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DOI 10.1007/s00216-005-0073-y
ORIGINAL PAPER
Nae Yoon Lee
.
Masumi Yamada
.
Minoru Seki
Development of a passive micromixer based on repeated fluid
twisting and flattening, and its application to DNA purification
Received: 15 June 2005 / Revised: 29 July 2005 / Accepted: 10 August 2005 / Published online: 20 September 2005
# Springer-Verlag 2005
Abstract We have developed a three-dimensional pas-
sive micromixer based on new mixing principles, fluid
twisting and flattening. This micromixer is constructed by
repeating two microchannel segments, a main channel
and a flattened channel, which are very different in size
and are arranged perpendicularly. At the intersection of
these segments the fluid inside the micromixer is twisted
and then, in the flattened channel, the diffusion length
is greatly reduced, achieving high mixing efficiency.
Several types of micromixer were fabricated and the
effect of microchannel geometry on mixing performance
was evaluated. We also integrated this micromixer with a
miniaturized DNA purification device, in which the
concentration of the buffer solution could be rapidly
changed, to perform DNA purification based on solid-
phase extraction.
Keywords Passive micromixer
.
Microfluidics
.
DNA
purification
.
Integration
Introduction
Effective mixing is a prerequisite for the success of
almost all chemical or biochemical reactions. In recent
years the micromixer has emerged as an indispensable
component for realization of micro total analysis systems
(TAS) or lab-on-a-Chip [13]. Various types of active
[48] and passive [920] micromixer have been devel-
oped and applied to microscale chemical or biochemical
reactions.
Active micromixers operate either as a batch or con-
tinuously and facilitate rapid mixing. These mixers are
complex in operation, however, and require outer fields or
devices, for example an acoustic wave [4, 5], a magnetic
force [6], or an electrokinetic field [7, 8]. Passive mic-
romixers, on the other hand, do not need these outer fields,
because mixing is achieved mainly as a result of the
shortened diffusion length. In addition, passive micromix-
ers are simple to operate because the mixing occurs
structurally, making them attractive and suitable for
integration with other kinds of miniaturized component.
Two types of mixing principle are usually adopted for
passive mixingchaotic advection and multi-lamination
[21]. Typically, micromixers based on chaotic advection
are effective at relatively high flow rates, although there
are exceptions. They are, therefore, not suitable for ex-
pensive samples or reagents, because a large volume of
liquid is required. On the other hand, the mixing effi-
ciencies of the multi-lamination mixers [1215] are high at
low flow rates, because the shortened diffusion length
facilitates mixing. The structures of these mixers tend to
be complex, however, requiring complicated fabrication
processes such as multi-layer stacking or multi-step
photolithography.
Considering that the time required for mixing is pro-
portional to the second power of the diffusion length,
shortening the distance between two or more kinds of liquid
is essential to achieve high mixing efficiency, irrespective
of the mixing principle. That is, use of a narrow chan-
nel accelerates the mixing of miscible liquids, even in
the absence of any specially designed mixing structures.
Narrow channels have very large hydrodynamic resis-
tances, however, and thus it is difficult to introduce liquids
into them. The use of a thin but wide microchannel is an
alternative means of reducing hydrodynamic resistance,
N. Y. Lee
.
M. Yamada
Department of Chemistry and Biotechnology,
School of Engineering,
The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8656, Japan
M. Seki (*)
Department of Chemical Engineering,
Graduate School of Engineering,
Osaka Prefecture University,
1-1 Gakuen-cho, Sakai,
Osaka 599-8531, Japan
e-mail: seki@chemeng.osakafu-u.ac.jp
Tel.: +81-72-2549296
Fax: +81-72-2549911
and thereby increasing throughput. It is, however, quite
difficult to stably stack multiple thin liquid layers in a thin
channel, and fabricating a microchannel with a high aspect-
ratio is also very difficult.
In this study, we have developed a three-dimensional
passive micromixer with relatively simple fabrication
processes and channel structures. The microdevices were
fabricated by bonding two polydimethylsiloxane (PDMS)
plates with different channel depths, a relatively common
technique used in microfabrication. By perpendicular
combination of microchannel segments with highly dif-
ferent aspect ratios, shortening of the diffusion length is
readily achieved, that is, we can obtain the same effect as is
achieved by use of a high aspect-ratio microchannel or
by stacking two thin liquid layers in a thin channel, but
without employing complex structures.
