Electrophoresis 2006, 27, 3297–3305 3297

Fu-Chun Huang1 Research Article

Chia-Sheng Liao2
Gwo-Bin Lee1, 2

1
Department of Engineering Science,
National Cheng Kung University,
Tainan, Taiwan
2
Institute of Micro-electro-
mechanical-system Engineering,
National Cheng Kung University,
Tainan, Taiwan
Received January 23, 2006
Revised March 2, 2006
Accepted March 2, 2006
An integrated microfluidic chip for DNA/RNA
amplification, electrophoresis separation and
on-line optical detection
This study presents an integrated microfluidic chip capable of performing DNA/RNA
(deoxyribonucleic acid/ribonucleic acid) amplification, electrokinetic sample injection
and separation, and on-line optical detection of nucleic acid products in an automatic
mode. In the proposed device, DNA/RNA samples are first replicated using a micro-
machine-based PCR module or reverse transcription PCR (RT-PCR) module and then
transported by a pneumatic micropump to a sample reservoir. The samples are sub-
sequently driven electrokinetically into a microchannel, where they are separated
electrophoretically and then detected optically by a buried optical fiber. The various
modules of the integrated microfluidic chip are fabricated from cheap bio-compatible
materials, such as PDMS, polymethylmethacrylate, and soda-lime glass. The func-
tionality of the proposed device is demonstrated through its successful application to
the DNA-based bacterial detection of Streptococcus pneumoniae and the RNA-based
detection of Dengue-2 virus. It is shown that the low thermal inertia of the PCR/RT-PCR
modules reduces the sample and reagent consumption and shortens the reaction time.
With less human intervention, the subsequent DNA separation and detection could be
performed in an automatic mode. The integrated microfluidic device proposed in this
study represents a crucial contribution to the fields of molecular biology, genetic anal-
ysis, infectious disease detection, and other biomedical applications.

Keywords: Capillary electrophoresis / Laser induced fluorescence / Microfluidics /

PCR / RT-PCR DOI 10.1002/elps.200600458

1 Introduction devices has facilitated the evolution of a wide range of

The microfabrication of miniature fluidic devices has
attracted considerable interest over the past decade.
MEMS (microelectromechanical systems) has proven
to be a fundamental enabling technology and has
revolutionized much of the existing analytical instru-
mentation used in the fields of molecular biology, genetic
analysis, disease diagnosis, and biomedicine. Due to
their ability to conduct the parallel processing of minute
amounts of biosamples, the development of microchip
Correspondence: Professor Gwo-Bin Lee, Engineering Science,
National Cheng Kung University, 1, University Road, Tainan, 701, Tai-
wan, China
E-mail: gwobin@mail.ncku.edu.tw
Fax: 1886-6-2761687
Abbreviations: cDNA, complementary DNA; DNA, deoxyribonucleic
acid; MEMS, microelectromechanical system; PMMA, polymethyl-
methacrylate; RT-PCR, reverse transcription PCR; SEM, scanning
electron microscope
rapid and efficient analytical techniques. Furthermore, the
concept of micro total analysis systems (m-TASs), in which
sample pretreatment, transportation, mixing, reaction,
separation, and detection functions are integrated on a
single miniature chip, can now be realized by combining
functional microfluidic components manufactured using
appropriate micromachining technologies [1].
Compared to the use of conventional analytical instru-
mentation, amplifying and analyzing deoxyribonucleic
acid (DNA) samples in a microchip format has significant
advantages. PCR is a well-developed nucleotide amplifi-
cation method for genetic identification and diagnosis,
and is recognized as one of the most essential proce-
dures currently performed in life-science laboratories.
Briefly, the PCR process involves amplifying the con-
centration of a certain segment of dsDNA by thermal
cycling. Recent advances in MEMS techniques have
enabled the fabrication of micro PCR chips. These chips,
which are generally fabricated on either silicon [2–5] or

