Anal. Chem. 1994,615,3485-3491
Electroosmotic Pumping and Valveless Control of Fluid Flow within a Manifold of Capillaries on a Glass Chip Kurt Seller, Zhonghul H. Fan, Karl Flurl, and D. Jed Harrison' Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Fluid flow was driven within a network of intersectingcapillaries integrated on a glass chip using electroosmotic pumping. Potentials could be applied to several capillaries simultaneously to quantitatively control the amount of each reagent stream delivered to an intersection of capillaries. An example of a simple dilution of sample with buffer is shown. Kirchhoffs rules for resistive networks were found to predict the currents and fluid flow within the capillaries. Leakage of sample from one channel to another at an intersection was shown to arise from both diffusive and hydrodynamic effects. Application of potentials to the intersecting channels could fully arrest such leakage. These phenomena were evaluated both qualitatively from photomicrographs of the fluid flow in the channels and quantitatively from measurements of electrophoretic separations of amino acids and fluoresceins. Electroosmotic flow can be used as a pumping mechanism within a We have shown that multiple, intersecting capillaries can be integrated on a planar device to form a manifold of flow channels in which capillary electrophoresis can be used for separation of sample components.48 Flow injection style sample handling can also be effected within the manifold.3 Separations on a time scale of seconds or even millisecondscan be achieved in such device^.^ It is also possible to control the flow of liquids at the intersection of channels with electric fields rather than valves or pumps, so that fluidic systems with no moving parts can be realized.3-5 The potential applications of such systems include miniaturized analytical systems that could compete with bench-top instruments or chemical sensors in terms of performance, analysis time, or l
To practically realize the use of electroosmotic pumping in a complex manifold of channels, it is necessary to develop an understanding of the factors that control flow within such ( I ) Manz, A.; Fettinger, J. C.; Verpoorte, E.; Lfidi, H.; Widmer, H. M.; Harrison, D. J. Trends Anal. Chem. 1991, 10, 144-149. (2) Capillary Electrophoresis: Theory and Practice; Grossman, P. D., Colburn, J. C., Eds.; Academic Press: New York, 1992. (3) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (4) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481-1488. ( 5 ) Harrison, D. J.; Manz, A.; Fan, Z.; Lfidi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (6) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A,; Liidi, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-258. (7) Harrison, D. J.; Glavina, P. G.;Manz, A. Sens. Actuators 1993, BIO, 107-
116.
(8) Harrison, D. J.; Fan, 2.;Seiler, K.; Manz,A.; Widmer, H. M. Anal. Chem. 1993, 283, 361-366. (9) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993,65,26372642. (10) Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. In Aduances in Chromatography; Brown, P. R., Grushka, E., Eds.; Marcell Dekker: New York, 1993; Chapter 1, pp 1-65. (11) Fan, 2.;Harrison, D. J. Anal. Chem. 1994, 66, 177-184.
0003-2700/94/0366-3485$04.50/0 0 1994 American Chemical Society
systems, particularly at the intersection of two channels containing different solutions. One of the best ways to achieve this is to visually image the flow process within the channels.3 In addition, the quantitative study of flow rates and study of the ability to control the direction of solvent flow using applied fields are required. In this report we present both images and quantitative studies of diffusional and convective mixing of solutions at channel intersections. We also show that judicious application of voltages to multiple channels within a manifold can be used to control such mixing and direct the flow of solvent and sample in the desired manner. This control can be used to reduce or eliminate unwanted leakage effect^^-^ and to provide controlled delivery of solutions and reagents within a complex manifold of intersecting capillaries, as might be required in flow injection analysis systems. When the electrolyte resistivities in each channel are either the same or known and the electroosmotic flow rates are the same in all channels, it is possible to quantitatively calculate the potential required to control the flow directions using a simple resistance network model of the