Electrokinetic Focusing in Microfabricated Channel Structures

Electrokinetic Focusing in Microfabricated. Channel Structures. Stephen C. Jacobson and J. Michael Ramsey*. Oak Ridge National Laboratory, P.O. Box 20...
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Anal. Chem. 1997, 69, 3212-3217

Electrokinetic Focusing in Microfabricated Channel Structures Stephen C. Jacobson and J. Michael Ramsey*

Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142

Glass microchips are fabricated to demonstrate twodimensional spatial confinement of ions and fluids using electrokinetic forces. Neutral and charged molecular samples can be electrokinetically focused to widths as narrow as 3.3 µm in a 18 µm long by 18 µm wide chamber. The electrokinetic focusing characteristics are tested using cationic, neutral, and anionic samples to evaluate the influence of electric fields on the sample stream. Focusing is achieved using electrophoresis, electroosmosis, or a combination of the two. In addition, concentration enhancement is observed in the focusing chamber by employing sample stacking, and the focusing chamber is multiplexed to confine spatially more than one sample stream simultaneously. There has been recent interest in utilizing micromachining techniques to fabricate miniaturized chemical instrumentation.1 Most of the reported work has dealt with liquid-phase chemical separation techniques. Electrically driven separations such as capillary electrophoresis,2-7 synchronized cyclic electrophoresis,8 free-flow electrophoresis,9 open-channel electrochromatography,10 micellar electrokinetic capillary chromatography,11 capillary gel electrophoresis,12-14 and a two-dimensional obstacle course for electrophoretic sizing of DNA fragments15 have been demonstrated. Structures that perform chemical reactions include arrays for solid-phase chemistry,16 reaction wells for polymerase chain (1) Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adv. Chromatogr. 1993, 33, 1. (2) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926. (3) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Lu ¨ di, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253. (4) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481. (5) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895. (6) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107. (7) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114. (8) Burggraf, N.; Manz, A.; Effenhauser, C. S.; Verpoorte, E.; de Rooij, N. F.; Widmer, H. M. J. High Resolut. Chromatogr. 1993, 16, 594. (9) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2858. (10) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369. (11) Moore, A. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184. (12) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949. (13) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11348. (14) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676. (15) Volkmuth, W. D.; Austin, R. H. Nature 1992, 358, 600. (16) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767.

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reactions,17 channels with immobilized enzymes for flow injection analysis,18 and stacked modules for flow injection analysis.19 The first monolithic devices that integrated reactions with analysis are precolumn20 and postcolumn derivatization21 with capillary electrophoresis and DNA restriction digestions with fragment sizing.22 Recently, a hybridized device for PCR amplification and capillary electrophoresis analysis was reported.23 Many of these devices have demonstrated advantages analogous to those of microelectronics including compact geometries and high speed analysis while having simple and reliable operation. Primary areas left to address are low-cost manufacturing and packaging and parallel architectures for solving large-scale problems. As the area of micromachined chemical instrumentation continues to grow, more sophisticated devices are being fabricated and tested that have increased functionality and, consequently, greater complexity. Fluid manipulations on-chip have been used for mixing of reagents, injection or dispensing of samples, and separations. Microchip devices are by no means limited to these functions. Flow cytometry has commonly been used for particle counting24 and, more recently, for sensitive fluorescence measurements.25 By incorporation of transverse spatial confinement of the sample, the probe volume of the detector can be reduced and isolated from container walls permitting more sensitive measurements to be made. Microfabricated sheath flow devices have been reported,26 but external pumps were used to deliver the focusing and sample flows. Unfortunately, on-chip mechanical pumps to deliver fluids have not been fabricated with great success, and other mechanisms of material transport such as electrokinetic phenomena, i.e., electroosmotic and electrophoretic transport, which are easily incorporated into microfabricated devices are of interest. We define electrokinetic focusing as the use of electrokinetic transport to confine spatially both fluids and ions. Electrokinetic focusing differs from hydrodynamic focusing, which uses pressure-driven flow to achieve confinement. The results (17) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 1815. (18) Murakami, Y.; Takeuchi, T.; Yokoyama, K.; Tamiya, E.; Karube, I.; Suda, M. Anal. Chem. 1993, 65, 2731. (19) Fettinger, J. C.; Manz, A.; Ludi, H.; Widmer, H. M. Sens. Actuators, B 1993, 17, 19. (20) Jacobson, S. C.; Koutny, L. B.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127. (21) Jacobson, S. C.; Hergenro¨der, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472. (22) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720. (23) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M.A. Anal. Chem. 1996, 68, 4081-4086. (24) Moldavan, A. Science 1934, 80, 188. (25) Dovichi, N. J.; Martin, J. C.; Jett. J. H.; Keller, R. A. Science 1983, 219, 845. Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553. (26) Sobek, D.; Senturia, S. D.; Gray, M. L. Proceedings from Solid-State Sensor and Actuator Workshop, Hilton Head, SC, 1994; p 260. S0003-2700(96)01093-1 CCC: $14.00

