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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-


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

Lin Chen received his M. Eng. microelectrodes as detectors


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

This journal is ß The Royal Society of Chemistry 2007 Lab Chip, 2007, 7, 1413–1423 | 1413
Fig. 1 Integrated PCR on microfluidic devices.

Genetic analysis employing m-TAS has elicited enormous readily lost given the limited availability of homogeneous
interest, and because the amount of original cellular matter (extraction-free) assay formats, the amplification of target
available for genetic analysis is often extremely limited and nucleic acids following careful sample manipulation including
separation processes are typically necessary steps. Thus, not
too surprisingly miniaturised in vitro gene amplification,
Philip Day graduated with a especially the polymerase chain reaction (PCR), has become
Ph.D. degree from the Wolfson synonymous with the development of miniaturisation and
Research Laboratories, microfluidics per se.5–9 Moreover, miniaturised PCR gives
University of Birmingham. other advantages such as high thermal cycling speed and low
From 1995–1997 at Oxford reagent consumption, which benefit from the intrinsic high
University he developed very surface-to-volume ratio. But so far, most of the reported
high throughput PCR for the miniaturised devices for nucleic acid amplification are stand-
Wellcome Trust in the Human
alone structures replacing only the role of the conventional
Genome Mapping Project. This
was followed by developments PCR thermocycler. The integration of nucleic acid amplifica-
into high throughput sequencing tion with other functional units, such as proximal sample
and gene micro-arrays with preparation and distil sequence analysis, on a single device is
Prof. Sir Edwin Southern, broadly accepted as the way forward and starts to emulate the
which he followed with the vision of m-TAS as discussed previously.9 Presently, integra-
Philip J. R. Day establishment of a Functional tion of miniaturised PCR is under rapid development, and
Genomics Unit, Kinderspital, PCR has been coupled with pre-PCR modules, such as sample
University of Zurich. His studies correlate innovative quantitative
purification and pre-concentration, and post-PCR modules,
measurements of nucleic acids to meaningful biomedical inter-
such as capillary gel electrophoresis (CGE) and DNA
pretation. He was appointed Reader in Genomics, University of
Manchester. In 2006 he was made Principal Investigator at the microarray, on single microdevices. The different approaches
Manchester Interdisciplinary Biocentre, and was later appointed to integrated gene analysis that encompasses in vitro gene
Professor of Applied Molecular Biology and Biochemistry at the amplification are relatively finite and are shown in Fig. 1 and
University of Dortmund, and with ISAS, Dortmund. 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 nucleic acids.10 Of equal importance and of high relevance to
other functionalities in a monolithic format for clinical m-TAS is the availability of biologically highly inert surfaces
diagnosis, encompassing solid, fluid and aerosol samples. that allow movement of bio-matter through the confines of
For a theoretical background into PCR and technical aspects microfluidic devices without losses incurred through non-
of microchip-based PCR, readers are referred to other specific association of the analyte or reagents with the device
published reviews for further details.5–9 itself. In this context, mirroring of the vascular transportation
system used for dispersing blood cells and associated serum
2. Integrated PCR constituents in organisms would provide many features of a
suitable conduit for moving PCR-related reaction constituents
2.1 Microdevice pre-conditioning and sample processing: pre- without compromising information retrieval. To date,
PCR reported passivation methods for the inner surfaces of PCR
The aim of integrated PCR is to analyse, within the confines microdevices include dynamic and static passivation. For static
of a usually portable miniaturised platform, real biological passivation, the surface of the PCR microdevice is always pre-
samples obtained from suspected aberrant tissues, fluidics coated with a PCR-friendly substance,11–14 which occurs
or locations. Any treatment or change influencing the before PCR, while dynamic passivation is realized by adding
sample prior to the analyte measurement procedure is the passivation reagents to the PCR cocktail solution, and the
critical since the quantitative assessment of the analyte coating ensues concurrently with the PCR process.15 Thus, we
biomarker can be irretrievably altered to produce a result that classify static passivation into the category of pre-PCR
may detract and lead to misinterpretation of the true biological treatment process. So far, there are two main types of static
situation. passivation, chemical silanisation11,12 and silicon oxide surface
coating.13,14 The first procedure employs deposition of a thin
2.1.1 Pre-conditioning. The characteristic seen for biological layer of silicon oxide onto the microchannel surface to enhance
systems is one of high specificity of interaction which has been the PCR compatibility, and the second method employs filling
particularly exploited in the case of proteins (antibodies) and the reaction chamber or channel with the silanising solution,

