micromixer1.pdf

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Anal Bioanal Chem (2005) 383: 776782DOI 10.1007/s00216-005-0073-y

O RIG IN A L PA PER

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, fluidtwisting and flattening. This micromixer is constructed by

repeating two microchannel segments, a main channeland a flattened channel, which are very different in sizeand are arranged perpendicularly. At the intersection ofthese segments the fluid inside the micromixer is twistedand then, in the flattened channel, the diffusion lengthis greatly reduced, achieving high mixing efficiency.Several types of micromixer were fabricated and theeffect of microchannel geometry on mixing performancewas evaluated. We also integrated this micromixer with aminiaturized DNA purification device, in which theconcentration of the buffer solution could be rapidlychanged, to perform DNA purification based on solid-

phase extraction.

Keywords Passive micromixer. Microfluidics . DNApurification. Integration

Introduction

Effective mixing is a prerequisite for the success ofalmost all chemical or biochemical reactions. In recent

years the micromixer has emerged as an indispensablecomponent 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 biochemicalreactions.

Active micromixers operate either as a batch or con-tinuously and facilitate rapid mixing. These mixers arecomplex in operation, however, and require outer fields ordevices, for example an acoustic wave [4, 5], a magneticforce [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 theshortened diffusion length. In addition, passive micromix-ers are simple to operate because the mixing occursstructurally, 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 advectionare effective at relatively high flow rates, although thereare exceptions. They are, therefore, not suitable for ex-

pensive samples or reagents, because a large volume ofliquid is required. On the other hand, the mixing effi-ciencies of the multi-lamination mixers [1215] are high atlow flow rates, because the shortened diffusion lengthfacilitates mixing. The structures of these mixers tend to

be complex, however, requiring complicated fabricationprocesses 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 liquidis essential to achieve high mixing efficiency, irrespectiveof the mixing principle. That is, use of a narrow chan-nel accelerates the mixing of miscible liquids, even inthe absence of any specially designed mixing structures.

Narrow channels have very large hydrodynamic resis-tances, however, and thus it is difficult to introduce liquidsinto them. The use of a thin but wide microchannel is analternative means of reducing hydrodynamic resistance,

N. Y. Lee. M. YamadaDepartment 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, Japane-mail: [email protected].: +81-72-2549296Fax: +81-72-2549911


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and thereby increasing throughput. It is, however, quitedifficult to stably stack multiple thin liquid layers in a thinchannel, and fabricating a microchannel with a high aspect-ratio is also very difficult.

In this study, we have developed a three-dimensionalpassive micromixer with relatively simple fabricationprocesses and channel structures. The microdevices werefabricated by bonding two polydimethylsiloxane (PDMS)

plates with different channel depths, a relatively commontechnique used in microfabrication. By perpendicularcombination of microchannel segments with highly dif-ferent aspect ratios, shortening of the diffusion length isreadily achieved, that is, we can obtain the same effect as isachieved by use of a high aspect-ratio microchannel or

by stacking two thin liquid layers in a thin channel, butwithout employing complex structures.

Several types of micromixer were fabricated and theeffect of microchannel dimensions on liquid behavior wasexamined. In addition, to assist in the application of thismicromixer to biochemical analysis the micromixer wasintegrated with a solid-phase extraction (SPE) [22] based

DNA purification device in which a stepwise change in saltconcentration is required.

Principle of mixing

Figure 1shows a schematic diagram of the passive mic-romixer and the arrangement for ideal mixing of fluids. Themicromixer is composed of two microchannel segments,a main channel and a flattened channel, which arearranged perpendicularly to each other.

First, two different liquids are continuously introducedinto the mixer from the two inlets. Before entering into the

flattened channel the virtual interface of the two liquids isdirected vertically. However, at the intersection of the twomicrochannel segments, fluid twisting occurs as a result ofthe introduction of the liquids from a relatively narrow anddeep channel into a wide and shallow channel, because theflattened channel is located below the main channel, andthose two are arranged perpendicularly. As mentionedabove, 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, thediffusion length becomes much smaller compared withthat in the main channel. Also, because of the large width

of the flattened channel, the fluid is retained for a relativelylong time, which facilitates mixing substantially. In ad-dition, by repeating the arrangement of the two channelsegments, the direction of twisting will alternate betweenleft and right, which compensates for the imperfection of asingle twist.