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3298 F.-C. Huang et al.
glass substrates [6, 7], have shown considerable potential
for rapid DNA amplification [2–8]. For example, Northrup
et al. [2, 3] presented a microfabricated, silicon-based
PCR chamber heated by boron-doped polysilicon resis-
tors positioned outside the PCR chamber. The low ther-
mal inertia of the developed microfluidic device enabled
successful amplification of DNA samples with reduced
sample and reagent consumption and a shorter reaction
time.
The use of MEMS-based micro CE chips for DNA sample
analysis has been extensively investigated [9]. Compared
to their conventional large-scale counterparts, micro CE
chips have the fundamental advantages of compactness,
low sample/reagent consumption, low cost, and high
detection limits. As a consequence, microchip-based
electrophoresis is currently employed in an increasing
number of DNA analysis procedures. Micro-scale elec-
trophoretic devices have typically been fabricated using
glass [10–12], fused-silica substrates [13], and plastics
[14–16].
Integrating PCR and electrophoresis devices provides the
potential for rapid amplification, separation, and identifi-
cation of DNA samples [5, 6, 17, 18]. For example, Wool-
ley et al. [5] presented a silicon-based PCR reactor inte-
grated with a glass-based CE chip and showed that the
PCR-CE analysis of a b-globin target could be success-
fully completed within 20 min. Waters et al. [19] demon-
strated a microchip device for cell lysis, multiplex PCR
amplification, and electrophoretic sizing analysis. Lagally
et al. [6, 7] developed an integrated PCR-CE system with
platinum temperature sensors located inside the reaction
chamber and heaters arranged outside the chamber.
Various researchers have reported success in developing
a two-stage DNA amplification/electrophoresis analysis
process. For example, Koh et al. [20] presented an inte-
grated plastic microfluidic device capable of PCR, valv-
ing, and electrophoretic separation to perform bacterial
detection and identification procedures. In their design,
screen-printed graphite ink resistors were used to con-
duct the thermal cycling required in the PCR. Following
the PCR operation, the PCR products were injected
electrokinetically through a gel valve and then separated
electrophoretically. The detection of Escherichia coli
O157 and Salmonella typhimurium was successfully
demonstrated using the proposed design. Similarly,
Rodriguez et al. [21] developed an integrated PCR-CE
system for genetic analysis. In their study, the PCR
chamber was fabricated using silicon and it incorporated
aluminum heaters and temperature sensors. Following
PCR, a large-scale external pumping device was used to
transport the amplified samples to a glass-based CE
chip, where DNA fragments differing in size by 18 bps
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2006, 27, 3297–3305
were successfully resolved. Although the pioneering de-
vices presented above have a proven ability to amplify
and analyze DNA samples, their application is limited in
practice since they generally involve some form of large-
scale optical detection system. Moreover, these devices
are unable to perform a fully automatic PCR/CE/detection
procedure, which is a pressing requirement in many bio-
medical and chemical analysis applications.
A technique commonly associated with the use of CE
chips is that of LIF detection. Briefly, in this technique, the
samples are labeled with a particular fluorescein, and the
fluorescence signals induced by a laser source are then
detected as the samples flow through the downstream
region of the separation channel. However, the conven-
tional LIF technique utilizes a bulky optical detection
apparatus comprising a microscope and various delicate
light coupling components. Therefore, the advantages
afforded by the miniaturization of the CE device are
somewhat lessened. Hence, the integration of miniature
optical detection systems with micro CE chips has enor-
mous appeal and provides the means of realizing the on-
line detection of bio-samples. Several approaches toward
integrating micro CE chips with optical detection devices
have been reported, including liquid core waveguides [22,
23], optic fibers [24], leaky waveguides [25, 26], and opti-
cal waveguides [27]. Recently, we developed a novel
design in which CE microfluidic devices were fabricated
with integrated buried optical waveguides for DNA/pro-
tein analysis applications [28]. In the developed chip,
optical detection was achieved by means of buried solid-
core optical waveguides formed by filling SU-8/SOG
(spin-on-glass) double layers within pre-etched wave-
guide channels. Alternatively, it has been reported that CE
detection can be achieved through the use of etched
optical fibers inserted directly into embedded channels,
orientated perpendicularly to the separation channel [29].
The diameter of etched optical fibers is 100 mm. This
arrangement was applied successfully to the separation
and detection of DNA samples. The chip fabrication pro-
cess was both cheap and effective. Hence, the proposed
design was a suitable candidate for the mass production
of disposable micro-CE chips with integrated on-line
optical detection mechanisms. Ro et al. [30] reported
integrated optical fibers for CE chip detection. In their
study, an additional slit channel between an optical fiber
and a fluid channel is used for highly efficient and sensi-
tive optical detection. Experimental data show that the
sensitivity greatly improved.
The present study develops an integrated microfluidic
chip capable of performing DNA/RNA (ribonucleic acid)
amplification, electrophoretic separation, and on-line
optical detection of DNA samples. In the proposed de-
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Electrophoresis 2006, 27, 3297–3305 Miniaturization 3299