channels. EXPERIMENTAL SECTION Device Fabrication. Figure l a shows the layout and dimensions of the principal glass device studied, which consisted of three capillary channels intersecting at right angles to form a T junction. It is referred to as COCE. The channels were 10 pm deep and 30 pm wide where shown as a single line width in the figure and 220 pm wide where shown as a heavier line. A cover plate, with 1.5-mm-diameter holes drilled ultrasonically or with a diamond bit, provided channel access points. It was thermally bonded to a glass plate in which channels were etched. A second device, Jet-1, is shown in Figure lb; the narrow lines are for channels 10 pm deep and 30 pm wide, while the heavier lines represent channels 300 pm wide. Devices were fabricated in Corning 7740 Pyrex glass at the Alberta Microelectronic Centre (AMC) using a modification of bulk silicon micromachining methods described elsewhere." Apparatus. A computer-controlled system was used to apply and switch the potentials on the device reservoirs and is described in detail el~ewhere.~.' Labview programs (National Instruments Corp., Austin, TX) written locally were used for data acquisition and instrument control. A 488-nm argon ion laser (Uniphase/Ionics Model 201 1) was used to excite fluorescence after being focused to about a 40-pm spot size in the device channel. The fluorescent emission was collected with a 1O:l microscope objective and directed onto a photomultiplier tube through a 1-mm pinhole at the image plane and an optical band pass filter (508-533 nm), as described elsewhere. For imaging the channels, the laser beam Analyticalchemistry, Vol. 66, No. 20, October 15, 1994
3405
intersection J and each of the reservoirs. Values of R and I are reported in Table 1 for both devices. Three intersecting channels with potentials applied to the solvent reservoirs of all three channels can be modeled as a simple network of three resistors, as illustrated in Figure 2. With two voltage sources and a near-ground potential connected to the channels, the current in each channel and the potential at the intersection are readily expressed using Kirchhoff's rules13 to give eqs 1-4,
COCE Layout
Jet-1 Layout
13 = V,/R,
(1)
+ I,
(4)
detector
I, = I, Inject. Waste
Sep. Waste
Flguro 1. (a) DeviceCOCE layout with reservoir labels, channellengths (Ix),and resistances, (R,) lndlcated. Values are given In Table 1. Overall dimensions were 2.8 X 7.0 cm. (b) Device Jet-1 layout with various labels. Values are In Table 1.
was expanded slightlyand directed onto the device. The device was mounted on a stage under an Olympus BH-2 microscope using a 5: 1 magnification, a long working distance objective, and a 1O:l eyepie~e.~ Photos were taken with a camera adaptor and an Olympus OM-2 camera body, while potentials were applied to the channels. Exposures were about 1.5 s long. The current in the channels was monitored from the potential drop across a 10-kQ resistor located between the channel reservoir and the power supply ground and recorded on a strip chart recorder. Materials and Reagents. Amino acids, fluorescein, and fluorescein 5-isothiocyanate (FITC) were used as received (Sigma). Amino acids were labeled with FITC.3-5,12Several pH 9.1 buffer solutions were used, including solutions 10,30, or 64 mM in carbonate ion or 10 mM in Na2B40~1OH20. The carbonate stock solution was prepared from a solution of 0.054 M NaHCO3 and 0.0096 M NazCO3. All chemicals were reagent grade; deionized, doubly-distilledwater was used for all solutions. Solutions were filtered using 0.22-pm pore size Millipore-GV sterile filter units (Millipore, Bedford, MA) before being injected into the capillaries on the chip in the manner previously d e ~ c r i b e d . ~ - ~ J I
RESULTS AND DISCUSSION Two devices were prepared with networks of intersecting channels in order to study the effect of applying potentials to multiple channels simultaneously on the behavior of fluid transport in the channel networks. The device COCE shown in Figure 1 consisted of three channels intersecting in a T configuration in which simple mixing experiments were performed, while Jet-1, also shown in Figure 1, had a slightly more complex geometry and was used for sample injection and separation. A labeling scheme is shown that is used to identify the channel resistances and lengths ( R and l ) by subscripts associated with the channel segment between the ~~
(12) Sweedler, J. V.;Shear, J. B.; Fishman, H. A.; Zare, R. N.; Scheller, R. H. Anal. Chem. 1991, 63, 496502.
