Anal. Chem. 1999, 71, 4455-4459
Microfluidic Devices for Electrokinetically Driven Parallel and Serial Mixing Stephen C. Jacobson,* Timothy E. McKnight, and J. Michael Ramsey
Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142
Microfabricated devices for parallel and serial mixing of fluids are demonstrated. To simplify the voltage control hardware, electrokinetic mixing is effected using a single voltage source with the channels dimensioned to perform the desired voltage division. In addition, the number of fluid reservoirs is reduced by terminating multiple buffer, sample, or analysis channels in single reservoirs. The parallel mixing device is designed with a series of independent T-intersections, and the serial mixing device is based on an array of cross intersections and sample shunting. These devices were tested by mixing a sample with buffer in a dilution experiment. Sample fractions of 1.0, 0.84, 0.67, 0.51, 0.36, 0.19, and 0 were generated for the parallel mixing device, and sample fractions of 1.0, 0.36, 0.21, 0.12, and 0.06 for the serial mixing device. Microfabricated devices for performing chemical and biochemical assays have garnered increased attention over the last several years.1-3 Considerable effort has been dedicated to developing functional elements to be incorporated into these labon-a-chip devices. A key issue is sample and reagent mixing. With electrokinetic material transport, reagents are mixed in proportions dictated by the applied potentials, the geometry of the channels, and the properties of the materials in those channels. To date, the proportioning of two or more fluids in different ratios has been accomplished by controlling the electric potentials applied to the fluidic reservoirs to effect dilution,4 reactions,5,6 and solvent programming.7,8 These have required voltage control external to the microchip, e.g., programmable power supplies. (1) Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. In Advances in Chromatography; Brown, P. R., Grushka, E., Eds.; Marcel Dekker: New York, 1993; Vol. 33, pp 1-66. (2) Colyer, C. L.; Tang, T.; Chiem, N.; Harrison, D. J. Electrophoresis 1997, 18, 1733-1741. (3) Jacobson, S. C.; Ramsey, J. M. In High-Performance Capillary Electrophoresis; Khaledi, M. G., Ed.; John Wiley & Sons: New York, 1998; Vol. 146, pp 613633. (4) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (5) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407-3412. (6) Salimi-Moosavi, H.; Tang, T.; Harrison, D. J. J. Am. Chem. Soc. 1997, 119, 8716-8717. (7) Kutter, J. P.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 51655171. (8) Kutter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297. 10.1021/ac990576a CCC: $18.00 Published on Web 09/08/1999
© 1999 American Chemical Society
Some simple integrated devices have combined precolumn9 and postcolumn10-12 derivatization reactions in conjunction with electrophoretic separations. In addition, restriction digestions13 and competitive immunoassays14 have been coupled to product analysis downstream. More features being integrated into a planar format have led to studying compact microchip designs,15 fabricating arrays of channels for DNA analysis,16,17 and multiple sample PCR with product analysis.18 Coupled to this increased integration is the added complexity of the hardware needed to control microfluidic operations. One approach to simplify the control hardware for electrokinetic manipulations is to design the fluidic channels to perform the appropriate voltage division for reagent mixing and reactions. In this paper, we describe microfluidic designs that simplify the voltage control necessary to effect parallel and serial electrokinetic mixing on microchips. If the fluidic channels provide the appropriate voltage division, only a single fixed voltage source is required to transport and mix material. This minimizes the highvoltage hardware necessary to operate the microfluidic chip. In an effort to make the microchip architecture compact, multiple buffer, sample, or analysis channels terminate in single reservoirs. To test the parallel and serial mixing schemes, a sample was diluted with buffer in proportions dictated by the channel geometries. EXPERIMENTAL SECTION The microchips were fabricated as described previously.19 Briefly, photomasks were fabricated by sputtering chrome films (50 nm) onto glass substrates (3011, Gold Seal Products), spinning (9) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132. (10) Jacobson, S. C.; Koutny, L. B.; Hergenro¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476. (11) Fluri, K.; Fitzpatrick, G.; Chiem, N.; Harrison, D. J. Anal. Chem. 1996, 68, 4285-4290. (12) Mangru, S. D.; Harrison, D. J. Electrophoresis 1998, 19, 2301-2307. (13) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723. (14) Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44, 591-598. (15) Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1998, 70, 3781-3789. (16) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181-2186. (17) Simpson, P. C.; Roach, D.; Woolley, A. T.; Thorsen, T.; Johnston, R.; Sensabaugh, G. F.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2256-2261. (18) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176. (19) Jacobson, S. C.; Ermakov, S. V.; Ramsey, J. M. Anal. Chem. 1999, 71, 32733276.
