Automated Control of Local Solution Environments in Open-Volume

Nov 15, 2007 - This technology offers the possibility to adjust the flow pattern ... Xinyu Zhang , Alix Grimley , Richard Bertram and Michael G. Roper...
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Anal. Chem. 2007, 79, 9286-9293

Automated Control of Local Solution Environments in Open-Volume Microfluidics Helen Bridle, Jessica Olofsson, Aldo Jesorka, and Owe Orwar*

Department of Chemical and Biological Engineering, and Microtechnology Centre, Chalmers University of Technology, SE-412 96 Go¨teborg, Sweden

We present an open-volume microfluidic system capable of on-line modification of a patterned laminar flow by using programmable inlet valves. Each separate solution environment in the flow pattern can be independently exchanged between different preloaded input solutions where each exchange requires 20 s. The number of flow patterns that can be generated by one device is Nn, where N represents the number of valve inlets and n the number of microchannels in the microfluidic system. Furthermore, the system can be operated as a combinatorial mixer, in which mixture of the different input solutions can be obtained independently in each channel. Since the flow patterns are generated in an open volume, they are accessible to many different detection methods and types of probes, e.g., microelectrodes, cells, or cell fragments. This technology offers the possibility to adjust the flow pattern composition in response to an output from a probe. This is the first step toward creating an automated feedback device controlled by, for example, biological cells. Microfluidic systems generating patterned laminar flows have been utilized in numerous applications including in-channel microfabrication,1,2 generation of surface gradients and surface patterning,3 studies of chemotaxis,4 and many more.5-13 However, * Corresponding author. E-mail: [email protected]. Phone + 46 31 772 30 60. Fax + 46 (0) 31 772 61 20. (1) Kenis, P. J. A.; Ismagilov, R. F.; Takayama, S.; Whitesides, G. M.; Li, S. L.; White, H. S. Acc. Chem. Res. 2000, 33, 841-847. (2) Takayama, S.; Ostuni, E.; Qian, X. P.; McDonald, J. C.; Jiang, X. Y.; LeDuc, P.; Wu, M. H.; Ingber, D. E.; Whitesides, G. M. Adv. Mater. 2001, 13, 570-+. (3) Li Jeon, N.; Dertinger, S. K. W.; Chiu, D. T.; Chiol, I. S.; Stroock, A. D.; Whitesides, G. M. Langmuir 2000, 16, 8311-8316. (4) Li Jeon, N.; Baskaran, H.; Dertinger, S. K. W.; Whitesides, G. M.; Van De Water, L.; Toner, M. Nat. Biotechnol. 2002, 20, 826-830. (5) Takayama, S.; Ostuni, E.; LeDuc, P.; Naruse, K.; Ingber, D. E.; Whitesides, G. M. Nature 2001, 411, 1016-1016. (6) Takayama, S.; Ostuni, E.; LeDuc, P.; Naruse, K.; Ingber, D. E.; Whitesides, G. M. Chem. Biol. 2003, 10, 123-130. (7) Costin, C. D.; Olund, R. K.; Staggemeier, B. A.; Torgerson, A. K.; Synovec, R. E. J. Chromatogr., A 2003, 1013, 77-91. (8) Weigl, B. H.; Kriebel, J.; Mayes, K. J.; Bui, T.; Yager, P. Mikrochim. Acta 1999, 131, 75-83. (9) Macounova, K.; Cabrera, C. R.; Holl, M. R.; Yager, P. Anal. Chem. 2000, 72, 3745-3751. (10) Macounova, K.; Cabrera, C. R.; Yager, P. Anal. Chem. 2001, 73, 16271633. (11) Sinclair, J.; Pihl, J.; Olofsson, J.; Karlsson, M.; Jardemark, K.; Chiu, D. T.; Orwar, O. Anal. Chem. 2002, 74, 6133-6138.

