miniature real time PCR on chip with multi-channel

DOI10.1007/s10544-007-9048-4

Miniature real time PCR on chip with multi-channelfiber optical fluorescence detection module

Q.Xiang·B.Xu·D.Li

C Springer Science+Business Media,LLC2007

Abstract This paper presents the design and implementa-tion of a miniature real time PCR system consisting of a disposable reactor chip,a miniature thermal cycler,and a multi-channelfiber opticalfluorescence excitation/detection module.The disposable PCR chip is fabricated by using soft photolithography by PDMS(Polydimethylsiloxane)and glass.The miniature thermal cycler has a thinfilm heater for heating and a fan for rapid cooling.Thefiber optical detec-tion module consists of laser,filter cube,photo-detector and 1×4fiber optical switch.It is capable of four-well real time PCR analysis.Real-time PCR detection of E.coli stx1has been demonstrated successfully with this system. Keywords Real-time PCR.Lab-on-a-chip. Miniaturization.E.coli

1Introduction

Polymerase chain reaction(PCR)is widely applied in clin-ical medicine,genetic disease diagnostics,forensic science, and evolutionary biology for both sequencing and genotyp-ing applications because of its specificity and capability of creating large amounts of copied DNA fragments from minute amounts of samples.Recently,miniaturized PCR chip devices have attracted great interest due to many advan-Q.Xiang·B.Xu

Department of Mechanical and Industrial Engineering,

University of Toronto,

5King’s College Road,Toronto,M5S3G8Canada

D.Li( )

Department of Mechanical Engineering,Vanderbilt University,

2301Vanderbilt Place,Nashville,TN37235-1592,USA

e-mail:dongqing.li@vanderbilt.edu tages over conventional PCR instruments,such as portabil-ity,higher thermal cycling speed,and significantly reduced reagents/sample consumption.

All real-time PCR systems consist of three components: a reaction chamber,a thermal cycler,and a PCR product analyzer.Both static chamber PCR chips(Lin et al.,2000; Giordano et al.,2001;Nagai et al.,2001;Lagally et al.,2001; Yang et al.,2002;Lee et al.,2003,2004a)and dynamicflow-through PCR chips(Hu et al.,2005;Kopp et al.,1998;Obeid et al.,2003;Gascoyne et al.,2004;Hashimoto et al.,2004; Wheeler et al.,2004;Bu et al.,2003;Liu et al.,2002a)were reported.PCR chips have been made by various materials such as silicon(Nagai et al.,2001;Matsubara et al.,2004, Lin et al.,2000),glass(Lagally et al.,2001),polycarbon-ate(Yang et al.,2002),polyamide(Giordano et al.,2001) and PMMA(Lee et al.,2004a).A single-well PCR chip and a multiple-well PCR chip(Nagai et al.,2001;Matsubara et al.,2004)were reported.Contact and non-contact heating (Giordano et al.,2001)as well as Joule heating(Hu et al., 2005)were used to power the thermal cycling.The analysis methods of the amplified PCR products include gel elec-trophoresis(Obeid et al.,2003;Lin et al.,2000;Lee et al., 2004a),fluorescence scan after PCR test(Matsubara et al., 2004,Yang et al.,2002)and real timefluorescence intensity detection by usingfluorescence microscope(Nagai et al., 2001;Lee et al.,2004b)or by a home-madefluorescence de-tection module(Belgrader et al.,2001;Higgins et al.,2003).

