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Microfluidic device for rapid (< 15 min) automated microarray hybridization

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 Papers in Press. First published August 17, 2005 as doi:10.1373/clinchem.2005.052845
Clinical Chemistry 51:10
000 – 000 (2005)
Microfluidic Device for Rapid (⬍15 min)
Automated Microarray Hybridization
Régis Peytavi,1 Frédéric R. Raymond,1 Dominic Gagné,1 François J. Picard,1
Guangyao Jia,2 Jim Zoval,2 Marc Madou,2 Karel Boissinot,1 Maurice Boissinot,1
Luc Bissonnette,1 Marc Ouellette,1 and Michel G. Bergeron1*
Background: Current hybridization protocols on mi-
croarrays are slow and need skilled personnel. Microflu-
idics is an emerging science that enables the processing
of minimal volumes of liquids to perform chemical,
biochemical, or enzymatic analyzes. The merging mi-
crofluidics and microarray technologies constitute an
elegant solution that will automate and speed up mi-
croarray hybridization.
Methods: We have developed a microfluidic flow cell
consisting of a network of chambers and channels
molded into a polydimethylsiloxane substrate. The sub-
strate was aligned and bound in reverse to the microar-
ray printed onto a standard glass slide to form a func-
tional microfluidic unit. The microfluidic units were
placed onto an engraved, disc-shaped support fixed on a
rotational device. Centrifugal forces drove the sample
and buffers directly onto the microarray surface.
Results: This microfluidic system allowed us to increase
the hybridization signal by ⬃10fold compared with a
passive system that made use of 10 times less sample. By
means of a 15–min automated hybridization process,
performed at room temperature, we demonstrated the
discrimination of 4 clinically relevant Staphylococcus
species that differ by as little as a single nucleotide
polymorphism (SNP). This process included hybridiza-
tion, washing, rinsing, and drying steps and does not
require any purification of target nucleic acids. This
1
Centre de Recherche en Infectiologie de l’Université Laval, Centre
Hospitalier Universitaire de Québec (Pavillon CHUL), Sainte-Foy, Québec,
Canada; 2 Department of Mechanical and Aerospace Engineering, University
of California, Irvine, CA
*Address correspondence to this author at: Dr. Michel G. Bergeron, Centre
de Recherche en Infectiologie de l’Université Laval, Centre Hospitalier Uni-
versitaire de Québec, Pavillon CHUL, 2705 Boul. Laurier, Sainte-Foy, Québec,
Canada, G1V 4G2. Fax 418-654-2715; e-mail Michel.G.Bergeron@crchul.
ulaval.ca.
Received April 21, 2005; accepted July 22, 2005.
Previously published online at DOI: 10.1373/clinchem.2005.052845
Copyright © 2005 by The American Association for Clinical Chemistry
Automation and
Analytical Techniques
platform was sensitive enough to detect 10 PCR-ampli-
fied bacterial genomes.
Conclusion: This microfluidic system for removing mi-
croarray hybridization onto glass slides is promising for
molecular diagnostics and gene profiling.
© 2005 American Association for Clinical Chemistry
In recent years, DNA microarrays have become powerful
tools for genomic research. Microarrays allow several
thousands of captured nucleic acid probes to be spotted
onto a small surface on a solid support, generally a glass
slide (1– 4 ). Efforts have been undertaken to adapt the
microarray technology for rapid identification of biomol-
ecules by means of signal transduction after binding to
specific probes attached to a solid support (5–10 ). Per se,
there is a need for a rapid (less than 1 h) and sensitive
microarray system, suitable for the molecular diagnosis of
infectious diseases, involving the detection of a wide
variety of microbial pathogens as well as associated
virulence genes such as antimicrobial resistance genes or
toxin genes (11, 12 ). Classic DNA microarray formats,
such as Affymetrix’s Genechip or custom microarrays on
glass slides, require hybridization times of 18 h for
detection of nucleic acids and are thus too slow for the
rapid diagnosis of infectious diseases. Microfluidics is an
emerging technology allowing the movement of mini-
mum volumes in microscopic channels and chambers that
are microfabricated into silicon, hard plastic, or soft
elastomer polydimethylsiloxane (PDMS) (13 ). Fluid pro-
pulsion and controlled valves have to be designed to
allow sequential displacement of liquids into the desired
channels and chambers (14 ). Microfluidic systems for
nucleic acid hybridization with micropumps (15 ), pneu-
matic pumps (16 ), and syringe pumps (6 ) have been
3
Nonstandard abbreviations: CD, compact disc; EOP, electroosmotic
pumping; PDMS, elastomer polydimethylsiloxane; SNP, single nucleotide
polymorphism.
