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A Valveless Pneumatic Fluid Transfer Technique Applied To Standard Additions on a Centrifugal Microfluidic Platform Matthew C. R. Kong and Eric D. Salin* Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal Quebec, Canada H3A 2K6 ABSTRACT: This paper demonstrates a valveless pneumatic fluid transfer technique applicable to centrifugal microfluidic platforms. The technique involves using compressed gas to generate a pneumatic force, which works together with the centrifugal force to control and direct fluid flow. Fluid can be pneumatically transferred from chamber to chamber, greatly decreasing the number of conventional valves required in a multistep process. By varying the rotational frequency of the centrifugal microfluidic platform while pneumatic force is applied, sequential fluid transfer steps can be achieved. The effectiveness of this fluid transfer method is demonstrated by performing a standard additions calibration. This technique is shown to be robust, easy to implement, and greatly reduces the design limitations traditionally associated with centrifugal microfluidic platforms.
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entrifugal microfluidics is a subset of conventional microfluidics, primarily involving the use of centrifugal force for pumping fluids. These microfluidic platforms are often shaped as disks which led to the term “Lab on a CD”.1 Centrifugal systems offer considerable advantages over many other microfluidic systems as the platforms do not require physical connections to pumps in order to initiate fluid flow.2 Centrifugal microfluidic platforms offer a convenient potential for parallel processing, because the centrifugal force is uniform at any given radius of the disk. As fluid pumping is achieved by centrifugal force, fluid flow does not rely on the properties of the fluids, as is the case with electro-osmosis based systems.1 In recent years, a growing focus of microfluidics has been the development of centrifugal micro-total analytical systems (μTAS),3 which can be used to perform quick and efficient environmental analyses or medical diagnostics. These analyses often require the integration of a large number of analytical steps on a single platform. To achieve this, the flow of samples and reagents on centrifugal platforms is initiated by centrifugal force and usually controlled in a stepwise fashion by valves. It is important to note that in centrifugal microfluidic applications, liquid usually flows “downward” or “outward” under the influence of the centrifugal force. A variety of valving techniques have previously been reported and used for centrifugal platforms.4 6 Among these, capillary and hydrophobic type valves7 are the most commonly used valve types. LaCroix-Fralish describes a typical application using capillary burst valves.8 A similar principle is used with hydrophobic valves, demonstrated by Haeberle in the development of a centrifugal blood plasma extraction system.9 Although common, these valving techniques can be quite labor intensive when manually fabricated and may be irreproducible. Siphon-based valves have also been used to control the flow of fluid on centrifugal microfluidic platforms.3,10,11 Although easy to design and fabricate, they require that the disk be stopped in order for the siphon to be primed. They also require that the surface of the siphon be properly treated (e.g., plasma treatment for hydrophilicity). Stopping the disk is sometimes undesirable as r 2011 American Chemical Society
the loss of centrifugal force may allow liquids to flow back into channels due to capillary action, while surface treatments may pose a longevity concern. A novel centrifugo-pneumatic valve has also been reported by Mark,12,13 but the design has some constraints that limit its general applicability. Valves can be one of the weak links in centrifugal platform design and construction. Flow control systems which involve a minimal number of valves may allow complex and previously difficult operational sequences of to be implemented more easily. In previous work, we reported a pneumatic technique offering the potential to vastly improve the capabilities of centrifugal microfluidic analytical systems by providing convenient flow switching at junctions and flow against centrifugal force (i.e., inward).14,15 This technique uses a stream of compressed air, directed at air vent holes already present on the platform, to influence fluid flow as desired. Building on that, we present here a valveless pneumatic fluid transfer technique that can change the fundamental design concepts of centrifugal microfluidic platforms, potentially opening doors to a new generation of centrifugal μTAS applications. We demonstrate the effectiveness of this technique by performing a standard additions calibration, which to date has not been applied to the field of centrifugal microfluidics. A powerful and commonly used analytical tool, standard additions is a multistep process that would be difficult to accomplish using only conventional valving techniques on centrifugal platforms.
’ METHOD AND MATERIALS Platform Fabrication and Reagents. A polycarbonate centrifugal platform was designed and fabricated using materials and techniques described in detail by LaCroix-Fralish.8 A fused-silica capillary with an inner diameter of 75 μm (Polymicro Received: August 22, 2011 Accepted: October 21, 2011 Published: October 21, 2011 9186
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Figure 1. Schematic depicting the instrument configuration. (a) Cylinder of compressed air. (b) Rotameter. (c) Solenoid valve. (d) Camera and lens. (e) Centrifugal microfluidic platform. (f) Servo motor.
