systems for chemical and biochemical reactions has ... biochemical reactions in microchips has several .... less than an average of 2 °C. The system precision.
TSINGHUA SCIENCE AND TECHNOLOGY ISSN 1007-0214 16/20 pp269 - 272 Volume 6, Number 3, August 2001
Temperature Control System for Biochemical Reactions in Microchip-Based Devices JING Gaoshan ( # I Ä J O \ ZHANG Jian (Jfc 3g·)1, ZHU Xiaoshan (^ΙΨΟΟ 1 , FENG Jihong ( 4 Ä * ) 1 , TAN Zhimin ( τ * * * * : ) 1 · 3 , LIU Litian ( ί | « Λ ) 1 , 3 , CHENG Jing ( « t)U2 1
Beijing National Biochip Research and Engineering Center; Jia 2 # Qinghua West Road, Beijing 100084, China; 2 State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084» China; 3 Institute of Microelectronics, Tsinghua University, Beijing 100084 , China Abstract:
A silicon-glass chip based microreactor has been designed and fabricated for biochemical reactions
such as polymerase chain reactions (PCR). The chip based microreactor has integrated resistive heating elements. The computer-controlled temperature control system is highly reliable with precise temperature control, excellent temperature uniformity, and rapid heating and cooling capabilities. The development of the microreaction system is an important step towards the construction of a lab-on-a-chip system. Key words:
temperature control; microreactor; microchip; polymerase chain reactions (PCR)
Introduction In recent years, the development of miniaturized systems for chemical and biochemical reactions has rapidly progressed1-1-1. Most of the microdevices for conducting these reactions are made of silicon or glass using conventional microfabrication methods used in the microelectronic industry or by modifying the processing technologies for micro electromechanical systems ( MEMS ). Several research groups have developed polymerase chain reactions ( P C R ) chips with various features to perform simple PCR [ 2 ] , real time PCR [ 3 ] , ligase chain reactions1-4-1, reverse transcription-PCR [5] , and degenerated oligonucleotide primed-PCR (DOP-PCR)1-6-1. Precise temperature control is very important to perform chemical or biomedical reactions in a miniaturized chamber. There are different ways for heating and cooling1-7-1. Compared with conventional approaches for performing biochemical reactions, performing biochemical reactions in microchips has several advantages. Mass production will significantly reduce the cost of the silicon based microdevices.
Reaction speed in the silicon-based microchips is fast because of small reagent volumes, high surface to volume ratio and excellent thermal properties (thermal conductivity is 157 W · m _ 1 · K _ 1 ) [ 8 ] . The reaction process can be readily controlled and monitored automatically through microsensors and actuators, and contamination can be reduced to minimum. The cost is low because of reduced consumption of reagents. Silicon based microreactors are especially suitable for biochemical reactions such as PCR where accurate temperature control is demanded. This paper describes the construction of a microreaction system, which will be an important part of a lab-on-a-chip system. The system is composed of a silicon microchip, a platinum thermal sensor for temperature monitoring and a computer-controlled thermoelectric cooler ( T E C ) for heating and cooling. Water, ethanol, and dimethylformamide ( D M F ) have been used as test liquids. All the system thermal requirements have been satisfied including heating and cooling speeds, temperature accuracy, and repeatability of thermal cycles.