2 Peytavi et al.: Rapid and Automated Microarray Hybridization
developed. However, these systems are complex, expen-
sive, and require special procedures for arraying bio-
probes and for detecting hybridization signals. In this
study, we have built and tested a removable microfluidic
structure allowing DNA hybridization onto a glass slide
microarray. After hybridization, microarrays can be ana-
lyzed externally with a standard scanner based on confo-
cal microscopy for glass slide microarray analysis. We
show that this microfluidic device allows the discrimina-
tion of single nucleotide polymorphisms (SNPs) to iden-
tify Staphylococcus species in 15 min.
Materials and Methods
capture probe hybridization
All chemical reagents were obtained from Sigma-Aldrich
and were used without further purification unless other-
wise noted. Oligodeoxyribonucleotide capture probes,
which were 5⬘-modified by the addition of two 9-carbon
spacers and an amino linker, were synthesized by Bio-
search Technologies. Four capture probes were used: (a),
a Staphylococcus aureus-specific probe (5⬘-CGTATTATCA-
AAAGACGAAG-3⬘); (b), an S. epidermidis-specific probe
(5⬘-CAIAGCTGAAGTATACGTAT-3⬘); (c), an S. hemolyti-
cus-specific probe (5⬘-CAAAATTTAAAGCAGACGTATA-
3⬘); and (d), an S. saprophyticus-specific probe (5⬘-AAAGCG-
GATGTTTACGTTTT-3⬘).
fabrication of the elastomeric flow cells
The microfluidic structures were fabricated using PDMS
replicating techniques reported by Duffy et al. (17 ). A
wide variation of PDMS structures can be molded using
microfabricated SU-8 micromolds (17–21 ). Two types of
photoresist (i.e., SU-8 25 and SU-8 100) available from
Microchem Inc. were used. SU-8 25 was used for the
microchannel structures, and SU-8 100 was used for the
reagent chambers. In the first step, SU-8 25 was processed
on a 15.24-cm reclaimed Si wafer (Addison Engineering)
to obtain the structures for the microchannels of 25 ␮m in
depth and the alignment marks for the second SU-8 25
layer. Subsequently, a thick layer (250 ␮m) of SU-8 100
was spin-coated over the substrate on which the molds for
the microchannels had been created. This thicker layer
was used to define the mold for the much larger reagent
reservoirs. Because crosslinked SU-8 photoresists have
lower optical transparency than their unexposed sur-
roundings, the alignment marks can be observed readily,
even when they are completely covered with a thick layer
of the unexposed photoresist. In the pattern design, we
compensated for possible alignment errors between the
two layers of photoresist. The channels and chambers
overlapped 50 ␮m in the connection areas to avoid
possible disconnections caused by misalignment. Six
identical molds were fabricated simultaneously onto the
15.24-cm Si wafer for faster replication.
PDMS was purchased from Dow Corning. For poly-
merization molding of the flow cell, the base (Sylguard
184 silicone elastomer) and the curing agent were thor-
oughly mixed in a weight proportion of 10:1. Because of
the thickness of the structures, low-temperature curing
(i.e., 65 °C) in a convection oven was preferred over
high-temperature baking. High temperature (e.g., 150 °C)
causes substantialt thermal stress at the interface between
the SU-8 patterns and the Si substrate that can actually
crack the substrate and peel off the SU-8 structures.