The difference in channel aspect ratios is one of the mostimportant factors for efficient mixing, because it is rea-sonable to assume that fluid twisting will never occur whenthe aspect ratios of both channel segments are verylow. 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 onthe mixing.

Materials and methods

Microdevice fabrication

PDMS microdevices were fabricated using conventionalsoft lithography and replica molding [23, 24]. Siliconwafers were coated with a negative photoresist, SU-8 5 orSU-8 50 (MicroChem, Newton, MA, USA) and SU-8structures (replica masters) were then created for mainchannels and flattened channels with different channel

depths. A 10:1 mixture of PDMS prepolymer and curingagent (Sylgard 184, Dow Corning, Midland, MI, USA) wasthen poured onto the replica masters, cured, and peeled off.After treatment of the surface of the PDMS replica with anoxygen plasma using a plasma reactor (PR 500; YamatoScientific, Tokyo, Japan), microchannel segments werealigned with the aid of methanol under a microscope, and

bonded. Silicone tubing, inner and outer diameter 1 and2 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 withoutcomplicated fabrication processes.

Fig. 1 Schematic diagrams showing the micromixer and the mixingprinciple. (a) Three-dimensional view of the micromixer. (b)Enlarged diagram showing intersections of the main and flattenedchannels. (c) Cross-sectional view of two liquids inside the main andflattened channels during one alternate cycle of twisting andflattening

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Micromixer design

The design of the micromixer is shown in Fig. 2a. Torealize a three-dimensional structure the main channelsare situated on the lower surface of the top PDMS plate andthe flattened channelsare placed on the upper surface ofthe bottom plate. In this study, several types of micromixerwere 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 typesof flattened channel are shown in Fig.2b and c. The size ofthe microdevice was 30 mm15 mm and the lengths of amain channel and flattened channel were 6.0 and 1.7 mm,respectively. There were eleven flattened channels in themicromixer and the total volumes of the micromixers were0.742.1 L.

To demonstrate the stepwise changes in the salt con-centration of a buffer solution for DNA purification basedon SPE, the micromixer was integrated with a DNA pu-

rification microcolumn. The design of the integratedmicrodevice and enlarged parts are shown in Fig. 2d.In this device, the width, depth, and length of a mainchannel 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 microcolumn400 m wide and 300 m deep was connected to the exit

of the micromixer and on the PDMS plate containingthe flattened channels there was a weir-structure (depth10 m) for holding the introduced glass powder (WakoPure Chemical, Osaka, Japan), the diameter of which was45 m.

Evaluation of mixing performance

To evaluate the mixing performance of the micromixers,10 mmol L1 Tris-HCl (pH 7.0) buffer solutions, onewith fluorescein (saturated in the buffer) and one with-out fluorescein, were prepared and introduced into the

micromixer through the two inlets by use of syringepumps (KDS 250, KD Scientific, New Hope, PA, USA).X-Y photographs (bitmap images) were captured in theflattened channels at intervals of 0.65 m along theZ-axis,using a confocal laser scanning microscope (Leica TCS

NT, Leica Microsystems, Heidelberg, Germany) with anexcitation wavelength of 488 nm from a Kr/Ar laser. Thehomogeneity of the fluid inside the microchannel, i.e. themixing or twisting efficiency, was evaluated by calculatingthe coefficient of variation (CV) of the green signalintensities of the cross-section of the microchannel, ob-tained from the multiple X-Y bitmap images. The CV isequal to the standard deviation of the signal intensities

divided by the average signal intensity. That is, a high CVvalue indicates that the signal intensity is not homoge-neous, and thus that the mixing efficiency is low. Todemonstrate the stepwise concentration change using theintegrated microdevice, a 0.5 mmol L1 aqueous solutionof aniline blue (Wako Pure Chemical) was used. Solutionswith and without the blue dye were introduced into themicrodevice using syringe pumps; the flow rates werecomputer-controlled.