1 Received: 2000-11-21
Materials and Methods
The microreaction system consists of a silicon
Tsinghua Science and Technology, August 2001, 6(3): 269 ~ 272
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based microreactor, a platinum temperature sensor, a TEC (DT 6 — 2. 5, Marlow Industries, Inc. , Dallas, T e x a s ) , a custom-made power amplifier circuit to drive the TEC and to convert the temperature signal into a voltage signal, a data acquisition ( DAQ ) card ( AT-MIO-16E-10, National Instruments, Austin, Texas) for online monitoring of the microreactor temperature, and a computer with a program generated using Lab VIEW (National Instruments, Austin, Texas) to control the whole microreaction system. The silicon based microreactor was fabricated by wet etching and photolithographic methods. Six reaction chambers were etched on a silicon wafer having diameter of 2 inch. After slicing, each reaction chip had a chamber size of 10. 8 juL and outside dimensions of 12 mm X 9 mm X 100 /xm. The reaction chamber must be airtight for the microreaction. The microreaction chamber was formed using an anodic bonding method. First, a Pyrex glass slide with dimensions of 16 mm X 11 mm was prepared with two drilled holes having diameters of 0. 6 mm. The silicon chip and the Pyrex glass were placed into ethanol ( 1 0 0 % ) overnight and then rinsed with deionized water. The Pyrex glass was then air-dried and placed on top of the dried silicon chip. The silicon-glass complex was heated to 500 °C on a hot plate (Model PC-200, Corning Inc. , Corning, New York) and then a 1000 V DC voltage was applied using a customized power supply with the silicon chip connected to the positive electrode and the glass connected to the negative electrode. Anodic bonding formed an airtight microreaction chamber in seconds. The two holes in the Pyrex glass cover are used as the inlet and outlet. Two plastic tubes ( Ellsworth Adhesive System, Germantown, Wisconsin) were attached to the holes using UV glue ( N O A 68, Norland Products, Inc. , New Brunswick, New Jersey), Fig. 1.
Fig. 1 Microreactor schematic diagram
The temperature change was measured by the change of the platinum sensor resistance. The linear correlation between the temperature and the resistance can be represented by : Ä=ÄoX[l+*(T-To)], where Rt is the sensor resistance ( Ω ) at temperature T C O > R0i$ the resistance (Ω) at the reference temperature T 0 C O and a is the temperature coefficient of resistance ( TCR, C O " 1 ) of Pt. For the currerit design, R0 is 1000 Ω, T 0 is 0°C and a is 0.0039 C O " 1 . The Pt temperature sensor and the microreactor were both placed on top of the T E C , Fig. 2. Both were glued to the TEC with thermal glue for good heat transfer. The TEC is a solid state heat pump which operates on the Peltier effect. The TEC can heat or cool the microreactor by changing the polarity of the DC voltage applied to the TEC. A power amplifier was used to drive the TEC. The LabVIEW based program acquired the temperature signal through the DAQ card and controlled the output of the power amplifier circuit. The input signal was amplified and processed through a normal feedback proportional integral ( P I ) algorithm for improved thermal cycling performance. Computer
DAQ card
*
I
A
Power amplifier circuit
\
Temperature sensor A
Thermoeletric CO()ler
Fig. 2 Microreactor control system configuration
2
Results and Discussion
Because the temperature measured by the sensor was not identical to the temperature in the microreactor chamber, the two temperatures had to be calibrated. A series of temperatures from the Pt sensors and the associated temperature in the reaction chamber were measured simultaneously with a precise thermal meter ( H H 2 3 , Omega Engineering, Inc. , Stamford, Connecticut). The thermal meter precision was + 0 . 1 °C. The two temperature values were calibrated using the LabVIEW-based program. Water, ethanol and DMF were used as the test liquids to test various thermal characteristics of the microreaction system
