Well-Less Capillary Array Electrophoresis Chip Using Hydrophilic

Oct 10, 2007 - In this work, simultaneous introduction of multiple samples onto well-less CAE channels arranged with 1-mm gaps was successfully ...
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Anal. Chem. 2007, 79, 9205-9210

Technical Notes

Well-Less Capillary Array Electrophoresis Chip Using Hydrophilic Sample Bridges Young Ho Kim, Inchul Yang, and Sang-Ryoul Park*

Health Metrology Group, Korea Research Institute of Standards and Science, Daejeon, Korea

An innovative sample introduction method that facilitates extreme simplification of the layout of a capillary array electrophoresis (CAE) chip is described. Multiple samples were directly injected onto CAE channels from sample loaders using hydrophilic sample bridges, thus obviating auxiliary components of sample wells and sampling channels of a typical CAE chip. Hydrophilic sample bridges were spontaneously formed in hydrophobic surroundings to connect sample loaders to corresponding CAE channels, through which electrosample injections were effectively made. Sample dispersion was intrinsically avoided due to the “sticky” nature of the bridges. Utilizing hydrophilic interactions, target spots for formation of sample bridges can be expanded from the actual openings of CAE channels, which reduces the burden of precise positioning of samples toward CAE channels. In this work, simultaneous introduction of multiple samples onto well-less CAE channels arranged with 1-mm gaps was successfully demonstrated. Well-less CAE chips, feasible now due to the hydrophilic sample bridges, would bring unprecedented advantages in both production and operation because of their extreme simplicity. Capillary array electrophoresis (CAE) technology has played a critical role in the successful progress of the Human Genome Project.1 The advantage of high-speed and high-resolution electrophoresis achievable with a capillary electrophoresis channel was greatly furthered in automated CAE systems, representatively ABI 7700 and GE MegaBace systems, that perform multiple analysis in parallel using arrays of 96 or even 384 capillary channels,2 providing sufficiently high gene-sequencing throughputs for timely accomplishment of the Human Genome Project. These days, low-cost gene sequencing services are readily available because of those automated CAE systems. In spite of great successes of the automated CAE systems in large-scale gene sequencing and in gene fingerprinting applications, traditional gel electrophoresis techniques that are slow and laborious still prevail in most biology laboratories. Current automated CAE systems are expensive but not versatile enough * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +82-42-868-5801. (1) Marshall, E. Science 1999, 284, 1439-1441. (2) Shibata, K.; et al. Genome Res. 2000, 10, 1757-1771. 10.1021/ac071115p CCC: $37.00 Published on Web 10/10/2007

© 2007 American Chemical Society

to perform various DNA analyses with high efficiency. This is especially true for analysis of PCR products, the most frequently performed DNA analysis in a typical biology laboratory. Lengths of typical CAE channels of current CAE systems are too long to rapidly carry out low-resolution electrophoresis of PCR products. CAE technology needs to be accordingly modified to gain desired versatility for widespread adoption in small- or medium-scale laboratories. Chip format is one promising direction of such modification of CAE technology. In a chip format, a great deal of flexibility is provided in creating compact CAE channels as required. In addition, more durable and handier CAE channels can be provided. To date, DNA Labchip-2100 Bioanalyzer from Agilent seems to be the most successful commercial product in this regard. However, this commercial product is not of true CAE technology as it uses a single CE channel to perform multiple electrophoresis in a sequential manner. In addition, manual pipetting is involved in sample loading. Versatile CAE chip systems are yet to be realized, especially for medium-scale laboratories where various DNA analyses need to be carried out in a rapid, convenient, and cost-effective manner.3,4 CAE chips have been enthusiastically developed as the simplest model system of “micro total analysis system” or “lab on a chip” since those terms were first introduced.5 In spite of numerous reports describing advances in CAE chip technology, however, the degree of complexity in both the structure and the operation of a typical CAE chip has not been substantially reduced, due mainly to the technical challenge in interfacing the macroscopic world of sample preparation with the microscopic CAE channels where samples are to be introduced. Initially, the “T” design6 was introduced for injection of a sharp sample band, and a number of variations have followed7-12 as summarized elsewhere.13 There (3) Obeid, P. J.; Christopoulos, T. K. Crit. Rev. Clin. Lab. Sci. 2004, 41, 429465. (4) Go ¨dde, R.; Akkad, D.-A.; Arning, L.; Dekomien, G.; Herchenbach, J.; Kunstmann, E.; Meins, M.; Wieczorek, S.; Epplen, J. T.; Hoffjan, S. Electrophoresis 2006, 27, 939-946. (5) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 1, 244288. (6) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (7) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 2637 - 2642. (8) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113.