DNA purification on an integrated microdevice

To perform DNA purification on an integrated micro-device, a suspension of the glass powder, which adsorbsDNA, was introduced from the sample inlet port. Thelength of the glass powder-packed region was 3 mm.Before introducing a sample containing DNA, the glass

powder-packed microchannel was pre-washed with50 mmol L1 MOPS buffer (pH 8.0) containing500 mmol L1 MgCl2 for better adsorption of DNA ontothe glass surface. As sample for this purification ex-

periment, a mixture containing DNA was obtained bydissolving a single hair root in 10 L DNA extractionsolution [25] at room temperature for 1 h. The mixture was

Fig. 2 Schematic illustrations of microdevice designs. (a) Thewhole micromixer. (b) The main channel cross-sections for differentaspect ratios (A, 0.33; B, 1.0; C, 3.0). (c) The flattened channelcross-sections for different depths (A, 10 m; B, 20 m). (d)An integrated microdevice for DNA purification comprising themicromixer and a solid-phase extraction (SPE)-based DNA puri-fication microcolumn

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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 sampleinlet port was closed.

Because DNA is negatively charged, it is strongly ad-sorbed by the glass surface under high-salt bufferconditions 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 saltconcentration is required. In this study the concentration ofMgCl2 was changed from 500 mmol L

1 to 15 mmol L1

by the micromixer, using 50 mmol L1 MOPS buffers withMgCl2 (1.0 mol L

1) and without MgCl2. To obtain abuffer containing 500 mmol L1 MgCl2 for washingadsorbed DNA, the mixing ratio of buffer with 1.0 mol L1

MgCl2 to that without MgCl2 was controlled at 1:1,whereas 15 mmol L1 MgCl2buffer for eluting DNA wasobtained by controlling the ratio at 1:66. Only one outletwas used in this experiment; the other outlet was closed.

Whether or not the eluted DNA was sufficiently pure tobe used directly as a template for PCR amplification wasthen examined. As a model, the D1S80 locus, which isused for individual identification [2628] and whose sizelies between 369 to 801 bp, was amplified by the usualPCR method. The primer sequences were [26]: 5-GAAACT GGC CTC CAA ACA CTG CCC GCC G-3and 5-GTC TTG TTG GAG ATG CAC GTG CCC CTT GC-3. Areaction solution containing 200 mol L1 of each dNTP(dNTP Mix, Promega, Madison, WI, USA), 1 mol L1 ofeach primer, 10 reaction buffer composed of 500 mmolL1 KCl, 100 mmol L1 Tris-HCl, 1% Triton X-100, and15 mmol L1 MgCl2, and 5 units L

1 polymerase (Taq

DNA polymerase, Promega) was used. The eluted DNAsolution (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 conventionalgel electrophoresis, and DNA was stained with SYBRGreen I.

Results and discussion

Effect of main channel aspect ratio on fluid twisting

First, the effect of the main channel aspect ratio on fluidtwisting was evaluated. Main channels with aspect ratios of0.33, 1.0, and 3.0 were fabricated and combined with a10 m-deep flattened channel. Top views of the firstintersection and cross-sectional fluorescence intensities ofthe first flattened channel are shown in Fig. 3. The flowrates for each inlet were 64 L min1, and the timesrequired for the fluid to flow from the confluence point tothe first flattened channel were 55.3, 18.5, and 55.3 ms forthe 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 wasmaintained in the vertical direction (Figs. 3A,a and3B,a),although it was slightly inclined when the main channelaspect ratio was 1.0 (Fig. 3B,b), indicating that fluidtwisting did not occur. When, on the other hand, the main

channel aspect ratio was 3.0, it can be seen that thepositions of the two liquids were changed from right-and-left to top-and-bottom (Fig. 3B,c), indicating that fluidtwisting occurred, although this twisting was not perfect. Inaddition, because the depth of the flattened channel wasonly 10 m, mixing of two liquids started immediately andthe interface between the two liquids became obscure.Fluid twisting would not have occurred if the depths of themain channels and flattened channels were uniform orif the two microchannel segments were connected not

perpendicularly but linearly, and the interface would havebeen kept in the vertical direction. Therefore, these results