Several types of micromixer were fabricated and the
effect of microchannel dimensions on liquid behavior was
examined. In addition, to assist in the application of this
micromixer to biochemical analysis the micromixer was
integrated with a solid-phase extraction (SPE) [22] based
DNA purification device in which a stepwise change in salt
concentration is required.
Principle of mixing
Figure 1 shows a schematic diagram of the passive mic-
romixer and the arrangement for ideal mixing of fluids. The
micromixer is composed of two microchannel segments,
a main channel and a flattened channel, which are
arranged perpendicularly to each other.
First, two different liquids are continuously introduced
into the mixer from the two inlets. Before entering into the
flattened channel the virtual interface of the two liquids is
directed vertically. However, at the intersection of the two
microchannel segments, fluid twisting occurs as a result of
the introduction of the liquids from a relatively narrow and
deep channel into a wide and shallow channel, because the
flattened channel is located below the main channel, and
those two are arranged perpendicularly. As mentioned
above, it is well known that the time required for mixing is
proportional to the second power of the diffusion length.
So, if the interface of the two liquids is directed hori-
zontally in the flattened channel as shown in Fig. 1c, the
diffusion length becomes much smaller compared with
that in the main channel. Also, because of the large width
of the flattened channel, the fluid is retained for a relatively
long time, which facilitates mixing substantially. In ad-
dition, by repeating the arrangement of the two channel
segments, the direction of twisting will alternate between
left and right, which compensates for the imperfection of a
single twist.
The difference in channel aspect ratios is one of the most
important factors for efficient mixing, because it is rea-
sonable to assume that fluid twisting will never occur when
the aspect ratios of both channel segments are very
low. Also, flattening of fluids after the twisting and rep-
etition of the twisting could become significant factors.
Therefore, we also examined the effects of these factors on
the mixing.
Materials and methods
Microdevice fabrication
PDMS microdevices were fabricated using conventional
soft lithography and replica molding [23, 24]. Silicon
wafers were coated with a negative photoresist, SU-8 5 or
SU-8 50 (MicroChem, Newton, MA, USA) and SU-8
structures (replica masters) were then created for main
channels and flattened channels with different channel
depths. A 10:1 mixture of PDMS prepolymer and curing
agent (Sylgard 184, Dow Corning, Midland, MI, USA) was
then poured onto the replica masters, cured, and peeled off.
After treatment of the surface of the PDMS replica with an
oxygen plasma using a plasma reactor (PR 500; Yamato
Scientific, Tokyo, Japan), microchannel segments were
aligned with the aid of methanol under a microscope, and
bonded. Silicone tubing, inner and outer diameter 1 and
2 mm, respectively, was inserted into the inlet and outlet
ports, diameter 2 mm, and then glued. In this way, three-
dimensional passive mixers were easily fabricated without
complicated fabrication processes.
Fig. 1 Schematic diagrams showing the micromixer and the mixing
principle. (a) Three-dimensional view of the micromixer. (b)
Enlarged diagram showing intersections of the main and flattened
channels. (c) Cross-sectional view of two liquids inside the main and
flattened channels during one alternate cycle of twisting and
flattening
777
Micromixer design
The design of the micromixer is shown in Fig. 2a. To
realize a three-dimensional structure the main channels
are situated on the lower surface of the top PDMS plate and
the flattened channels are placed on the upper surface of
the bottom plate. In this study, several types of micromixer
were constructed by combining different types of main
channel and flattened channel. The cross-sectional dimen-
sions of the three types of main channel and the two types
of flattened channel are shown in Fig. 2b and c. The size of
the microdevice was 30 mm15 mm and the lengths of a
main channel and flattened channel were 6.0 and 1.7 mm,
respectively. There were eleven flattened channels in the
micromixer and the total volumes of the micromixers were
0.742.1 L.
To demonstrate the stepwise changes in the salt con-
centration of a buffer solution for DNA purification based
on SPE, the micromixer was integrated with a DNA pu-
rification microcolumn. The design of the integrated
microdevice and enlarged parts are shown in Fig. 2d.