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they suffer from a major drawback, namely, that the composition of the flow pattern is constant. To our knowledge, there is currently no microfluidic device capable of generating a flow containing separate solution environments in which the content of each solution segment can be individually updated over time. We have developed a system that is able to rapidly, on the order of tens of seconds, switch between numerous laminar flow patterns. This is achieved via the connection of a pair of external, computer-controlled valves to each microfluidic channel in a commercially available microfluidic chip. External valves integrated with flow systems have previously been used to control and pump fluid flows,14 to generate sequential exposures,15-17 and to mix.14,15 While incorporation of valves inside microfluidic systems allows for reduction of the dead volume, it is not trivial to achieve. The methods so far reported for creating valves in elastomeric microfluidic devices with pressure-driven flows generally involve hydrogels,18,19 actuated by either pH or temperature, which influence fluid properties and have slow response times, or deformation, requiring complicated 3D microfabrication and microchannels with a bell-shaped cross section.20-22 For each input channel, we chose to use a pair of external valves, where one is used to control the input into each channel and the other is used for fast exchange of the dead volume. The system can switch between Nn different patterns, where N represents the number of valve inlets and n the number of microchannels. We utilize valves with 2 inlets interfaced to 8 microchannels, and therefore, switching between 256 different patterns is possible. However, by increasing the number of valve inlets or the number of channels utilized, the number of patterns, which can be generated with the system, grows exponentially. Pattern switching is computer(12) Olofsson, J.; Bridle, H.; Sinclair, J.; Granfeldt, D.; Sahlin, E.; Orwar, O. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8097-8102. (13) Choban, E. R.; Markoski, L. J.; Wieckowski, A.; Kenis, P. J. A. J. Power Sources 2004, 128, 54-60. (14) Gu, W.; Zhu, X.; Futai, N.; Cho, B. S.; Takayama, S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15861-15866. (15) Fabio, R. P. R.; Reis, B. F.; Elias, G, A., Z.; Lima, J. L. F. C.; Lapa, R. A. S.; Santos, J. L. M. Anal. Chim. Acta 2002, 468, 119-131. (16) Dolmetsch, R. E.; Xu, K.; Lewis, R. S. Nature 1998, 392, 933-936. (17) Hsu, C. H.; Chen, C.; Folch, A. Lab Chip 2004, 4, 420-424. (18) Yu, Q.; Bauer, J. M.; Moore, J. S.; Beebe, D. J. Appl. Phys. Lett. 2001, 78, 2589-2591. (19) Folk, C., Chen, X., Wudl, F., Ho, C. M. ho.seas.ucla.edu/publications/ conference/2003/232.pdf, 2003. (20) Unger, M. A.; Chou, H. P.; Thorsen, T.; Scherer, A.; Quake, S. R. Science 2000, 288, 113-116. (21) Ismagilov, R. F.; Rosmarin, T. D.; Kenis, J. A.; Chiu, D. T.; Zhang, W.; Stone, H. A.; Whitesides, G. M. Anal. Chem. 2001, 73, 4682-4687. (22) Samel, B. S., Griss, P., Stemme, G. J. Microelectromech. Syst. 2006, 16. 10.1021/ac0712087 CCC: $37.00

