1914 IEEE SENSORS JOURNAL, VOL. 12, NO. 6, JUNE 2012

Glass-Based Continuous-Flow PCR Chip With aPortable Control System for DNA Amplification

Ning Xue, Student Member, IEEE, and Weiping Yan

Abstract A glass-based continuous-flow polymerase chainreaction (PCR) chip has been designed and fabricated. The deviceconsists of a glass microfluidic channel, three NiCr heaters, andthree Ni thermometers on the silicon substrate. An intelligenttemperature-control circuit system has been designed to achievedesirable temperature control (95, 72, and 55 C) at the threetemperature zones of the PCR chip. Simulation underneath themicrofluidic channel using the finite element method shows thatthe temperature distribution through the three temperature zonesare relatively uniform. A mixture of DNA samples for PCR wasallowed to flow through the microfluidic channel under differentflow rates. The amplified sample of the target DNA obtainedfrom the PCR chip was then separated by electrophoresis andwas analyzed using an ultraviolet analyzer. The result indicatesthat DNA amplification can be achieved and that its amplificationfactor depends greatly on the injection rate of the sample. Theoptimum sample-flow rate is 0.6 l/min.

Index Terms DNA amplification, microelectromechanicalsystems (MEMS), Ni thermometer, NiCr heater, polymerase chainreaction (PCR) chip, temperature control system.

I. INTRODUCTION

RECENTLY, miniaturization of analytical systems forapplication in biomedicine and clinic diagnosis havereceived much attention due to its advantages such as lowreagents consumption, short reaction time, and low cost. Thepurpose of lab-on-a-chip system is to integrate the functionsof biosample preconditioning, chemical reaction, separation ofsamples and detection of the constituents into a single chip.Thus, research related to a polymerase chain reaction (PCR)chip is a vital part for lab-on-a-chip system [1][5]. Duringthe PCR reaction process, DNA molecules are amplified inthe presence of an enzyme, respective primes and dNTPsin three alternate temperature zones, consisting of a high-temperature denaturation zone (95 C), a low-temperatureannealing zone (5060 C), and a medium-temperature exten-sion zone (72 C). Usually, PCR chips microfabricated intwo structures can carry out the amplification reaction: amicro-chamber PCR chip and a continuous-flow PCR chip.

Manuscript received November 27, 2011; accepted December 20, 2011.Date of publication January 3, 2012; date of current version April 25, 2012.The associate editor coordinating the review of this paper and approving itfor publication was Prof. Elliot R. Brown.

N. Xue is with the Department of Electrical Engineering, Universityof Texas at Dallas, Richardson, TX 75080 USA (e-mail:nxx083000@utdallas.edu).

W. Yan is with the School of Electronic Science and Technology,Dalian University of Technology, Dalian 116024, China (e-mail:yanwp@utdallas.edu).

Color versions of one or more of the figures in this paper are availableonline at http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/JSEN.2011.2182047

In micro-chamber PCR chip, DNA mixtures are reacted andDNAs are amplified in a micro-chamber made of materialssuch as silicon, glass and polymers. In the continuous-flowPCR chip, under the action of a certain force, the PCRmixtures flow through the serpentine channel maintained atthree different temperatures. Commonly, the channel of thischip is designed for 1530 amplification cycles. Each cycle isexpected to finish one DNA-molecules-amplification process.Compared to microchamber PCR chip, a continuous-flow PCRchip has the advantage of short reaction time, and rapid andsteady temperature control. In addition, it can be integratedwith capillary electrophoresis (CE) and yield PCR-CE chips.Table 1 lists the comparison among three types of PCR chipsin terms of the required sample volume, reaction time, cost,and integration to other lab-on-a-chip systems [6]-[10].

Glass, poly(dimethylsiloxane) (PDMS), and silicon arethree common materials used to compose the PCR chips. Kimdeveloped a PDMS-based microchamber PCR chip, whereone temperature cycle was set to 70 seconds [11]. Howeververy few papers have reported a continuous-flow PCR chipmade of PDMS because the latters high hydrophobic naturecan impede the flow of samples in the microchannel. Siliconis a common material used in both microelectromechanicalsystems, popularly known as MEMS, and the semiconductorfield. Hence, many previous microfabricated PCR chips haveused silicon as their structural or channel material. However,silicon has certain undesirable properties such as opacityand least bio-compatibility which have limited its furtherdevelopment as the material of choice for lab-on-a-chipsystems. Glass is an ideal material to compose lab-on-a-chipsystem, because it is rigid, transparent, bio-compatible andhydrophilic [1], [5], [12].

