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

Available online at www.sciencedirect.com

Sensors and Actuators B 130 (2008) 848–856

Enhancement of thermal uniformity for a microthermal cycler and its application for polymerase chain reaction夽 Tsung-Min Hsieh, Ching-Hsing Luo, Fu-Chun Huang, Jung-Hao Wang, Liang-Ju Chien, Gwo-Bin Lee ∗ Department of Electrical Engineering, National Cheng Kung University, Tainan, Taiwan Received 17 August 2007; received in revised form 16 October 2007; accepted 25 October 2007 Available online 20 February 2008

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. © 2007 Elsevier B.V. All rights reserved. Keywords: MEMS; Micro reactors; PCR; Thermal uniformity

1. 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

夽 The preliminary results of the current paper had been presented at the Transducers 2007, June 10–14, 2007, Lyon, France. ∗ Corresponding author at: Department of Engineering Science, National Cheng Kung University, Tainan, Taiwan. Tel.: +886 6 2757575x63347; fax: +886 6 2761687. E-mail address: [email protected] (G.-B. Lee).

0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.10.063

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 [6–8]. The temperature in a micro chamber is precisely adjusted to achieve PCR cycling. Another kind of micro PCR thermal

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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 [15–17,24,25] since glass is relatively lowcost 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

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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 [6,7,15–17] 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 basepairs (bps) with a higher sensitivity is successfully amplified when compared with old microthermal cyclers and bench-top PCR machines. 2. Materials and methods 2.1. Design The major contribution of this paper is a new design for arraytype 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

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Fig. 1. (a) Original microthermal cycler chip with block-type microheaters. (b) Block-type microheaters with AC units. (c) Array-type microheaters with AC units. The dimensions of each block in (a) are 2900 ␮m × 6000 ␮m, which are divided into grids (100 ␮m × 100 ␮m) with a spacing of 100 ␮m in (c).

are used for comparison [19]. Two block microheaters are used to heat up the reaction area since one temperature sensor has to be placed in the middle of the reaction chamber for temperature sensing (Fig. 1a). The resistance of the microheaters and the sensor are 10 and 280 , respectively. In this case, a significant temperature gradient around the edge region of the microheaters occurs when the ambient temperature is much lower than the operating temperature for PCR process. In order to reduce heat loss from the edges of the microheaters, two narrow heaters are first designed to form thermal compensation units. The dimensions and resistances of these two thermal compensation units are the same as that of the temperature sensor (Fig. 1b). However, a locally low temperature field caused by the temperature sensor is still observed. Therefore, block-type microheaters are first divided into several strip-shaped microheaters in one axis. Then these strip-shaped microheaters are further separated into array-type microheaters by gold leads in another axis to become more symmetrical array-type microheaters (Fig. 1c). With this approach, the equivalent resistance of the total heaters is kept almost the same, making it suitable to use the same impedance matched thermal cycler control system and the same low supply voltage and power. The resistance of each strip-shaped microheater is measured to be 11  after the gold lead deposition and patterning. The AC heaters can supply a tunable power to compensate for the thermal loss at the edges of the heating area. The new microthermal cyclers are fabricated using the same process without using more complicate controllers. A similar approach can be made to compensate for the temperature gradients near the other edges if the AC units are used two-dimensionally. 2.2. Fabrication A typical microthermal cycler is essentially composed of microheaters and a micro temperature sensor. In this study, platinum is used as material for the microheaters and the temperature sensors [19]. A simplified fabrication process is shown in Fig. 2. First, a layer of 90-nm platinum (Pt) is deposited and patterned on a soda-lime glass substrate (G-Tech Opto-electronics Corp.,

Fig. 2. Simplified fabrication process of the microthermal cycler chip: (a) deposition and patterning of a micro temperature sensor and microheaters using Pt/Ti on a glass substrate; (b) deposition and patterning of electrical leads using Au/Ti. (c) A glass slide is bonded on top of the reaction area. (d) A PDMS open chamber is attached on top of the glass slide.

