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PCR - Lab on Chip - LOC
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.
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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