Department of Electrical and Computer Engineering, Duke University, Durham, NC USA
ABSTRACT
We report on performing parallel processing on electrowetting-based digital microfluidic chips by taking advantage of reconfigurable architecture combined with programmable control, scalability, and contamination-free liquid handling.
The opportunity is to increase throughput of processes such as sample preparation, biomolecular extraction, and cytotoxicity screening through on-chip resource sharing and without the need for large microfluidic chip I/O.
KEYWORDS : Droplets, Digital Microfluidics, Electrowetting, Lab-on-a-chip
INTRODUCTION
Electrowetting-on-dielectric (EWD) microfluidics is based on the actuation of droplet volumes from picoliters up to several microliters using the principle of modulating the interfacial tension between a liquid and an electrode coated with a dielectric layer. An electric field established in the dielectric layer creates an imbalance of interfacial tension if the electric field is applied to only one portion of the droplet, which forces the droplet to move. Droplets are usually sandwiched between two parallel plates with a filler medium, such as silicone oil.
Investigators have conducted extensive research on the basic principles and operations underlying the implementation of electrowetting-based digital microfluidic systems. The result is a subs tantial “microfluidic toolkit” that enables biomedical applications where the transport and processing of biological liquids and beads is required.
It is well recognized that most commercial microfluidic chips are either limited to single flow path operation or require large control boxes to provide external pressure for liquid transport and valve control in parallel systems.
Another key challenge is the need to prepare samples off-chip in laboratory well plates, and then to interface the well plates to a small form factor chip for analysis. Such procedures can be done in a research laboratory, but are incompatible with point-of-care applications or field use. In this paper we will describe the requirements for high throughput, point-of-care microfluidic chips that can process raw physiological samples from input to answer.
The key attributes of a high throughput point-of-care microfluidic chip are as follows: 1) low number of input/output
(I/O) ports and on-chip reagent storage; 2) flexible chip architecture for efficient use of fluidic processing elements; 3) programmable electronic control; 4) parallel or multiplexed operation; 5) low cross-contamination to allow resource sharing; and 6) scalability. Each of these requirements is described below.
SYSTEM REQUIREMENTS
I/O and Reservoirs: Recent advances in lab-on-a-chip technology include the ability to perform multiplexed chemistry to accelerate screening speed and reduce reagent consumption. Wang et al [1] produced over a thousand reactions on a chip with about 80 I/O tubes for chemical/pressure access and significant supporting equipment for pneumatic valve control, positive pressure peristaltic pumps, and vacuum-assisted liquid transport. (Fig. 1a) A similar large I/O problem occurs in the use of microfluidic chips for cytotoxicity screening. Wang et al. [2] recently fabricated a microfluidic device that traps cells to form a 24 × 24 array (Fig. 1b). The array of cultured cells subsequently could be used for screening the cytotoxicity of different compounds. The chip also was bound by fluidic inputs. And, Fluidigm ’ s
TOPAZ X.96 series of screening chips have 48 reagent inlets plus eight other sample loading ports. (Fig. 1c)
Assay
Reagent
Dilution
(2x4 mixer + splitter)
Detector
Cell Mix
(2x4 mixer + splitter)
Waste
Fig. 1a. Optical image of a Fig. 1b. Micrograph of microfluidic Fig. 1c. Floor plan of a Fluidigm
’ s microfluidic device for chip for cytotoxicity screening TOPAZ X.96 screening chip.
performing highly parallel (Wang et al. 2007)
reactions. (Wang et al. 2009)
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Miniaturized Systems for Chemistry and Life Sciences
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The large I/O problem can be addressed in two ways: 1) on-chip reagent reservoirs and dispensing, and 2) on-chip parallel sample or reagent preparation. A digital microfluidic biochip has been proposed to perform cytotoxicity testing
[3]. The proposed chip would dilute the compound to be tested over a range of concentrations (user defined) so that the cytotoxic behavior of the compound could be characterized in parallel reactions. The chip would mix the compound dilutions with a cell suspension and then incubate the mixture for a user specified period of time. Following incubation, the chip would add the cytotoxicity assay reagent(s) to the mixture and then optically detect the amount of cell death.
