligonucleotide, S:C is the hybridized duplex, kf and kb are the respective rate constants for hybridization and

Figure 3. Fluorescence profiles of extension products captured at select temperatures and voltages. Profiles are shown after complete capture of 30 nL of unpurified sequencing sample at 40, 45, and 50 °C under different driving potentials. Indicated potentials are between the cathode and waste ports. Profiles are measured 30 s after removal of the electric field. The affinity-capture matrix comprises 5% linear polyacrylamide copolymerized with 20 µM capture probe in 1× TTE.

Figure 2. False-color fluorescence images of inline sample plug formation and injection. Cooler colors indicate lower intensity, warmer colors higher intensity. Microfluidic channels are outlined in white, the scale indicates displacement in micrometers from the sample arm junction, and arrows indicate the direction of sample plug migration. (A) Image of the sample plug (1) and residual reagents (2) 200 s after plug formation was initiated at 45 °C by applying 25 V between the cathode and waste ports. (B) The negatively charged sample plug and capture matrix migrate 400 µm (3) toward the waste port after 15 min of electrophoretic washing. (C) Heating the microdevice to 72 °C releases the sample plug from the capture matrix and shifts it toward the cathode (4) due to separation channel gel expansion. (D) Application of the separation electric field (100 V/cm) between the cathode and anode causes the sample plug to elongate (5) as it begins to size-fractionate.

denaturation, µs is the mobility constant, and E is the electric field in space, x, and time, t.21 Accessible parameters for optimization are kf and kb through probe sequence design and microdevice temperature, [C] in the capture matrix, and the applied E. Sample plug optimization begins with rational capture matrix design. Provided that the matrix meets specificity and binding capacity requirements, iterative reformulation is not necessary as tuning of hybridization stringency is readily accomplished through external thermal and electric gradients. Theoretical melting temperature calculations were used to select an extensionfragment-specific, high TM probe oligonucleotide (TM ) 62 °C) to enhance kf while still allowing denaturation and sample injection at the CE separation temperature (72 °C).30 Elevated probe (28) Blazej, R. G.; Paegel, B. M.; Mathies, R. A. Genome Res. 2003, 13, 287293. (29) Paegel, B. M.; Emrich, C. A.; Weyemayer, G. J.; Scherer, J. R.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 574-579. (30) Owczarzy, R.; Vallone, P. M.; Gallo, F. J.; Paner, T. M.; Lane, M. J.; Benight, A. S. Biopolymers 1997, 44, 217-239.

concentration favors forward binding kinetics, increased binding capacity and reduced sample plug size. However, gel electromigration effects also increase with probe concentration. The probe was therefore copolymerized at a moderate 20 µM concentration in 5% w/v acrylamide.4,20,21 The capability of this matrix formulation to create inline sample plugs suitable for high-resolution separation was investigated within the microdevice by capturing unpurified sequencing sample under various binding conditions. Figure 3 illustrates the effect of temperature and driving potential on the fluorescence profile of hybridized extension fragments within the channel. For a constant 25 V driving potential, binding efficiency increases initially with increased temperature due to Arrhenius behavior; the fluorescence profile created at 45 °C produces a narrower sample plug with higher peak intensity compared to capture performed at 40 °C. At 50 °C, however, kb[S:C] is increased relative to kf [S][C] resulting in sample plug broadening. Similar adjustment of hybridization stringency is achieved by modulating the driving potential. Doubling the capture voltage to 50 V at 45 °C results in sample plug broadening due to a higher electrophoretic flow acting in opposition to binding kinetics. Voltage reduction below that required for efficient extension fragment movement into the capture matrix does not significantly improve sample plug characteristics as seen by the similar peak shapes generated at 12 and 25 V at 45 °C. Additional data on temperature and driving potential effects are quantitated in Table 1. Log-normal fits to each fluorescence profile were used to model boundary condition hybridization at the free-solution/capture-matrix interface. Capture was performed either at 12 V for 480 s, 25 V for 240 s, or 50 V for 120 s. The smallest sample plugs were obtained at 45 °C with a driving potential of 25 V or less, while suboptimal capture conditions result in approximate plug size doubling. To illustrate matrix dynamics, values are given 30 s after removal of the indicated voltage (step A) and under applied voltage during the electrophoretic wash step Analytical Chemistry, Vol. 79, No. 12, June 15, 2007

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Table 1. Effect of Temperature and Voltage on Sample Plug Formation temp (°C)

voltage (V)

35

25

40

25

45 45

12 25

45 50

50 25

stepa

sizeb

dispc

cap.d

[peak]e

corref

A B (15) A B (15) A A B (5) B (15) A A B (15)