Among the static chamber PCR chip devices,Yang et al. (2002)and Liu et al.(2002b)reported a micro PCR system in which the temperature of the micro reactor was controlled by two Peliter thermoelectric devices sandwiching the reactor. The PCR chip is made of polycarbonate,and fabricated by a direct laser writing method.A commercialfluorescence analyzer was used to detect the amplified products after the thermal cycling.Lin et al.(2000)and Lee et al.(2004a)Biomed Microdevices

developed a PCR system with a reaction well fabricated in a silicon wafer sealed with a glass substrate and placed a heater at the bottom of the silicon wafer.In their design,a small reaction volume was used to increase the temperature uniformity.Gel electrophoresis was employed to analyze the amplifications.Nagai’s group(2001)and Matsubara’s group (2004)presented micro array PCR chips patterned on silicon wafer.A commercial thermal cycler was used to conduct the PCR and afluorescence microscope or micro scanner was employed to measure thefluorescence intensity of the PCR products.Nano-liter∼pico-liter PCR was achieved.

In dynamicflow-through PCR devices,PCR reactants were heated and cooled by transporting the reactants through different temperature zones.A typicalflow-through thermal cycler was presented in literature with thinfilm platinum heaters and sensors patterned onto a silicon wafer to gen-erate three different temperature zones(Kopp et al.,1998; Schneegass et al.,2001;Obeid et al.,2003;Gascoyne et al., 2004;Hashimoto et al.,2004;Bu et al.,2003).PCR reac-tions were also achieved in a continuousflow mode in a ring chamber with controlled temperature regions(West et al., 2002;Liu et al.,2002a;Sun et al.,2002).Comparing with the static chamber PCR systems,theflow-through PCR can reduce the heating and cooling time and thus shorten the total time of PCR reaction.However,it is difficult to insulate the different temperature zones,to exam the PCR results and to collect the PCR product for further analysis.

Real-time PCR is highly attractive because it can detect PCR results and quantify the template through the real time analysis offluorescent signals generated during the reac-tion,without the conventional post-PCR processes such as gel electrophoresis.Although there are many publications on PCR chips or micro PCR devices,only few researchers have integrated afluorescence detection unit for the real time PCR test in their works(Belgrader et al.,2001;Higgins et al., 2003;Lee et al.,2004b).The compact real time PCR systems reported by Belgrader et al.(2001)and Higgins et al.(2003) include a real timefluorescence sensing unit,however,need tens of microliters of the reaction mixture to perform the PCR.Lee et al.(2004b)developed a miniature spectrom-eter capable of detecting a spectrum offluorescence from DNA labeled SYBR green dye.However,they used a com-mercial thermal cycler,and hence the overall system is not much different from a conventional real time PCR system. Xiang et al.(2005)reported on-chip real-time PCR using a miniature thermal cycler to detecting E.coli;however,the real-time detection was done by using a desktopfluorescent microscope.

In this paper,to explore the great potential of the miniature real time PCR,a new chip-based real-time PCR system was developed.It consists of a PDMS reactor chip,a miniaturized thermal cycler and afiber opticalfluorescence excitation and detection module.Comparing with the commercial real time PCR system,the designed miniature real time PCR system is inexpensive and portable.Compared to silicon or glass PCR chips which need photolithography,metal film deposition,dry or wet etching,and oxidation processes during the fabrication,our PCR chip needs only the simple soft photolithography and replica processes.Hence,the chip is disposable after a single use.The miniature thermal cycler was built with a thinfilm heater for heating and a fan for rapid cooling.Thefiber opticalfluorescence module is composed of a laser,afilter cube,a photo-detector and a1×4fiber optical switch.It is capable of four-well real time PCR signal detection.The application offiber optics eliminates the need of precise alignment and stabilization of optical components.Opticalfiber switch can quickly turn on or off the light in a specific channel(well).For a specific well,the light is turned on for only1.5s in every cycle;thus the photo bleach is greatly reduced.In addition,detecting more wells in a chip can be easily achieved by increasing the number of channels of the optical switch without any other changes in the system.This real-time PCR device has been applied successfully to perform real time tests of E.coli stx1. 2Experimental methods

2.1PCR chip

The PCR chip is composed of two layers of0.15mm glass with a layer of PDMS in between,as shown in Fig.1.The PDMS is a0.5mm thick square-shaped sheet.It has4mm holes in diameter punched through on the stage to form either one well or four wells for holding the reagents.

