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Contamination-Free Continuous Flow Microfluidic Polymerase Chain Reaction for Quantitative and Clinical Applications Kevin D. Dorfman,† Max Chabert,† Jean-Hugues Codarbox,† Gilles Rousseau,† Patricia de Cremoux,‡ and Jean-Louis Viovy*,†
Laboratoire Physicochimie-Curie, UMR/CNRS 168, and Unite´ de Pharmacologie, Institut Curie Section de Recherche, 26 Rue d’Ulm, F-75248 Paris Cedex, France
We present a method for performing polymerase chain reaction (PCR) using isolated droplets flowing in an immiscible fluorinated solvent system. Thanks to an optimized control of interfacial properties, we could achieve in this capillary-based system reproducible amplification factors, without any detectable contamination between neighboring droplets. The system is readily amenable to further miniaturization and automation and serves as the first step toward a clinically viable, highthroughput, quantitative continuous flow PCR apparatus. Recent advances in genetics present a pressing need for increasing polymerase chain reaction (PCR) throughput, in particular for clinical applications such as mutation detection.1-3 In addition to genomic applications, fields such as virology and microbiology are adopting PCR, in particular quantitative PCR (QPCR) and quantitative reverse-transcription PCR, as an alternative to traditional culture-based analyses.4-9 Many research groups have begun to address the need for high-throughput PCR via microfluidics, as recently reviewed in ref 10. The most straightforward method is simply shrinking the PCR sample volume by fabricating micro/nano/picoliter reservoirs.11-17 Flow-based pro* To whom correspondence should be addressed. E-mail: Jean-Louis.Viovy@ curie.fr. Phone: (33) 1 42 34 67 52. Fax: (33) 1 40 51 06 36. † Laboratoire Physicochimie-Curie. ‡ Unite´ de Pharmacologie. (1) Mashal, R. D.; Sklar, J. Curr. Opin. Gen. Dev. 1996, 6, 275-280. (2) Nollau, P.; Wagener, C. Clin. Chem. 1997, 43, 1114-1128. (3) Weber, J.; Barbier, V.; Pages-Berhouet, S.; Caux-Moncoutier, V.; StoppaLyonnet, D.; Viovy, J.-L. Anal. Chem. 2004, 76, 4839-4848. (4) Bretagne, S.; Durand, R.; Olivi, M.; Garin, J.-F.; Sulahian, A.; Rivollet, D.; Vidaud, M.; Deniau, M. Clin. Diagn. Lab. Immunol. 2001, 8, 828-831. (5) Bogard, M. et al. J. Clin. Microbiol. 1997, 35, 3298-3300. (6) Grundy, J. E.; Ehrnst, A.; Einsele, H.; Emery, V. C.; Hebart, H.; Prentice, H. G.; Ljungman, P. J. Clin. Microbiol. 1996, 34, 1166-1170. (7) Sto ¨cher, M.; Berg, J. J. Clin. Microbiol. 2002, 40, 4547-4553. (8) Malorny, B.; Hoorfar, J.; Bunge, C.; Helmuth, R. Appl. Environ. Microbiol. 2003, 69, 290-296. (9) Hoorfar, J.; Cook, N. Methods Mol. Biol. 2003, 216, 51-64. (10) Aroux, P.-A.; Koc, Y.; de Mello, A.; Manz, A.; Day, P. J. R. Lab Chip 2004, 4, 534-546. (11) Wilding, P.; Shoffner, M. A.; Kricka, L. J. Clin. Chem. 1994, 40, 18151818. (12) Woolley, A. T.; Hadley, D.; Landre, P.; de Mello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (13) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484-487.