Taiwan) by using electron-beam evaporation and standard liftoff processes (Fig. 2a). Prior to this process, a layer of 15-nm titanium (Ti) is deposited and used as an adhesion layer. Then, a layer of 200 nm gold (Au) is deposited and is patterned as electrical leads (Fig. 2b). Next, a 100-␮m-thick glass slide (Marienfeld Corp., Germany) is bonded on top of the glass substrate by using ultraviolet (UV) sensitive glue, which forms an electrical isolation layer (Fig. 2c). Finally, a molded PDMS open chamber is bonded on top of the glass slide to form a reaction chamber [18] (Fig. 2d). The chamber is 5.2 mm in diameter and 2 mm in height, respectively. The volume of the whole chamber is calculated to be about 40 ␮l. Finally, the microthermal cycler is packaged and connected to a custom-made thermal cycler controller. A scanning electron microscope (SEM) image of the array-type microheater with AC units is shown in Fig. 3. The dimensions of the heating area are measured to be 6 mm × 6 mm. 2.3. Thermal cycler control system A thermal cycler control system is designed and schematically shown in Fig. 4. It consists of two feedback control loops, namely, a main temperature control loop and a compensation temperature control loop. The main temperature control loop is composed of a readout circuit and a micro controller (ATMEGA8535, ATMEL Corp., USA). Note that the readout circuit, which is composed of instrument amplifiers and filters, can transform temperature signals to electrical signals for the subsequent operation. The micro controller is used to provide a 10-bit analog-to-digital converter (ADC) and a 10-bit pulsewidth-modulation (PWM) module, which can be programmed with embedded control schemes. A tunable PWM duty cycle is used to provide a tunable heating effect [8]. Similarly, the compensation temperature control loop uses the micro controller to control the AC units. When the temperature set point is changed, the micro controller provides a specific power to the AC units to reduce the heat loss from the edges of the main heaters. The

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2.4. PCR reagents In this study, a PCR process was performed in a 20-␮l total volume with a mixture with 40 ng of template DNA, 400 pM of specific primers for the detection gene, 0.2 mM deoxynucleoside triphosphates (dNTP), 1.25 mM MgCl2 , and 1× of Taq polymerase. A gene (lytA) with a length of 273 base-pairs associated with the detection of S. Pneumoniae was used. The details for the primer design and DNA amplification process can be found in our previous work [19]. 2.5. Experimental setup

Fig. 3. A SEM image of the array-type microheaters.

heating effect of the AC units using different power consumption was characterized prior to the PCR experiments. It was determined that the AC units were providing extra power of 240, 120, 160 mW for set temperatures of 94, 57 and 72 ◦ C, respectively, to efficiently reduce the heat loss from the edges. At the different thermal set points, the AC units provide different power to the central main heaters. When the AC heaters improve the edge thermal uniformity of the main heaters at a set temperature, the required power from the AC units is now defined as the optimize compensation power. In other words, the power consumption was calibrated such that the temperature uniformity can be enhanced.

Before performing the biological experiments, the temperature of the reaction chamber was first calibrated. In this study, a PDMS open chamber was attached to the microthermal cycler during the calibration process. 20-␮l of the sample mixture and 20-␮l of mineral oil were first loaded into the PCR chamber to prevent the evaporation of the DNA samples. Then a thermocouple (Tung Kwang Scientific Instrument Inc., Taiwan) with a diameter of 100 ␮m was placed in the chamber and connected to a thermometer (model-3003, DER EE Inc., Taiwan). While activating the microheaters with different power levels, the actual temperature in the chamber was detected by the thermocouple and was used to calibrate the micro temperature sensor. For the PCR experiments, a pipette was used to load samples (20 ␮l) into the chamber of the PCR chip. Again, in order to prevent evaporation of the samples, 20-␮l of mineral oil was used. In this study, the spatial thermal uniformity of the microthermal cyclers was verified by an infrared imager (Infrared Thermography TVS-200N, Nippon Avinics Co. Ltd., Japan). The spatial resolution of the IR imager is 29 ␮m. The accuracy of the temperature measurement is ±1 ◦ C. A conventional PCR machine (MyCycler, Bio-Rad, USA) was also used for DNA amplification. The amplification efficiency of the microthermal cyclers was compared with the one from the conventional PCR machine. 3. 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). 3.1. Three types of microheaters without heat sinks

Fig. 4. Schematic diagram of the microthermal cycler control system consisting of a thermal cycler controller and a microthermal cycler chip.

Three types of the microheaters were first operated without heat sinks underneath the substrate. Fig. 5 presents 2-dimemsional (2-D) and 3-dimemsional (3-D) temperature profiles for each microthermal cycler while operating at a denaturing temperature of 94 ◦ C. The 2-D temperature profiles for

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Fig. 5. Infrared images of each microthermal cycler without heat sinks at the denaturing temperature. (a) 2-D temperature profile of the block-type microheaters. (b) 2-D temperature profile of the block-type microheaters with AC units. (c) 2-D temperature profile of the array-type microheaters with AC units. (d) 3-D temperature profile of the block-type microheaters. (e) 3-D temperature profile of the block-type microheaters with AC units. (f) 3-D temperature profile of the array-type microheaters with AC units. (The IR image has 320 × 240 pixels and the resolution of each pixel is 29 ␮m. The dotted line shows the location of the PCR reaction chamber).