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Analytlcal Chemistty, Vol. 66,No. 20, October 15, 1994
where the assumed directions of the currents ZI,ZZ,and Z3 are shown in Figure 2. The 10-kQ resistor has been omitted in eq 1, as it is much smaller than R3 in our applications. Solving for VJ and I3gives eqs 5 and 6, Vj = (VIR@,
+ V2RlRJ/(RiR2 + RzR3 + RlRJ
(5)
While in general the potentials V Iand Vzcan have anypolarity, in this study V I was always positive and VZalways negative. Equation 6 shows that a plot of Z3 versus V Iwill be linear when V2 is held constant; however, Z3 may be positive or negative depending on the potentials applied and the resistance of each channel. Note that a switch in sign of the current will also mean a change in the direction of electroosmotic flow. During the course of experimentation, the polarity of potentials applied to each of the device reservoirs was varied. The correlations of device channel resistances with the values in eqs 5 and 6 are provided in the text or figure captions. Electrical Characteristics. Table 1 gives the measured lengths and resistances between each pair of reservoirs identified in Figure 1 for both devices. The ratios of these values to those for the reservoir pair A-B or the channel length 1, are also given for the two different devices. The resistance of each channel should be proportional to the length of the channel and its cross-sectional area, as well as the electrolyte resistivity.14 Some segments were either 220 or 300 pm wide, while the standard channels were 30 pm wide at the top surface, so that the segment resistances varied along their length. For convenience an equivalent length (1,) was calculated for the wider segments using the formula 1, = 'trueA30/Atrue
(7)
where A30 and Atmeare the cross-sectional areas of the 30-pm and the wider channels, respectively. It is these equivalent lengths that are given in Table 1. The resistances and their (13) Halliday, D.; Resnick, R. Physics; J. Wiley and Sons: New York, 1960. (14) The relationship between R and geometry is R = pl/A, where p depends on a given electrolyte's resistivity and I and A are the channel's length and cross sectional area, respectively.
TaMe 1. Comparieon of Channel Lengths and Experlmentally Determined Reslstances for Devlces COCE and JET-1’ A-Bb A-C C-B A B
distance (mm, f0.05 mm) resistance (Ma)’ distance ratio to A-B resistance ratio to A-B distance ratio to I , resistance ratio to R,
138.3 1035f2 1 1
distance (mm, *0.05 mm) resistance (MQ)Csd distance ratio to A-B resistance ratio to A-B distance ratio to ,/ resistance ratio to R,
30.85 526 f 3 1 1
Device COCE 53 101.3 396f4 797 f 9 0.383 f 0.010 0.732 f 0.013 0.382 f 0.005 0.770 f 0.01 1
45 320 f 20
93.3 720 f 20
Device Jet-ld 4.7 29.35 81.4 f 0.5 502 f 3 0.152 f 0.002 0.951 f 0.002 0.155 f 0.001 0.954 f 0.008
8 79 f 8
2.07 f 0.03 2.3 f 0.2
1 1
27.75 473 f 2
3.1 53 f 2
0.18 f 0.01 0.25 f 0.08 1.6 29 f 2 0.52 f 0.06 0.55 f 0.04
9.0 f 0.1 8.9 f 0.3
1 1
C
a Errors (standard deviation) in resistance between reservoirs are determined from least-square fits of current-voltage curves, while errors in lengths are f0.05 mm based on an estimate of robable alignment errors. All other errors are calculated using error propagation analysis. b Labels A, B, and C and A-B, etc. refer either to the individual channels originating from the reservoirs and terminating at the intersection or to the channel lengths between the indicated reservoir pairs, respective1 See Figure 1A for the reservoir labels. The resistance of a given channel segment and its length are given. Ratios are given relative to the lengtg or resistance of the segment between reservoirs A and B or between A and the intersection point, as indicated. For Jet-1, no calculations were performed using the resistance of channel D, so those data are omitted for brevity (note ld = 20.3 mm).
.
A
R,
“1
=
R,
7
recorder
10kG
Gnd Flgure 2. Equivalent circuit for intersection of three channels with controlled potentials.
standard deviations were determined from the slopes of linear current-voltage curves obtained with a potential applied between each reservoir pair. Table 1 also gives the lengths, la, lb, and I,, and the corresponding resistances, Ra, Rb, and Rcr for each of the channel segments measured from the intersection point to each reservoir. The error in the channel lengths arises from the error in positioning the reservoir access holes in the cover plate over the channels and is taken as f0.5 mm, although this may be an overestimate. The data show that the resistances Ra, Rb, and R, are indeed proportional to the equivalent channel lengths within experimental error. This would be expected providing there is no defect in the bonding of the glass cover plate to the etched piece. The results are consistent with our previous observations with other devi~es.~-sJ The behavior of COCE with potentials applied to all three channels was evaluated according to the scheme indicated in the inset of Figure 3. The current was measured from the potential drop across a 10-kQ resistor between ground and reservoir C, with V I and V2 applied to reservoirs A and B, respectively. In this configuration, the resistances for eqs 1-6 were R1 = RarR2 = Rb, and R3 = R,. The current was indeed linear in VIwhen V2 was held constant. Figure 3 shows the data obtained for three different values of VZ.It was possible to calculate the current using eq 6 and the measured channel
0
500
1000
1500
2000
Voltage V i (V) Flgure 3. Current as a function of VI applied to reservoir A, with the Indicated values of Vz applied to reservoir B. Symbols are for experimental data: solid lines are for current calculated from eq 6 usingthe data in Table 1. The inset shows the applied potential scheme.
