Monolithically integrated PCR biochip for DNA amplification

Sensors and Actuators A 108 (2003) 162–167

Monolithically integrated PCR biochip for DNA amplification Zhan Zhao a,∗ , Zheng Cui b , Dafu Cui a , Shanhong Xia a a

State Key Lab of Transducer Technology, Institute of Electronics, Chinese Academy of Sciences, Beijing 100080, China b Rutherford Appleton Laboratory, Chilton, Didcot, Oxon OX11 0QX, UK Received 29 July 2002; received in revised form 7 April 2003; accepted 9 May 2003

Abstract A monolithically integrated microliter PCR (polymerase chain reaction) biochip has been designed and fabricated for DNA amplification. It consists of a vessel made in silicon, platinum thin film heater deposited on to the bottom of the vessel and a temperature sensor. A miniaturized thermal cycling system was designed with a PIC microcontroller to provide heating power and control of thermal cycling. The volume of PCR vessel is approximately 2 ␮l. It has a low time constant of thermal cycling, with the maximal heating rate over 15 ◦ C/s and cooling rate at around 10 ◦ C/s. This paper reports the PCR vessel structure, chip package problems and solutions, its thermal uniformity analysis, thermal cycling properties and the result of amplification. The result has demonstrated that the integrated PCR biochip can provide rapid heat generation and dissipation and improved temperature uniformity in DNA amplification. © 2003 Elsevier B.V. All rights reserved. Keywords: Polymerase chain reaction; Biochip; Integrated

1. Introduction Polymerase chain reaction (PCR) has been a popular technique for DNA amplification with wide applications in DNA sequencing and biomedical diagnostics. A single DNA molecule can be copied a billion times to allow easy detection. The process of PCR involves 25–30 heat cycles. Each cycle involves three steps of heating and cooling. A double stranded seed DNA is first separated into two single strands at an elaborated temperature (∼95 ◦ C). Oligonucleotide primers that flank the DNA region to be amplified are then annealed at a low temperature (∼55 ◦ C). Finally these DNA strands are extended in the presence of a polymerization enzyme and deoxynucleotide triphosphates (dNTPs) at an intermediate temperature (∼75 ◦ C). Each cycle doubles the amount of DNA. Commercial PCR instruments have been developed by companies such as Perkin Elmer, Roche, etc., and equipped in many hospitals and gene laboratories. These instruments has PCR taking place in glass or plastic tubes which require relatively large volume of reagents (>50 ␮l), need longer time to reach thermal balance and much of the reagents is wasted. In recent years, microstructured PCR chips have demonstrated much higher ∗ Corresponding author. Tel.: +86-10-62658751; fax: +86-10-62636165. E-mail address: [email protected] (Z. Zhao).

0924-4247/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0924-4247(03)00258-9

thermal cycling speed and efficient amplification of DNA. These microPCR chips can be batch fabricated in large quantity. Therefore, PCR microchips are potentially low cost, disposable and possible for handheld instruments to be manufactured. There are in general two types of microchip based PCR systems, continuous flow PCR chip and microvessel PCR chip. A continuous flow PCR was fabricated on a glass, which have three well-defined zones to keep at three different temperatures by means of thermostatic copper blocks [1]. The chip provided a fast DNA amplification procedure. However, it lacks the adjustment of cycle times such as denature, annealing and extension time. The microvessel PCR systems, on the other hand, use silicon based material. These silicon PCR chips employ a vessel structure and integrated heater to perform the thermal cycling [2,3]. With the silicon micromachining technology readily available, realization of a microstructured PCR vessel is not difficult. However, as the scale of reaction reduces in the PCR chip, many issues arise in performing the DNA amplification in such a small chip, such as temperature uniformity, thermal stress effect, controlling of thermal cycle, sample filling, sealing of the vessel and detection of reaction products. It is these practical issues which is preventing the widespread use of the microPCR chips. In this paper, a monolithically integrated silicon PCR chip is demonstrated. It is a single vessel chip with integrated

Z. Zhao et al. / Sensors and Actuators A 108 (2003) 162–167

163

thin film heater and temperature sensor. A portable thermal cycling kit has been developed for testing the chip. Practical issues in chip design and test have been discussed, such as the temperature distribution and thermal stress, control of thermal cycle, chip packaging and sealing, sample filling and detection. The PCR chip with its unique packaging and sealing methods offers many advantages over traditional PCR devices and other microstructured PCR vessels. It has fast thermal cycling speed, low consumption of reagent. The chip can be produced in batch and it has a low cost packaging and disposable.