the block-type microheaters, the block-type microheaters with AC units, and the array-type microheaters with AC units are shown in Fig. 5(a–c), respectively. In order to provide physical insight into the temperature distribution, 3-D temperature profiles of the three microheaters are also shown in Fig. 5(d–f), respectively, for comparison. It is noted that the temperature distributions for all three microheaters have a valley in the center of the microthermal cycler since there is a micro temperature sensor located at this position. It is obvious that the locally low temperature field has been significantly alleviated by the array-type heaters. For block-type microheaters, there also exists a slight heating difference between the two block microheaters due to the slight difference in resistance (0.2 ) caused by non-uniformity of the chip fabrication process. In addition to the slight non-uniformity in the center region, a significant temperature gradient exists near the edges of the microheaters. The temperature uniformity inside the reaction chamber is greatly improved after adding the AC units (Fig. 5e). The array-type microheaters were observed to possess the most uniform temperature distribution when compared to the other two designs. In order to get physical insight into these temperature distributions, cross-sectional temperature profiles through the center of each microthermal cycler at each temperature set point (94, 57, and 72 ◦ C) are investigate and shown in Fig. 6. In Fig. 6(a), a significant temperature difference up to 6 ◦ C exists at the edges of the block-type microheaters at a denaturing temperature of 94 ◦ C. The AC units can reduce the heat loss from the edges of the main heaters and reduce this temperature difference. The array-type microheaters with AC units provide the best thermal uniformity in the reaction chamber. For the other two operating temperatures (57 and 72 ◦ C), the block-type microheaters with the AC units and the array-type microheaters still provide similar effects as shown in Fig. 6(b and c).

3.2. Three types of microheaters with heat sinks In practical applications, PCR thermal cyclers providing a high cooling rate can significantly reduce the total reaction time. Therefore fans or heat sinks for enhancement of thermal convection or conduction are commonly used. In this study, a heat sink made of aluminum (46.3 mm × 23.1 mm × 40.9 mm) is placed under the bottom of the PCR chip to enhance the cooling rate. The cooling rate without heat sinks is usually less than 1 ◦ C/s. It can be enhanced to up to 10 ◦ C/s when the heat sinks are used.

Fig. 6. Cross-sectional temperature profiles through the center of the reaction chamber for microheaters without heat sinks: (a) at the denaturing temperature (94 ◦ C); (b) at the annealing temperature (57 ◦ C); and (c) at the extension temperature (72 ◦ C).

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Fig. 7. IR images of each microthermal cycler equipped with heat sinks at a denaturing temperature. (a) 2-D temperature profile of the block-type microheaters. (b) 2-D temperature profile of the block-type microheaters with AC units. (c) 2-D temperature profile of the array-type microheaters with AC units. (d) 3-D temperature profile of the block-type microheaters. (e) 3-D temperature profile of the block-type microheaters with AC units. (f) 3-D temperature profile of the array-type microheaters with AC units. (The IR image has 320 × 240 pixels and the resolution of each pixel is 29 ␮m. The dotted line shows the location of the PCR reaction chamber).

With this approach, the temperature inside the reaction chamber can be well controlled at a higher cooling rate. Fig. 7 presents the 2-D and 3-D temperature profiles for each microthermal cycler equipped with a heat sink operating at a denaturing temperature (94 ◦ C). Again, the array-type microheaters with AC units provide a better thermal uniformity than the other two block-type microheaters. Cross-sectional temperature profiles through the center of each microthermal cycler at each of the three operating temperatures also reveal similar observations (Fig. 8). In addition to a significant temperature gradient near the edges of the microheaters, over-heating has been observed for these two block-type microheaters (Fig. 8). The non-uniformity of the temperature distribution is greatly improved by using the array-type microheaters. Table 1 lists the percentage of the area with a uniform set point temperature for three types of microheaters without heat sinks within a total area of 27.04 mm2 (5.2 mm × 5.2 mm). The area of

temperature uniformity is defined as where the temperature difference between the set point temperatures is less than 1 and/or 2 ◦ C [22]. At a set point temperature of 94 ◦ C, block-type microheaters without AC units only provide a 53.4% uniform area within ±2 ◦ C, which is about 27.0% less than the uniform area maintained by the array-type microheaters (80.4%). For ±1 ◦ C uniformity, the percentage drops to 38.6% for the block-type microheaters while the array-type microheaters can still provide a uniform area of 63.6%. Table 2 is for the microheaters with heat sinks. It is observed that the temperature uniformity inside the reaction chamber degenerates by using the heat sinks even though the cooling rate has been enhanced. At a set point temperature of 94 ◦ C, the block-type microheaters without AC units only provides a ±2 ◦ C uniform area of 41.1%, which is about 25.8% less than the one provided by the array-type microheaters Table 1 Comparison of the thermal uniformities for three types of microheaters (block w/o AC: block-type microheaters without AC; block w/AC; block-type with AC units; array w/AC: array-type microheaters with compensation units, respectively) (sampling points: 32,400 within an area of 27.04 mm2 ) Uniform area (%) ±1 ◦ C

Fig. 8. Cross-sectional temperature profiles for microheaters equipped with heat sinks: (a) at the denaturing temperature (94 ◦ C); (b) at the annealing temperature (57 ◦ C); and (c) at the extension temperature (72 ◦ C).