Leveling of the PDMS on the substrate is required to
achieve a uniform thickness over the entire flow cells.
preparation of glass slides
The glass surface was functionalized based on the chem-
ical reactions described by Joos et al. (22 ). All chemical
reactions were carried out in polypropylene jars. Surfaces
used were 25 ⫻ 75-mm microscope glass slides (VWR
International). After sonication (Branson 1210 ultrasonic
cleaner from Branson Ultrasonics Corporation) for 1 h in
deionized water, the slides were sonicated for 1 h in 40
mL of 10% NaOH, washed several times with deionized
water, and dried under a stream of nitrogen. The slides
were then sonicated in an aminopropyltrimethoxysilane
solution (2 mL water, 38 mL methanol, and 2 mL ami-
nopropyltrimethoxysilane) for 1 h, washed with metha-
nol, dried, and baked for 15 min at 110 °C. The amine-
modified slides were activated by sonication overnight in
40 mL of 1,4-dioxane containing 0.32 g (2 mmol) of
carbonyldiimidazole as a coupling agent, washed with
dioxane and diethyl ether, and dried under a stream of
nitrogen.
microarray production
Microarrays were fabricated based on the method previ-
ously reported by Schena et al. (23 ). Oligonucleotide
probes at 10 ␮mol/L in phosphate buffered saline (pH 7.4,
Sigma-Aldrich) supplemented with 1 mmol/L EDTA
were diluted 2fold by the addition of Array-it Microspot-
ting Solution Plus (Telechem International). Capture
probes were spotted in duplicate, using a Virtek SDDC-2
arrayer (Bio-Rad Laboratories) equiped with SMP2 pins
(Telechem International). Each spot had a volume of 0.6
nL and a diameter of 60 – 80 ␮m. Subsequently, the slides
were dried overnight, washed by immersion in boiling
0.1% Igepal CA-630 for 5 min, rinsed in boiling ultrapure
water for 5 min, and dried by centrifugation for 5 min
under vacuum (SpeedVac plus from Thermo Savant). The
slides were stored at room temperature in a dry and
oxygen-free environment.
pcr amplification and amplicon labeling
Amplicons of 368 bp were generated either by standard
PCR or by asymmetrical PCR amplification of purified
staphylococcal genomic DNA using the Staphylococcus-
specific primers previously described (24 ). Genomic
DNAs were purified from strains S. aureus ATCC 43300, S.
epidermidis ATCC 14990, S. hemolyticus ATCC 29970, and
S. saprophyticus ATCC 35552, as previously described (24 ).
Fluorescent Cy dyes were incorporated during asymmet-
 Clinical Chemistry 51, No. 10, 2005 3

rical PCR amplification (25 ). Cy-labeled dUTP nucleo-

tides (Amersham Biosciences) were used at a concentra-
tion of 0.02 mmol/L in a 25 ␮L PCR mixture containing
0.02 mmol/L dATP, 0.05 mmol/L dCTP, 0.05 mmol/L
dGTP, 0.05 mmol/L dTTP, 5 mmol/L KCl, 1 mmol/L
Tris-HCl (pH 9), 0.01% Triton X-100, 2.5 mmol/L MgCl2,
0.5 unit of Taq DNA polymerase (Promega), 0.2 ␮mol/L
of primer TstaG765, primer TstagG422 at 0.2 ␮mol/L for
standard PCR or at 0.005 ␮mol/L for asymmetric PCR,
and 1 ⫻ 10-4 to 1 ng of purified staphylococcal genomic
DNA (equivalent of 10 to 1 ⫻ 105 genome copies per PCR
reaction). Thermal cycling for PCR amplification (180 s at
94 °C followed by 40 cycles of 5 s at 95 °C, 30 s at 55 °C,
and 30 s at 72 °C) was carried out on a PTC-200 DNA
Engine thermocycler (MJ Research). For analytical detec-
tion limit assays, PCR amplifications were performed
using the equivalent of 1, 10, 100, 1000, and 10 000 genome
copies purified from S. aureus strain ATCC 43300 to
evaluate the minimal number of bacterial genome copies
that can be detected using the microfluidic platform.
Approximately 9 Cy-labeled nucleotides per amplicon
were incorporated during PCR.