JING Gaoshan (#] it) ^ ) et al : Temperature Control System for Biochemical Reactions such as t e m p e r a t u r e precision, heating and cooling s p e e d s , and repeatability of the t h e r m a l cycles. T h e testing was designed to stimulate the t h e r m a l cycles of conventional P C R reactions. Each cycle was divided into three t e m p e r a t u r e z o n e s : 55 °C for 30 s , 72 °C for 30 s , and 95 °C for 30 s. The temperature was monitored and controlled by a PI a l g o r i t h m , so the P I p a r a m e t e r s played a vital role in the t e m p e r a t u r e response of the microreaction s y s t e m . T o obtain b e t t e r t e m p e r a t u r e c o n t r o l , specific P I p a r a m e t e r s were chosen for each predetermined t e m p e r a t u r e value. Ziegler-Nichols T u n i n g [ 9 ] was used to adjust these p a r a m e t e r s . T h e p a r a m e t e r s for the different set t e m p e r a t u r e s could be adjusted w h e n needed. Important information about the thermal characteristics of the microreaction s y s t e m was obtained using this way. T h e s y s t e m t e m p e r a t u r e precision is ± 0 . 1 °C. Considering t h e precision of the t h e r m a l m e t e r , t h e absolute value of the s y s t e m precision is no b e t t e r t h a n 0. 2 °C , which just met the prerequisite for a highly t e m p e r a t u r e sensitive P C R reaction. T e m p e r a t u r e r a m p i n g is a n o t h e r i m p o r t a n t t h e r m a l characteristic of the microreaction s y s t e m . T h e r a m p i n g speeds were different in different t e m p e r a t u r e zones. F r o m 72 °C to 95 °C> the average heating speed was 3 ° C # s _ 1 , w h e r e a s from 55 °C to 72 °C the average heating speed w a s 6 °C # s - 1 . T h e average cooling speed was 3 °C » s - 1 . Compared w i t h r e s u l t s reported by other g r o u p s , our s y s t e m r a m p i n g rate w a s relatively low [ 1 0 _ 1 2 ] , and unlike other s y s t e m s w h e r e the t e m p e r a t u r e sensor and the h e a t e r / c o o l e r were integrated into one p a r t , our s y s t e m was relatively simple. T h e cost of the microreactor allows it to be disposable in future practical u s e . O v e r s h o o t of the set t e m p e r a t u r e m u s t be controlled in an effective microreaction s y s t e m . T h e overshoot values differed for the different set points. For 55 °C the overshoot value was 2 °C while for 72 °C and 95 °C the overshoot values were less t h a n 1 °C. Different controlling strategies were used to adjust the parameters of different set p o i n t s . In adjusting the normal PI p a r a m e t e r s , a conflict occured between the t e m p e r a t u r e response and the o v e r s h o o t . Increasing Kpor increasing ^ ( h e r e , K{ is the reciprocal of conventional integral p a r a m e t e r ) resulted in a faster t e m p e r a t u r e r e s p o n s e b u t larger overshoot. O n the c o n t r a r y , decreasing Kp or decreasing K{ resulted in a m u c h s l o w e r t e m p e r a t u r e response b u t much less
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overshoot at the set point. F o r t e m p e r a t u r e s above 90 °C , the overshoot had to be minimized so t h a t t h e reagent in the microreaction chamber would not evaporate or boil which would lead to the disastrous result of enzyme inactivation. For t e m p e r a t u r e s below 80 °C, the p a r a m e t e r s were t u n e d for a high r a m p i n g rate with some overshoot. T h e results s h o w n in Fig. 3 d e m o n s t r a t e the good repeatability of the t h e r m a l c y c l e s , while the results in Fig. 4 d e m o n s t r a t e the accurate t e m p e r a t u r e control and rapid heating and cooling speeds. 100
Temperature control
p 80 g' 60
t40
I 20 01
301 601 901 1201 1501 1801 2101 2401 Time (unit 0.1 s) Fig. 3 Three temperature cycles Temperature control
301 401 501 601 701 Time (unit 0.1 s)
801 901
Fig. 4 Temperature curve for one thermal cycle
3
Conclusions
A silicon chip-based microreaction s y s t e m was constructed and the system t h e r m a l characteristics were studied by choosing w a t e r , e t h a n o l , and D M F as test liquids. T h e PI p a r a m e t e r s were carefully chosen for accurate t e m p e r a t u r e control. T h e microreaction s y s t e m has relatively rapid heating and cooling speeds which could reach 3 to 6 °C · s _ 1 . T h e overshoot value was constrained to less t h a n an average of 2 °C. T h e s y s t e m precision w a s h i g h , i. e. , less t h a n 0. 2 °C , in general. Compared with similar microreaction s y s t e m s , the microchip was easier to fabricate and the s y s t e m was compact and easy to control. A real P C R reaction will need additional w o r k such as surface After passivation and other effects1-2,13'14-1. successful simulation of the P C R reaction
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Acknowledgements We are grateful to Mr. Xu Junquan for his help.
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