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Table 1. Channel Dimensions, Calculated and Experimental Sample Fractions, n, for the Parallel Mixing Device channel
Ls (mm)a
A1 A2 A3 A4 A5 A6 A7
4.9 5.0 9.6 9.5 24.8 15.0
Lb (mm)a
La (mm)a
n (calc)b
n (exp)
14.7 24.5 10.4 10.5 4.8 4.7
30.1 39.7 39.8 39.9 39.8 39.7 30.1
0 0.83 0.68 0.52 0.34 0.16 1.0
0 0.84 0.67 0.51 0.36 0.19 1.0
a The average channel width and depth are 54 and 10 µm, respectively. b The sample fraction, n, is calculated using eq 2.
Table 2. Channel Dimensions, Calculated and Experimental Sample Fractions, n, for the Serial Mixing Device channel
length (mm)a
n (calc)b
n (exp)
A1 A2 A3 A4 A5
12.0 11.9 11.8 11.9 12.0
1.0 0.37 0.22 0.12 0.052
1.0 0.36 0.21 0.13 0.059
a Other channel lengths: channel D is 5.7 mm, channel S is 15.6 1 1 mm, channels D2-D5 are 0.2 mm, channels B1-B5 are 0.1 mm, channels S2-S5 are 0.4 mm, and channel S6 is 5.0 mm. The average channel width and depth are 24 and 5.5 µm, respectively. b The sample fraction, n, is calculated using eq 6.
positive photoresist (1811, Shipley) onto the chrome films, and exposing the microchip designs into the photoresist using a CAD/ CAM laser machining system (Ar+, 457 nm). Subsequently, the chrome films were etched (CeSO4/HNO3; Transene Co.). The channel designs were then transferred onto the glass substrates using positive photoresist and UV flood exposure (360-440 nm for 30 s; UXM-501MA, Ushio). After the photoresist was developed (MF319, Shipley), the channels were etched into the substrate in a dilute, stirred HF/NH4F bath (Transene Co.); 2-mm-diameter channel access holes were drilled in the cover plates (3011; Gold Seal Products). To form the closed network of channels, the cover plates were bonded to the substrates over the etched channels by hydrolyzing the surfaces (NH4OH/H2O2; J. T. Baker/EM Science), bringing the cover plates into contact with the substrates, and annealing to 500 °C. Cylindrical glass reservoirs were then affixed on the cover plates using epoxy. The microchip dimensions are listed in Tables 1 and 2 for the parallel and serial mixing devices, respectively. The output of a single programmable high-voltage power supply (10A12-P4, Ultravolt) was applied directly to the sample and buffer reservoirs with the waste reservoir(s) grounded. Input to the power supply was computer controlled using a multifunction I/O card (NB-MIO16XL-42, National Instruments) and Labview 4.1 (National Instruments). Fluid manipulations were monitored by fluorescence detection using rhodamine B (40 µM for the parallel mixing experiments and 100 µM for the serial mixing experiments; Exciton). Twodimensional (2D) images were acquired using an optical microscope (Nikon) and a CCD camera (Princeton Instruments). The spatial uniformity of the excitation source was calibrated by flowing 4456
Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 1. Schematic of mixing at a T-intersection with a sample channel, a buffer channel, and an analysis channel. Equations 1 and 2 describe operation. Arrows depict direction of flow.
an equal concentration of dye through each channel of the microchip. The relative intensity of the fluorescence was used to correct the signal in the mixing experiments for nonuniform illumination. The buffer in all reservoirs was 10 mM sodium tetraborate (pH 9.2; EM Science) for the parallel mixing experiments and 20 mM sodium tetraborate (pH 9.2) for the serial mixing experiments. RESULTS AND DISCUSSION Parallel Mixing Device. Electrokinetic mixing of two reagents at a T-intersection for homogeneous buffer and channel properties is depicted in Figure 1, where the arrows indicate the direction of fluid flow. The current flowing through the three channels satisfies,
iSj + iBj ) iAj
(1)
where iX is the current in the sample (S), buffer (B), and analysis (A) channels and j is the channel number. For these experiments, the microchips were designed with channels having similar cross-sectional areas; thus channel resistance is proportional to channel length. In addition, the same electrical potential is applied to both the sample and buffer reservoirs. With homogeneous buffer and channel properties, the fluid flow in each channel is proportional to the current. The flow of sample into the analysis channel relative to the total fluid flow in the analysis channel is the sample fraction, n, and can be estimated,
nj )
iSj iSj + iBj
)
LBj LSj + LBj
(2)
where LX is the channel length. For example, when the sample and buffer channels have the same length, the sample fraction is 0.5. These T-intersections can be easily replicated to produce an array of independent mixing devices each having two inputs and one output. The channel resistances of the inputs can be designed to proportion fluids in either similar or dissimilar mixing ratios, allowing an array of mixing elements to be controlled with a single voltage source. Figure 2 shows a microchip with an array of seven analysis channels and five T-intersections to produce seven different sample fractions between 0 and 1. The sample fraction in channels B1 and A1 was 0 and in channels S7 and A7 was 1.0. The sample fraction in the analysis channels A2-A6 depended on the relative lengths (resistances) of the respective sample and buffer channels.