dna microarray hybridization and data

acquisition
Five ␮L of amplified PCR reaction mixture containing the
Cy-labeled PCR amplicons were mixed with 15 ␮L of
hybridization buffer (8⫻SSPE (OmniPur, EM Sciences),
0.04% polyvinylpyrrolidone and 40% formamide). Passive
hybridization was performed in self-sticking, 20 ␮L, 15 ⫻
13-mm Hybri-well hybridization chambers (Sigma-Al-
drich). Amplicons produced by standard PCR were dena-
tured by heating at 95 °C for 5 min. Hybridization buffer
containing the labeled sample was introduced into the
chambers, and hybridization was conducted for 5 min at
room temperature. After hybridization, the microarrays
were washed with 2⫻SSPE containing 0.1% sodium do-
decyl sulfate for 5 min at room temperature and rinsed
once with 2⫻SSPE for 5 min. The microarrays were then
dried by centrifugation at 1350g for 3 min. The slides were
then scanned using a ScanArray 4000XL (Packard Bio-
science Biochip Technologies), and fluorescent signals
were analyzed using its QuantArrayTM software.
For flow-through hybridization, a unit consisting of a
glass slide and our homemade flow cell was used. This
unit was placed onto a prototype plastic disc support, and
the whole platform was fixed on the hub of a motor
controlled by a computer (Fig. 1C). The labeled sample
was prepared in the same way as for passive hybridiza-
tion. Two ␮L of sample and 10 ␮L of washing and rinsing
buffer were loaded onto the microfluidic unit just before
spinning the disc. The disc was spun at different speeds to
sequentially burst the centrifugal valves and to allow the
sample (12g), washing buffer (44g), and rinsing buffer
(50g) to flow through the hybridization chamber. The disc
was spun at high speed (100g) for 1 min to dry the slide
Fig. 1. Schematic representation of our prototype microfluidic system
for microarray hybridization.
(A), Molded PDMS microfluidic unit juxtaposed on a glass slide consisting of
chambers 2 (3.5 ␮L), 3 (12 ␮L), and 4 (10 ␮L), which allow the movement of
reagents through a middle microchannel 5 to reach the hybridization chamber 1
(140 nL). (B) Schematic view of the hybridization chamber of 400 ⫻ 3600 ␮m
showing the area of the chamber that can accommodate up to 150 nucleic acid
capture probes spotted onto a glass slide. (C) The microfluidic system molded in
PDMS is applied to a glass slide onto which the capture probes are arrayed. The
glass slide is placed on a CD support that can hold up to 5 slides overlaid with
PDMS. The hybridization reagents are positioned to be pumped sequentially
through the hybridization chamber by centrifugal force starting with chamber 2.
and then scanned as described above for passive hybrid-
ization.
Results
design of the hybridization units
This research is aimed at developing a simple, automated,
and affordable DNA hybridization unit using centrifugal
force for moving reagents. This system should be rapid,
discriminative, and sensitive enough to be used for direct
detection of microbes directly from clinical specimens.
4 Peytavi et al.: Rapid and Automated Microarray Hybridization
Amino-linker oligonucleotides specific to different staph-
ylococcal species are used as capture probes. The capture
probes are immobilized on 4 linear arrays of 5 ⫻ 70-␮m
spots on a standard 75 ⫻ 25-mm glass slide. A self-
contained hybridization process is performed into a flow
cell designed for the compact disc (CD) platform. The
flow cell consists of a hybridization column aligned with
the DNA microarrays spotted onto the glass slide, a
sample chamber, a washing buffer chamber, and a rinsing
buffer chamber. The reagent chambers are connected to
the hybridization column with a 50-␮m wide and 25-␮m
deep microchannel. The flow cell is aligned to adhere to
the glass slide to form a nucleic acid hybridization unit.
The microfluidic PDMS units and the glass slide with the
capture probes array are pressed together, and hydropho-
bicity of both materials allows leakage-free microfluidic
channels. With this prototype, numerically controlled
machining (Fig. 1). The reagents are positioned to be
displaced through the hybridization column by centrifu-
gal force in a sequence beginning with chamber 2, up to
chamber 4. Capillary valves contain the reagent in their
specific reservoir. The physical principle involved in
capillary valves is based on the surface tension, which
develops when the cross section of a hydrophilic capillary
expands abruptly (26, 27 ). The flow sequence is achieved
by shifting the balance between the capillary forces and
centrifugal pressure (28 ). By varying the angular velocity
of the driving motor, the flow rate of the different
solutions can be controlled. The sample containing the
labeled amplicons (chamber 2) is released first and flows
over the 140-nL hybridization chamber (chamber 1),
where the oligonucleotide capture probes are spotted
onto the glass support. The wash buffer (chamber 3) and
the rinsing buffer (chamber 4) flow sequentially at higher
angular velocities and are used to wash the nonspecifi-
cally bound targets after the hybridization step. After
running through the hybridization chamber, waste liq-
uids are collected in a furrow engraved in the CD plastic
platform.
