CRITICAL REVIEW www.rsc.

org/loc | Lab on a Chip

Total nucleic acid analysis integrated on microfluidic devices

Lin Chen,a Andreas Manza and Philip J. R. Day*ab
Received 1st June 2007, Accepted 12th July 2007
First published as an Advance Article on the web 9th August 2007
DOI: 10.1039/b708362a

The design and integration of microfluidic devices for on-chip amplification of nucleic acids from
various biological samples has undergone extensive development. The actual benefit to the
biological community is far from clear, with a growing, but limited, number of application
successes in terms of a full on-chip integrated analysis. Several advances have been made,
particularly with the integration of amplification and detection, where amplification is most often
the polymerase chain reaction. Full integration including sample preparation remains a major
obstacle for achieving a quantitative analysis. We review the recently described devices
incorporating in vitro gene amplification and compare devices relative to each other and in terms
of fully achieving a miniaturised total analysis system (m-TAS).

1. Introduction advantages. The often quoted advantages related to imple-

Micro total analysis system (m-TAS), also known as ‘‘lab-on-a-
chip’’, was proposed in the early 1990s, and has been
enthusiastically embraced by analytical specialists wishing to
instigate whole processes on microfluidic platforms.1–4 Many
research groups have expended much effort to construct
various analytical components, such as hydrodynamic (micro-
pump and micro-valve), thermodynamic (micro-heater),
electro-dynamic (micro-electrode) and detection units (micro-
sensor and micro-detector) onto silicon, glass or polymer
microchip substrate materials. Compared to conventional
methods, integrated m-TAS platforms offer several remarkable
a
Institute for Analytical Sciences, Bunsen-Kirchhoff Str. 11, D-44139
Dortmund, Germany
b
The Manchester Interdisciplinary Biocentre, University of Manchester,
131, Princess Street, Manchester, UK M1 7ND.
E-mail: Philip.j.day@manchester.ac.uk; Fax: +44-161-275-1617;
Tel: +44-161-275-1621
menting m-TAS include; low cost, high speed, enhanced
sensitivity and automation of nearly all necessary processes
from sample preparation to outcome of analysis results.1–4
However, whilst some or indeed all of these may have high
importance for bioanalytics, the over-riding factor relating to
the implementation of m-TAS is more likely to be associated
with increased quality of assays with respect to sample tracking,
reproducibility and producing results that can be gauged
quantitatively, where the same cannot be readily achieved using
a connected series of current analytical procedures. This
scenario is particularly prevailing in the situation surrounding
gene-based measurements which are correlated to titred
presence and abundance of pathogen, disease or marker nucleic
acids. Notably, the in-vitro diagnostic market has been slow to
take-up miniaturised PCR devices, seemingly because the
current integration of processes to achieve m-TAS for nucleic
acid measurements endures at least the limitations associated
with conventional PCR assay formats.

Lin Chen received his M. Eng. microelectrodes as detectors

in Applied Chemistry (2004)
from Shanghai Jiao Tong
University, P. R. China. He is
now reading for a Ph.D. under
the supervision of Professor
Andreas Manz and Professor
Philip Day at the Institute for
Analytical Sciences (ISAS) in
Dortmund, Germany. His
research focuses on the devel-
opment of an integrated micro-
fluidic platform for nucleic acid
preparation, amplification and
Lin Chen real-time analysis to contribute
towards quantitative bioassays
k
employing -TAS.
Andreas Manz obtained his Ph.D. from the Swiss Federal
Institute of Technology (ETH) Zurich, Switzerland, with
Professor W. Simon. His thesis dealt with the use of
for picolitre-size volumes. He
spent one year at Hitachi
Central Research Lab in
Tokyo, Japan, as a postdoc-
toral fellow and produced
liquid chromatography column
on a chip. At Ciba-Geigy,
Basel, Switzerland, he devel-
oped the concept of miniatur-
ized total analysis systems and
built a research team on chip-
based analytical instrumenta-
tion from 1988–1995. He was
Andreas Manz professor for analytical chem-
istry at Imperial College in
London from 1995–2003. Since 2003, he has been the head of the
ISAS in Dortmund, Germany, and a Professor of Analytical
Chemistry at the University of Dortmund. His research interests
include fluid handling and detection principles for chemical
analysis, bioassays, and synthesis using microfabricated devices.