The Fig.1Schematic diagram of the one well and four well PCR chip design.They are composed of two layers of thin glass(0.15mm)and a layer of PDMS.The PDMS is a0.5mm thick square-shaped sheet with4mm holes in diameter to form either one well or four wells for the containment of reagents.The smaller glass cover is placed onto the chip to seal PCR reagents

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two pieces of glass were spin-coated with PDMS before use.The PDMS layer and the glass base (the bigger glass piece in Fig.1)are permanently bonded by bringing both surfaces together after plasma treatment.A certain amount of PCR mixture was added into the wells with a pipette.The internal surface of the well is hydrophilic due to the plasma treatment,which makes the well fully filled with the PCR mixture.This also prevents bubble formation during thermal cycling.The smaller glass covers are placed onto the chip after the well has been filled with PCR reagents.The chip containing PCR reagents is covered by a 1mm glass slice (VWR International),and then clamped onto a thermally controlled substrate by a specially designed clamper.2.2Miniature thermal cycler

A miniature thermal cycler was designed and built to provide different temperature levels required for PCR.It consists of a thin film heating element for heating and a fan for rapid cooling.A thin film heater is sandwiched between two thin metal plates to form the heating unit,and a thermocouple is placed between the top metal plate and thin film heater to control the temperature of the top surface by adjusting the heating power using the feedback information from the ther-mocouple.The heating unit was then fixed onto a specially designed stage.The PCR chip was clamped on the top of the heater to ensure good contact between the PCR chip and the top surface of the heater.The temperature control was accomplished by using a computer system through a data acquisition card (PCI-DAS1001,Measurement Computing Corporation,Middleboro,Massachusetts).

The liquid temperature in the reaction well is lower than that of the substrate,and was calibrated by using a calibra-tion chip that has a thermocouple embedded directly in the reaction well.By adjusting the temperature of the substrate,the ideal temperature profile in the reaction well can be ob-tained.Figure 2presents a photograph of the actual thermal cycler.

2.3Fluorescence detection

The fluorescence detection module is schematically shown in Fig.3.It consists of a laser,a filter cube,an optical switch and a photo-detector.All are fiber coupled devices.Collinear excitation-collection geometry is used for fluorescence ex-citation and collection,similar to a standard fluorescence microscope.Light from the laser is fed into the reflection port (port 1)of the filter cube through a fiber,and reflected by the dichroic filter.The fiber at the input port (port 2)of the optical switch receives the laser light and guides it to the sample through the output fibers of the optical switch.These fibers also collect the fluorescence emission that propagates back to the optical switch,passes through the dichroic filter

Fig.2Photograph of the thermal cycler.A thin film heater was used for heating and a fan was used for cooling.A thermocouple is sandwiched between the metals plates to measure and control the temperature

and band pass filter,and then reaches the detector connected to the transmission port (port 3).After the operation ampli-fier,the signal is fed to data acquisition card.The laser is a single mode fiber coupled diode laser with a wavelength of 0nm (Blue Sky Research,CA,USA).The photo-detector is a fiber coupled Si-PIN photo-detector (Silicon Lightwave Technology Inc,CA,USA).The optical switch is a 1×4multimode fiber optical switch (Piezosystem Jena GmbH,Germany)with the fiber core diameter of 200µm.A pro-gram in a computer controls the light switching between channels.

The glass cover on the top of the PDMS chip forms a gap between the end of the collection fibers and the PCR solution.For the fiber fluorescence probes,previous works showed that the smaller the gap,the higher the fluorescence collection efficiency (Pfefer et al.,2001).To improve the fluorescence excitation and collection efficiency,two meth-ods were developed and tested as presented in Fig.3(b)and (c).In Fig.3(b),the fiber is embedded into the PDMS chip and the fiber end is directly inserted into the reaction well.The second method is using a fiber coupled two-lens device shown in Fig.3(c).This is a telecentric lens system;the fiber end and the sample are placed on the focal planes of the lens.