2. Chip fabrication and thermal cycling kit 2.1. Fabrication process The standard silicon micromachining process for MEMS devices has been used for PCR chip fabrication. The fabrication started with a double side polished silicon wafer of 270 ␮m thickness. Silicon nitride film of 2 ␮m thickness was deposited on both sides of wafer. The standard size of the PCR chip is 8 mm × 4 mm, with a 4 mm × 2 mm PCR vessel etched on one side of the wafer and thin film platinum heater deposited on the other side of wafer. Wet chemical etching with KOH was used for the vessel. The etching stopped at the nitride film. Therefore the depth of vessel is the thickness of the wafer. The total volume of the vessel is about 2 ␮l. The thin film heater is in direct contact with the nitride layer, which provides immediate heating to the reagent in the vessel. After etching a 100 nm thermal oxide layer was deposited on the inner surface of vessel wall and the bottom side of wafer. The thin film heater, together with an integrated temperature sensor, was patterned by photolithography and platinum lift-off process. Apart from the vessel, two channels with 1 mm length was etched on both sides of the vessel to provide inlet and outlet of reagent. The chip was covered with a 0.2 mm thick borosilicate glass with two holes of 0.5 mm diameter aligned to the inlet and outlet channel. Fig. 1(a) shows the schematic layout and cross-section of the PCR chip. After fabrication, the chip was mounted on a PCB with patterned electrodes connected to the thin film heater and temperature sensor to facilitate the thermal cycling test. The complete PCR chip for test is shown in Fig. 1(b).

Fig. 1. (a) Schematic layout and cross-section of the PCR chip; (b) the mounted chip ready for test.

was provided by a pulse-width-modulation (PWM) power source at frequency of 19.53 kHz. The advantage of PWM heating is that it can improve the thermal uniformity inside the vessel because the nitride membrane will vibrate under the pulse heating, which can stir up the molecule movement of the reagent and speed up the thermal diffusion process.

2.2. Portable thermal cycler A portable thermal cycling kit has been developed for the PCR chip. The heat cycler consists of four parts: heating power supply circuit, temperature detection circuit, display unit and keypad. Fig. 2(a) shows the diagram of the controlling units and the actual size of the thermal cycling kit is shown in Fig. 2(b). The cycler was controlled by a microcontroller. Active proportional integral derivative (PID) control was used to regulate the temperature. The heating

Fig. 2. (a) Block diagram of thermal cycler; (b) a photo of the thermal cycler.

164

Z. Zhao et al. / Sensors and Actuators A 108 (2003) 162–167

3. Practical issues in microPCR experiment 3.1. Thermal analysis of PCR vessel For silicon based PCR chips, there are two ways to construct the amplification vessel. The liquid vessel can be made from etching a silicon wafer to the depth of ∼400 ␮m and leaving ∼50 ␮m thickness of silicon at the bottom as the supporting floor. The chamber can also be made by completely etching through the silicon wafer, leaving a few micrometer oxide or nitride layer as the supporting membrane. The membrane type PCR chip can have much higher thermal cycling rate due to the good thermal isolation of the chip design [4]. The penalty for the much higher thermocycling rate is the fragility of this very thin supporting membrane. Earlier design of the PCR chips with 2 ␮m thickness nitride membrane often resulted in membrane crack, causing the failure of test. Thermal modeling of PCR chips with MEMS design software IntelliSuite has revealed that there are hot spots of high thermal stress across the membrane, as shown in the modeling result in Fig. 3(a) [5]. The localized high thermal stress can be reduced either by improved heater design or simply use thicker membrane. Fig. 3(b) shows the thermal stress distribution for 8 ␮m thickness nitride membrane. The hot spots of locally high thermal stress have all disappeared.