Technologies, AZ) was used as a burst valve for the metering chamber. The capillary dimension was determined experimentally to burst well above 500 rpm. Solutions of 60 and 30 ppm of bromophenol blue (Fisher Scientific, NJ) dissolved in deionized water were used as the demonstrative sample and standards, respectively, for the standard additions experiment. Data Acquisition and Instrumentation. The experiment was carried out with the pneumatic instrument configuration described in detail by Kong15 but augmented with an open/close solenoid valve (SV3309, Omega, QC, Canada) to allow for precise timing control over the air flow. The solenoid valve remains switched on whenever a pneumatic operation is required. This configuration is shown in Figure 1. High-speed digital images were obtained using a motorized stage and strobe system developed by Duford16 but enhanced as described by Kong.15 The motorized stage, strobe, camera, and solenoid valve were controlled by a custom LabVIEW program (LabVIEW 8.6, Developer Version, National Instruments, QC, Canada). The centrifugal microfluidic platform was spun in a clockwise fashion for all experiments. For spectral measurements, the disk sample cell (path length of 1.4 mm) was placed in the optical path of an absorbance measurement system built from optical components including an iris (SM1D12SZ, Thorlabs, NJ), piano-convex lenses (LA1951, Thorlabs, NJ) and fiber optic holders (Thorlabs, NJ). Visible source radiation from a tungsten-halogen/deuterium lamp (DTMINI-2-GS, Ocean Optics, FL) was transmitted through a 300 μm 1 m fiber optic (QP300-1-SR, Ocean Optics, FL). A 600 μm 1 m detection fiber optic (QP600-2-UV vis, Ocean Optics, FL) was connected to a USB compatible miniature photodiode array spectrometer (USB4000-UV vis, Ocean Optics, FL). Platform Design and Procedure. A schematic of the platform design used to perform standard additions is shown in Figure 2, and a schematic depicting how pneumatic fluid transfer works is shown in Figure 3. With pneumatic pressure applied
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Figure 2. Schematic of disk design used to demonstrate the standard additions calibration.
while the disk is rotating at 380 rpm, liquid in the inner chamber (labeled Standard 1, Figure 2) is transferred into the metering chamber (Figure 3d(i)). The liquid can then be drained into the detection chamber at a high spin rate (Figure 3d(ii)). With pneumatic pressure applied while the disk is rotating at 260 rpm, liquid in the outer chamber (labeled Standard 2, Figure 2) is transferred into the metering chamber (Figure 3f(i)). This is then drained, at a high spin rate, into the detection chamber (Figure 3f(ii)). The two chambers containing the standard have air vents occupying equal radial positions. This pneumatic transfer configuration is new, as pneumatic pressure would be effected at several different radii in previous configurations. Although the air vents of the standard introduction chambers occupy the same radial position, addition of the two volumes of standard is selective by changing the rotational frequency of the disk. At 380 rpm, only the first volume of standard is transferred to the metering chamber, because the pneumatic force is unable to overcome the centrifugal force on the liquid in the lower chamber. At 260 rpm, the pneumatic force is sufficient to overcome the centrifugal force, allowing the transfer of the second volume of standard to the metering chamber. The standard additions calibration was performed by pipetting 70 μL of the demonstration sample (60 ppm bromophenol blue) and two 70 μL volumes of the standard (30 ppm bromophenol blue) into the sample introduction chamber and standard introduction chambers, respectively. The sequence of operation steps during the standard additions calibration experiment is shown as strobed images in Figure 4 and described in Table 1. With the recorded absorbance measurements, a ratiometric calculation method described in detail by LaCroix-Fralish was used to estimate the blank.17 The absorbing wavelength range used was 592.06 598.83 nm, and the nonabsorbing wavelength range used for the ratiometric calculation was 456.03 463.12 nm. A threepoint calibration curve was constructed using the absorbance data obtained, and a linear least-squares regression was performed to estimate the concentration of the sample (Figure 5). 9187
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Figure 3. Schematic showing sequential fluid flow during the standard additions experiment. (a b) Sample and standards are loaded onto the disk. The disk is spun and liquid is transferred from the sample chamber to the metering chamber. Excess liquid flows down the side channel to the waste chamber. (b c) The metered volume of sample is transferred to the detection chamber by spinning the disk at an increased rate sufficient to burst the capillary valve. (d e) Pneumatic flow is initiated. Fluid is transferred from the upper reservoir labeled Standard 1 to the metering chamber where it is metered. The first metered volume of the standard is transferred to the detection chamber by spinning the disk at an increased rate. (f g) Pneumatic flow is initiated. Fluid is transferred from the lower reservoir labeled Standard 2 to the metering chamber where it is metered. The second metered volume of the standard is transferred to the detection chamber by spinning the disk at an increased rate.