© 1997 American Chemical Society

Figure 1. Schematic of a microchip with an electrokinetic focusing chamber. The circles depict reservoirs which contain fluids labeled by function.

however are comparable, as discussed below. Observation of focusing effects using electroosmotic transport was previously observed on microchips.6 Here, we describe the fabrication of a two-dimensional focusing chamber in a glass substrate and the characterization of its performance using electrically driven sample and focusing streams. Samples are confined using electrophoresis, electroosmosis, or a combination of the two, and cationic, neutral, and anionic dyes are used to evaluate these various conditions. EXPERIMENTAL SECTION The microchips are fabricated using standard micromachining techniques as previously described.6 The channel design, e.g., Figure 1, is transferred onto the substrates using a positive photoresist, photomask, and UV exposure. The channels are etched into the substrate using a dilute, stirred HF/NH4F bath. To form the closed network of channels, a cover plate is bonded to the substrate over the etched channels by hydrolyzing the surfaces, bringing them into contact with each other, and processing thermally to 500 °C. Four different microchips with the architecture shown in Figure 1 were investigated. Microchip A had channels with a width of 18 µm and a depth of 6.4 µm; microchip B had channels with a width of 64 µm and a depth of 9.0 µm; microchip C had channels with a width of 50 µm and a depth of 10.4 µm; and microchip D had channels with a width of 61 µm and a depth of 8.5 µm. A multiplexed electrokinetic focusing structure, microchip E, had sample, focusing, and waste channels with a width of 42 µm and a depth of 8.6 µm and a focusing chamber with a width of 360 µm, a length of 71 µm, and a depth of 8.6 µm. (The architecture of the multiplexed microchip is described in greater detail below.) These dimensions are measured using a stylus-based surface profiler, and the channel widths and lengths are reported at half-depth. The reservoirs are affixed with epoxy at the point where the channel extends beyond the cover plate. The electroosmotic mobility for microchips A, B, C, and E is that of native glass, but electroosmotic mobility is minimized for microchip D by covalent immobilization of linear polyacrylamide.27 Electrical contact with the reservoirs is made using platinum wire. (27) Hjerten, S. J. Chromatogr. 1985, 347, 191.

Figure 2. Schematic of the focusing process. Arrows depict direction of fluid transport or electrostatic forces on positive ions.

Microchip performance is monitored by laser-induced fluorescence (LIF) using a charge-coupled device (CCD) for imaging. For CCD imaging, the argon ion laser beam (514.5 nm, 100 mW) is expanded to ≈5 mm diameter at the microchip surface using a lens. The fluorescence signal is collected using an optical microscope, filtered spectrally (550 nm cut-on), and measured by the CCD. The buffer is 10 mM sodium tetraborate, and the samples are rhodamine 6G (40 µM), rhodamine B (40 µM), and disodium dichlorofluorescein (60 µM) in 10 mM buffer. For the stacking experiment, the sample is rhodamine 6G (40 µM) in 50 µM buffer. These experiments are performed under continuous infusion of the sample through the focusing chamber. The microchip is capable of performing injections, but only the transport characteristics of the chamber are discussed here. For the experiments involving the focusing of a single sample stream, the potentials at the sample, focusing 1, and focusing 2 reservoirs are controlled by individual power supplies, and the waste reservoir is grounded. No potential is applied to the buffer and sample waste reservoirs; i.e., the reservoirs are floated. The relative potentials at the reservoirs are varied to enhance or diminish the degree of lateral focusing. For the focusing of multiple sample streams, each sample and focusing reservoir is controlled with an individual power supply, and the waste reservoir is grounded. Figure 2 shows a schematic of the sample focusing where the sample and focusing channels intersect perpendicular to each other as in the microchip design in Figure 1. The sample is introduced from the top of the vertical channel in a continuous fashion. Transport from the two side channels, focusing 1 and 2, confines the sample stream. The sample stream reaches a minimum and then broadens because of diffusion. Small differences in channel lengths and widths require that the voltages applied to the focusing 1 and 2 reservoirs differ slightly to balance the field strengths and to obtain a symmetric fluid transport in the focusing chamber. The sample field strength is defined as the electric field strength in the sample channel, and correspondingly, the focusing field strength is the electric field strength in the focusing channels. All sample stream profiles were measured at full width at half-maximum (fwhm) at the exit of the focusing chamber (inlet of the waste channel), and three images were taken for each set of applied voltages. With the magnification of the Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

