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Biotechnology and Bioprocess Engineering 2007, 12: 634-639

Microfluidic Lab-on-a-chip for Microbial

Identification on a DNA Microarray
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Department of Chemical Engineering, Myongji University, Yongin 449-728, Korea
2
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA

^Äëíê~Åí= A lab-on-a-chip for the rapid identification of microbial species has been developed for a water monitoring system. We em-
ployed highly parallel DNA microarrays for the direct profiling of microbial populations in a sample. For the integration and
minimization of the DNA microarray protocols for bacterial identification, rRNA was selected as a target nucleotide for
probe:target hybridization. In order to hybridize target rRNA onto the probe oligonucleotide, intact rRNA extracted from bK=
Åçäá rRNA was fragmented via chemical techniques in the lab-on-a-chip platform. The size of fragmented rRNA was less
than 400 base pairs, which was confirmed by polyacrylamide gel electrophoresis. The fragmented rRNA was also labeled
using fluorescent chemicals. The lab-on-a-chip for fragmentation and labeling includes a PDMS chaotic mixer for efficient
mixing, operated by flow pressure. In addition, the fragmented rRNA was hybridized successfully on a DNA microarray with
sample recirculation on a microfluidic platform. Our fragmentation and labeling technique will have far-reaching applications,
which require rapid but complicated chemical genetic material processing on a lab-on-a-chip platform. © KSBB

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A rapid microbial-detection system has an advantage over
the existing time-consuming culture-based procedures for a
variety of applications. The current approaches to microbial
monitoring, for example in the clinical field, involve tradi-
tional plate culturing techniques, which provide results in
several days. In addition, these techniques are not sufficient
for the comprehensive monitoring of all potentially problem-
atic microbes [1]. Recently, DMS (DNA Microarray Sensor)
technology has been used for rapid response time and a
highly parallel analysis of target microbes [2]. For the effec-
tive detection of target microbes with the DMS, PCR (Poly-
merase Chain Reaction), or other target nucleotide amplify-
cation schemes have been employed. However, these ampli-
fication methods require more time and effort for their proc-
esses [3]. In addition, the amplifications often introduce bi-
ases for the quantification of natural bacterial populations.
Bacterial 16S ribosomal RNA (rRNA) has been utilized as
a target nucleotide for its identification. The level of sp-
ecificity of the bacterial identification with 16S rRNA is
sufficient for differentiation between very closely related
microorganisms. Besides its specificity, rRNA itself is the
G`çêêÉëéçåÇáåÖ=~ìíÜçê
Tel: +82-31-330-6392 Fax: +82-31-337-1920
e-mail: hyunho@mju.ac.kr
most abundant genetic material within microorganisms.
Therefore, the use of naturally amplified rRNA in bacterial
detection schemes would be clearly advantageous [4-6].
Typically, the purification of nucleic acids from speci-
mens, most notably bacterial cultures, has traditionally been
both laborious and time-consuming. Moreover, the typical
method requires several pre-treatment and centrifugation
steps [7]. However, there are many situations in which pre-
treatment and centrifugation are not feasible, for example,
when portable instruments must be utilized for analysis.
Therefore, a substitute technique for pre-treatment and cen-
trifugation should be devised, and a microfluidic system is
currently the most promising candidate [3,7].
Microfluidics is a technology which enables many new
experimental sciences, including microbiology [7-10]. Mi-
crofluidics allows for the use of small volumes [7,8], avoids
the need for reagents and reduces waste costs, and creates
new types of assays which are impossible at the macroscopic
scale. Recently, experimental designs involving the rapid
fabrication of a versatile laminate-based technology have
allowed for the integration of multiple detection processes on
dedicated microfluidic systems [8].
The coupling of microfluidics with DNA microarrays us-
ing rRNA is a more simple process than other more classi-
cally used techniques, as it requires no additional PCR-based
amplification of genetic materials. Instead, the size of rRNA
targeted for the DNA microarray hybridization should be

shortened, to enable effective probe:target hybridization
[4,6].
On a microfluidic platform, when two laminar flows meet,
a transverse diffusive transport is required for mixing. Un-
fortunately, the laminar nature of these low Reynolds num-
ber (Re) flows involves diffusion only as the primary trans-
verse transport. As the diffusive timescale is far slower than
that of the flow, undesired long channels are often necessary
in order for full mixing to occur. Recently, a wonderful ap-
proach has been developed to overcome these limitations
(e.g. chaotic advection) [11]. By the introduction of a her-
ringbone structure beneath a straight smooth microchannel,
the mixing length, where transverse elements stretch and
fold volumes of fluid over the cross section of the micro-
channel, could be reduced [11]. In addition, via the simple
introduction of curvature and size changes in a cross section
of the microchannel, it was reported that enhanced mi-
cromixing could be achieved [12].