In this device, the width, depth, and length of a main
channel were 100 m, 300 m, and 1.5 mm, respectively,
whereas those of the flattened channel were 300, 10,
and 635 m, respectively. On the PDMS plate contain-
ing the main channels a DNA purification microcolumn
400 m wide and 300 m deep was connected to the exit
of the micromixer and on the PDMS plate containing
the flattened channels there was a weir-structure (depth
10 m) for holding the introduced glass powder (Wako
Pure Chemical, Osaka, Japan), the diameter of which was
45 m.
Evaluation of mixing performance
To evaluate the mixing performance of the micromixers,
10 mmol L
1
Tris-HCl (pH 7.0) buffer solutions, one
with fluorescein (saturated in the buffer) and one with-
out fluorescein, were prepared and introduced into the
micromixer through the two inlets by use of syringe
pumps (KDS 250, KD Scientific, New Hope, PA, USA). X-
Y photographs (bitmap images) were captured in the
flattened channels at intervals of 0.65 m along the Z-axis,
using a confocal laser scanning microscope (Leica TCS
NT, Leica Microsystems, Heidelberg, Germany) with an
excitation wavelength of 488 nm from a Kr/Ar laser. The
homogeneity of the fluid inside the microchannel, i.e. the
mixing or twisting efficiency, was evaluated by calculating
the coefficient of variation (CV) of the green signal
intensities of the cross-section of the microchannel, ob-
tained from the multiple X-Y bitmap images. The CV is
equal to the standard deviation of the signal intensities
divided by the average signal intensity. That is, a high CV
value indicates that the signal intensity is not homoge-
neous, and thus that the mixing efficiency is low. To
demonstrate the stepwise concentration change using the
integrated microdevice, a 0.5 mmol L
1
aqueous solution
of aniline blue (Wako Pure Chemical) was used. Solutions
with and without the blue dye were introduced into the
microdevice using syringe pumps; the flow rates were
computer-controlled.
DNA purification on an integrated microdevice
To perform DNA purification on an integrated micro-
device, a suspension of the glass powder, which adsorbs
DNA, was introduced from the sample inlet port. The
length of the glass powder-packed region was 3 mm.
Before introducing a sample containing DNA, the glass
powder-packed microchannel was pre-washed with
50 mmol L
1
MOPS buffer (pH 8.0) containing
500 mmol L
1
MgCl
2
for better adsorption of DNA onto
the glass surface. As sample for this purification ex-
periment, a mixture containing DNA was obtained by
dissolving a single hair root in 10 L DNA extraction
solution [25] at room temperature for 1 h. The mixture was
Fig. 2 Schematic illustrations of microdevice designs. (a) The
whole micromixer. (b) The main channel cross-sections for different
aspect ratios (A, 0.33; B, 1.0; C, 3.0). (c) The flattened channel
cross-sections for different depths (A, 10 m; B, 20 m). (d)
An integrated microdevice for DNA purification comprising the
micromixer and a solid-phase extraction (SPE)-based DNA puri-
fication microcolumn
778
then introduced directly into the glass powder-packed mic-
rochannel from the sample inlet port, without any further
pretreatment. After introduction of the DNA the sample
inlet port was closed.
Because DNA is negatively charged, it is strongly ad-
sorbed by the glass surface under high-salt buffer
conditions whereas the binding forces for other contami-
nants, for example proteins or sugars, are relatively weak,
enabling selective DNA adsorption. On the other hand,
adsorbed DNA can be eluted and collected under low-salt
buffer conditions, and thus a stepwise change in salt
concentration is required. In this study the concentration of
MgCl
2
was changed from 500 mmol L
1
to 15 mmol L
1
by the micromixer, using 50 mmol L
1
MOPS buffers with
MgCl
2
(1.0 mol L
1
) and without MgCl
2
. To obtain a
buffer containing 500 mmol L
1
MgCl
2
for washing
adsorbed DNA, the mixing ratio of buffer with 1.0 mol L
1
MgCl
2
to that without MgCl
2
was controlled at 1:1,
whereas 15 mmol L
1
MgCl
2
buffer for eluting DNA was
obtained by controlling the ratio at 1:66. Only one outlet
was used in this experiment; the other outlet was closed.