Figure 4. Series of experimental strobed images during the standard additions demonstration. Operations a g and the experimental conditions are described in detail in Table 1.
’ RESULTS AND DISCUSSION Pneumatic Fluid Transfer. In general, flow is controlled on centrifugal microfluidic platforms by increasing rotational frequency until liquid “bursts” through a restricting valve. In the design process and fabrication process, each subsequent valve must be more resistive (i.e., usually smaller). The need for precise smaller and smaller valves can become a fabrication concern while higher spin rates may also lead to leakages. As the transfer of the two volumes of standard was effected by pneumatic force, valves to keep the standards in their chambers were not required in this design of the disk. Also, a consequence of the use of the pneumatic fluid transfer technique is that the
platform is largely immune to high spin rates, as demonstrated when the standards remain in their chambers during the 2600 rpm rotation steps. The 2600 rpm rotation provides a high centrifugal field which ensures proper drainage of the metering and detection chambers. Another major advantage of this pneumatic technique is that a fluid transfer within the platform can be performed while the disk is rotating at relatively low frequencies, which may be convenient in a more complex system consisting of many reaction steps. With two parameters available to adjust (rotational frequency and air flow rate), the pneumatic technique illustrated here offers greater flexibility in terms of disk design and specific requirements of an experiment. 9188
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Table 1. Sequence of Operations Performed during the Standard Additions Demonstration Figure
Duration/rpm
Operation
4a
0 s/0 rpm
introduction of sample and standards into platform.
4b
45 s/500 rpma
meter sample in metering chamberd.
4c
45 s/2600 rpmb
drain sample into detection chamber. absorbance measurementse made through detection chamber.
4d 4e
meter first volume of standard in metering chamber (pneumatic transferc).
c
45 s/380 rpm
b
45 s/2600 rpm
drain first volume of standard into detection chamber. absorbance measurementse made through detection chamber.
4f 4g
c
45 s/260 rpm 45 s/2600 rpmb
meter second volume of standard in metering chamber (pneumatic transfer f). drain second volume of standard into detection chamber. absorbance measurementse made through detection chamber.
a
Rotational rate based on the required burst valve frequency of the sample chamber. b High rotational rate arbitrarily chosen to ensure proper drainage of the metering chamber. c Rotational rates for pneumatic transfer determined experimentally to complement the selected air flow rate. d Excess solution is drained to waste chamber during metering. e Absorbing wavelengths between 592.06 and 598.83 nm. f Air flow rate of 1.0 SCFM (standard cubic feet per minute) (∼28 L min 1).
Figure 5. Linear regression curve used to estimate the concentration of the sample.
In this demonstrative prototype, the microfluidic features on the disk were made relatively large to facilitate the experimental and measurement processes. However, it has been shown in previous work that this pneumatic technique is also applicable to smaller volumes of 10 or 15 μL.15 Upon further miniaturization, certain aspects of the apparatus (e.g., the inner diameter of the tube) and experimental conditions (e.g., air flow rate) will have to be adjusted accordingly. However, until a physical limit is reached when the channels become too resistive for air to enter, the functionality of this pneumatic technique should be retained. Application: Standard Additions. The method of standard additions is an extremely useful calibration method that is lacking in the area of centrifugal microfluidics. We chose standard additions as a demonstrative application to show that a reaction process requiring a series of steps and a variety of operations can be implemented easily using the pneumatic fluid transfer technique. We also wanted to show that the use of valves can be kept to a minimum and that the rotational frequencies of the system do not have to be sequentially increased, thereby making the platform easy to fabricate and offering more flexible experimental conditions. This disk design implements a common metering chamber used for the aliquoting of the sample and the standards, increasing the reproducibility of the volumes added. Similarly, sequential absorbance measurements, after the addition of each standard, can be made through the same detection chamber. The standard additions calibration method was successfully performed using known concentrations of sample and standard. The relative standard deviations (RSDs) for the absorbance