Fig. 3 Effect of the main channel aspect ratio on twisting. (A) Topviews of the first intersection of the main and flattened channels. (B)Cross-sectional fluorescence intensities measured along the X-Xlines of the first flattened channel. In (A) and (B), a, b, and ccorrespond to main channel aspect ratios, which were 0.33, 1.0, and3.0, respectively, as shown in Fig. 2b. The depths of the flattenedchannels were 10 m for these micromixers

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signify that significant fluid twisting occurs at theperpendicular intersection of the two microchannel seg-ments, but only when the difference between the aspectratios of two microchannel segments is large.

The effect of flow rate on fluid twisting was evaluatedfor main channels with aspect ratios of 0.33 and 3.0(Fig. 4). Flow rates for each inlet were varied from 2 to64 L min1. For quantitative estimation of fluid twisting,

the fluorescence signal intensities of the cross-section ofthe first flattened channel were visualized along the X-Xline in Fig.3, and CVs were calculated. As shown in Fig.4,when the main channel aspect ratio was 0.33, the CVs weremore than 0.5 for all flow rates, indicating that the signalintensities were not homogeneous, and fluid twisting didnot occur. Under the low flow rate conditions (2 and4 L min1), the CVs were relatively low (0.5), whichmay have been because of diffusion. On the other hand,when the main channel aspect ratio was 3.0, the CVs werelower than 0.2, irrespective of flow rate. This indicates thatfluid twisting occurred, and mixing of the two liquidsfollowed, even though the retention times from the con-

fluence point to the detection point were very short. Theseresults revealed that fluid twisting was not affected by theflow rates of the liquids.

Effect of the depth of the flattened channel on twisting

Second, the effect of the depth of the flattened channel ontwisting was evaluated. A micromixer whose flattenedchannel depth and main channel aspect ratio were 20 mand 3.0, respectively, was fabricated, and the results for thismicromixer were compared with those for a micromixerwhose flattened channel depth and main channel aspect

ratio were 10 m and 3.0, respectively. The signalintensities of the cross-section of the first flattened channelwere also visualized, and the CVs were calculated.

As can be seen from Fig.5, the CVs in the 20 m-deepflattened channel were higher than those in the 10 m-deepflattened channel for all flow rates, indicating that the fluidtwisting was not effective for the 20 m-deep flattenedchannel. This tendency was especially apparent under thehigh flow rate conditions. Under these conditions, the

retention time was very short, suggesting that the twoliquids were not sufficiently mixed by diffusion. It was thusconfirmed 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 flattenedchannels 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 min1, irrespectiveof the depths of the flattened channel. This suggests thatfluid twisting was slightly enhanced by increasing the flowrate up to a certain value. It was therefore considered thatfluid twisting would not effectively occur when the flowrate is too low. It can, however, be said that the effect offlow rate on fluid twisting was much smaller than the effectof channel geometry.

Effect of repeated turns on mixing

Next, the effect of the number of perpendicular turns onmixing was examined. Mixing efficiencies were measuredat five different detection points (points 3, 5, 7, 9, and 11 inFig.6), at different flow rates. In this experiment a CV of

Fig. 4 CV of fluorescence intensities measured at the first flattenedchannels at varying flow rates when the main channel aspect ratioswere 0.33 and 3.0. The depths of the flattened channels were 10 mfor these conditions

Fig. 5 CV of fluorescence intensities measured in the first flattenedchannels at different flow rates when the flattened channel depthswere 10 m and 20 m. The main channel aspect ratios were 3.0 forthese conditions