Whether or not the eluted DNA was sufficiently pure to
be used directly as a template for PCR amplification was
then examined. As a model, the D1S80 locus, which is
used for individual identification [2628] and whose size
lies between 369 to 801 bp, was amplified by the usual
PCR method. The primer sequences were [26]: 5-GAA
ACT GGC CTC CAA ACA CTG CCC GCC G-3 and 5-
GTC TTG TTGGAG ATG CAC GTG CCC CTT GC-3. A
reaction solution containing 200 mol L
1
of each dNTP
(dNTP Mix, Promega, Madison, WI, USA), 1 mol L
1
of
each primer, 10 reaction buffer composed of 500 mmol
L
1
KCl, 100 mmol L
1
Tris-HCl, 1% Triton X-100, and
15 mmol L
1
MgCl
2
, and 5 units L
1
polymerase (Taq
DNA polymerase, Promega) was used. The eluted DNA
solution (5 L) was mixed with this reaction solution
(45 L). Amplification was conducted using a conven-
tional thermocycler for 29 cycles under the conditions:
94C for 1 min, 65C for 1 min, and 72C for 1 min.
Amplification results were confirmed by conventional
gel electrophoresis, and DNA was stained with SYBR
Green I.
Results and discussion
Effect of main channel aspect ratio on fluid twisting
First, the effect of the main channel aspect ratio on fluid
twisting was evaluated. Main channels with aspect ratios of
0.33, 1.0, and 3.0 were fabricated and combined with a
10 m-deep flattened channel. Top views of the first
intersection and cross-sectional fluorescence intensities of
the first flattened channel are shown in Fig. 3. The flow
rates for each inlet were 64 L min
1
, and the times
required for the fluid to flow from the confluence point to
the first flattened channel were 55.3, 18.5, and 55.3 ms for
the main channels with aspect ratios of 0.33, 1.0, and 3.0,
respectively. As can be seen, when the main channel aspect
ratio was 0.33, the interface of the two liquids was
maintained in the vertical direction (Figs. 3A,a and 3B,a),
although it was slightly inclined when the main channel
aspect ratio was 1.0 (Fig. 3B,b), indicating that fluid
twisting did not occur. When, on the other hand, the main
channel aspect ratio was 3.0, it can be seen that the
positions of the two liquids were changed from right-and-
left to top-and-bottom (Fig. 3B,c), indicating that fluid
twisting occurred, although this twisting was not perfect. In
addition, because the depth of the flattened channel was
only 10 m, mixing of two liquids started immediately and
the interface between the two liquids became obscure.
Fluid twisting would not have occurred if the depths of the
main channels and flattened channels were uniform or
if the two microchannel segments were connected not
perpendicularly but linearly, and the interface would have
been kept in the vertical direction. Therefore, these results
Fig. 3 Effect of the main channel aspect ratio on twisting. (A) Top
views of the first intersection of the main and flattened channels. (B)
Cross-sectional fluorescence intensities measured along the X-X
lines of the first flattened channel. In (A) and (B), a, b, and c
correspond to main channel aspect ratios, which were 0.33, 1.0, and
3.0, respectively, as shown in Fig. 2b. The depths of the flattened
channels were 10 m for these micromixers
779
signify that significant fluid twisting occurs at the
perpendicular intersection of the two microchannel seg-
ments, but only when the difference between the aspect
ratios of two microchannel segments is large.
The effect of flow rate on fluid twisting was evaluated
for main channels with aspect ratios of 0.33 and 3.0
(Fig. 4). Flow rates for each inlet were varied from 2 to
64 L min
1
. For quantitative estimation of fluid twisting,
the fluorescence signal intensities of the cross-section of
the first flattened channel were visualized along the X-X
line in Fig. 3, and CVs were calculated. As shown in Fig. 4,
when the main channel aspect ratio was 0.33, the CVs were
more than 0.5 for all flow rates, indicating that the signal
intensities were not homogeneous, and fluid twisting did
not occur. Under the low flow rate conditions (2 and
4 L min
1
), the CVs were relatively low (0.5), which
may have been because of diffusion. On the other hand,
when the main channel aspect ratio was 3.0, the CVs were
lower than 0.2, irrespective of flow rate. This indicates that
fluid twisting occurred, and mixing of the two liquids
followed, even though the retention times from the con-
fluence point to the detection point were very short. These
results revealed that fluid twisting was not affected by the
flow rates of the liquids.