We introduce a glass-channel PCR chip in this study.Based on the simulation using the finite element analysis(FEM), a continuous-flow PCR chip has been designed andfabricated, including a micro-reaction glass channel, heaters,and thermometers on a silicon substrate. The three constant-temperature zones were controlled by a portable circuit system.

II. EXPERIMENTA. Design

The microfluidic circulation channel was etched on a soda-lime glass substrate. Another glass lid with the microfluidicchannel was covered on the glass substrate. Three heaters andthermometers were fabricated on an oxidized silicon substrate.Finally, the glass substrate and silicon chip were bonded toform the PCR chip, as shown in Fig. 1.

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XUE AND YAN : GLASS-BASED CONTINUOUS-FLOW PCR CHIP WITH A PORTABLE CONTROL SYSTEM FOR DNA AMPLIFICATION 1915

TABLE ICOMPARISON AMONG THREE TYPES OF PCR DEVICES

Minimumrequiredsamplevolume

Reactiontime

(30 cycles)

Costof

device

Integrationwith other

micro-chips

ConventionalPCR machine 20 l 30-60 min High No

Microfabricatedcontinuous-flow

PCR chip< 10 l 2-30 min Low Easy

MicrochamberPCR chip 10-50 l 15-30 min Low Difficult

Cover-glassNot to scale

Substrate-glass

Thermometers and heaters

Fig. 1. Schematic view of cross-section of the PCR chip.

1 cm

2.5 cm

1 cm

1 cm

Sample inlet Sample outlet

DNA annealing zone

DNA denaturation zone

DNA extension zone

Fig. 2. Micro-circulation channel of continuous-flow PCR chip.

To obtain high DNA-amplification efficiency, the durationof denaturation and annealing should be shorter, whereas theextension time should be longer. As a result, in our continuous-flow PCR chip, different lengths of the microreaction channelwere designed in each of the temperature zones. In addition,to ensure DNA strands fully unfastened, the DNA should bepreheated before the DNA amplification starts. This processcan be achieved by extending the length of the denaturationchannel before the first circle.

A micro-channel with 20 cycles was designed in the PCRchip (Fig. 2). The length ratio of the channels with threedifferent temperature zones is 2:2:5, and the total length ofthe microchannel is approximately 1.9 m. The width anddepth of the microchannel are 120 and 30 m, respectively.Moreover, to have a smooth liquid flow, the bending area ofthe channel was designed in an arc. The three temperaturezones are marked in Fig. 2.

As a heater, a NiCr thin film has relatively high heatingefficiency and good stability, whereas a Ni film is usedas the material for temperature sensor because of its high

2.8 cm2.7 mm

4 mm1 cm

NiCr heater Ni thermometer

Fig. 3. Schematic layout of a heater and a thermometer.

(a) (b)

(c) (d)

Fig. 4. Thermal distribution analysis of continuous-flow PCR chip. Thick-nesses of glass are (a) 0.6 mm, (b) 1.4 mm, and (c) 2.2 mm. (d) Cross-sectionalthermal distribution of 1.4-mm-thick glass.

temperature coefficient of resistance (TCR) [13]. Three heaterswere designed in a serpentine shape to enlarge the heatingarea and increase the resistance. To maintain the accuracy oftemperature measurement, three thermometers were placed atthe center of each heater. The design of one pair of heater andthermometer is shown in Fig. 3, in which the trace width ofthe NiCr heater is 400 m and the gap between two traces ofthe heater is 300 m; similarly, the trace width is 75 m andgap is 100 m for the Ni thermometer.

B. FEM SimulationIn our PCR chip, the heaters and thermometers are under-

neath the glass substrate, thus a uniform thermal distrib-ution on the surface of the channel is the main concern.Thermal distributions of the glass substrate with differentthicknesses (0.6, 1.4 and 2.2 mm) were studied using theANSYS software. Three different temperatures (95, 72, and55 C) were applied at the three bottom zones of the glasssubstrate. The simulated thermal-distribution profiles on thesurface of the channel are depicted in Fig. 4 (a through c).Nonuniformity of thermal distribution for the 2.2-mm-thickglass obstructs the performance of the PCR process; whereas0.6 and 1.4-mm-thick glasses are suitable for use because ofthe observation of uniform thermal distribution on the surfaceof the channel. Moreover, a temperature difference existsbetween the channel and the heater. A cross-section view ofthe thermal distribution across a 1.4-mm-thick glass substrateis shown in Fig. 4d, showing that an average difference of2 C exists between the heater and the surface of channel.