Figure 3. (a) Fluorescence image for parallel mixing of the sample with buffer. A voltage of 1.0 kV was applied to the buffer and sample reservoirs with the waste reservoir grounded. Arrow depicts direction of flow. (b) Average fluorescence signal in the analysis channels from the image in (a). See Table 1 for calculated and experimental sample fractions.
Figure 2. Schematic of the microchip for parallel electrokinetic mixing. The circles depict the sample, buffer, and waste reservoirs, the sample, buffer, and analysis channels are labeled “S”, “B”, and “A”, respectively, and the T-intersections are labeled “T”. The channels are represented by double lines, and two channels emanate from each sample and buffer reservoir. See Table 1 for channel dimensions. The signal presented in Figure 3 is obtained from the region enclosed by the rectangle. Drawing is not to scale.
In Table 1, the lengths of each of the channels are listed with the calculated sample fraction. The microchip was designed such that each T-intersection and analysis channel operate independently from the others and the electric field strengths in all of the analysis channels are nearly constant. Having similar field strengths in the analysis channels simplifies calibration and analysis of the fluorescence signal because photobleaching of the sample is similar. The parallel mixing device in Figure 2 was tested by mixing a sample with buffer in a dilution experiment and monitoring the relative fluorescence signal after dilution. In Figure 3a, a CCD image shows the relative intensities of the fluorescence from the sample for different sample fractions. A voltage of 1.0 kV was applied to the sample and buffer reservoirs, and the CCD images were taken just below where channels B1 and S7 join channels A1 and A7, respectively (see Figure 1). The brighter regions indicate a higher sample fraction in the analysis channel. In Figure 3b, the average fluorescence signal is plotted for the CCD image in Figure 3a, and from this plot, the measured sample fractions were determined (see Table 1). The values in Table 1 are corrected for the nonuniform illumination of the excitation source. The calculated and experimental sample fractions correspond well to each other. The largest discrepancy is for channels A5 and A6 where the sample fraction is slightly higher than expected. This discrepancy can be attributed to the widths of channels B5 and B6 being narrower than the other channels, resulting in a 5%
Figure 4. Schematic of mixing at a cross intersection with sample and buffer input channels and sample and analysis output channels. Equations 3-5 describe operation. Arrows depict direction of flow.
higher resistance than estimated from their channel lengths. The difference in channel widths occurred during writing of the photomask used for fabricating the microchip. With this correction, the calculated sample fractions, n, for channels A5 and A6 are 0.35 and 0.17, respectively, and fall within experimental error. Moreover, these microchips can easily be calibrated following fabrication to handle subtle differences in performance by measuring either the electrical resistances in the channels or the optical response for a test mixture. Serial Mixing Device. The cross intersection is the basic unit for the serial mixing device and in Figure 4 is composed of two inputs, sample (Sk) and buffer (Bk) channels, and two outputs, sample (Sk+1) and analysis (Ak) channels. As depicted in Figure 4, the mixing of two reagents (sample and buffer) at the cross intersection for a homogeneous buffer and channel system can be described by,
iSk + iBk ) iAk + iSk+1
(3)
where k is the channel number. In addition, one of the two Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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Figure 5. Schematic of the microchip for serial electrokinetic mixing. The circles depict the sample, buffer, waste-1, and waste-2 reservoirs, and the sample, buffer, and analysis channels are labeled “S”, “B”, and “A”, respectively. The channels are represented by double lines. See Table 2 for channel dimensions. The signal presented in Figure 6c is obtained from the region enclosed by the rectangle. Drawing is not to scale.