shows the layout of the platinum thin film heater deposited on the nitride film. Because silicon is a good heat conductor, the line format of heater was designed unevenly. The simulation result of heat distribution was showed in Fig. 5. In the middle of vessel, the temperature was fairly uniform. Because the silicon frame which is of high thermal conductivity, the temperature gradient from the center to the edge is high. It can be improved by making the vessel suspended by only a few silicon beams instead of the solid frame [4,6]. The temperature uniformity can be further improved by using PWM heating, as mentioned in Section 2.2.

3.2. Heater format and thermal uniformity

3.3. Compatibility of PCR with silicon vessel

The best results of thermal uniformity would be obtained if one carefully designed the form of the heater and considered the heat transfer properties of the substrate. Fig. 4

A problem with silicon PCR chip is that bare silicon vessel is not compatible with PCR amplification. Experiment using silicon powder has been proved that silicon can prohibit the DNA amplification [3]. Therefore the surface of vessel has to be passivated with silanizing agents so that a high amplification rate can be obtained. Alternatively the surface can be coated with oxide for passivation. In our PCR chip, the oxide film was coated, which is compatible with PCR process.

Fig. 4. The heater and temperature sensor pattern on the chip mounted in PCB.

3.4. Calibration of the temperature sensor Accurate control of temperature is a key for PCR process. To control the temperature accurately, the temperature has to

Fig. 3. Thermal stress distribution of (a) thin membrane and (b) thick membrane.

Fig. 5. Simulation of thermal uniformity of the PCR vessel.

Z. Zhao et al. / Sensors and Actuators A 108 (2003) 162–167

165

be measured accurately. Conventional systems can have the temperature accuracy of ±0.5 ◦ C. In our PCR chip, the temperature sensor was fabricated by deposited platinum thin film on nitride film and patterned. Because the thin film deposition conditions may vary, the temperature sensor may not have identical value for different chips. Therefore, the temperature sensor has to be calibrated for each chip. There are two methods to calibrate the sensor, one is modulation by a laser that give every chip same value. Another is to calibrate the sensor under two known temperature conditions, for example, at ice point and boiling point, so that a relation between sensor’s resistance and temperature can be correctly calibrated. The temperature sensor in our PCR chip has measurement accuracy better than ±0.5 ◦ C. 3.5. Sealing of inlet and outlet PCR amplification is a heat cycling process. When the temperature increases to the denaturing DNA at 95 ◦ C, the solution of the sample will evaporate gradually. Therefore it is very important that the two inlet and outlet holes are sealed properly. Pinch of filling tubes or cover with drop of oil have been used by others for sealing [4,6]. In our case, the inlet and outlet holes are sealed by tape. In addition the sample solution filled in the vessel should not be completely full. A small air bubble should be left behind for liquid expansion when it be heated to the high temperature. The ideal situation is to keep the bubble near the exit of the holes. 3.6. Air bubbles during thermal cycling As mentioned, keeping one small bubble in the vessel during sample filling is convenient for sealing up the hole. However when temperature raises to 95 ◦ C for denaturing of DNA, the volume of the solution expanded. Some liquid may exude. When temperature decreases, air bubbles may be generated. If the bubbles remain at the two sides of the vessel, continuing heat cycling would be successful. If it occurs in the center of the vessel (Fig. 6), the heat cycling would fail, because the temperature sensor was fabricated in the middle of the chip, which may disable the temperature control.