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tocols, using either forced18,19 or natural20-24 convection, are also useful for improving the process efficiency. In all of these cases,11-24 the amplification is a batch process and occurs in a closed system. It is a basic tenant of chemical engineering practice that batch processes are preferential for small-scale operations, such as conventional laboratory PCR, while continuous processes are required for large-scale operations, such as mutation screening. In the context of PCR, a batch-to-continuous transition requires moving the sample through different temperature zones in an open system. Manz and co-workers25 presented the first continuous flow system for PCR, pumping the sample through an open microchannel while maintaining different regions of the channel at the appropriate temperatures for PCR. However, their samples were not self-contained and dispersed throughout the flow. Clinical applications of this technique would result in significant contamination between samples. Performing continuous flow PCR for high-throughput clinical and quantitative applications requires encapsulating the samples and preventing contamination between them. In principle, this can be accomplished in two ways: either the sample can be a droplet entrained in an immiscible solvent,26 or numerous aqueous sample plugs can be separated by plugs of air.27-29 In the latter, wall treatments are necessary to prevent adsorption of DNA to the (14) Northrup, M. A.; Benett, W.; Hadley, D.; Landre, P.; Lehew, S.; Richards, J.; Stratton, P. Anal. Chem. 1998, 70, 918-922. (15) Lee, D.-S.; Park, S.-H.; Yang, H.; Chung, K.-H.; Yoon, T. H.; Kim, S.-J.; Kim, K.; Kim, Y. Y. Lab Chip 2004, 4, 401-407. (16) Krishnan, M.; Burke, D. T.; Burns, M. A. Anal. Chem. 2004, 76, 65886593. (17) Liu, J.; Hansen, C.; Quake, S. R. Anal. Chem. 2003, 75, 4718-4723. (18) Liu, J.; Enzelbeger, M.; Quake, S. Electrophoresis 2002, 23, 1531-1536. (19) Chiou, J.; Matsudaira, P.; Sonin, A.; Ehrlich, D. Anal. Chem. 2001, 73, 2018-2021. (20) Krishnan, M.; Ugaz, V. M.; Burns, M. A. Science 2002, 298, 793. (21) Braun, D.; Goddard, N. L.; Lichaber, A. Phys. Rev. Lett. 2003, 91, 158103. (22) Wheeler, E. K.; Benett, W.; Stratton, P.; Richards, J.; Chen, A.; Christian, A.; Ness, K. D.; Ortega, J.; Li, L. G.; Weisgraber, T. H.; Goodson, K.; Milanovich, F. Anal. Chem. 2004, 76, 4011-4016. (23) Chen, Z.; Qian, S.; Abrams, W. R.; Malamud, D.; Bau, H. H. Anal. Chem. 2004, 76, 3707-3715. (24) Krishnan, M.; Agrawal, N.; Burns, M. A.; Ugaz, V. M.; Anal. Chem. 2004, 76, 6254-6265. (25) Kopp, M. U.; de Mello, A. J.; Manz, A. Science 1998, 280, 1046-1048. (26) Curico, M.; Roeraade, J. Anal. Chem. 2003, 75, 1-7. (27) Park, N.; Kim, S.; Han, J. H. Anal. Chem. 2003, 75, 6029-6033. (28) Obeid, P. J.; Christopoulos, T. K. Anal. Chim. Acta 2003, 494, 1-9. 10.1021/ac050031i CCC: $30.25
© 2005 American Chemical Society Published on Web 04/23/2005
walls and subsequent transfer between samples. So far, however, wall treatments were not sufficient to completely eliminate contamination,27 so it was necessary to include a “wash” droplet between samples. The wall contamination problem is a significant drawback to this technique, since (i) the wall coating is not permanent and adds to the fabrication complexity and (ii) the wash droplet halves the throughput of the device. Moreover, bubbles in the stream may introduce irregularities in the thermal history of the droplets, making this technique less attractive for quantitative applications. The flowing droplet system26 does not suffer from either of these drawbacks, since the walls are uncoated and there are no bubbles in the system. However, the initial work on this system26 suffered from contamination between droplets, which was attributed to the formation of small satellite drops in the flow. We present here a detailed study of the problems encountered in earlier biphasic liquid flow PCR systems26,27 and propose an improved system for performing continuous flow PCR using droplets in an immiscible, fluorinated solvent flowing in Teflon capillaries. Our solvent system prevents the droplet breakage observed by Curcio and Roeraade,26 thereby eliminating the contamination between droplets. We have also developed a compact heating system in the spirit of Park et al.27 for performing the amplification. We demonstrate that our system produces adequately reproducible amplification between different drops without any detectable contamination between them. We estimate that we could perform 6000 PCR/day, with mix and sample volumes smaller than 1 µL, using the unoptimized prototype discussed here with automated injection and detection. This number could be substantially increased by further miniaturization.