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controlled and requires 20 s. Thus, with a hold time of 10 s per pattern, all 256 patterns could be sampled in just over 2 h. Furthermore, our system can be operated as a combinatorial mixer where mixing occurs independently in each channel. Mixing is inherently problematic within microfluidic systems due to low Reynolds and high Peclet numbers.23 Numerous strategies to enhance mixing have been presented. However, the use of complicated device geometries, such as complex channel networks (both 2D and 3D) or the inclusion of grooves, herringbone structures, or surface modifications, requires complex microfabrication and results in only one fixed flow pattern.14,24-28 Mixing by control of flow rates can result in uneven flow rates,14,29 and the exploitation of external fields may interfere with other onchip processes or detection methods.30,31 Valves have also been used for mixing.14,32,33 In our system, mixing is achieved by cycling of the inlet valve between the two different input solutions to generate a series of flow plugs that mix within the dead volume. The main advantage of our mixing system is that it is possible to obtain combinatorial mixtures of different substances arranged in any desired spatial pattern. Since, in our system, the patterned laminar flow is generated in an open volume, it is accessible to probes, such as microelectrodes, cells, or cell fragments. The microfluidic chip part of the system is mounted on a computer-controlled scanning stage. Thus, programmable translation of probes in the patterned flow is possible and results in a probe experiencing sequential exposure to different solutions with rapid exchange times (typically ∼1012 ms34 although this depends upon factors such as probe size and translation velocity35). Previously demonstrated applications of the microfluidic chip have exploited this rapid solution exchange and access to several different solution environments to study rapidly desensitizing ion channels as well as generate concentration variations, mimicking physiological conditions.12,34,36 The system presented here is unique in the combination of rapid solution exchange with access to numerous different flow patterns, the content of which can be updated over time. Furthermore, operation of the system, including both switching of the valves and probe scanning, is fully automated and programmable enabling rapid, automated acquisition of the response of a probe to sequential exposures to the different patterns. (23) Ottino, J. M.; Wiggins, S. Philos. Trans., A: Math. Phys. Eng. Sci. 2004, 362, 923-935. (24) Bringer, M. R.; Gerdts, C. J.; Song, H.; Tice, J. D.; Ismagilov, R. F. Philos. Trans., A: Math. Phys. Eng. Sci. 2004, 362, 1087-1104. (25) Pihl, J.; Sinclair, J.; Sahlin, E.; Karlsson, M.; Petterson, F.; Olofsson, J.; Orwar, O. Anal. Chem. 2005, 77, 3897-3903. (26) Stroock, A. D.; Dertinger, S. K.; Ajdari, A.; Mezic, I.; Stone, H. A.; Whitesides, G. M. Science 2002, 295, 647-651. (27) Stroock, A. D.; Dertinger, S. K.; Whitesides, G. M.; Ajdari, A. Anal. Chem. 2002, 74, 5306-5312. (28) Neils, C.; Tyree, Z.; Finlayson, B.; Folch, A. Lab Chip 2004, 4, 342-350. (29) Glasgow, I.; Aubry, N. Lab Chip 2003, 3, 114-120. (30) Grumann, M.; Geipel, A.; Riegger, L.; Zengerle, R.; Ducree, J. Lab Chip 2005, 5, 560-565. (31) Lu, L. H.; Ryu, K. S.; Liu, C. J. Microelectromechan. Syst. 2002, 11, 462469. (32) Li, N.; Hsu, C. H.; Folch, A. Electrophoresis 2005, 26, 3758-3764. (33) Paegel, B. M.; Grover, W. H.; Skelley, A. M.; Mathies, R. A.; Joyce, G. F. Anal. Chem. 2006, 78, 7522-7527. (34) Granfeldt, D.; Sinclair, J.; Millingen, M.; Farre, C.; Lincoln, P.; Orwar, O. Anal. Chem. 2006, 78, 7947-7953. (35) Sachs, F. Biophys. J 1999, 77, 682-690. (36) Sinclair, J.; Granfeldt, D.; Pihl, J.; Millingen, M.; Lincoln, P.; Farre, C.; Peterson, L.; Orwar, O. J. Am. Chem. Soc. 2006, 128, 5109-5113.

Specific examples for applications of the system reported here include the possibility to sequentially expose a probe to different flow patterns to, for example, investigate ion channel kinetics or oscillation decoding.12,34,36 Additionally, this system offers the ability to adjust inputs in response to the output from a probe, such that feedback from a probe could control subsequent pattern generation. The work presented in this paper is the first step toward creating an automated, real-time feedback system capable of spatiotemporal control of the environment around a wide variety of different probes with access to a plethora of detection methods. MATERIALS AND METHODS Experimental Setup. The microfluidic chip was purchased from Cellectricon AB (Gothenburg, Sweden). A detailed description of the chip can be found elsewhere.12,37 The chip is made of PDMS and glass and consists of a number of microchannels individually connecting solution inlets to an open volume. We utilized a 16-channel chip, which had channels with a center-tocenter distance of 72 µm (channel width 50 µm and wall width 22 µm) and a height of 60 µm. The channels are of equal length (50 mm) and exit as a tightly packed array into the open volume. Valves, tubing, and other assembly parts are all from Bio-Chem Valve/Omnifit (Cambridge, United Kingdom). Valves were connected to the middle eight solution inlets/channels of the chip with in-house-built, tapered, brass connectors with a diameter, at the narrowest end, of 5.5 mm. The remaining eight solution inlets were not used. Each connector couples two valves to a solution inlet: one that is used to switch between different inputs and one that acts to speed up exchange of the dead volume. The setup is illustrated in Figure 1 and described under the System section of the Results and Discussion. The open volume of the chip is filled with buffer solution so that the flows from the channels enter a solution-filled bath. When the system is operated in the low Reynolds number regime, using fluids that are sufficiently homogeneous, a patterned laminar flow comprising separate solution environments is generated in the open volume outside the channel outlets.12,37 The microfluidic chip is placed upon a scanning stage so that probes (nano- and microsized electrodes, open tips, and patch-clamped patches or whole cells have previously been tested but the system could also be used with other nano- or micro-sized probes) can be translated between the separate solution environments, with solution exchange times of tens of milliseconds. The solution exchange time depends upon many factors including probe size and translation velocity; with this microfluidic chip, for a ∼10-µm cell translated at a speed of several millimeters per second it has been reported to be 10-12 ms.34 The microfluidic chip is mounted on an inverted microscope stage (Leica DM IRB, Wetzlar, Germany) equipped with a motorized scanning stage (Scan IM 120 × 100, Martzhauser Wetzlar GmbH & Co. KG, Wetzlar-Steindorf, Germany). The motorized scanning stage we employed has a travel distance of 120 × 100 mm and a maximum translation velocity of 180 mm/s. For the used load, the maximum acceleration was ∼1 m/s2. Control Software. Switching of the valves is controlled via a script programmable USB interface. Thus, sequences of valve switches can be programmed as well as mixing algorithms (37) Olofsson, J.; Pihl, J.; Sinclair, J.; Sahlin, E.; Karlsson, M.; Orwar, O. Anal. Chem. 2004, 76, 4968-4976.