In this study, a lab-on-a-chip system featuring chaotic her-
ringbone structures with multiple curvatures for rRNA frag-
mentation and labeling was constructed andevaluated. The
major accomplishment of the lab-on-a-chip platform was
that complicated chemical processes for rRNA fragmenta-
tion could be integrated into a credit-card sized platform. In
addition, the platform was embedded with a passive mixer,
which could enhance the yield of the lab-on-a-chip chemical
reaction. Following the rRNA fragmentation and labeling on
the first lab-on-a-chip, microbial identification was con-
ducted on the second lab-on-a-chip, which was a recirculat-
ing DNA microarray microfluidic system [13]. The sensitiv-
ity of the microbial identification was increased by the recir-
culating microfluidic system.
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For the fabrication of the PolyDiMethyl Siloxane (PDMS)
(Sylgard 184, Dow Corning, Midland, MI, USA) passive
mixer, a 4-inch silicon wafer was employed as a mold master.
The pattern of the silicon master was determined by photo-
lithography with SU-8 (Microchem, Newton, MA, USA).
The herringbone structure was constructed by two-step li-
thography with SU-8. The straight channel was first pat-
terned with a width of 400 μm and a height of 150 μm. The
herringbone pattern was then patterned on the straight chan-
nel pattern with a ridge amplitude of 50 μm. For a perfect
mixing test, a PDMS microchannel device with 14 cycles of
herringbone units with two curvatures was prepared and, for
the fragmentation and labeling on the lab-on-a-chip, a mi-
crochannel with 25 cycles of herringbone units with 6 curva-
tures was fabricated. To prepare the PDMS microchannel, a
freshly prepared mixture of PDMS prepolymer and curing
agent at a ratio of 10:1 (v:v) was thoroughly mixed and de-
gassed under vacuum. Then, 5 mL of a prepolymer-curing
agent mixture was poured onto the silicon template and
cured in an oven for 2 h at 65oC. The whole assembly was
Biotechnol. Bioprocess Eng. SPR=
^= = = = = = = = = = = = = = = = = = = = = _
cáÖK=NK (A) A microscopic image of the PDMS chaotic mixer and
(B) a photograph of the mixing examination of the chaotic
passive mixer for rRNA fragmentation and labeling. For
the test mixing of two laminar flows, the PDMS chaotic
mixer consists of 14 herringbone units, which feature 20
ridges and 2 curvatures, as shown in Fig. 1B.
removed from the oven and cooled, after which the PDMS
was peeled off the master, thereby generating a replica with
the designed topographical layout (Fig. 1). The PDMS rep-
lica was treated with oxygen plasma and irreversibly at-
tached onto 1′ × 1′ slide glass. The slide glass was drilled
with a hole, which was then connected to the Mylar laminate
microfluidic module.
c~ÄêáÅ~íáçå=çÑ=i~ÄJçåJ~JÅÜáé
The fragmentation and labeling lab-chip was constructed
of seven laminated devices made from Mylar with pressure-
sensitive adhesive on either side (Fralock Inc., San Carlos,
CA, USA). A rectangular channel and reaction chamber was
then cut into the Mylar layer using a programmable CO2
laser cutting system (Universal Laser Systems Inc., Scotts-
dale, AZ, USA), thereby yielding a straight channel. A
schematic diagram of the layered Mylar structure is shown in
Fig. 2.
The recirculating DNA microarray microfluidics was also
fabricated with Mylar laminate layers. Each device was fab-
ricated of standard glass microscope slides for the top and
bottom layers, and a middle Mylar layer. Its detailed con-
figuration and fabrication was conducted almost identically
with that of a recirculation device, as reported previously
[11].
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A cell pellet (E. coli KB91) obtained from 1 mL of log-
phase culture was lysed by bead beating, extracted with phe-
nol-chloroform, and ethanol-precipitated using a kit designed
from the Qbiogen FastPrep® Kit and instrument (Irvine, CA,
USA). The kit is a single-reagent extraction technique de-
signed to quickly and efficiently isolate the total cellular
RNA from bacteria. The average RNA yield from 1010 bac-
teria is approximately 50 μg. In order to confirm successful
SPS=
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_ After reduction with NaCNBH3 and ethanol precipitation,
cáÖK=OK= Electropherogram of (A) RNA size marker and (B) ex-
tracted bK= Åçäá= KB91 rRNA by Agilent RNA 6000 Nano
Assay.
cáÖK=PK Diagram and picture of fragmentation and labeling lab-
on-a-chip embedding two serpentine reservoirs of
fragmentation chemicals and rRNA solution.