Figure 1. Schematic of the continuous flow PCR device. The exposed face is the side opposite the turbine. Ventilation, heating, and thermocouple sensor holes are depicted. The capillary entry and exit holes are not included in the figure.
EXPERIMENTAL SECTION Droplet Train Stability. We created trains of droplets by using a Y-connector (Upchurch Scientific) connected to an electropinch valve (NResearch, Caldwell NJ). One entry to the Y-connector was filled with TBE 5× buffer (0.45 M Tris base, 0.45 M boric acid, and 0.01 M EDTA; Sigma) dyed with a trace amount of bromophenol blue for ease of observation. The other side of the Y-connector and the test capillary (PFA; i.d. 800 µm, Upchurch Scientific) were primed with either the bulk fluorinated oil FC-40 (3M) or FC-40 containing various amounts of a fluoro alcohol surfactant (1H,1H,2H,2H-perfluorodecan-1-ol, Fluorochem). Droplet trains were created by cycling the electrovalve with a LabView program while aspirating from the other end of the capillary with a computer-controlled Harvard milliliter module syringe pump. A typical cycle consists of 6 s of aspiration from the TBE line and 8 s of aspiration from the FC-40 line. The droplets were observed with a binocular microscope (Olympus) and recorded using a CCD camera (Hitachi) and WinTV. In a typical experiment, we make at least 200 droplets and the capillary is sufficiently long to contain ∼50 drops along its length. Interfacial tension measurements were made using a homemade drop-volume tensiometer. The FC-40/surfactant drop is dispensed into a reservoir of TBE 5× buffer from a 0.8-mm-i.d. Teflon capillary. Using a 50-µL Hamilton gastight syringe and a Hamilton PSD/2 syringe pump at maximum resolution, we were
able to increase the drop volume in 25-nL increments with arbitrary waiting times between steps. To allow for equilibration of the surfactant, we typically waited 30 s between steps. The tension measurements are the average of at least 15 different drops. The calculation follows the procedure of ref 30, which allows us to correct for the wetting of the Teflon tip. Design of the Heater. The device, depicted schematically in Figure 1, consists of a 4-cm-diameter copper cylinder machined into three pieces corresponding to the denaturing, annealing, and elongation regions. The elongation regime is twice the size of the other two zones, thus occupying half the cylinder. The three zones are isolated one from another by thermally insulating sheets, which are affixed between the pieces of the copper cylinder. The two ends of the heater are capped with polycarbonate cylinders to provide structural stability while maintaining isolation between the temperature zones. Three small holes per quarter cylinder were drilled through the entire device to provide vents for air cooling. Two small holes (for the thermocouples) and one larger central hole (for the heater) were drilled partially through the cylinder in each temperature zone. The heating and thermocouple holes are not open to the air-cooling side of the heater. The capillary bottom is in direct contact with the copper surface. Each zone is then covered with a thin layer of aluminum foil whose edges are in contact with a raised piece of the copper cylinder corresponding to that zone. The aluminum provides heating from above and improves temperature uniformity inside the capillary. The foil layers are covered by a polycarbonate shell and a layer of cotton insulator, thereby insulating the cylinder and providing a uniform temperature across the capillary. A small turbine blows ambient air through three flow-through ventilation holes per quarter-cylinder (different from the thermocouple and heater holes), allowing for a short time constant for temperature decrease in the event of a temperature spike. This provides better temperature control and uniformity. Each zone includes a resistance heater and two Pt-100 thermocouples. The resistance heaters are located in the center of their respective zone, while the thermocouples are located near the interface between zones. Thermocouples can be moved inside the copper elements to probe temperature uniformity. The heating elements and thermocouples are connected to custom electronics. The thermocouples communicate with a custom PID program control written in LabView via a Keithley 2701 multimeter with a
(29) Obeid, P. J.; Christopolous, T. K.; Crabtree, H. J.; Backhouse, C. J. Anal. Chem. 2003, 75, 288-295.