2.4Regents and sample preparation

To demonstrate the developed real time PCR chip and sys-tem,a 150-bp segment of Escherichia coli O157:H7stx1was amplified by using TaqMan polymerase chain reaction.E.coli DNA was extracted from cells using protocol and reagents from the QIAGEN Blood and Cell Culture DNA Kit.The primer set (Gene Link,Hawthorne,New York)is:for-ward,5 -GAC TGC AAA GAC GTA TGT AGA TTC G-3 ,and reverse,5 -ATC TAT CCC TCT GAC ATC AAC TGC-3 .The TaqMan probe (Gene Link,Hawthorne,New York)

lens

(a)

(b)(c)

Fig.3(a)A schematic

diagram offiber optical

fluorescence excitation and

detection system.Afiber optical

switch was used to realize the

multiple well detection.(b)PCR

chip with afiber embedded in

the reaction well.(c)

Fiber-lens-coupling device

was labeled with AlexaFluor7reporter dye and BHQ3

quencher dye with the following sequence:AlexFluor7

5 -TGA ATG TCA TTC GCT CTG CAA TAG GTA CTC-3

BHQ3.The excitation and emission peak of AlexFluor7

are650nm and670nm respectively.

Every100µl PCR mixture contains10µl of10×buffer,

1.2µl of each primer(25µM),

2.0µl of probe(10µM),

8.0µl of dNTPs(0.625mM of each),3.0µl of MgCl2,1.0µl

of TaqMan polymerase(5U/µl)and an appropriate volume

of H2O and DNA template.All reagents were purchased

from SIGMA-ALDRICH.Each cycle was comprised of three

stages:denaturing at94◦C for20s,annealing at55◦C for

30s,and extension at72◦C for30s.Each PCR run began

with a hot start at94◦C for5min,and ended with afinal

extension at72◦C for10min.

In this work,gel electrophoresis was used to confirm the

results of our on-chip real time PCR detection.The well

volume of the gel pad is10µl,thus a relatively larger volume

of PCR wells is necessary.The chip well with a diameter

of4mm was chosen in the experiment,whose volume is

about6µl.A2%agarose gel with0.04%ethidium bromide

was used and the results were visualized with a UV camera

(Bio-Rad Gel Doc1000,Bio-Rad Laboratories,Hercules,

California).

3Results and discussion

The experiments used TaqMan polymerase chain reaction

techniques to amplify the stx1segment of E coli O157:H7.

As described above,its length is150-bp.The results demon-

strate that this system can generate the correctly amplified

PCR product and perform complete real time PCR detection.

Additionally,the real time PCR experiments in four wells can

be done simultaneously using four-well PDMS chips.

3.1Single well PCR

Two detection methods have been demonstrated for single

well chip.During PCR test,the optical switch turns on for

1.5s in the last5s of the annealing period(55◦C,30s

in this experiment).Figure4(a)shows the meanfluores-

cent intensity curve for two different template DNA con-

centrations using thefiber-in-well detection method.The

observed characteristic intensity profiles for the two cases

indicate that the PCR was carried out successfully.It can be

clearly seen that the measuredfluorescent intensity starts to

increase exponentially at different cycle numbers for dif-

ferent amounts of initial DNA template.Although DNA

quantification is beyond the scope of this research,the

Biomed Microdevices

010203040

Cycle Number

F l u o r e s c e n c e I n t e n s i t y (a .u )

(a)

0.46

0.470.480.490.50.510.52

010203040

Cycle Number

F l u o r e s c e n c e I n t e n s i t y (a .u )

(b)