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Figure 1. Schematics of device design. (A) A 3D schematic drawing of the microfluidic chip and external components. Highlighted in red are the 16 microchannels, each of which originates in a solution reservoir, or connection well, and exits into the open volume as a tightly packed array. Three connectors linking 3 of the channels to external valves are shown. The 3 channels for which connectors are shown are marked 1, 2, and 3 and correspond to the segment of the device illustrated in (B). (B) Illustration of a 3-channel segment of the system. (C) Side view of the chip, press fit connector, and valves. (D) Operation of the system during solution switching.

involving several switches per valve per second. Translation of the chip relative to the probe is also fully programmable. The velocity and trajectory over time are software controlled. Therefore, sequential scans and switching of the valves can be preprogrammed such that a probe can be exposed to a series of different 9288

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flow patterns in an entirely automated manner. Both programs are currently separate, and operation of the complete system requires manual simultaneous start of both programs. Electrochemistry. Solutions containing varying concentrations of hexaamineruthenium(III) chloride in a buffer of 25 mM

KH2PO4, 25 mM K2HPO4, and 0.1M KCl were loaded into the containers as shown in the figures or described in the text. A carbon fiber electrode having a cylindrical electroactive area (5 µm in diameter and 30 µm in length; ProCFE, Dagan Corp., Minneapolis, MN) was positioned 20 µm outside the channel outlets with micromanipulators. The symmetry axis of the electrodes was aligned in the flow direction in order to optimize the spatial resolution of the measurements. A potential of -0.3 V versus Ag/AgCl was applied to the electrode using a Heka EPC10 triple patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). The current resulting from reduction of the hexaamineruthenium(III) complex is proportional to the concentration of hexaamineruthenium(III) chloride, and this was measured using the same amplifier. The electrode was scanned along a trajectory, perpendicular to the channel outlets, by moving the microchip relative to the electrode with the motorized scanning stage. The scans occurred in a plane 30 µm above the bottom of the chip. The maximum scan speed utilized was 0.05 mm/s. The sampling frequency was 200 Hz, and all curves were low-pass filtered at 100 Hz for analysis and presentation. The traces were not corrected for electrode fouling. Fluorescence Experiments. The fluorescence microscopy was carried out using an inverted microscope (Leica DM IRB, Leica, Wetzlar, Germany) equipped with an Hg lamp as the excitation source. The excitation light was sent through a 10× objective (Leica PL Fluotar) and collected through a Y3 filter cube. Fluorescence micrographs were obtained using a Nikon D200 digital SLR camera. Every other channel/container was loaded with 50 µM fluorescein, and the dilution buffer contained 10 mM HEPES, 20 mM NaCl, 5 mM KCl, 1 mM CaCl2, and 1 mM MgCl2 and was pH adjusted to 7.4 with KOH. All chemicals used in both the electrochemistry and fluorescence experiments were purchased from Sigma-Aldrich Ltd. or Merck. Finite Element Method Simulations. To simulate the flow pattern immediately outside the channel outlets, we used the finite element simulation program COMSOL (Comsol, Stockholm, Sweden). Navier-Stokes equations, for incompressible Newtonian fluid, were solved for a 2D geometry (see Figure 3A). The inflow velocity was set to either 5 or 10 mm/s and the pressure was set to zero on the boundaries confining the open volume. All other boundaries were set to no slip. The fluid density was set to 1000 kg/m3 and the viscosity to 1 mPa. RESULTS AND DISCUSSION System. We used a commercially available microfluidic chip, for which the design, fabrication, and fluidic properties have been fully described in previous publications.11,12,37 In Figure 1A, a schematic of the chip is shown. The chip is made of PDMS and glass and consists of an array of microchannels individually connecting solution inlets to an open volume. Input solutions are delivered to the chip through valves coupled to the on-chip solution inlets from containers suspended 0.5 m above the microfluidic chip. Flow through the system is driven by hydrostatic pressure. The valves are connected to the on-chip solution inlets by 0.8-mm-inner diameter tubing linked to tapered brass connectors that attach into these connection wells via a press fit connection. The diameter of the connectors (5.5 mm) is 20% larger than that of the on-chip connection holes to ensure a good quality