(30) Earnshaw, J. C.; Johnson, E. G.; Carroll, B. J.; Doyle, P. J. J. Colloid Interface Sci. 1996, 177, 150-155.
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pair of model 7706 Multiplexer cards, and voltage signals to the heaters are returned by the PID program through the same multimeter. The temperature of each zone can be set arbitrarily. For the experiments discussed here, we used a denaturing temperature of 94 °C, an annealing temperature of 55.5 °C, and an elongation temperature of 72 °C. With our design, we typically maintained a temperature difference of (0.2 °C around the set point at a fixed point in each zone and (1.0 °C along the cylinder axis in a given zone. A 4.5-m-long transparent PFA capillary (i.d. 800 µm, Upchurch Scientific) enters the cylinder through a groove in the denaturing region, providing an initial denaturation step of ∼1 min. The capillary is then wound 35 times around the cylinder, corresponding to 35 PCR cycles. The capillary exits the heater through a hole in the extension segment, providing ∼30 s of additional extension on the 35th cycle. PCR Amplification The template is a 2823-base pair DNA fragment of Litmus 28i (New England Biolabs). This fragment is amplified on 572 base pairs from base 2008 to base 2580 (lower primer 5′-CGC-ATT-GCG-GTA-TCT-AGA-ACC-GGT-GAC-GTC-3′, upper primer 5′-AGC-TTG-GAG-CGA-ACG-ACC-3′, Eurogentech Oligold). A 50-µL PCR mix is prepared using the Ready Mix Taq reaction mixture (Sigma) according to the manufacturer’s specifications with the maximum concentration of template and primers. The carrier fluid is a bulk fluorinated oil FC-40 (3M) containing 0.5-1.0 wt % fluoro alcohol surfactant (1H,1H,2H,2H-perfluorodecan-1-ol, Fluorochem). The surfactant prevents the transient adsorption of droplets to the capillary walls. Prior to use, the carrier fluid is degassed in a vacuum for at least 30 min to minimize the amount of dissolved gas. Initially, the capillary inlet is open and its outlet is connected to a Hamilton PSD/2 pump and a 100-µL Hamilton gastight syringe. Multiple 2-µL aqueous droplets, separated from one another by 5-µL FC-40 spacers, are injected at the capillary inlet by aspirating from appropriate fluid reservoirs. After injecting the desired number of droplets and spacers, the outlet is disconnected from the Hamilton PSD/2 pump and placed in a waste reservoir. The inlet is then connected to a computer-controlled Harvard milliliter-module pump with a 5-mL Hamilton gastight syringe. The latter pump pushes the droplets at a speed of 0.1 cm/s. Each droplet is collected at the outlet and its full 2-µL volume is analyzed by gel electrophoresis on a 1 wt % agarose gel in 0.5× TAE buffer. A control amplification sample is made by amplifying the remaining volume of the 50 µL of PCR mix in a classic PCR thermal cycler (Perkin-Elmer) with a cycle of 1 min at 94 °C, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. (Our thermal cycler only permits integer values of temperature. We found that our capillary-based PCR was most effective at 55.5 °C, so we could not make an exact control without compromising the efficiency of our new system.) This mimics the cycling in our continuous flow PCR, although the lag time for heating and cooling the classic cycler means that the total amplification time is approximately twice as long as our flow device. A 2-µL aliquot of the amplified control system is used for the gel electrophoresis. After electrophoresis, the gels are stained with ethidium bromide and photographed under UV light. 3702 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005
Figure 2. Interfacial tension between TBE 5× buffer and FC-40/ fluoro alcohol surfactant as a function of fluoro alcohol surfactant weight percent. The error bars are the size of the data points.
