Total Nucleic Acid Analysis Integrated on Microfluidic Devices

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

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

advantages. The often quoted advantages related to imple-

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.

aInstitute for Analytical Sciences, Bunsen-Kirchhoff Str. 11, D-44139Dortmund, GermanybThe Manchester Interdisciplinary Biocentre, University of Manchester,131, Princess Street, Manchester, UK M1 7ND.E-mail: [email protected]; Fax: +44-161-275-1617;Tel: +44-161-275-1621

Lin Chen received his M. Eng.in Applied Chemistry (2004)from Shanghai Jiao TongUniversity, P. R. China. He isnow reading for a Ph.D. underthe supervision of ProfessorAndreas Manz and ProfessorPhilip Day at the Institute forAnalytical Sciences (ISAS) inDortmund, Germany. Hisresearch focuses on the devel-opment of an integrated micro-fluidic platform for nucleic acidpreparation, amplification andreal-time analysis to contributetowards quantitative bioassaysemploying k-TAS.

Andreas Manz obtained his Ph.D. from the Swiss FederalInstitute of Technology (ETH) Zurich, Switzerland, withProfessor W. Simon. His thesis dealt with the use of

microelectrodes as detectorsfor picolitre-size volumes. Hespent one year at HitachiCentral Research Lab inTokyo, Japan, as a postdoc-toral fellow and producedliquid chromatography columnon a chip. At Ciba-Geigy,Basel, Switzerland, he devel-oped the concept of miniatur-ized total analysis systems andbuilt a research team on chip-based analytical instrumenta-tion from 1988–1995. He wasprofessor for analytical chem-istry at Imperial College in

London from 1995–2003. Since 2003, he has been the head of theISAS in Dortmund, Germany, and a Professor of AnalyticalChemistry at the University of Dortmund. His research interestsinclude fluid handling and detection principles for chemicalanalysis, bioassays, and synthesis using microfabricated devices.

Lin Chen Andreas Manz

CRITICAL REVIEW www.rsc.org/loc | Lab on a Chip

This journal is � The Royal Society of Chemistry 2007 Lab Chip, 2007, 7, 1413–1423 | 1413

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

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

Philip Day graduated with aPh.D. degree from the WolfsonR e s e a r c h L a b o r a t o r i e s ,University of Birmingham.From 1995–1997 at OxfordUniversity he developed veryhigh throughput PCR for theWellcome Trust in the HumanGenome Mapping Project. Thiswas followed by developmentsinto high throughput sequencingand gene micro-arrays withProf. Sir Edwin Southern,which he followed with theestablishment of a FunctionalGenomics Unit, Kinderspital,

University of Zurich. His studies correlate innovative quantitativemeasurements of nucleic acids to meaningful biomedical inter-pretation. He was appointed Reader in Genomics, University ofManchester. In 2006 he was made Principal Investigator at theManchester Interdisciplinary Biocentre, and was later appointedProfessor of Applied Molecular Biology and Biochemistry at theUniversity of Dortmund, and with ISAS, Dortmund.

Philip J. R. Day

Fig. 1 Integrated PCR on microfluidic devices.

1414 | Lab Chip, 2007, 7, 1413–1423 This journal is � The Royal Society of Chemistry 2007

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

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,

Fig. 2 Schematic design of a nucleic acid m-TAS device for point-of-care applications.


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

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

Table 1 Pre-PCR integrated with PCR on a microchip

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

Stationarychamber

Glass Whole blood 202-bp DNA fragmentof dystrophin gene

Microchip filter for whiteblood cells isolation

50 20

Stationarychamber

Silicon/glass Whole blood 226-bp regions of humancoagulation Factor V gene

White blood cells isolation on thefilter section of the microchip

12 21

Stationarychamber

PC Whole blood 221-bp fragment fromE.coli K12 specific gene

Immunomagnetic bead-basedcell capture,

20 22

Stationarychamber

PDMS/silicon Listeriamonocytogenscells

544-bp DNA segment fromListeria monocytogens

DNA bound to silica-coated pillars inthe presence of the chaotropic saltguanidinium isothiocyanate

50 23,24

Stationarychamber

Silicon/glass E.coli BL21 16S-rRNA region ofbacterial genome

Cell lysis by laser-irradiated magneticbead system, and magnetic beadsfor removing denatured proteins