RNA extraction, an RNA 6000 Nano Assay (Agilent, Palo
Alto, CA, USA) was utilized, and the extracted rRNA was
identified (Fig. 3).
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All reagents were prepared freshly in DEPC-treated water.
For microtube-scale fragmentation and labeling, the total
volume of the labeling-fragmentation reaction was 130 μL.
The reaction mixture contained 10 μg of extracted rRNA, 20
mM sodium phosphate (pH 7.0), 5 mM O-phenanthroline
hydrochloride monohydrate (OP) (Fluka, Ronkonkoma, NY,
USA), 0.5 mM CuSO4 × 5H2O (Cu, Sigma-Aldrich, St.
Louis, MO, USA) (stock solutions of 150 mM OP and 15
mM Cu may be stored for at least one year at RT), 2 mM
freshly prepared and filtered Lissamine Rhodamine B ethyl-
ene diamine (LissRhod) (Molecular Probes, Eugene, OR,
USA). The solution was then added to a final concentration
of 4.5 mM H2O2 and heated for 5 min at 95oC. Reduction
was conducted by the addition of a final concentration of 23
mM sodium cyanoborohydride and incubated for 10 min at
95oC. The mixture was then ethanol-treated in order to pre-
cipitate and recover the fragmented rRNA. For indirect la-
beling (labeling with a non-fluorescent chemical) with OP-
Cu, ethylenediamine (10 μL, 0.5 M) was used rather than the
the RNA pellet was vacuum-dried and dissolved in 100 μL
of DEPC water for both direct and indirect labeling.
i~ÄJçåJ~JÅÜáé=cê~ÖãÉåí~íáçå=~åÇ=i~ÄÉäáåÖ
All liquid handling components were provided in the lab-
on-a-chip system, which included laser-cut fluid microchan-
nels, pneumatic valvings, and sample/reagent reservoirs. The
components were integrated in a MicroFlow system (Mic-
ronics, Redmond, WA, USA). Liquid handling automation
was achieved using control software (Micronics’ micro-
Scripts) and a benchtop pumping system through a card
manifold interface.
For the rRNA sample inlet, 10 μg/mL of extracted rRNA
in DI water was introduced by the MicroFlow system at 0.2
μL/s. For the reagent inlet, 20 mM sodium phosphate (pH
7.0), 10 mM OP, 1.0 mM CuSO4 × 5H2O, 4 mM freshly
prepared and filtered LissRhod, 9.0 mM H2O2 and 45 mM
sodium cyanoborohydride were introduced through the same
flow system. Two inlet flows were merged at an entrance of
the chaotic mixer. The mixed solution was then supplied into
a fragmentation and labeling chamber. The mixture was
heated at 70 or 85oC.
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The microfluidic device was mounted on the stage of a
Zeiss ICM-405 inverted, epifluorescence microscope (Carl
Zeiss Inc., Thornwood, NY, USA) and imaged the sensor
through a 10 × objective. A rhodamine filter set (excitation
bandpass: 520~560 nm; dichroic beamsplitter: 580 nm;
emission bandpass: 575~635 nm) was used to condition the
light. To obtain the images after hybridization, the microflu-
idic microarray was affixed to thermo-table mounted on the
stage of a custom-designed epifluorescence microscope
(State Optical Institute, St. Petersburg, Russia). Hybridiza-
tions were conducted at room temperature (20oC) for 2.5 h.
After hybridization, the DNA microarray was washed with a
solution of 0.9 M NaCl, 20 mM Tris-HCl (pH 8.0), and 40%
formamide.
obpriqp=^ka=afp`rppflk=
Fig. 1 shows (A) a microscopic image of the PDMS cha-
otic mixer and (B) a photograph of the mixing examination
of the chaotic mixer. The master structures on the Si wafer
were constructed with a two-step photolithography proce-
dure in an SU-8 photoresistance system. The first layer of
photolithography defined the microchannel structure; the

second layer defined the pattern of ridges. The pattern of
ridges was aligned to lie atop the channel structure in the
first layer. The microfluidic device is constructed by mold-
ing in polydimethyl-siloxane (PDMS) using soft lithography
techniques. It readily bonds to any hydroxyl-terminated sur-
face (such as glass) after a brief oxygen plasma treatment.
As is shown in Fig. 1A, by introduction of herringbone
units atop a straight microchannel, an effective mixing unit
could be fabricated. Each herringbone unit has 20 ridges
with a 45o orientation. The amplitude of ridges (αh) is 50 μm
with a channel height (h) of 150 μm.