vice, the DNA/RNA samples are replicated using a micro

PCR (or reverse transcription PCR (RT-PCR)) module. It is
notable that the integrated microfluidic chip is capable of
RT-PCR for detection of RNA-based virus. The resulting
products are transported by a pneumatic micropump to
the sample buffer channel of a CE chip. Using an electro-
kinetic driving scheme, the samples are injected into the
CE chip and separated electrophoretically in the separa-
tion channel. Finally, the samples are detected optically
by an embedded optical fiber located in the downstream
region of the separation channel. The novel combination
of pneumatically driven and electrokinetically driven
transportation mechanisms ensures the injection of pre-
cise amounts of DNA/RNA samples into the CE chip and
the segregation of PCR reagents and CE buffer solutions.
To the best of our knowledge, this study represents the
first reported attempt to utilize such a hybrid driving
scheme in integrated microfluidic systems designed for
DNA- and RNA-based applications. With less human
intervention, the DNA/RNA amplification, separation, and
detection could be performed in an automatic mode.

2 Materials and methods

2.1 Design

Miniature devices for the rapid PCR-based analysis of

DNA samples are crucial for genetic applications. Inte-
grated PCR and electrophoresis devices are ideal candi-
dates for rapid DNA analysis. These devices are not only
capable of rapid amplification of DNA samples, but can
also conduct the subsequent DNA separation and sizing
analysis tasks. This study develops a novel integrated
microfluidic device capable of performing DNA/RNA
amplification, separation, and on-line detection in an
automatic mode. Figure 1a shows a conventional PCR/
CE integrated chip [31]. One of the CE reservoirs was
used as a PCR chamber. Thus PCR reagents may be
mixed with CE buffers. EDTA in CE buffer solutions is
known to be one of the PCR inhibitors [32]. As a result,
PCR efficiency could be decreased. Besides, high tem-
perature near the PCR chamber could cause the drying of
the CE buffers, thus affecting the CE performance. Fig-
ure 1b presents a schematic illustration of the proposed
microfluidic chip. The device comprises three major
modules, namely a micro PCR (or RT-PCR) chip with three
chambers and micropumps, a cross-shaped CE channel
for the electrokinetic injection and separation of the
amplified DNA samples, and a buried optical fiber to carry
out on-line DNA detection. As shown in Fig. 1c, the inte-
grated microfluidic chip is fabricated by bonding together
the three separate glass-, polymethylmethacrylate
(PMMA)-, and PDMS-based chips. In most of the report-
Figure 1. Schematic illustration of (a) a conventional
PCR/CE integrated chip, (b) a proposed microfluidic chip
capable of DNA/RNA amplification, electrophoretic injec-
tion, separation, and on-line detection of DNA samples,
and (c) cross-sectional view of the proposed microfluidic
chip comprising glass-, PMMA-, and PDMS-based mod-
ules.
ed micro PCR devices, the sensors and heaters required
for the PCR process are located outside the PCR cham-
ber. This leads to an inaccurate temperature measure-
ment of the DNA sample and a low heating/cooling rate
during thermal cycling. Accordingly, in the proposed
design, two microheaters and one temperature sensor
are deposited on a glass substrate and are located within
the PCR chamber. It has been shown previously that this
arrangement improves the precision of the temperature
measurement and delivers a more efficient heating/cool-
ing performance [33]. The developed microchip employs
electrokinetic forces to carry out sample injection and
separation in a crossshaped CE channel. In fabricating
this channel, a simple and reliable method involving pho-

