Anal. Chem. 2005, 77, 5706-5710
Free-Flow Electrophoresis on an Anodic Bonded Glass Microchip Bryan R. Fonslow and Michael T. Bowser*
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455
A micro-free-flow electrophoresis chip has been fabricated into a glass wafer etched with 20-µm-deep channels. Wafers were bonded anodically using an intermediate amorphous silicon film. Electric fields as high as 283 V/cm were applied across the separation channel to obtain baseline resolution of fluorescent standards in 4.8 s. The effect of electric fields ranging from 0 to 283 V/cm on the separations and resulting resolutions were examined. Resolution was shown to increase linearly with the applied electric field. Joule heating was not significant under the conditions tested. Instead, the generation of electrolysis products at higher currents proved to be the limiting factor preventing higher separation potentials from being used. Free-flow electrophoresis (FFE) is a preparative separation technique used to continuously separate charged analytes.1 Thinlayer free solution electrophoretic separations were first introduced in 1940,2 and FFE followed in the early 1960s.3,4 The mechanism of separation is shown schematically in Figure 1A. A thin sample stream is introduced into a separation channel with buffer running in parallel. An electric field is applied perpendicularly across the separation channel, and charged analytes are separated laterally based on their electrophoretic mobility. FFE has been well studied for separation of cells, cellular components5-9 and more recently proteomics applications.10,11 FFE was originally developed as a preparative separation technique. Relatively large volume separation beds (∼25 mL) were used to keep throughput high. Unfortunately, the low surface areato-volume ratio of these beds limited heat dissipation. Joule heating was significant and ultimately limited the electric field that could be applied and the efficiency of the separation.12 * To whom correspondence should be addressed. E-mail: bowser@ chem.umn.edu. (1) Roman, M. C.; Brown, P. R. Anal. Chem. 1994, 66, 86A-94A. (2) Philpot, J. S. L. Trans. Faraday Soc. 1940, 36, 38-46. (3) Barrolier, V. J.; Watzke, E.; Gibian, H. Z. Naturforshung 1958, 13B, 754. (4) Hannig, K. Z. fuer Anal. Chem. 1961, 181, 244-254. (5) Hoffstetter-Kuhn, S.; Kuhn, R.; Wagner, H. Electrophoresis 1990, 11, 304309. (6) Hoffstetter-Kuhn, S.; Wagner, H. Electrophoresis 1990, 11, 451-456. (7) Hoffstetter-Kuhn, S.; Wagner, H. Electrophoresis 1990, 11, 457-462. (8) Kessler, R.; Manz, H.-J. Electrophoresis 1990, 11, 979-980. (9) Poggel, M.; Melin, T. Electrophoresis 2001, 22, 1008-1015. (10) Zischka, H.; Weber, G.; Weber, P. J. A.; Posch, A.; Braun, R. J.; Buehringer, D.; Schneider, U.; Nissum, M.; Meitinger, T.; Ueffing, M.; Eckerskorn, C. Proteomics 2003, 3, 906-916. (11) Zuo, X.; Lee, K.; Speicher, D. W. Proteome Anal. 2004, 93-118.
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Figure 1. (A) Mechanism of FFE separations. Sample is continuously introduced into a separation buffer flowing perpendicular to an electric field. Sample components are separated laterally based on electrophoretic mobility differences. (B) Schematic of the µ-FFE mask with the following features: (1) separation buffer inlet, (2) sample inlet channel, (3) electrode buffer inlet channel, (4) Au electrode, (5) separation channel, (6) electrode buffer outlet channel, and (7) fraction collection outlets.