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 Fig. 1 Integrated PCR on microfluidic devices.
Genetic analysis employing m-TAS has elicited enormous
interest, and because the amount of original cellular matter
available for genetic analysis is often extremely limited and
Philip Day graduated with a
Ph.D. degree from the Wolfson
Research Laboratories,
University of Birmingham.
From 1995–1997 at Oxford
University he developed very
high throughput PCR for the
Wellcome Trust in the Human
Genome Mapping Project. This
was followed by developments
into high throughput sequencing
and gene micro-arrays with
Prof. Sir Edwin Southern,
which he followed with the
Philip J. R. Day establishment of a Functional
Genomics Unit, Kinderspital,
University of Zurich. His studies correlate innovative quantitative
measurements of nucleic acids to meaningful biomedical inter-
pretation. He was appointed Reader in Genomics, University of
Manchester. In 2006 he was made Principal Investigator at the
Manchester Interdisciplinary Biocentre, and was later appointed
Professor of Applied Molecular Biology and Biochemistry at the
University of Dortmund, and with ISAS, Dortmund.
1414 | Lab Chip, 2007, 7, 1413–1423
readily lost given the limited availability of homogeneous
(extraction-free) assay formats, the amplification of target
nucleic acids following careful sample manipulation including
separation processes are typically necessary steps. Thus, not
too surprisingly miniaturised in vitro gene amplification,
especially the polymerase chain reaction (PCR), has become
synonymous with the development of miniaturisation and
microfluidics per se.5–9 Moreover, miniaturised PCR gives
other advantages such as high thermal cycling speed and low
reagent consumption, which benefit from the intrinsic high
surface-to-volume ratio. But so far, most of the reported
miniaturised devices for nucleic acid amplification are stand-
alone structures replacing only the role of the conventional
PCR thermocycler. The integration of nucleic acid amplifica-
tion with other functional units, such as proximal sample
preparation and distil sequence analysis, on a single device is
broadly accepted as the way forward and starts to emulate the
vision of m-TAS as discussed previously.9 Presently, integra-
tion of miniaturised PCR is under rapid development, and
PCR has been coupled with pre-PCR modules, such as sample
purification and pre-concentration, and post-PCR modules,
such as capillary gel electrophoresis (CGE) and DNA
microarray, on single microdevices. The different approaches
to integrated gene analysis that encompasses in vitro gene
amplification are relatively finite and are shown in Fig. 1 and
2. In this article, we concisely review the recent development of
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 Fig. 2 Schematic design of a nucleic acid m-TAS device for point-of-care applications.
in vitro gene amplification (primarily PCR) integrated with
other functionalities in a monolithic format for clinical
diagnosis, encompassing solid, fluid and aerosol samples.
For a theoretical background into PCR and technical aspects
of microchip-based PCR, readers are referred to other
published reviews for further details.5–9
2. Integrated PCR
2.1 Microdevice pre-conditioning and sample processing: pre-
PCR
The aim of integrated PCR is to analyse, within the confines
of a usually portable miniaturised platform, real biological
samples obtained from suspected aberrant tissues, fluidics
or locations. Any treatment or change influencing the
sample prior to the analyte measurement procedure is
critical since the quantitative assessment of the analyte
biomarker can be irretrievably altered to produce a result that
may detract and lead to misinterpretation of the true biological
situation.
2.1.1 Pre-conditioning. The characteristic seen for biological
systems is one of high specificity of interaction which has been
particularly exploited in the case of proteins (antibodies) and
This journal is ß The Royal Society of Chemistry 2007
nucleic acids.10 Of equal importance and of high relevance to
m-TAS is the availability of biologically highly inert surfaces
that allow movement of bio-matter through the confines of
microfluidic devices without losses incurred through non-
specific association of the analyte or reagents with the device
itself. In this context, mirroring of the vascular transportation
system used for dispersing blood cells and associated serum
constituents in organisms would provide many features of a
suitable conduit for moving PCR-related reaction constituents
without compromising information retrieval. To date,
reported passivation methods for the inner surfaces of PCR
microdevices include dynamic and static passivation. For static
passivation, the surface of the PCR microdevice is always pre-
coated with a PCR-friendly substance,11–14 which occurs
before PCR, while dynamic passivation is realized by adding
the passivation reagents to the PCR cocktail solution, and the
coating ensues concurrently with the PCR process.15 Thus, we
classify static passivation into the category of pre-PCR
treatment process. So far, there are two main types of static
passivation, chemical silanisation11,12 and silicon oxide surface
coating.13,14 The first procedure employs deposition of a thin
layer of silicon oxide onto the microchannel surface to enhance
the PCR compatibility, and the second method employs filling
the reaction chamber or channel with the silanising solution,
Lab Chip, 2007, 7, 1413–1423 | 1415
and incubating the chip for a period of time, followed by
washing and drying.
2.1.2 Sample processing. Amongst all the pre-treatment
methods for PCR analysis, cell identification, cell capture,
nucleic acids extraction, sample/analyte purification and pre-
concentration are all essential, since PCR requires relatively
pure nucleic acid samples that are free from reaction inhibiting
contaminants. Increased stringency of sample treatments to
safeguard sample purity and performance in PCR are
heightened requirements for quantitative assessment of PCR
amplified products.
The difficulty in manipulating micro-scale liquid-carried
samples renders most DNA pre-treatment microdevices devoid
of downstream microfluidic integration. Microchip-based
DNA purification was described first by Christel et al.16 In
their study, pillars were created in a micro-channel to increase
the contact surface area. The extraction and concentration of
DNA from samples were accomplished utilizing silicon fluidic
microchips with high surface area-to-volume ratios. Wolfe
et al.17 developed sol-gel immobilized silica particles in a
micro-channel for purification of DNA as a low-cost
alternative. Later, the performance of the solid phase
extraction (SPE) device on the microchip was thoroughly
examined using human genomic DNA from whole blood and
bacterial DNA from colony samples and spores.18 A micro-
fabricated electrophoretic bioprocessor integrated with DNA
sequencing, sample desalting, template removal, preconcentra-
tion and CGE analysis was demonstrated by Mathies’ group.19
A novel chamber geometry, capture process, and affinity
capture chemistry were developed for the purification of DNA
fragments and integrated with high-speed microdevice sequen-
cing. Sample immobilization, pre-concentration, and desalting
were completed in only 120 s, approximately a 10-fold
reduction in time and 100-fold reduction in reagent volume.
Microfluidic devices coupling pre-PCR components to
downstream PCR has also been studied. Literature reporting
pre-PCR processes integrated with PCR on microchips are
summarized in Table 1.20–25 Wilding et al.20,21 isolated white
blood cells from whole blood by constructing a series of 3.5 mm
filters in silicon-glass microchips. Genomic DNA from the
whole blood cells isolated on the filters was directly amplified
using PCR. The on-chip sample preparation reported by Liu
et al.22 started with mixing and incubating blood and a
solution containing immunomagnetic beads in a sample
Table 1 Pre-PCR integrated with PCR on a microchip
PCR type Substrate material Source Template DNA
Stationary Glass Whole blood 202-bp DNA fragment
chamber of dystrophin gene
Stationary Silicon/glass Whole blood 226-bp regions of human
chamber coagulation Factor V gene
Stationary PC Whole blood 221-bp fragment from
chamber E.coli K12 specific gene
Stationary PDMS/silicon Listeria 544-bp DNA segment from
chamber monocytogens Listeria monocytogens
cells
Stationary Silicon/glass E.coli BL21 16S-rRNA region of
chamber bacterial genome
1416 | Lab Chip, 2007, 7, 1413–1423
storage chamber to ensure target cell capture from blood.
The sample mixture is then pumped through into the PCR
chamber, where target cell capture and pre-concentration
occur as the bead-bacteria conjugates are trapped by the
magnet. The washing buffers were consecutively pumped
through the PCR chamber to purify the captured cell. After
the PCR reagents were transferred into the PCR chamber, on-
chip thermal cell lysis and PCR were performed. Cady et al.23,24
reported a poly(dimethylsiloxane) (PDMS)–silicon microde-
vice consisting of a microfabricated channel in which silica-
coated pillars were etched. DNA was selectively bound to these
pillars in the presence of the chaotropic salt guanidinium
isothiocyanate, followed by washing with ethanol and elution
with water. Simultaneous pumping of a concentrated PCR
master mix through a second inlet port allowed for parallel
flow of eluted DNA and master mix into the PCR reaction
chamber for real-time analysis. A method combining laser-
irradiation and magnetic beads was developed for rapid cell
lysis and DNA isolation on microchips.25 By using an 808 nm
laser and carboxyl-terminated magnetic beads, the authors
demonstrated that pathogens can be lysed by a single laser
pulse of 40 s inside a 4 ml chamber, and subsequently real-time
PCR for pathogen detection was performed using the same
microchip.
2.2 Real-time quantitative PCR
Recently, integration of real-time PCR on microfluidic devices
has gained in popularity. In this process, the amplification of
specific gene sequences is coupled to quantification of the
original target DNA, which is typically monitored through
intercalation of a dye or fluorescence of a probe.26,27 Real-time
detection is achieved by recording the increase in fluorescence
resulting from the stochastically associated measurement of
fluorescence with increased dsDNA production after each
round of thermocycling. Once the yield of fluorescence-
associated PCR products exceeds the background, the forma-
tion of reaction products can be monitored as the geometric
reaction proceeds, which contrasts with measuring the gross
amplified product at the end of a fixed number of cycles. The
number of cycles that are needed to reach the detection
threshold (often termed crossing point (Cp), or cycle threshold
value (Ct)) is proportional to the negative logarithm of the
initial concentration of target DNA. Thus, the Ct values from
different initial concentrations of target DNA can be used to
Pre-treatment technique Volume/mL Ref.
Microchip filter for white 50 20
blood cells isolation
White blood cells isolation on the 12 21
filter section of the microchip
Immunomagnetic bead-based 20 22
cell capture,
DNA bound to silica-coated pillars in 50 23,24
the presence of the chaotropic salt
guanidinium isothiocyanate
Cell lysis by laser-irradiated magnetic 4 25
bead system, and magnetic beads
for removing denatured proteins
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 Ref.
determine unknown amounts of sample or calculate the PCR

