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Lab on Chip PCR - LOC PCR (1) Lab on Chip PCR - LOC PCR (2) Lab on Chip PCR - LOC PCR (3)
Lab
on a Chip
NATURE Vol 442 27 July 2006 Agenda by Rosamund Daw, Senior Editor & Joshua Finkelstein, Associate Editor The ability to perform laboratory operations on a small scale using miniaturized (labon-a-chip) devices is very appealing. Small volumes reduce the time taken to synthesize and analyse a product; the unique behaviour of liquids at the microscale allows greater control of molecular concentrations and interactions; and reagent costs and the amount of chemical waste can be much reduced. Compact devices also allow samples to be analysed at the point of need rather than a centralized laboratory. Initially, however, pioneers of the field asked in Chimia whether their ideas about miniaturization would be “next century’s technology or just a fashionable craze”. The advantages are compelling, but designing and making devices of reduced size that operate effectively is challenging. The pioneers recognized the huge financial input and research effort needed to realize the full potential of the concept. Now, well into that next century, it is clear that labs on chips are here to stay. Physicists and engineers are creating exciting functionality, and are starting to construct highly integrated compact devices. Chemists are using such tools to synthesize new molecules and materials, and biologists are using them to study complex cellular processes. Furthermore, labs on chips offer point-of-care diagnostic abilities that could revolutionize medicine. Such devices may find uses in other areas, including a range of industrial applications and environmental monitoring. Commercial exploitation has been slow, but is gaining pace, with some products now on the market. A technology for this century? The signs are looking good. In this Insight, we present a collection of topical Reviews that discuss the history, design, application and future of lab-on-a-chip technologies, focusing on microfluidic flow devices. A little goes a long way Faster, safer and easier to control — chemical reactions in microreactors are taking off in the lab. Now industry is being seduced by the charms of the lab on a chip. by Jenny Hogan Scaling and the design of miniaturized chemical-analysis systems Dirk Janasek, Joachim Franzke & Andreas Manz Micrometre-scale analytical devices are more attractive than their macroscale counterparts for various reasons. For example, they use smaller volumes of reagents and are therefore cheaper, quicker and less hazardous to use, and more environmentally appealing. Scaling laws compare the relative performance of a system as the dimensions of the system change, and can predict the operational success of miniaturized chemical separation, reaction and detection devices before they are fabricated. Some devices designed using basic principles of scaling are now commercially available, and opportunities for miniaturizing new and challenging analytical systems continue to arise. Microfluidic diagnostic technologies for global public health Paul Yager, Thayne Edwards, Elain Fu, Kristen Helton, Kjell Nelson, Milton R. Tam & Bernhard H. Weigl The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world. Control and detection of chemical reactions in microfluidic systems Andrew J. deMello Recent years have seen considerable progress in the development of microfabricated systems for use in the chemical and biological sciences. Much development has been driven by a need to perform rapid measurements on small sample volumes. However, at a more primary level, interest in miniaturized analytical systems has been stimulated by the fact that physical processes can be more easily controlled and harnessed when instrumental dimensions are reduced to the micrometre scale. Such systems define new operational paradigms and provide predictions about how molecular synthesis might be revolutionized in the fields of high-throughput synthesis and chemical production. Nanodroplet real-time PCR system with laser assisted heating Hanyoup Kim, Sanhita Dixit, Christopher J. Green and Gregory W. Faris Molecular Physics Laboratory and Biosciences Division, SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025, USA  Abstract: We
report the successful application of low-power (~30 mW) laser
radiation as an optical heating source for high-speed real-time polymerase
chain reaction (PCR) amplification of DNA in nanoliter droplets
dispersed in an oil phase. Light provides the heating, temperature measurement,
and Taqman real-time readout in nanoliter droplets on a disposable
plastic substrate. A selective heating scheme using an infrared laser
appears
ideal for driving PCR because it heats only the droplet, not the oil
or plastic
substrate, providing fast heating and completing the 40 cycles of
PCR in 370
seconds. No microheaters or microfluidic circuitry were deposited
on
the substrate, and PCR was performed in one droplet without affecting
neighboring droplets. The assay performance was quantitative and its
amplification efficiency was comparable to that of a commercial instrument.
