Anal. Chem. 2004, 76, 6053-6057
Strategy for Repetitive Pinched Injections on a Microfluidic Device Christopher D. Thomas, Stephen C. Jacobson,† and J. Michael Ramsey*
Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142
A microfluidic valve was fabricated with a cross intersection and two tee intersections in close proximity and evaluated for repetitive pinched injections. Electrokinetic forces were used to mobilize the sample and control diffusive transport at a cross intersection to produce sample plugs of short axial extent in an analysis channel similar to the standard pinched valve. The addition of a tee intersection in the sample channel maintained the sample close to the injection valve under “pullback” conditions allowing more rapid loading into the cross intersection. A second tee intersection allowed unidirectional transport in the analysis channel enabling loading of subsequent injections during an analysis. The two tee intersections were each located 80 µm from the cross intersection. Injection frequencies of 1, 2.5, 5, and 10 Hz were tested with a duty cycle of 0.5 for sample loading and dispensing. With 1 kV applied to the microchip during dispensing, the relative standard deviation of the peak areas for 15 injections was 1.6%. The peak width (4σ) for the repetitive injections increased from 71 to 96 µm compared to a standard pinched injection due to the presence of the tee intersection in the analysis channel. With the continued development of microfluidic technologies,1,2 more features are being integrated into a planar format to perform chemical and biochemical assays. This increased level of integration requires assay performance to be optimized and microchip architectures to be designed with increased sophistication and efficiency. Central to fluid manipulations on microfabricated devices is sample and reagent dispensing. Basic valve designs include a tee intersection,3 double tee intersection,4 and cross intersection.5,6 The cross intersection has been used for both the * To whom correspondence should be addressed. E-mail: jmramsey@ mail.unc.edu. Present address: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599. † Present address: Department of Chemistry, Indiana University, Bloomington, IN 47405. (1) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (2) Auroux, P.-A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (3) Harrison, D. J.; Manz, A.; Fan, Z.; Luedi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (4) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642. (5) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113. (6) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132. 10.1021/ac035475y CCC: $27.50 Published on Web 09/14/2004
© 2004 American Chemical Society
constant-volume valve, i.e., pinched injection,5 and the variable volume valve, i.e., gated injection.6 The small axial length of the injected samples using these valving techniques has enabled highperformance, electrokinetically driven separations to be performed including capillary electrophoresis,4,7-10 synchronized cyclic electrophoresis,11 capillary gel electrophoresis,12-14 open channel electrochromatography,15 and micellar electrokinetic chromatography.10,16,17 More recently, chromatographic and electrophoretic separations have been serially coupled for analyzing peptides using two-dimensional separations.18-20 The small dead volume connections between the first- and second-dimension separation channels and use of the gated valve have contributed to the high performance of these two-dimensional separations. In these applications and others, dispensing performance for the gated injection is essentially limited by the rise and fall times of the high-voltage power supplies. Compared to the gated valve, the pinched valve has the advantage of constant-volume dispensing resulting in decreased sample bias and is preferable for analyzing samples that have components with markedly different electrophoretic mobilities while minimizing the axial extent of the injected plug. However, a disadvantage of the pinched injection is the time required to reload the sample for subsequent injections. During dispensing of the pinched injection, the sample in the sample channel is withdrawn from the intersection to minimize excess sample (7) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (8) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118. (9) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476-3480. (10) Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2000, 72, 5814-5819. (11) Burggraf, N.; Manz, A.; Effenhauser, C. S.; Verpoorte, E.; de Rooij, N. F.; Widmer, H. M. J. High Resolut. Chromatogr. 1993, 16, 594-596. (12) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949-2953. (13) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (14) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680. (15) Kutter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297. (16) Kutter, J. P.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 51655171. (17) von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (18) Rocklin, R. D.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 52445249. (19) Gottschlich, N.; Jacobson, S. C.; Culbertson, C. T.; Ramsey, J. M. Anal. Chem. 2001, 73, 2669-2674. (20) Ramsey, J. D.; Jacobson, S. C.; Culbertson, C. T.; Ramsey, J. M. Anal. Chem. 2003, 75, 3758-3764.