±2 ◦ C

94 ◦ C Block w/o AC Block w/AC Array w/AC

38.6 60.7 63.6

53.4 76.3 80.4

72 ◦ C Block w/o AC Block w/AC Array w/AC

57.6 63.7 79.6

76.3 82.0 93.7

57 ◦ C Block w/o AC Block w/AC Array w/AC

88.4 92.1 96.6

99.4 100.0 100.0

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Table 2 Comparison of the thermal uniformities after using a heat sink to improve the cooling rate Uniform area (%) ±1 ◦ C

±2 ◦ C

94 ◦ C Block w/o AC Block w/AC Array w/AC

20.8 34.1 41.9

41.1 53.8 66.9

72 ◦ C Block w/o AC Block w/AC Array w/AC

41.0 47.4 59.4

60.7 68.9 74.0

57 ◦ C Block w/o AC Block w/AC Array w/AC

58.7 67.2 77.8

81.6 86.1 92.3

(66.9%). For ±1 ◦ C uniformity, the percentage drops to 20.8% for block-type microheaters while the array-type microheaters can still provide a uniform area of 41.9%. For other operating temperatures (72 and 57 ◦ C), similar trends are observed.

Fig. 9. A typical PCR thermal cycling profile. The heating/cooling rate is up to 20/10 ◦ C/s, and the temperature variation is less than 0.2 ◦ C at each temperature set point.

3.3. PCR verification In order to characterize the microthermal cyclers, a gene (lytA) associated with the detection of S. Pneumoniae was amplified using a PCR process [19]. The thermal cycling for this PCR process is described as follows. It was first heated to 94 ◦ C for 90 s and then with the following 30 thermal cycles are performed with set point temperatures at 94 ◦ C for 20 s, at 57 ◦ C for 30 s, and at 72 ◦ C for 30 s, respectively, for each thermal cycle. Finally, the PCR chip was maintained at 72 ◦ C for 4 min after the thermal cycling. The thermal cycling profile detected by the built-in temperature sensor with the array-type heaters is shown in Fig. 9. The heating and cooling rates are measured to be about 20 and 10 ◦ C/s, respectively, thanks to the small thermal inertia of the microthermal cyclers. The temperature variation, as measured by the integrated temperature sensor, was measure to be less than 0.2 ◦ C at each operating temperature. Fig. 10 is the electropherograms of the PCR products from using the block-type microheaters without AC units, and the array-type microheaters with AC units, and a bench-top PCR machine. Lane L is a 100-bp ladder DNA marker (Yeastern Biotech Corp., Taiwan). Signals in lanes P, B and A represent the PCR products obtained by using the PCR machine for positive control, the block-type microheaters without AC units, and the array-type microheaters with AC units. After 30 thermal cycles, the lytA gene can be successfully amplified by the PCR machine, the block-type microheaters, and the array-type microheaters as well. Both microthermal cyclers exhibit a better performance than the PCR machine. Especially, the array-type heaters have the best efficiency due to their high temperature uniformity. For the PCR verification, three separate experiments are performed. For each experiment, two PCR processes are repeated.

Fig. 10. Slab gel electropherograms of a detection gene (lytA, 273 bps) associated with the detection of S. Pneumoniae. Lane L: 100-bp ladder DNA markers; signals in lanes P, B and A represent PCR products for the positive control, the block-type heaters, and the array-type heaters, respectively.

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[4] [5]

[6]

[7]

[8] Fig. 11. The relative fluorescence densities for the bench-top PCR machine, the block-type microthermal cycler, and the array-type microthermal cycler, respectively.

[9] [10]

Fig. 11 shows the relative fluorescence densities for these three cases with quantification values of 0.66, 0.75, and 1.37, respectively. The reference fluorescence density is from 500-bp DNA markers (75 ng). The relative fluorescence density of the PCR products from using the array-type microheaters is statistically higher than the block-type ones (p < 0.01, Student’s t-test, n = 6). It is observed that the PCR efficiency is enhanced by the improvement of thermal uniformity.

[11]

[12]

[13]

[14]

4. Conclusion [15]

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 detection of S. Pneumoniae using a PCR process. The development of the microthermal cyclers is crucial for further micro-total-analysis system applications. 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.

[16]

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[18]

[19]

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[22]

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Biographies 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.