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INSTITUTE OF PHYSICS PUBLISHING

JOURNAL OF MICROMECHANICS AND MICROENGINEERING

J. Micromech. Microeng. 12 (2002) 813–823

PII: S0960-1317(02)35363-4

Precise temperature control and rapid thermal cycling in a micromachined DNA polymerase chain reaction chip Dae Sung Yoon1, You-Seop Lee2, Youngsun Lee1, Hye Jung Cho3, Su Whan Sung4, Kwang W Oh1, Junhoe Cha1 and Geunbae Lim1 1 Biochip Project Team and MEMS Laboratory, Samsung Advanced Institute of Technology, PO Box 111, Suwon, Korea 2 Computational Science and Engineering Center, Samsung Advanced Institute of Technology, PO Box 111, Suwon, Korea 3 MEMS Laboratory, Samsung Advanced Institute of Technology, PO Box 111, Suwon, Korea 4 Department of Chemical and Biomolecular Engineering and Center for Ultramicrochemical Process Systems, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yuseong-gu, Taejon, 305-701, Korea

E-mail: [email protected] (Dae Sung Yoon) and [email protected] (Geunbae Lim)

Received 28 March 2002, in final form 23 July 2002 Published 3 October 2002 Online at stacks.iop.org/JMM/12/813 Abstract We have fabricated Si-based micromachined DNA polymerase chain reaction (PCR) chips with different groove depths. The platinum thin-film micro heater and the temperature sensor have been integrated on the chip. The volume of the PCR chamber in the chip is about 3.6 µl and the chip size is 17 × 40 mm2. The effects of groove geometry, including width, depth and position, on the thermal characteristics of the PCR chip have been investigated by numerical analysis and experimental measurement. From the results, the power consumption required for the PCR chip is reduced with the increase of groove depth. Compared with results for the case of no groove, the power consumption of the chip with a groove of 280 µm is reduced by 24.0%, 23.3% and 25.6% with annealing, extension and denaturation, respectively. The heating rate is increased rapidly with the increase of the groove depth. In particular, it is revealed that this effect is predominant for depths in the region above 280 µm. For a more precise control of chip temperature, the nonlinear feedback proportional-integral control scheme is used. The obtained heating and cooling rates are about 36 ◦ C s−1 and 22 ◦ C s−1, respectively. The overshoot and the steady state error are less than 0.7 ◦ C and ±0.1 ◦ C, respectively. In the experiment, the effects of the PCR buffer and the bubbles in the chamber on the temperature uniformity have also been studied. From the temperature measurement, it is revealed that the temperature difference between the thin-film sensor (on the lower plate) and the PCR buffer can be neglected if there is no air bubble in the PCR buffer. With such a high performance control scheme, we could implement a remarkable thermal cycling of conducting 30 cycles for 3 min. Finally, the chip PCR of plasmid DNA was successfully performed with no additives using the temperature control system.

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1. Introduction The polymerase chain reaction (PCR) is a well-known DNA amplification technique, which became one of the most important techniques for genetic identification and gene diagnostics during the last decade [1–3]. The method uses a repeated thermal cycling involving three PCR steps: denaturation (95 ◦ C), annealing (55 ◦ C) and extension (72 ◦ C). A single cycle for a selective amplification of a certain segment of a double stranded DNA is done in about 4 min when using a conventional thermal cycling device. Then, almost two hours are required to perform the full PCR process including pre-heat treatment, PCR cycling and final extension. In general, such a long PCR is very exhausting as well as undesirable because the activity of Taq DNA polymerase decreases with time. Recently, microfabricated PCR devices made of silicon, glass or polymeric materials have been developed in a number of laboratories [4–24]. For the case of the micromachined PCR chip, it is possible to use rapid thermal cycling due to its low heat capacity. Therefore, using the chip PCR it can be possible to conduct high throughput and high speed DNA amplification for gene diagnostics. Recently, rapid thermal cycling has been demonstrated near 30 s per cycle [16, 19], and even 12 s per cycle [7]. The short cycle times were achieved by decreasing PCR dwell times consisting of three PCR steps as well as temperature ramp times. Especially, temperature ramp times such as heating and cooling should be minimized for rapid thermal cycling. From previous studies, temperature ramp rates of 2–10 ◦ C s−1 [8, 10, 12, 19, 22] or even 40–74 ◦ C s−1 [6, 24] for cooling, and of 2–10 ◦ C s−1 [7, 8, 10, 12, 13, 19] or even 80–90 ◦ C s−1 [6, 24] for heating have been reported. For rapid thermal cycling, the low heat capacity and thermal isolation of the PCR chamber become primary key factors. The heat sink for cooling and the power control algorithm for temperature regulation also greatly affect PCR cycling. Several research groups have reported on PCR chips with special structures for the thermal isolation around the reaction chambers. Chou et al [5] have demonstrated the continuous PCR chip with an air gap for thermal isolation of the reaction zone. From their experiments, it is observed that the cross talking between reaction chambers was prevented. Daniel et al [6] demonstrated a rapid PCR chip with small thermal mass. In their study, the complete etching around the chamber was done for small heat capacity and thermal isolation. These structures allow rapid thermal cycling by preventing the horizontal heat conduction from the reaction chamber toward the plate. However, the above thermal isolation pattern has several difficulties on application to the lab-on-a-chip (LOC) or the micro total analysis system (µ-TAS). Generally, since LOC or µ-TAS are based on a microfluidic network, the silicon part around the chamber should remain and can be micromachined for fluid passages. Also, the upper plate anodically bonded with the silicon substrate inherently inhibits the thermal isolation compared to the air gap structure without the upper plate. In this study, we have manufactured a micromachined PCR chip and analyzed its physical properties. We used a Pt thin-film heater as a heat source instead of the external heating block of conventional PCR machines to avoid the disadvantages of a large heat capacity and a low rate of 814