RESULTS AND DISCUSSION We first tested the stability of droplet trains in our fluidic system. In previous work on this type of PCR,26 cross-contamination between droplets was attributed to droplet instability and the formation of small satellite droplets. In the present work, we wanted not only to look for individual droplet breakage but also to observe the overall stability of droplet trains over several hundred droplets. The stability of such trains is essential for highthroughput applications of our technique. When the droplets are entrained in pure FC-40, we observed that they occasionally stick to the walls. This is unexpected, since both the walls and the carrier fluid are fluorinated, and we would expect that the walls will be strongly wetted by the FC-40. We attribute the sticking to imperfections (either roughness or chemical inhomogeneities) in the capillary walls, which would be expected in bulk capillaries manufactured by an extrusion process. The layer of FC-40 between the droplets and the capillary wall is very thin, and small perturbations to the wall surface could disrupt the lubrication flow between the droplets and the wall. In any event, once one droplet becomes entrained on the wall, even temporarily, the train as a whole loses its stability. The trailing droplet collides with the entrained droplet and exchanges fluid, the entrained droplet is released from the wall, and the trailing droplet becomes entrained. This process proceeds ad infinitum and would be catastrophic in any PCR application. We then performed the same experiments using 0.5-3.0 wt % fluoro alcohol surfactant in FC-40. We never observed any transient pinning to the walls, satellite droplet formation, or instabilities in the droplet train, even for trains containing over 200 droplets. The improved stability can be attributed to lowering the droplet/FC-40 interfacial tension relative to the droplet/wall tension. To determine a suitable surfactant concentration, we measured the interfacial tension as a function of the weight fraction of surfactant. As depicted in Figure 2, even a modest addition of surfactant dramatically reduces the droplet/oil surface tension relative to the clean FC-40 value of 51.8 ( 0.2 dyn/cm. We found
Figure 3. Gel electrophoresis of the reproducibility experiment. Lane 1: 1-kbp DNA ladder (New England Biolabs). Lane 2: 2-µL control sample of mix with DNA. Lanes 3: droplet 1. Lane 4: droplet 2. Lane 5: droplet 3. Lane 6: droplet 4. Lane 7: droplet 5. Each droplet is 2 µL.
Figure 4. Gel electrophoresis of the contamination experiment. Lane 1: 1-kbp DNA ladder (New England Biolabs). Lane 2: 5-µL control sample of mix without DNA. Lane 3: 2-µL control sample of mix with DNA. Lane 4: droplet 1 (no template). Lane 5: droplet 2 (no template). Lane 6: droplet 3 (with template). Lane 7: droplet 4 (no template). Lane 8: droplet 5 (no template). Each droplet is 2 µL.
that concentrations around 0.5-1.0 wt % were sufficient to provide train stability. As Figure 2 indicates, we would not expect much improvement in performance at weight percents greater than 1.5%, since the interfacial tension reaches a plateau there. The tension values from our drop volume technique compare favorably in magnitude and error to pendant drop measurements on a similar system.31 Having determined that our fluidic system produces stable droplet trains, we proceeded to test the reproducibility of the amplification between different drops. We made a train of five droplets, each one containing the PCR mix and template. The result of this successful amplification is depicted in Figure 3. The degree of amplification is comparable to that obtained in the conventional thermal cycler but appears to vary somewhat between droplets. We repeated this test numerous times (results not shown) and found that there was some variation in band intensity both within and between experiments. (For example, consider the difference in band intensity between Figures 3 and 4.) There are three factors that could contribute to the variation: (i) the inherent error in using gel electrophoresis as a quantitative measure of DNA concentration; (ii) imperfect recovery of the droplets at the outlet; and (iii) variations in droplet size. Temperature nonuniformity may also play some role, although its effect should be small within a given experiment since the droplets are closely spaced and feel essentially the same temperature fluctuations. In case iii, we note that, before aspirating the PCR mix, we first aspirate a 5-µL spacer of FC-40/fluoro alcohol surfactant mixture. This wets the capillary tip with FC-40, so that during the subsequent step, we aspirate both the wetting fluorocarbon mixture and the PCR mix. Variability in the amount of wetting (31) Tice, J. D.; Song, H.; Lyon, A. D.; Ismagilov, R. F. Langmuir 2003, 19, 9127-9133.