1916 IEEE SENSORS JOURNAL, VOL. 12, NO. 6, JUNE 2012

(a)

(b)

(c)

(d)

(f)

(g)

SiN

SiliconSiO2

(h)

Glass lid

NiCr NiCrNi

Glass substrate

Glass lid

GlassCr

PhotoresistMask

Glass substrate

(e) Not to scale

Fig. 5. Fabrication process of continuous-flow PCR chip. (a)(e) Glassfabrication: (a) Cr patterning, (b) Cr etching, (c) glass etching, (d) protectionlayers removal, (e) thermal bonding of two glasses to form sealed channel.(f)(g) Silicon fabrication: (f) SiN deposition, (g) Ni thermometer and NiCrheater patterning. (h) Bonding of glass and silicon.

700

600

500

400

100

Tem

pera

ture

(C)

0

Time (min)0 100 200 300 400 500 600 700

Fig. 6. Temperature curve for glass thermal bonding.

Accordingly, this temperature difference must be consideredin the design of the temperature control system. We chose1.4-mm-thick glass because it is a commercially availableglass product and has good uniformity of thermal distributionin the channel.

C. FabricationA 1.4-mm-thick glass, coated with 145-nm-thick Cr /

570-nm-thick photoresist, was used to fabricate themicro-circulation channel. The fabrication process ofthe micro-circulation channel is shown in Fig. 5. First, thephotoresist was patterned, followed by Cr-etching. A 30-m-thick glass channel was then anisotropically etched usingHF:HNO3:H2O = 5 ml:10 ml:80 ml, with an etching rate of0.5 m/min. Another unprocessed glass was used as the lid;3-mm-diameter inlet and outlet holes were drilled through theglass lid using an ultrasonic driller. To seal the microfludicchannel, the two glass plate were placed in contact with eachother with a heavy load on top and were thermally treated ina programmable air convection oven. And the temperature-control curve is shown in Fig. 6. Uniform firm bonding canbe achieved after 34 cycles of temperature control.

For the silicon fabrication process, a 170-nm-thick layerof silicon nitride was deposited on a 2-m-thick oxidized

Fig. 7. Photograph of packaged PCR chip.

Amplifier andA/D

PE couplerand amplifier

55 C

72 C

95 C

Microprocesser

LCD display

Keypad control

Fig. 8. Diagram of temperature-detection-control system of PCR chip.

silicon wafer that is used as the electrical isolation layer.Subsequently, thin films of NiCr and Ni thin films weredeposited and patterned as the heater and the thermometer,respectively. Finally, the three pieces of heaters andthermometers were attached to the bottom of the glasssubstrate by thermally conductive glue. The photograph ofa packaged fabrication device is shown in Fig. 7, where thepads of the heaters and thermometers are connected to thepins on the printer circuit boards.

D. Temperature-Control SystemThe hardware temperature-control system was designed to

implement the function of temperature sensing and tempera-ture control. To achieve rapid and stable constant-temperatureconditions (95, 72, and 55 C) at the various temperaturezones, a proportional-integral-differential (PID) algorithm wasadopted to calculate the heating time, while the analysis ofthe complete dataset was accomplished in the microprocessor.The diagram of the hardware system is presented in Fig. 8,and it mainly includes temperature data-collection module(Wheatstone bridge, voltage amplifier, and analog-to-digitalconverter) to acquire temperature information from the ther-mometers, 8052 microprocessor, power output-driving module(photoelectric coupler and power amplifier) that turns on/offthe heaters voltage, keypad control, liquid crystal display(LCD) display, and RS232 communication module. Finally,this control circuit was connected to the personal computer(PC) interface for real-time temperature monitoring.

XUE AND YAN : GLASS-BASED CONTINUOUS-FLOW PCR CHIP WITH A PORTABLE CONTROL SYSTEM FOR DNA AMPLIFICATION 1917

Tem

pera

ture

(C)

120

140

100

80

60

40

20

Voltage (V)0 10 20 30 40 50

Fig. 9. Curve of voltage-temperature of NiCr heater.