378 280 379 243 142 156 91 147 311 285 267

1.84 2.38 1.59 2.06 1.60 1.56 1.65 1.92 1.63 1.60 2.22

1.45 1.17 1.17 1.06 1.19 1.18 1.13 1.13 0.98 0.87 0.80

0.63 0.70 0.50 0.73 1.40 1.26 2.07 1.29 0.49 0.50 0.50

0.986 0.997 0.970 0.970 0.992 0.956 0.991 0.975 0.985 0.981 0.992

a Process step as shown in Figure 2 (electrophoretic wash time in minutes). b Full-width at half-maximum in micrometers. c Sample plug displacement in mm from the sample arm junction. d Amount of captured extension fragments in femtomoles. e Peak captured extension fragment concentration in micromolar. f Correlation coefficient (r2) of the log-normal fit used in (b)-(d).

(step B). The effects on sample plug size are shown in detail for the 45 °C, 25 V case. Under the no-field condition in step A, the matrix is in a relaxed state with an initial 156-µm sample plug. Once the potential is applied in step B, the plug compresses due to the axial electric field. After 5 min of electrophoretic washing, the sample plug is reduced to 91 µm. Continued washing for 15 min causes sample plug broadening to 147 µm due to equilibrium hybridization kinetics and electromigration-induced matrix distortion. The extent of electromigration is given by the difference in displacement from the sample arm junction where the initial position is a function of matrix contraction during cooling from 72 °C and the capture temperature, voltage, and time. Calibration against a fluorescence standard containing 200 nM concentrations of each ET sequencing primer in 1× TTE was used to estimate the amount of bound extension fragments at each step in Table 1. Values for step A are anomalously high because residual, unbound fluorescent primers remain in the sample plug region until electrophoretic washing in step B. The total amount of bound material ranged from 1.17 to 0.80 fmol with lower amounts occurring under higher stringency capture conditions. This decrease is somewhat unexpected as the capture matrix is in large molar excess and should bind all available extension fragments given a long enough capture channel. One possible explanation is that under unfavorable binding conditions the sample plug tails considerably, dropping below detectable limits. Peak capture concentrations reach ∼2 µM, 10-fold less than saturation binding and significantly lower than previous studies utilizing tapered capture chambers to geometrically reduce the field in the capture region.4,21 A tapered inline injection capture region is infeasible in the present application because band broadening would occur as the sample plug injects into the narrow separation channel. Nonetheless, sufficiently small and concentrated sample plugs were created at 45 °C, 25 V in 240 s to permit diffusion-limited CE analysis, indicating that further plug size reduction is unnecessary. Attomole-Scale Sanger Sequencing. Mitochondrial sequences are widely used in molecular evolutionary studies and 4504 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007

DNA testing.31,32 Hypervariable region II (HVII) was selected for sequencing in this study to demonstrate the adaptability of DNAbased affinity capture to a chosen sequence of interest in addition to common cloning vector sequences.4,21 The optimized binding conditions described above were used for sample plug formation and inline injection of 30-nL unpurified sequencing reaction containing 100 amol of a 455-bp amplicon spanning the HVII region. The total amount of bound product for these capture conditions is 1.13 fmol (Table 1) corresponding to ∼1.6 million extension fragments per band. Four-color sequence data were collected on a radial scanner in 32 min and processed with the Cimarron 3.12 base caller to generate automatic base calls. Total sample processing time including pre-electrophoresis, sample plug formation, electrophoretic washing, and CE is ∼1 h 10 min. The processed electropherogram and base-called data generated on the microdevice is presented in Figure 4. Using a 99% accuracy cutoff with the known mitochondrial sequence, automatic base calls produce a read length of 365 bases. The probe oligonucleotide is internal to the 455-bp amplicon and hybridizes extension fragments 52 bases or longer from the 18-base sequencing primer initiation site for a total possible read length of 386 bases. Three consecutive runs performed under identical conditions exhibited excellent uniformity generating 371 (σ ) 2.6) correct base calls. Importantly, unlike cross-injected CE separations, no optimization of injection timing was necessary to achieve this base-call consistency because separation is initiated by direct application of the CE field to the inline sample plug. The repetitive motif at base 300 (CCCCCCCCCTCCCCCC) is well-resolved; however, false stops likely caused by this difficult to sequence homopolymer region result in an excess peak that prevents the software from making a base call following the T peak and in reduced signal intensity after this region. Subsequent errors occur near the end of the sequence read as signal intensity falls, making base calling progressively more difficult. While the sequencing results achieved in this inline-injection microdevice are remarkable in terms of approaching the molecular limits of Sanger sequencing, overall read length falls below that previously demonstrated for short-channel microdevice separations.21,29,33 To investigate sources of this discrepancy, resolution measurements were made on C traces extracted from unprocessed four-color electropherogram data. Figure 5 plots resolution measurements of neighboring singlet peaks for separations performed at 100 and 122 V/cm. Achieved resolution is consistent with published microfluidic and capillary instruments, but the inflection point occurs at a smaller fragment size.33,34 The base call errors that accumulate after 350 bases are primarily a result of falling signal intensity since adequate peak resolution is maintained until the end of the run at 100 V/cm. Increasing the field to 122 V/cm results in an initial increase in resolution consistent with a diffusion-limited separation.35 However, the steep decline in resolution after 150 bases is inconsistent with Joule (31) Larsson, N. G.; Clayton, D. A. Annu. Rev. Genet. 1995, 29, 151-178. (32) Pakendorf, B.; Stoneking, M. Annu. Rev. Genomics Hum. Genet. 2005, 6, 165-183. (33) Salas-Solano, O.; Schmalzing, D.; Koutny, L.; Buonocore, S.; Adourian, A.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 2000, 72, 3129-3137. (34) Zhou, H. H.; Miller, A. W.; Sosic, Z.; Buchholz, B.; Barron, A. E.; Kotler, L.; Karger, B. L. Anal. Chem. 2000, 72, 1045-1052. (35) Luckey, J. A.; Norris, T. B.; Smith, L. M. J. Phys. Chem. 1993, 97, 30673075.