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Figure 3. CCD images of focusing for rhodamine 6G with (b) 3.9 kV, (c) 2.9 kV, and (d) 1.9 kV applied to the sample reservoir of microchip A. The potentials applied to the focusing 1, focusing 2, and waste reservoirs are 2.4, 2.6, and 0 kV, respectively. Arrows depict direction of transport.

Figure 4. Variation of sample stream width (rhodamine 6G) (O) and focusing field strength (0) versus sample field strength in microchip A with 2.4 and 2.6 kV applied to focusing 1 and 2 reservoirs, respectively.

microscope used in most of the experiments, the width of a pixel of the CCD camera corresponded to a length of 0.3 µm on the microchip. Assuming the experimental conditions, e.g., relative conductivities of the buffers, applied voltages, and electroosmotic flow, remained unchanged, the variability of the stream width over time was within a pixel width in all cases measured. RESULTS AND DISCUSSION The operation of the two-dimensional focusing is shown visually in Figure 3 using CCD images. The sample stream was focused to widths of 9.2, 5.6, and 3.3 µm in panels b-d, respectively, of Figure 3, using microchip A. The focusing chamber is the region where the two channels intersect, and for microchip A, the chamber is 18 µm long by 18 µm wide. The brighter regions in the images show the fluorescence from the sample, rhodamine 6G. For these images the potential at the focusing 1 and 2 reservoirs are held constant with the voltage at the sample reservoir varied. As the potential at the sample reservoir is decreased, the sample field strength decreases, and the focusing field strength increases resulting in a tighter focus of the sample stream, as shown in Figure 4. In Figure 3b-d, the sample field strengths are 1.12, 0.61, and 0.11 kV/cm, respectively, 3214 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

Figure 5. Sample stream concentration profiles taken 3 µm upstream from the focusing chamber (profile 1), 9 µm into the chamber (profile 2), 18 µm into the chamber and at the tightest focus (profile 3), and 60 µm downstream of the chamber (profile 4). These profiles are extracted from the image in Figure 3d, and the amplitudes are normalized to compare sample stream widths. The focusing chamber begins at the top of the intersecting channels.

and the focusing field strengths are correspondingly 0.85, 1.01, and 1.16 kV/cm. Sample transport through the focusing chamber stops when the potential at the intersection exceeds the potential applied to the sample reservoir; i.e., the direction of transport in the sample channel reverses. In Figure 5, the intensity profiles are extracted from the image in Figure 3d. The profiles are normalized in order to compare relative peak widths. Profile 1 is taken before the sample enters the focusing region and is 3 µm upstream from the intersection. This profile is similar for any cross-section taken along the sample channel. Profile 2 is 9 µm into the chamber, i.e., in the center of the focusing chamber, and is partially focused. Profile 3 is 3.3 µm wide (fwhm) at the exit of the chamber. Profile 4 is 60 µm downstream of the focusing chamber. Profile 4 is dispersed slightly relative to profile 3 due to diffusion of the sample stream into the adjacent focusing streams. In Figure 6, the variation of the sample stream width is plotted with increasing sample field strength for four different sets of potentials applied to the focusing 1 and 2 reservoirs. Essentially, as the field strength in the focusing channels increases, the degree of focusing increases. For example, with focusing potentials of 0.3 kV, the focusing field strength is 0.15 kV/cm, yielding a sample stream width of 5.8 µm. With ≈2.5 kV applied to the focusing reservoirs, the sample stream width is 3.3 µm and the focusing field strength is 1.20 kV/cm. A 1.8-fold decrease in the sample stream width is observed for an 8-fold increase in the focusing field strength. In addition, only for the highest focusing potential (≈2.5 kV) does the stream width appear to be asymptotically approaching a minimum in Figure 6. The stream width is also dependent upon the width of the sample channel. In Figure 7 the stream widths are compared for two microchips with sample channel widths of 18 µm for microchip A and 64 µm for microchip B. The sample stream widths are 3.3 µm for microchip A and 6.3 µm for microchip B. For a 3.6-fold decrease in channel width, the sample stream width is 1.9 ()x3.6) times narrower for microchip A than for microchip B. Comparable focusing field strengths of 1.20 and 1.25 kV/cm are used for

