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View Online PAPER www.rsc.org/loc | Lab on a Chip Actuation of elastomeric microvalves in point-of-care settings using handheld, battery-powered instrumentation† Kweku A. Addae-Mensah, Yuk Kee Cheung, Veronika Fekete, Matthew S. Rendely and Samuel K. Sia* Downloaded by Columbia University on 06 May 2011 Published on 12 April 2010 on http://pubs.rsc.org | doi:10.1039/C002349C Received 3rd February 2010, Accepted 16th March 2010 First published as an Advance Article on the web 12th April 2010 DOI: 10.1039/c002349c Although advanced fluid handling using elastomeric valves is useful for a variety of lab-on-a-chip procedures, their operation has traditionally relied on external laboratory infrastructure (such as gas tanks, computers, and ground electricity). This dependence has held back the use of elastomeric microvalves for point-of-care settings. Here, we demonstrate that microfabricated microvalves, via liquid-filled control channels, can be actuated using only a handheld instrument powered by a 9 V battery. This setup can achieve on–off fluid control with fast response times, coordinated switching of multiple valves, and operation of a biological assay. In the future, this technique may enable the widely used elastomeric microvalves (made by multilayer soft lithography) to be increasingly adopted for portable sensors and lab-on-a-chip systems. Introduction In recent years, the use of multilayer soft lithography for making microfabricated pneumatic valves has provided a reliable and versatile platform for advanced fluid handling in lab-on-a-chip (LOC) devices, offering advantages of rapid prototyping and biocompatibility compared to silicon-based MEMS. Such pneumatic valves have been used for a wide range of research applications taking place in centralized facilities.1–4 A drawback in the most widely adopted implementation of pneumatic valves is that they require laboratory infrastructure such as gas tank, computers, and ground electricity. Hence, advanced fluid handling with pneumatic valves is difficult to accomplish for point-of-care (POC) systems in health diagnostics, epidemiology, autonomous sensors, and homeland security. This limitation applies especially to resource-limited settings such as developing countries, where even reliable external power is lacking. For example, while a variety of valves (such as screwbased valves,5 stimuli-responsive hydrogels,6,7 passive valves,8 and burst valves9) are useful for different applications performed in a research lab, they typically do not have the combination of automated operation, fast response times, resistance to leakage, independence of external infrastructure, and ability to be used multiple times for an ideal POC system. Previous studies on implementing valves for remote settings include a system used on the planet Mars,10,11 although fabrication of the chip was relatively complex. In another approach pioneered by Takayama’s group, moving pins from a Braille Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace, 1210 Amsterdam Ave, New York, NY, 10027, USA. E-mail: [email protected] † Electronic supplementary information (ESI) available: Fig. S1, Circuit diagram of the microcontroller; Fig. S2, Circuit diagram for measuring the response times of microvalves; Fig. S3, Circuit diagram for measuring the power drawn by solenoids; Fig. S4, Response time of valves using ionic liquid inside control channels; Movie S1, A movie of colored dye moving through two parallel channels in a microfluidic chip with the valves actuated in an arbitrary sequence described in Fig 3. See DOI: 10.1039/c002349c 1618 | Lab Chip, 2010, 10, 1618–1622 display12,13 driven by a computer (running a text editor or Braille screen reading software) have been used to successfully demonstrate hydraulic-principle based two-layer valve control.14 However, the requirement for the computer may introduce limitations for use at the POC. Here, we develop a handheld instrument to operate membrane-based microvalves, based on the hydraulic principle for actuation.14 We sought to replace the requirement for external instrumentation and a computer with a portable device that can maintain high control over valve operation. Materials and methods Microfluidic chamber and control layer fabrication The microfluidic flow and control layers were fabricated using standard methods as described by Unger et al.,15 with a push-up configuration where the control layer is located below the fluid flow layer. The control layer was filled with water, which served as the hydraulic fluid. Before filling the control layer, we sonicated the water for 15 minutes to eliminate micro-bubbles. With the control channels being closed at one end, they were filled by placing the chip under vacuum for 20 minutes, while water droplets covered the control layer inlets. Once