 A
polymer lab-on-a-chip for reverse transcription (RT)-PCR based
point-of-care clinical diagnostics
Soo Hyun Lee, Sung-Woo Kim, Ji Yoon Kang and Chong H. Ahn Lab Chip, 2008, 8, 2121 - 2127 An innovative polymer lab-on-a-chip (LOC) for reverse transcription (RT)-polymerase chain reaction (PCR) has been designed, fabricated, and characterized for point-of-care testing (POCT) clinical diagnostics. In addition, a portable analyzer that consists of a non-contact infrared (IR) based temperature control system for RT-PCR process and an optical detection system for on-chip detection, has also been developed and used to monitor the RT-PCR LOC. The newly developed LOC and analyzer have been interfaced and optimized for performing RT-PCR procedures and chemiluminescence assays in sequence. As a clinical diagnostic application, human immunodeficiency virus (HIV) for the early diagnosis of acquired immune deficiency syndrome (AIDS) has been successfully detected and analyzed using the newly developed LOC and analyzer, where the primer sets for p24 and gp120 were used as the makers for HIV. The developed polymer LOC and analyzer for RT-PCR can be used for POCT for the analysis of HIV with the on-chip RT-PCR and chemiluminescence assays in shorter than one hour with minimized cross-contamination.  Microchip-based
one step DNA extraction and real-time PCR in one chamber for rapid
pathogen identification
Jeong-Gun Lee, Kwang Ho Cheong, Nam Huh, Suhyeon Kim, Jeong-Woo Choib and Christopher Koa Lab Chip, 2006, 6, 886–895 Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system. PCR
thermal management in an integrated Lab on Chip
Janak Singh & Mayang Ekaputri J. Phys.: Conf. Ser. (2006) 34 222-227 1 A-STAR Institute of Microelectronics, 11 Science Park Road, Singapore Science Park II, Singapore - 117685; 2 School of Electrical and Computer Engineering, National University of Singapore, 21 Lower Kent Ridge Road, Singapore - 119077 Thermal management modelling and simulations of a polymerase chain reaction (PCR) device to be integrated on a lab on chip (LOC) have been carried out and presented. A typical MEMS PCR in symmetrical configuration is the base model for this study. When the PCR device is integrated on a fluidic chip with many other bio-analysis components such as DNA extraction, RNA extraction, electro-chemical sensor, flow through components and channels etc., thermal symmetry required for uniform temperature across the PCR chamber is normally lost. In this paper, ANSYS 8.0 simulations in varying conditions and corresponding physical basis have been investigated and presented. Model optimizations are carried out when PCR chamber is placed, one, in the centre (symmetry) and two, in the corner (asymmetry) of the integrated chip. In both cases, temperature uniformity within ±0.5 °C variation is obtained. Clockwork PCR
including sample preparation.
Pipper J, Zhang Y, Neuzil P, Hsieh TM. Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669 Angew Chem Int Ed Engl. 2008;47(21): 3900-3904 Masahiko
Hashimoto,b Pin-Chuan Chen,a Michael W. Mitchell,a Dimitris E.
Nikitopoulosa
Steven A. Soperb and Michael C. Murphya a Department of Mechanical Engineering, Louisiana State University, Baton Rouge LA 70803, USA b Department of Chemistry, Louisiana State University, Baton Rouge LA 70803, USA Lab Chip , 2004 , 4 , 638 – 645 Continuous
flow polymerase chain reaction (CFPCR) devices are compact reactors
suitable for microfabrication
and the rapid amplification of target DNAs. For a given reactor design,
the amplification time can
be reduced simply by increasing the flow velocity through the
isothermal zones of the device; for flow velocities
near the design value, the PCR cocktail reaches thermal equilibrium at
each zone quickly, so that near
ideal temperature profiles can be obtained. However, at high flow
velocities there are penalties of an increased
pressure drop and a reduced residence time in each temperature zone for
the DNA/reagent mixture, that
potentially affect amplification efficiency. This study was carried out
to evaluate the thermal and biochemical
effects of high flow velocities in a spiral, 20 cycle CFPCR device.
Finite element analysis (FEA) was
used to determine the steady-state temperature distribution along the
micro-channel and the temperature of the
DNA/reagent mixture in each temperature zone as a function of linear
velocity. The critical transition was between
the denaturation (95 uC) and renaturation (55 uC–68 uC) zones; above 6
mm s21 the fluid in a passively-cooled
channel could not be reduced to the desired temperature and the
duration of the temperature transition
between zones increased with increased velocity. The amplification
performance of the CFPCR as a function
of linear velocity was assessed using 500 and 997 base pair (bp)
fragments from l-DNA. Amplifications at
velocities ranging from 1 mm s21 to 20 mm s21 were investigated. The
500 bp fragment could be observed in a
total reaction time of 1.7 min (5.2 s cycle21) and the 997 bp fragment
could be detected in 3.2 min (9.7 s cycle21).