Figure 6. Variation of sample stream width with sample field strength for rhodamine 6G with 0.31 and 0.33 kV (4), 0.61 and 0.65 kV (0), 1.2 and 1.3 kV (]), and 2.4 and 2.6 kV (O) applied to focusing reservoirs 1 and 2, respectively, of microchip A.

Figure 7. Variation of sample stream width with sample field strength for microchip A with a channel width of 18 µm (O) and for microchip B with a channel width of 64 µm (]). Rhodamine 6G is the sample.

microchips A and B, respectively. This quadratic dependence between sample stream width and channel width can be understood if the transverse focusing force is assumed to be constant over the width of the channel and that this force is opposed by diffusion. The sample and focusing channels are assumed to be of equal width, and the electric field strengths to be equivalent, as in our experiments. The focusing force is applied for a proportionally longer time with wider channels, and likewise, diffusion has a longer period over which to spread the sample stream with the diffusion distance depending on the square root of time. Also, the focusing for the wider channel of microchip B does not appear to reach a minimum stream width as it does with microchip A. In Figure 8, the focusing of a cationic dye, rhodamine 6G, and a neutral dye, rhodamine B, is compared. These samples are used to compare electrokinetic focusing by purely electroosmotic transport (rhodamine B) and both electroosmotic and electrophoretic transport (rhodamine 6G). Using a focusing field strength of 1.20 kV/cm, the sample stream widths are 3.3 µm for rhodamine 6G and 3.9 µm for rhodamine B. The 0.6 µm difference

Figure 8. Variation of sample stream width with sample field strength for rhodamine 6G (0) and rhodamine B (O) using microchip A.

Figure 9. Variation of sample stream width with sample field strength for rhodamine 6G (4) and dichlorofluorescein (]) using microchip C with electroosmotic transport and for rhodamine 6G (0) and dichlorofluorescein (O) using microchip D without electroosmotic transport. For dichlorofluorescein the polarity of the high-voltage power supplies is reversed to effect anionic electrophoretic flow.

is presumably due to the electrophoretic contribution in the case of the cationic dye. In Figure 9, sample focusing is performed with and without electroosmotic transport for a cationic dye (rhodamine 6G) and an anionic dye (dichlorofluorescein). In the case where electroosmotic transport is minimized by the presence of covalently immobilized polyacrylamide in the channels, electrophoretic forces are used to effect the focusing using voltages of appropriate polarity. For microchip C with electroosmotic flow, the sample channel width is 50 µm and the focusing field strength is 0.3 kV/cm. These conditions result in sample stream widths of 9.7 µm for rhodamine 6G and 11.2 µm for dichlorofluorescein. The reduced focusing observed for the anionic dye is expected for the cathodic electroosmotic flow conditions employed because the electrophoretic force on the anions will tend to spread the ions rather than focus them. For microchip D without electroosmotic flow, the sample channel width is 61 µm and the focusing field strength is 0.3 kV/cm. These conditions give sample stream widths of 15.0 µm for rhodamine 6G and 15.6 µm for dichlorofluorescein. As expected, cations and anions of similar electroAnalytical Chemistry, Vol. 69, No. 16, August 15, 1997

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Figure 10. (a) CCD image of focusing under stacking conditions for rhodamine 6G (45 µM in 50 µM buffer) with a 10 mM focusing buffer using microchip A. The potentials applied to the sample (S), focusing 1 (F1), focusing 2 (F2), and waste (W) reservoirs are 3.0, 2.4, 2.6, and 0 kV, respectively. (b) Trace from the CCD image showing the observed concentration enhancement of 2.9 times.