24

25

28

29

30

31
32
33

34
efficiency.26,27 Stationary microchip PCR can be readily
adapted into miniaturised real-time PCR with some minor

10 for HPV 16 and

changes, such as PCR reagent formulation and an additional

50 for ssDNA
on-line fluorescence analysis, which is well-established for

0.9, 1, 3 and 7
microchip CGE and PCR-CGE. For continuous flow micro-

Volume/mL
chip PCR, the optical detection is either movable or split into

y0.42
several identical parts to permit simultaneous detection of

N/A
50

50

50

25
different amplification reactions. Reported literature relating

4
to real-time PCR miniaturised platforms are summarized in
Table 2.24,25,28–34

16S-rRNA region of bacterial genome

Northrup et al. first developed a miniaturised analytical

150-bp DNA segment of E.coli stx1

150-bp DNA segment of E.coli stx1
thermal cycling instrument for real-time PCR detection.28,29 A

294-bp human b-actin and HIV

266-281-bp segments of HA gene;

Campylobacter jejuni cadF gene

Hantavirus, Borrelia burgdorferi,

113-bp DNA segment of DNA

micro-machined silicon reaction chamber was integrated with

73-bp segment of C6 gene

HPV 16 and 118-bp ssDNA
544-bp DNA segment from
heaters and electronics for controlling temperature. The device

398-bp DNA fragment of

Listeria monocytogens
is a scaling-down of a conventional real-time PCR instrument
and was successfully used for the detection of single-base

plasmids pMU 15
differences in viral and human DNA. Their studies indicate

Template DNA
that real-time PCR can also be performed in a portable
format. A real-time nucleic acid sequence-based amplification
(NASBA) platform in nanolitre volume was developed in a
silicon–glass microchip. NASBA is isothermal and con-
sequently no thermocycling was needed, therefore it simplifies
both the microchip design and the instruments specifications.30
Later, Cady et al.24 developed integrated miniaturised real-