In Fig. 1B, two laminar flows with red and green dyes
were mixed thoroughly after passage of the chaotic mixer.
The flow rate was 0.2 μL/s. Two differently dyed buffers
were separately supplied at the entrance of the mixer and the
final flow was split after the passage of the mixer, as is
shown in Fig. 1B. By comparing the mixed color of the split
flows, it was shown that perfect mixing occurred during the
passage of the mixer. Even at a flow rate as high as 0.5 μL/s,
the two differently colored liquids were mixed completely
through the passive mixer.
Without an external power source, the ability to achieve
effective mixing in a microchannel between two parallel
streams is crucial to a variety of applications. Unfortunately,
the laminar nature of these low Reynolds number (Re) flows
involves only diffusion as the primary process of lateral
transport. This frequently results in an undesired long chan-
nel in order for diffusive mixing to occur. In order to reduce
the mixing length, there must be transverse components over
the cross-section of the microchannel. For a straight mixer,
the downstream distance over which liquids must traverse to
be fully intermixed, or Δym, is calculated by Eq. (1)
d2
Δy m ~ V × = Pe × d (1)
D peroxide and radical generating coordination complex, 1,10-
where V is the average flow velocity, D is the diffusion coef-
ficient for rRNA, d is a length scale associated with the
channel diameter, and Pe is a Peclet number [11]. However,
an introduction of chaotic stirring flows by the herringbone
units is expected to reduce the downstream distance, Δym,
which is calculated as Eq. (2)
Δym ~ λ ln(Pe) (2)
where λ is a characteristic length determined by the geometry
and trajectory in the chaotic flow [11]. Comparing Eq. (1)
and (2), it can be observed that the downstream mixing length
increases linearly by Peclet number without chaotic mixing,
but increases logarithmically by Peclet number with chaotic
mixing. Therefore, for a high Peclet number flow, the mixing
length will be shorter with the mixer than without it.
Fig. 2 shows the electropherogram of the (A) size marker
of RNA and the (B) extracted E. coli KB91 rRNA. Fig. 2
proves the successful extraction or preparation of rRNA by
extraction. Intact 16S rRNA was detected clearly with the
bead beating rRNA extraction method.
Fig. 3 shows the diagram and picture of the fragmentation
and labeling lab-on-a-chip embedding of two serpentine res-
Biotechnol. Bioprocess Eng. SPT=
^= = = = = = = = = = = = = = = = = = = = = = _
=
cáÖK=QK Electrophoresis of labeled and fragmented bK= Åçäá KB91
rRNA under denaturing conditions (7 M urea) on 8%
polyacrylamide gel.
ervoirs of peroxide chemical mixture and rRNA solution.
Fig. 3 also shows a detailed component for the lab-on-a-chip
system. The mixer unit was integrated into a polymeric unit,
which was fabricated with a CO2 laser cutter using acrylate
and mylar. To ensure perfect mixing with a possible high
flow rate requirement, such as > 2.0 μL/s, 25 herringbone
units and 6 curvatures were designed for the PDMS chaotic
mixer. The mixer, fragmentation chamber, and purification
filters can be connected by an external silicon tube, which
were interfaced with the O-ring inside of the lab-on-a-chip.
The reagent for the fluorescent labeling and fragmentation
is an oxidant with free radical characteristics [4,6]. It is a
phenanthroline-Cu(II) (OP-Cu). Therefore, a random non-
specific reaction by a chemical nuclease proceeds to a sin-
gle-strand nucleotide, i.e., rRNA, in the reaction chamber.
As the total volume of liquid contained in the reaction
chamber is 150 μL, the retention time of the reaction mixture
within the reactor is quite dependent upon the flow rate of
0.05~0.4 μL/s. At high temperatures, i.e., 95oC, the lab-on-a-
chip fractured after several usages. Therefore, all the ex-
periments were conducted at temperatures of below 95oC, as
is shown in Fig. 3.