The benefits of miniaturizing electrophoretic separations have been demonstrated quite convincingly with the development of capillary electrophoresis.13 Performing separations in capillaries made higher voltages, increased efficiencies, and faster separations possible. More recently, researchers have moved toward miniaturizing FFE in the hopes of seeing similar improvements. Raymond et al. were the first to fabricate a FFE device in a microfluidic chip (µ-FFE).14 The µ-FFE chip was fabricated by etching 50-µmdeep features into a silicon substrate and anodically bonding a glass cover plate. The separation channel was 1 cm wide × 5 cm long with a reported peak capacity of ∼8 bands/cm. Miniaturization effectively decreased the effects of Joule heating. Theoretical broadening effects in µ-FFE were studied, and baseline resolution of selected proteins was reported. Unfortunately, fabrication using a semiconductor substrate limited the effectiveness of the device. (12) Clifton, M. J.; Jouve, N.; Balmann, H. d.; Sanchez, V. Electrophoresis 1990, 11, 913-919. (13) Jorgenson, J. W.; Lukacs, K. D. Science 1983, 222, 266-272. (14) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1996, 68, 25152522. 10.1021/ac050766n CCC: $30.25
© 2005 American Chemical Society Published on Web 08/02/2005
Notably, separation fields higher than 100 V/cm could not be applied due to the breakdown potential of silicon. Recent work has focused on fabrication of µ-FFE devices using substrates that will allow higher separation potentials to be applied. Zhang and Manz fabricated a µ-FFE chip with 10-µm-deep channels in a poly(dimethylsiloxane) (PDMS) substrate.15 Using PDMS allowed electric fields as high as 270 V/cm to be applied across the separation channel. Weak adhesion between PDMS and the glass base made it necessary to use vacuum to pull fluid through the device instead of the more common pressure-driven flow. The elasticity of the PDMS made it necessary to incorporate support pillars into the separation channel to prevent the applied vacuum from collapsing the device. Kobayashi et al. have fabricated a µ-FFE chip in thermally bonded Pyrex.16 Separations and isolation of two proteins and DNA from a mixture were demonstrated. The separation channel was 30 µm deep, 5.65 cm wide, and 3.5 cm long. The design has a high surface area-to-volume ratio and fabrication is in an insulating substrate, both characteristics necessary for high-voltage applications. The thermal bonding used for wafer bonding may complicate the fabrication of shallower separation channels though. Sagging in the large separation channel is likely during hightemperature annealing. Although this was not investigated, earlier work reported by Pamme and Manz suggested that a similar device used for magnetophoresis, with a 20-µm-deep separation channel, required pillars to provide support during thermal bonding.17 An ideal µ-FFE device would allow the application of high voltages, dissipate Joule heating, and have channel geometries that limit band broadening. Further reduction of channel dimensions is an obvious approach to minimizing Joule heating. A glass µ-FFE device would seem ideal, but thermal bonding of glass could collapse the separation channel at depths smaller than 30 µm. Shinohara et al. have reported the fabrication of µ-FFE device in Pyrex using anodic bonding.18 Anodic bonding eliminates hightemperature annealing, which may require the introduction of support pillars in shallower separation channels. Typically, anodic bonding is performed between a conductive substrate and an ionic glass at a temperature high enough to make the glass conductive, but below the glass transition temperature.19 Upon application of 100-1000 V, a sodium-depleted region of the glass at the wafer/ wafer interface is generated and an electrostatic interaction between the wafers is created. Their intimate contact facilitates anodic oxidation of the silicon and formation of chemical bonds between the wafers. To bond glass to glass, a sodium ion barrier and an oxidizable material are necessary.20 Amorphous silicon (aSi) has been shown to fulfill both requirements.20,21 Here we describe the fabrication of a µ-FFE device in a Borofloat glass (15) Zhang, C.-X.; Manz, A. Anal. Chem. 2003, 75. (16) Kobayashi, H.; Shimamura, K.; Akaida, T.; Sakano, K.; Tajima, N.; Funazaki, J.; Suzuki, H.; Shinohara, E. J. Chromatogr., A 2003, 990, 169-178. (17) Pamme, N.; Manz, A. Anal. Chem. 2004, 76, 7250-7256. (18) Shinohara, E.; Tajima, N.; Suzuki, H.; Funazaki, J. Anal. Sci. 2001, 17, i441443. (19) van Helvoort, A. T. J.; Knowles, K. M.; Holmestad, R.; Fernie, J. A. Philos. Mag. 2004, 84, 505-519. (20) Berthold, A.; Nicola, L.; Sarro, P. M.; Vellekoop, M. J. Sens. Actuators, A 2000, 82, 224-228. (21) Choi, W. B.; Ju, B. K.; Lee, Y. H.; Jeong, S. J.; Lee, N. Y.; Sung, M. Y.; Oh, M. H. J. Electrochem. Soc. 1999, 146, 400-404.