Analytical Chemistry, Vol. 76, No. 20, October 15, 2004 6053
Figure 1. Schematic of microfluidic device used for repetitive pinched injections. The lines represent microfabricated channels, and the shaded circles represent fluid reservoirs. Dashed lines highlight the region used for the images in Figures 2 and 3. The letters r and s indicate the detection points for the repetitive and standard injections, respectively.
leaking into the analysis channel. Reloading the sample for subsequent injections requires the sample be returned to the cross intersection. Furthermore, electrokinetic flow in the analysis channel is reversed during sample loading to confine the sample to the intersection and potentially allows previously injected samples to travel back to the cross intersection. The gated valve, on the other hand, keeps the sample at the cross intersection enabling rapid, repetitive injections, while maintaining unidirectional electrokinetic flow of the injected samples in the analysis channel. In this paper, we discuss the design and operation of a modified pinched injection that allows for rapidly reloading the sample and maintains unidirectional electrokinetic flow in the analysis channel for analyzing previously dispensed samples. The valve was evaluated with applied potentials of 0.5, 1.0, and 2.0 kV and frequencies of 1, 2.5, 5, and 10 Hz using two-dimensional imaging and detection at a single point. EXPERIMENTAL SECTION The microchip shown in Figure 1 was designed and fabricated using standard micromachining methods. Briefly, a positive photomask was commercially fabricated on a chrome-coated soda lime glass substrate (HTA Photomask, San Jose, CA) from a CAD design (Vectorworks; Nemetschek North America, Columbia, MD). The microchip substrate (SL-4006-2C-AR3-AZ1350; Hoya, Inc., Shelton, CT) was white crown glass covered with a thin chrome film (100 nm), an antireflective coating, and a positive photoresist. The microchip design was then transferred onto the substrate by UV flood exposure. The exposed photoresist was developed (MF-319; Shipley, Marlborough, MA), the chrome film etched (Chromium Etchant; Transene Co., Danvers, MA), and the substrate immersed in a dilute sulfuric acid solution as a cleaning step. Channels were etched into the substrate using a dilute, stirred buffered oxide etchant (10:1; Transene Co.). Access 6054
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holes were ultrasonically drilled (Sonic Mill, Albuquerque, NM) at the ends of the etched channels, and remaining photoresist and chrome film were removed. The drilled substrate and an uncoated coverplate were then hydrolyzed, joined, thermally ramped, and held at 550 °C for 10 h to permanently anneal the substrate and coverplate. Short segments of glass tubing (1/4-in. o.d. × 3/16-in. i.d. × 1/4 in.) were epoxied to the drilled side of the bonded substrate to serve as fluid reservoirs. Microchip channels were treated with 1 N NaOH prior to use. Prior to bonding, the channel dimensions were measured using a stylus-based profiler (P-10; Tencor, Santa Clara, CA) and were 10.4-µm deep and 24.0-µm wide at half-depth. Channel lengths were measured using a linear measuring system (2-LMH and MicroCode II; Boeckeler Instruments, Tucson, AZ) mounted on the stage of an inverted microscope (TE300; Nikon, Melville, NY). For resistance measurements, the channels were filled with a 2 mM sodium tetraborate and 100 mM sodium chloride buffer, and a potential of 0.3 kV from a high-voltage power supply with six independent high-voltage sources (2866A; Bertan High Voltage, Hicksville, NY) was applied between each pair of reservoirs. The resulting current measured by the power supply was digitized using a multifunction I/O card (PCI-MIO-16XE-50; National Instruments, Austin, TX) and read into LabView (6i; National Instruments). The channel resistances and field strengths were then calculated using Ohm’s Law and Kirchoff’s Rules.21 The resistances in channels c1 and c2 (see Figure 2) were estimated by multiplying the channel length by the specific resistivity determined in the other channels and dividing by the cross sectional area. The channel lengths, channel resistances, applied potentials, and electric field strengths are summarized in Table 1. The electric field strengths reported were calculated using the measured resistances, channel lengths, and applied potentials. The percent electroosmotic flows in various channels were then calculated from the electric field strengths. Optical images were obtained using the inverted microscope equipped with a 10× objective lens, a high-pressure mercury lamp, and a CCD camera (MicroMAX:512BFT; Roper Scientific, Trenton, NJ). The CCD exposure time was 50 ms. For detection at a single point in a channel with laser-induced fluorescence, an argon ion laser beam (514.5 nm, ∼7.7 mW; 543R-AP-A01; Melles Griot, Carlsbad, CA) was focused onto the channel. Fluorescence was collected with a 40× objective (CD-240-M40; Creative Devices, Neshanic Station, NJ), spatially filtered with a 1-mm pinhole, spectrally filtered with a 580-nm band-pass filter (580DF30; Omega Optical, Battleboro, VT), and detected with a photomultiplier tube (PMT; R928; Hamamatsu, Bridgewater, NJ). The PMT signal was amplified (SR570; Stanford Research Systems, Sunnyvale, CA) and digitized using the multifunction I/O card. For the repetitive injections, the detection point was positioned 200 µm from the cross intersection in the analysis channel, and for the standard pinched injection the detection point was positioned 200 µm from the cross in the buffer-1 channel. The detection points for the repetitive and standard injections are indicated in Figure 1. For the imaging and detection at a single point, the buffer was 20 mM boric acid and 100 mM Tris (tris(hydroxymethyl)aminomethane), and rhodamine B (50 µM in the buffer; Eastman Kodak, Roch(21) Seiler, K.; Fan, Z. H.; Fluri, K.; Harrison, D. J. Anal. Chem. 1994, 66, 34853491.