In the flow-through system, the total amount of targets
transported onto the spot from the bulk solution was
approximated using diffusion layer theory (29 ). Under
laminar flow conditions, when a sample solution flows
over the capture arrays, the target DNA in a layer close to
the spot surface will be depleted during the hybridization
reaction. Close to the surface of the chamber, the velocity
of the fluid approaches zero and can be regarded as
forming a diffusion boundary layer having a thickness
inversely proportional to the cubic root of the stream
velocity (29 ). Within this layer, depletion to some degree
is expected despite the mass transport caused by convec-
tion (30 ). The “diffusion layer thickness” can be estimated
from Equation (1). The equation is derived from a case of
diffusion layer inside a circular tube (29 ) and has been
adapted for laminar flow between two parallel plates of
infinite width shown in Fig. 2,
Fig. 2. A schematic illustration of the laminar flow between 2 parallel
plates.
H represents the depth of the hybridization chamber, x is the distance from the
edge of the measured region along flow direction, and v0 is the maximum velocity
of the fluid, which is the velocity at the center of the chamber. ␦ represents the
diffusion layer thickness. A parabolic velocity profile is expected in the vertical
direction. Because the width (the transverse dimension, i.e., w0 in Equation no.
2) of the plate is substantialtly larger than the height, a flat velocity profile and,
consequently, a uniform accumulation of targets are expected in width direction
of the hybridization surface (400 ⫻ 3600 ␮m).
␴⫽
1 DHx
冉 冊
0.67 2v 0
1/3
(1)
where D is the diffusion coefficient of the target mole-
cules, H represent the depth of the hybridization chamber,
x is the distance from the edge of the measured region
along flow direction, and v0 is the maximum velocity of
the fluid, which is the velocity at the center of the
chamber.
Total accumulation of captured DNA molecules on the
surface over a certain amount of time (which is propor-
tional to the inverse of the maximum flow velocity when
the sample volume is constant), is then obtained from
Equation (2), which was adapted for our self-contained
flow cells from the equation for flux in a circular tube (29 ),
3w
F flow ⫽ c V(2D 2x2)1/3 H ⫺4/3v 0⫺2/3 (2)
4w 0 0
where Fflow is the total molar concentration of target
molecules on the hybridization surface, w0 is the width of
the hybridization chamber, w is the diameter of the spots,
c0 is the sample concentration, and V is the sample
volume. The specification of the microfluidic design and
operational parameters was guided by this equation. Note
that, if all the other parameters remain constant, the depth
of the hybridization chamber is the most sensitive factor
affecting the accumulation of captured DNA. Smaller
depths yield higher accumulation.
For passive hybridization, the accumulation of targets
on the hybridization surface can be predicted using Equa-
tion 3 (31 ),
F psv ⫽ 2cA0wx 冑 Dt
␲
(3)
where Fpsv is the accumulation of the targets, cA0 is the
concentration of target at the surface, and t is the time.
 Clinical Chemistry 51, No. 10, 2005
flow-through hybridization in 15 mins
The flow-through hybridization unit combines a PDMS
flow cell juxtaposed with a glass slide a few min before
the experiment without performing any surface treatment
or adhesion step. This microfluidic system allows robust
control of valve openings to sequentially release the
contents of different chambers. Indeed, Fig. 3 shows that
the bursting range in rotation per min for each of the 3
centrifugal valves of our CD microfluidic system do not
overlap.