resistances given in Table 1, and the calculated response is shown as the solid lines in Figure 3. Thevery good agreement illustrated between theory and experiment shows that the model is accurate. It can be used to determine potentials and currents within a complex network of capillaries when several voltage sources are applied simultaneously. A practical limitation of most high-voltage power supplies should be noted here. The high-tension terminal of a power supply presents a very high impedance path to ground, so current will not normally flow into this terminal when there is an alternate route to ground. For example, if both VIand VZin Figure 2 were positive supplies, then the potential at the junction would be positive. If VZwas set sufficiently large, the intersection potential VJ would exceed the value of V I . AnalyticalChemistry, Vol. 66,No. 20, October 15, 1994
3487
This would demand that VIact as a sink for current, but since the resistance network provides a path to ground the power supply would usually not. Consequently, no current would flow in resistance (i.e., channel) 1, and VIwould no longer control the potential along that channel. This puts limitations on the design of networks that can be utilized and may define where the ground terminal can be located. Mixing of Solutions Using Voltage Control. When electroosmotic flow is present in a capillary,the linear flow velocity, u, is given by eq 8,
where pe0 and pep are the electroosmotic and electrophoretic mobilities, respectively, and E is the electric field a~p1ied.l~ Theoverall mobility, p, is thevector sum of these two mobilities, and pe0 is generally larger than pep in absolute terms. In the following discussions it is helpful to note that with negatively charged capillary walls, the direction of electroosmotic flow of the solvent will be in the same direction as the current flow. This is the case for glass at pH 9. The velocity of species i in each of the channels is given by eqs 9-1 1,
where VJ is given by eq 5, and the various Ui refer to the velocity of species i in each channel of length 1. The subscripts 1,2, and 3 refer to the velocity and channel length associated with the potential sources V I ,V2, and ground, respectively. These expressions indicate that controlled mixing of solutions should be possible at the intersection of the three channels by adjustment of the potentials VI and V2. This phenomenon was explored both quantitativelyand qualitatively,as described below. Equations 9-1 1 express the electrokineticallydriven flow. Since the channel cross sections are equal at the intersection, the sum of u1 and u2 for the linear flow rate of the solvent must equal u3 in order to have mass balance. Substitution of pe0 for pi in eqs 9-1 1 shows that the mass balance condition will be met by electroosmoticflow alone only when pe0is the same in each channel. This will require that the ionic strength and pH be the same in each channel. If this is not the case, then the excess electroosmoticflow in the channel with the highest pm will induce additional flow in the other channels so that mass balance will occur. In this work, the pH and ionic strength were the same in all channels. The COCE device was mounted under a microscope equipped with a camera, and the region near the intersection was illuminated with 488-nm light. Any striations visible in the fluorescent dye stream seen in Figure 4 and later photos are due to nonuniformity in the illumination or collection efficiences, as it is the tendency of the intensity to fade at the edges of the photos. The exposures were about 1.5 s long and so capture the steady-stateflow of the fluid stream. Following (15) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981,53, 1298-1302. (16) Lauer, H. H.; McManigill, D. Anal. Chem. 1986, 58, 166-170.
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Flgure 4. Photomicrographs with arrows showing the direction of flow during controlled mixing of 10 mM carbonate buffer, pH 9.1, and 100 pM fluorescein in the same buffer. Channels are 30 pm wide and provide the scale for the photo. The buffer reservoir (Buf) was at grounded, and the sample reservoir (S) had the Indicated positive potentials applied, while the waste reservoir (W) was at -3.75 kV.
