Coordinating Multiple Droplets in Planar Array Digital Microfluidic Systems
Eric J. Griffith  & Srinivas Akella
Department of Computer Science, Rensselaer Polytechnic Institute, Troy, NewYork 12180, USA
The International Journal of Robotics Research
Vol. 24, No. 11, November 2005, pp. 933-949

In this paper we present an approach to coordinate the motions of droplets in digital microfluidic systems, a new class of lab-on-a-chip systems for biochemical analysis. A digital microfluidic system typically consists of a planar array of cells with electrodes that control the droplets. The primary challenge in using droplet-based systems is that they require the simultaneous coordination of a potentially large number of droplets on the array as the droplets move, mix, and split. In this paper we describe a general-purpose system that uses simple algorithms and yet is versatile. First, we present a semi-automated approach to generate the array layout in terms of components. Next, we discuss simple algorithms to select destination components for the droplets and a decentralized scheme for components to route the droplets on the array. These are then combined into a reconfigurable system that has been simulated in software to perform analyses such as the DNA polymerase chain reaction. The algorithms have been able to successfully coordinate hundreds of droplets simultaneously and perform one or more chemical analyses in parallel. Because it is challenging to analytically characterize the behavior of such systems, simulation methods to detect potential system instability are proposed.

Performance Characterization of a Reconfigurable Planar-Array Digital Microfluidic System
Eric J. Griffith, Srinivas Akella, Member, IEEE, and Mark K. Goldberg

This paper describes a computational approach to designing a digital microfluidic system (DMFS) that can be rapidly reconfigured for new biochemical analyses. Such a “lab-on-a-chip” system for biochemical analysis, based on electrowetting or dielectrophoresis, must coordinate the motions of discrete droplets or biological cells using a planar array of electrodes. The authors have earlier introduced a layout-based system and demonstrated its flexibility through simulation, including the system’s ability to perform multiple assays simultaneously. Since array-layout design and droplet-routing strategies are closely related in such a DMFS, their goal is to provide designers with algorithms that enable rapid simulation and control of these DMFS devices. In this paper, the effects of variations in the basic array-layout design, droplet-routing control algorithms, and droplet spacing on system performance are characterized. DMFS arrays with hardware limited row-column addressing are considered, and a polynomial-time algorithm for coordinating droplet movement under such hardware limitations is developed. To demonstrate the capabilities of our system, we describe example scenarios, including dilution control and minimalist layouts, in which our system can be successfully applied.

Microcontact Printing of DNA Molecules
Sebastian A. Lange, Vladimir Benes, Dieter P. Kern, J. K. Heinrich Ho rber, and Andre Bernard,
Indigon GmbH, Sindelfingerstrasse 3, 72070 Tübingen, Germany, European Molecular Biology Laboratory,
Meyerhofstrasse 1, 69117 Heidelberg, Germany, University of Tu¨bingen, Auf der Morgenstelle 10,
72076 Tu¨bingen, Germany, and Department of Physiology, Wayne State University School of Medicine,
540 East Canfield Avenue, Detroit, Michigan 48201

The controlled placement of DNA molecules onto solid surfaces is the first step in the fabrication of DNA arrays. The sequential deposition of tiny drops containing the probe DNA fragments using arrays of spotting needles or ink jet nozzles has become a standard. However, a caveat of liquid spotting is the drying of the deposited drop because this creates the typical inhomogeneities, i.e., rims around the spot. Another drawback is that each DNA array is an original and has to be fabricated individually. Microcontact printing is a versatile technique to place proteins onto different target surfaces in uniformly patterned monolayers with high lateral resolution. Here, we show for the first time that DNA can also be printed with equally high resolution in the submicrometer range using an elastomeric stamp with chemically tailored surface. Two regimes for the transfer of the molecules were observed. Finally, microcontact printing of an array of DNA probes onto a solid support and its use in a subsequent hybridization assay was demonstrated.

Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes
Pavel Neuzil*, Chunyan Zhang, Juergen Pipper, Sharon Oh and Lang Zhuo
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos,Singapore 138669
Nucleic Acids Research, 2006, Vol. 34, No. 11 e77

We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the system’s thermal constant of
0.27 s, we have achieved a heating rate of 175° s1 and a cooling rate of 125C s1. A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput.