Fig.4Fluorescence intensity of two runs of real time PCR with different concentration of the initial DNA template (150-bp E.coli O157:H7stx1).(a)Fiber-in-well detection method was used during the test.The intensity started to increase earlier for higher initial DNA concentration than lower initial DNA concentration.(b)Fiber-lens-coupling detection method was used during the tests.The results are similar to that using fiber-in-well detection method

correct trend is shown in these results:A larger amount of initial template DNA corresponds to an earlier onset of the exponential phase,or an earlier detectable increase in intensity.The fluorescent intensity started to increase at ap-proximately the 17th cycle in the case of 12.5ng/µl initial template DNA,the 20th cycle for the case of 1.25ng/µl.The second detection method using a fiber-lens-coupling approach has been tested as well.In this case,the lens was vertically held in a holder above the chip as shown in Fig.2.The holder and the chip clamper were designed to make the chip on the front focal plane of the lens once the chip is clamped on the mini thermal

cycler.Figure 4(b)shows the mean fluorescent intensity curve with the same template DNA concentrations as in Fig.4(a).Similar to the fiber-in-well detection method,the characteristic intensity profiles observed for the two cases indicate that the PCR was carried

50 bp

150 bp 300 bp 500 bp LADDER

Fig.5Gel electrophoresis results of four PCR tests of 150-bp E.coli O157:H7stx1as described in Fig.4(a)and (b).The position of bright band of the samples corresponds to the 150-bp of the ladder,which indicated that the increase of the fluorescence intensity in Fig.4(a)and (b)is from the amplification of E.coli stx1

out successfully.The fluorescent intensity started to increase at approximately the 17th cycle in the case of 12.5ng/µl ini-tial template DNA,the 20th cycle for the case of 1.25ng/µl.To verify the PCR amplification results,gel electrophore-sis was also done for the four samples tested above.The results are presented in Fig.5.Since the DNA sample of E.coli O157:H7stx1is 150-bp,the gel results should show a distinct band corresponding to the 150-bp marker in the PCR ladder.As shown in Fig.5,the first column is PCR ladder,the second to the fifth column is the results of the on-chip PCR reactions.As expected,the bright band of PCR product for each sample corresponds to the marker’s band at 150-bp.The gel results further proved that amplification of the correct PCR product was achieved.3.2Four-well PCR

Multiple concurrent reactions can validate the repeatability of the same PCR protocol,or can be used to complete the serial dilution curves required for the quantification of the amount of DNA in the sample efficiently.Four-well chips were tested in this work to verify the repeatability of the system.Although the volume of the wells (4mm in diameter and 0.5mm in thickness)is 6.3µL,in the experiments,7µL reaction mixture was applied to each well in the four-well chip.The well was slightly over-filled so that no air was left after putting on the cover glass slide to prevent bubble formation during thermal cycling.The actual mixture volume in the well is about 6µL.The extra mixture is squeezed out when cover glass slide is placed on the top of the well.

In our studies,we have successfully conducted real-time PCR with a well volume as small as 1or 2µL.However,for the results reported in this paper we used the relatively larger wells (∼6.3µL)in order to accumulate sufficient solutions to perform gel electrophoresis to verify the on-chip results.The fluorescent intensity of each well was monitored using the system described above.Figure 6shows the

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0.42

0.44

0.46

0.48

0.5

0.52

010203040

Cycle Number

F l u o r e s c e n c e I n t e n s i t y (a .u )

Fig.6Fluorescence intensity results from a four-well chip real time PCR of 150-bp E.coli O157:H7stx1.There is no fluorescence intensity increase from negative control.The fluorescence intensity started to increase at almost the same cycle (20th)for well 2and 3which have the same initial template DAN concentration,while,the fluorescence intensity started to increase earlier for well 1(17th cycle)due to the higher initial DNA concentration