4 25


determine unknown amounts of sample or calculate the PCR

efficiency.26,27 Stationary microchip PCR can be readily

adapted into miniaturised real-time PCR with some minor

changes, such as PCR reagent formulation and an additional

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

microchip CGE and PCR-CGE. For continuous flow micro-

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

several identical parts to permit simultaneous detection of

different amplification reactions. Reported literature relating

to real-time PCR miniaturised platforms are summarized in

Table 2.24,25,28–34

Northrup et al. first developed a miniaturised analytical

thermal cycling instrument for real-time PCR detection.28,29 A

micro-machined silicon reaction chamber was integrated with

heaters and electronics for controlling temperature. The device

is a scaling-down of a conventional real-time PCR instrument

and was successfully used for the detection of single-base

differences in viral and human DNA. Their studies indicate

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-

time PCR detection equipped with microprocessor, pumps,

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

cence excitation/detection. Monolithic DNA purification and

real-time PCR enable fast detection of Listeria monocytogenes

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

templates, and both were able to amplify the 150-bp DNA

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

time PCR inside a PDMS-based microchannel using the Joule

heating effect.32 Their method applied Joule heating generated

by the current of the thermal source, therefore smartly

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

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

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

for continuous flow was developed recently by using laser

beam scanning within the temperature annealing region of the

device.33 Both thermal and polymer waveguide optical

detection systems were integrated inside a SU-8 chamber,

which is an important step towards a portable tool for real-

time quantitative PCR.34

2.3 Post-PCR

Post-PCR product analysis is singularly the most developed

area encompassing PCR integration within a single micro-

device. This is most likely attributed to complexities associated

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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

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


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

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


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

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

via multiparallelisation. Recently, the BioMarkTM

48.48 System

(Dynamic Arrays and Digital Arrays) for real-time PCR


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).

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.

References

1 P. A. Auroux, D. Iossifidis, D. R. Reyes and A. Manz, Anal.Chem., 2002, 74, 2637–2652.

2 D. R. Reyes, D. Iossifidis, P. A. Auroux and A. Manz, Anal.Chem., 2002, 74, 2623–2636.

3 T. Vilkner, D. Janasek and A. Manz, Anal. Chem., 2004, 76,3373–3385.

4 P. S. Dittrich, K. Tachikawa and A. Manz, Anal. Chem., 2006, 78,3887–3908.

5 L. J. Kricka and P. Wilding, Anal. Bioanal. Chem., 2003, 377,820–825.

6 P. A. Auroux, Y. Koc, A. deMello, A. Manz and P. J. R. Day, LabChip, 2004, 4, 534–546.

7 M. G. Roper, C. J. Easley and J. P. Landers, Anal. Chem., 2005,77, 3887–3893.

8 C. S. Zhang, J. L. Xu, W. L. Ma and W. L. Zheng, Biotechnol.Adv., 2006, 24, 243–284.

9 P. J. Day, Expert Rev. Mol. Diagn., 2006, 6, 23–28.10 H. Kitano, Science, 2002, 295, 1662–1664.11 M. U. Kopp, A. J. Mello and A. Manz, Science, 1998, 280,

1046–1048.12 P. J. Obeid, T. K. Christopoulos, H. J. Crabtree and

C. J. Backhouse, Anal. Chem., 2003, 75, 288–295.13 T. B. Taylor, E. S. Winn-Deen, E. Picozza, T. M. Woudenberg and

M. Albin, Nucleic Acids Res., 1997, 25, 3164–3168.14 H. Nagai, Y. Murakami, Y. Morita, K. Yokoyama and E. Tamiya,

Anal. Chem., 2001, 73, 1043–1047.15 B. C. Giordano, E. R. Copeland and J. P. Landers, Electrophoresis,

2001, 22, 334–340.16 L. A. Christel, K. Petersen, W. McMillan and M. A. Northrup,

J. Biomech. Eng., 1999, 121, 22–27.17 K. A. Wolfe, M. C. Breadmore, J. P. Ferrance, M. E. Power,

J. F. Conroy, P. M. Norris and J. P. Landers, Electrophoresis,2002, 23, 727–733.

18 M. C. Breadmore, K. A. Wolfe, I. G. Arcibal, W. K. Leung,D. Dickson, B. C. Giordano, M. E. Power, J. P. Ferrance,S. H. Feldman, P. M. Norris and J. P. Landers, Anal. Chem., 2003,75, 1880–1886.

19 B. M. Paegel, C. A. Emrich, G. J. Wedemayer, J. R. Scherer andR. A. Mathies, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 574–579.