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and incubating the chip for a period of time, followed by storage chamber to ensure target cell capture from blood.
washing and drying. The sample mixture is then pumped through into the PCR
chamber, where target cell capture and pre-concentration
2.1.2 Sample processing. Amongst all the pre-treatment occur as the bead-bacteria conjugates are trapped by the
methods for PCR analysis, cell identification, cell capture, magnet. The washing buffers were consecutively pumped
nucleic acids extraction, sample/analyte purification and pre- through the PCR chamber to purify the captured cell. After
concentration are all essential, since PCR requires relatively the PCR reagents were transferred into the PCR chamber, on-
pure nucleic acid samples that are free from reaction inhibiting chip thermal cell lysis and PCR were performed. Cady et al.23,24
contaminants. Increased stringency of sample treatments to reported a poly(dimethylsiloxane) (PDMS)–silicon microde-
safeguard sample purity and performance in PCR are vice consisting of a microfabricated channel in which silica-
heightened requirements for quantitative assessment of PCR coated pillars were etched. DNA was selectively bound to these
amplified products. pillars in the presence of the chaotropic salt guanidinium
The difficulty in manipulating micro-scale liquid-carried isothiocyanate, followed by washing with ethanol and elution
samples renders most DNA pre-treatment microdevices devoid with water. Simultaneous pumping of a concentrated PCR
of downstream microfluidic integration. Microchip-based master mix through a second inlet port allowed for parallel
DNA purification was described first by Christel et al.16 In flow of eluted DNA and master mix into the PCR reaction
their study, pillars were created in a micro-channel to increase chamber for real-time analysis. A method combining laser-
the contact surface area. The extraction and concentration of irradiation and magnetic beads was developed for rapid cell
DNA from samples were accomplished utilizing silicon fluidic lysis and DNA isolation on microchips.25 By using an 808 nm
microchips with high surface area-to-volume ratios. Wolfe laser and carboxyl-terminated magnetic beads, the authors
et al.17 developed sol-gel immobilized silica particles in a demonstrated that pathogens can be lysed by a single laser
micro-channel for purification of DNA as a low-cost pulse of 40 s inside a 4 ml chamber, and subsequently real-time
alternative. Later, the performance of the solid phase PCR for pathogen detection was performed using the same
extraction (SPE) device on the microchip was thoroughly microchip.
examined using human genomic DNA from whole blood and
bacterial DNA from colony samples and spores.18 A micro- 2.2 Real-time quantitative PCR
fabricated electrophoretic bioprocessor integrated with DNA
sequencing, sample desalting, template removal, preconcentra- Recently, integration of real-time PCR on microfluidic devices
tion and CGE analysis was demonstrated by Mathies’ group.19 has gained in popularity. In this process, the amplification of
A novel chamber geometry, capture process, and affinity specific gene sequences is coupled to quantification of the
capture chemistry were developed for the purification of DNA original target DNA, which is typically monitored through
fragments and integrated with high-speed microdevice sequen- intercalation of a dye or fluorescence of a probe.26,27 Real-time
cing. Sample immobilization, pre-concentration, and desalting detection is achieved by recording the increase in fluorescence
were completed in only 120 s, approximately a 10-fold resulting from the stochastically associated measurement of
reduction in time and 100-fold reduction in reagent volume. fluorescence with increased dsDNA production after each
Microfluidic devices coupling pre-PCR components to round of thermocycling. Once the yield of fluorescence-
downstream PCR has also been studied. Literature reporting associated PCR products exceeds the background, the forma-
pre-PCR processes integrated with PCR on microchips are tion of reaction products can be monitored as the geometric
summarized in Table 1.20–25 Wilding et al.20,21 isolated white reaction proceeds, which contrasts with measuring the gross
blood cells from whole blood by constructing a series of 3.5 mm amplified product at the end of a fixed number of cycles. The
filters in silicon-glass microchips. Genomic DNA from the number of cycles that are needed to reach the detection
whole blood cells isolated on the filters was directly amplified threshold (often termed crossing point (Cp), or cycle threshold
using PCR. The on-chip sample preparation reported by Liu value (Ct)) is proportional to the negative logarithm of the
et al.22 started with mixing and incubating blood and a initial concentration of target DNA. Thus, the Ct values from
solution containing immunomagnetic beads in a sample different initial concentrations of target DNA can be used to

Table 1 Pre-PCR integrated with PCR on a microchip


PCR type Substrate material Source Template DNA Pre-treatment technique Volume/mL Ref.