Each staphylococcal amplicon at 10 nmol/L generated
by asymmetrical PCR was hybridized using both passive
and flow-through hybridizations. For flow-through hy-
bridization, loading of the reagents was performed imme-
diately before spinning the disc platform to avoid reagent
evaporation. A spin speed was selected so as to obtain a
sample flow rate of 0.4 ␮L/min in the hybridization
chamber, which corresponds to a hybridization time of 5
min considering that 2 ␮L of sample were loaded onto the
microfluidic unit. This hybridization time is identical to
the one used in the passive hybridization experiments.
After the hybridization step, the spin speed of the plat-
form was increased to sequentially burst the centrifuga-
tion valves, releasing 10 ␮L of washing buffer and 10 ␮L
of rinsing buffer, respectively, into the hybridization
chamber. These two buffers flowed through the hybrid-
ization chamber with an mean flow rate of 2.2 ␮L/min in
⬃9 min. Finally, there was a 1-min drying step (high spin
speed). Therefore, the total time for the entire hybridiza-
tion process was ⬃15 min. Subsequently, the PDMS
microfluidic flow cells were pealed off, and the hybrid-
ized microarrays were scanned. Plotting the fluorescence
intensity revealed that flow-through hybridization in a
140-nL chamber was more sensitive than passive hybrid-
Fig. 3. Bursting range for each of the 3 centrifugal valves of the CD
microfluidic system.
The bursting range in RPM for each valve was determined by performing
flow-through hybridizations using 15 independent microfluidic units.
5
ization in the 20-␮L chamber. Five min of hybridization
with 10 nmol/L of amplicon generated from S. aureus, S.
epidermidis, S. hemolyticus, and S. saprophyticus showed
ratios of 9.5, 13.5, 18.7, and 6.9, respectively, between
flow-through and passive hybridizations (Fig. 4). Hybrid-
izations of amplicons generated by standard PCR ampli-
fication from the equivalent of 1, 10, 100, 1000, or 10 000
genome copies were performed using the flow-through
hybridization system. It was found that the equivalent of
as little as 10 genome copies of starting material was
sufficient to produce an unambiguous hybridization sig-
nal (Fig. 5).
discrimination of 4 clinically important
Staphylococcus species
Staphylococcus-specific PCR primers targeting the tuf gene
were used to amplify a 368-bp fragment from S. aureus, S.
epidermidis, S. hemolyticus, and S. saprophyticus purified
genomic DNAs. Species-specific capture probes targeting
these 4 staphylococcal species were arrayed onto glass
slides and hybridized with the 4 different staphylococcal
amplicons. The results demonstrated that it was possible
to detect and discriminate the 4 different staphylococcal
tuf amplicons (Fig. 6). The S. epidermidis-specific oligonu-
cleotide probe was designed in a specific area of its
genome that differs from the S. aureus sequence by only a
an SNP. A nucleotide analog was added at a strategic
location in the S. epidermidis probe to make it more
discriminative for the S. aureus amplicon. Using this
strategy, hybridization of S. aureus amplicons gave a
fluorescence signal ⬃6 times stronger with the S. aureus
probe compared with the S. epidermidis probe (Fig. 6). The
other oligonucleotide capture probes had at least 3 nucle-
otide mismatches with the nonhomologous Staphylococcus
amplicons, and no substantialt cross-hybridization was
observed. In addition, a mixture of the 4 Staphylococcus
amplicons (each at 1 nmol/L), hybridized on the same
Fig. 4. Comparison between the sensitivity of labeled staphylococcal
amplicon detection in passive hybridization vs flow-through hybridiza-
tion.
Both passive and flow-through hybridizations were carried out for 5 min at room
temperature with 10 nmol/L of 368 bp staphylococcal Cy-labeled amplicons. The
graph shows the mean fluorescence intensity in units for each species-specific
capture probe. Standard deviations are for the results of 5 hybridizations.
6 Peytavi et al.: Rapid and Automated Microarray Hybridization

Fig. 5. Analytical detection limit assays using the prototype microfluidic system.
Hybridization to microarrays of an S. aureus-specific capture probe was performed using Cy-labeled tuf gene amplicons generated by standard PCR amplification of 1,
10, 100, 1000, or 10 000 genome copies of S. aureus. The graph shows the mean fluorescence intensity for each bacterial genome copy number tested. Standard
deviations are for the results of 4 hybridizations.