Figure 2. Fabrication schematic of the µ-FFE device.
substrate using an anodic bonding method that incorporates a deposited a-Si film. EXPERIMENTAL SECTION Reagents and Chemicals. Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Deionized water (18.3 MΩ, Barnstead, Dubuque, IA) was used for all preparations unless otherwise noted. Separation buffer consisted of 25 mM HEPES, adjusted to pH 7.0 using 1 M NaOH (Mallinckrodt, Paris, KY), filtered through a 0.2-µm membrane filter (Fisher Scientific, Fairlawn, NJ), and degassed with argon (Airgas, Radnor, PA) for 30 min. Stock solutions of fluorescent standards were prepared in ethanol (Fisher Scientific, Fairlawn, NJ), and dilutions were made in separation buffer. Piranha solutions (4:1 H2SO4/H2O2, Ashland Chemical, Dublin, OH) were used to clean glass wafers and etch deposited Ti. GE-6 (Acton Technologies, Inc., Pittston, PA) was used to etch or remove Au, and CR-12S (Cyantek, Fremont, CA) was used to etch or remove Cr. Concentrated HF (Ashland Chemical) was used to etch the glass wafers. Silver conductive epoxy (MG Chemicals, Surrey, BC, Canada) was used to make electrical connections to the chip. Chip Fabrication. The fabrication process for the µ-FFE chip is outlined in Figure 2. The chip was fabricated using two 1.1mm-thick Borofloat wafers (Precision Glass & Optics, Santa Ana, CA). Wafers were cleaned for 5 min in piranha solution prior to Analytical Chemistry, Vol. 77, No. 17, September 1, 2005
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all deposition steps. Then, 100 nm of Cr followed by 100 nm of Au was deposited on both sides of wafers using a Temescal electron beam evaporation instrument. Standard photolithography was performed on one side using Shipley 1813 positive photoresist (Rohm & Haas, Mission Viejo, CA), a chromium mask (University of Minnesota Nanofabrication Center, Minneapolis, MN), and a MA-6 aligner (Karl Suss, Munich, Germany). Manufacturer’s procedures for spinning, baking, exposing, and developing of the photoresist were used. Following photolithography, the glass was unmasked with Cr and Au etchants. Channels were etched into the unmasked regions for 1 min to a depth of 20 µm. Metal masks were removed, and the glass wafer was cleaned in piranha solution. Electron beam deposition was repeated laying down 20and 150-nm layers of Ti and Au, respectively. Photolithography was repeated with a mask for patterning electrodes into the side channels, and unwanted metal was removed. Access holes (1-mm diameter) were drilled by the UMN Electrical Engineering/ Computer Science (EE/CS) Machine Shop in an unprocessed wafer with an ultrasonic diamond drill oscillating at 25 kHz at a feed of 1/8 in./min. Drilled wafers were cleaned with piranha solution, and a-Si was deposited using plasma enhanced chemical vapor deposition (PECVD, Plasma-Therm, St. Petersburg, FL). A diced rectangular wafer was placed on the separation channel region to prevent deposition of a-Si in the separation channel. The drilled, a-Si deposited wafer was aligned with the etched, electrodedeposited wafer on the wafer chuck of a SB-6 bonder (Karl Suss, Munich, Germany), and 900 V was applied for 2 h at 450 °C and 5 µbar. The voltage was applied to the a-Si deposited wafer with the other wafer grounded. If bonding was not complete, the wafer was rotated and bonding was repeated. Chips were diced into rectangles with a 1.5-cm border around access holes. Nanoports (Upchurch Scientific, Oak Harbor, WA) were attached to the access holes using manufacturer’s procedures. Electrodes were connected to wires using silver conductive epoxy (MG Chemicals). The chip was perfused with 0.1 M NaOH until the channels were clear (150 min) to remove unwanted a-Si. SEM Characterization. A 50-nm Ti layer was deposited on an etched wafer using electron beam evaporation. A 1 × 1 cm square region centered on the sample channel inlet into the separation channel was diced. The