Molecular beacon probe

time PCR detection equipped with microprocessor, pumps,

Ethidium bromide &

SYTOX Orange and

Detection technique

TaqMan1 probe
thermocylcer and light emitting diodes (LEDs)-based fluores-

probe

TaqMan1 probe

TaqMan1 probe
TaqMan1 probe
TaqMan1 probe
cence excitation/detection. Monolithic DNA purification and

TO-PRO-3
real-time PCR enable fast detection of Listeria monocytogenes SYBR green
1
TaqMan
cells (104 to 107) within 45 min. Xiang et al.31 reported real-
time detection of a 150-bp DNA segment of E.coli stx1 on a
well-based PDMS microchip using fluorescent hydrolysis
(TaqMan1) probes. Single-well and three-well real-time PCR
were tested with different initial concentrations of DNA

heater and temperature sensor

Aluminium block with cartridge
templates, and both were able to amplify the 150-bp DNA
Thermoelectric heater/cooler

segment of E.coli stx1. The same group performed this real-

Integrated Pt heater array

time PCR inside a PDMS-based microchannel using the Joule Thin film heater and fan
GeneSpector Micro

heating effect.32 Their method applied Joule heating generated

by the current of the thermal source, therefore smartly
Thin film heater

Thin film heater

PCR machine
Thermal source

Peltier element
1

avoiding the necessity to build-up a thermocycler. Under an

Joule heating

electric field, DNA fragments migrate from the anode to the

cathode, thus in order to keep the DNA fragments inside the
PCR cocktail solution, the direction of the applied current
Table 2 Real-time PCR integrated on a microchip

needs to be changed at a certain frequency. The applied

voltage needs to be adjusted if the composition of the PCR
solution changes. A real-time on-line PCR microfluidic device
Device substrate

for continuous flow was developed recently by using laser

PDMS/silicon

Silicon/glass

Silicon/glass

beam scanning within the temperature annealing region of the

device.33 Both thermal and polymer waveguide optical
PDMS
PDMS
PDMS
Silicon

Silicon

SU-8

detection systems were integrated inside a SU-8 chamber,

which is an important step towards a portable tool for real-
time quantitative PCR.34
Stationary chamber
Stationary chamber

Stationary chamber

Stationary chamber

Stationary chamber

Stationary chamber
Stationary channel

2.3 Post-PCR
Continuous flow
Stationary well
(NASBA)

Post-PCR product analysis is singularly the most developed

PCR type

area encompassing PCR integration within a single micro-

device. This is most likely attributed to complexities associated
with sample handling during pre-PCR treatments and

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 Table 3 Post-PCR analysis integrated on a microchipa
PCR type Substrate material Template DNA Thermal source Volume/mL Detection method Ref.

Stationary chamber PC 221-bp E.coli gene Resistive heater 20 DNA microarray 22

Stationary chamber Glass 268-bp b-globin Polysilicon heater y20 Microchip CGE 35
Stationary chamber Glass 500-bp regions of l DNA and 154-, 264-, 346-, Commercial thermocycler 10–25 Microchip CGE 36
410-and 550-bp regions of E. coli from lysed cell

1418 | Lab Chip, 2007, 7, 1413–1423

Multiple stationary chamber Glass 199- and 500-bp regions of l DNA, 346- and Commercial thermocycler y12 Microchip CGE 37
410-bp regions of E. coli
Stationary chamber Glass 121-bp and 107-bp for D4Mit,142, and 149 bp and Commercial thermocycler 12.5 Microchip CGE 38
114 bp for D8Mit9 from genomic mouse DNA
Stationary chamber Glass 136-bp DNA of M13/pUC19 cloning vector Thin film heater 0.28 Microchip CGE 39
Stationary chamber Glass 136-bp DNA of M13/pUC19 cloning vector, 231-bp Resistive heater 0.28 Microchip CGE 40
fragment of human genomic DNA
Stationary chamber Glass 157-bp and 200-bp fragment of X and Integrated Ti/Pt heating 0.2 Microchip CGE 41
Y chromosome elements with RTD
Stationary chamber PDMS/glass 500-bp regions of l DNA Peltier element 30–50 Microchip CGE 42
Stationary chamber Silicon/glass CHD-W and CHD-Z genes Aluminium thin film resistors y3 or y10 Microchip CGE 43
Stationary chamber Poly (cyclic olefin) Genomic DNA from E. coli O157 or Graphite ink-based heating 0.029–0.084 Microchip CGE 44
S. typhimurium resistor, fan and RTD
Stationary chamber Glass Vero-E6 cell and nasopharyngeal swab Peltier thermoelectric module 3.5 y 4 Microchip CGE 45
Stationary chamber PDMS/glass BK virus Thermoelectric module y2 Microchip CGE 46
Stationary chamber Glass chip for PCR, 240-bp region of Streptococcus pneumoniae; Ti/Pt thin layer heater 10 Microchip CGE 47
PMMA chip for CGE 419-bp of Degue-2 virus
Multiple stationary Glass 197-bp from pUC19, 230-bp from M13 mp18 Ti/Pt resistance temperature 0.38 Multiple microchip 48
chamber bacteriophage and 237-bp from from E.coli K12 heater and sensors CGE
Stationary chamber PMMA 136-bp fragment of PSA cDNA; 132-bp l-DNA Block incubator 10 Microchip CGE 49
(isothermal)
Stationary chamber PC 221-bp fragment of E. coli gene; 195-bp Peltier element 38 DNA hybridization 50
E. faecalis gene assay
Stationary chamber Silicon/glass 5S-rRNA gene Resistance sensor and heater y3 DNA microarray 51
Stationary chamber PC 305-bp fragment of B. cereus genomic DNA Thermoelectric module y8 Immuno-assay 52
Stationary chamber Silicon/glass 5S-rRNA gene Platinum temperature 8 Electrochemical 53
sensors and heaters detection
Stationary chamber Silicon/glass E. coli and Bacillus subtilis cells. Platinum temperature 8 Electrochemical 54
sensors and heaters detection
a
CGE = capillary gel electrophoresis, CHD = chromodomain-helicase-DNA, NASBA = nucleic acid sequence-based amplification, PSA = prostate-specific antigen, RTD = resistance temperature
detector, PC = polycarbonate, PMMA = poly(methyl methacrylate); PDMS = poly(dimethylsiloxane)