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3300 F.-C. Huang et al.
toresist as an etch mask is used to fabricate a glass tem-
plate of the various microchannels [12]. A hot-embossing
method is then used to transfer the inverse micro-
structures onto a PMMA substrate. A second PMMA
cover plate with predrilled via holes is thermally bonded to
this substrate to seal the microfluidic channels [16]. An
etched optical fiber is inserted into an embedded channel
perpendicular to the separation channel to detect the LIF
signals [29]. Finally, the pneumatic micropumps used to
control the flow of the reagents and samples in the PCR
process, and to transport the amplified products to the
CE sample buffer, each comprise three individual PDMS
membranes, which are driven sequentially by external
compressed air in order to generate a peristaltic pumping
action [34]. The PDMS membranes can be deflected by
compressed air to such an extent that they block the
microchannels completely. Furthermore, EDTA in CE buf-
fer solutions is known to be a PCR inhibitor. In Fig. 1b,
three micropumps consisting of three PDMS membranes
were designed. When one pump is activated, the other
two serve as valves simultaneously. Therefore, the PDMS
membranes can provide a valving function to ensure a
proper isolation of the PCR reagents and the CE buffers.
Besides, the distance between the PCR chambers and
CE channels provides a good thermal isolation, such that
drying of the CE buffers could be alleviated.
The proposed microfluidic chip design has a number of
key advantages. First, the RT-PCR chamber allows the
reverse transcription reaction of messenger RNA (mRNA)
to be performed if required. In the reverse transcription
process, precise amounts of RNA reagents are trans-
ported by the pneumatic micropumps to the PCR cham-
ber from the RT-PCR reagent chamber. Synthesized
complementary DNA (cDNA) samples are then further
amplified after pumping PCR reagents from the PCR
reagent chamber. This two-step RT-PCR amplification
process provides a reliable operation for RNA samples. If
it is only necessary to amplify DNA samples, the reverse
transcription process is simply omitted. Second, the
pneumatic micropumps also provide a micro-valving
function such that the PCR chambers and the CE buffer
reservoir can be effectively isolated. It has been shown
experimentally that mixing of the reagents and buffer has
a detrimental effect on the DNA amplification process
[32]. Additionally, the high-temperature field (.957C) in
the PCR chamber can cause a drying of the CE buffer, and
hence can affect the injection of the amplified DNA sam-
ples into the CE channel. Third, the novel combination of
pneumatic and electrokinetic pumping mechanisms pro-
posed in the current design represents a promising
approach for PCR/CE applications. Micro-CE chips are
typically driven by electrokinetic forces. However, when
amplified DNA samples and reagents are transported to
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2006, 27, 3297–3305
CE reservoirs by using electrokinetic forces, they will
move at different speeds due to different charge-to-mass
ratios. It may affect the following injection and separation
process. The proposed design overcomes this problem
by using a series of pneumatic micropumps to manipulate
the DNA/RNA samples prior to the electrophoretic
separation process, and then using conventional electro-
kinetic forces to conduct sample injection and separation
once the DNA/RNA samples have been amplified. Finally,
the proposed design incorporates an optical fiber buried
at a downstream location in the CE separation channel to
detect the induced fluorescence signals. The intensity of
the optical signals is enhanced in the current design by
means of a side channel filled with index-matching oil
[35].
2.2 Fabrication
Micro PCR chips are fabricated on glass substrate.
Reaction chambers and micropumps are fabricated by
using PDMS. Micro CE chips are made up of PMMA. The
reasons for using these materials are low cost, bio-com-
patible and mature manufacturing methods. Figure 2
presents an overview of the fabrication process employed
for the integrated microfluidic chip. Briefly, the micro CE