application of the voltages, it usually required a few seconds or less for the steady-state flow to be established. Figure 4 shows the dye stream when the buffer reservoir (Buf) was maintained at ground and the waste reservoir (W) at -3.75 kV, while the potential on the sample reservoir (S) varied from +325 to +8550 V. The potentials applied to the three reservoirs of COCE drove 10 mM carbonate buffer, pH 9.1, from the left (marked GND(Buf)) of Figure 4a and 100 pM fluorescein sample (S) from the right (+375 V(S)) toward the waste reservoir (-V(W)). Figure 4a shows the mixing of the two streams as they move downstream of the intersection point in the direction of the arrows. Figures 4b and c establish that increasing the potential on the sample reservoir increases the amount of dye relative to buffer downstream of the intersection. However, Figure 4c also illustrates that the direction of flow in the channel connected to ground can be reversed as the potential at the channeljunction, VJ,changes from negative to positive. Figure 4ccaptures this change in flow direction within 5 s of increasing the potential to 8550 V. The potential VI required for such a reversal of the buffer flow was calculated using eq 5 , with R1 = Rb, R2 = Ra, and R3 = Rc for the COCE device. The potential VJis predicted to be negativewhen the samplechannel potential VI is less than 8400 V, causing flow from the buffer
A n n n
3000
1 cz,
1
II
"L,
Grid
Sample
' i
2000
1000
2000
Figure 6. (a) Photomicrograph showing flow of dye around a comer with no potential applied (FI) to the buffer reservoir (Buf) (10 mM carbonate, pH 9.1). The sample reservoir (S) was grounded (100 pM fluorescein in the same buffer), and the sample was driven toward the waste reservoir (W) at -10 kV. Arrows show the flow direction, and the channelswere 30 pm wide. (b) Effect of disconnectingall potentials for 15 s after the profile in part a was established.
1000
0 0
10
20
Time (min) Figure 5. Fluorescence intensity downstream of the T intersection as a function of time, while the potential on the sample reservoir was decreased in -100 V steps every 60 s. Polarities are indicated in the inset and are the same as those for Figure 4 with V2 = -2 kV (30 mM carbonate, pH 9.1 buffer, and 10 pM fluorescein sample in the same buffer).
reservoir towards the junction, while VJshould be +5 V when VI is 8550, causing dye to flow into the buffer channel, as was observed in Figure 4c. A similar experiment was performed in a quantitative fashion using a detector located about 1 cm downstream of the mixing intersection. The potentials were applied with the same polarity as in Figure 4 and are shown in the inset of Figure 5. The intensity of the focused laser beam was reduced to the point where photobleaching of fluorescein did not occur, so the intensity of the signal was not a function of the solution velocity. Figure 5 shows the intensity decreased in a stepwise fashion as the potential applied to the sample reservoir was decreased; however, there was a lag time of about 10 s. The decreased intensity arose from the decreased amount of dye relative to buffer downstream of the intersection. The lag time is in agreement with the expected transit time between the intersection point and the detector, confirming that the decrease in signal is due to dilution of the dye. Imagingof Leakage. We have previously discussedleakage effects at the intersection of c h a n n e l ~ . ~ -In ~ Jparticular, if a side channel is left floating while a potential is applied to cause flow in the main channel, there will be leakageof solution from the side channel, contaminating the main channel. We have indirectly shown that both hydrodynamic and diffusion effects contribute to this mixing or leakage effect at the
intersection.ll In previously studied devices with various layouts, the concentration of sample leaking into the main channel was about 1-396 of that in the sample ~hannel.~JJl Figures 6 and 7 illustrate the leakage effect at the intersection in COCE. In Figure 6, sample (S) was directed from the grounded reservoir (GND(S)) toward the negative waste reservoir (-V( W)), while the remaining reservoir which contained buffer (Buf),was left floating (Fl). Figure 6a shows that the sample turned the corner at the intersection without significantly spreading back into the buffer channel. All reservoirs were then left floating (Le., with no applied potentials) for 15 s to allow diffusion to occur, creating the dye distribution seen in Figure 6b. This illustrates the significant extent of diffusion that occurs at the uncontrolled intersection and exemplifies the diffusion effects discussed by Dose and G~ioch0n.l~ However, when the samplewas flowing, as in Figure 6a, the dye distribution was quite sharp. This must mean that buffer was drawn out of its channel and swept the diffusingdyedownstream, leading to the sharpdistribution observed at the corner. This flow of the buffer likely arises from the viscous drag of the flowing dye stream on the stagnant solutionwithin the buffer channel.18 It is equivalent in nature to the forces causing aspiration. A more graphic demonstration of the leakage phenomenon in COCE is shown in Figure 7. In this case, the grounded buffer solution (GND( Buf)) was driven toward the negative waste reservoir (-V(W)), while the dye sample reservoir was left floating (Fl(S)). The flow of dye into the intersection from the side channel and subsequent mixing downstream are (17) Dose, E. V.; Guiochon, G. Anal. Chem. 1992,64, 123-128. (1 8) (a) Gerhart, P. M.; Gross, R. J. Fundamentals of Fluid Mechanics;AddisonWesley: Reading, MA, 1985. (b) Jeppson, R. W. Analysis of Flow in Pipe Nerworks, Ann Arbor Science Pub.: Ann Arbor, MI, 1976.