Fig. 6. Bubbles generated in the center of the vessel: (a) temperature at 55 ◦ C; (b) temperature at 95 ◦ C.

assay technique so that a fluorescent can be detected. The DNA fluorescent detecting was under the Olympus BX51 fluorescent microscope. The dye SYBR Green I can bind to the minor groove of the DNA double helix. In solution, the unbound dye exhibits very little fluorescence, however, fluorescence is greatly enhanced upon DNA binding. The

3.7. DNA amplification with PCR biochip The ideal detection method for the PCR microchip is on chip readout, which means that a DNA hybridization sensor is integrated on the chip. At present there are a number of methods to detect the amplified results. One is using capillary electrophoresis integrated into PCR chip to analyze the product [7]. The most mature and well developed method is laser induced fluorescence detection, which was the method in this experiment. A standard hepatitis B (HB) testing reagent test box was used as PCR amplification thermal cycling materials. The experiment employed the fluorescent dye SYBR Green I

Fig. 7. Fluorescence of DNA double helix labeled with dye SYBR Green I.

166

Z. Zhao et al. / Sensors and Actuators A 108 (2003) 162–167

more DNA double helix is formed the more enhanced fluorescence will be detected. Fig. 7 shows the fluorescence result when 30 cycles have finished on the PCR biochip.

4. Conclusion A monolithically integrated PCR biochip has been fabricated and tested. The device contains on one chip the chamber, heater and temperature sensor. A miniaturized thermal cycling system was designed with a PIC microcontroller to provide heating power and control of thermal cycling. The volume of PCR vessel is approximately 2 ␮l. It has a low time constant of thermal cycling, with the maximal heating rate over 15 ◦ C/s and cooling rate at around 10 ◦ C/s. This silicon based PCR microchip has smaller volume and faster heating and cooling rate than the plastic based one [8]. Although microchip based PCR is of high thermal cycling speed and lower cost, it is more difficult to operate. One may encounter many practical issues in order to have a successful DNA amplification with PCR chips. To have a true miniaturized DNA amplification system, the key problem is that an integrated detection technique should be developed instead of readout under fluorescence microscopy. Recently an integrated diode detector and optical fibers with PCR chip was reported, it has advantages of fast reaction and real time detection [9]. Some have developed integrated PCR chip with capillary electrophoresis analysis. Further development may be including pump, valve and fluidic systems integrated on chips [10].

[4] J.H. Daniel, S. Iqbal, R.B. Milinton, D.F. Moore, C.R. Lowe, D.L. Leslie, M.A. Lee, M.J. Pearce, Silicon microchambers for DNA amplification, Sens. Actuators A 71 (1998) 81–88. [5] Z. Cui, Z. Zhao, S. Xia, Electrothermal modelling of silicon PCR chips, SPIE 4407 (2001) 275. [6] S. Poser, T. Schulz, U. Dilner, V. Baier, J.M. Koehler, D. Schimkat, G. Maye, A. Siebert, Chip elements for fast thermocycling, Sens. Actuators A 62 (1997) 672. [7] L.C. Waters, S.C. Jacobson, N. Kroutchinia, J. Khandurina, R.S. Foote, J.M. Ramsey, Multiple sample PCR amplification and electrophoretic analysis on a microchip, Anal. Chem. 70 (1998) 5172– 5176. [8] H. Yu, P. Sethu, T. Chan, N. Kroutchinina, J. Blackwell, C.H. Mastrangelo, P. Grodzinski, A miniaturized and integrated plastic thermal chemical reactor for genetic analysis, in: Proceedings of the Micro Total Analysis Systems 2000 Conference, Enschede, The Netherlands, May 14–18, 2000, pp. 545–548. [9] C.G.J. Schabmueller, J.R. Pollard, A.G.R. Evans, J.S. Wilkinson, G. Ensell, A. Brunnschweiler, Integrated diode detector and optical fibres for in situ detection within micromachined polymerase chain reaction chips, J. Micromech. Microeng. 11 (2001) 329–333. [10] M.A. Burns, B.N. Johnson, S.N. Brahmasandra, K. Handique, J.R. Webster, M. Krishnan, T.S. Sammarco, P.M. Man, D. Jones, D. Heldsinger, C.H. Mastrangelo, D.T. Burke, An integrated nanoliter DNA analysis device, Science 282 (1998) 484–487.