Res

istan

ce (k

)

0.680.70

0.660.640.620.600.580.560.540.520.500.48

Temperature (C)

Resistance 1Resistance 2Resistance 1 36 h laterResistance 2 36 h later

20 40 60 80 100

Fig. 10. Resistance response of Ni thin film as a function of temperatureand time.

III. RESULTS AND DISCUSSIONA. Characterization of Heater and Thermometer

An excellent heater should maintain a stable resistance evenunder changing of temperatures. Lower power input to obtaina high temperature is also preferable. We have proved that theresistance of the thin film of NiCr alloy can remain constanteven under conditions of variable temperatures [13] - [14]. Thecurve of voltage-temperature of the fabricated NiCr heater inthe PCR chip is shown in Fig. 9. Within the voltage rangeof 545 V, the corresponding temperature range is 2595 C.Because the highest temperature is 95 C for the PCR chip,the fabricated NiCr film can satisfy the requirement as a heaterin a PCR chip.

The sensitivity of the thermometer corresponds to the slopof the resistance-temperature curve. From Fig. 10, the resis-tance of the Ni thin film has a linear temperature response,and its TCR is 4.0103/C.

B. System SetupThe system setup is presented in Fig. 11. Saline (0.9 %

NaCl) solution was injected into the inlet of the microchannelusing a syringe pump, and the flow rate was set to 1 l/min.The temperature values of the three zones (55, 72, and 95 C)were preset through the PC program. As the program started,

100110120

8090

6070

20304050

010

55 C

Time/min

72 C95 C

T/CData window

0 1 2 3 4 5

Fig. 11. PCR chip testing setup with control circuit and PC interface control.

1 2 3

1 2 3 1 2 3

1 2 3

Fig. 12. Electrophoresis diagram of amplified DNA samples under differentflow rates (a) 7.6 l/min, (b) 3.1 l/min, (c) 1.2 l/min, (d) 0.6 l/min.(Bars in column 1 presents the standard plant DNA, bars in columns 2 and 3presents the PCR results by conventional PCR machine and microfabricatedcontinuous flow PCR chip, respectively.)

the preset temperature data were sent to the microprocessorfor analysis and the heating intervals of the three heaterswere calculated from the PID control program. In Fig. 11,the window of real-time monitoring from the PC programshows that the response time of the heater was approximately20 seconds. The control circuit can maintain the temperaturesufficiently stable, with a small variation of 1 C during thesample flow.

C. In Vitro TestingTo verify the feasibility of DNA amplification in our

continuous-flow PCR chip, 2 L of plant DNA, 5 L of for-ward primer, 5 L of reverse primer, 8 L of dNTP, 10 L ofphosphate buffer solution, 0.5L of TaKaRa EX Taq enzymeand 69.5L of distilled water were mixed to prepare a DNAmixture sample. The sample flowed through the fabricatedPCR chip, and the temperatures were controlled at constantlevels. Meanwhile, the above DNA mixture sample was ampli-fied 30 cycles in a commercial PCR thermal cycler machine(TP-600, Dalian, China), as a control. After the PCR process,both the DNA analytes were subjected to electrophoresis underan electric field of 2 V/cm in an electrophoresis apparatus (30III, Beijing Liuyi Instrument Factory, China) to separate theDNA molecules in the mixture. Finally, an ultraviolet analyzer(JY02S, Junyi-Dongfang Electrophoresis Equipment, China)was used to obtain the electrophoresis diagram. The results ofDNA amplification under different sample flow rates comparedwith the DNA amplification result from a conventional PCRmachine are shown in Fig. 12. In Fig. 12, Column 1 shows thedifferent standard plant DNA samples; Column 2 contains theDNA-separation result from the commercial PCR machine,

1918 IEEE SENSORS JOURNAL, VOL. 12, NO. 6, JUNE 2012

and Column 3 presents the DNA-amplification result obtainedfrom our PCR chip. It verifies that our microfabricatedcontinuous-flow PCR system can accomplish amplificationof the DNA samples. Furthermore, the figure shows that aslower sample flow rate can enhance the efficiency of DNAamplification. The optimum flow rate is 0.6 l/min.

