Anal. Chem. 2005, 77, 7933-7940
Microchip Separations in Reduced-Gravity and Hypergravity Environments Christopher T. Culbertson,* Yogesh Tugnawat, Amanda R. Meyer, Gregory T. Roman, J. Michael Ramsey,† and Steve R. Gonda‡
Department of Chemistry, 111 Willard Hall, Kansas State University, Manhattan, Kansas 66506
Microfabricated fluidics technology, e.g., lab-on-a-chip devices, offers many attractive features for performing chemistry and biochemistry on space-based platforms. We have constructed a portable, battery-operated microfluidic platform that was tested under reduced gravity and hypergravity conditions that would be experienced in space flight and launch. This device consisted of a microchip, microchip holder, two 0-8-kV high-voltage power supplies, a high-voltage switch, a solid-state diodepumped green laser, an optical train, a channel photomultiplier, and an inertial mass measurement unit all under the control of a laptop computer and powered by 10 D-cell alkaline batteries. The unit was tested on NASA’s reduced gravity research aircraft at gravity levels that are relevant to NASA’s intended use of bioreporterbased microchips for environmental monitoring of space and planetary environments on manned and unmanned spacecraft. Over the course of two flights, 834 fast electrophoretic separations of four amino acids were performed under a variety of gravitational environments including zero-g, Martian-g, lunar-g, and ∼1.8-g. All separations were performed in less than 12 s and automatically analyzed. After correction with an internal migration standard, the migration time reproducibilities were all 40 periods where the gravitational pull is effectively 1.8g. Mechanical and acoustical vibrations can be severe on the aircraft with the operating noise levels in the cabin at ∼80 dB. In addition, the aircraft and, therefore, the instrumentation is not level during parabolic flight. Going into or coming out of each parabola, the aircraft is generally 45° nose up or nose down with minor variations in both pitch and yaw as the aircraft is under “automatic” pilot control. The parabolic flight environment creates hydrodynamic pressures in a microfluidic device that are amplified by increased gravitational forces during the “pull-outs” after the microgravity portions of the flight. Not only does the gravitational force change multiple times during a flight but it does so rapidly generally going from ∼0 to ∼1.8g in just over 2 s. These acceleration gradients can affect the equilibrium flow conditions in the chip and, therefore, the injections of analytes. Negative g-spikes also occur, and this can affect the fluid and gas distribution in the microchip reservoirs leading to the potential introduction of gas bubbles into the microchip channels. Other nonoptimal conditions for analysis include slight variations in the atmospheric pressure while transitioning through the parabolic maneuvers, no humidity control, and large temperature variations from ground to flight level. (The temperature and humidity variations are not unique to this aircraft but rather caused by the typical weather conditions found in the Houston, TX, region.) Finally, because instruments must be powered down for takeoff and there is little time for warm-up prior to the initiation (25) Chabinyc, M. L.; Chiu, D. T.; McDonald, J. C.; Stroock, A. D.; Christian, J. F.; Karger, A. M.; Whitesides, G. M. Analytical Chemistry 2001, 73, 44914498. (26) Mayers, B. T.; Vezenov, D. V.; Vullev, V. I.; Whitesides, G. M. Anal. Chem. 2005, 77, 1310-1316. (27) 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. (28) Skelley, A. M.; Scherer, J. R.; Aubrey, A. D.; Grover, W. H.; Ivester, R. H.; Pascale, E.; Grunthaner, F. J.; Bada, J. L.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1041-1046. (29) Renzi, R. F.; Stamps, J.; Horn, B. A.; Ferko, S.; Vandernoot, V. A.; West, J. A. A.; Crocker, R.; Wiedenman, B.; Yee, D.; Fruetel, J. A. Anal. Chem. 2005, 77, 435-441.
7934 Analytical Chemistry, Vol. 77, No. 24, December 15, 2005
Figure 1. Schematic of microchip channel manifold. Critical channel lengths are indicated.