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Figure 1. Model well-less 24-channel capillary array electrophoresis chips. (A) A typical CAE chip. (B) CAE chips of various lengths prepared from a chip mold: (1) 3, (2) 6, (3) 10, and (4) 20 cm. (C) Installation of a CAE chip on a two-piece chip receptacle with an adjustable gap.

also have been a number of reports regarding sample introduction techniques onto CAE chips including on-column sample stacking.14-19 Nevertheless, all these methods still require implementation of sample wells or sampling channels on a CAE chip, in addition to electrophoresis channels. The additional components unavoidably consume substantial space on the chip and lead to rather complex channel layouts for CAE chips. The chip disk approach that features radial alignment of CAE channels20,21 would be the most effective in accommodating the additional components. In this approach, the components for sample introduction are implemented on the outer side of the disk where more space is available than the central area. When all necessary components were included, 384 electrophoresis channels could be laid out on a single disk.21 This approach, however, requires an unconventional (rotating) detection strategy to fit with the arrangement of the electrophoresis channels, which may not be desirable for reliable detector operation. In this work, we attempted to realize a CAE chip of an extremely simplified layout by removing all components from the chip except the electrophoresis channels. Although the simplification brought substantial advantages in both production and (9) Fu, L.-M.; Yang, R.-J.; Lee, G.-B. Anal. Chem. 2003, 75, 1905-1910. (10) Slentz, B. E.; Penner, N. A.; Regnier, F. Anal. Chem. 2002, 74, 48354840. (11) Lapos, J. A.; Ewing, A. G. Anal. Chem. 2000, 72, 4598-4602. (12) Lin, C. H.; Yang, R. J.; Tai, C. H.; Lee, C. Y.; Fu, F. M. J. Micromech. Microeng. 2004, 14, 630-646. (13) Wenclawiak, B.; Pu ¨ schi, R. Anal. Lett. 2005, 39, 3-16. (14) Bu ¨ ttgenbach, S.; Wilke, R. Anal. Bioanal. Chem. 2005, 383, 733-737. (15) Fang, Q.; Xu, G.-M.; Fang, Z.-L. Anal. Chem. 2002, 74, 1223-1231. (16) Zhang, L.; Yin, X. F. J. Chromatogr., A 2006, 1137, 243-248. (17) Kim, D. K.; Kang, S. H. J. Chromatogr., A 2005, 1064, 121-127. (18) Jung, B.; Bharadwaj, R.; Santiago, J. G. Electrophoresis 2003, 24, 34763483. (19) Jung, B.; Bharadwaj, R.; Santiago, J. G. Anal. Chem. 2006, 78, 2319-2327. (20) Shi, Y.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361. (21) Emrich, C. A.; Tian, H.; Medintz, I. L.; Mathies, R. A. Anal. Chem. 2002, 74, 5076-5083.