20 P. Wilding, L. J. Kricka, J. Cheng, G. Hvichia, M. A. Shoffner andP. Fortina, Anal. Biochem., 1998, 257, 95–100.

21 P. K. Yuen, L. J. Kricka, P. Fortina, N. J. Panaro, T. Sakazumeand P. Wilding, Genome Res., 2001, 11, 405–412.

22 R. H. Liu, J. N. Yang, R. Lenigk, J. Bonanno and P. Grodzinski,Anal. Chem., 2004, 76, 1824–1831.

23 N. C. Cady, S. Stelick and C. A. Batt, Biosens. Bioelectron., 2003,19, 59–66.

24 N. C. Cady, S. Stelick, M. V. Kunnavakkam and C. A. Batt, Sens.Actuators, B, 2005, 107, 332–341.

25 J. G. Lee, K. H. Cheong, N. Huh, S. Kim, J. W. Choi and C. Ko,Lab Chip, 2006, 6, 886–895.

26 S. A. Bustin, J. Mol. Endocrinol., 2000, 25, 169–193.27 S. A. Bustin, J. Mol. Endocrinol., 2002, 29, 23–39.28 M. A. Northrup, B. Benett, D. Hadley, P. Landre, S. Lehew,

J. Richards and P. Stratton, Anal. Chem., 1998, 70, 918–922.29 M. S. Ibrahim, R. S. Lofts, P. B. Jahrling, E. A. Henchal,

V. W. Weedn, M. A. Northrup and P. Belgrader, Anal. Chem.,1998, 70, 2013–2017.

30 A. Gulliksen, L. Solli, F. Karlsen, H. Rogne, E. Hovig,T. Nordstrom and R. Sirevag, Anal. Chem., 2004, 76, 9–14.

31 Q. Xiang, B. Xu, R. Fu and D. Li, Biomed. Microdev., 2005, 7,273–279.

32 G. Q. Hu, Q. Xiang, R. Fu, B. Xu, R. Venditti and D. Q. Li, Anal.Chim. Acta, 2006, 557, 146–151.

33 T. Nakayama, Y. Kurosawa, S. Furui, K. Kerman, M. Kobayashi,S. R. Rao, Y. Yonezawa, K. Nakano, A. Hino, S. Yamamura,Y. Takamura and E. Tamiya, Anal. Bioanal. Chem., 2006, 386,1327–1333.

34 Z. Wang, A. Sekulovic, J. P. Kutter, D. D. Bang and A. Wolff,Electrophoresis, 2006, 27, 5051–5058.

35 A. T. Woolley, D. Hadley, P. Landre, A. J. deMello, R. A. Mathiesand M. A. Northrup, Anal. Chem., 1996, 68, 4081–4086.

36 L. C. Waters, S. C. Jacobson, N. Kroutchinina, J. Khandurina,R. S. Foote and J. M. Ramsey, Anal. Chem., 1998, 70, 158–162.

37 L. C. Waters, S. C. Jacobson, N. Kroutchinina, J. Khandurina,R. S. Foote and J. M. Ramsey, Anal. Chem., 1998, 70, 5172–5176.

38 W. C. Dunn, S. C. Jacobson, L. C. Waters, N. Kroutchinina,J. Khandurina, R. S. Foote, M. J. Justice, L. J. Stubbs andJ. M. Ramsey, Anal. Biochem., 2000, 277, 157–160.

39 E. T. Lagally, P. C. Simpson and R. A. Mathies, Sens. Actuators,B, 2000, 63, 138–146.

40 E. T. Lagally, I. Medintz and R. A. Mathies, Anal. Chem., 2001,73, 565–570.

41 E. T. Lagally, C. A. Emrich and R. A. Mathies, Lab Chip, 2001, 1,102–107.

42 J. W. Hong, T. Fujii, M. Seki, T. Yamamoto and I. Endo,Electrophoresis, 2001, 22, 328–333.

43 I. Rodriguez, M. Lesaicherre, Y. Tie, Q. B. Zou, C. Yu, J. Singh,L. T. Meng, S. Uppili, S. F. Y. Li, P. Gopalakrishnakone andZ. E. Selvanayagam, Electrophoresis, 2003, 24, 172–178.

44 C. G. Koh, W. Tan, M. Q. Zhao, A. J. Ricco and Z. H. Fan, Anal.Chem., 2003, 75, 4591–4598.

45 Z. M. Zhou, D. Y. Liu, R. T. Zhong, Z. P. Dai, D. P. Wu,H. Wang, Y. G. Du, Z. N. Xia, L. P. Zhang, X. D. Mei andB. C. Lin, Electrophoresis, 2004, 25, 3032–3039.

46 G. V. Kaigala, R. J. Huskins, J. Preiksaitis, X. L. Pang,L. M. Pilarski and C. J. Backhouse, Electrophoresis, 2006, 27,3753–3763.

47 F. C. Huang, C. S. Liao and G. B. Lee, Electrophoresis, 2006, 27,3297–3305.

48 C. N. Liu, N. M. Toriello and R. A. Mathies, Anal. Chem., 2006,78, 5474–5479.

49 Y. Hataoka, L. H. Zhang, Y. Mori, N. Tomita, T. Notomi andY. Baba, Anal. Chem., 2004, 76, 3689–3693.

50 Y. J. Liu, C. B. Rauch, R. L. Stevens, R. Lenigk, J. N. Yang,D. B. Rhine and P. Grodzinski, Anal. Chem., 2002, 74, 3063–3070.