fluorescent intensity results of four simultaneous reactions with various initial DNA concentrations.The concentration is 12.5ng/µl in well 1,1.25ng/µl in well 2and 3,and 0.0ng/µl in well 4(negative control).Similar to the results of single well chips,the curves have the characteristic profile of a typical real time PCR.For the cases with the same ini-tial concentration of a template DNA of 1.25ng/µl (well 2and well 3),the measured fluorescent intensity started to in-crease at essentially the same cycle number,the 20th cycle.The fluorescence intensity started to increase earlier (17th cycle)for well 1than well 2and 3due to its higher DNA concentration (12.5ng/µl).In addition,the curve for the negative control is flat,which is expected.The plateau phase intensity of well 1is different from that of well 2(well 3).This may be due to the optic loss difference of fiber optical switch from channel to channel.In real-time PCR,it is the slope of the intensity ∼cycle number curve that is important.The loss difference does not affect the slop of the curve,and affect only the final intensity value.

Comparing the initial DNA concentration and critical cy-cle number of PCR runs conducted in the multiple-well chip in Fig.6and the single-well chip in Fig.4(a)and (b),the results indicated that the multiple-well PCR chip has the same efficiency as that of the single well chip,and also in-dicated that the multiple-well PCR is repeatable,and can be used to generate simultaneous serial dilution curves for quantification.3.3Standard curve

The standard curve and amplification efficiency of the developed system were examined using

fiber-lens-coupling

202224262830

-1.9

-1.7-1.5-1.3-1.1-0.9

Log(initial DNA concentration(ng/µl))

T h r e s h o l d c y c l e n u m b e r

Fig.7Standard curve of the developed PCR system.Fiber-lens-coupling detection method was used for this curve.The concentration of the four templates DNA for the standard curve is 0.093,0.047,0.023and 0.016ng/µl respectively.The amplification efficiency of the system calculated based on this curve is 1.63

detection method.The concentration of the original template DNA is 9.3ng/µl.It was diluted 100,200,400and 600times.That is,the concentration of the four templates DNA used is 0.093,0.047,0.023and 0.016ng/µl respectively.The standard curve is shown in Fig.7.The amplification efficiency calculated based on this standard curve is 1.63.Generally,the amplification efficiency depends on both the PCR system and the PCR protocol.In this experimental study,our objective is limited to demonstrate the feasibility of the on-chip real-time PCR,we didn’t tempt to optimize the PCR protocol.We believe the efficiency can be improved by optimizing the PCR protocol in the future.

4Conclusion

We developed a new miniature real time PCR system that consists of a disposable PDMS/glass PCR chip,a miniature thermal cycler and a fiber optical multi-well fluorescence detection module.The chips are cost effective and dispos-able.A fiber optical switch was used to realize multiple-well real-time PCR detection.Laser illuminates the PCR mix-ture only for 1.5s in every cycle,which greatly reduced the photobleach of the fluorescent dye.In addition,the optical components in the fluorescence detection module do not need precise alignment and stabilization due to the application of fibers.Moreover,increasing the number of channels of the optical switch can increase the number of detectable wells without any other changes in the detection module.For real time PCR system with high density well format,the well-to-well distance is limited by the diameter of fiber detection head (lens).In this study,an aspheric lens with a diameter of 5mm was used.However,there is other smaller diame-ter lens available and also compatible with fiber assembly,

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such as GRIN lens.Typical GRIN lens diameter is1.8mm. It was experimentally demonstrated that this system could successfully amplify and detect in real time E.coli O157:H7 stx1gene in both one well and four well chips.The results showed that the efficiency of the multiple well chips is the same as that of the single well chips.7µL PCR mixture was used in this experiment,due to the volume requirement to run gel electrophoresis to verify the results.Otherwise,the PCR solution(well)volume could be significantly smaller. This system has the potential to be developed into a compact and portable real time PCR device.

References

P.Belgrader,S.Young, B.Yuan,M.Primeau,L.A.Christel,

F.Pourahmadi,and M.A.Northrup,Anal.Chem.73,286–2

(2001).