dispensing for the repetitive and standard injections are listed in Table 1, and the values listed are multiplied by 0.5, 1, or 2 for the different experiments. Fifteen replicate injections were performed at 1, 2.5, 5, and 10 Hz, and at each frequency, the duty cycle was 0.5 with equal times spent in the sample loading and dispensing steps. In addition, the influence of the electroosmotic flow from the buffer-2 channel into the analysis channel during the dispense mode was evaluated by applying 0.6, 0.65, and 0.7 kV to the buffer-2 reservoir, while maintaining all other potentials as listed in Table 1.
Figure 2. White light image of (a) the repetitive pinched injection valve and fluorescence images of (b) sample loading and (c, d) dispensing. In (b) the arrows indicate the direction of the electroosmotic flow in all channels during sample loading, and in (c, d) the arrows indicate the change in the direction of the electroosmotic flow in channels c1 and c2 during dispensing. For the applied potentials and field strengths, multiply the values in Table 1 by 0.5. Table 1. Channel Lengths (L), Channel Resistances (R), Applied Potentials (V), and Electric Field Strengths (E) for Repetitive and Standard Pinched Injections dispense load
repetitive
standard
L R V E V E V E channel (mm) (MΩ) (V) (V/cm) (V) (V/cm) (V) (V/cm) sample waste-1 buffer-1 waste-2 analysis buffer-2 c1 c2
26.0 11.3 5.7 15.8 6.1 11.9 0.08 0.08
80 900 35 300 18 400 49 0 19 0 36 1100 0.25 0.25
200 60 60 230 600 630 150 30
800 0 1000 0 0 650
150 370 1000 270 680 200 220 490
800 0 0 0 1000 650
150 360 720 260 950 190 210 1200
ester, NY) served as the sample. Rhodamine B is neutral in this buffer and, thus, is a molecular tracer that follows the electroosmotic flow. For performing the repetitive injections, four of the six independent sources on the high-voltage supply were used to actively control the potentials applied to the reservoirs and were under computer control. The waste-2 and analysis reservoirs were held at ground. The potentials applied for sample loading and
RESULTS AND DISCUSSION Figure 1 shows a schematic of the microchip used for repetitive pinched injections, and the channel lengths, channel resistances, applied potentials, and electric field strengths are listed in Table 1. The microchip was designed with two additional tee intersections 80 µm from the cross intersection (center-to-center): intersection t1 located in the sample channel and intersection t2 located in the analysis channel (see Figure 2a). Intersection t1 in the sample channel allowed for sample to be routed toward the waste-1 reservoir during the dispensing step instead of being shuttled back toward the sample reservoir. This also permitted rapid reloading of the sample for subsequent injections, leaving only the 80-µm gap (channel c1) for the sample to traverse for the next injection. The addition of intersection t2 enabled unidirectional electrokinetic flow toward the analysis reservoir throughout all microchip functions, and thus an injected plug could be analyzed while new sample was being loaded and spatially confined in the cross intersection. Buffer from the buffer-2 channel was split between channel c2 and the analysis channel during the sample loading mode. Figure 2 shows fluorescence images of an injection cycle with arrows depicting the direction of the electroosmotic flow. The sample was electroosmotically transported from the sample channel into the waste-1 and waste-2 channels during sample loading (Figure 2b). Approximately 30% of the sample was diverted into the waste-1 channel during loading using the potentials shown in Table 1. Buffer from the buffer-1 and buffer-2 channels served to electrokinetically confine the sample in the cross intersection. Applied potentials were then switched to dispense mode, and the sample plug was launched into the analysis channel (Figure 2c). During dispensing, the direction of the electrokinetic flow in channel c2 was reversed, and the field strength in the buffer-2 channel was dropped from 630 to 200 V/cm (Table 1) to allow the sample to pass intersection t2. The tailing resulted from sample passing through an axially asymmetric electric field at intersection t2. This band asymmetry remained slightly visible in Figure 2d, although to a lesser extent, as the sample continued to move electroosmotically down the analysis channel. This asymmetry resulted in the additional band dispersion observed in the single point detection measurements discussed below. The electroosmotic flow from the buffer-2 channel was imaged by filling the buffer-2 reservoir with the rhodamine B sample and placing buffer in the sample reservoir. The fluorescence images in Figure 3 were taken during sample loading (Figure 3b) and dispensing (Figure 3c) under the conditions listed in Table 1. In Figure 3b, the electroosmotic flow from buffer-2 was split 5% into channel c2 and 95% into the analysis channel. In Figure 3c, the electroosmotic flow from the buffer-2 channel was only routed Analytical Chemistry, Vol. 76, No. 20, October 15, 2004
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Figure 5. Variation of the peak width (4σ) and area for standard and repetitive pinched injections. For the standard injection, the sample was dispensed toward the buffer-1 reservoir. For the repetitive injections, 24%, 29%, and 34% correspond to 0.60, 0.65, and 0.70 kV applied to the buffer-2 reservoir, respectively. For the applied potentials and field strengths, see Table 1. The error bars are (σ.
