Enhancement of thermal uniformity for a microthermal cycler and its application for polymerase chain reaction

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Abstract

Thermal uniformity is essentially important for micro reactors which require precise control of critical reaction temperatures. Accordingly, we report a new approach to increase the temperature uniformity inside a microthermal cycler, especially for polymerase chain reaction (PCR). It enhances the thermal uniformity in the reaction region of a PCR chip by using new array-type microheaters with active compensation (AC) units. With this approach, the edges of the microthermal cyclers which commonly have significant temperature gradients can be compensated. Significantly, the array-type microheaters provide higher uniformity than conventional block-type microheaters. Besides, experimental data from infrared (IR) images show that the percentages of the uniformity area with a thermal variation of less than 1 °C are 63.6%, 96.6% and 79.6% for three PCR operating temperatures (94, 57 and 72 °C, respectively) for the new microheaters. These values are significantly better than the conventional block-type microheaters. Finally, the performance of this proposed microthermal cycler is successfully demonstrated by amplifying a detection gene associated with Streptococcus Pneumoniae (S. Pneumoniae). The PCR efficiency of the new microthermal cycler is statistically higher than the block-type microheaters. Therefore, the proposed microthermal cycler is suitable for DNA amplification which requires a high temperature uniformity and is crucial for micro reactors with critical thermal constraints.

Introduction

Recently, micro-electro-mechanical-systems (MEMS) technology and micromachining techniques have been popular in miniaturization of biomedical devices and systems. The micromachined biomedical system has several advantages over its large-scale counterparts, including low-cost, disposability, low consumption of reagents and samples, portability, and low power consumption. More importantly, functionality and reliability of the micro biomedical devices can be improved by integrating the mature integrated-circuit (IC) logic technology and other microfluidic devices. Among these micro biomedical systems, micro reactors are crucial for a variety of applications in biomedical and chemical fields. For example, polymerase chain reaction chips [1], [2], bio-reactors [3], cell culture chips [4], and chemical synthesis reactors [5] require micro reactors to maintain a precise and critical temperature inside a micro chip for efficient biomedical and chemical reactions. Among them, micro reactors requiring a repetitive thermal cycling (usually referred as microthermal cyclers) for nucleotide amplification is promising for replacing large-scale equipment. Thermal cyclers to perform PCR for genetic identification and diagnosis purposes represent one of the most fundamental analytical procedures in life-science laboratories. Microthermal cyclers using MEMS techniques have attracted considerable interest and shown great potential for DNA/RNA amplification. One kind of microthermal cycler is called temporal PCR devices [6], [7], [8]. The temperature in a micro chamber is precisely adjusted to achieve PCR cycling. Another kind of micro PCR thermal cycler is spatial devices. Instead of regulating temperature in a chamber, DNA samples are driven by pumps [9], [10] or circular close-loop ferrofuild [11] to proceed thermal cycling. Generally speaking, PCR thermal cycles consist of three operating procedures, specifically denaturation, annealing, and DNA extension, respectively. Typically, PCR utilizes temperatures in the ranges of 90–95 °C for the denaturation of the double-stranded DNA, 50–65 °C for the hybridization of the primers, and 70–75 °C for extension. During the PCR procedure, the concentration of a certain segment of double-stranded DNA is doubled through a thermal cycling process involving these three different temperatures.

