2. William L. Hwang †, Fei Su ‡, Krishnendu Chakrabarty Department of Electrical & Computer Engineering Duke University, Durham, NC 27708, USA Automated Design of Pin-Constrained Digital Microfluidic Arrays for Lab-on-a-Chip Applications † Department of Physics, University Of Oxford ‡ Intel Corporation

3. Motivation for Biochips Transfer conventional biochemical laboratory methods to lab-on-a-chip (LoC), or microfluidic biochips Potential to revolutionize biosensing, clinical diagnostics, drug discovery –Small size and sample volumes, O(nL) –Lower cost –Higher sensitivity Conventional Biochemical Analyzer Shrink Digital Microfluidic Biochip

4. Microfluidic Biochips Based on precise control of very small volumes of liquids Integrate various fluid-handling functions such as sample prep, analysis, separation, and detection Most commercially available microfluidic devices are continuous-flow –Permanently etched microchannels, pumps, and valves (University of Michigan) 1998 (Duke University) 2002

5. Microfluidic Biochips Digital microfluidic biochips (DMBs) –Manipulate discrete droplets (smaller volumes) –Electrical actuation –No need for cumbersome micropumps and microvalves –Dynamic reconfigurability (virtual routes) –Architectural scalability and greater automation –System clock controls droplet motion; similar in operation to digital microprocessor (University of Michigan) 1998 (Duke University) 2002

6. Electrowetting Novel microfluidic platform invented at Duke University Droplet actuation achieved through an effect called electrowetting No Potential A droplet on a hydrophobic surface originally has a large contact angle. Applied Potential The droplet’s surface energy increases, which results in a reduced contact angle. The droplet now wets the surface.

7. Actuation Principle Droplets containing samples travel inside filler medium (e.g., silicone oil), sandwiched in between glass plates Bottom plate – patterned array of control electrodes Top plate – continuous ground electrode Surfaces are insluated (Parylene) and hydrophobic (Teflon AF)

8. Actuation Principle Droplet transport occurs by removing potential on current electrode, applying potential on an adjacent electrode Interfacial tension gradient created

9. PCB Microfluidic Biochips Rapid prototyping and inexpensive mass-fabrication Copper layer for electrodes (coplanar grounding rails) Solder mask for insulator Teflon AF coating for hydrophobicity Disposable PCB biochip plugged into controller circuit board, programmed and powered with USB port

10. OUTLINE What are digital microfluidic biochips (DMBs)? Pin-Constrained Digital Microfluidic Biochips –Background –Pin Assignment Problem Minimum Number of Pins for Single Droplet Pin-Assignment Problem for Two Droplets –Virtual Partitioning Scheme Impact of Partitioning on PCNI Evaluation Example: Multiplexed Bioassays Summary and future outlook

11. Direct Addressing Most design and CAD research for DMBs has been focused on directly-addressable chips Suitable for small/medium-scale microfluidic electrode arrays (e.g., with fewer than 10 x 10 electrodes) For large-scale DMBs (e.g., > 100 x 100 electrodes), multi-layer electrical connection structures and complicated routing solutions are needed for that many control pins

12. Pin-Constrained DMBs Product cost is major marketability driver due to disposable nature of most emerging devices Multiple metal layers for PCB design may lead to reliability problems and increase fabrication cost Reduce number of independent control pins (pin-constrained DMBs) –Reduce input bandwidth between electronic controller and microfluidic array while minimizing any decrease in performance

13. Pin-Constrained DMBs Pin-constrained array –Advantage: Reduce number of independent pins for n x m array from n x m to k ≤ n x m k = 5 is fewest # of control pins to control single droplet –Disadvantage: Potential for unintentional interference when multiple droplets are present Example: There is no way to concurrently move D i to position (1,2) and D j to position (4,4) 1235 3541 4123 2354 DiDi DjDj

14. Pin-Constraint Problem First, examine interference for two droplets For multiple droplets, the interference problem reduces to two droplet problem by examining all possible pairs of droplets Assumptions –Any sequence of movements for multiple droplets can occur in parallel, controlled by a clock –In a single clock cycle a droplet can move a maximum of one edge length –Assume no diagonal adjacent effect (experimentally verified for smaller electrode sizes)

15. Pin-Constraint Problem In some situations, we would like both droplets to move to another cell at the next clock edge. If this is not possible without interference, then a contingency plan would be to have one droplet undergo a stall cycle (stay on its current cell) and only move a single droplet at a time.

16. Pin-Constraint Problem Notation Droplets i and j are denoted Di and Dj The position of droplet i at the time t is given by Pi(t) The directly adjacent neighbors of a droplet as a function of time is denoted Ni(t), where Ni(t) is a set of cells The operator is the set of pins (no redundancies) that control the set of cells Formulation We examine the general problem of two droplets moving concurrently, which reduces to the problem of one droplet moving and one droplet waiting if we set Pj(t) = Pj(t+1): –Di moves from Pi(t) to Pi(t+1) –Dj moves from Pj(t) to Pj(t+1)

17. Interference vs. Mixing Interference constraints are designed to prevent “long-range” interference between the desired paths of droplets Fluidic constraints are necessary to avoid “short-range” interference in the form of inadvertent mixing Interference is a manifestation of the sharing of control pins between cells anywhere on the array while mixing (i.e., when fluidic constraints are violated) is a result of physical contact between droplets.