channels and optical fiber channels were replicated on a
PMMA substrate from a glass template using hot-
embossing methods (Fig. 2a). A cover PMMA plate with
predrilled via holes was then thermally bonded to the
substrate to form the sealed micro-CE chip (Fig. 2b).
The microheaters and temperature sensor were fabri-
cated on a soda-lime glass substrate (Central Glass Tai-
wan Trading, Hsin Chu, Taiwan). Initially, a thin layer of
titanium (0.02 mm) was deposited on the glass substrate
to form an adhesion layer for the subsequent deposition
of a 0.1 mm platinum layer for the electron-beam eva-
porator. The platinum layer was then patterned as a
resistor using standard lift-off processes (Fig. 2c). Note
that platinum resistors were used for both the tempera-
ture sensor and the two heating elements in order to sim-
plify the fabrication process. The resistances of the sen-
sor and heaters were measured to be 400 and 30 O,
respectively. A conventional gold metallization (0.4 mm)
process was then employed to form electrical leads. A
cover glass slide (100 mm thick) was attached to the glass
substrate to form an electrical isolation layer (Fig. 2d).
Finally, via-holes were drilled (Fig. 2e).
The pneumatic micropumps in the integrated microfluidic
chip comprise two PDMS structures replicated on silicon
substrates from SU-8 templates (Figs. 2f, 3g). The upper
PDMS structure incorporates air chambers for the deliv-
ery of compressed air to deform the PDMS membranes,
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Electrophoresis 2006, 27, 3297–3305
sensor and heaters on glass substrate for micro PCR device, followed by deposition/patterning of gold as electrical leads,
(d) bonding of cover slide for electric insulation, (e) drilling of via-holes, (f) replication of PDMS membranes using SU-8 tem-
plate on silicon substrate, (g) replication of PDMS fluid channels using SU-8 template on silicon substrate, and (h) peeling of
PDMS structures and bonding of two PDMS layers to form micropumps.
while the lower PDMS structure contains the fluidic
channels. In fabricating the micropumps, PDMS elasto-
mers and curing agents (Sylgard 184 Silicone Elastomer
Kit, Dow Corning, USA) were mixed in the ratio of 10:1
and then cured at 757C for 180 min. The two PDMS
structures were then peeled off mechanically and bonded
in an oxygen plasma treatment (Fig. 2h).
The final integrated microfluidic chip was formed by
bonding the glass PCR chip to the PMMA CE chip using
UV-sensitive glue. The PDMS micropumps chip was then
bonded on top of the glass PCR chip using an oxygen
plasma treatment. The assembly process was completed
by inserting an etched optical fiber into the buried channel
through a coupling device under a microscope [35]. Then,
the optical fiber was fixed in position by UV glue.
2.3 Experimental
The process of DNA/RNA amplification, electrophoresis
separation, and on-line optical detection conducted in the
present study can be briefly described as follows
(Fig. 1a). Initially, RNA or DNA templates were placed in
reservoir 2 (PCR chamber) and the crossshaped CE
channel was filled with CE buffer (a mixture of
1.5% HPMC (hydroxypropyl methyl cellulose) in TBE (tris-
borate-EDTA) and 1% YO-PRO®-1 fluorescence dyes
(Molecular Probes, USA)) [36]. RT-PCR and PCR reagents
were then loaded in reservoirs 1 and 3, respectively. Note
that in this study, the 10 mL RT-PCR reagents contained
1 mg of RNA, 0.5 mL of 10 mM dNTP (deoxynucleoside
triphosphates (Yeastern Biotech, Taiwan)), 2 mL of
56 reaction buffer (250 mM Tris-HCl, pH 8.3,
375 mM KCl, and 15 mM MgCl2), 0.5 mL of 10 mM primer
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Miniaturization 3301
Figure 2. Simplified fabrication
process of the integrated micro-
fluidic chip. (a) Hot embossing of
PMMA plates using glass tem-
plate with inverse images of CE
channels, (b) drilling of via-holes
in the second PMMA plate and
bonding, (c) deposition/pattern-
ing of platinum temperature
(Merck Taiwan, Taiwan), 1 mL of 0.1 M DTT (Promega,
USA), and 0.5 mL of moloney murine leukemia virus RT
(200 U/mL, Gibco BRL, MD, USA). Similarly, the 10 mL
PCR reagents contained 0.2 mM each of deoxy-
adenosine triphosphate (dATP), deoxycytidine triphos-