The longer amplification time required for detection of the 997 bp
fragment was due to the device being
operated at its enzyme kinetic limit (i.e., Taq polymerase
deoxynucleotide incorporation rate).
DNA amplification: does ‘small’ really mean ‘efficient’ ? Andrew J. de Mello reviews developments in DNA amplification Lab on a Chip, 2001, 1, 24N–29N Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis Eric T. Lagally, Charles A. Emrich and Richard A. Mathies* Lab on a Chip, 2001, 1, 102–107 A
fully integrated genomic
analysis microsystem including microfabricated heaters, temperature
sensors, and PCR chambers directly connected
to capillary electrophoretic separation channels has been constructed.
Valves and hydrophobic
vents provide controlled and sensorless sample
positioning and immobilization into 200 nL PCR chambers.
The use of
microfabricated heating and temperature sensing elements improves the
heating and cooling rates for the PCR reaction
to 20 °C s21. The amplified
PCR product, labeled on-column with an intercalating fluorescent
dye, is
injected into the gel-filled capillary
for electrophoretic analysis. Successful sex determination using
a multiplex PCR
reaction from human genomic DNA is demonstrated in less than 15 min.
This device is an important step toward a
microfabricated genomic microprocessor for use in forensics and
point-of-care molecular medical diagnostics.
Single-molecule DNA
amplification and analysis in an integrated microfluidic device.
Lagally ET, Medintz I, Mathies RA. Department of Chemistry, University of California, Berkeley 94720, USA. Anal Chem. 2001 Feb 1;73(3):565-70. Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages. Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection Thomas Ming-Hung Lee, Maria C. Carles and I-Ming Hsing* Lab Chip, 2003, 3, 100–105 Microfabricated
silicon/glass-based devices with functionalities of simultaneous
polymerase chain reaction (PCR) target amplification
and
sequence-specific electrochemical
(EC) detection have been successfully developed. The microchip-based
device has
a reaction chamber (volume of 8
µl) formed in a silicon substrate sealed by bonding to
a glass substrate.
Electrode materials such as gold and
indium tin oxide (ITO) were patterned on the glass substrate
and served as EC
detection platforms where DNA probes were immobilized. Platinum
temperature sensors
and heaters were patterned on top of the silicon substrate for
real-time, precise and rapid thermal cycling of the
reaction chamber as
well as for efficient target amplification by PCR. DNA analyses in the
integrated PCR-EC
microchip start with the asymmetric PCR amplification to produce
single-stranded target amplicons, followed by immediate
sequence-specific recognition of the
PCR product as they hybridize to the probe-modified electrode.
Two
electrochemistry-based detection techniques
including metal complex intercalators and nanogold particles
are employed in
the microdevice to achieve a sensitive detection of target DNA
analytes. With the integrated PCR-EC
microdevice, the detection of trace amounts of target DNA (as few as
several hundred copies) is demonstrated. The
ability to perform DNA amplification and EC sequence-specific product
detection simultaneously
in a single reaction chamber is a great leap towards the realization of
a truly portable and integrated DNA analysis
system.
Removal of PCR inhibitors using dielectrophoresis as a selective filter in a microsystem I. R. Perch-Nielsen, D. D. Bang, C. R. Poulsen, J. El-Alia and A. Wolff* Lab Chip, 2003, 3, 212–216 Diagnostic
PCR has been
used to analyse a wide range of biological
materials. Conventional PCR consists of several steps
such as
sample preparation, template purification, and
PCR amplification. PCR is often inhibited by contamination
of DNA
templates. To increase the sensitivity of the PCR,
the removal of PCR inhibitors in sample preparation
steps is
essential and several methods have been published.
The methods are either chemical or based on filtering.
Conventional
ways of filtering include mechanical filters
or washing e.g. by centrifugation. Another way of
filtering is the use
of electric fields. It has been shown that
a cell will experience a force when an inhomogeneous
electric
field is applied. The effect is called
dielectrophoresis (DEP). The resulting force depends on
the difference between
the internal properties of the cell and the
surrounding fluid. DEP has been applied to manipulate
cells in many
microstructures. In this study, we used DEP as
a selective filter for holding cells in a microsystem
while the PCR
inhibitors were flushed out of the system.