resulting sample was mounted on a stage and loaded into a JEOL JSM-6500F field emission scanning electron microscope (Peabody, MA) for imaging. Instrumentation and Data Collection. A SMZ 1500 stereomicroscope (Nikon Corp., Tokyo, Japan) mounted with a 100-W Hg lamp (Nikon Corp.) and Spot Insight Qe CCD camera (Diagnostic Instruments, Sterling Heights, MI) was used for fluorescence imaging. The microscope was equipped with an Endow GFP long-pass emission filter cube (Nikon Corp) containing a band-pass filter (450-490 nm), dichroic mirror (495-nm cutoff), and a long-pass filter (500-nm cutoff). A 1.6× objective was used for collection at 0.75× zoom for all experiments. The entire 1-cm cross section of the separation channel could be imaged at this magnification. MetaVue software (Downington, PA) was used for image collection and processing. Analysis of the electropherograms was performed using Cutter 5.0.22 (22) Shackman, J. G.; Watson, C. J.; Kennedy, R. T. J. Chromatogr., A 2004, 1040, 273-282.
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Figure 3. SEM image of the sample inlet channel (center vertical channel) intersection with the separation channel. Connection channels to the separation buffer inlet (left of sample inlet channel) and the electrode channel (right of sample inlet channel) are also shown.
µ-FFE Separations. Separation buffer was pumped into the separation channel inlet at 25-50 µL/min and the electrode buffer inlets at 10-20 µL/min using syringe pumps (Harvard Apparatus, Holliston, MA). Flow rates were chosen to give nearly equal linear velocities within the separation and electrode channels. Fluorescent standards were pumped at 1% of the separation buffer flow rate (i.e., 0.25-0.5 µL/min). Separation voltages (0-562 V) were applied at the left electrode using a CZE1000R high-voltage power supply (Spellman High Voltage Co, Hauppauge, NY), and the resulting currents were recorded. The electric field in the separation channel ranged from 0 to 283 V/cm at these voltages. Analyte detection was performed in the separation channel, 2.6 cm downstream from the sample inlet. Safety Considerations. Caution: piranha solution self-heats to ∼70 °C and is extremely caustic. RESULTS AND DISCUSSION Wet chemical etching was used to generate the features of the µ-FFE chip shown in Figure 1B. The key features of the chip from a fabrication standpoint are the 1 cm × 5 cm × 20 µm separation channel, the inlet channel, and the membrane channels at the top and sides of the separation channel. The purpose of the side channels is to isolate flow over the electrodes from that traveling through the separation channel. Similar channels could be incorporated into the outlet of the separation channel to facilitate fraction collection. There was some concern about being able to incorporate features of such varying sizes on the same device. SEM was used to characterize the etching process. Figure 3 is an SEM image of the upper right corner of the 1 × 5 cm separation channel (see Figure 1B). All of the microscale features of the chip are represented in this region. The sample inlet channel and side channels were 20 µm deep × 50 µm wide, the expected aspect ratio for isotropic etching. The etched channels are uniform and smooth aside from a small, infrequent defect in the sample inlet channel. The sample channel and the individual side channels are completely isolated from one another. One of the motivations for fabricating a µ-FFE chip in Borofloat was to allow higher voltages to be applied than was feasible on earlier devices fabricated in silicon. Electric breakdown through
Figure 4. (A) Fluorescence image of an analyte stream containing 500 µM fluorescein, 1 mM rhodamine 110, and 1 mM rhodamine 123 in the absence of an electric field. (B) µ-FFE separation of the same sample stream at 259 V/cm into four components: (1) fluorescein, (2) rhodamine 110 impurity, (3) rhodamine 110, and (4) rhodamine 123.