press fit connection.38 The connection wells have a diameter of 4.5 mm, a height of 5 mm, and hold 80 µL of solution when employed as solution reservoirs, as in previous work.11,12,37,39 The connector occupies ∼80% of the connection well, and thus, the dead volume when interfaced to a connector is 16 µL. The total dead volume of the system, including the dead volume of the valve, a 12-cm length of 0.8-mm (I.D.) tubing linking the valve and the connection well, is 126 µL. Exchange of this dead volume at a flow rate of 10 mm/s through the microchannel would take about 70 min. Therefore, each connector contains two segments of tubing, each of which is linked to a valve. The first valve (referred to as the inlet valve) controls the input to the microchannel whereas the second valve (referred to as outlet valve) is employed to speed up exchange of the dead volume. Every time switching of an inlet valve occurs, the corresponding outlet valve is opened for 15 s to ensure complete exchange of the dead volume. The setup is illustrated in Figure 1B. The design and operation of the connectors and valves are shown in Figure 1C and D, respectively. The pressure difference driving the flow was calculated to be 4.9 kPa. The volume flow, when the outlet valve is closed, has been calculated to be 3 × 10-5 mL/s using the Poiseuille equation.40 This corresponds to an average flow velocity in the microchannel of 10 mm/s. When the outlet valve is opened, the volume flow is observed to be 0.05 mL/s (from the rate of decrease of the solution in the containers). Thus, although the dead volume is 126 µL, the amount of solution consumed in one switch of an inlet valve is 750 µL, since opening the outlet valve results in a flow rate of 0.05 mL/s maintained for 15 s. The inchannel flow rate when the outlet valve is opened is discussed later under Hydrodynamic Effects. Some variability in the flow rate over time is expected as the solution level in the containers drops. The maximum difference in flow rate has been calculated to be 0.5 mm/s. However, during electrochemistry and fluorescence imaging, undertaken to characterize the system, this variation was too small to be observed. Additionally, the flow rate variability can be minimized by continual manual refilling of the containers and could be further reduced by using wider containers. Time Required for Solution Switching. We used amperometry in order to measure how the concentration outside a channel is altered over time when the inlet valve is switched. Figure 2 shows the amperometric response, from a 5-nm-diameter carbon fiber electrode, held at -0.3 V, measured following switching of the inlet valve from an electroactive substance, hexaamineruthenium(III) chloride, to buffer. The electrode was positioned 20 µm outside the channel exits, at a height of 30 µm. The black arrow indicates the time between switching of the inlet valve and the onset of solution switching in the bath chamber, which is 10.5 s and is due mainly to the time required to clear the channel of the hexaamineruthenium(III) chloride solution. Additionally, one can observe that when the outlet valve is opened, the amperometric response decreases due to a lower in-channel flow rate and thus decreased mass transport to the electrode. From the time taken (38) Christensen, A. M.; Chang-Yen, D. A.; Gale, B. K. J. Micromech. Microeng. 2005, 15, 928-934. (39) Sinclair, J.; Olofsson, J.; Pihl, J.; Orwar, O. Anal. Chem. 2003, 75, 67186722. (40) Beebe, D. J.; Mensing, G. A.; Walker, G. M. Annu. Rev. Biomed. Eng. 2002, 4, 261-286.