Even thought micro PCR chips have been demonstrated successfully, keeping a uniform temperature distribution inside a micro PCR chamber still remains challenging [12], [13]. An appreciable non-uniformity in the temperature fields was reported while heating DNA samples using the microheaters outside the chambers, causing a low duplication efficiency of DNA. A straight-forward approach is to increase the dimensions of the microheaters such that they can cover an area much larger than that of the reaction chamber. However, it may increase the dimensions of the PCR chips and increase the cost of each chip. Besides, it also increases the power consumption. More importantly, it may cause serious cross-talk issues for neighboring microheaters if multiple reaction chambers are required. Therefore, it remains an issue on how to increase the temperature uniformity while keeping the dimension of the microheaters the same. For example, an integrated PCR-CE system comprising platinum temperature sensors inside the reaction chamber and heaters located outside the chamber was reported [12]. Experimental data show that significant non-uniformity of temperature fields still exists since the volume percentage that is 0–5 °C below the set temperatures was more than 25%. An M-shape microheater was thus fabricated inside a micro PCR chamber to increase the temperature uniformity [13]. Selection of materials with different thermal conductivities is one of the critical issues for maintaining a uniform temperature distribution for micro PCR chips. Typically, silicon-based thermal cyclers are commonly used for micro PCR applications. Not only do they provide high heating and cooling rates, but they can also be easily fabricated using compatible micromachining techniques. These silicon-based thermal cyclers are usually operated by using built-in microheaters [6], [7] or by external heaters [8], [14] with an incorporated micro temperature sensor for feedback control. By using these two heating methods, they can precisely perform thermal cycling for PCR applications. However, the cost of these microthermal cyclers may be relatively high and may hinder their practical applications in disposable devices. Alternatively, PCR thermal cyclers can be made on silicon-glass hybrid materials [15], [16], [17], [24], [25] since glass is relatively low-cost as well as highly biocompatible. Furthermore, glass-based thermal cyclers and low-cost reaction chambers were reported. For example, polydimethylsiloxane (PDMS) [10], [18], [19], polyimide [20], thick photoresist [21] or ceramic [22] materials are popularly used to form a micro reaction chamber due to their low-cost, disposability and capability to be integrated with subsequent microfluidic components. In this paper, glass-PDMS hybrid materials were adopted to fabricate the microthermal cyclers.

In order to precisely control the operating temperature during the DNA amplification process, a microthermal cycler typically consists of microheaters and a built-in temperature sensor. The microheaters are used to precisely heat up a specific area inside the reaction chamber without external heating equipment. Then, the temperature sensor is used to detect the temperature inside the reaction area and can feedback a precise signal to the microheaters. However, the edge regions inside the reaction chamber still exhibit a significant temperature gradient caused by the lower temperature of the ambient environment. This needs to be compensated for micro reactors which require a precise and critical reaction temperature. Furthermore, some DNA amplification processes with specific primer designs require extremely precise thermal control in order to enhance yield rates [21]. Therefore, microthermal cyclers with different microheater patterns such as blocks [10], [19] or serpentine-shapes [6], [7], [15], [16], [17] have been reported in the literature to improve the temperature uniformity. Other approaches to improve thermal uniformity were also reported for the PCR chips. For example, a thermal control chip to reduce heat loss from the side heaters by using 3-sets of heaters was demonstrated [23]. However, it still requires a larger area, more complicated control units, and a higher power consumption. Alternatively, a microthermal cycler with fence-like heaters to improve the thermal uniformity was reported [21]. Attempts to improve the thermal uniformity of the PCR chip by means of edged heaters or suspended structures [24], [25] have also been reported. These approaches can successfully increase the temperature uniformity inside the PCR chip to some extent. However, they usually require additional fabrication processes and a more complicated control scheme. Moreover, some fabrication processes lead to fragile suspended structures that can hinder its practical applications.

In this study, a new approach using a combination of symmetrical distributed microheaters and active compensation (AC) units is proposed to enhance thermal uniformity of the reaction area on the micro PCR chips. With this approach, only the heater layout is required to change to compensate for the edge regions of the PCR chip without using a complicated fabrication process. Besides, no fragile structures and complicated control schemes are required. With the new microthermal cyclers and their higher thermal uniformity, a detection gene with a length of 273 base-pairs (bps) with a higher sensitivity is successfully amplified when compared with old microthermal cyclers and bench-top PCR machines.