IV. CONCLUSIONBased on FEM analysis of the thermal distribution over the

surface of a microfluidic channel, a glass-based continuous-flow PCR chip was successfully designed and fabricated.A portable temperature-control/detection circuit was alsodesigned based on a 8052 microprocessor. The temperaturevariation was only 1 C during sample injection. Finally, theDNA-amplification performance of the fabricated PCR chipwas established. The sample of DNA mixture flows throughthe PCR chip at different flow rates. The result verifies thefeasibility of DNA amplification by our continuous-flow PCRdevice, which is of great importance for the future study of aPCR-CE system.

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[2] C. T. Wittwer, G. C. Fillmore, and D. J. Garling, Minimizing thetime required for DNA amplification by efficient heat-transfer to smallsamples, Anal. Biochem., vol. 186, no. 2, pp. 328331, May 1990.

[3] E. T. Lagally, P. C. Simpson, and R. A. Mathies, Monolithic integratedmicrofluidic DNA amplification and capillary electrophoresis analysissystem, Sensors Actuat. B, vol. 63, no. 3, pp. 138146, May 2000.

[4] M. A. Northrup, L. A. Christel, W. A. McMillan, K. Petersen, F.Pourahmadi, L. Western, S. Young, A. I. Michael, H. G. David, andJ. S. John, A new generation of PCR instruments and nucleic acidconcentration systems, in PCR Applications. San Diego, CA: Academic,1999, pp. 105125.

[5] Z. Q. Zou, X. Chen, Q. H. Jin, M. S. Yang, and J. L. Zhao, A novelminiaturized PCR multi-reactor array fabricated using flip-chip bondingtechniques, J. Micromech. Microeng., vol. 15, no. 8, pp. 14761481,Aug. 2005.

[6] K. Sun, A. Yamaguchi, Y. Ishida, S. Matsuo, and H. Misawa, A heater-integrated transparent microchannel chip for continuous-flow PCR,Sensors Actuat. B, vol. 84, nos. 23, pp. 283289, May 2002.

[7] T. Fukuba, T. Naganuma, and T. Fujii, Microfabricated flow-throughPCR device for underwater microbiological study, in Proc. Int. Symp.Underwater Technol., 2002, pp. 101105.

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[10] W. Li, J. A. Abad, R. D. French-Monar, J. Rascoe, A. Wen, N. C.Gudmestad, G. A. Secor, I.-M. Lee, Y. Duan, and L. Levy, Multiplexreal-time PCR for detection, identification and quantification of Can-didatus Liberibacter solanacearum in potato plants with zebra chip, J.Microbiol. Methods, vol. 78, no. 1, pp. 5965, Jul. 2009.

[11] J. A. Kim, S. H. Lee, H. Park, J. H. Kim, and T. H. Park, Microheaterbased on magnetic nanoparticle embedded PDMS, Nanotechnology,vol. 21, no. 16, p. 165102, Apr. 2010.

[12] G. Voskerician, M. S. Shive, R. S. Shawgo, H. von Recum, J. M.Anderson, M. J. Cima, and R. Langer, Biocompatibility and biofoulingof MEMS drug delivery devices, Biomaterials, vol. 24, no. 11, pp.19591967, May 2003.

[13] W. P. Yan, N. Xue, X. H. Shi, J. S. Liu, and J. H. Guo, Study on metalmembrane temperature sensor and microheater for PCR chip, Surf. Rev.Lett., vol. 15, no. 1, pp. 183187, Feb. 2008.

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Ning Xue (S11) received the B.S. and M.S. degrees in electrical engineeringfrom the Dalian University of Technology, Dalian, China, in 2005 and2008 respectively. He has worked in the bioMEMS microfluidc devices forhis master research. He is currently pursuing the Ph.D. degree with theDepartment of Electrical Engineering, University of Texas, Dallas.

He joined Micro-Nano Devices and Systems Laboratory, Microelectro-mechanical Systems (MEMS) Research Group, University of Texas at Dallas,Richardson, TX, in 2009. His current research interests include MEMS fabri-cation, microfluidic device, bioMEMS for wireless implantable applications,pressure sensors, and radio frequency (RF) MEMS of on-chip inductor andcapacitor.

Weiping Yan received the M.S. degree in physics and devices of semiconduc-tors from Jilin University, Changchun, China, in 1975 and 1989, respectively.

She was with the Department of Electronic Science, Jilin University, from1975 to 1990. She has been with the Department of Electronic Engineering,Dalian University, Dalian, China, since 1990. Her current research interestsinclude the fabrication of the integrated biochip, design, and fabrication ofsemiconductor sensors.

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