of the experiments, therefore, electrical component warm-up, i.e., laser and photomultiplier tube, issues can also arise. Successful operation of a microfluidic device under such conditions should be a good measure of its general robustness. In this paper, we report on the development and testing of a prototype portable microfluidic system for use in reduced and hypergravity situations. EXPERIMENTAL METHODS Chemicals and Sample Derivatization. Sodium tetraborate, sulfuric acid, hydrochloric acid, ammonium hydroxide, and amino acids were obtained from Acros Organics (Morris Plains, NJ). 1/10 Buffered Oxide Etch (BOE) and Chromium Mask Etchant were obtained from Transene Co., Inc. (Danvers, MA). Microposit Developer was obtained from Shipley (Marlborough, MA). 5-Carboxytetramethylrhodamine succidimidyl ester (TAMRA-SE) was obtained from Molecular Probes (Eugene, OR). All of the chemicals were used as received. The amino acids were individually derivatized with TAMRA as described by Molecular Probes protocols.30 All solutions were made using distilled, deionized water from a Barnstead Nanopure System (Dubuque, IA) and then filtered through 0.45-µm Acrodiscs (Gelman Sciences; Ann Arbor, MI). Microchip Design and Fabrication. The channel manifold layouts for the microfluidic devices were designed using AutoCAD2000LT (Thomson Learning; Albany, NY). Precision laser photoplots were generated from these designs by The Photoplot Store (www.photoplotstore.com; Colorado Springs, CO) at 8000 dpi. The channel design and channel lengths are shown in Figure 1. Microfluidic devices were fabricated from soda lime glass (Telic Co.; Santa Monica, CA) using standard photolithographic and wet chemical etching techniques modified from that described previously.31 In brief, a chrome and photoresist-coated 10.12 cm × 10.12 cm × 0.152 cm soda lime plate was covered with the photoplot. These photoplots consisted of eight individual 5.08 cm × 2.54 cm chip designs. The photoresist on the plate below the clear areas in the mask was exposed to UV light for 10 s at a (30) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes: Eugene, OR, 1996. (31) Jacobson, S. C., Hergenroder, R., Koutny, L. B., Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118.
Figure 2. Photograph of portable microfluidic device flown on NASA’s microgravity research aircraft.
power of 45 mJ/cm2 using a flood exposure system (ThermoOriel; Stratford, CT). The exposed plate was submerged in a stirred solution of Microposit Developer for 90 s, followed by a thorough rinse with 18 MΩ‚cm water. The plate was then submerged in the stirred Chrome Mask Etchant for 3 min, rinsed with 18 MΩ‚ cm water, and dried with inert gas. The channels were wet chemically etched using the stirred BOE. Prior to use, the BOE was diluted with 18 MΩ‚cm water and mixed with concentrated 12 M HCl in the volumetric proportions 1:4:2, respectively. The channel dimensions were measured periodically during etching using a stylus-based surface profiler (Ambios Technology; Santa Cruz, CA). Once the channels reached the desired depth, the remaining photoresist was removed by immersing the plate in the stirred Chrome Mask Etchant for 10 min. The plate was then rinsed with 1 M sulfuric acid followed by18 MΩ‚cm water. The etched plate was cut into the eight individual chips using a dicing and cutting saw (model EC-400; MTI Corp.). Holes were mechanically drilled into the 10.12 cm × 10.12 cm × 0.152 cm cover plates prior to bonding (Technical Glass Inc.; Aurora, CO). These cover plates were then cut into eight individual plates to match the substrates with the etched channels. The eight individual chips were cleaned with acetone and rinsed thoroughly with ethanol followed by 18 MΩ‚cm water. Next, the chips were submerged in a stirred 3.5 M sulfuric acid solution for 5 min, rinsed with 18 MΩ‚cm water, and then immersed in a Versa-Clean Liquid soap solution (Fisher Scientific; Pittsburgh, PA). The chips were sonicated (3510 Branson) for 15
min in the soap solution, rinsed with 18 MΩ‚cm water, and dried with inert gas. The chips were placed in the above-described dilute BOE solution for 10 s, rinsed with 18 MΩ‚cm water, and placed into a dilute hydrolysis solution for 12 min at 60 °C. The dilute hydrolysis solution consisted of 1 part NH4OH, 1 part H2O2, and 2 parts water. Following hydrolysis, the chips were sonicated in flowing 18 MΩ‚cm water for 60 s before joining. The chips were removed from the flowing water and placed on a Cleanroom Wiper (DURX 670; Great Barrington, MA). The cover plates were removed from the flowing water and placed on top of the respective chip. Binder clips were fastened on the perimeter of the chip to ensure contact between the two surfaces. The joined chip was then placed in the oven at 95 °C for 15 min to drive out any water in the channels. After the channels were dry, the chips were placed into an oven at 565 °C to generate an irreversibly sealed device. Finally, reservoirs and epoxy rings were obtained from Upchurch Scientific (Oak Harbor, WA) and attached as specified by the supplier. The channel depth and widths for the chip used to generate the data reported below were 11 µm deep and 36 µm wide at halfdepth for the narrow channels and 11 µm deep and 230 µm wide at half-depth for the wide channels indicated in Figure 1. Portable Device Design. The portable microfluidic instrument was enclosed in an ABS/PC blended box (NBA-10148; Bud Industries Inc.; Willoughby, OH). The box was 28 cm wide × 38.4 cm tall × 16 cm deep and hinged on the front as shown in Figure 2. The box was designed so that it could be powered through Analytical Chemistry, Vol. 77, No. 24, December 15, 2005