Stationary Glass Whole blood 202-bp DNA fragment Microchip filter for white 50 20
chamber of dystrophin gene blood cells isolation
Stationary Silicon/glass Whole blood 226-bp regions of human White blood cells isolation on the 12 21
chamber coagulation Factor V gene filter section of the microchip
Stationary PC Whole blood 221-bp fragment from Immunomagnetic bead-based 20 22
chamber E.coli K12 specific gene cell capture,
Stationary PDMS/silicon Listeria 544-bp DNA segment from DNA bound to silica-coated pillars in 50 23,24
chamber monocytogens Listeria monocytogens the presence of the chaotropic salt
cells guanidinium isothiocyanate
Stationary Silicon/glass E.coli BL21 16S-rRNA region of Cell lysis by laser-irradiated magnetic 4 25
chamber bacterial genome 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

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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)

This journal is ß The Royal Society of Chemistry 2007


contrasts with the highly characterised detection methods such amplification product derived from the M13/pUC19 cloning
as CGE, DNA micro-array and immunoassay which have vector and a 231 bp product amplified from a human genomic
previously been adapted and applied to chip format (see DNA control sample.40 Furthermore, valves and hydrophobic
Table 3).22,35–54 vents were integrated on this device for sample positioning and
immobilization into 200 nL PCR chambers. Successful sex
2.3.1 Capillary gel electrophoresis (CGE). CGE is a long- determination employing a multiplex PCR reaction from
established and commonly used method for post-PCR human genomic DNA was demonstrated in less than 15 min
analysis, and it has been intensively and extensively studied using this fully integrated PCR-CGE microchip.41 A combined
in chip format, but is usually employed in stand-alone devices. PCR-CGE hybrid PDMS–glass microchip together with a
Since the late 1990s, numerous attempts have been made to temperature control system for PCR was described.42 PCR of
directly apply CGE analysis after on-chip DNA amplification a 500 bp l DNA (1 ng per 100 mL) target was successfully
on a single microfluidic device.56–58 The coupling of PCR and performed in 30–50 mL chambers, followed by subsequent
CGE on microchip attempts to exploit both procedures by analysis of the product in the same chip. Compared to
achieving a sensitive and rapid analysis and reduced reagent microchips fabricated from silicon and glass, PDMS-based
consumption. Moreover, manipulation of samples is contin- microchips are well suited for a single-use device for wide
uous in a single device, thus it avoids contamination with PCR application of genetic analysis, due to its relative simple and
amplified matter, which is a critical issue. Normal CGE inexpensive fabrication processes. Rodriguez et al.43 developed
microfluidic devices can be straightforwardly changed into a a PCR-CGE microdevice which combined a silicon-based
PCR-CGE monolithic platform, where the sample reservoirs PCR chamber and CGE glass microchip to analyze genomic
can serve as PCR amplification chambers. After on-chip PCR, DNA from bird species. A poly(cyclic olefin)-based plastic
the amplified products can be directly injected into the CGE microchip equipped with electrophoretically permeable micro-
separation channel for detection. valves, PCR chambers down to 29 nL, screen-printed heaters,
A microfabricated PCR reactor and CGE microchip were and CGE driving electrodes was demonstrated by Koh et al. in
first coupled to form an integrated DNA analysis system by 2003.44 The device was used for bacterial detection and
Woolley et al.35 The device was composed of a planar CGE identification based on amplification of several of their unique