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contrasts with the highly characterised detection methods such
as CGE, DNA micro-array and immunoassay which have
previously been adapted and applied to chip format (see
Table 3).22,35–54
2.3.1 Capillary gel electrophoresis (CGE). CGE is a long-
established and commonly used method for post-PCR
analysis, and it has been intensively and extensively studied
in chip format, but is usually employed in stand-alone devices.
Since the late 1990s, numerous attempts have been made to
directly apply CGE analysis after on-chip DNA amplification
on a single microfluidic device.56–58 The coupling of PCR and
CGE on microchip attempts to exploit both procedures by
achieving a sensitive and rapid analysis and reduced reagent
consumption. Moreover, manipulation of samples is contin-
uous in a single device, thus it avoids contamination with PCR
amplified matter, which is a critical issue. Normal CGE
microfluidic devices can be straightforwardly changed into a
PCR-CGE monolithic platform, where the sample reservoirs
can serve as PCR amplification chambers. After on-chip PCR,
the amplified products can be directly injected into the CGE
separation channel for detection.
A microfabricated PCR reactor and CGE microchip were
first coupled to form an integrated DNA analysis system by
Woolley et al.35 The device was composed of a planar CGE
chip for the electrophoretic separation and a polypropylene
reaction chamber in a polysilicon heating mantle, both of
which were connected through the cross injection channels that
served as an ‘‘electrophoretic valve’’. The rapid thermal cycling
capabilities of early microfabricated PCR devices (10 uC s21
heating, 2.5 uC s21 cooling) and high-speed DNA separations
of microfabricated CGE chips enabled a fast assay for
Salmonella genomic DNA, which required less than 45 min
from the initiation of PCR to the completion of the separation.
In 1998, Ramsey’s group performed cell lysis, multiplex PCR
amplification and CGE sizing on a single monolithic glass
microchip.36 A cross-shape CGE microchip was used as the
platform and reservoirs employed as PCR chambers. After
amplification, an intercalating dye and DNA sizing ladder
were added to PCR products, which were then eletrophor-
etically loaded into the main channel for CGE analysis. They
used a standard PCR protocol to amplify a 500 bp region of l
phage DNA and 154, 264, 346, 410 and 550 bp regions of
E. coli from lysed cells, and then CGE separation of the
products was executed in less than 3 min. Later on, the same
group developed multiple PCR-CGE analysis for up to 4
simultaneous PCR reactions and then direct CGE analysis.37
Following the initial study of PCR-CGE microfluidic
devices, further applications were carried out. Dunn et al.38
used a single PCR-CGE glass microchip for analysis of a
simple sequence length polymorphism in mouse DNA. A
miniaturised thermal system (thin film heater) was directly
inserted into a PCR chamber on PCR-CGE microchip, which
significantly improved the thermal cycling efficiency and
heating and cooling rate, indicating that a PCR-CGE
microchip can exploit this form of heater.39 Based on this
device, eight 280 nL PCR chambers were interfaced with CGE
microchannels and single-molecule level DNA amplification
and analysis were observed with multiplex PCR of a 136 bp
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amplification product derived from the M13/pUC19 cloning
vector and a 231 bp product amplified from a human genomic
DNA control sample.40 Furthermore, valves and hydrophobic
vents were integrated on this device for sample positioning and
immobilization into 200 nL PCR chambers. Successful sex
determination employing a multiplex PCR reaction from
human genomic DNA was demonstrated in less than 15 min
using this fully integrated PCR-CGE microchip.41 A combined
PCR-CGE hybrid PDMS–glass microchip together with a
temperature control system for PCR was described.42 PCR of
a 500 bp l DNA (1 ng per 100 mL) target was successfully
performed in 30–50 mL chambers, followed by subsequent
analysis of the product in the same chip. Compared to
microchips fabricated from silicon and glass, PDMS-based
microchips are well suited for a single-use device for wide
application of genetic analysis, due to its relative simple and
inexpensive fabrication processes. Rodriguez et al.43 developed
a PCR-CGE microdevice which combined a silicon-based
PCR chamber and CGE glass microchip to analyze genomic
DNA from bird species. A poly(cyclic olefin)-based plastic
microchip equipped with electrophoretically permeable micro-
valves, PCR chambers down to 29 nL, screen-printed heaters,
and CGE driving electrodes was demonstrated by Koh et al. in
2003.44 The device was used for bacterial detection and
identification based on amplification of several of their unique
identifying DNA sequences. The limit of detection was about
6 copies of target DNA. Glass-based PCR-CGE microchips
were applied for determination of severe acute respiratory
syndrome (SARS)-coronavirus specimens from clinic SARS
patients, and displayed the great potential of a PCR-CGE
microdevice for fast clinical diagnoses.45 PDMS–glass hybrid
PCR-CGE microchips were used for assessing the risk of BK
virus-associated nephropathy in renal transplant recipients,
implying likely wider applications of microchip-based systems
in clinical fields.46 An integrated PCR-CGE microchip
composed of different modules is reported by Huang et al.47
DNA/RNA samples were first replicated in a PCR or reverse
transcription PCR (RT-PCR) module micro-machined in a
glass microchip, and then transferred to a poly(methyl
methacrylate) (PMMA) microchip for CGE detection by a
PDMS-based pneumatic pump. The device was used for DNA-
based bacterial detection and RNA-based virus detection.
More recently, a four-lane fully integrated PCR-CGE array
microdevice was developed to amplify femtogram amounts of
DNA in 380 nL volumes followed by the direct CGE
separation of PCR amplicons in less than 30 min.48 More
recently, this device was applied to RT-PCR.55 Improved
parallelism of the microdevice was demonstrated to be well-
suited for high-throughput genetic differentiation assays.