Fig. 4 shows the results of the electrophoresis of labeled
and fragmented E. coli KB91 rRNA under denaturing condi-
tions (7 M urea) on 8% polyacrylamide gel. Fig. 4A is the
fluorescence image prior to ethidium bromide staining and
Fig. 4B is an ethidium bromide-stained image. Therefore, for
Fig. 4A, unless the rRNA was labeled with a fluorescent
chemical, no band was detected. Lane 1 shows the single
strand marker without fluorescent labeling, lane 2 shows the
fluorescent (Oregon Green) labeled oligonucleotides, 18-
and 42-mer, lane 3 shows the fragmented rRNA with ethyl-
ene diamine rather than LissRhod (indirect labeling) at 85oC
on the lab-on-a-chip system, lane 4 shows the fragmented
and labeled rRNA with LissRhod (direct labeling) at 85oC on
SPU=
q~ÄäÉ=NK DNA oligonucleotide probes detected in this study
Probe DNA Sequence (5′ to 3′)
Eub338 5′-GCTGCCTCCCGTAGGAGT
Eub338-g11c 5′-GCTGCCTCCCCTAGGAGT
Eub338-III 5′-GCTGCCACCCGTAGGTGT
Eub336 5′-TGCCTCCCGTAGGAGTCT
Spir344 5′-CTTARCTGCTGCCTCCCG
Spir344-g8c 5′-CTTARCTCCTGCCTCCCG
R is the IUPAC symbol for the presence of adenine or guanine.
cáÖK=RK= A hybridization image after continuous recirculation (0.1
mL/min) of the hybridization buffer including lab-on-a-chip
fragmented rRNA.
the lab-on-a-chip system, lane 5 shows the fragmented
rRNA with ethylene diamine rather than LissRhod at 70oC
on the lab-on-a-chip system, and lane 6 shows intact E. coli
KB91 rRNA without any treatment. All samples in Fig. 4
were ethanol-precipitated and rRNA was recovered after the
lab-on-a-chip fragmentation.
As is shown in Fig. 4, regardless of the existence of
ethidium bromide, fluorescently labeled rRNA evidenced
bands which are shown in both Figs. 4A and 4B. Lane 4
shows bands in both Figs. 4A and 4B, indicating a success-
fully fragmented and fluorescently labeled rRNA. The size
of the fragmented rRNA size was measured to be approxi-
mately 300~400 base pairs. However, lane 5 shows only one
band in Fig. 4B with ethidium bromide. Therefore, we de-
termined that 85oC is a more appropriate fragmentation con-
dition than 70oC on the lab-on-a-chip system.
Fig. 5 shows the image data collected on-line following
continuous recirculation (0.1 mL/min) of the hybridization
buffer, including the same fragmented rRNA (10 μg/mL), as
is shown in lane 4 of Fig. 4. All the experimental details of
the recirculation system for the DNA microarray are illus-
trated in the previous report [13]. After 2.5 h of continuous
recirculation, we noted a significant hybridization of target
rRNA to the probes Eub338, Eub336, Eub338-g11c, Spir344,
and Spir344-g8c (reflected by profound fluorescent intensi-
ties). The sequences of the oligonucleotideswhich represent
the genetic sequences of the bacterial rRNA, are shown in
Table 1. Eub338 targets general Bacteria 16S rRNA, Eub-
336 targets another general Bacteria 16S rRNA, Eub338-
g11c targets Bacteria 16S rRNA harboring a mutation at the
11th position with C rather than G, Spir344 targets Spiro-
chetes 16S rRNA, and Spir344-g8c targets Spirochetes 16S
rRNA harboring a mutation at the 8th position with C rather
than G [6,14]. As is shown in Fig. 5, the fragmented E. coli
rRNA was hybridized successfully on a microfluidic lab-on-
a-chip DNA microarray. For Eub338-g11c, Spir344, and
Spir344-g8c, there should be one or two mismatched base-
pairs between the fragmented and labeled E. coli rRNA and
the probes [6,14]. In addition, to drastically enhance the sen-
sitivity of the fragmented E. coli rRNA, a technique using
magnetic bead as well as recirculation will be helpful by
reducing non-complementary target:probe hybridizations
[15].
`lk`irpflk=
In this study, we have demonstrated the advantages of a
fragmentation and labeling lab-on-a-chip and microfluidic
recirculation DNA microarray system. High molecular
weight rRNA could be fragmented into approximately 400
basepaired rRNA, and they could be hybridized on a DNA
microarray with a recirculating system. From the denaturing
PAGE analysis of the processed rRNA, 85oC was deter-
mined to be the optimal temperature for the lab-on-a-chip
platform, with a flow rate of 0.2 μL/s. To realize a more effi-
cient system, lab-on-a-chips for rRNA fragmentation and
DNA microarray detection can be assembled into one robust
lab-on-a-chip system.
^ÅâåçïäÉÇÖÉãÉåíë The authors would like to thank
Professor D. A. Stahl at the Department of Environmental
Engineering at the University of Washington, Seattle, USA.
The authors also would like to thank the Argonne National
La-boratory for the MAGIChip. This research was supported
by NASA JPL (MSMT-2004-0045-0066).
Received July 5, 2007; accepted November 1, 2007
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