the a-Si layer deposited during anodic bonding was a potential concern. Resistivity measurements of various dimension a-Si wires deposited on a glass wafer were made in order to confirm that the 90-nm a-Si intermediate film was not conductive. Voltages as high as 15.5 kV were applied to a 90 nm thick × 8 cm long × 1 cm wide a-Si wire without any detectable current. Based on these measurements, the resistivity of the a-Si layer can be estimated to be >475 kΩ‚cm, significantly higher than the 3.9 kΩ‚cm of the separation buffer. No evidence of significant current through the a-Si layer was observed in any electrophoresis experiments performed here. A group of fluorescent standards were used to characterize the fully fabricated device. Figure 4A shows the analyte stream observed when a mixture of fluorescein, rhodamine 110, and rhodamine 123 is introduced into the µ-FFE device in the absence of any applied voltage. A smooth sample stream was observed as the analyte was carried through the separation channel under laminar flow conditions. The angle of the stream is determined by the relative flow rates of the buffer entering the separation chamber and the electrode channels. The slight drift toward the center of the separation channel observed in Figure 4A indicates that the pressure exerted by the electrode buffer is higher than that of the separation buffer at the flow rates used in this example. In the current experiment, this proved to be a useful way of moving analytes toward the middle of the separation channel to facilitate simultaneous detection of cationic and anionic analytes. In future designs, it may prove more reliable to move the sample inlet channel to the center of the separation channel if both anions and cations are to be analyzed simultaneously. Figure 4B demonstrates a µ-FFE separation of the same three fluorescent standards when 259 V/cm is applied across the separation channel. The maximum current of the high-voltage power supply (310 µA) was reached at this voltage, preventing the examination of higher separation potentials. The separation order follows the trend expected considering the charge of the analytes. Fluorescein bears a -2 charge at pH 7 and is deflected toward the anode. Similarly, rhodamine 123 is cationic and is deflected toward the cathode. Rhodamine 110 is neutral and is only minimally affected by the applied voltage. A fluorescent
Figure 5. Line scans of µ-FFE separations of (1) fluorescein, (2) rhodamine 110 impurity, (3) rhodamine 110, and (4) rhodamine 123 at increasing electric fields. The linear velocity of the buffer in the separation channel was 2.7 mm/s during separations, yielding a residence time in the electric field before detection of 9.6 s.
Figure 6. Resolution as a function of electric field in the separation channel. Baseline resolution was achieved for all fluorophore pairs and showed a linear trend. The linear velocity of separations was 5.4 mm/s, yielding a residence time of 4.8 s.
impurity was detected as well and by its migration order can be inferred to bear a -1 charge. An important difference between CE and µ-FFE is that µ-FFE allows the separation to be monitored continuously as analytes stream past the detector. As shown in Figure 5, a line scan across the analyte streams can be used to generate electropherograms analogous to those observed in CE. The time between subsequent scans is only limited by the imaging rate of the CCD (typically 1 Hz). Figure 5 also demonstrates the effect of varying the electric field in the separation channel. Baseline resolution of the fluorescent standards was observed at fields as low as 82 V/cm. Figure 6 plots resolution of the fluorescent standards as a function of the electric field in the separation channel. As expected, resolution increases linearly with increasing electric field. It should be noted that the electric field is not constant across the µ-FFE chip. The side channels used to isolate fluid flow in the separation channel from that in the electrode channel change the cross-sectional area of the buffer, creating a high-resistance region. Consequently, the electric field gradient is higher in the side channels than in the Analytical Chemistry, Vol. 77, No. 17, September 1, 2005
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Figure 7. Ω plot of voltage vs current measured while pumping 25 mM HEPES buffer through the µ-FFE chip at various linear velocities.