Fig. 6 Effect of the number of perpendicular turns on mixing atdifferent flow rates. For the micromixer used the main channelaspect ratio and flattened channel depth were 3.0 and 10 m,respectively. The flow rates for each inlet were varied from 2 to64 L min1. CV of fluorescence intensities was measured at fivedetection points (points 3, 5, 7, 9, and 11) of the flattened channelsas indicated on the right

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0.015 was considered as an indicator of a homogeneoussolution. As shown in Fig. 6, at relatively low flow rates,for example 2 and 4 L min1 (the retention times insidethe micromixer were 32 s and 16 s, respectively) mixingwas complete even when the liquids did not pass throughthe whole mixer. At relatively high flow rates, however, forexample 8 and 64 L min1 (retention times 8 s and 1 s,respectively), CVs gradually decreased as liquids passed

through the turns and, eventually, at detection point 11, theCVs were almost the same as those at relatively low flowrates. This indicates that alternate repetition of the per-

pendicular turns assisted fluid mixing. A small organic dyemolecule with a molecular weight of 330 requires 0.2 s todiffuse 10 m [29], whereas it requires 20 s to diffuse100 m. The molecular weight of fluorescein, the dye usedin this experiment, is 332.3. So if the depths of the mainchannels and flattened channels were uniform, completemixing would not be achieved at high flow rates. Inthis study the two liquids were completely mixed within1 s, showing the rapid mixing performance of thismicromixer.

Mixer operation on the integrated microdevice

A micromixer with a reduced volume was fabricated andintegrated with a DNA purification microcolumn as shownin Fig.2d. The lengths of the main channels were simplyreduced four-fold, because almost complete mixing wasachieved in the previous micromixers, and the widths anddepths were the same as in the previous micromixer, shownin Fig. 2b. The changes in the concentrations of theintroduced 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 glasspowder). The total flow rate was controlled at 2.5 L min1; first, the mixing ratio of buffer containing 1.0 mol L1

MgCl2 to that without MgCl2 was controlled at 1:1(1.25 L min1 each), and then the ratio was changed to1:66 (0.0373:2.46 L min1). These conditions corre-sponded to the DNA washing and elution conditions,respectively, when buffer solutions containing 1.0 mol L1

MgCl2and without MgCl2were introduced from each inletfor the stepwise concentration change. The retention timein the micromixer was 14 s.

Figure 7 shows the results from measurement of theconcentration changes of the aniline blue dye solution. As

can be seen in Fig.7a, the two liquids were almost perfectlymixed after passing through the micromixer, and mixingcould be achieved even when the difference between flowrates was large (1:66). Figure7b shows the time course ofthe concentration change at the entrance of the weir-structure. Blue signal intensity of the buffer solutionwithout the dye was adjusted to be zero, whereas that of the1:1 mixture of the dye and buffer solutions was 110. Onthe other hand, the average intensity calculated from 400 to600 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 signalintensities was achieved within 30 s after the change in theflow rates from the syringe pumps, which demonstrates theimmediate response of this micromixer, because the timerequired for complete concentration change was only abouttwice its retention time. This response was faster than thatof a typical straight microchannel, in which the flow ratedistribution 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 intothe microchannel, 50 mmol L1 MOPS buffers, one withand one without MgCl2, were introduced, and their flowrates 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 ofthe weir-structure, when the mixing ratios of 50 mmol L1 MOPSbuffer containing 1.0 mol L1 MgCl2 to that without MgCl2 were1:1 and 1:66. (b) Change in the blue signal intensities of the mixedsolution, measured at the entrance of the weir-structure

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was washed with the buffer solution containing500 mmol L1 MgCl2 for 5 min, and then the MgCl2concentration was changed to 15 mmol L1 for DNAelution. During these operations 10 L of the washoutsolution with 500 mmol L1 MgCl2was collected and, 40 safter the concentration change, 5 L of the solutioncontaining eluted DNA was also collected. The DNA in

each solution was amplified. The overall process fromsample introduction to DNA elution was completed within8 min.