Figure 3. White light image of (a) the repetitive pinched injection valve and fluorescence images with rhodamine B in the buffer-2 reservoir during (b) sample loading and (c) dispensing. Arrows depict the direction of the electroosmotic flow of the rhodamine B doped buffer. For the applied potentials and field strengths, multiply the values in Table 1 by 0.5.
Figure 4. Five sequential injections at 5 Hz. The short arrows indicate the times the microchip was switched to sample loading, and the tall dashed arrows indicate the times the microchip was switched to dispensing. The laser was focused 200 µm downstream from the cross intersection in the analysis channel. For the applied potentials and field strengths, multiply the values in Table 1 by 2.
into the analysis channel and constituted 29% of the flow in that channel. During dispensing (Figure 3c), the electroosmotic flow at intersection t2 appeared similar to the asymmetry observed for the sample plug during injection (Figure 2c). The injection process was then tested at frequencies of 1, 2.5, 5, and 10 Hz. Figure 4 shows the temporal profiles generated at 5 Hz and detected 200 µm from the cross intersection in the analysis channel. The short arrows mark the time the potentials 6056 Analytical Chemistry, Vol. 76, No. 20, October 15, 2004
were switched to sample loading (see Figures 2b and 3b), and the tall dashed arrows mark the time the potentials were switched to dispensing (see Figure 2c,d and Figure 3c) resulting in the launch of the sample plug into the analysis channel. The profiles exhibited a small amount of tailing, but the amount was similar to the standard (pinched) injection. To compare the band profiles of the repetitive pinched injections with standard injections, the applied potentials were reconfigured to perform standard injections into the buffer-1 channel. Peak widths (4σ) and areas for the two injection schemes are plotted in Figure 5. Data points for peak width and area at 0% electroosmotic flow represent the standard injections into the buffer-1 channel. For the repetitive pinched injections dispensed into the analysis channel, the electroosmotic flow from the buffer-2 channel was varied by adjusting the voltages applied to the buffer-2 reservoir. The relative electroosmotic flow from the buffer-2 channel to the analysis channel was 24%, 29%, and 34% for 0.60, 0.65, and 0.70 kV, respectively, applied to the buffer-2 reservoir. The peak areas for the repetitive and standard injections were nearly constant indicating similar amounts of material were dispensed. Therefore, the amount of sample injected was not impacted by either the presence of the buffer-2 channel or by varying the potential applied to the buffer-2 reservoir. Also, varying the electroosmotic flow in the buffer-2 channel from 24% to 34% did not substantially alter the peak width. The peak width (4σ), however, increased for repetitive pinched injections by 35% from 71 to 96 µm compared to the standard pinched injections. Although an increase was observed, the peak widths for the repetitive injections compare favorably with pinched injections reported previously22 where the peak widths ranged from 60 to 160 µm depending on the potentials applied during sample loading and dispensing. Also, an injection plug length of 150 µm4 has been reported for double tee injections, and injection plug lengths of 100 µm20 and 160 µm10 for gated injections. The separations with (22) Alarie, J. P.; Jacobson, S. C.; Culbertson, C. T.; Ramsey, J. M. Electrophoresis 2000, 21, 100-106.
Figure 6. Variation of peak area with injection frequency. For the applied potentials and field strengths, multiply the values in Table 1 by 2. The error bars are (σ.
the double tee and gated valves have the smallest plate heights on microfluidic devices reported to date, and the injection plug lengths were minor contributions to the total plate heights. To test the maximum operating frequency of the repetitive pinched injection with 2 kV applied during dispensing (multiply the applied potentials and field strengths in Table 1 by 2), frequencies of 1, 2.5, 5, and 10 Hz were tested for 15 cycles of
sample loading and dispensing. Figure 6 shows the variation in peak area with the injection frequency, and as the injection frequency increased, the peak area decreased. This decrease at faster cycle times was due to incomplete sample loading at the cross intersection at the time of dispensing. Higher injection frequencies can be obtained by increasing the applied potentials (>2 kV), reducing the lengths of channels c1 and c2 (
