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Sensors and Actuators A 136 (2007) 178183

Thermal control of micro reverse transcription-polymerasechain reaction systems

Nan-Chyuan Tsai , Chung-Yang SueDepartment of Mechanical Engineering, National Cheng Kung University, No.1, Ta-Hsueh Road, Tainan 701, Taiwan

Received 8 June 2006; received in revised form 13 October 2006; accepted 24 October 2006Available online 4 December 2006

bstract

Being beneficial from dramatic progress in biochemical and micro-electromechanical technologies, traditional DNA manipulation devices tend toe miniaturized to speed up detection processing. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR)re two typical examples of them. In this report, micro RT-PCR (RT-PCR) is designed to quantitatively detect viruses. Test samples reservoirs,T-PCR, and capillary electrophoresis are integrated on a SU-8 based monolithic chip. A high-precision temperature control unit is well developed

y embedding amplification circuits and an Intel 8051 microprocessor. The integrated system exhibits high efficacy of heat transfer, exemptionf fluid flow clogging and adequate sensitivity to, by feedback, control the cycle of detection for the infected malignant tissues via intensiveimulations.

2006 Elsevier B.V. All rights reserved.

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eywords: RT-PCR; Capillary electrophoresis; SU-8; Feedback control

. Introduction

Polymerase chain reaction (PCR) in DNA amplification wasver profoundly studied and reported by Mullis et al. in 1985 [1].CR is mainly to duplicate a certain fragment sequence of DNA

n million times precisely. The amplification process of PCRan be divided into three consecutive temperature sectors: (A)enaturation: the double-strand DNA segment is disassemblednto two singles at high temperature 94 C, (B) annealing: theingle-strand DNA is attached to a specific primer at 54 C, (C)xtension: the associated complementary DNA (cDNA) singlesith primers are extended into double-strand DNAs. This paper

s an extended research report from PCR to reverse transcriptionCR (RT-PCR). With RNA as the template of RT-PCR, the firsttrand of the corresponding cDNA is synthesized by a set ofrtificial primer and certain particular enzyme. Right afterward,he cDNA is used as the template for PCR, thus a great deal of

NA fragments are duplicated via appropriate transformation

riggered by Taq DNA polymerase. RT-PCR can be appliedor detection of RNA virus, mass production of cDNA, and

Corresponding author. Tel.: +886 6 2757575x62137; fax: +886 6 2369567.E-mail address: nortren@mail.ncku.edu.tw (N.-C. Tsai).

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924-4247/$ see front matter 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.sna.2006.10.057uantitative analysis of infection caused by DNA virus. A fewllnesses do not stem from DNA virus (e.g., Hepatitis C [2]) sohat PCR detection process cannot be directly employed. Theyave to be reversely transcribed to the first-strand cDNA at first,efore PCR process can be undertaken. The manipulation typesf Chip-PCR can be categorized into continuous-flow [27] andtationary [8,9] PCR. The type of stationary PCR is processedn a chamber that can provide heating and cooling cycles butamples are not flowing at all. The samples for the type ofontinuous-flow PCR dose flow through a few heated zones,ndividually with specified temperature curves, to complete theull reaction cycle. The first Chip-PCR was reported by Northrupt al. and it was basically a stationary type [9]. They utilizedilicon as the substrate and included heater, sensor, and reactionhamber in a single chip by MEMS technologies. In 1998, Koppnd Manz proposed a continuous-flow PCR by heating threeeaction zones from externally-added heating blocks [7]. Theontinuous-flow micro RT-PCR (RT-PCR) chips, reported inhis paper, are to integrate internally-embedded heaters, thermalensors, RT-PCR module, and capillary electrophoresis into

single detection unit so that it is more compact, efficient,

nd reliable. In RT-PCR, it is extremely crucial to controlemperature to follow the required trajectory precisely. Only ifhe required temperature is satisfied in each reaction zone, can

mailto:nortren@mail.ncku.edu.twdx.doi.org/10.1016/j.sna.2006.10.057

nd Actuators A 136 (2007) 178183 179

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Toasystem is shown in Fig. 3. The heaters and thermal sensors arefabricated in the top layer, as shown in Fig. 4, to control the tem-perature of the fluidic flowing in the microchannels fabricatedin the lower layer.