The results are shown in Fig.8. Lanes 2 and 3 show theamplification results of the DNA washout solution andeluted DNA solution, respectively, when DNAwas purifiedwith the same buffers using a discrete DNA purificationmicrocolumn packed with glass powder. Lanes 4 and 5show the results of the DNA washout solution and elutedDNA solution, respectively, purified using the integratedmicrodevice. No bands were observed in lanes 2 and 4,demonstrating that adsorbed DNA was not washed offwhen the salt concentration was relatively high. On the

other hand, DNA was eluted under the low-salt conditions,and PCR was successfully performed, as shown in lanes 3and 5. It was also confirmed that the same bands wereobtained using both the discrete DNA purification micro-column and the integrated microdevice, revealing that theDNA solution, prepared using the integrated microdevice,was sufficiently pure to be used directly as a template forPCR 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 enableseffective and rapid mixing on the basis of repeated fluidtwisting and flattening. This mixing structure was easilyfabricated simply by bonding two PDMS plates containingmicrochannel segments of different depth. This micromixeris therefore highly suitable for integration as one of theunits on a microdevice, and is appropriate for versatileapplications. As an application, this micromixer wasintegrated with an SPE-based DNA purification micro-

column, and DNA purification from a biological samplewas demonstrated by rapidly generating a stepwise changeof salt concentration. With this integrated microdevice thetime-consuming and labor-intensive DNA preparation

process was greatly simplified, and we expect the processcould be further automated, facilitating a variety of clinicalapplications 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 ofEducation, Science, Sports and Culture of Japan, and by the ResearchAssociation 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 377:8208253. Bilitewski U, Genrich M, Kadow S, Mersal G (2003) Anal

Bioanal Chem 377:556569

4. Zhu X, Kim ES (1998) Sens Actuators A 66:355

3605. Yang Z, Goto H, Matsumoto M, Maeda R (2000) Electropho-

resis 21:1161196. Lu LH, Ryu KS, Liu C (2002) J Microelectromech Syst

11:4624697. Oddy MH, Santiago JG, Mikkelsen JC (2001) Anal Chem

73:582258328. Wu HY, Liu CH (2005) Sens Actuators A 118:1071159. Liu RH, Stremler MA, Sharp KV, Olsen MG, Santiago JG,

Adrian RJ, Aref H, Beebe DJ (2000) J Microelectromech Syst9:190197

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 6:99

10213. Bessoth FG, deMello AJ, Manz A (1999) Anal Commun36:213215

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) IndEng Chem Res 38:10751082

16. Johnson TJ, Ross D, Locascio LE (2002) Anal Chem 74:455117. Chung YC, Hsu YL, Jen CP, Lu MC, Lin YC (2004) Lab Chip

4:707718. Jeon MK, Kim JH, Noh J, Kim SH, Park HG, Woo SI (2005) J

Micromech Microeng 15:34635019. Kirner T, Albert J, Gnther M, Mayer G, Reinhckel K, Khler

JM (2004) Chem Eng J 101:657420. Kim DJ, Oh HJ, Park TH, Choo JB, Lee SH (2005) Analyst

130:293298

21. Nguyen NT, Wu Z (2005) J Micromech Microeng 15:R1

R1622. Tian H, Huhmer AFR, Landers JP (2000) Anal Biochem283:175191

23. Lee NY, Yamada M, Seki M (2004) Anal Sci 20:48348724. Yamada M, Seki M (2004) Anal Chem 76:89589925. Gill P, Jeffreys AJ, Werrett DJ (1985) Nature 318:57757926. Budowle B, Chakraborty R, Giusti AM, Eisenberg AJ, Allen

RC (1991) Am J Hum Genet 48:13714427. Roy R (1997) Forensic Sci Int 87:637128. Hayes JM, Budowle B, Freund M (1995) J Forensic Sci

40:88889229. 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 DNAwashout solution and eluted DNA solution, respectively, obtainedusing a discrete DNA purification microcolumn packed with theglass powder. Lanes 4 and 5: amplification results of the DNAwashout solution and eluted DNA solution, respectively, obtainedusing the integrated microdevice

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