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operation of the chip, a number of problems had to be overcome for successful sample introduction onto CAE channels without the help of sample wells and sampling channels. We finally solved those problems by forming hydrophilic sample bridges between the sample loaders and corresponding CAE channels in the hydrophobic surroundings. Therefore, a well-less CAE chip of unprecedented simplicity now is a feasible chip format with the sample introduction technique using hydrophilic sample bridges. EXPERIMENTAL SECTION Preparation of a Model Well-Less CAE Chip. Uncoated (internally) fused-silica capillary tubing (100-µm i.d., 375-µm o.d.) was purchased from Polymicro Technologies (Tucson, AZ). The external protective polyimide coating was burnt to be sublimed at 600 °C for 1 h using an electric furnace (REX-P100; Ahjeon Industrial, Seoul, Korea). Twenty-four pieces were aligned in parallel with gaps of 1 mm, with the assistance of a pair of capillary holding objects. The assembled capillaries were molded using epoxy resin (YD128, epoxy polymer, and Japamin D230, curing agent; Kukdo Chemical, Seoul, Korea) as the assembly was placed on a plastic half-case. Epoxy was cured at room temperature for 24 h while vacuum (∼1 Torr) was applied to remove trapped bubbles. The completely cured capillary assembly was cut using a diamond-wheel saw to produce a number of 3-cm-long CAE chips. A fabricated CAE chip is shown in Figure 1A. A number of CAE chips of various lengths were also produced from a single preparation of chip mold (Figure 1B). Apparatus for Electrophoresis. A CAE chip was assembled onto a plastic receptacle to form an electrophoresis unit (Figure 1C). The plastic receptacle consisted of two components, which facilitated accommodation of CAE chips of various lengths. The plastic receptacles also served as buffer reservoirs together with the edges of the CAE chip. Two Pt wires (26720-1; Aldrich, Milwaukee, WI) were implemented on the corners of the buffer reservoirs as an anode and a cathode for electrophoresis. The electrophoresis channels of the CAE chip were filled with a

Figure 2. Electrophoresis of a DNA mixture consisting of 60, 200, 300, 400, 480, 600, 700, 800, 900, and 1000 bp DNAs (from right to left in panel 4) using the model CAE chip: (1) injected sample bands; (2) focused sample bands; (3) early stage of separation. (4) complete separation (see also movie S-1).

polymer sieving medium by applying negative pressure to the sample-introduction side reservoir while the other side of the reservoir was filled with the polymer medium. An electric field for electrophoresis (67 V/cm for both sample injection and separation) was provided by a high-voltage power supply (2197; LKB, American Instrument Exchange, Haverhill, MA). Migration of DNA was visualized by laser-induced fluorescence imaging, and an Ar ion laser (95; Lexel, Fremont, CA) was used as an excitation source (488 nm). The fluorescence images of stained DNA bands were captured by a microscope system (model SZ61; Olympus, Tokyo, Japan) supplemented with a digital camera (DP70; Olympus). Sample Introduction. To assess the uniformity of separation (Figure 2), the sample introduction side of the buffer reservoir was filled with a DNA sample to be injected into all 24 electrophoresis channels simultaneously. For simultaneous introduction of different samples onto individual electrophoresis channels, a set of sample loaders made of stainless steel tubing (755064, 125µm i.d., 510-µm o.d.; Nilaco, Tokyo, Japan) was used to deliver and inject samples to the designated electrophoresis channels. The sample loader set was prepared with 1-mm gaps to match the gaps of electrophoresis channels. The sample introduction sequence is depicted in Figure 3A. The sample introduction side of the reservoir was filled with silicone oil prior to sample loading, and using a multiple channel syringe pump (220; KD Scientific, Holliston, MA), sample drops (400 ( 100 µm in diameter or 32 ( 8 nL in volume) were formed at the tips of the loaders while the tips were immersed in silicone oil. Sample bridges were formed by positioning the sample drops in contact with the entrances of