Effect of the depth of the flattened channel on twisting
Second, the effect of the depth of the flattened channel on
twisting was evaluated. A micromixer whose flattened
channel depth and main channel aspect ratio were 20 m
and 3.0, respectively, was fabricated, and the results for this
micromixer were compared with those for a micromixer
whose flattened channel depth and main channel aspect
ratio were 10 m and 3.0, respectively. The signal
intensities of the cross-section of the first flattened channel
were also visualized, and the CVs were calculated.
As can be seen from Fig. 5, the CVs in the 20 m-deep
flattened channel were higher than those in the 10 m-deep
flattened channel for all flow rates, indicating that the fluid
twisting was not effective for the 20 m-deep flattened
channel. This tendency was especially apparent under the
high flow rate conditions. Under these conditions, the
retention time was very short, suggesting that the two
liquids were not sufficiently mixed by diffusion. It was thus
confirmed that there was less fluid twisting in the 20 m-
deep flattened channels than in the 10 m-deep channels.
These results also indicate that a large difference between
the aspect ratios of the main channels and flattened
channels is a prerequisite for effective twisting.
It was also observed that the CVs decreased slightly as
the flow rates increased from 2 to 8 L min
1
, irrespective
of the depths of the flattened channel. This suggests that
fluid twisting was slightly enhanced by increasing the flow
rate up to a certain value. It was therefore considered that
fluid twisting would not effectively occur when the flow
rate is too low. It can, however, be said that the effect of
flow rate on fluid twisting was much smaller than the effect
of channel geometry.
Effect of repeated turns on mixing
Next, the effect of the number of perpendicular turns on
mixing was examined. Mixing efficiencies were measured
at five different detection points (points 3, 5, 7, 9, and 11 in
Fig. 6), at different flow rates. In this experiment a CVof
Fig. 4 CVof fluorescence intensities measured at the first flattened
channels at varying flow rates when the main channel aspect ratios
were 0.33 and 3.0. The depths of the flattened channels were 10 m
for these conditions
Fig. 5 CVof fluorescence intensities measured in the first flattened
channels at different flow rates when the flattened channel depths
were 10 m and 20 m. The main channel aspect ratios were 3.0 for
these conditions
Fig. 6 Effect of the number of perpendicular turns on mixing at
different flow rates. For the micromixer used the main channel
aspect ratio and flattened channel depth were 3.0 and 10 m,
respectively. The flow rates for each inlet were varied from 2 to
64 L min
1
. CV of fluorescence intensities was measured at five
detection points (points 3, 5, 7, 9, and 11) of the flattened channels
as indicated on the right
780
0.015 was considered as an indicator of a homogeneous
solution. As shown in Fig. 6, at relatively low flow rates,
for example 2 and 4 L min
1
(the retention times inside
the micromixer were 32 s and 16 s, respectively) mixing
was complete even when the liquids did not pass through
the whole mixer. At relatively high flow rates, however, for
example 8 and 64 L min
1
(retention times 8 s and 1 s,
respectively), CVs gradually decreased as liquids passed
through the turns and, eventually, at detection point 11, the
CVs were almost the same as those at relatively low flow
rates. This indicates that alternate repetition of the per-
pendicular turns assisted fluid mixing. A small organic dye
molecule with a molecular weight of 330 requires 0.2 s to
diffuse 10 m [29], whereas it requires 20 s to diffuse
100 m. The molecular weight of fluorescein, the dye used
in this experiment, is 332.3. So if the depths of the main
channels and flattened channels were uniform, complete
mixing would not be achieved at high flow rates. In
this study the two liquids were completely mixed within
1 s, showing the rapid mixing performance of this
micromixer.
Mixer operation on the integrated microdevice
A micromixer with a reduced volume was fabricated and
integrated with a DNA purification microcolumn as shown
in Fig. 2d. The lengths of the main channels were simply
reduced four-fold, because almost complete mixing was
achieved in the previous micromixers, and the widths and
depths were the same as in the previous micromixer, shown
in Fig. 2b. The changes in the concentrations of the
introduced and mixed liquids were estimated using aniline
blue dye solution, and blue signal intensities were detected
at the entrance of the weir-structure (not packed with glass
powder). The total flow rate was controlled at 2.5 L min
1
; first, the mixing ratio of buffer containing 1.0 mol L
1
MgCl
2
to that without MgCl
2
was controlled at 1:1
(1.25 L min
1
each), and then the ratio was changed to
1:66 (0.0373:2.46 L min
1
). These conditions corre-
sponded to the DNA washing and elution conditions,
respectively, when buffer solutions containing 1.0 mol L
1
MgCl
2
and without MgCl
2
were introduced from each inlet
for the stepwise concentration change. The retention time
in the micromixer was 14 s.