51 D. Trau, T. M. Lee, A. I. Lao, R. Lenigk, I. M. Hsing, N. Y. Ip,M. C. Carles and N. J. Sucher, Anal. Chem., 2002, 74, 3168–3173.

52 J. Wang, Z. Y. Chen, P. Corstjens, M. G. Mauk and H. H. Bau,Lab Chip, 2006, 6, 46–53.

53 T. M. H. Lee, M. C. Carles and I. M. Hsing, Lab Chip, 2003, 3,100–105.

54 S. W. Yeung, T. M. Lee, H. Cai and I. M. Hsing, Nucleic AcidsRes., 2006, 34, e118.

55 N. M. Toriello, C. N. Liu and R. A. Mathies, Anal. Chem., 2006,78, 7997–8003.

56 L. Chen and J. Ren, Comb. Chem. High Throughput Screening,2004, 7, 29–43.

57 V. Dolnik and S. Liu, J. Sep. Sci., 2005, 28, 1994–2009.58 V. Dolnik, S. Liu and S. Jovanovich, Electrophoresis, 2000, 21,

41–54.59 M. J. Heller, Annu. Rev. Biomed. Eng., 2002, 4, 129–153.60 Z. Chen, M. G. Mauk, J. Wang, W. R. Abrams, P. L. Corstjens,

R. S. Niedbala, D. Malamud and H. H. Bau, Ann. N. Y. Acad. Sci.,2007, 1098, 429–436.

61 C. J. Easley, J. M. Karlinsey, J. M. Bienvenue, L. A. Legendre,M. G. Roper, S. H. Feldman, M. A. Hughes, E. L. Hewlett,T. J. Merkel, J. P. Ferrance and J. P. Landers, Proc. Natl. Acad.Sci. U. S. A., 2006, 103, 19272–19277.

62 L. Zhu, Q. Zhang, H. Feng, S. Ang, F. S. Chau and W. T. Liu, LabChip, 2004, 4, 337–341.

63 P. Sethu, A. Sin and M. Toner, Lab Chip, 2006, 6, 83–89.64 J. S. Marcus, W. F. Anderson and S. R. Quake, Anal. Chem., 2006,

78, 3084–3089.65 P. Gascoyne, C. Mahidol, M. Ruchirawat, J. Satayavivad,

P. Watcharasit and F. F. Becker, Lab Chip, 2002, 2, 70–75.66 P. Gascoyne, J. Satayavivad and M. Ruchirawat, Acta Trop., 2004,

89, 357–369.67 E. T. Lagally, S. H. Lee and H. T. Soh, Lab Chip, 2005, 5,

1053–1058.68 J. El-Ali, P. K. Sorger and K. F. Jensen, Nature, 2006, 442,

403–411.69 C. E. Sims and N. L. Allbritton, Lab Chip, 2007, 7, 423–440.70 B. Huang, H. Wu, D. Bhaya, A. Grossman, S. Granier,

B. K. Kobilka and R. N. Zare, Science, 2007, 315, 81–84.71 J. Ueberfeld, S. A. El-Difrawy, K. Ramdhanie and D. J. Ehrlich,

Anal. Chem., 2006, 78, 3632–3637.72 L. A. Legendre, J. M. Bienvenue, M. G. Roper, J. P. Ferrance and

J. P. Landers, Anal. Chem., 2006, 78, 1444–1451.73 J. E. Prest, S. J. Baldock, P. J. Day, P. R. Fielden, N. J. Goddard

and B. J. Treves Brown, J. Chromatogr., A, 2007, 1156, 154–159.74 L. Chen, J. E. Prest, P. R. Fielden, N. J. Goddard, A. Manz and

P. J. Day, Lab Chip, 2006, 6, 474–487.75 S. S. Yeung, T. M. Lee and I. M. Hsing, J. Am. Chem. Soc., 2006,

128, 13374–13375.76 http://www.fluidigm.com/biomark_qPCR.htm.77 E. A. Ottesen, J. W. Hong, S. R. Quake and J. R. Leadbetter,

Science, 2006, 314, 1464–1467.78 M. Curcio and J. Roeraade, Anal. Chem., 2003, 75, 1–7.79 N. Park, S. Kim and J. H. Hahn, Anal. Chem., 2003, 75,

6029–6033.80 K. D. Dorfman, M. Chabert, J. H. Codarbox, G. Rousseau,

P. de Cremoux and J. L. Viovy, Anal. Chem., 2005, 77, 3700–3704.


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Total Nucleic Acid Analysis Integrated on Microfluidic Devices