Figure 3. Schematic illustrations (not to scale) of the sequence of the proposed sample introduction method and the sample injection setup. (A) Sample introduction sequence: (1) collection of samples by applying negative pressure; (2) formation of sample drops at the loader tips by applying positive pressure; (3) formation of sample bridges and subsequent electrokinetic injection (refer to panel B); (4) replacement to the buffer and initiation of electrophoresis. (B) Setup for electrokinetic sample injection: 1. metallic sample loader; 2. cathodic buffer reservoir filled with silicone oil; 3. sample drop/bridge; 4. high-voltage power supply; 5. anode; 6. anodic buffer reservoir; 7. capillary wall; 8. capillary EP channel; 9. EP chip.

designated electrophoresis channels (Figure 3B). The samples were injected onto electrophoresis channels by applying 67 V/cm (between the loader tips and the anode) for 20 s. The silicone oil was replaced with electrophoresis buffer for the following electrophoresis. Reagents and DNA Sample. Sieving medium was composed of 2% poly(ethylene oxide) consisting of equal amounts of 1 and 8 MDa (Aldrich, Milwaukee, WI) in 0.5× tris-borate-EDTA (TBE) buffer (Bioneer, Daejeon, Korea). The polymer solution was stored in the dark to avoid photolysis. TBE buffer (0.5×) was used as the electrophoresis buffer, and DNA was stained with SyBr Gold (Molecular Probes, Eugene, OR). Silicone oil used for sample introduction was purchased from Sigma (175633; St. Louis, MO). The DNA sample was a mixture of PCR-amplified double-strand DNAs of 10 different sizes (60, 200, 300, 400, 480, 600, 700, 800, Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Figure 4. Electrokinetic sample introduction in a nonaqueous environment (silicone oil medium). (A) A fine conduction path formed by a focused electric field in a highly resistive medium: 200 V was applied between the loader tip and the anode placed in the other end of the electrophoresis channel. (B) Complete electrical isolation of neighboring electrophoresis channels due to high resistance of the surrounding medium: there was no sign of migration of previously injected sample bands during sequential electrosample injections using a single sample loader.

900, 1000 bp). Individual PCR products were mixed without purification. To confirm successful introduction of individual samples, samples lacking specific sized DNAs were prepared separately (see the caption for Figure 6). RESULTS AND DISCUSSION Performance of the Model CAE Chip. Satisfactory performance of the model CAE chip was confirmed by the reproducible DNA separation pattern across all CAE channels (Figure 2; Supporting Information movie S-1). As commercial fused-silica tubing of a long successful application history in DNA electrophoresis was used as electrophoresis channels, the separation patterns were normal and acceptable. One thing noteworthy is self-focusing of DNA bands in the initial stage of electrophoresis (Figure 2, panel 1 f panel 2). The spontaneous focusing of sample bands is likely due to transient isotachophoresis (TITP),19,22-24 where chloride ions in the sample matrix (1× PCR buffer) and borate ions in the electrophoresis buffer (0.5× TBE buffer) act as the leading ions and the terminating ions, respectively. The suggested TITP mechanism for the self-focusing of DNA bands was supported by the following observations. Either removal of chloride ions from the sample matrix or their addition to the electrophoresis buffer eliminated the self-focusing of DNA bands. Therefore, it is highly likely that fast-migrating chloride ions in the sample matrix function as the leading ions of isotachophoresis, which cannot be established if chloride ions are continuously (22) Auriola, S.; Jaaskelainen, I.; Regina, M.; Urtti, A. Anal. Chem. 1996, 68, 3907-3911. (23) Krˇiva´nkova´, L.; Pantu˚cˇkova´, P.; Gebauer, P.; Bocˇek, P.; Caslavska, J.; Thormann, W. Electrophoresis 2003, 24, 505-517. (24) Xu, Z.; Nishine, T.; Arai, A.; Hirokawa, T. Electrophoresis 2004, 25, 38753881.