separation channel. Considering the dimensions of the side channels (average width 35 µm, depth 20 µm, and length 1 mm), 50% of the voltage difference applied between the electrodes will occur in the separation channel with the remaining 50% occurring within the side channels. This compares well with previous designs where only 4.45% of the applied voltage was experienced in the separation channel.15 This is important considering that resolution improves with increasing electric field. A total of 515 V had to be applied to achieve 259 V/cm in the separation channel. Other µ-FFE designs require higher separation potentials to be applied to achieve the same electric field. Many CE separations are limited by the Joule heating experienced as the separation voltage is increased. A similar effect might be expected to limit µ-FFE, especially considering the higher cross-sectional area of the separation channel and the higher currents that this gives rise to. An Ω plot was generated to determine whether significant Joule heating was occurring (see Figure 7). Joule heating is detected in an Ω plot as a positive deviation from the ideal linear plot of current versus voltage. This deviation is the result of a decrease in buffer viscosity and, therefore, resistance caused by Joule heating. No positive deviation is observed in Figure 7, suggesting that Joule heating is not significant at the electric fields studied here. On the contrary, a negative deviation was actually observed, suggesting that resistance was increasing with increasing electric field. This can be attributed to the generation of electrolysis products. The currents observed in µ-FFE are larger than those typically observed in CE and generate O2 and H2 bubbles at the anode and cathode, respectively. The bubbles disrupt both fluid flow and the electric field. As shown in Figure 7, increasing the flow rate through the electrode channels decreased bubble formation and gave rise to more linear Ω plots. (23) Lukacs, K. D.; Jorgenson, J. W. J. High. Resolut. Chromatogr. 1985, 8, 407411. (24) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 28582865.
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As noted previously, application of the electric field only had a modest effect on the neutral rhodamine 110 stream. This suggests that little if any electroosmotic flow (EOF) was generated in the µ-FFE chip. This was surprising considering that Pyrex is known to yield an EOF of ∼5 × 10-4 cm2/V‚s at pH 7.23 Borofloat is a similar borosilicate glass and would be expected to exhibit a comparable EOF. Previous µ-FFE chips exhibited EOF when fabricated in Pyrex24 and PDMS.15 The EOF observed in our device was ∼2 orders of magnitude lower. A possible explanation is that the borosilicate channel surfaces are modified during anodic bonding. This is unlikely considering that the chips are rinsed with NaOH before use, which would be expected to regenerate the channel surfaces. A second possibility is that the flow through the µ-FFE channels is fast enough to destabilize the double layer necessary to generate EOF. CONCLUSIONS We have demonstrated the fabrication of a µ-FFE device using insulating substrates. Fabrication was facilitated by anodically bonding two glass wafers with a 90-nm a-Si intermediate bonding film. Baseline separation of three fluorescent standards and an impurity differing by a single charge was achieved even when only modest potentials were applied. Bubble formation from water electrolysis proved to be the limiting factor of the device, preventing higher potentials from being applied. This limitation would appear to be surmountable, and we are currently exploring next-generation µ-FFE designs that will minimize the effects of electrolysis, allowing higher electric fields to be applied. If this limitation is overcome, µ-FFE is a technique with great potential. Purification of microliter samples is an obvious application. While interesting, as is the case with other miniaturized techniques, the analytical applications of µ-FFE are likely to have greater impact than preparative applications. µ-FFE should prove particularly attractive to monitoring applications that require high-speed separations. Because there is no discrete injection in µ-FFE, changes in analyte concentration can be monitored continuously over time. ACKNOWLEDGMENT Funding for this research was provided by the National Institute of Health (GM 063533). Special thanks are given to Anthony Ratkovich for his SEM expertise, the University of Minnesota Nanofabrication Center (NFC) for use of instrumentation and equipment necessary for the µ-FFE chip fabrication, and the following NFC staff: Kathy Burklund for lithography and metal deposition training, Kevin Roberts for wafer dicing training, Tony Whipple for PECVD, ellipsometry, and Tylan for furnace training, and Suzanne Miller for many useful fabrication comments. Received for review May 4, 2005. Accepted June 30, 2005. AC050766N