phoretic mobilities focus to similar extents using potentials of opposite polarities. Clearly, a mixture of cations and anions cannot be focused simultaneously, and neutrals are not focused at all using only electrophoresis. Electrokinetic focusing can also be performed with sample and focusing buffers having different conductivities. By having a sample in a buffer with a lower conductivity than that of the focusing buffer, stacking of the sample at the boundary between the two buffers occurs. Consequently, a concentration enhancement can be observed in addition to confinement of the sample by the focusing buffers. Figure 10a shows an image of an electrokinetic focusing experiment with sample stacking (the image is rotated 90° relative to the images in Figure 3). Microchip A is used with 3.0 kV applied to the sample reservoir and with 2.4 and 2.6 kV applied to the focusing 1 and 2 reservoirs. The brighter region indicates more intense fluorescence. Figure 10b is a plot of the signal intensity of the CCD pixels along a line going through the center of the sample channel in Figure 10a. For these conditions, the fluorescence signal increases by a factor of 2.9 as the sample traverses the center of the focusing cell. At the exit of the focusing chamber and point of tightest focus, the concentration of the sample is 2.4 times greater than that under nonstacking conditions. Sample stacking does not continue downstream of the focusing chamber probably because the concentration gradient between the sample and focusing buffers disappears due to diffusion, and dilution of the sample by the focusing streams occurs, as is the case for the other experiments. The focusing chamber can also be multiplexed on a planar surface to achieve lateral spatial confinement of more than one sample stream. In Figure 11a, two sample channels (S1 and S2) terminate in the focusing chamber and the focusing channels (F1, F2, and F3) are positioned adjacent to the sample channels. Lateral confinement occurs when the flow of buffer in the focusing channels is greater than the flow of sample in the sample channels. The focusing channel F2 in the middle of the microchip serves to confine the sample streams in the two adjacent sample channels conserving the total number of focusing channels needed. This design can obviously be extended to larger numbers of sample streams as necessary. In Figure 11b, two sample streams are focused simultaneously to an average width of 8 µm using the multiplexed focusing 3216 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

Figure 11. (a) CCD image of a multiplexed focusing chamber with two sample channels (S1, S2) and three focusing channels (F1, F2, F3). (b) CCD image of focusing for two rhodamine 6G streams using microchip E with field strengths of 40 V/cm in S1 and S2, 400 V/cm in F1 and F3, and 750 V/cm in F2.

microchip. The sample channels have a center-to-center spacing of 160 µm for this microchip. The average field strength in the two sample channels (S1 and S2) is 40 V/cm, the average field strength in the two outside focusing channels (F1 and F3) is 400 V/cm, and the field strength in the center focusing channel (F2) is 750 V/cm. The field strength in the center focusing channel is almost twice as high as that in the outside focusing channel because this channel helps focus two sample streams. The potentials at each reservoir are individually controlled in order to tune the focusing and achieve a symmetrically focused sample stream. This allows greater flexibility for studying flow dynamics but would not be necessary once a microchip design is finalized. Another multiplexed microchip design was initially studied which had a similar span for the focusing chamber but had a length of 5 mm instead of 70 µm as in Figure 11a. Focusing was achieved with the open focusing chamber design, but spatial confinement improved ≈1.6 times by having the three channels converge into a closely spaced waste channel; i.e., F1, S1, and F2 converge into W1, and F2, S2, and F3 converge into W2. In conclusion, two-dimensional spatial confinement of materials has been demonstrated using electrokinetic forces in a microfabricated channel structure. The goal of this work was to show that a microfabricated element similar to a sheath flow cuvette could potentially be monolithically integrated with other microchip elements such as chemical separations. Fluid dynamics calculations are underway to help determine the optimum geometry for the focusing chamber. Focusing may improve by introducing the focusing streams at acute angles relative to the sample channel, increasing the width of the focusing channels relative to the sample channel, or constricting the focusing chamber width. Also, devices which incorporate three-dimensional electrokinetic focusing should be considered. This would allow further reduction of the probe volume and thus the background signal from the substrate material. Applications for the electrokinetic focusing

effects reported in this paper include flow cytometry and ultrasensitive fluorescence measurements.

96OR22464. The authors thank Justin E. Daler for assistance in fabricating the microchips.

ACKNOWLEDGMENT This research was sponsored by the Department of Energy Office of Research and Development. Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under Contract DE-AC05-

Received for review October 24, 1996. Accepted May 29, 1997.X AC961093Z X

Abstract published in Advance ACS Abstracts, July 1, 1997.

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