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supplied from the electrophoresis buffer that follows a sample band. Self-focusing was observed with a wide range of chloride concentrations (20-300 mM) in the sample matrix, where time required for completion of focusing elongated as chloride concentration increased. Self-focusing was observed with a variety of slowly migrating anions (borate, glycine, and dodecyl sulfate) in the electrophoresis buffer as they functioned as the terminating ions, but disappeared with a fast migrating anion such as acetate. Xu et al. also reported isotachophoretic concentration of DNA in between chloride ions (the leading ions) and glycine (the terminating ions).24 Self-focusing of sample bands is beneficial for both high sensitivity of detection and high resolution of separation of excessively injected samples and was utilized in this work. Problems in Direct Sample Introduction onto a Well-Less CAE chip. The 1-mm channel spacing was given to the model CAE chip to demonstrate the compatibility of the proposed sample introduction method with densely aligned CAE channels on chip formats. Tight arrangement of the CAE channels is advantageous as the channels can be covered with a compact detection system. With the given spacing, however, the ultimate challenge in operating the model CAE chip was the introduction of each sample onto the corresponding electrophoresis channel without contaminating the neighboring channels. No physical barrier (a sample well) was implemented to prevent mixing of samples (see the electrokinetic sample introduction scheme in Figure 3A). Dispersion and diffusion of a sample from the loader tip could be avoided by filling the sample-introduction side reservoir with a viscous polymeric medium, such as 10% poly(vinylpyrrolidone). Electrokinetic injection efficiency was considered poor if a sample drop did not precisely contact the entrance of the corresponding electrophoresis channel, as the strength of an electric field across

Figure 5. “Sticky” sample bridges formed between sample loaders and CAE channels: (1) loader tips approaching CAE channels; (2) sample drops suspended to the loader tips; (3) formation of sample bridges; (4) sample loaders pulled to the right; (5) sample loaders pulled to the left; (6) sample loaders pulled backward; (7) sample bands injected using the sample bridges.

a wide conduction path is low. The poor injection efficiency led to extension of an injection time, which in turn caused gas evolution at the metallic loader tip, and gas evolution often agitated sample drops to disperse them toward other electrophoresis channels. Precise positioning of sample drops to ensure contact with the openings of electrophoresis tubing (100 µm in diameter) would not be practical, particularly for simultaneous and repetitive transfer of multiple samples for routine applications. Furthermore, previously injected samples in other electrophoresis channels migrated inward from the starting point in response to the electric field applied for a new sample batch injection, which hampered synchronous start of electrophoresis. Sample Introduction Using Hydrophilic Sample Bridges. All the above-mentioned obstacles were addressed by filling the sample-introduction side of the reservoir with hydrophobic, nonaqueous medium (silicone oil in this work) during sample introduction. Hydrophilic-hydrophobic repulsion between the sample drops and the surrounding medium was strong enough to prevent the sample drops, suspended from the loader tips, from dispersing despite rapid moving of the loaders around the reservoir. Surrounded with the highly electrically resistive medium, the applied electric field was focused on the gap between a sample drop and the entrance of the electrophoresis channel, which resulted in a fine conduction path across the gap (Figure 4A). Therefore, samples could successfully be injected against physical gaps between the sample drops and the entrances of electrophoresis channels. Electrokinetic injection through the conduction path was sufficiently efficient to avoid gas evolution. High electrical resistance of the surrounding medium also isolated other electrophoresis channels from the applied electric field (Figure 4B); thus, a synchronous start of electrophoresis was possible regardless of the injection order. (Electrophoresis is