chip for the electrophoretic separation and a polypropylene identifying DNA sequences. The limit of detection was about
reaction chamber in a polysilicon heating mantle, both of 6 copies of target DNA. Glass-based PCR-CGE microchips
which were connected through the cross injection channels that were applied for determination of severe acute respiratory
served as an ‘‘electrophoretic valve’’. The rapid thermal cycling syndrome (SARS)-coronavirus specimens from clinic SARS
capabilities of early microfabricated PCR devices (10 uC s21 patients, and displayed the great potential of a PCR-CGE
heating, 2.5 uC s21 cooling) and high-speed DNA separations microdevice for fast clinical diagnoses.45 PDMS–glass hybrid
of microfabricated CGE chips enabled a fast assay for PCR-CGE microchips were used for assessing the risk of BK
Salmonella genomic DNA, which required less than 45 min virus-associated nephropathy in renal transplant recipients,
from the initiation of PCR to the completion of the separation. implying likely wider applications of microchip-based systems
In 1998, Ramsey’s group performed cell lysis, multiplex PCR in clinical fields.46 An integrated PCR-CGE microchip
amplification and CGE sizing on a single monolithic glass composed of different modules is reported by Huang et al.47
microchip.36 A cross-shape CGE microchip was used as the DNA/RNA samples were first replicated in a PCR or reverse
platform and reservoirs employed as PCR chambers. After transcription PCR (RT-PCR) module micro-machined in a
amplification, an intercalating dye and DNA sizing ladder glass microchip, and then transferred to a poly(methyl
were added to PCR products, which were then eletrophor- methacrylate) (PMMA) microchip for CGE detection by a
etically loaded into the main channel for CGE analysis. They PDMS-based pneumatic pump. The device was used for DNA-
used a standard PCR protocol to amplify a 500 bp region of l based bacterial detection and RNA-based virus detection.
phage DNA and 154, 264, 346, 410 and 550 bp regions of More recently, a four-lane fully integrated PCR-CGE array
E. coli from lysed cells, and then CGE separation of the microdevice was developed to amplify femtogram amounts of
products was executed in less than 3 min. Later on, the same DNA in 380 nL volumes followed by the direct CGE
group developed multiple PCR-CGE analysis for up to 4 separation of PCR amplicons in less than 30 min.48 More
simultaneous PCR reactions and then direct CGE analysis.37 recently, this device was applied to RT-PCR.55 Improved
Following the initial study of PCR-CGE microfluidic parallelism of the microdevice was demonstrated to be well-
devices, further applications were carried out. Dunn et al.38 suited for high-throughput genetic differentiation assays.
used a single PCR-CGE glass microchip for analysis of a Integration of isothermal amplification and CGE was reported
simple sequence length polymorphism in mouse DNA. A by Hataoka et al. using PMMA microchips.49 The relatively
miniaturised thermal system (thin film heater) was directly low and single temperature required by isothermal amplifica-
inserted into a PCR chamber on PCR-CGE microchip, which tion avoids any complex temperature cycling and control
significantly improved the thermal cycling efficiency and systems for efficient operation of the microdevice, and thus
heating and cooling rate, indicating that a PCR-CGE makes it a very promising tool for on-chip nucleic acid
microchip can exploit this form of heater.39 Based on this amplification and analysis.
device, eight 280 nL PCR chambers were interfaced with CGE
microchannels and single-molecule level DNA amplification 2.3.2 Hybridization assay. Microchip-based DNA hybridiza-
and analysis were observed with multiplex PCR of a 136 bp tion array is a widely used detection technology in genome