phate (dCTP), deoxyguanosine triphosphate (dGTP), and
deoxythymidine triphosphate (dTTP) (Yeastern Biotech),
1 mL of 106PCR buffer (15 mM MgCl2, 500 nM KCl,
1.5 M Tris-HCl, pH 8.7), 200 nM of the appropriate paired
primers, and 1 U Taq DNA polymerase (Amersham, UK)
[37]. In the RT-PCR process, the RT-PCR reagents were
pumped from reservoir 1 to reservoir 2 by the pneumatic
micropumps. The RNA template was then synthesized to
cDNA at a temperature of 437C and maintained for
30 min. Process conditions of 657C for 10 min were then
implemented to prevent nonspecific binding prior to the
Taq DNA polymerase addition. Following the synthesis of
cDNA, 2 mL of cDNA was left in reservoir 2 for the sub-
sequent PCR process. PCR reagents were then trans-
ported from reservoir 3 to reservoir 2 to conduct the
amplification of cDNA over 20 thermal cycles of 947C for
10 s, 527C for 20 s, and 727C for 20 s. Note that the final
cycle included an additional thermal stage of 727C for
1 min. Following cDNA amplification, the DNA samples
were transported to reservoir 4, i.e., the CE sample
reservoir. The amplified DNA samples were then pumped
electrokinetically into the separation channel, where they
were separated electrophoretically and then detected by
the buried optical fiber.
Details about the experimental setup employed for the
electrokinetic injection/separation and LIF detection
could be found in our previous work [38]. Briefly, a con-
ventional single crossedchannel injection method was
utilized to perform sample injection via the appropriate
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3302 F.-C. Huang et al.
switching of a high-voltage power supply. Prior to injec-
tion of the amplified DNA samples, the CE channel was
filled with CE buffer. When the DNA samples were driven
into the CE channel, they were dyed simultaneously. In
addition to the integrated microfluidic chip, the whole
system needs an additional temperature controller, an air
compressor and micropump controller. These peripheral
components were installed inside a box with a dimension
of 2161268.5 cm3.
3 Results and discussion
Figure 3a presents a photograph of the completed
microfluidic chip. The chip measures 65645 mm2, and
the width and depth of the micro CE channels are 100 and
30 mm, respectively. The separation channel has a total
length (as measured from the intersection) of 50 mm and
the buried optical fiber is located at a distance of 5 mm
Figure 3. (a) Photograph of the integrated microfluidic
chip. The three layers of the integrated chip are com-
posed of a PMMA CE chip, a glass micro PCR chip and
PDMS micropumps (bottom-up). The size of chip is
measured to be 6.563.960.8 cm3. The channel is of
100 mm (width)630 mm (depth). The volumes of the PCR
and reagent chambers are 11.25 mL. (b) Close-up SEM
image of the buried optic fiber channel. Note the side
channel filled with index-matching oil.
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Electrophoresis 2006, 27, 3297–3305
from the downstream end of the channel. The volumes of
PCR and reagent chambers are 11.25 mL. Figure 3b pro-
vides a close-up view of the micro CE channel and the
optical fiber channel. In practical applications, air exists
between optical fibers and CE channels, which could
affect the optical coupling of the excitation and detection
of light. Thus index-matching oil was used to fill a side
channel, thus enhancing the optical detection [35]. Fig-
ure 4 shows a scanning electron microscope (SEM)
image of the temperature sensor and the two heaters. As
discussed previously, these resistors are located within
the PCR chamber in order to improve the precision of the
temperature measurement and to provide a more efficient
heating/cooling performance.
In this study, a PWM (pulse width modulation) controller
and an ASIC (application specific integrated circuit) were
used to control the temperature field inside the PCR
chamber [8]. During operation, the micro temperature
sensor provided real-time measurements of the temper-
ature field, while the micro heaters regulated the temper-
ature of the sample inside the PCR chamber during ther-
mal cycling. The capability of the micro PCR module was
demonstrated by conducting a typical PCR cycle. The