ITO-coated glass/polydimethylsiloxane continuous-flow PCR chip
Seung-Ryong Joung, Jaewan Kim, Y. J. Choi, C. J. Kang and Yong-Sang Kim*
Proceedings of the 2nd IEEE International
Conference on Nano/Micro Engineered and Molecular Systems
January 16 - 19, 2007, Bangkok, Thailand

Abstract-We propose a continuous-flow polymerase chain reaction (PCR) chip using indium-tin-oxide (ITO)-coated glass/ polydimethylsiloxane (PDMS) materials for DNA amplification. The continuous-flow PCR chip enables fast thermal cycling and series amplification, which are difficult to achieve in a conventional PCR or micro-chamber PCR chip. Six heaters of ITO thin films were fabricated on glass for the thermal cycling of the flowing PCR sample. The PDMS microchannel was fabricated using a negative molding method. The width and depth of the microchannel are 250 gm and 200 gm, respectively, with a total channel length of 1340 mm. The PCR chip can perform 20 cycles of ampliflcations. The ratio of the channel lengths for three different temperature zones, namely denaturation, annealing, and extension, is 2:2:3, respectively. Using the fabricated continuous-flow PCR chip, two DNA plasmids (720-bp pKS-GFP and 300-bp PG-noswsi) were successfully amplifled.

Z. Wang1, A. Sekulovic1 ,2, J.P. Kutter1, D.D. Bang3 and A. Wolff1
1MIC – Department of Micro and Nanotechnology, Technical University of Denmark
2Department of Biotechnology, Technical University of Delft
3Department of Poultry, Fish and Fur Animals, Danish Institute for Food and Veterinary Research
MEMS 2006, Istanbul, Turkey, 22-26 January 2006

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. By using the integrated optical system of the real-time PCR chip, cadF – a virulence gene of Campylobacter jejuni, could specifically be detected. Two different DNA binding dyes, SYTOX Orange and TO-PRO-3, were added to the PCR mixture to realize the real-time PCR. The presented approach shows reliable real-time quantitative information of the PCR amplification of the targeted gene.

An integrated fluorescence detection system for lab-on-a-chip applications
Lukas Novak, Pavel Neuzil,* Juergen Pipper, Yi Zhang and Shinhan Lee
Lab Chip, 2007, 7, 27–29 27

We present a low-cost miniaturized fluorescence detection system for lab-on-a-chip applications with a sensitivity in the low nanomolar range; a built-in lock-in amplifier enables measurements under ambient light.

Nucleic acid based biosensors: The desires of the user
Sinuhe Hahn, Susanne Mergenthaler, Bernhard Zimmermann, Wolfgang Holzgreve
Laboratory for Prenatal Medicine, University Women’s Hospital/Department of Research, University of Basel, Spitalstrasse 21, CH 4031 Basel, Switzerland
Bioelectrochemistry 67 (2005) 151– 154

The need for nucleic acid based diagnostic tests has increased enormously in the last few years. On the one hand, this has been stimulated by the discovery of new hereditary genetic disease loci following the completion of the Human Genome Project, but also by the presence of new rapidly spreading viral threats, such as that of the SARS epidemic, or even micro-organisms released for the purpose of biological warfare. As in many instances rapid diagnoses of specific target genetic loci is required, new strategies have to be developed, which will allow this to be achieved directly at the point-of-care setting. One of these avenues being explored is that of biosensors. In this review, we provide an overview of the current state of the art concerning the high-throughput analysis of nucleic acids, and address future requirements, which will hopefully be met by new biosensor-based developments.

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection
Evgeni A. Kabotyanskia, Inna L. Botchkina b, Olga Kosobokova a, Galina I. Botchkina b,
Vera Gorfinkel a, Boris Gorbovitski c
a Department of Electrical and Computer Engineering, State University of New York at Stony Brook, Stony Brook, NY 11794, USA
b Department of Surgery and Surgical Oncology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA
c BioPhotonics Corporation, Stony Brook, NY 11794, USA
Biosensors and Bioelectronics 21 (2006) 1924–1931

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially
enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 104 cells at 29 cycles and over 105 cells at 25 cycles. In contrast, the SPD-based CE–LIF has revealed 6-base repeats in samples with as low as 102 cells after 29 cycles and 103 cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.

Parallel Picoliter RT-PCR Assays Using Microfluidics
Joshua S. Marcus, W. French Anderson, and Stephen R. Quake
Option in Biochemistry and Molecular Biophysics, Department of Applied Physics, California Institute of Technology,
MS 128-95, Pasadena, California 91125, and Gene Therapy Laboratories, Keck School of Medicine,
University of Southern California, Los Angeles, California 90033
Anal. Chem.2006, 78,956-958

The development of microfluidic tools for high-throughput nucleic acid analysis has become a burgeoning area of research in the post-genome era. Here, we have developed a microfluidic chip to perform 72 parallel 450-pL RTPCRs. We took advantage of Taqman hydrolysis probe chemistry to detect RNA templates as low as 34 copies. The device and method presented here may enable highly parallel single cell gene expression analysis.