Figure 7 shows the results from measurement of the
concentration changes of the aniline blue dye solution. As
can be seen in Fig. 7a, the two liquids were almost perfectly
mixed after passing through the micromixer, and mixing
could be achieved even when the difference between flow
rates was large (1:66). Figure 7b shows the time course of
the concentration change at the entrance of the weir-
structure. Blue signal intensity of the buffer solution
without the dye was adjusted to be zero, whereas that of the
1:1 mixture of the dye and buffer solutions was 110. On
the other hand, the average intensity calculated from 400 to
600 s was 2.0. Although the noise was relatively large,
this value corresponded well to the desired concentration,
demonstrating that the concentration could be properly
adjusted. In addition, almost a complete change in signal
intensities was achieved within 30 s after the change in the
flow rates from the syringe pumps, which demonstrates the
immediate response of this micromixer, because the time
required for complete concentration change was only about
twice its retention time. This response was faster than that
of a typical straight microchannel, in which the flow rate
distribution is parabolic. By reducing the length of the main
channels in the micromixer, this time lag could be reduced,
accomplishing a steeper concentration change for conduct-
ing different chemical and biochemical reactions.
DNA purification on the integrated microdevice
After introducing a sample solution containing DNA into
the microchannel, 50 mmol L
1
MOPS buffers, one with
and one without MgCl
2
, were introduced, and their flow
rates were adjusted. First, DNA-adsorbing glass powder
Fig. 7 Evaluation of mixer performance on an integrated micro-
device by monitoring the color changes of aniline blue dye solution.
(a) Photographs showing the confluence point and the entrance of
the weir-structure, when the mixing ratios of 50 mmol L
1
MOPS
buffer containing 1.0 mol L
1
MgCl
2
to that without MgCl
2
were
1:1 and 1:66. (b) Change in the blue signal intensities of the mixed
solution, measured at the entrance of the weir-structure
781
was washed with the buffer solution containing
500 mmol L
1
MgCl
2
for 5 min, and then the MgCl
2
concentration was changed to 15 mmol L
1
for DNA
elution. During these operations 10 L of the washout
solution with 500 mmol L
1
MgCl
2
was collected and, 40 s
after the concentration change, 5 L of the solution
containing eluted DNA was also collected. The DNA in
each solution was amplified. The overall process from
sample introduction to DNA elution was completed within
8 min.
The results are shown in Fig. 8. Lanes 2 and 3 show the
amplification results of the DNA washout solution and
eluted DNA solution, respectively, when DNAwas purified
with the same buffers using a discrete DNA purification
microcolumn packed with glass powder. Lanes 4 and 5
show the results of the DNA washout solution and eluted
DNA solution, respectively, purified using the integrated
microdevice. No bands were observed in lanes 2 and 4,
demonstrating that adsorbed DNA was not washed off
when the salt concentration was relatively high. On the
other hand, DNAwas eluted under the low-salt conditions,
and PCR was successfully performed, as shown in lanes 3
and 5. It was also confirmed that the same bands were
obtained using both the discrete DNA purification micro-
column and the integrated microdevice, revealing that the
DNA solution, prepared using the integrated microdevice,
was sufficiently pure to be used directly as a template for
PCR amplification. Using this microdevice, the DNA
preparation procedures were greatly simplified, signifi-
cantly shortening the overall processing time.
Conclusions
We have developed a passive micromixer that enables
effective and rapid mixing on the basis of repeated fluid
twisting and flattening. This mixing structure was easily
fabricated simply by bonding two PDMS plates containing
microchannel segments of different depth. This micromixer
is therefore highly suitable for integration as one of the
units on a microdevice, and is appropriate for versatile
applications. As an application, this micromixer was
integrated with an SPE-based DNA purification micro-
column, and DNA purification from a biological sample
was demonstrated by rapidly generating a stepwise change
of salt concentration. With this integrated microdevice the
time-consuming and labor-intensive DNA preparation
process was greatly simplified, and we expect the process
could be further automated, facilitating a variety of clinical
applications and rapid disease diagnosis.