Fig. 2. Desired temperature control curve.N.-C. Tsai, C.-Y. Sue / Sensors a

he expected DNA amplification be achieved. In contrast, if theequired reaction temperature is deviated far away, the desiredeaction in each zone would not be completed and the exam-nation result might be incorrect. Since the measured signal,rovided by the thermal sensor, is very weak in magnitude, anfficient amplification circuit is needed to overcome the possiblyost fidelity. In addition, the embedded heater also needs anfficient control circuit to regulate required drive current that isuantitatively decided by a microprocessor Intel 8051. Finally,heoretical analysis and simulation results are addressed andiscussed.

. Design and fabrication

.1. Design concept

The sample and the reagent together are designed to flowhrough four different temperature zones. Because these fourones require different time durations to complete individualhemical reaction, the net channel length of each zone is pur-osely designed to define the time duration for the fluid to passhrough. In this work, the time duration ratio for the four reac-ion zones is set as 4.5:1:1:2 for RT, denaturation, annealing, andxtension, respectively.

Since the reported RT-PCR unit is composed of two layersonded together (the heater in the upper layer but micro fluidichannels in lower layer), sufficient thermal conductivity, fromeater to fluid, has to be verified and examined. The heater isxed but the fluid in the channel flows so that the heat energy is

ransferred by both conduction and convection. Before the RT-CR unit is really fabricated, it has to be qualified, in advance,y commercial software IntelliSuite to ensure the heat transferfficacy from heater to microfluidic channels. The simulationesults will be shown and discussed later.

.2. Fabrication process

Roughly speaking, the fabrication can be divided into twoortions: (A) heaters and sensors and (B) reservoirs and reac-ion meanders. At first, poly-methyl methacrylate (PMMA) isssigned to serve as the material of the substrate of reservoirs andeaction meanders and the SU-8 photo-resist is used to make theain frame of structure. SU-8 is coated on the PMMA surface

nd then patterned to construct the reservoirs and microchan-els. Heaters and thermal sensors are made of Ti and Pt/Cr,espectively on the Pyrex 7740 wafer. Both of them are fabri-ated by means of lift-off process. Finally, SU-8 photo-resists used to serve as the isolation between individual heater blocksnd sensor blocks as well. The fabrication addressed above isor the upper layer of the RT-PCR unit. Once the lower layers also finished, the SU-8 photo-resist would serve as the bond-ng material between two layers. Traditionally, thermal diffusiononding process is undertaken at 650 C to bond two individ-

al layers. However, severe damage to micro devices, such asrack or fracture, might be seeded. In order to avoid the riskf high-temperature sandwiching, SU-8 bonding is employed inhis work Fig. 1.ig. 1. Schematic diagram of designed Chip-RT-PCR: (a) reservoir of RT, (b)eservoir of PCR, (c) reaction meanders of RT, (d) reaction meanders for denatur-ng, (e) reaction meanders for annealing, and (f) reaction meanders for extension.

. Temperature control system

The desired temperature curve for RT-PCR is shown in Fig. 2.he so-called temperature control system of RT-PCR consistsf heaters, thermal sensors, amplifiers, a microprocessor, andssociated control circuits. The schematic diagram of controlFig. 3. Schematic diagram of temperature control system

180 N.-C. Tsai, C.-Y. Sue / Sensors and Actuators A 136 (2007) 178183

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Fig. 4. Allocation of heaters and sensors.

.1. Amplification circuit for sensors

The electrical resistance of any material is varying as tem-erature changes. The relation of them can be described by:

T = R0[1 + (T T0)] (1)

here is the temperature coefficient of resistance (TCR) in/C. R0 is the reference resistance at T0. In this work, the tem-erature sensors are made of Pt/Cr and the dimension each is400 m 100 m 1500 m.

The TCR curve of thermal sensor is calibrated by experimentsnd shown in Fig. 5(a). The associated amplification circuit is

esigned and shown in Fig. 6. The resistor R4 is behaving ashe thermal sensor. The heater is made of titanium (Ti) withimension 1 cm 3.5 cm 1500 m. The corresponding TCRurve of the heater is shown in Fig. 5(b). The ideal heating poweran be expressed as follows:

ig. 5. (a) R/T curve of the thermal sensor (Pt) and (b) R/T curve of the heaterTi).

c

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Fig. 6. Amplification circuit of the thermal sensor.