Section snippets

Design

The major contribution of this paper is a new design for array-type microheaters with AC units. It significantly enhances the thermal uniformity in the reaction region of a PCR chip. The new microthermal cycler consists of two kinds of microheaters, one for the main heaters, and another for AC units. Fig. 1 shows three designs used in this study. First, original block-type microheaters are used for comparison [19]. Two block microheaters are used to heat up the reaction area since one

Results and discussion

Block-type microheaters are commonly used in PCR chips. However, the uniformity for this type of the microheater is not satisfactory, especially at the edges of the microheaters. Hence, a new microthermal cycler design capable of enhancing the temperature uniformity in the reaction region is reported in this study. Three types of designs, including block-type microheaters, block-type microheaters with AC units, and array-type microheaters with AC units are explored (Fig. 1).

Conclusion

This study reports a new microheater design to enhance the thermal uniformity inside a chemical reaction chamber, which is crucial for micro reactors which require precise control of a critical reaction temperature. Based on this new design, the uniformity inside the reaction chamber is significantly improved without using a complicated fabrication process and delicate controllers. The performance of the new microthermal cycler is verified by amplifying a detection gene associated with the

Acknowledgements

The authors would like to thank financial support from the National Science Council in Taiwan. Also, the authors would like to thank Prof. C.C. Chang in Kaoshiung Medical University for providing useful discussion for PCR experiments.

Tsung-Min Hsieh received the BS and MS degrees in Electrical Engineering from National Cheng Kung University, Tainan, Taiwan, in 2001 and 2003, respectively. He is now pursuing his PhD degree in Department of Electrical Engineering from National Cheng Kung University. His research fields include design and fabrication techniques of biochips, embedded system design and system instrumentation.

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    Tsung-Min Hsieh received the BS and MS degrees in Electrical Engineering from National Cheng Kung University, Tainan, Taiwan, in 2001 and 2003, respectively. He is now pursuing his PhD degree in Department of Electrical Engineering from National Cheng Kung University. His research fields include design and fabrication techniques of biochips, embedded system design and system instrumentation.

    Ching-Hsing Luo received the BS degree in electrophysics from National Chaio Tung University and the MS degree in electrical engineering from the National Taiwan University in 1982 and in biomedical engineering from John Hopkins University in 1987. He received PhD degree in biomedical engineering from Case Western Reserve University in 1991. He is a full professor in Department of Electrical Engineering, National Cheng Kung University in Taiwan since 1996 and honored as a distinguished professor in 2005. His research interests include biomedical instrumentation-on-a-chip, assistive tool implementation, cell modeling, signal processing, home automata, RFIC, gene chip, and quality engineering.

    Fu-Chun Huang received his BS degree in Department of Engineering Science from National Cheng Kung University in 2002. After spending one year in MS program, he joined a PhD program directly in 2003. He received his PhD in Department of Engineering Science from National Cheng Kung University in 2007. He is currently a post-doctorate researcher in Department of Engineering Science at National Cheng Kung University. His research interests lie on microfluidics and its biomedical applications.

    Jung-Hao Wang received his MS degree in Department of Engineering Science from National Cheng Kung University in 2004. He is currently a PhD candidate in Department of Engineering Science at National Cheng Kung University. His research interests mainly are focused on pneumatic microdevices and their applications for disease diagnosis.

    Liang-Ju Chien received her BS degree in Department of Bioenvironmental Systems Engineering from National Taiwan University in 2006. She is currently a graduate student in Department of Engineering Science at National Cheng Kung University. Her research interests lie on microfluidics, micro polymerase chain reaction (PCR), and micropumps.

    Gwo-Bin Lee received his BS and MS degrees in Department of Mechanical Engineering from National Taiwan University in 1989 and 1991, respectively. He received his PhD in Mechanical & Aerospace Engineering from University of California, Los Angeles, USA in 1998. He is currently a full Professor in the Department of Engineering Science at National Cheng Kung University. His research interests lie on microfluidics, bio-sensing, diagnosis, lab-on-a-chip, nanobiotechnology and its biomedical applications.

    The preliminary results of the current paper had been presented at the Transducers 2007, June 10–14, 2007, Lyon, France.

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