18. Interference Constraints Interference constraints for two droplets moving simultaneously on a two-dimensional array

19. Fluidic Constraints Fluidic constraints for two droplets on the same two- dimensional array

20. Pin-Constrained Non-Interference Index Objective: Given k independent pins, maximize the number of independent movements that a droplet can undertake from each position of the array while not interfering with another droplet on the same array. Need useful, application-independent index representing the independence of movement for two droplets on an array Easily extended to multiple droplets

21. Pin-Constrained Non-Interference Index Let Φ be the set of all possible pin configurations using k pins for an n x m array. For a particular pin configuration c Φ using k-pins in our 2-droplet system, can develop algorithm to obtain a pin-constrained non- interference index (PCNI) The situation of one droplet moving and one droplet waiting is the “safe” contingency plan if two droplets moving concurrently cause interference. We therefore examine this case here.

22. Pin-Constrained Non-Interference Index The output value, index, is a value between 0 and 1 that is the fraction of legal moves for two droplets (one moving, one waiting) on a n x m array with each cell having its own dedicated control pin that are still legal with pin layout c Φ and k < n x m pins,

23. Examples of PCNI Layout 1 1238 8765 5491 Layout 2 1671 2582 3493 Layout 3 1479 7258 9836

24. Maximizing PCNI Qualitatively speaking, better layouts seem to loosely obey two principles: –Spread out placement of pins used multiple times –Place multiply-used pins on cells that have fewer neighbors (e.g. sides and corners) Most assays cannot even be completed as scheduled on pin-constrained arrays (functionality problem, not just throughput)

25. Virtual Partitioning Alternative: partition array into regions in which only one droplet will be present at any given time With partitioned array, # of droplets that can be transported simultaneously without interference is equal to the number of partitions since partitions do not share any control pins (no interference between partitions possible) Fluidic constraints still must be satisfied so that inadvertent mixing does not occur.

26. Virtual Partitioning When would droplets in each partition need to be near each other? When they need to be mixed! It would be a relatively simple matter to create central partition(s) of the array for mixing purposes. Sometimes not necessary. When mixing is complete, the merged droplet can be moved to the appropriate partition for further processing without fear of interference with other droplets.

27. Non-Partitioned ArrayVirtually-Partitioned Array For full control of single droplet, need k ≥ 5 regardless of array size For full control of single droplet in each partition, need k = 5 x p, where p = # of partitions (assuming 3x3 partitions or larger) A lot of interference even with k significantly larger than 5 (most assays cannot properly run from start to finish) No interference if partitioning is such that there is one droplet per partition at any given time Throughput severely reducedNo change in throughput despite significantly reduced input bandwidth Fixed pin layoutReconfigure pin layouts one time before an assay is performed (only need to reconfigure if a new assay is performed) Virtual Partitioning

28. 16381 27492 385103 49274 5 165 Non-partitioned I(10,5,5) = 0.2626 Dynamically divide the array into two partitions such that two droplets will never have the potential to interfere - Only the fluidic constraints need to be considered 12367 45189 234106 51678 4289 Yellow Partition: pins 1-5 Green Partition: pins 6-10 I(10,5,5) = 0.4041 Examples of Partitioned Arrays

29. I(20,10,10) = 0.7331 Four partitions can accommodate up to four droplets simultaneously. 41352968107 352418 796 241357968 1352468 79 52413 7968 14111315121916182017 13151214111820171916 12141113151719161820 11131512141618201719 15121411132017191618 Multiple Partitions

30. 15x15 array with depiction of droplet paths for multiplexed glucose and lactase assays Multiplexed Bioassays

31. With 225 control pins (i.e., fully addressable array), schedule was devised to be: Step/Time Elapsed (seconds) Operation Step 1 / 0Sample 2 and reagent 2 start to move towards the mixer. Step 2 / 0.8 Sample 2 and reagent 2 begin to mix together and turn around in the 2  3-array mixer. Step 3 / 6.0Sample1 and reagent 1 start to move towards the mixer. Sample 2 and reagent 2 continue the mixing. Step 4 / 6.8Sample 2 and reagent 2 finish the mixing and product 2 leaves the mixer to optical detection location 2. Sample 1 and reagent 1 begin to mix in the 2  3-array mixer. Step 5 / 12.8Sample 1 and reagent 1 finish the mixing and product 1 leaves the mixer to the optical detection location 1. Product 2 continues the absorbance detection. Step 6 / 19.8Product 2 finishes optical detection and leaves the array to the waste reservoir. Product 1 continues the absorbance detection. Step 7 / 25.8Product 1 finishes optical detection and leaves the array to the waste reservoir. One procedure of the multiplexed bioassays ends. Multiplexed Bioassays

32. Can reduce input bandwidth while maintaining same throughput (true of most assays). Only need 5 partitions and 25 pins (11.11% of original input bandwidth). Throughput would be significantly reduced with a non-partitioned array with k ≥ 25 and in many instances, assay cannot be finished. In many instances, substantial rerouting and rescheduling is required to finish the assay. Multiplexed Bioassays

33. For pin-constrained arrays, virtual partitioning reduces interference when arrays are used randomly, and removes interference when one droplet per partition rule is followed. Partitioning Advantage

34. Summary Addressed an important problems in automating design of DMBs –New design method for pin-constrained digital microfluidics involving virtual partitioning to reduce input bandwidth without sacrificing schedule functionality and throughput