= V2

R(2)

here V is the applied voltage and R is the resistance of heater.is dependent of material and dimensions:

= LA

(3)

here is the resistivity ( cm); L, the length of heater andis its cross section area. In general, the thickness of heaters is

onstant, therefore Eq. (3) can be rewritten as follows:

= t

L

W= Rs L

W(4)

here Rs is the sheet resistance. From Eq. (4), it can bebserved that the resistance is mainly related to geometry ofhe heater. Therefore, the resistance can be designed in geom-try in advance. On the other hand, the thermal transfer can bebtained by the finite element (FE) model:[kt

][0]

[0] [k]

] {{T }{V }

}

={{Qnd}{Ind}

}+

{{Qc}{0}

} {{Qc}{0}

}+

{{Qg}{0}

}+

{{Qj}{0}

}

(5)

here [kt]: thermo-conduction matrix, [kv]: electric conductivityatrix, {T}: node temperature vector, {Qnd}: node temperature

ector, {Ind}: node current vector, {Qc}: surface convection,V}: node potential vector.

In order to evaluate the performance of designed heaters, Eq.5) is used to examine and verify the transformation from appliedontrol current to resulting temperature by commercial software,NSYS.A high-resolution thermal sensor amplification circuit is pre-

ented and shown in Fig. 6. The circuit consists of an OP AD620

nd a voltage regulator IC AD581 that provides a precise outputt 10 V from an unregulated input level ranging from 12 to 30 V.his sensor amplification circuit plays the role of feedback toicrocontroller by an ADC. The output voltage versus resistance

N.-C. Tsai, C.-Y. Sue / Sensors and Actuators A 136 (2007) 178183 181

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Fig. 7. Resolution of thermal sensor circuit.

urve is shown in Fig. 7. The resolution of sensor amplify circuits about 0.1 (V/C). This resolution is far higher (five times) thanonventional design such as electrical bridge. In addition, it isufficiently sensitive for a 8-bit ADC (the minimum resolutionequirement of 8-bit ADC is about 0.02 V/Step).

. Simulations

Since the reported RT-PCR is a continuous-flow type andhe chain reactions are subjected to specified temperatures,ffective flow of the fluid, carrying DNA samples, and correctemperature distribution of four heated zones are the most impor-

ant two issues to be ensured. The designed RT-PCR, whoseealistic picture is shown in Fig. 8.

The temperature distribution of the top layer is simulated andhown in Fig. 9, where four heated zones present different tem-

Fig. 8. RT-PCR Chip.

Fig. 9. Temperature distribution of the top layer of RT-PCR unit.

l

q

w0tr

{wmIb

((0cC

Fig. 10. Thermal entry length of three reaction zones.

erature portraits in color. For each reaction zone, there existsso-called thermal entry length before the desired temper-

ture is saturated, as shown in Figs. 10 and 11. The thermalntry length is mainly function of temperature and flow rate.he higher temperature to be saturated, the longer the thermalntry length will be. The thermal entry length becomes shorterf the flow rate is lower. If the heat transfer from the heaters tohe fluid is concerned, the bounding layer, made of photo-resistU-8, has to be examined for its transfer efficacy. From Fouriers

aw:

= kcAc Tn

(6)

here q is the heat flux; kc, thermal conductivity of SU-8 (about.2 W/mK); Ac, the cross section of heat flux; and T/n is theemperature gradient in normal direction. Eq. (6) can be furtherewritten in matrix form:

Qnd} = [Kt]{T } (7)here {Qnd} is heat flux vector, [Kt] is thermal conductivityatrix and {T} is the temperature vector. Commercial software

ntelliSuite is used to simulate the distribution of temperature,ased on proper boundary conditions and parameters.

A few assumptions have been made for simulations, such asa) flow rate: 50 nl/s, (b) thermal conductivity: 0.643 W/mm C,

c) density of fluid: 1000 kg/m3, and (d) viscosity of fluid:.0005 kg/ms. After the heat is transferred transversely, heatonvection then succeeds along the microchannels by Newtonooling Law:

Fig. 11. Temperature distribution in heated zone.

182 N.-C. Tsai, C.-Y. Sue / Sensors and Actuators A 136 (2007) 178183

Fig. 12. Micrograph of microchannels.

q

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Fig. 13. Sideview of flow field within microchannels.

=hAs(T s T) (8)here q is the heat flux; h, the thermal conductivity; As, the

urface of fluid; Ts, the surface temperature; and T is theteady temperature input. The simulation on heat convections conducted by commercial software CFD-RC.