the control channels were completely filled, we sealed their inlets with a thin PDMS membrane as follows. First, we coated a cured PDMS membrane (about 3 mm2 in area) with PDMS prepolymer. We then placed the membrane on the water droplet covering the control channel inlet, and gently pressed the membrane down until it covered the inlet and was firmly in place, while ensuring that we did not trap any air bubbles between the membrane and the control channel inlet. To cure the sealing membrane, we placed the completed device in a humid chamber (Petri dish containing water-soaked Kim wipes) for 48 hours at room temperature. We did not detect any loss of water from the control channels for up to 7 days when the device was kept in the sealed Petri dish. We also repeated the above experiment using ionic This journal is ª The Royal Society of Chemistry 2010 View Online liquid (1-butyl-3-methylimidazolium tetrafluoroborate, Acros Organics) instead of water as the fluid in the control channels. Design and construction of solenoid-containing portable actuation unit Downloaded by Columbia University on 06 May 2011 Published on 12 April 2010 on http://pubs.rsc.org | doi:10.1039/C002349C Solenoids (F110C-6V GeePLus, Inc) with cylindrical plungers of diameter 1.2 mm were used to drive the hydraulics. They can be operated between 6.5 and 9 volts, and are rated for force between 0.07 and 1.2 N and stroke lengths from 0.25 to 1.25 mm. They were modified by inserting springs (rates between 0.014 and 0.17 N mm1) between the plunger and the solenoid body (Fig. 1B) to provide a return mechanism for the solenoid plungers. These modified solenoids were attached to a movable plate which could be moved freely in the vertical direction such that the plungers aligned with the positions of the access holes punched for the control layer (Fig. 1). To ensure alignment of the inlets in the control layer with the positions of the solenoids, we imported the design drawings used for making the movable plate into the designs used for making the fabrication masks for the control layer. Design of microcontroller-based electronic circuit The solenoids were controlled by an Atmel Mega32 microcontroller along with peripheral circuitry (Fig. S1†). The actuation sequence of the solenoids and valves could be pre-programmed into the microcontroller, which contained 32 programmable input/output lines. The operating voltage for the microcontroller is 4.5 to 5.5 V. The microcontroller-based circuit also controls a small micropump (Hargraves Advanced Fluidic Solutions E219-12) with operating voltage of 3–6 V. A liquid trap was also built to interface the micropump to the microfluidic network to prevent liquid from destroying the pump. The trap consisted of a 500 mL well with two outlets one connected to the micropump and the other connected to the outlet of the microfluidic network. The speed/strength of the micropump could be adjusted by setting a value in the microcontroller which determined the final voltage applied to the pump using a pulse width modulation (PWM) algorithm. The speed was adjusted to account for the latency introduced by the liquid trap. The overall footprint of the device was 13 7.6 5.8 cm, and could be operated by a 9 V dry-cell battery. Operation of sample biological assay: detection of horseradish peroxidase (HRP) Fig. 1 Schematic diagram, picture and mode of operation of the actuation system. (A) Simplified schematic and picture of instrument. A simplified schematic diagram of the instrument showing the main components. A cross-sectional view of a single solenoid showing a typical arrangement of the coil assembly, and plunger is also shown. The picture shows the fully constructed instrument and a close up of the interface between the solenoids and the microfluidic chip. The whole unit is designed to be battery operated with a microcontroller and peripheral electronic components assembled on a custom made printed circuit board. (B) Schematic showing how the hydraulic valves operate. The solenoids are modified by inserting solenoid return springs between the solenoid body and the plunger. This journal is ª The Royal Society of Chemistry 2010 To demonstrate the fluid handling capability of the microcontroller based electronic control system, we ran an assay to detect horseradish peroxidase (as a mock sample) using Amplex Red. The Amplex Red reagent was prepared in 1 reaction buffer to a final concentration of 100 mM, and mixed with 2 mM H2O2. We drew 200 mL of the resulting solution into 5 cm long Tygon tubing (0.05000 0.09000 OD), and connected it to the input port of the microfluidic network. Similarly, we connected tubings containing 100 mL of sodium phosphate buffer (pH z 7.4) and horseradish peroxidase in 1 reaction buffer to two other inlet ports. We connected a small battery-powered micropump to the outlet and used to draw