Figure 4. Inline-injected sequence data from 30 nL of sequencing sample containing 100 amol (60 million molecules) of DNA template. Sanger sequencing extension fragments from a 455-bp human mitochondrial PCR amplicon are resolved in 32 min at 72 °C, 100 V/cm using the inline-injection microdevice. Automatic base calls by the Cimarron base caller and base numbers are indicated above the electropherogram. The 365 bases are read with 99% accuracy compared to the known sequence. Arrows indicate base call errors; correct calls are given below. “x” indicates an inserted base.

heating or reduced matrix selectivity effects and therefore implies an additional source of band dispersion.33,36 Movement of the separation matrix induced by temperature oscillations has been shown to significantly degrade resolution.37 In our inline injection system, the charged capture matrix exerts an axial force on the separation matrix during electrophoresis and may produce laminar dispersion effects that increase with applied field. Improved signal intensity and elimination of capture matrix electromigration will be necessary in order to fully realize the CE separation capability of our inline injection system. Dye-primer chemistry was chosen for initial evaluation due to lower concentrations of residual fluorescent molecules after cycle sequencing compared to dye-terminator chemistry. However, standard dyeprimer chemistry reaction conditions utilize limiting amounts of primer and can display false stops, whereas dye-terminator chemistries are not primer-limited and unlabeled false stops do not interfere with base calling. Theoretically, the cycling conditions used in this study should generate 2.45 fmol of extension fragments in 30 nL assuming 70% efficiency at each cycle. As seen in Table 1, a maximum of 1.19 fmol was generated from a limiting 2.25 fmol of available primer present at a standard 75 nM concentration. The additional primer available in dye-terminator (36) Harke, H. R.; Bay, S.; Zhang, J. Z.; Rocheleau, M. J.; Dovichi, N. J. J. Chromatogr. 1992, 608, 143-150. (37) Voss, K. O.; Roos, H. P.; Dovichi, N. J. Anal. Chem. 2001, 73, 1345-1349.

sequencing reactions should result in improved reaction yield and higher signal intensity, but excess fluorescent unincorporated

Figure 5. Resolution as a function of base number for inline injection separations performed at 100 (triangles, bold line) and 122 V/cm (circles, thin line) in 3.8% (w/v) LPA at 72 °C. Inline sample plugs were formed at 45 °C, 25 V for 240 s in 5% (w/v) LPA containing 20 µM probe oligonucleotide. Lines are second-order fits to the resolution measurements.