Integration of isothermal amplification and CGE was reported
by Hataoka et al. using PMMA microchips.49 The relatively
low and single temperature required by isothermal amplifica-
tion avoids any complex temperature cycling and control
systems for efficient operation of the microdevice, and thus
makes it a very promising tool for on-chip nucleic acid
amplification and analysis.
2.3.2 Hybridization assay. Microchip-based DNA hybridiza-
tion array is a widely used detection technology in genome
Lab Chip, 2007, 7, 1413–1423 | 1419
analysis projects. A sample preparation process usually
precedes PCR, which is followed by the hybridization of the
amplified gene fragment to oligonucleotide probes (gene array)
immobilised to a solid support. The conventional method of
hybridisation is relatively slow and requires manual liquid
transfer, which also necessitates larger reagent and sample
volumes. The high concentration of both PCR fragments and
tethered DNA probes resolve these drawbacks by hastening
the rate of hybridisation. Compared to the combination of
PCR and CGE on microchips, which can only identify PCR
products by their length difference, more information related
to the PCR amplicon product sequence can be obtained by
PCR-DNA micro-array microchips, and with a much higher
throughput.59 Asymmetrical PCR amplification and subse-
quent hybridization of both E. coli and E. Faecalis genes were
demonstrated in disposable polycarbonate (PC)-based mono-
lithic microdevices.50 The PC surface of the hybridization
channel was first immobilized with oligonucleotide probes.
After on-chip PCR amplification in the serpentine chamber,
the amplified sample solution was continuously introduced
into the hybridization channel for analysis. Controlling of the
fluids was achieved by integrating Pluronics phase change
valves. Trau et al.51 developed a silicon-based micro-DNA
amplification and analysis device (m-DAAD) consisting of a
multiple PCR micro-reactor with an integrated DNA micro-
array. The authors also demonstrated its application for the
genotyping of Chinese medicinal plants on the basis of
differences in the non-coding region of the 5S-rRNA gene.
The genetic material was first amplified and the fluorescently-
labelled amplicons were consecutively detected by the inte-
grated oligonucleotide probes.
2.3.3 Immunoassay. Wang et al.52 reported a PC-based
microfluidic cassette for a lateral flow (LF) immunoassay
directly after on-chip PCR amplification. Firstly, the DNA
target (a specific 305 bp DNA fragment from B. cereus) was
amplified, and then the PCR amplicons were mixed and
incubated with ‘‘up-converting’’ phosphor particles (UPT).
Finally, the DNA-UPT complexes were propelled through the
LF strip, bound to the immobilised ligands in the test zone and
detected by infrared laser scanning. The fluids were controlled
by integrated temperature-sensitive hydrogel valves. The
valves were used to seal the chamber that served as the on-
chip PCR reactor. Integration of immunoassays and up-stream
PCR on microchips has great potential for the detection of free
nucleic acids in body fluids,60 especially for rapid detection of
pathogens at the point of care.
2.3.4 Electrochemical detection. To date, most reported
integrated PCR microchips employed laser excited fluores-
cence detection for post-PCR detection. However, optical
systems are difficult to miniaturise onto a microchip platform
and these are separate from the chip which requires careful
alignment between optics and the microfluidic devices, and size
requirements limit certain applications. To achieve the goal of
a fully integrated genetic analysis, alternative detection
methods merit investigation, and electrochemical detection
has been developed for post-PCR analysis directly after on-
chip PCR. Lee et al.53 fabricated a silicon–glass-based
1420 | Lab Chip, 2007, 7, 1413–1423
microdevice equipped with PCR-electrochemical detection
for simultaneous DNA amplification and detection. The
microdevice consists of a reaction chamber in a silicon
substrate and an electrochemical sensor fabricated onto a
glass substrate. Heaters and temperature sensors were fash-
ioned on the top of the PCR chamber for thermal cycling. Two
electrochemical detection techniques including metal complex
intercalators and gold nanoparticles were applied in the
microdevice. Asymmetric PCR was first performed to produce
single-stranded target amplicons complementary to the probe-
modified electrode. Finally, the reporter is bound to the
hybridized amplicons, the amount of which is electrochemi-
cally determined. Liu et al.22 developed a self-contained fully
integrated sample preparation, PCR and DNA micro-array
PC-based disposable microchip. The micro-array was also
subjected to electrochemical detection. After on-chip PCR, the
sample solution was mixed with hybridization buffer on chip
and moved over a micro-array chamber. The chamber was
incubated at 35 uC for hybridization and the electrochemical
signals corresponding to hybridization were collected by AC
voltammetry. The implementation of electrochemical detection
directly after on-chip PCR, together with sample preparation
in a single microchamber recently has been used for multi-
plexed pathogen identification.54 This integrated PCR plat-
form offers a cost-effective and sample-to-answer technology
for on-site monitoring.
3. Assessment
To succeed with full on-chip genetic analyses, general
protocols for biological sample treatment are well-established,
which normally involve nucleic acids extraction (e.g. tissue,
blood, cell, etc.), amplification (e.g. PCR and RT-PCR) and
product analysis (real-time quantitative PCR, gel electro-
phoresis, CGE, DNA array). m-TAS shows a great capability
for assembling different functional components for genetic
analysis of various samples or diverse purposes. For current
miniaturised PCR, several procedures, such as sample pre-
treatment, delivery, reaction efficiency, and detection sensitiv-
ity, need optimisation, which will greatly facilitate the use of
m-TAS for genetic research. A prototype of a fully integrated
PCR system consisting of SPE for extracting DNA from a
whole blood sample, PCR amplification chamber and micro-