Ana!yfkalChemistry, Vol. 66, No. 20, October 75, 7994
3489
-~
~~
Flgure 8. Immobilizationof sample dye solution (S) at an intersectlon by the application of potentialto all three channels. The buffer reservoir (Buf) was at 525 V, the sample reservoir (S) was at ground, and the buffer flowed toward the waste reservoir (W) at a potential of -3.75 kV. Solutions were the same as in Figure 6. Arrows show the flow direction, and the channels were 30 pm wide.
II
Injection/
Flgure 7. Leakage of sample dye solution (S) from a floating sMe channel (FI) when flow was directed from the grounded buffer reservoir (Buf) to the waste reservoir (W) with (a) -1.25 kV applied and (b) -10 kV applied to W. Solutions were the same as in Figure 6. Arrows show the flow direction, and the channels were 30 pm wide.
3490 Ana&ticalChemistry, Vol. 66,No. 20, October 15, 1994
GND
IRttf\
.-ul very clearly seen. The flow velocity increased with increasing potential, and this increased both the extent of leakage and the distance of travel required for mixing downstream of the intersection, as seen in Figure 7. This demonstrates that hydrodynamic flow effects lead to leakage, in addition to any diffusion-driven transport. In comparing the leakage effects observed within COCE, Jet- 1,and other devices4v5J* studied previously, it became clear that the magnitude of the effect can vary greatly: from 1 to 3% in some layouts and up to 21% in COCE. The principal difference in the layouts was the relative lengths of the intersecting channels. When a separation channel was much longer than a side channel, leakage was more pronounced. This is presumably related to the resistance to flow associated with a channel's geometry and a need to balance these resistances. We do not yet have a clear picture of the factors governing leakage, but it is clearly desirable to eventually put our present understanding on a more quantitative basis. LeakageControl. Control of the leakage at an intersection can be achieved by controlling the potential of three channels simultaneously. Figure 8 shows that dye can be forced to sit at the intersection in COCE without leaking into the flowing channel with the appropriate choice of potentials. The dye appeared immobilized for over 60 s, and then buffer slowly crept into the sample channel over several minutes. For this experiment, the potential was adjusted to achieve stasis, but it is possible to calculate the required values with reasonable accuracy, as illustrated quantitatively below for device Jet- 1. Figure 9 shows data obtained in device Jet- 1with the layout and reservoir labeling indicated in Figure 1. The separation of fluorescein isothiocyanate (F1TC)-labeled arginine (Arg) and tyrosine (Tyr) is shown. For these experiments, sample solution (S) was present in reservoir C, which was held at ground. As shown in the diagram at the top of Figure 9,
I
-3 KV
10001
Arg-FITC
FITC
Tyr-FITC
a,
Y
0
20
0
20
40
60
80
40
60
80
Time (s) Flgure9. Separationof 20 pM Arg-FITC and 40 pM Tyr-FITC in 0.064 M carbonate buffer, pH 9.1, with potentials applied to three reservoirs of device Jet-1. The injection procedure is shown at the top, and separations withtwo different values of VI applied to the buffer reservoir are shown. The dashed lines indicate the background level when the sample channel was instead left floating during separation.
samples were injected across a "double T" injector9 from (S) toward the injection waste reservoir (inj W), which was at -3 kV. This created a sample plug in the separation channel defined by the offset of the double T geometry, about 150 pm in length. To evaluatethe abilityto control leakageduring a separation we applied potentials to three reservoirs simultaneously. The voltages of the buffer (Buf), sample (S),and separation waste (W) reservoirs (A, C, and B, respectively, in Figure 1) were controlled,while the injection waste reservoir was left floating (Le., no electrical contact), as shown in the insets of Figure 9. In the first electropherogram of Figure 9, VI= 284 V was applied to the buffer reservoir during the separation. This resulted in a high background fluorescence compared to the background seen when the sample channel was instead left floating (dashed line). The potential at the sample to separation channel intersection, VJ,was calculated to be -1 7
0
A
9
5
G
s.