the fluids through the microfluidic network. After mixing, the valves were closed to confine the mixed reagents within the detection zones. We measured fluorescence by analyzing mean intensities of light emanating from three rectangular regions within the detection zone. We subtracted the background from the signal region to obtain corrected values of fluorescence. Results and discussion We constructed a handheld instrument for actuating elastomeric microvalves as follows. Four linear pull-type solenoids for controlling the microvalve system were attached to a plate that could move vertically (in order to accommodate a microfluidic chip) relative to a fixed base plate (Fig. 1A). The positions of the Lab Chip, 2010, 10, 1618–1622 | 1619 Downloaded by Columbia University on 06 May 2011 Published on 12 April 2010 on http://pubs.rsc.org | doi:10.1039/C002349C View Online solenoids were designed such that they aligned with positions of inlets into the control layer of the microfluidic chip (Fig. 1A). The positions of the inlets were spaced apart (at least 12 mm) to accommodate interfacing with multiple solenoids. The microfluidic channels in the control layer were filled with water. Each solenoid was a linear pull-type solenoid, consisting of a plunger (movable iron component) and a coil assembly (Fig. 1A). Initial contact of the solenoid plungers with PDMS membranes (250 mm thick) covering the inlets to the control channels resulted in the downward deflection of the membranes, thereby transmitting force through the fluid-filled control channels to the thin membrane (30 mm) between the control and the fluid layer. As a result, the microvalves in the fluid layer closed. When a voltage was applied to the coil assembly, a magnetic attractive force was produced between the plunger and the coil, pulling the plunger into the metal housing; in turn, the microvalves in the fluid layer opened (Fig. 1B). Fig. 2 Demonstration of operation of the valve. (A) Images of the valve in the closed and open state. Colored dye was flowed through the microfluidic channel using a Kent Scientific pump operating at 1 mL min1 and the valve was opened and closed at various times. The images represent bright field images of the valve closed at 90 seconds and opened at 5 seconds. The rectangular area represents the region of interest that is processed for generation of mean intensity values used for the plot in (B). (B) Valve opening and closing with time. A graph showing the opening and closing of the valve recorded over 100 s (16 cycles). (C) Time response of the valve. Time response of valves 100 mm 100 mm 12 mm and 8.6 mm long filled with water which is used as the hydraulic fluid. The average time for the valve to close for devices filled with water was 53.4 ms. 1620 | Lab Chip, 2010, 10, 1618–1622 In these commercially available solenoids, the plunger stayed in the metal housing even after switching off the voltage. We inserted a return spring between the plunger and the metal housing (Fig. 1B), such that the plunger moved out of the housing (closing the microvalves) immediately after switching off the voltage. This design resulted in two useful features: (i) the microvalves would be closed in the absence of voltage supplied to solenoids, and (ii) the plunger was primed for movement upon re-application of the voltage. We first characterized the ability of the solenoids to actuate the valves to high precision. In order to monitor the response times of the valves, we used a MATLAB script to apply voltage to the solenoids (Fig. S2 and S3† show electronic circuit and connections) and a data acquisition and control card that control image Fig. 3 Parallel operation of multiple valve locations. (A) Bright field images of multiple valve locations. Bright field images taken with a QImaging Retiga 2000R 16bit monochrome camera showing multiple valve locations. The images represent snapshots of the fluid and control channels at 19 and 56 seconds with the valves designated as grey, dashed black, and black. (B) A plot showing valve opening and closing with time. We pumped colored dye through two parallel microfluidic channels and closed and opened the valves in the following arbitrary sequence: open grey (t ¼ 3 s), open dashed black (t ¼ 4 s), open black (t ¼ 5 s), hold all three valves open (t ¼ 5 to 9 s), close black (t ¼ 9 s), close dashed black (t ¼ 10 s), close grey (t ¼11 s), hold all three valves closed (t ¼ 11 to 13 s), and repeat the cycle. We used a Kent Scientific syringe pump operated as a vacuum pump with a 30 mL BD syringe. The fluid flow rate was approximately 4 mL min-1. This journal is ª The Royal Society of Chemistry 2010 Downloaded by Columbia University on 06 May 2011 Published on 12 April 2010 on http://pubs.rsc.org | doi:10.1039/C002349C View Online acquisition on the camera. Two bright field images (Fig. 2A) show colored