microarray using our microfluidic system, showed fluo-
rescence hybridization signals of intensity similar to those
shown in Fig. 6 for each of the 4 species-specific capture
probes.
Discussion
In recent years, microarrays have become tools of choice
for gene expression profiling. The expression of thou-
sands of genes can be monitored in a single experiment
with this technology. Several investigators have at-
tempted to adapt this technology to rapidly detect infec-
tious agents in clinical specimens for diagnostic purposes
(6, 8, 32–34 ). However, such systems are still in their
infancy, and most of them require technologically com-
plex systems with integrated heating/cooling (6, 8, 16 ).
This study reports the merging of standard microarray
glass slide technology with a simple, low-cost microflu-
idic device. We demonstrated that nanoliter volumes of
liquid can be moved precisely into channels and cham-
bers on the glass slide surface created with a microfluidic
elastomeric flow cell juxtaposed above the slide. This
custom microarray hybridization microfluidic platform is
easy to use, automated, and rapid. It uses standard glass
slides compatible with commercial arrayers and scanners
found in most academic departments. In this removable
microfluidic system, the hybridization chamber is com-
posed of a microfluidic network engrafted onto a dispos-
able low-cost elastomeric material. This elastomeric ma-
terial reversibly sticks without any adhesive or chemical
reaction to the glass slide, forming together the microflu-
idic unit. Placed onto a plastic CD-like support, the
microfluidic units are spun at different speeds to control
fluid movements. To simplify hybridization experiments
using this device, buffer compositions and capture probe
sequences are optimized to be compatible with room
temperature hybridizations to avoid the need for a heat-
ing device. Furthermore, this microfluidic system allows a
drastically reduction in the volume of reagents needed for
microarray hybridizations and does not require a PCR
amplicon purification step, which may be time-consum-
ing.
Among various pumping methods attempted in re-
search of flow-through DNA chips, one is electroosmotic
pumping (EOP) (35– 40 ). However, DNA has a high
negative electrophoretic mobility because of the large
number of negative charges carried by its phosphate
 Clinical Chemistry 51, No. 10, 2005 7

Fig. 6. Microarray hybridization applying the prototype microfluidic system.

Hybridization to microarrays of species-specific capture probes targeting staphylococcal tuf sequences were performed using Cy-labeled tuf gene amplicons generated
by asymmetric PCR amplification of 1 ng of genomic DNA purified from 4 staphylococcal species. (A) Crude images of microarray, hybridized 5 min at room temperature,
with the 4 different staphylococcal labeled amplicons. (B) Graphs showing the mean fluorescence intensity in units for each capture probe. Standard deviations are
for the results of 5 hybridizations.

groups at most pH values. Consequently, EOP of DNA
requires a high electroosmotic mobility buffer to over-
come the negative electrophoretic mobility of the DNA
molecules. Furthermore, DNA hybridization buffers often
contain high concentrations of salt that reduce the elec-
troosmosis effect and make the EOP a less effective
approach (16 ). Pumping with mechanical pressure pre-
sents some advantages over the EOP approach as it is
insensitive to pH, to the charge of moving molecules, and
to salt concentration. However, high back pressures can
be generated because of the high flow resistance caused
by the small dimensions of the microchannels. Conse-
quently, leakage is often a problem if the unit is not
sealed. With the CD platform, the reagents are delivered
by centrifugal force generated over the entire length of the
liquid element. Therefore, local high pressure is avoided,
and as a result, interface sealing is readily accomplished.
In fact, no leakage was observed with this approach. In
the present study, a PDMS was selected to make the
microchambers and channels. PDMS is a low-cost mate-
rial that can be easily prototyped and can make reversible
and watertight seals with glass slides. Each microfluidic
unit, composed of the molded PDMS juxtaposed on a
glass slide, was placed into a custom-made plastic disc
support (Fig. 1). Centripetal force was used to move the
liquid into the microfluidic chambers and channels as
previously described by us (41, 42 ). The rapidity of the
hybridization reactions prevents reagent evaporation
problems that may be encountered in slower standard
microarray hybridization methods.