Now, let us consider the fluid flow. The worst thing to conductowing in microchannels is clogging that often results from

ncorrect design of channel curvature at corners. The dimensionsf microchannel of RT-PCR are 100 m in width and 50 m

n height respectively, as shown in Fig. 12. The simulation, fromideview, of flow field in the microchannels is shown in Fig. 13.he flow field at the turning corners is shown in Fig. 14, where

Fig. 14. Flow field around turning corner.

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ig. 15. Gel electrophoresis for Y40 PCR amplification product (marker: 3 l;ample: 3 l).

he flow is slowed down at the corner but retains a practicallycceptable flow speed. That is, clogging problem can be avoided.

. Conclusion

A full RT-PCR unit is designed, fabricated and verifiedy intensive experiments simulations. The advantage of theeported RT-PCR is that the fluid flow, carrying DNA orNA samples, can continuously travel along purposely designedicrochannels that are heated and regulated at specified tem-

eratures, controlled by a microprocessor. In this work, a bitf test sample Y40 is to be amplified. The preliminary exper-mental result of RT-PCR is shown in Fig. 15. It is notedhat M represents the marker which behaves like the scaler ruler. From this gel electrophoresis analysis (only Columnis used in this work), it can be clearly seen how serious

he virus defection is by quantity. Intensive simulations areonducted via commercial softwares, ANSYS, CFD-RC, andntelliSuite to ensure efficient heat transfer and effective fluidow, especially at the turning corners of microchannels. Thever-heated and insufficient heat transfer are both carefullyxamined and ruled out. The clogging problems in microchan-els can be avoided since the flow speed is retained above aertain level. A temperature control system, that consists ofensors, heaters, of a microprocessor, and related amplificationircuits, is integrated as a compact module. The overall through-uts of the continuous-flow RT-PCR can be greatly increasedn addition to much improvement of precise temperatureontrol.

cknowledgementsThe authors would like to thank the Center for Micro/Nanoechnology Research,National Cheng Kung University, Tainan,aiwan, and National Nano Devices Laboratory (NDL) forquipment access and technical support. This research was par-

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ially supported by National Science Council (Taiwan) withrant NSC94-2212-E-006-054.

eferences

1] R.K. Saiki, S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, N.Arnheim, Enzymatic amplification of beta-GLOBIN genomic sequence andrestriction site analysis for diagnosis of sickle cell anemia, Science 230(1985) 13501354.

2] Y.C. Lin, M.Y. Huang, K.C. Young, T.T. Chang, C.Y. Wu, A rapid micro-polymerase chain reaction system for hepatitis C virus amplification, Sens.Actuators B 71 (2000) 28.

3] E.T. Lagally, C.A. Emrich, R.A. Mathies, Fully integrated PCR-capillaryelectrophoresis microsystem for DNA analysis, Lab. Chip 1 (2) (2001)102107.

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situ detection within micromachined polymerase chain reaction chips, J.Micromech. Microeng. 11 (1990) 329333.

6] M.A. Northrup, B. Benett, D. Hadley, P. Landre, S. Lehew, J. Richards,P. Stratton, A miniature analytical instrument for nucleic acids based onmicromachined silicon reaction chambers, Anal. Chem. 70 (1998) 918922.

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iographies

an-Chyuan Tsai was born in Taiwan in 1963. He received his BS degreerom National Cheng Kung University in 1986 and MS degree in mechani-al engineering and electrical engineering from PENN STATE University in991 and 1993, respectively. In 1995 he received his PhD degree in Mechanicalngineering from PENN STATE University. He has been an assistant professort National Cheng Kung University since 2003. His research interests includeEMS/NEMS Technology, Mechatronics, Control Engineering, Active Mag-

etic Bearings and Biochip applications.

hung-Yang Sue was born in Taiwan in 1980. He received his BS degree

rom Kun Shan University in 2003 and MS degree from National Cheng Kungniversity 2005 both in mechanical engineering. He has been in the PhD program

n the field of Bio-MEMS technologies at National Cheng Kung Universityince 2005. His research interests include design, fabrication and experimentsf MEMS/NEMS sensors and actuators.

Thermal control of micro reverse transcription-polymerase chain reaction systemsIntroductionDesign and fabricationDesign conceptFabrication process

Temperature control systemAmplification circuit for sensors

SimulationsConclusionAcknowledgementsReferences