dye in the fluid channel, with a valve in closed and open states. As expected, we saw fluid flow in the open state, and saw no flow in the closed state, indicating that the microvalves could be controlled by movement of the plungers. We estimated the degree of ‘‘valve opening’’ by expressing the mean pixel intensity value of the dye region as a percentage between the maximum (255 for 8-bit grayscale values) and minimum (0) pixel intensities. The valve opened and closed consistently over at least 16 cycles (Fig. 2B). The response time of microvalves is an important factor in determining its use. For instance, hydrogel-based microvalves that respond in seconds to minutes are well-suited for drug delivery and other applications where slow delivery and control of fluid flow are required. By contrast, faster response times (on the order of seconds or less) are needed for analytical systems where sorting, splitting and merging of fluid are prevalent. We measured the response time of the valve by measuring the difference in time for when the control signals were sent to the valve and when a change in the valve opening state was observed (see ESI†). For water-filled control channels, we observed a response time of 53 5 ms (Fig. 2C). (Since a previous study also reported the use of ionic liquids in the control layer,14 which can potentially be kept longer at room temperature without evaporation through the PDMS, we also measured the response time of our microvalves with control channels filled with ionic liquid. The response time was 128 14 ms, which is also fast. In this study, we used water for the rest of the experiments.) Next, we tested the ability to coordinate the operation of multiple microvalves via solenoid-driven control. We tested the ability of three solenoids to control six microvalves (each solenoid controlled two valves). As in the previous experiment, we used a data acquisition module in order to accurately monitor the response times of the valves. We flowed color dye through two parallel microchannels in the fluid layer; three microvalves regulated flow at different sections of a single microchannel (Fig. 3A). With the three valves designated as grey, dashed black, and black (Fig. 3A), we sought to operate the valves in an arbitrary sequence (Fig. 3). The microvalves operated as designed (see Fig. 3A and Movie S1, ESI†). We also measured the degree of opening and closing of the three valves, and observed a high degree of control over operation (Fig. 3B). Fig. 4 Detection of horseradish peroxidase. (A) Design of the microfluidic network. Outline of microfluidic network showing the fluid and control layers, inlets and outlets and an image of the microfluidic chip filled with the fluid and control channels filled with food coloring making them easier to visualize. The regions within the detection zones that are imaged and analyzed have been shown. We programmed the MCU to turn on the solenoids in the following sequence: turn on the micropump (t ¼ 0 s), open valves 1, 2, 3 and 4 (t ¼ 5 s) and keeping them open (t ¼ 5 to 25 s) to allow mixing of the fluorescent dye with a negative control (i.e. sodium phosphate buffer), and close valves 1, 2, 3, and 4 (t ¼ 25 s) to confine the mixed solution to the detection zone. The MCU repeated this operating sequence with valves 5, 6, 7 and 8 to mix the fluorescent dye with the sample (horseradish peroxidase), and confine the mixed sample to the detection zone. Finally the MCU would turn off the micropump one second later, and the valves would be closed off during a 30 minute incubation period. (B) Epifluorescence images. Images of the detection zone showing epifluorescence produced from mixing 100 mM Amplex Red reagent, 2 mM H2O2 and 20 mU of HRP in 50 mM sodium phosphate buffer pH 7.4 and from mixing 100 mM Amplex Red reagent, 2 mM hydrogen peroxide and 50 mM sodium phosphate buffer pH 7.4 which is used as a negative control. The mixtures were incubated for 30 minutes confined within the respective detection zones. The rectangular regions of interest analyzed for mean intensity values for the plot in (C) are also shown. (C) Plot of relative intensities. A plot of the relative intensity (l ¼ 585 nm) of the fluorescence produced by the reaction of Amplex red, hydrogen peroxide and HRP and for a no-HRP (sodium phosphate buffer) control reaction. Background fluorescence, determined for the no-HRP control region, was subtracted from the HRP values. Table 1 The solenoids used to drive the hydraulics are rated to operate from 6 V to 12 V with a power rating of 2.3 W below a 50% duty cycle (maximum on time of 100 seconds at 8.5 V). From calculations based on data sheets provided by manufacturer, the current drawn by each solenoid is about 250 mA. For a typical assay, we assume that the valves are open for 10% of the time for an assay that runs for 20 minutes Component