In a passive hybridization system, a hybridization
event requiring collision between a capture probe and the
analyte relies solely on diffusion. In such systems, detec-
tion limit is increased by means of long hybridization
periods (43, 44 ). One advantage of flow-through hybrid-
ization is that, because of the shallow architecture of the
microchannels, the probability of collision between the
probe and the analyte is increased by the much shorter
diffusion distance. This allows fluid movement of nano-
liter volumes over the capture probes, thereby accelerat-
ing the hybridization kinetics (44 – 46 ). Using a microflu-
idic system, Chung et al. (47 ) have shown a 6fold increase
in hybridization efficiency with the flow-through hybrid-
ization in a 33 ␮L chamber compared with the passive
hybridization performed in a chamber of the same vol-
ume. In our work, we increased even further the kinetic of
hybridization with a much smaller hybridization chamber
(140 nL) combined with flow-through hybridization.
Flow-through hybridization in such a small hybridization
chamber allowed a substantialt reduction of the reagent
and sample volumes (1/10) while reducing hybridization
time to 5 min. This rapid hybridization by means of our
microfluidic system increased the kinetic of hybridization
by an average of 10.5fold compared with passive hybrid-
ization using 368 bp PCR amplicons as nucleic acid
targets. By contrast, 30 min was required for hybridiza-
8 Peytavi et al.: Rapid and Automated Microarray Hybridization
tion in the flow-through hybridization system described
by Chung et al. (47 ). Furthermore, there is no need to
purify the target PCR amplicons before hybridization,
thereby reducing the overall time of the assay.
To be useful for the diagnosis of infectious diseases in
clinical laboratories, a molecular test should be highly
sensitive, specific, and ideally, rapid and inexpensive. Our
system showed a detection limit of 500 amol of amplified
target. This detection limit is comparable to those ob-
tained with microfluidic devices that are more complex to
manufacture (mainly irreversibly bounded plastic devices
actuated with active valves) and requiring much longer
hybridization times (6, 48 ). One system using chemilumi-
nescence shows a detection limit of 250 amol, but requires
a 3-h hybridization period (49 ), too long for practical use
in clinical diagnostics (50 ). Here, we report a microfluidic
system that allows detection of PCR amplicons generated
from the amplification of 10 bacterial genome copies,
which is at least 1000 times more sensitive than results
obtained by other groups showing microarray hybridiza-
tion using microfluidic devices (15 ). The addition of an
amplicon concentration step, as well as the use of a
microfluidic device for faster PCR thermocycling, should
allow detection of even lower copy numbers.
In terms of specificity, this simple system discrimi-
nated among 4 different Staphylococcus species with a
post-PCR hybridization protocol of only 15 min. An
artificial mismatch created by the addition of an inosine
was incorporated in the S. epidermidis capture probe to
further destabilize binding events with the S. aureus
amplicons differing by only an SNP. The nucleotide
analog was located 4 nucleotides upstream from the SNP
present in the S. aureus genomic DNA sequence. Our
actual device design can accommodate up to 150 spots in
the 140-nL hybridization chamber. This should be suffi-
cient for some practical applications in the diagnostic
field, but future improvements in spot density and hy-
bridization chamber geometry will substantialtly increase
the number of spots in the microfluidic hybridization
chamber.
In conclusion, this microfluidic microarray system could
become a tool of choice for the rapid identification of
nucleic acid sequences in fields such as molecular diag-
nostics, detection of bioterrorism agents, food quality
survey, and forensic analysis. This prototype paves the
way toward the development of micro-total analysis
system (m-TAS) on a compact disc support, where sample
preparation, PCR amplification, and microarray hybrid-
ization and detection could be accomplished with a single
portable device for point-of-care diagnostic applications.
We thank Yves Lechasseur for technical help in setting up
the rotational platform, Eric Martel for mathematical
assistance, and Guy Boissinot for work on the plastic
support. This research project was supported by grants
from “Valorisation de la Recherche du Québec”, from
“Génome Québec” and Genome Canada, and from the
Canadian Institutes of Health Research (PA-15586). M.O.
is a Burroughs Welcome Fund scholar in molecular par-
asitology and the holder of a Canada Research Chair in
antimicrobial resistance.
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