chip gel electrophoresis for amplicons size information was
recently reported.61 It has a sample-in–answer-out capability
that shows a promising future for miniaturised integrated
genetic analyses as point-of-care devices.
3.1 Pre-PCR
Cell isolation is always needed since a typical biological sample
contains different types of cells. Cell isolation can be
accomplished according to the size of the cells, where filters
of appropriate dimension are required.62,63 For cells of similar
size, hydrodynamic64 or electrodynamic65–67 methods are
available for cell isolation and sorting, and both have the
potential to enhance the rapidity and selectivity.
Cell counting has been applied on microdevices, but not
coupled with PCR so far. The amount of cells used for
subsequent PCR is crucial to acquire an average level of a
This journal is ß The Royal Society of Chemistry 2007
certain molecule per cell. Although the amount of nucleic acids
obtained in pre-PCR can be determined using spectrometric or
fluorescence detection, quantitative information of molecules
per cell is not possible due to the difficulty in cell handling and
loss of the cells and their constituents during transportation.
When the genetic analysis is focused at the single cell level,
the inherent dimensions of m-TAS are suitable for single cell
analysis, and m-TAS probably could find its ‘‘killer applica-
tion’’ in this field.68,69 Furthermore, the results for single cell
PCR can be used as quantitative information since all the
results originate from one cell,70 thus eliminating the need for
cell sorting and counting. Nucleic acids extraction from
isolated single cells has been reported on a microfluidic device
recently.64 Therefore, it follows that a next development will
see the coupling of single cell lysis to on-chip PCR.
After samples were collected, nucleic acid extraction is a
necessary step to obtain target DNA or RNA of good quality.
The most popular method is SPE. For a solid phase, such as
membranes and beads, particles with a very high affinity for
DNA/RNA and a very low affinity for proteins, was
embedded inside the microdevices. As fluid containing nucleic
acids passes through, the DNA/RNA selectively binds to the
beads, and later is released and eluted by buffers with a
different polarity.71,72 Isotachophoresis (ITP) is a well-
established technology for on-chip sample pre-treatment.73,74
Analytes can be purified and concentrated simultaneously by
choosing adequate leading and terminating electrolytes. ITP
could be a potential tool for on-chip nucleic acids purification,
as it can be readily adapted to all chips where electric fields are
employed.
The current method for transferring nucleic acids to
subsequent PCR is mainly by manual transportation or
feeding with PCR reagents. Both methods prove difficult to
provide reliable quantitative information and are highly prone
to contamination. In order to overcome these potential
problems, the future microdevices require the ability to mix
reagents on-site and produce small identical reaction volumes.
As shown in Fig. 1, to date, pre-PCR has not been
extensively studied and no quantitative information has been
obtained before sending the analytes to PCR. The quantitative
information from pre-PCR is useful for absolute quantifica-
tion in many fields, where the amounts of specific molecules
per cell and range of molecules across a cell population are
both needed for a complete genetic diagnosis.
3.2 PCR
Due to significantly increased surface to volume ratio and
together with the surface properties, the literature inevitably
mentions that miniaturised PCR can not be a success without
surface modification of microdevices, when using current
available materials for microdevices (silicon, glass and poly-
mers). Such surface chemistry alteration processes, happening
either before PCR or during PCR, will obviously increase the
cost and time for miniaturised PCR. Furthermore, the
consistency or stability will also effect the quantitative
information of miniaturised PCR. Thus, new materials which
are PCR-friendly and meet the demands of massive micro-
fabrication need to be explored. On the other hand, an
This journal is ß The Royal Society of Chemistry 2007
alternative way is to develop a surface modification method
which has a reproducible good performance or good long-term
stability. Dynamic coating during PCR is a good choice for
surface passivation of a single-use device, as it is simply and
straightforwardly achieved by adding passivation agents such
as bovine serum albumin (BSA), poly(ethylene glycol) (PEG)
and polyvinylpyrrolidone (PVP) into PCR solutions to reduce
the undesired adsorption of enzyme and DNA. Chemical
modification before PCR is a better way if the device is
designed to be re-usable. Silanization is a well-established
method as it introduces aprotic organic groups onto the
microdevice surface to enhance the PCR compatibility.
It is worthy to note that lots of the microchip chamber
stationary PCR experiments are actually carried out in
conventional PCR instruments, using nearly the same thermal
conditions for conventional PCR. This is mainly due to the
difficulty and high-cost of fabricating miniaturised thermal
systems directly on microchips. Indium-tin-oxide (ITO) is a
generally used material for heating films on chip due to its
transparency and relatively low cost. Another alternative way
is directly employing electric field upon PCR solution, using its
Joule heating effect as a thermal source.32
3.3 Real-time PCR and post-PCR
As the volume needs for miniaturised PCR goes down to ynL
or ypL order, highly sensitive detection is required to achieve
accurate quantitative information. The common adapted
instrument for current real-time or post-PCR analysis is based
on fluorescence detection. Generally, an external source, such
as mercury, tungsten or xenon lamps and lasers, is needed to
provide a high intensity and stable excitation light. But
unfortunately, most of the currently available sources are
bulky bench-top instruments, which severely inhibit the
portability of miniaturised PCR devices. So far, there are
few reports concerning miniaturised detection systems. Light