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analysis projects. A sample preparation process usually microdevice equipped with PCR-electrochemical detection
precedes PCR, which is followed by the hybridization of the for simultaneous DNA amplification and detection. The
amplified gene fragment to oligonucleotide probes (gene array) microdevice consists of a reaction chamber in a silicon
immobilised to a solid support. The conventional method of substrate and an electrochemical sensor fabricated onto a
hybridisation is relatively slow and requires manual liquid glass substrate. Heaters and temperature sensors were fash-
transfer, which also necessitates larger reagent and sample ioned on the top of the PCR chamber for thermal cycling. Two
volumes. The high concentration of both PCR fragments and electrochemical detection techniques including metal complex
tethered DNA probes resolve these drawbacks by hastening intercalators and gold nanoparticles were applied in the
the rate of hybridisation. Compared to the combination of microdevice. Asymmetric PCR was first performed to produce
PCR and CGE on microchips, which can only identify PCR single-stranded target amplicons complementary to the probe-
products by their length difference, more information related modified electrode. Finally, the reporter is bound to the
to the PCR amplicon product sequence can be obtained by hybridized amplicons, the amount of which is electrochemi-
PCR-DNA micro-array microchips, and with a much higher cally determined. Liu et al.22 developed a self-contained fully
throughput.59 Asymmetrical PCR amplification and subse- integrated sample preparation, PCR and DNA micro-array
quent hybridization of both E. coli and E. Faecalis genes were PC-based disposable microchip. The micro-array was also
demonstrated in disposable polycarbonate (PC)-based mono- subjected to electrochemical detection. After on-chip PCR, the
lithic microdevices.50 The PC surface of the hybridization sample solution was mixed with hybridization buffer on chip
channel was first immobilized with oligonucleotide probes. and moved over a micro-array chamber. The chamber was
After on-chip PCR amplification in the serpentine chamber, incubated at 35 uC for hybridization and the electrochemical
the amplified sample solution was continuously introduced signals corresponding to hybridization were collected by AC
into the hybridization channel for analysis. Controlling of the voltammetry. The implementation of electrochemical detection
fluids was achieved by integrating Pluronics phase change directly after on-chip PCR, together with sample preparation
valves. Trau et al.51 developed a silicon-based micro-DNA in a single microchamber recently has been used for multi-
amplification and analysis device (m-DAAD) consisting of a plexed pathogen identification.54 This integrated PCR plat-
multiple PCR micro-reactor with an integrated DNA micro- form offers a cost-effective and sample-to-answer technology
array. The authors also demonstrated its application for the for on-site monitoring.
genotyping of Chinese medicinal plants on the basis of
differences in the non-coding region of the 5S-rRNA gene. 3. Assessment
The genetic material was first amplified and the fluorescently-
labelled amplicons were consecutively detected by the inte- To succeed with full on-chip genetic analyses, general
grated oligonucleotide probes. protocols for biological sample treatment are well-established,
which normally involve nucleic acids extraction (e.g. tissue,
2.3.3 Immunoassay. Wang et al.52 reported a PC-based blood, cell, etc.), amplification (e.g. PCR and RT-PCR) and
microfluidic cassette for a lateral flow (LF) immunoassay product analysis (real-time quantitative PCR, gel electro-
directly after on-chip PCR amplification. Firstly, the DNA phoresis, CGE, DNA array). m-TAS shows a great capability
target (a specific 305 bp DNA fragment from B. cereus) was for assembling different functional components for genetic
amplified, and then the PCR amplicons were mixed and analysis of various samples or diverse purposes. For current
incubated with ‘‘up-converting’’ phosphor particles (UPT). miniaturised PCR, several procedures, such as sample pre-
Finally, the DNA-UPT complexes were propelled through the treatment, delivery, reaction efficiency, and detection sensitiv-
ity, need optimisation, which will greatly facilitate the use of
LF strip, bound to the immobilised ligands in the test zone and
m-TAS for genetic research. A prototype of a fully integrated
detected by infrared laser scanning. The fluids were controlled
PCR system consisting of SPE for extracting DNA from a
by integrated temperature-sensitive hydrogel valves. The
whole blood sample, PCR amplification chamber and micro-
valves were used to seal the chamber that served as the on-
chip gel electrophoresis for amplicons size information was
chip PCR reactor. Integration of immunoassays and up-stream
recently reported.61 It has a sample-in–answer-out capability
PCR on microchips has great potential for the detection of free
that shows a promising future for miniaturised integrated
nucleic acids in body fluids,60 especially for rapid detection of
genetic analyses as point-of-care devices.
pathogens at the point of care.

3.1 Pre-PCR
2.3.4 Electrochemical detection. To date, most reported
integrated PCR microchips employed laser excited fluores- Cell isolation is always needed since a typical biological sample
cence detection for post-PCR detection. However, optical contains different types of cells. Cell isolation can be
systems are difficult to miniaturise onto a microchip platform accomplished according to the size of the cells, where filters
and these are separate from the chip which requires careful of appropriate dimension are required.62,63 For cells of similar
alignment between optics and the microfluidic devices, and size size, hydrodynamic64 or electrodynamic65–67 methods are
requirements limit certain applications. To achieve the goal of available for cell isolation and sorting, and both have the
a fully integrated genetic analysis, alternative detection potential to enhance the rapidity and selectivity.
methods merit investigation, and electrochemical detection Cell counting has been applied on microdevices, but not
has been developed for post-PCR analysis directly after on- coupled with PCR so far. The amount of cells used for
chip PCR. Lee et al.53 fabricated a silicon–glass-based subsequent PCR is crucial to acquire an average level of a