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Figure 2. Amperometric trace demonstrating a complete solution switch outside one channel of the microchip. The blue panel indicates how long the outlet valve was open. Opening of the outlet valve and switching of the inlet valve occurred simultaneously. The black arrow indicates the time from when the inlet valve was switched until the new solution reached the bath chamber. The blue arrow shows the solution exchange time experienced by the electrode, positioned outside the channel. The green arrow indicates the time from when the outlet valve was shut until solution exchange of the flow segment outside the channel was completed. The black vertical mark in the trace shows when the electrode was translated to outside an adjacent buffer channel.

to clear the channel (50 mm long), we estimate that the in-channel flow rate is reduced by 50% to ∼5 mm/s. After the onset of solution switching, the electrode experiences a gradual solution exchange over 10 s (see the blue arrow in Figure 2). The change of slope observed in this exchange segment corresponds to the flow rate increase associated with shutting the outlet valve. The time taken from when the outlet valve is closed to completion of the solution switching in the bath chamber is 5.5 s (the green arrow). Thus, the total time required for solution switching is 20.5 s (blue panel plus green arrow) whereas the solution exchange time experienced by a probe in the open volume is 10 s (the blue arrow). Experiments indicated that 15 s was the optimal opening time for the outlet valve since shorter times resulted in incomplete exchange (results not shown). To confirm that complete solution switching occurs with a 15-s opening time, an adjacent channel was loaded with buffer, and after the 20.5 s required for solution switching, the electrode was translated, using the programmable motorized scanning stage, into the flow segment emanating from this buffer channel. As can be seen after the black mark in Figure 2, no concentration difference was measured by the electrode between the switched and the control solution. Thus, it was confirmed that complete solution exchange had occurred. By dividing the dead volume by the flow rate of 0.05 mL/s, when the outlet valve is open, the solution contained in the tubing and the connection well should be exchanged in 2.5 s. The difference between the calculated and experimentally determined times may be explained by the presence of an unstirred layer at the edges of the solution reservoir, exchange of which occurs only by diffusion.23 It was also considered that setup variations such as the tube length between the inlet valve and the connector and the height placement of the connectors, for which it is difficult to ensure exact reproducibility, could contribute to the exchange time discrepancy. However, even if the dead volume was quadrupled to 504 µL (corresponding to, for example, a dead volume of 40 µL in the connection well and a tube length of 0.82 m), it 9290 Analytical Chemistry, Vol. 79, No. 24, December 15, 2007

Figure 3. Effect of opening the outlet valve on the flow pattern. (A) Simulations of the patterned laminar flow. The flow velocity can be read out using the color bar. Streamlines emanating from the edges of adjacent channels indicate the position of the interface between the solution segments exiting these channels. Left panel: outlet valves shut in all channels and thus the in-channel mean flow velocity is set to 10 mm/s. Right panel: outlet valves opened in channels 2 and 6 and therefore the flow rate is decreased by 50% to 5 mm/s in these channels. The simulation does not take diffusion into account. (B) Fluorescence images. Left panel: outlet valves are shut in all. Right panel: outlet valves opened in channels 2 and 6. (C) An amperometric trace corresponding to a scan across 8 channels. The loading pattern is shown in the yellow box under the trace where the upper panel shows the concentration of hexaamineruthenium(III) chloride and the lower the channel number (the inlet valves were not switched). Two scans are shown: one with outlet valves are shut in every channel (black trace) and one where the outlet valves are open in the buffer channels (red trace). The resulting narrowing of the flow from these channels in the red trace is observed as a slight shift in the relative lengths of the different sections of the amperometric trace.

would only take 10 s to exchange. This is less than the observed time of 15 s, suggesting that the unstirred layer is the limiting factor and thus that small variations in the setup, such differences in tube length or connector placement, will not affect the performance of the system. Hydrodynamic Effects. The role of the outlet valve is to speed up the exchange of the dead volume by providing an extra outlet, thus increasing the flow through the system. However, the inchannel flow rate is decreased when the outlet valve is opened. By comparing the resistances40 of the exit tubing to the outlet valve and the channel (5 × 109 Pa‚s/m3 compared to 1.6 × 1014 Pa‚s/m3), we have calculated that 0.03% of the flow will pass through the channel, which corresponds to an in-channel flow rate of 5 mm/s and thus a decrease of 50%. This agrees well with the electrochemistry results since the time until onset of switching in the open volume (see the black arrow in Figure 2) divided by the channel length gives an estimate of the in-channel flow rate of ∼5 mm/s. For applications of this system, the important factor is whether this flow rate reduction disturbs the laminar flow pattern. Evidently, if the flow is reduced in only one channel, the flow segment emanating from this channel will be narrowed. COMSOL simulations as well as imaging and electrochemistry experiments