Acknowledgements This research was supported in part by Grant-
in-Aids for JSPS Fellows, Scientific Research (B) (No. 16310101),
and Priority Areas (A) (No. 13025216) from the Ministry of
Education, Science, Sports and Culture of Japan, and by the Research
Association of Micro Chemical Process Technology, Japan.
References
1. Burns MA, Johnson BN, Brahmasandra SN, Handique K,
Webster JR, Krishnan M, Sammarco TS, Man PM, Jones D,
Heldsinger D (1998) Science 282:484487
2. Kricka LJ, Wilding P (2003) Anal Bioanal Chem 377:820825
3. Bilitewski U, Genrich M, Kadow S, Mersal G (2003) Anal
Bioanal Chem 377:556569
4. Zhu X, Kim ES (1998) Sens Actuators A 66:355360
5. Yang Z, Goto H, Matsumoto M, Maeda R (2000) Electropho-
resis 21:116119
6. Lu LH, Ryu KS, Liu C (2002) J Microelectromech Syst
11:462469
7. Oddy MH, Santiago JG, Mikkelsen JC (2001) Anal Chem
73:58225832
8. Wu HY, Liu CH (2005) Sens Actuators A 118:107115
9. Liu RH, Stremler MA, Sharp KV, Olsen MG, Santiago JG,
Adrian RJ, Aref H, Beebe DJ (2000) J Microelectromech Syst
9:190197
10. Stroock AD, Dertinger SKW, Ajdari A, Mezi I, Stone HA,
Whitesides GM (2002) Science 295:647651
11. Park SJ, Kim JK, Park J, Chung S, Chung C, Chang JK (2004)
J Micromech Microeng 14:614
12. Schwesinger N, Frank T, Wurmus H (1996) J Micromech
Microeng 6:99102
13. Bessoth FG, deMello AJ, Manz A (1999) Anal Commun
36:213215
14. Mae K, Maki T, Hasegawa I, Eto U, Mizutani Y, Honda N
(2004) Chem Eng J 101:3138
15. Ehrfeld W, Golbig K, Hessel V, Lwe H, Richter T (1999) Ind
Eng Chem Res 38:10751082
16. Johnson TJ, Ross D, Locascio LE (2002) Anal Chem 74:4551
17. Chung YC, Hsu YL, Jen CP, Lu MC, Lin YC (2004) Lab Chip
4:7077
18. Jeon MK, Kim JH, Noh J, Kim SH, Park HG, Woo SI (2005) J
Micromech Microeng 15:346350
19. Kirner T, Albert J, Gnther M, Mayer G, Reinhckel K, Khler
JM (2004) Chem Eng J 101:6574
20. Kim DJ, Oh HJ, Park TH, Choo JB, Lee SH (2005) Analyst
130:293298
21. Nguyen NT, Wu Z (2005) J Micromech Microeng 15:R1R16
22. Tian H, Huhmer AFR, Landers JP (2000) Anal Biochem
283:175191
23. Lee NY, Yamada M, Seki M (2004) Anal Sci 20:483487
24. Yamada M, Seki M (2004) Anal Chem 76:895899
25. Gill P, Jeffreys AJ, Werrett DJ (1985) Nature 318:577579
26. Budowle B, Chakraborty R, Giusti AM, Eisenberg AJ, Allen
RC (1991) Am J Hum Genet 48:137144
27. Roy R (1997) Forensic Sci Int 87:6371
28. Hayes JM, Budowle B, Freund M (1995) J Forensic Sci
40:888892
29. Weigl BH, Yager P (1999) Science 283:346347
Fig. 8 Amplification results of the D1S80 locus. Lane 1: 100 bp
DNA ladder. Lanes 2 and 3: amplification results of the DNA
washout solution and eluted DNA solution, respectively, obtained
using a discrete DNA purification microcolumn packed with the
glass powder. Lanes 4 and 5: amplification results of the DNA
washout solution and eluted DNA solution, respectively, obtained
using the integrated microdevice
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