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terminators will require longer electrophoretic washing with concomitant increases in matrix electromigration. Alternatively, charge manipulation of the dye-terminators that imparts a net positive charge on unincorporated terminators has been demonstrated and would significantly reduce electrophoretic washing time.38 Physically immobilizing the capture matrix through in situ cross-linked photopolymerization using riboflavin as the photoinitiator is one approach to provide additional control over sample plug localization and to eliminate electromigration.20 Grafting of polyacrylamide to glass microfluidic channels, following previously described methods,39 is possible using bifunctional (γ-methacryloxypropyl)trimethoxysilane and riboflavin photoinitiation.40 In microdevices where an easily replaceable matrix is desired, capture matrix immobilization may be reversibly achieved with thermoresponsive polymers that exhibit low viscosity at room temperature but high viscosity at elevated temperature.41,42 Additionally, electromigration due to the native capture matrix charge may be removed by replacing the DNA probe with a chargeneutral peptide nucleic acid (PNA) affinity capture molecule.43,44 As well as exhibiting enhanced hybridization kinetics and higher sequence specificity, PNA-DNA hybrids can be formed at lower ionic strengths, a significant advantage since inline sample plugs must form in relatively low ionic strength electrophoresis buffer. The use of PNA probes in a replaceable matrix or in combination with in situ photopolymerization should result in significantly faster and narrower sample plug formation as well as mitigation of electromigration issues. The large binding capacity provided by immobilized oligonucleotides in a polymer network has enabled inline purification, preconcentration, and defined sample plug formation suitable for Sanger sequencing analysis. The methods presented here are compatible with fully integrated microfluidic systems utilizing electrokinetic transport to manipulate minute analyte quantities and a thermally switchable affinity-capture matrix for precise external control of sample capture and release. Unlike the inherent sample excess required for cross-injection, an integrated affinitycapture inline injection system can theoretically operate near 100% (38) Finn, P. J.; Bull, M. G.; Xiao, H. G.; Phillips, P. D.; Nelson, J. R.; Grossmann, G.; Nampalli, S.; McArdle, B. F.; Mamone, J. A.; Flick, P. K.; Fuller, C. W.; Kumar, S. Nucleic Acids Res. 2003, 31, 4769-4778. (39) Zangmeister, R. A.; Tarlov, M. J. Langmuir 2003, 19, 6901-6904. (40) Wang, T. L.; Bruin, G. J.; Kraak, J. C.; Poppe, H. Anal. Chem. 1991, 63, 2207-2208. (41) Buchholz, B. A.; Doherty, E. A. S.; Albarghouthi, M. N.; Bogdan, F. M.; Zahn, J. M.; Barron, A. E. Anal. Chem. 2001, 73, 157-164. (42) Kan, C. W.; Doherty, E. A. S.; Buchholtz, B. A.; Barron, A. E. Electrophoresis 2004, 25, 1007-1015. (43) Nielsen, P. E.; Egholm, M.; Buchardt, O. Bioconjugate Chem. 1994, 5, 3-7. (44) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X. H.; Shiraishi, H.; Dontha, N.; Luo, D. B.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 76677670. (45) Dressman, D.; Yan, H.; Traverso, G.; Kinzler, K. W.; Vogelstein, B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8817-8822. (46) Margulies, M.; Egholm, M.; Altman, W. E.; et al. Nature 2005, 437, 376380.

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efficiency with losses limited to nonspecifc adsorption. Here we exploit this efficiency to generate sequence data from a 30-nL sample containing only 100 amol of template DNA. This achievement establishes the feasibility of performing Sanger sequencing from constrained template sources such as microbeads that can maximally bind PCR amplicons in the 100-amol range.45,46 Since clonal PCR microbeads can be prepared using highly parallel emulsion methods, library creation and template preparation steps for genome-scale sequencing are greatly simplified. The ability to perform Sanger sequencing using clonal microbeads will enable the development of a high-throughput microbead integrated DNA sequencer capable of automatically parsing template-carrying microbeads into thermal cycling chambers for long-read sequencing, thereby eliminating the need for discrete pipetting and other complex sample manipulation steps.4 CONCLUSIONS We have demonstrated an affinity-capture-based inline purification, preconcentration, and injection microdevice capable of operating at the template limits of Sanger sequencing. The ubiquitous cross-injector concept, so central to µCE development despite its inherent inefficiency and dependence on injection timing, has been replaced by an efficient, time-independent inline injector. Thermally and electrically tunable hybridization kinetics rather than channel geometry are used to define the sample plug while simultaneously providing the critical sample purification and preconcentration that are needed for high-performance, highsensitivity CE analysis. Read lengths of 365 bases with 99% accuracy were achieved from a 30-nL sequencing sample containing 100 amol of human mitochondrial HVII template. Factors necessary to extend read lengths to state-of-the-art Sanger sequencing have been identified and presented. As microdevices evolve into integrated total analysis systems, the inefficient crossinjector is a fundamental barrier to reaching theoretical limits of miniaturization. The inline injection microdevice presented here will enable us to push past this barrier, allowing integrated microdevices to operate at their ultimate limits. ACKNOWLEDGMENT We gratefully acknowledge discussions on hybridization kinetics with Brian Paegel, and on inline injection with Chung Liu and Nicholas Toriello. Microfabrication was carried out at the University of California Berkeley Microfabrication Laboratory. This research was supported by grants from the National Institutes of Health (HG003583 via Microchip Biotechnologies Inc.). R.A.M. has a financial interest in Microchip Biotechnologies, Inc. that is commercially developing aspects of the technologies presented here. Received for review January 22, 2007. Accepted April 3, 2007. AC070126F