Figure 4. Switching between different patterned flows. Upper panel: fluorescence images of a series of flow patterns, consisting of 50 µM fluorescein and buffer, sampled every 45 s. Lower panel: an amperometric trace showing back and forth scans (each starting in channel 1) across the four patterned flows A-D shown in the upper panel but with the 50 µM fluorescein buffer exchanged for 100 µM hexaamineruthenium(III) chloride solution. During the time frames marked with green in the color panel under the trace, the electrode was scanned back and forth across the current flow pattern (marked A-D, which refer to the flow patterns shown in the upper panel). The time frames marked with blue in the color panel under the amperometric trace represent periods of switching of the valves. During these periods, the electrode was positioned outside channel 1.

were undertaken to characterize the effect of opening the outlet valve on the patterned laminar flow. Although, both the simulations and imaging results (Figure 3A,B) indicate a narrowing of flow segments emanating from channels in which the outlet valve is open, the integrity of the pattern close to the channel exits is maintained. This is also shown in Figure 3C where the form of an amperometric trace, obtained by scanning an electrode through the patterned flow at a distance of 20 µm from the channel exits, is only slightly altered by opening of the outlet valve in certain channels. Generation of Numerous Flow Patterns. Utilizing switching between preloaded input solutions, the system is capable of online switching between Nn different patterned flows, where N is the number of valve inputs and n, the number of microchannels. Here, we employed valves with two input ports and a common output connected into 8 channels of a 16-channel chip. Thus, we have increased the number of input combinations, corresponding to different flow patterns, for this 8-channel segment from 1 to 28, i.e., 256. Four different patterns are illustrated in Figure 4 where the patterned flow outside the channel outlets is sampled every 45 s using fluorescence imaging (upper panel) and amperometry (lower panel). A back and forth amperometric scan across eight channels was performed for each pattern, with switching of the valves to change patterns, occurring between the scans. The entire scan and switching of the valves was fully automated. A 25-s wait between each scan was employed, and each scan took 20 s at a scanning velocity of 57 µm/s. Additionally, a fluorescence image was taken of each pattern. The number of flow patterns could be further increased by utilizing valves with a greater number of inlets as well as more channels. For example, using valves with 3 input ports interfaced to all 16 channels of the chip generates over 40 million different flow patterns. Additionally, although the number of input combina-

tions remains constant with a two-input valve system, the fact that the system is open allows for manual exchange of solutions in between experiments so that the container loading pattern and, thus, the content of the flow patterns is easily altered. Mixing. Mixing of the inlet solutions can be performed by rapid switching between the different inlets. This generates alternating plugs of the different solutions, which become perfectly mixed within the connection well. The mixing procedure is schematically illustrated in Figure 5A. The outlet valve is opened and the inlet valve is rapidly cycled (on the order of a few hundred of milliseconds) between the two different inputs to generate a series of small flow plugs (ten of microliters). With a 1:1 cycling ratio, a series of flow plugs of equal size are created thus resulting in a 1:1 mixture of the two input solutions. After 30 s, complete mixing of the connection well is obtained. The outlet valve is then shut, but the cycling of the inlet valve is continued to ensure a constant input into the connection well of the same concentration. To ensure such a constant input, the flow plugs should be small enough that they have time to completely mix within the tubing before they enter the connection well. Since the flow rate is significantly lower when the outlet valve is closed, sufficiently small flow plugs will be formed even with slightly slower cycling rates (seconds). We diluted a solution of hexaamineruthenium(III) chloride by mixing with buffer. Figure 5B shows how different concentrations can be obtained by varying the ratios of the plug sizes. We successfully diluted a solution by 33, 50, and 66% with an accuracy of within (5%. Due to the uncertainty in using the amperometric response to monitor concentration over time, we could not verify the mixing more accurately than (5%. For example, changes in the flow rate due to refilling/depletion of the solution in the solution containers as well as electrode fouling can alter the measured response. Note that the electrochemistry traces have Analytical Chemistry, Vol. 79, No. 24, December 15, 2007