following criteria must be met for the output sample channel to contain a mixture of sample and buffer,
iSk > iAk or iBk < iSk+1
(4)
The output sample channel, similar to the analysis channel of the T-intersection in Figure 1, contains a mixture of the two input channels. Unlike the T-intersection, a portion of the input sample is shunted away for analysis and the extent of shunting determines the relative fractions of the two input materials in the sample output channel. The analysis channel has the dual function of providing a measurement region and acting as a sample shunt to assist in the delivery of a lower flow rate of sample from the input sample channel to the output sample channel. This concept can be extended by making the sample output the input to another cross intersection, repeating as often as desired to generate a series of reagent fractions in parallel analysis channels. A serial mixing device using five cross intersections is shown in Figure 5. The number of cross intersections and resistances of the input and output channels can be tailored for the desired mixing scenario. The serial mixing device schematically depicted in Figure 5 is pictured in Figure 6a. The sample flow from channel S1 is split between channels A1 and S2, and buffer from channel B1 is mixed with sample in channel S2 (arrows depict flow direction). The sample concentration in channels A1 and S1 is equal. The sample 4458 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 6. (a) White light image of the serial mixing manifold. (b) Fluorescence image of the serial mixing of the sample with buffer. A voltage of 0.4 kV was applied to the buffer and sample reservoirs with the waste-1 and waste-2 reservoirs grounded. Arrows depict direction of flow. (c) Average fluorescence signal in the analysis channels from the image in Figure 4b with 0.4 kV applied (solid line) and with 1.6 kV applied (dashed line). See Table 2 for calculated and experimental sample fractions.
in channel S2 is then split between channels S3 and A2, and buffer from channel B2 is mixed with sample in channel S3. The sample concentration in channels A2 and S2 is equal. Channels D1-D5 distribute the buffer to each of the cross intersections. The flow splitting and mixing continues across the channel manifold in a similar fashion, and in this example, the sample is further diluted with buffer at each cross intersection. The microchip in Figure 5
has five analysis channels, but the series of tee and cross intersections can continue for as many mixing intersections as needed. The sample fraction, n, in the output sample channel of a single cross intersection can be estimated by,
nk+1 )
iSk - iAk iSk+1
)1-
iBk iSk+1
(5)
for k g 1. The sample fraction in channel A1 is 1.0, and the sample fraction in channels A2-A5 can be estimated by, m
nm+1 )
∏ k)1
( ) 1-
iBk
iSk+1
(6)
The currents in each of the channels for the microchip in Figure 5 were modeled using SPICE. Again, in these experiments the channels have approximately the same cross-sectional areas, so the resistance was varied by adjusting the channel length. Also, use of narrow channels allowed more rapid equilibration of the mixed sample and buffer streams in channels S2-S5. To test the serial mixing device in Figure 6b, a sample was diluted with buffer. Figure 6b shows a fluorescence image of the sample being mixed with buffer with 0.4 kV applied to the sample and buffer reservoirs with the waste-1 and -2 reservoirs grounded. The fluorescence signal decreases from A1 to A5 due to mixing of the sample with buffer. Figure 6c shows the average fluorescence signal for the analysis channels taken from Figure 6b, and the calculated and experimental sample fractions are listed in Table 2. The calculated sample fractions are corrected for a 15% difference between widths of channels S2-S5 and channels D2D5 incurred during fabrication of the photomask used for the microchip. These dimensions were obtained using a stylus-based surface profiler. In addition, the experimental sample fractions in Table 2 are corrected for nonuniform excitation for the 2D fluorescence images. In Table 2, the calculated and experimental sample fractions compare well and are within experimental error.
As seen in Figure 6b, the sample and buffer combined in channels S2-S5 require enough time to equilibrate diffusively before being mixed at the subsequent cross intersection. If sufficient time to equilibrate is not allowed, then the sample fraction in the succeeding channels is incorrect. This can be observed in Figure 6c (dashed line) when a voltage of 1.6 kV is applied to the microchip resulting in a 4-fold increase in the electrokinetic velocity in all channels. Because the sample is introduced on the lower half of channel S2, a disproportionate amount of this sample is shunted down channel A2, leading to an inflated average signal. Because the total sample entering the channel manifold is conserved, analysis channels A3-A5 consequently have a lower than projected sample fraction. In conclusion, the voltage control hardware is simplified for electrokinetic mixing by using a single voltage source and channels having appropriate geometries. The parallel mixing device was designed using a series of T-intersections, and the serial mixing device is based on an array of cross intersections and sample shunting. The parallel mixing device, as designed here, has the advantage that each set of channels is independent of the others. Consequently, a failure in one set of channels does not affect the operation of the others. The dynamic range of the serial mixing device is larger especially if the sample shunting is carried out with additional cross intersections added to the channel manifold beyond the fifth cross intersection. Both mixing scenarios enable parallel chemical reaction kinetics to be determined and could find numerous applications in chemical and biochemical analysis. ACKNOWLEDGMENT This research is sponsored by the U.S. Department of Energy, Office of Research and Development. Oak Ridge National Laboratory (ORNL) is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy under Contract DE-AC0596OR22464. The authors thank S. Shane Frank for assistance with SPICE and Justin E. Daler, Christopher D. Thomas, and John W. Cockfield for fabrication of the microchips. Received for review June 1, 1999. Accepted July 30, 1999. AC990576A
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