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either typical 110 V/60 Hz line voltage or 10 D-cell batteries. The power source was selectable through the actuation of a front panel switch. In either case, the voltage was downregulated to 12, 5, or 3 V as necessary to power the individual components in the box. A master power switch (emergency kill switch) and individual power switches for the laser and channel photomultiplier were also mounted on the front panel. The box was outfitted with two 0-8 kV power supplies (C80; EMCO High Voltage Corp.; Sutter Creek, CA) and a high- voltage switch (K81C; Kilovac; Santa Barbara, CA) to enable electrophoretic separations with gated injections to be performed. The supplies and switch were mounted on the PC control board on the inside of the door of the portable device and controlled using LabVIEW through a PCI-6036E multifunction I/O card (National Instruments, Inc.; Austin, TX) in a Dell laptop computer (Round Rock, TX). The laser-induced fluorescence detection system was mounted on the rear inside wall of the portable device. The excitation source consisted of a 5-mW, 532-nm solid-state diode pumped (SSDP) green laser pointer (The Laser Guy; Houston, TX). The battery compartment of the laser was removed and the leads connected to two D-cell batteries. The laser beam was reflected off a longpass dichroic mirror (560DRLP; Omega Optical; Brattleboro, VT) and focused using a 40× objective (CD-240-M40X, Creative Devices; Mechanic Station, NJ) into a small spot in the separation channel 2.0 cm below the cross-intersection. The fluorescence emission was collected with the same objective, passed through the dichroic mirror, spatially filtered with a 1-mm pinhole (Oriel; Stratford, CT), and spectrally filtered using a 595-nm band-pass filter (595AF60); Omega Optical). The signal was then detected and amplified using a channel photomultiplier (cPMT) module (MD972; Perkin-Elmer Optoelectronics; Santa Clara, CA). The gain on the cPMT module was controlled through a 10-turn potentiometer on the front panel of the portable device as suggested by the manufacturer. The signal from the cPMT module was sampled at 200 Hz using the same PCMCIA 6036E card that was used to control the high-voltage power supplies and switch. To be able to accurately synchronize the separation data with the “effective” gravitational environment, a three-axis inertial mass measurement unit (“accelerometer,” ADXL105EM-3; Analog Devices; Norwood, MA) was incorporated into the portable instrument on the same PC board as the HV power supplies and switch. The three outputs of the accelerometer were sampled at the same rate as the cPMT output using the same LabVIEW program and data acquisition card. In addition to the cPMT signal and the three outputs from the accelerometer, the cPMT gain and the return current to the power supplies were also sampled and acquired by LabVIEW using the same PCMCIA 6036-E card. Sampling of the return current, i.e., the current from the chip, was used to monitor the continuity of the fluidic connections and to monitor the system for Joule heating effects. All data analysis was performed using in-house-written LabVIEW code and Igor Pro (Wavemetrics; Oswego, OR). Microchip Holder. The microchips were held in place over the microscope objective in a custom-machined cartridge on an x-y positioner (XY1; Thor Labs). This cartridge also held the highvoltage power lines needed for the electrophoretic separations. To prevent liquid spills, all of the reservoirs were covered with 7936
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silicone rubber. Platinum electrodes were inserted through the rubber to make contact with the buffer solutions in the reservoirs. In addition, to prevent any pressure buildup in the reservoirs, they were all punctured with 26-gauge syringe needles. Separations. Prior to each flight, microchips were prepared, loaded into the instrument, and optically aligned and the separations tested on the ground. Chip preparation consisted of flushing a 50/50 (v/v) 1 M NaOH/methanol solution through the channels, followed by water and then run buffer. The solutions were pulled through for 5 min each. Just prior to flight, the reservoirs were loaded with 80 µL of sample or buffer and then covered with silicone. The electrophoretic separations were carried out in a pH 9.3, 25 mM sodium tetraborate buffer. Gated injections between 0.025 and 0.1 s were performed to introduce the sample into the separation channel. The separation distance was 2.0 cm, and the field strength in the separation channel was ∼800 or ∼1000 V/cm. During flight, runs were made consecutively and no reservoir refilling was performed. Laser light scattering off of the channel walls was used to align the device. Data Analysis. To analyze the data obtained on the two flights, a LabVIEW program was written. Peak locations and heights were identified using the built-in LabVIEW peak find subvi after each electropherogram was smoothed using a Butterworth filter. Peak widths were determined from the first-derivative plot of the electropherogram. Peak area and the number of theoretical plates generated were calculated from this information based upon the assumption that the peaks were Gaussian in nature. RESULTS AND DISCUSSION The experiments reported below were performed over the course of two flights on NASA’s reduced gravity program’s KC135 research aircraft. On each flight ∼40 parabolas were performed. The flights lasted for 1.25-1.5 h. Near the apex of each parabola, ∼25 s of ∼0g were experienced. Between these ∼0g episodes, periods of 1-1.8 effective g were experienced. As experiment operator incapacitation due to motion sickness is not uncommon on the KC-135, the experiments were automated in order to ensure that all of the ∼0g opportunities/flight could be used. This required that a simple sample be chosen so that a separation could be completely accomplished from injection to detection in