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Figure 5. Mixing. (A) Schematic of the mixing procedure. (B) Upper trace: 1:1 cycling ratio results in a 50% dilution of a 200 µM hexaamineruthenium(III) chloride solution. The outlet valve was open between A-B, C-D, E-F, and G-H, and the inlet valve was cycled between C-E. Lower trace: 2:1 and 1:2 cycling ratios gives dilution of the solution to 33 and 66%, respectively. The outlet valve was open between A-B, C-D, E-F, G-H, I-J, K-L, and M-N, and the inlet valve was cycled between E-G and K-M.

not been corrected for fouling. One contribution to the discrepancy between our mixing results and the expected values may be explained by differences in the relative heights of solution in the containers from which the two solutions were mixed. This source of error could be reduced by using a more uniform method of solution delivery, e.g., by using large solution containers or micropumps. For the 1:1 mixture, we used a mixing algorithm comprising 0.2-s switches between the different inputs whereas for the 2:1 switches the relative opening times for each inlet were 0.4 and 0.2 s. When the outlet valve was shut, the switching times were increased 5-fold. It is straightforward to upscale the mixing procedure with the same microfluidic design by generalization of the mixing algorithm to obtain a wider range of different concentrations or by addition of valves with more inlets to facilitate mixing of more complex solution compositions. The main advantage of this mixing system is that each solution segment in a patterned flow can be individually mixed and additionally be changed over time. There9292

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fore, it is possible to obtain combinatorial mixtures of several substances arranged in any desired spatial pattern. Furthermore, the ability to mix increases the number of input combinations, Nn, to an almost infinite number since it increases N, from a fixed number of inlet ports to a wide range of possible concentrations that one could mix. Applications. Previously demonstrated examples of applications of the patterned laminar flow generated by the microfluidic section of this system include investigations of ligand-ion channel interactions and the generation of complex concentration variations (oscillations) around microscopic objects.12,34,36 Solution exchange on the order of tens of milliseconds occurs around a probe translated across narrow interfaces in a laminar flow,35 and this has been essential in these applications to study rapidly desensitizing ion channels and to generate high-resolution oscillations mimicking physiological rhythms. Solution exchange on the order of hundreds of milliseconds can be observed in scanning segments of Figure 4, and this could be further improved by

Figure 6. Waveform created via scanning back and forth outside 8 channels where the flow patterns are switched between the forward and backward scans. The flow patterns can be considered as piecewise constant approximations. One approximation generates the steplike form, and the other corresponds to buffer in all channels. Automated switching (blue panels) between the two piecewise linear approximations was correlated with automatic probe scanning (green panels) to generate this waveform.

increasing the translation velocity. One of the advantages of the system reported in this paper is the combination of rapid solution exchange with the ability to update the flow pattern over time. This could be applied to increase throughput in patch-clamp by facilitating exposure to larger number of solution environments, while retaining the ability to achieve high-quality data; to expose a cell, or cell fragment, to a series of oscillations with varying amplitude, frequency, and shape for decoding studies; and to offer the ability to use a series of piecewise constant approximations to generate an oscillation, as illustrated in Figure 6. This latter application has many advantages in creating oscillations; generation of nonmirror symmetric oscillations, greater control of the relative phases of overlapping oscillations, and the ability to utilize a greater number of channels in the approximation as a probe no longer merely has a choice between two different solution environments as its neighboring environments are temporally dynamic. However, the strategy of interfacing to external valves, via press fit connectors, could also be applied to other microfluidic systems, and within these, the ability to rapidly alter the flow pattern may open up many other applications. One exciting possibility, using our system, is that on-line modifications of the spatial pattern can be adopted in response to the output from a probe, thereby creating a feedback system. The

next challenge is to develop a system in which the feedback is automatic and in real time, i.e., a system that responds to the signal recorded from the probe and relays this information back to the inputs such that they can be adjusted in an appropriate manner. An automated feedback device is likely to prove useful in many different experiments; two biological examples are that a patchclamped cell could autonomously determine EC50 values by sequential narrowing of a concentration span or a cell could report on optimal signaling characteristics by self-scanning a series of exposures to oscillations of varying amplitude, frequency, and shape. ACKNOWLEDGMENT This work was supported by the Swedish Research Council (VR), the Swedish Foundation for Strategic Research (SSF), the NanoX, and Biomics programs and the Go¨ran Gustafsson Foundation.

Received for review June 8, 2007. Accepted July 17, 2007. AC0712087

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