M.Bu,T.Melvin,G.Ensell,J.S.Wilkinson,and A.G.R.Evans,J.

Micromech.Microeng.13,S125–S130(2003).

P.Gascoyne,J.Satayavivad,and M.Ruchirawat,Acta Tropica, 357–369(2004).

B.C.Giordano,J.Ferrance,S.Swedberg,A.F.R.Huhmer,and J.P.

Landers,Anal.Biochem.291,124–132(2001).

M.Hashimoto,P.C.Chen,M.W.Mitchell,D.E.Nikitopoulos,S.A.

Soper,and M.C.Murphy,Lab.Chip.4,638–5(2004).

J.A.Higgins,S.Nasarabadi,J.S.Karns,D.R.Shelton,M.Cooper,A.

Gbakima,and R.P.Koopman,Biosens.Bioelectron.18,1115–1123(2003).

G.Hu,Q.Xiang,R.Fu,B.Xu,R.Venditti,and D.Li,Anal.Chim.

Acta.557,146–151(2005).

M.U.Kopp,A.J.Mello,and A.Manz,Science280,1046–1048(1998).E.T.Lagally,I.Medintz,and R.A.Mathies,Anal.Chem.73,565–570

(2001).

D.S.Lee,S.H.Park,H.S.Yang,K.H.Chung,T.H.Yoon,S.J.Kim,

K.W.Kim,and Y.T.Kim,Lab Chip.4,401–407(2004a).

D.S.Lee,M.H.Wu,U.Ramesh,C.W.Lin,T.M.Lee,and P.H.Chen,

Actuat.B-Chem.100,401–410(2004b).

T.M.Lee,M.C.Carles,and I.Hsing,Lab Chip.3,100–105(2003). Y.C.Lin,C.C.Yang,and M.Y.Huang,Sensor.Actuat.B-Chem.71, 127–133(2000).

J.Liu,M.Enzelberger,and S.Quake,Electrophoresis23,1531–1536 (2002a).

Y.Liu,C.B.Rauch,R.L.Stevens,R.Lenigk,J.Yang,D.B.Rhine,and P.Grodzinski,Anal.Chem.74,3063–3070(2002b).

Y.Matsubara,K.Kerman,M.Kobayashi,S.Yamamura,Y.Morita, Y.Takamura,and E.Tamiya,Anal.Chem.76,34–39 (2004).

H.Nagai,Y.Murakami,K.Yokoyama,E.Tamiya,and Y.Morita,Anal.

Chem.73,1043–1047(2001).

P.J.Obeid,T.K.Christopoulos,H.J.Crabtree,and C.J.Backhouse, Anal.Chem.75,288–295(2003).

T.J.Pfefer,K.T.Schomacker,M.N.Ediger,and N.S.Nishioka,IEEE J.

Quantum Elect.7,1004–1012(2001).

I.Schneegass,R.Brautigam,and J.M.Kohler,Lab Chip.1,42–49

(2001).

K.Sun,A.Yamaguchi,Y.Ishida,S.Matsuo,and H.Misawa,Sensor.

Actuat.B-Chem.84,283–2(2002).

J.West,B.Karamata,B.Lillis,J.P.Gleeson,J.Alderman,J.K.Collins, W.Lane,A.Mathewson,and H.Berney,Lab.Chip.2,224–230 (2002).

E.K.Wheeler,W.Benett,P.Stratton,J.Richards,A.Chen,A.Christian,

K.D.Ness,J.Ortega,L.G.Li,T.H.Weisgraber,K.Goodson,and

F.Milanovich,Anal.Chem.76,4011–4016(2004).

Q.Xiang,B.Xu,R.Fu,and D.Li,Biomed.Microdev.7,273–279 (2005).

J.Yang,Y.Liu,C.B.Rauch,R.L.Stevens,R.Liu,R.Lenigk,and P.

Grodzinski,Lab.Chip.2,179–187

(2002).

Springer