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences
Xiaochuan Zhou 3,4, Shiying Cai 3, Ailing Hong 3,4, Qimin You 3, Peilin Yu 1, Nijing Sheng 1, Onnop Srivannavit 2, Seema Muranjan 3, Jean Marie Rouillard 2, Yongmei Xia 2, Xiaolin Zhang 3,4, Qin Xiang 3, Renuka Ganesh 1,4, Qi Zhu 1, Anna Matejko 1, Erdogan Gulari 2 and Xiaolian Gao 1
1 Department of Chemistry, University of Houston, Houston, TX 77004-5003, USA,
2 Department of Chemical
Engineering, University of Michigan, Ann Arbor, MI 48109, USA
3 Xeotron Co. 8275 El Rio, Suite 130, Houston,
TX 77054, USA
4 Atactic Technologies Inc., 2575 W. Bellfort, Suite 270, Houston, TX 77054, USA

Nucleic Acids Research, 2004, Vol. 32, No. 18 5409–5417

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, highthroughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.

Separation of DNA fragments for fast diagnosis by microchip electrophoresis using programmed field strength gradient
Seong Ho Kang, Mira Park, Keunchang Cho
Department of Chemistry, Chonbuk National University, Jeonju, South Korea
Digital Bio Technology, SKC Central Research Institute, Suwon, South Korea
Electrophoresis 2005, 26, 3179–3184

We evaluated a novel strategy for fast diagnosis by microchip electrophoresis (ME), using programmed field strength gradients (PFSG) in a conventional glass double-T microfluidic chip. The ME-PFSG allows for the ultrafast separation and enhanced resolving power for target DNA fragments. These results are based on electric field strength gradients (FSG) that use an ME separation step in a sieving gel matrix poly-(ethylene oxide). The gradient can develop staircase or programmed shapes FSG over the time. The PFSG method could be easily used to increase separation efficiency and resolution in ME separation of specific size DNA fragments. Compared to ME that uses a conventional and constantly applied electric field (isoelectrostatic) method, the MEPFSG achieved about 15-fold faster analysis time during the separation of 100 bp DNA ladder. The ME-PFSG was also applied to the fast analysis of the PCR products, 591 and 1191 bp DNA fragments from the 18S rRNA of Babesia gibsoni and Babesia caballi.

On-Chip Nanoliter-Volume Multiplex TaqMan Polymerase Chain Reaction from A Single Copy Based on Counting Fluorescence Released Microchambers
Yasutaka Matsubara, Kagan Kerman, Masaaki Kobayashi, Shouhei Yamamura, Yasutaka Morita, Yuzuru Takamura, and Eiichi Tamiya
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi,
Ishikawa, 923-1292 Japan
Anal. Chem.2004, 76,6434-6439

A novel method for multiplex TaqMan PCR in nanoliter volumes on a highly integrated silicon microchamber array is described. Three different gene targets, related to ‚-actin, sex-determining region Y (SRY), and Rhesus D (RhD) were amplified and detected simultaneously on the same chip by using three different types of human genomic DNA as the templates. The lack of crosscontamination and carryover was shown using alternate dispensing of mineral oil-coated microchambers containing template and those without template. To confirm the
specificity of our system to ‚-actin, SRY, and RhD genes, we employed the larger volume PCR samples to a commercial real-time PCR system, SmartCycler. The samples were cycled with the same sustaining temperatures as with the microchamber array. Instead of the conventional method of DNA quantification, counting the number of the fluorescence released microchambers in consequence to TaqMan PCR was employed to our chip. This simple method of observing the end point signal had provided a dynamic quantitative range. Stochastic amplification of 0.4 copies/reaction chamber was achieved. The microfabricated PCR chip demonstrated a rapid and highly sensitive response for simultaneous multiple-target detection, which is a promising step toward the development of a fully integrated device for the “lab-on-a-chip” DNA analysis.

Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems
Vasile I. Furdui and D. Jed Harrison
Department of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2.
Lab Chip, 2004 , 4 , 614 – 618

A one-step immunomagnetic separation technique was performed on a microfluidic platform for the isolation of specific cells from blood samples. The cell isolation and purification studies targeted T cells, as a model for low abundance cells (about 1:10,000 cells), with more dilute cells as the ultimate goal. T cells were successfully separated on-chip from human blood and from reconstituted blood samples. Quantitative polymerase chain reaction analysis of the captured cells was used to characterize the efficiency of T cell capture in a variety of flow path designs. Employing many (4–8), 50 mm deep narrow channels, with the same overall cross section as a single, 3 mm wide channel, was much more effective in structuring dense enough magnetic bead beds to trap cells in a flowing stream. The use of 8-multiple bifurcated flow paths increased capture efficiencies from y20 up to 37%, when compared to a straight 8-way split design, indicating the value of ensuring uniform flow distribution into each channel in a flow manifold for effective cell capture. Sample flow rates of up to 3 mL min21 were evaluated in these capture beds.