PCR module is capable of performing typical temperature
cycles. The heating and cooling rates of the system were
measured to be approximately 207C/s and 107C/s,
respectively, with a variation of 0.27C. Such accuracy is
important for most experimental protocols and is critical
to the scientific communication of reproducible results.
Prior to conducting DNA amplification, the performance
of the pneumatic micropumps was characterized. Fluid
pumping was achieved via the sequential activation of the
Figure 4. SEM image of the temperature sensor and
heaters.
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Electrophoresis 2006, 27, 3297–3305
three PDMS membranes in the micropump. A peristaltic
pumping action was induced by changing the phase and
frequency of the driving pressure and applying a sequen-
tial control to the individual membranes. The experi-
mental data confirmed that the pump was capable of
successful sample transportation. Moreover, these PDMS
membranes could be used as microvalves while they are
deflected completely. Therefore, they can provide a valv-
ing function to ensure a proper isolation of the PCR
reagents and the CE buffers. Experimental data showed
that PCR efficiency could be greatly affected if the PCR
reagents were mixed with the CE buffers. In practical
applications, DNA/RNA could not be successfully ampli-
fied without proper isolation of the PCR reagents and the
CE buffers. As shown in Fig. 5, the pumping rate can be
controlled simply by changing the frequency of the
applied driving signal. It is noted that compressed air is
supplied at a constant pressure of 20 psi in this figure. It
could be clearly seen that an increase in activation fre-
quency significantly increases the pumping rate of the
peristaltic micro-pumps. The pumping rate will eventually
reach a saturation value limited by the maximum driving
frequency of the electromagnetic valves (about 15 Hz).
Although not shown here, the experimental data revealed
that the pumping rate can be increased by increasing the
driving air pressure [34].
Figure 5. Relationship between the pumping rate of the
pneumatic micropump and driving frequency. Note com-
pressed air pressure is 20 psi. An increase in activation
frequency significantly increases the pumping rate. The
pumping rate will eventually reach a saturation value lim-
ited by the maximum driving frequency of the electro-
magnetic valves (about 15 Hz).
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Miniaturization 3303
Aftercharacterizing the PCR module and the pneumatic
pumps, the developed microfluidic chip was used to
perform the DNA-based bacterial detection of Strepto-
coccus pneumoniae (S. pneumoniae) and the RNA-
based viral detection of Dengue fever (Type 2). In the
bacterial detection trial, a gene of length 240 bps is
amplified for detection of the resistance of S. pneumo-
niae to the penicillin antibiotic in 20 thermal cycles. The
concentration of the PCR products increases with ther-
mal cycles [8]. Figure 6 presents a slab-gel electro-
phoregram of the amplified PCR products. The micro
PCR operation was completed within 15 min and con-
sumed a total sample volume of just 10 mL. To prevent
evaporation of PCR reagents, mineral oil was used to
cover the PCR reagents. For comparison purposes, the
PCR process was also performed using a conventional
large-scale PCR machine. This traditional technique
required 2 h to complete and consume 25 mL of sample.
In Figure 6, the fluorescence signals in the first lane cor-
respond to the 100 bp DNA ladders. Lane (C) shows the
fluorescence signal obtained from the micro PCR chip,
and Lane (M) shows the PCR product obtained using the
bench-top PCR machine (PCR Sprint HBSP02, Thermo
Electron, USA). The results confirm the ability of the
developed PCR microchip to perform rapid and accurate
PCR operations.
Figure 6. Slab-gel electropherogram showing successful
amplification of DNA samples using developed micro
PCR module. (Lane L: 100 bp DNA ladders; Lane C: PCR
products using micro PCR chip; Lane M: PCR products
using conventional PCR machine.)
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3304 F.-C. Huang et al. Electrophoresis 2006, 27, 3297–3305