measurements of the sample, first standard, and second standard were 5%, 6%, and 8%, respectively (n = 10). The imprecision is due to the cell-to-cell variation when making absorbance measurements through our disks. This may be due to differences in path length and reflectivity of the plastics used to fabricate these disks. To confirm this, a study of the cell-to-cell variation was carried out with a standard solution of bromophenol blue. This study of the absorbance measurements made through different cells resulted in a RSD of 6% (n = 10), corresponding closely with the RSDs calculated during the standard additions experiment. An F-test confirmed that there is no statistical difference in the precision of the absorbance measurements made during the standard additions experiment, at a 95% confidence limit. This indicates that the pneumatic fluid transfer technique used for the addition steps did not significantly affect the precision of the measurements and that the cell-to-cell variation was the main source of error. To further confirm this, repeated absorbance measurements were made through one detection cell which was repositioned in the apparatus in the same manner used for different cells. The measurements yielded an RSD of 0.3%, significantly lower than the 6% for measurements made through multiple cells. Another source of imprecision could be the fabrication process of these platforms. For example, the capillary valves used were manually inserted into the platform, and varying lengths or positions would be a contributing factor to the error observed. This is to be expected in a prototyping environment and could probably be eliminated with an automated fabrication process. Upon extrapolating the curve by linear regression (Figure 5), the calculated concentration of the sample was determined to be 59 ppm with an RSD of 11%. This corresponds closely with the 12% calculated using the uncertainties associated with the slopes and intercepts of the linear regression plots.18
’ CONCLUSIONS The pneumatic fluid transfer technique described here offers a potential solution to the persistent problem of fabricating sequential valves on centrifugal microfluidic platforms. It was applied to a platform designed for performing standard additions and was successfully demonstrated as a viable alternative for fluid flow control. Integrating this technique with centrifugal systems simplifies the design and fabrication process of centrifugal 9189
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microfluidic platforms and offers greater flexibility in terms of experimental parameters.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Phone: +1-514-398-6236. Fax: +1-514-398-3797.
’ ACKNOWLEDGMENT The authors gratefully acknowledge Discovery Grant support from the National Sciences and Engineering Research Council of Canada. The authors wish to thank David Duford for his assistance with programming and instrumentation. ’ REFERENCES (1) Madou, M.; Zoval, J.; Jia, G.; Kido, H.; Kim, J.; Kim, N. Annu. Rev. Biomed. Eng. 2006, 8, 601–628. (2) Mark, D. H.; Stefan, S.; Roth, G.; Stetten, F. v.; Zengerle, R. Chem. Soc. Rev. 2009, 39 (3), 1153–1182. (3) Gorkin, R.; Clime, L.; Madou, M.; Kido, H. Microfluid. Nanofluid. 2010, 541–549. (4) Gorkin, R.; Park, J.; Siegrist, J.; Amasia, M.; Lee, B. S.; Park, J. M.; Kim, J.; Kim, H.; Madou, M.; Cho, Y. K. Lab Chip 2010, 10, 1758–1773. (5) Moore, J. L.; McCuiston, A.; Mittendorf, I.; Ottway, R.; Johnson, R. D. Microfluid. Nanofluid. 2011, 10, 877–888. (6) Hwang, H.; Kim, H. H.; Cho, Y. K. Lab Chip 2011, 11, 1434–1436. (7) Madou, M. J.; Lee, L. J.; Daunert, S.; Lai, S.; Shih, C.-H. Biomed. Microdevices 2001, 3, 245–254. (8) LaCroix-Fralish, A.; Templeton, E. J.; Salin, E. D.; Skinner, C. D. Lab Chip 2009, 9, 3151–3154. (9) Haeberle, S.; Brenner, T.; Zengerle, R.; Ducree, J. Lab Chip 2006, 6, 776–781. (10) Ducree, J.; Haeberle, S.; Lutz, S.; Pausch, S.; von Stetten, F.; Zengerle, R. J. Micromech. Microeng. 2007, 17, S103–S115. (11) Siegrist, J.; Gorkin, R.; Clime, L.; Roy, E.; Peytavi, R.; Kido, H.; Bergeron, M.; Veres, T.; Madou, M. Microfluid. Nanofluid. 2010, 9, 55–63. (12) Mark, D.; Metz, T.; Haeberle, S.; Lutz, S.; Ducree, J.; Zengerle, R.; von Stetten, F. Lab Chip 2009, 9, 3599–3603. (13) Mark, D.; Weber, P.; Lutz, S.; Focke, M.; Zengerle, R.; Von Stetten, F. Microfluid. Nanofluid. 2011, 10, 1279–1288. (14) Kong, M. C. R.; Salin, E. D. Anal. Chem. 2010, 82, 8039–8041. (15) Kong, M. C. R.; Salin, E. D. Anal. Chem. 2011, 83, 1148–1151. (16) Duford, D. A.; Peng, D. D.; Salin, E. D. Anal. Chem. 2009, 81, 4581–4584. (17) LaCroix-Fralish, A.; Clare, J.; Skinner, C. D.; Salin, E. D. Talanta 2009, 80, 670–675. (18) Bader, M. J. Chem. Educ. 1980, 57, 703–706.
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