Although the chip design was directed for use in cytotoxicity assays, the chip could be easily interfaced with other digital microfluidic biochip capabilities as well.
Flexible Architecture: The digital microfluidic architecture shown below in Fig. 2 [4] capitalizes on the flexibility of a unit flow grid array. At any given time, the array can be partitioned into “cells” that perform fluidic functions, such as storage, mixing, or transport. If the array is actuated by a clock that can change the voltage at each electrode on the array in one clock cycle, then the architecture has the potential for dynamically reconfiguring the functional cells at least once per clock cycle. Thus, once the fluidic function defined by a cell is completed, the cell electrode voltages can be reconfigured for the next function.
Figure 2. Two-dimensional electrowetting electrode array used in digital microfluidic architecture.
Programmable Electronic Control: Digital microfluidic architecture also can be operated under software-driven electronic control. Whereas the flexibility of a unit flow grid array is depicted in Fig. 2, this capability under software control also allows the system to run multiple parallel applications, which potentially can increase sample processing throughput. Significant economies of scale are also possible if the droplets don’t contaminate the surfaces of the chip, which then allow the use of shared transport paths and shared dispensing reservoirs.
Arcihtectural flexibility and program control leverage microfluidic designs into multiple applications, which is the basis for the lab-on-a-chip concept. As it is with the equipment in a life sciences laboratory, each fluidic component performs a canonical operation. In this way, the complexity of the diverse set of biomedical applications can be reduced to a manageable set of fluidic operations. These operations can then be configured in various ways to realize a desired application. To accomplish such a lab-on-a-chip architecture requires a hierarchical taxonomy. At the top level, applications are scaled to a microfluidic platform. The second level describes the microfluidic operations. And the third level describes the components to perform the operations. This manageable design approach has been previously described. [5] The modularity of the architecture allows flexibility of creating and choosing a collection of fundamental operations that meet the specific needs of a particular user. This modularity also allows one to perform multiplexed applications on a single platform.
Parallel or Multiplexed Operation: A key to parallel or multiplexed processing in point-of-care applications is utilizing shared resources. Sharing resources, such as reservoirs and transport paths, is enabled by controlling crosscontamination. In an electrowetting chip, a spacer separates the top and bottom plates, resulting in a fixed gap height.
The gap is usually flooded with silicon oil, which acts as a filler fluid.
An example of multiplexed chip operation using shared resources is an experimental electrowetting chip designed by
Advanced Liquid Logic and used to perform research in pyrosequencing. [6] Sequencing-by-synthesis methods involve enzymatic extension by polymerase through the iterative addition of labeled nucleotides, often in an array format.
Pyrosequencing of DNA has been performed on a digital microfluidic platform [6]. To improve throughput, a multiplexed architecture was implemented. In multiplexed operation, several DNA sequencing operations are performed simultaneously, where the bead wash region and mixing region are unique to each sequencing operation. In addition, the reagent storage can be common for all sequencing sites. The detection region can be either common for all sites or independent, though the latter approach would require multiple detectors. Advanced Liquid Logic has developed the experimental chip shown in Fig. 3. This chip has 12 different wash and mixing (reaction) lanes and a set of common reservoirs for nucleotides, enzymes and wash buffer. This design allows the user to perform 12 pyrosequencing reactions simultaneously under software control. After the reactions, the beads containing the DNA duplexes are retained using magnets underneath and the droplets containing pyrophosphate or ATP are then marched sequentially to the detection spot. Subsequently, they will be discarded to the waste reservoir and the beads will be washed and reset for the next base addition. The results of four-plex sequencing are shown in Fig. 4, where four lanes on the chip in Fig. 3 were used to run the same sequence assay, one of which was a control with no DNA present. It can be seen that the assays run in each lane produced similar results.