Voltage/V Current/mA Quantity Power/W Avg % on time per run Usage/W Usage/mA h Microcontroller Solenoid 4N35 Optoisolator TIP 31 Switching Transistor 2N3902 Switching Transistor Totals 5.0 9.0 1.2 9.0 5.0 12 233a 10 — — 255 1 4 4 4 4 0.06 8.40 0.05 — — 8.51 100 10 10 10 10 0.06 0.83 0.01 — — 0.90 4.0 31.1 0.3 — — 35.4 a Measured value. All other current values are estimates from manufacturer’s data sheets. This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10, 1618–1622 | 1621 Downloaded by Columbia University on 06 May 2011 Published on 12 April 2010 on http://pubs.rsc.org | doi:10.1039/C002349C View Online Finally, we sought to use the solenoid-control system to conduct a biological assay. In the previous experiments, we had used a data acquisition module in order to measure the response times. Here, we substituted the data acquisition and control module card (and the accompanying computer needed for running the MATLAB scripts) with a microcontroller unit (MCU) pre-programmed with a solenoid-actuation sequence. We translated the schematic diagram of the electronic circuit (Fig. S1†) into a printed circuit board layout (6.5 6.5 cm), in order to minimize the footprint of the device (Fig. 1A). In this manner, we could operate the solenoids without computer control, and since the solenoids and MCU could be powered by a 9 V battery, without ground electricity. In addition, a small micropump (also controlled by the microcontroller and also powered by the 9 V battery), rather than an external syringe pump, was used to pump the fluids through the microfluidic chip (Fig. 1A). We tested the use of this compact setup to operate the valves in the microfluidic chip. We programmed the MCU to turn on the solenoids in a predetermined sequence (Fig. 4, legend). We first tested the sequence of valve operation by using water and colored dyes as substitutes for the actual reagents, and observed the expected fluid movement. For the biological assay, we used buffer as negative control and horseradish peroxidase as sample, and measured the fluorescence intensity in the two detection regions. The assay produced a strong fluorescent signal for the case with enzyme and a weak background signal for the case of the negative control (Fig. 4B), with significant differences in the fluorescence intensities (Fig. 4C), as expected. Since the instrument is designed to operate without ground electricity, we sought to minimize power consumption of the electrical and mechanical components. We believe that an important feature of this design is that the solenoids can close the microvalves without a continuous supply of power, since many biological assays feature incubation times where valves would need to stay closed to confine samples or reagents. Moreover, we estimated how many assays can be run using a single 9 V battery, by calculating the power requirements of all the components in our device. We determined the current drawn by a single solenoid (using a circuit described in Fig. S3†) for 33 consecutive on–off cycles to be an average of 233 mA per cycle. Using estimates of power consumption from the manufacturers’ data sheets for other components, our estimate of energy consumption for a 20 minute biological assay is 35 mA hour (Table 1). Assuming 80% of capacity of a 9 V dry-cell battery rated for 625 mA hour, our handheld instrument for controlling the pneumatic microvalves can operate for 36 assays on a single battery. Previous valve technologies10–14 that require a computer will consume at least about 11 W h for a 30 minute assay, considering only the power consumed by the computer such as a laptop. This power requirement is substantially higher than our current design (0.45 W h). Over a long time period, water-filled control channels in PDMS may pose problems due to evaporation of water through the PDMS. Future designs using PDMS could make use of ionic liquids rather than water in the control channels,14 or other combinations of fluids and elastomeric materials where fluid adsorption to the material is low.16 In conclusion, we developed a setup that retained the advantages of PDMS-based pneumatic microvalves, but enable their use for POC diagnostics, including in resource-limited settings.17,18 1622 | Lab Chip, 2010, 10, 1618–1622 Acknowledgements We acknowledge an NIH STTR grant (5R41NR010753) and the Wallace H. Coulter Foundation for financial support. We thank Professor Bruce Land (Cornell University) whose layout of the printed circuit board we adapted for our design. We also thank Keith Yeager for help with machining and construction of the instrument. References 1 C. C. Lee, G. Sui, A. Elizarov, C. J. Shu, Y. S. Shin, A. N. Dooley, J. Huang, A. Daridon, P. Wyatt, D. Stout, H. C. Kolb, O. N. Witte, N. Satyamurthy, J. R. Heath, M. E. Phelps, S. R. Quake and H. R. 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