Haemoglobin and heparin – natural components of blood –
were
selected as PCR inhibitors, since the
inhibitory effects of these components to PCR have been
well documented.
The usefulness of DEP in a microsystem to
withhold baker’s yeast (Saccharomyces cerevisiae) cells
while the
PCR inhibitors haemoglobin and
heparin are removed will be presented and factors that influence
the effect of DEP
in the microsystem will be discussed. This
is the first time dielectrophoresis has been used as a
selective filter
for removing PCR inhibitors in a microsystem.
Miniaturized flow-through PCR with different template types in a silicon chip thermocycler Ivonne Schneegaß, Reiner Bräutigam and Johann Michael Köhler Lab on a Chip, 2001, 1, 42–49 Flow-through
chip
thermocyclers can be used in miniaturized rapid
polymerase chain reaction (PCR) despite their high
surface to volume
ratio of samples. We demonstrated that a
thermocycler made of silicon and glass chips and containing
thin film
transducers for heating and temperature control
can be adapted to the amplification of various DNA
templates of different
sources and properties. Therefore, the
concept of serial flow in a liquid/liquid two-phase
system was
combined with a surface management of
inner side walls of the microchannel and an adaptation
of PCR mixture
composition. In addition, the process
temperatures and the flow rates were optimized. Thus, a
synthetic template
originating from investigations
on nucleic acid evolution with 106 base pairs [cooperative
amplification
of templates by cross hybridization
(CATCH)], a house keeping gene with 379 base pairs
[glutaraldehyde
3-phosphate dehydrogenase (GAPDH)] and a zinc
finger protein relevant in human pathogenesis with 700
base
pairs [Myc-interacting zinc finger
protein-1, knock-out (Miz1-KO)] were amplified successfully.
In all three
cases the selectivity of priming and
amplification could be shown by gel electrophoresis. The
typical amplification
time was 1 min per temperature cycle. So, the
typical residence time of a sample volume inside the 25
cycle device
amounts to less then half an hour. The
energy consumption of the PCR chip for a 35 min PCR
process amounts to
less than 0.012 kW h.
Rosanne M. Guijt, Arash Dodge, Gijs W. K. van Dedem, Nico F. de Rooija and Elisabeth Verpoorte Lab Chip, 2003, 3, 1–4 Microfluidic
devices are a
promising new tool for studying
and optimizing (bio)chemical reactions and analyses. Many (bio)chemical
reactions
require accurate temperature control, such as
for example thermocycling for PCR. Here, a new
integrated temperature
control system for microfluidic
devices is presented, using chemical and physical processes to
locally regulate
temperature. In demonstration experiments, the
evaporation of acetone was used as an endothermic process
to cool a
microchannel. Additionally, heating of a
microchannel was achieved by dissolution of concentrated
sulfuric acid
in water as an exothermic process.
Localization of the contact area of two flows in a microfluidic channel
allows control of
the position and the magnitude of the thermal
effect.
High sensitivity PCR assay in plastic micro reactors Jianing Yang, Yingjie Liu, Cory B. Rauch, Randall L. Stevens, Robin H. Liu, Ralf Lenigk and Piotr Grodzinski Lab Chip, 2002, 2, 179–187 Small
volume operation and
rapid thermal cycling have been
subjects of numerous reports in micro reactor chip development.
Sensitivity
aspects of the micro PCR reactor have not been
studied in detail, however, despite the fact that
detection of rare
targets or trace genomic material from
clinical and/or environmental samples has been a great
challenge for
microfluidic devices. In this study, a
serpentine shaped thin (0.75 mm) polycarbonate plastic PCR
micro reactor was
designed, constructed, and tested for not only
its rapid operation and efficiency, but also its
detection sensitivity
and specificity, in amplification of
Escherichia coli (E. coli) K12-specific gene fragment. At
a template concentration
as low as 10 E. coli cells (equivalent to
50 fg genomic DNA), a K12-specific gene product (221
bp) was
adequately amplified with a total of 30 cycles in
30 min. Sensitivity of the PCR micro reactor was
demonstrated
with its ability to amplify K12-specific gene
from 10 cells in the presence of 2% blood. Specificity
of the
polycarbonate PCR micro reactor was also proven
through multiplex PCR and/or amplification of different
pathogen-specific genes. This is, to our knowledge, the
first systematic study of assay sensitivity and specificity
performed in
plastic, disposable micro PCR devices.
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