2000
'0 C
z 0
&)
Y 0
m m
1000
I
Sample (S) grounded Sample (S) left floating
:\ -
(s)
GND
-3KV
Consequently, leakage from the injection waste channel was expected to be much smaller than that from the sample channel. The low background levels achieved show that this was the case, further confirming that the resistance of a channel to flow is a significant design parameter in controlling leakage effects. However, the data also show that applied potentials can be used to actively control leakage and that the values required can be predicted quantitatively from a simple impedance network.
-*--.a-
CONCLUSIONS 1 These experiments show that simultaneous control of the
0 ZOO
300
400
Voltage
500
V,
600
700
(V)
Flgure 10. Effect of control of three voltages as in Figure 9 on the background level, shown as a function of VI, the potential applied to the buffer reservoir, with other conditions as in Flgure 9.
f 12 V using eq 5 and the measured resistances of the channels in Table 1, with R1 = RarR 2 = R b , and R3 = R,. Since the sample channel was at ground, the result should have been a net flow out of the sample channel during the separation and an increased background, as was observed. With VI = 360 V and the sample channel grounded, the background fluorescence dropped well below the level observedwhen the sample channel was left floating (dashed line in Figure 9). The potential VJ was calculated to be 8 f 10 V, indicating that buffer would flow into the sample channel during the separation or the sample would be held stagnant at the intersection, either way preventing leakage of the dye. The decrease in background in Figure 9 confirms that the flow in the samplechannel was either stagnant or reversed. The longer injection times required as the separation potential VI was increased to 360 V indicate a need to compensate for backflow into the sample channel during separation and demonstrate that sample flow was reversed. Figure 10 shows the intensity of background fluorescence during separation as a function of the buffer potential. About 350 V was required on the buffer reservoir to prevent leakage from the side channel. This compares favorably with the value of 334 f 14 V that is predicted to be needed to stop flow from the sample channel, given the measured channel resistances. The slight discrepancy between these numbers is most likely experimental error, although it may reflect any additional voltage required to reduce leakage from the side channel that arises from the effects of diffusion and the viscous drag caused by flow in the main channel. For the scheme described above and shown in Figure 9, leakage from the injection waste channel, Le., from reservoir D, could still occur. The length of the waste channel (20.3 mm) relative to the sample (1.6 mm) and buffer (3.1 mm) channels meant it had a comparatively high resistance to flow.
potential of multiple channels in a manifold is possible. The channels behave as resistors in terms of their electrical characteristics, so the potential at any point within a channel can be estimated by application of Kirchhoff's rules if the channel geometries are known and the solution resistivity is the same in all channels or the resistivities are known. This makes it possible to predict the direction and magnitude of solvent flow at intersections. The applied voltages can be used to control the flow, so that a trulyvalveless fluidic control system can beachieved. Thedemonstration of sample dilution presented here shows that the addition of reagents for pH adjustment, derivatization, complexation or masking of interferences, as would be performed in flow injection analysis, can be achieved within a chip. The behavior of flow within a manifold can be qualitatively understood. The images clearly show that both hydrodynamic and diffusional effects contribute to mixing and leakage at intersections. However, not all of the behavior is completely understood, and it will be necessary to develop a more quantitative description of the influence of the resistance to flow of intersecting channels on phenomena such as leakage or mixing at intersections. Qualitatively, when a short side channel feeds into a long main channel, there will be a relatively high induced flow from the side channel when flow is present in the main channel. When the intersecting channels have similar flow resistances, the hydrodynamically induced leakage will be substantially reduced. These results and the ability to image the flow processes in situ will aid greatly in the design of channel manifolds. ACKNOWLEDGMENT We thank the Natural Sciences and Engineering Research Council of Canada and Ciba-Geigy, Basel, Switzerland, for support of this research. We thank the Alberta Microelectronic Centre (AMC) for use of their facilities and assistance in device fabrication. Z. F. thanks AMC for a graduate stipend. Received for review November 11, 1993. Accepted June 17, 1994.' @Abstractpublished in Aduunce ACS Absvucrs. August 1, 1994.
Analytical Chemistry, Vol. 66,No. 20, October 15, 1994
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