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
IEEE
TRANSACTIONS ON COMPUTER-AIDED DESIGN OF INTEGRATED CIRCUITS AND
SYSTEMS, VOL. 25(2) 2006
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.
REAL-TIME PCR USING A
PCR MICROCHIP WITH INTEGRATED THERMAL SYSTEM AND
POLYMER WAVEGUIDES
FOR THE DETECTION OF CAMPYLOBACTER JEJUNI
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.
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