fluorocarbon mixture of the tip between aspiration steps (i.e., from evaporation) would then lead to differences in the PCR droplet sizes. We checked for the variance in droplet size by making a train of five droplets and then taking images of the droplets with a binocular microscope and a CCD camera. Using Scion Image (NIH) to analyze the images, we estimate the droplet sizes to be 1.84, 1.66, 1.76, 1.94, and 1.91 µL, respectively. Although our present system results in some variance in the final mass of DNA in each droplet, we believe that this variance could be minimized with an automated injection and detection system. From the numerous series of tests we performed, it appears that the first drop injected in the system often yields a smaller amplification than the other drops, which statistically cannot be accounted for by variability in injection, recovery, or gel electrophoresis. Rather, the first drop entering the system creates a perturbation to the temperature field in front of it because its thermal conductivity is different from that of FC-40. Consequently, the thermal history of the first droplet is different from that of subsequent droplets. This “start-up” phenomenon should not be an issue for a high-throughput continuous flow system intended to amplify thousands of droplets per day. It might be eliminated by a “dummy” plug of buffer preceding the first sample droplet to avoid this phenomenon. We also tested our system for cross-contamination between droplets. In this experiment, we made two separate PCR mixes; a first mix contains the template, primers, and Ready Mix reaction mixture and the second mix is identical except that it does not contain any template. We aspirated five droplets, where only the third droplet contains the template. To avoid contamination from the tip itself, we washed it in distilled water between each droplet injection. Figure 4 shows the gel electrophoresis result from this experiment. There is no observable contamination between different dropletssthe only droplet exhibiting any DNA amplification is the third droplet, which contained the template DNA at the outset. CONCLUSION We have presented a compact solid cylindrical system for continuous flow PCR in a two-phase system, where the samples to be amplified are droplets entrained in an immiscible bulk fluorinated oil. The combination of PFA capillaries and a fluorinated oil/fluorosurfactant solvent system provides droplet train stability and prevents contamination between successive droplets. Moreover, we have demonstrated adequately reproducible amplification between droplets. The estimated throughput of 6000 PCR reactions/day with the present system is already at least 6-fold greater than a typical 96-well PCR cycler and comparable to the state-of-the-art large-scale cyclers. The device can be easily miniaturized, leading to even much higher throughput. This work represents the first step toward a continuous flow PCR system for clinical applications. However, several further developments will be necessary to build a routine apparatus. First, we need to develop automated injection, which would allow a better reproducibility between samples and reduce reagent volumes. Another strong advantage of the droplet continuous flow system is that mixing of the sample and PCR mix prior to injection can be avoided by injecting droplets with only the template and then coalescing them with reagent droplets before they enter the Analytical Chemistry, Vol. 77, No. 11, June 1, 2005
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heater. We have demonstrated elsewhere32 an electrocoalescence method for accomplishing this task. This in-capillary mixing approach not only provides the route to higher automation but it will also permit reactions with much smaller reagents consumption than presented here. Typically, PCR reactions in microtiter plates should be performed on volumes of ∼10 µL for good reproducibility, whereas DNA sequencers, for instance, actually use only a few tens of nanoliters of solution. In our system, reproducible amplification was achieved with volumes of the order of 1 µL, and this could be easily reduced to ∼100 nL by downscaling capillary diameter. In-capillary mixing of droplets is a powerful way of working at such scales, using only the quantity of PCR mix actually required for detection. By further integrating automated injection with on-line optical detection, it will also in the future be possible to turn the present system into a real-time quantitative continuous flow PCR machine. As our template (∼600 bp) is ∼6 times longer than those used in
current Q-PCR detection protocols, we do not expect any difficulties in the amplification, even at relatively high copy numbers. Automatic droplet recovery, sorting, and analysis of the amplified samples are other options to open a wide range of applications in the form of a completely integrated DNA analysis system.
(32) Chabert, M.; Dorfman, K. D.; Viovy, J.-L. Submitted.
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ACKNOWLEDGMENT We thank Aure´lien Bancaud, Je´re´mie Weber, Renaud Fulconis, Johan Roeraade (Royal Institute of Technology, Stockholm Sweden), and particularly Claus Fu¨tterer for numerous discussions and assistance. K.D.D. acknowledges the support of a postdoctoral fellowship from the International Human Frontier Science Program Organization. This work was supported by a grant from the 6th European Framework (Contract NMP4-CT-2003-505311, NABIS). Received for review January 7, 2005. Accepted April 1, 2005.