After amplifying the DNA, we pipette DNA markers in

reservoir 4. Then, amplified DNA samples were pumped
to reservoir 4 and mixed with DNA markers. After the
mixing process, the mixture of DNA markers and ampli-
fied DNA samples was injected and separated in the CE
channels and then detected by the integrated optical
fiber. This trial was conducted using a CE buffer of
1.5% HPMC in TBE with 1% YO-PRO-1 fluorescence
dye. Sample injection was performed by applying a volt-
age of 200 kV for 0.5 min, while separation was con-
ducted under a voltage of 1.3 kV applied for 3.5 min. For
comparison purposes, Hae III digested fx-174 DNA
markers with a concentration of 10 ng/mL were mixed with
the PCR products and separated at the same time. DNA
fluorescence signals (509 nm wavelength) were induced
using a Hg excitation light with a 491 nm wavelength.
Fluorescence signals were collected by the buried optical
fiber downstream of the separation channel and amplified
via a PMT (photomultiplier tube) module (C3830, R928,
Hamamatsu, Japan). The amplified analog signals were
converted to digital signals by 24-bit ADC (Model 0224-2,
SISC, Taipei, Taiwan), and recorded.

Figure 7a presents an electrophoregram of the mixture of

DNA markers and PCR products obtained during the
detection of S. pneumoniae. It can be seen that all the
11 peaks of the DNA markers and the single peak of the
PCR product (240 bps) from the S. pneumoniae bacteria
were separated successfully within 1.4 min. In addition to
fast analysis and efficient separation, chip-base electro-
phoresis has been known to have a higher detection limit
while compared to conventional slab-gel electrophoresis
(about one order of magnitude improvement). Figure 7b
shows the electrophoregram corresponding to the PCR
product (419 bps) obtained from the Dengue-2 RNA virus.
Again, all the 11 peaks of the DNA markers and the single
peak of the RT-PCR product (419 bps) were separated
successfully within 1.4 min. These two tests confirm the
capability of the proposed device for DNA/RNA amplifi-
cation and detection. Typically, LIF detection requires
complicated optics for focusing excitation light on the
microchannel and collecting induced fluorescence sig-
Figure 7. (a) Electropherogram of amplified DNA prod-
nals back to a sensitive photo-detector. In this study,
ucts associated with detection gene (240 bps) of
optical fiber can guide fluorescence light from the chip to
S. pneumoniae. (b) Electropherogram of amplified RNA
a PMT without using any complicated focus lens, pin- products associated with detection gene (419 bps) of
holes, and alignment stages. It is an improved design and Dengue-2 virus.
a user-friendly process to acquire optical signals directly.

tion in an automatic mode. The micromachine-based

4 Concluding remarks
This study has presented an integrated microfluidic chip
capable of performing DNA/RNA amplification, sample
transportation, CE separation, and on-line optical detec-
PCR (or RT-PCR) chip was fabricated using established
MEMS-based techniques; and consumes minimum
reagent and sample volumes, and provides higher heat-
ing/cooling rates together with a more precise tempera-
ture control. A clever design was used to ensure a proper

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2006, 27, 3297–3305
isolation of the PCR reagents and the CE buffers. The
micropump can be used to transport the PCR products
automatically. The ability of the integrated microfluidic
device to perform the successful amplification of the
detection genes for S. pneumoniae bacteria and Dengue-
2 viruses, and the ability to carry out the injection and
separation of the amplified PCR products have been
demonstrated. The ultimate goal of the study is to use
“optical-fiber only” structures for light in/out of the chip
and a fully-integrated biochip for detection of pathogens.
The authors gratefully acknowledge the financial support
provided to this study by the National Science Council,
Taiwan (grant number NSC 92-2323-B-006-010) and the
MOE Program for Promoting Academic Excellence of
Universities (grant number EX-91-E-FA09-5-4). The
authors also thank the Center for Micro/Nano Technology
Research, National Cheng Kung University for access
provided to major fabrication equipments. Finally, the
authors would like to extend their thanks to Dr. Che-Hsin
Lin, Mr. Tsung-Min Hsieh, and Dr. Ching-Hsing Luo for
their valuable input to project discussions, and to
Dr. Jiunn-Jong Wu, Dr. Chih-Ching Chang, and Dr. Hsiao-
Sheng Liu for their kind assistance in the preparation of
DNA/RNA samples.
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