Biographies Zhan Zhao received his bachelor degree in Physics in 1982 from the Shanxi University and his master degree and his doctoral degree PhD in Physical Electronics and Devices in 1987 and 2003, respectively, both from the Institute of Electronics, Chinese Academy of Sciences (IECAS). Since 1994 he has been with the State Key Laboratory of Transducer Technology (SKLTT) at IECAS. He was a Visiting Scholar in Rutherford Appleton Laboratory, UK, in 2001. Since 2000 he is a Professor for integrated sensor and microsystem at the SKLTT. His research subjects concern MEMS-based integrated biochip, integrated sensors, microactuators (microneedles and pumps) and microsystem design and technology.

Acknowledgements This work has been supported by National Natural Science Foundation of China (69936010 and 60071008), the Chinese National Key Basic Research Development Program (973) (G1999033102), State Key Laboratory of Transducer Technology and partly supported by the Royal Society of UK. The authors gratefully thank Li Wang for technical support, Dr. Xiang Chen for PCR test reagent preparation and valuable discussions.

References [1] M.U. Kopp, A.J. De Mello, A. Manz, Chemical amplification: continuous-flow PCR on a chip, Science 280 (1998) 1046–1048. [2] T.B. Taylor, E.S. Winn-Deen, E. Picozza, T.M. Woudenberg, M. Albin, Optimization of the performance of the polymerase chain reaction in silicon-based microstructures, Nucl. Acids Res. 25 (1997) 3164–3168. [3] M.A. Shoffner, J. Cheng, G.E. Hvichia, L.J. Kricka, P. Wilding, Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR, Nucl. Acids Res. 24 (1996) 375–379.

Zheng Cui is a Principal Scientist and Group Leader, responsible for the Microsystem Technology Centre at the Central Microstructure Facility, Rutherford Appleton Laboratory, UK. He participated in six European projects, with two of the projects as the Coordinator. He has been the Coordinator of European Competence Centre for Microactuators (CCMicro) since 2000. He has expertise in a wide range of microfabrication technologies and interests in microsystem design and modelling. Recent research activities include development of microbioreactors for cell engineering and microactuators for flow control. He is a Member of Technical Advisory Board for the UK Microsystem and Nanotechnology Professional Network, an Associate Editor for the Journal of Microlithography, Microfabrication and Microsystems and a Visiting Professor at the Institute of Electronics, the Chinese Academy of Sciences. Dafu Cui graduated from the Department of Radio and Electronics, Chinese University of Science and Technology in 1964. Then he joined the Institute of Electronics, Chinese Academy of Sciences (IECAS). In 1992 he became a Professor. He was a Visiting Scholar in the Molecular Electronics Research Centre, Durham University, in 1989, a Royal Society Visiting Scientist in 1994 and a JSPS Exchange Visitor, Yokohama National University in 1997. Now he is a Vice Leader of the State Key Laboratory of Transducer Technology. His present research interests are biochemical microsensors, microsystem and bioMEMS. Shanhong Xia received her BSc degree from the Electronic Engineering Department, Tsinghua University, China, in 1983, her MSc degree

Z. Zhao et al. / Sensors and Actuators A 108 (2003) 162–167 from the Institute of Electronics, Chinese Academy of Sciences (IECAS), Beijing, China in 1986, and her PhD degree in Electrical Engineering from Cambridge University, UK, in 1994. She joined the IECAS in 1986 and worked there till 1990 when she went to the Cambridge University Engineering Department, UK. Following her PhD she worked as a post-doctoral worker at Cambridge and returned to the IECAS in 1995.

167

Since then, she has been engaged in the research on vacuum microelectronic devices, sensors and microsystem technologies. She is now Professor and Vice-Director of the IECAS, a Senior Member of Directorate of the Chinese Association of Electronics, a Senior Member of the IEEE and Vice-Chairman of the IEEE Electron Devices Society Beijing Chapter.