1420 | Lab Chip, 2007, 7, 1413–1423 This journal is ß The Royal Society of Chemistry 2007
certain molecule per cell. Although the amount of nucleic acids alternative way is to develop a surface modification method
obtained in pre-PCR can be determined using spectrometric or which has a reproducible good performance or good long-term
fluorescence detection, quantitative information of molecules stability. Dynamic coating during PCR is a good choice for
per cell is not possible due to the difficulty in cell handling and surface passivation of a single-use device, as it is simply and
loss of the cells and their constituents during transportation. straightforwardly achieved by adding passivation agents such
When the genetic analysis is focused at the single cell level, as bovine serum albumin (BSA), poly(ethylene glycol) (PEG)
the inherent dimensions of m-TAS are suitable for single cell and polyvinylpyrrolidone (PVP) into PCR solutions to reduce
analysis, and m-TAS probably could find its ‘‘killer applica- the undesired adsorption of enzyme and DNA. Chemical
tion’’ in this field.68,69 Furthermore, the results for single cell modification before PCR is a better way if the device is
PCR can be used as quantitative information since all the designed to be re-usable. Silanization is a well-established
results originate from one cell,70 thus eliminating the need for method as it introduces aprotic organic groups onto the
cell sorting and counting. Nucleic acids extraction from microdevice surface to enhance the PCR compatibility.
isolated single cells has been reported on a microfluidic device It is worthy to note that lots of the microchip chamber
recently.64 Therefore, it follows that a next development will stationary PCR experiments are actually carried out in
see the coupling of single cell lysis to on-chip PCR. conventional PCR instruments, using nearly the same thermal
After samples were collected, nucleic acid extraction is a conditions for conventional PCR. This is mainly due to the
necessary step to obtain target DNA or RNA of good quality. difficulty and high-cost of fabricating miniaturised thermal
The most popular method is SPE. For a solid phase, such as systems directly on microchips. Indium-tin-oxide (ITO) is a
membranes and beads, particles with a very high affinity for generally used material for heating films on chip due to its
DNA/RNA and a very low affinity for proteins, was transparency and relatively low cost. Another alternative way
embedded inside the microdevices. As fluid containing nucleic is directly employing electric field upon PCR solution, using its
acids passes through, the DNA/RNA selectively binds to the Joule heating effect as a thermal source.32
beads, and later is released and eluted by buffers with a
different polarity.71,72 Isotachophoresis (ITP) is a well- 3.3 Real-time PCR and post-PCR
established technology for on-chip sample pre-treatment.73,74
As the volume needs for miniaturised PCR goes down to ynL
Analytes can be purified and concentrated simultaneously by
or ypL order, highly sensitive detection is required to achieve
choosing adequate leading and terminating electrolytes. ITP
accurate quantitative information. The common adapted
could be a potential tool for on-chip nucleic acids purification,
instrument for current real-time or post-PCR analysis is based
as it can be readily adapted to all chips where electric fields are
on fluorescence detection. Generally, an external source, such
employed.
as mercury, tungsten or xenon lamps and lasers, is needed to
The current method for transferring nucleic acids to
provide a high intensity and stable excitation light. But
subsequent PCR is mainly by manual transportation or
unfortunately, most of the currently available sources are
feeding with PCR reagents. Both methods prove difficult to
bulky bench-top instruments, which severely inhibit the
provide reliable quantitative information and are highly prone
portability of miniaturised PCR devices. So far, there are
to contamination. In order to overcome these potential
few reports concerning miniaturised detection systems. Light
problems, the future microdevices require the ability to mix
emitting diodes (LEDs) are one good option, as they have
reagents on-site and produce small identical reaction volumes.
several obvious advantages, such as low cost, high efficiency,
As shown in Fig. 1, to date, pre-PCR has not been
small size and considerable durability. LEDs have been used as
extensively studied and no quantitative information has been
miniaturised excitation sources for real-time PCR detection.24
obtained before sending the analytes to PCR. The quantitative
An alternative for portable detection instruments is using
information from pre-PCR is useful for absolute quantifica-
electrochemical detection for miniaturised PCR.75 The small
tion in many fields, where the amounts of specific molecules
size of electrodes and no need of an external optical source
per cell and range of molecules across a cell population are
make electrochemical detection another attractive option. But
both needed for a complete genetic diagnosis.
the problems of electrode contamination and relative low
sensitivity need further improvements.
3.2 PCR
Handling a tiny volume of sample is a big challenge for real-
Due to significantly increased surface to volume ratio and time or post-PCR analysis, as manipulation is difficult using
together with the surface properties, the literature inevitably current technologies. Multi-layer microfluidic devices have
mentions that miniaturised PCR can not be a success without shown their capabilities of manipulating liquid of ypL
surface modification of microdevices, when using current volume, which makes them promising tools for real-time
available materials for microdevices (silicon, glass and poly- PCR detection or post-PCR sample delivery to different units
mers). Such surface chemistry alteration processes, happening for diverse analysis purposes. Several commercial PCR-based
either before PCR or during PCR, will obviously increase the micro-devices have been developed, but these have encoun-
cost and time for miniaturised PCR. Furthermore, the tered temperature control, surface passivation and integration
consistency or stability will also effect the quantitative difficulties, as seen in academic groups. Costs of devices are
information of miniaturised PCR. Thus, new materials which high, and can be deferred by achieving quantitative m-TAS or
TM
are PCR-friendly and meet the demands of massive micro- via multiparallelisation. Recently, the BioMark 48.48 System
fabrication need to be explored. On the other hand, an (Dynamic Arrays and Digital Arrays) for real-time PCR