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Correct sequence: TAAG GCGG GATC GCTT TGAC
Lane 9
Lane 7
Lane 5 (blank)
Lane 3
0
G T AA C C GG C
Fig. 4. Four-plex multiplexed pyrosequencing results Fig. 3 Layout of the new chip architecture for multiplexed
pyrosequencing illustrating the location of different reservoirs and other functional elements on the chip.
Scalability: Parallelization of fluidic processes puts pressure on microfluidic chip size. Thus, it is important that the liquid volumes utilized be as small as possible, given the limitations on molecular detection. We have demonstrated that electrowetting actuators can be scaled to 35pl droplet operation. [7] In addition, we have demonstrated picoliter enzyme chemistry performed on chip. [8]
With regard to on-chip sample preparation for controlling I/O, multiplexed biomolecule extraction from raw physiological samples will provide significant gains preparing samples for diagnostics. It has been estimated that 90% of the cost and 95% of the time needed to obtain molecular diagnostic data today is sample preparation, including collection and transportation. [9] The primary challenge in traditional continuous-flow microfluidic systems has been the complexity of performing nucleic acid extraction. We believe that extraction using a programmable digital microfluidic platform overcomes the complexity barrier for integrated systems while providing increased sample throughput with multiplexed operation. Once a sample is prepared, it also is efficient then to perform multiple on-chip diagnostic tests.
In summary, a wide diversity in biomedical applications can be parsed into manageable components and assembled into an architecture proving the advantages of being programmable, parallel, and efficient in the use of shared resources.
This capability opens the possibility of handling all of the protocols that a given laboratory or a class of applications would require at the point-of-care in a reasonably-sized chip.
CONCLUSION
Electrowetting-based digital microfluidics has advantages in performing multiplexed assays and sample preparation processes based on the flexibility of the unit flow architecture, programmable control, on-chip reagent storage, and contamination-free liquid handling.
REFERENCES
[1] Y. Wang, W-Y Lin, K. Liu, R.J. Lin, M. Selke, H.C. Kolb, N. Zhang, X-Z Zhao, M.E. Phelps, C. Chen, K.F. Faull, and HR Tseng, “An integrated microfluidic device for l arge-scale in situ chemistry screening,” Lab Chip, 9, 2281 -
2285 (2009)
[2] Z. Wang, M.C.Kim, M. Marquex and T. Thorsen, “ High-density microfluidic arrays for cell cytotoxicity analysis .
Lab Chip, 7 , 740-745 (2007).
[3] Y. Zhao, A. Wang, Y. Yamanaka (2007) unpublished.
[4] J. Ding, K. Chakrabarty and R.B. Fair, Scheduling of microfluidic operations for reconfigurable two-dimensional electrowetting arrays, IEEE Transactions on Computer-Aided Design of Integrated Circuits & Systems , 29, 1463-
1468 (2001).
[5] R.B. Fair, “Digital Microfluidics: is a true lab -on-achip possible?” J. Microfl. and Nanofl.
, vol. 3, 245-281 (2007).
[6] P. Thwar, J.L. Rouse, A.E. Eckhardt, P. Griffin, M.G. Pollack and R.B. Fair, “Digital microfluidic DNA
[7] s equencing,” Advance s in Genome Biology and Technology (AGBT) Meeting, Marco Island, Florida, Feb., 2009.
J
.H. Song, R. Evans, Y.-Y. Lin, B.N. Hsu, and R.B. Fair, “A scaling model for electrowetting-on-dielectric microfluidic actuators, ” Microfluid Nanofluid, vol. 7, 75-89 (2009).
[8] E. Welch (2010), unpublished.
[9] P.R.C. Gascoyne, and J.V. Vykoukal, “Dielectrophoresis based sample handling in general -purpose programmable diagnostic instruments,” Proc. IEEE , 92, 22-42 (2004)
CONTACT:
R.B. Fair: tel. 1-919-660-5277; rfair@ee.duke.edu
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