emitting diodes (LEDs) are one good option, as they have
several obvious advantages, such as low cost, high efficiency,
small size and considerable durability. LEDs have been used as
miniaturised excitation sources for real-time PCR detection.24
An alternative for portable detection instruments is using
electrochemical detection for miniaturised PCR.75 The small
size of electrodes and no need of an external optical source
make electrochemical detection another attractive option. But
the problems of electrode contamination and relative low
sensitivity need further improvements.
Handling a tiny volume of sample is a big challenge for real-
time or post-PCR analysis, as manipulation is difficult using
current technologies. Multi-layer microfluidic devices have
shown their capabilities of manipulating liquid of ypL
volume, which makes them promising tools for real-time
PCR detection or post-PCR sample delivery to different units
for diverse analysis purposes. Several commercial PCR-based
micro-devices have been developed, but these have encoun-
tered temperature control, surface passivation and integration
difficulties, as seen in academic groups. Costs of devices are
high, and can be deferred by achieving quantitative m-TAS or
TM
via multiparallelisation. Recently, the BioMark 48.48 System
(Dynamic Arrays and Digital Arrays) for real-time PCR
Lab Chip, 2007, 7, 1413–1423 | 1421
detection was released by Fluidigm based on integrated
channels, chambers and valves.76 These devices are capable
of on-chip division of sample and/or reagents into 100s to
1000s of identical aliquots (Digital Arrays), or full mixing of
samples and reagents in a manner permitting highly uniform
reagent-to-sample ratios. Furthermore, the real-time PCR can
be performed in a matrix architecture (N samples 6 M
reagents) using these devices, and has been demonstrated for
multigene analyses of massive target numbers in a study
screening individual environmental bacteria.77 An alternative
way to massively generate small volumes for on-chip PCR is
via the formation of droplets containing all the reaction
components inside microfluidic channels.78–80 Improved
methods to produce samples with identically defined tiny
volumes are also likely to be embraced for reliable quantitative
nucleic acid based information retrieval and avoid the innate
sample heterogeneity associated with biological matter.
Therefore, despite the 9 log concentration range that PCR
can operate, rarely are more than a few thousand copies of a
particular gene sequence present within a cell, and the lower
detection capacity of PCR may be more important for nucleic
acid quantification.
Although quantitative studies have been reported regarding
real-time PCR and post-PCR (Fig. 1), the majority of them
started with known amounts of nucleic acids. The applications
with unknown samples are limited to the qualitative level due
to the uncertainty relating to the amount of nucleic acids
entering PCR. Thus, in order to comprehensively realize
quantitative studies for genet-based diagnoses, the functional
components that collectively represent an integrated PCR
microfluidic device will have to be developed synergistically
and in multiple directions, such as: detection units, sample
delivery, increasing sensitivity and handling of populations of
cells.
4. Conclusions
The amplification of nucleic acids using integrated micro-
fluidic-based devices has benefited from many innovative
developments, but, as yet, its incorporation into fully working
(quantitative) nucleic acid assays remains essentially an
unresolved challenge given the caveats related to sample
handling. Because gene amplification delivers sufficient gene-
specific fragments to enable a very high level of analyte
detection sensitivity, and due to the ubiquitous rules applying
to nucleic acid hybridisation and action of modification
enzymes, the category of the nucleic acid analyte provides an
ideal start-point for biomarker detection in an integrated
m-TAS format. The motivation for miniaturisation of bioas-
says draws much from a desire to link life processes to a
carefully measured response, to predict and then permit
interaction to control or indeed circumvent disease outcome.
The challenge is therefore extreme, given that this correlation
has not been achieved in more conventional molecular biology
laboratory settings. The requirement of m-TAS in the context
of nucleic acid analysis is not only a re-packaging, scaling-
down exercise, but more an assertive step towards the bridging
of sampled cells to a quantitative calculation of disease or
condition-related nucleic acid sequences (Fig. 1).
1422 | Lab Chip, 2007, 7, 1413–1423
There have been a number of notable successes in m-TAS in
this respect, with most relating to post-nucleic acid treatments,
enzymology and related analyte measurements. The biggest
drawback relates to the linking of a raw sample to the output
of an on-chip amplification process; and this reflects exactly
the current situation within the typical molecular biology
laboratory. In other words, to fully reach quantitative clinical
diagnosis, the sample has to be better defined to permit highly
characterised regions or multi-parallelised populations of
single cells to enter PCR amplification, and facilitate a move
towards perceiving cell activity in terms of molecules of nucleic
acids per cell type. This strategy therefore avoids analysis
anomalies associated with gross measurements from hetero-
geneous cellular samples and errors of sampling in the case of
clinical biopsies. Perhaps, whilst miniaturisation offers the
potential to improve sample analysis through enhanced cell
recognition or selection, this may not be achieved until the
dimensions of the channel are fully exploited to move cells as
discrete units into homogeneous assay formats devoid of any
assay losses or measurement aberrations. In this context,
m-TAS developments are synonymous with facilitating abso-
lute nucleic acid quantification. For now, the more qualitative
high throughput nucleic acid amplification as seen for
pathogen detection is a best measure of current achievement
of m-TAS.
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