This journal is ß The Royal Society of Chemistry 2007 Lab Chip, 2007, 7, 1413–1423 | 1421
detection was released by Fluidigm based on integrated There have been a number of notable successes in m-TAS in
channels, chambers and valves.76 These devices are capable this respect, with most relating to post-nucleic acid treatments,
of on-chip division of sample and/or reagents into 100s to enzymology and related analyte measurements. The biggest
1000s of identical aliquots (Digital Arrays), or full mixing of drawback relates to the linking of a raw sample to the output
samples and reagents in a manner permitting highly uniform of an on-chip amplification process; and this reflects exactly
reagent-to-sample ratios. Furthermore, the real-time PCR can the current situation within the typical molecular biology
be performed in a matrix architecture (N samples 6 M laboratory. In other words, to fully reach quantitative clinical
reagents) using these devices, and has been demonstrated for diagnosis, the sample has to be better defined to permit highly
multigene analyses of massive target numbers in a study characterised regions or multi-parallelised populations of
screening individual environmental bacteria.77 An alternative single cells to enter PCR amplification, and facilitate a move
way to massively generate small volumes for on-chip PCR is towards perceiving cell activity in terms of molecules of nucleic
via the formation of droplets containing all the reaction acids per cell type. This strategy therefore avoids analysis
components inside microfluidic channels.78–80 Improved anomalies associated with gross measurements from hetero-
methods to produce samples with identically defined tiny geneous cellular samples and errors of sampling in the case of
volumes are also likely to be embraced for reliable quantitative clinical biopsies. Perhaps, whilst miniaturisation offers the
nucleic acid based information retrieval and avoid the innate potential to improve sample analysis through enhanced cell
sample heterogeneity associated with biological matter. recognition or selection, this may not be achieved until the
Therefore, despite the 9 log concentration range that PCR dimensions of the channel are fully exploited to move cells as
can operate, rarely are more than a few thousand copies of a discrete units into homogeneous assay formats devoid of any
particular gene sequence present within a cell, and the lower assay losses or measurement aberrations. In this context,
detection capacity of PCR may be more important for nucleic m-TAS developments are synonymous with facilitating abso-
acid quantification. lute nucleic acid quantification. For now, the more qualitative
Although quantitative studies have been reported regarding high throughput nucleic acid amplification as seen for
real-time PCR and post-PCR (Fig. 1), the majority of them pathogen detection is a best measure of current achievement
started with known amounts of nucleic acids. The applications of m-TAS.
with unknown samples are limited to the qualitative level due
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