initiated once the hydrophobic medium is replaced with an electrophoresis buffer, as well as when an electric field is applied to the wire electrode in the buffer reservoir.) A more remarkable feature of sample introduction in a hydrophobic medium is the formation of sample bridges between the loader tips and the entrances of electrophoresis channels. As a sample drop was placed to come in contact with the exposed cross-sectional area of the corresponding capillary tubing, a sample bridge covering the entire cross-sectional area formed from the loader tip. Considering the hydrophobic nature of the epoxy mold of the CAE chip and the hydrophilic nature of fused silica, this bridge is likely formed from hydrophilic attractions while expelled by the hydrophobic surroundings. The sample bridges were “sticky” enough to maintain connections despite intentional (but gentle) pulling motions of the loader tip away from the entrance (Figure 5). Thus, cross-contamination of neighboring channels through sample dispersion was easily avoidable. The sample bridges provided excellent conduction paths for electrokinetic injection of the samples. Simultaneous sample introduction onto multiple electrophoresis channels without contaminating neighboring channels is shown in Figure 6 (see also movie S-2). As shown in Figure 5, sample introduction was successfully carried out even with visibly noticeable poor alignment of the loader array. The success was likely possible due to the sample bridges. Sample bridges are formed with a nearby hydrophilic spot, which could be larger than the actual opening of an electrophoresis channel. Therefore, the burden of precise positioning of samples toward CAE channels can be effectively reduced. In the case of the model CAE chip, the target area expands ∼13-fold as the sample drop comes in contact with any portion of the exposed cross section of the fused-silica capillary (360 µm in diameter) instead of the actual opening of the capillary Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Linear extension of parallel channel alignment of a well-less CAE chip may also be beneficial in widening applicability of a chip system. Samples of tens of base pair spacing such as PCR products can be analyzed in a few minutes using a short CAE chip (Figure 6). Chips with longer CAE channel lengths are needed for higher resolution DNA analysis. As CAE channels aligned in parallel are linearly extended, an identical detector can be used with suitable repositioning by simple linear movements. (We are currently developing a high-resolution detection system to demonstrate high-resolution electrophoresis.) Using a two-piece chip receptacle with an adjustable gap (Figure 1C), CAE chips of various lengths can be accommodated by a single system.

Figure 6. Successful introduction of individual samples facilitated by the sample bridges, by which cross-contamination was avoided. The numbers (1-10) represent DNA bands of 60, 200, 300, 400, 480, 600, 700, 800, 900, and 1000 bp, respectively. The sample introduced onto lane c lacked specific size (200, 700, 800 bp) DNAs whereas the sample introduced onto lane d lacked the 60, 500, and 700 bp DNA fragments (see also movie S-2).

(100 µm in diameter). Contact with any hydrophilic spot results in the entire area, including the opening, being covered, which enables successful electrokinetic injection. Potential Advantages of Well-Less CAE Chips. The suggested well-less CAE chips would bring substantial advantages in production because of their simple structures. CAE channels can be densely aligned because no additional components are attached. More important, no interconnections among channels are needed, which allow adoption of various fabrication strategies including rather low-end technologies as demonstrated in this work. As channels are aligned in a parallel and linear fashion, chips of various lengths can be produced in a mass production manner from a single preparation of a long mold (Figure 1B).

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CONCLUSIONS This work demonstrates the feasibility of operation of a wellless CAE chip with a novel sample introduction technique using hydrophilic sample bridges. The hydrophilic sample bridges not only provided satisfactory electrosample injection conditions for a well-less CAE chip but also effectively reduced the burden of accurate positioning of samples toward CAE channels. Because of extremely simplified layouts, well-less CAE chips would bring substantial advantages in both their production and application. Combined with the automatic sample-transfer mechanism, the reported CAE chip technology could be advanced to realize a novel high-throughput electrophoresis platform. ACKNOWLEDGMENT This work was supported by the Innovation in Measurement Technology Program of the Korea Research Institute of Standards and Science, and the research project of New Technologies for Establishment of Measurement Standards in Biosciences of the Korea Research Council of Public Science and Technology. SUPPORTING INFORMATION AVAILABLE Movie S-1. performance text of a model well-less CAE chip with a DNA mixture (refer to Figure 2 for experimental details). Movie S-2. introduction and electrophoresis of four independent DNA samples (refer to Figure 6 for experimental details). This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review May 29, 2007. Accepted August 30, 2007. AC071115P