Figure 1. Layout of a PCR chip: (a) top view and (b) cross-sectional view. The chip has one reaction chamber, two grooves, one heater and one sensor. The heater and the sensor are designed to have the resistances of 100 and 400 , respectively.

thermal conduction. Grooves with various depths around the reaction chamber were etched in the PCR chips. We evaluated their effects on temperature responses and power consumption and we recognized that they contributed to a high heating/cooling rate and a reduction of power consumption. For high performance temperature control, we constructed a self-made temperature control system consisting of a nonlinear proportional-integral (PI) control algorithm and other hardware for data acquisition and power supply. To improve the cooling rate, we adopted the forced cooling method using a fan and heat sinks. The chip PCR of plasmid DNA was performed successfully using the temperature control system.

2. Fabrication and instrumentation of PCR chips Figure 1 shows a layout and a cross-sectional view of the fabricated PCR chip. The fabrication process for the PCR chip consists of two photolithography steps for silicon and glass. The starting wafer is a double-sided polished silicon (100) substrate with a diameter of four inches and a thickness of around 300 µm. After wet oxidation of the wafer for growing a silicon oxide layer with a thickness of 700 nm, the oxide layer is patterned by photolithography and wet etched down to the silicon. With the removal of the photoresist, the silicon is etched to a depth of 100 µm by deep reactive ion etching (DRIE) to generate the reaction chamber (600 µm × 600 µm × 100 µm) and the microchannels (50 µm × 100 µm × 15 mm). To pattern the groove around the reaction chamber, the above three processes such as wet oxidation, photolithography and

Precise temperature control and rapid thermal cycling in a micromachined PCR chip

DRIE are repeated. After cleaning the wafer, we obtain the micromachined grooves with various depths of 100, 146, 224 and 280 µm. The width of the groove is fixed as 1000 µm from the preliminary numerical analysis, which can be seen in a later section. In order to form the Pt thin-film heater and sensor on the bottom of the Si wafer, a thermal oxide film is required for electrical insulation between the silicon and the Pt film. The thermal oxide film with a thickness of 500 nm is re-grown and the wafer is patterned by photolithography. This thermal oxide film serves as an electrical insulation layer. The pattern alignment of the heater and the sensor on the back side of the wafer to the chamber on the front of the wafer is acquired by an EV620 aligner (Electronic Visions Inc.). In general, for the PCR experiment, the inner surface of the reaction chamber has to be passivated to avoid any nonspecific adsorption [4, 18] of the reagents, enzyme and DNA used in the PCR. A Ti film of 30 nm and a Pt film of 500 nm are deposited on the patterned bottom side of wafer by dc off-axis magnetron sputtering. Finally, the heater and the sensor patterns are developed by the lift-off technique and the wafer is rinsed with deionized water. The glass substrate, with a diameter of four inches and a thickness of 300 µm, is cleaned and laminated by a BF410 film photoresist (Tokyo Ohka Kogyo Co. Ltd). The photoresist is patterned by photolithography to form holes with a diameter of 1 mm. The holes serve as the inlet and outlet fluid passages. By using a sand blast, holes for fluid transport are formed on the glass. After alignment, the etched silicon wafer is anodically bonded with the glass substrate. Then the wafer is diced into individual PCR chips. To operate and control the PCR, the micro-thermal cycle system is constructed, as shown in figure 2. It consists of six parts: silicon-based micro PCR chip, cooling fan, amplification circuit, data acquisition system, external power supplier, software for the automatic control algorithm and graphic user interface. The Pt thin-film sensor on the chip is used to measure the temperature of the microchamber. The measured voltage of platinum sensor is amplified by the amplification circuit and transferred to the analog input of the data acquisition system. The amplification contributes to the increase in resolution in reading the temperature and suppresses the measurement noise due to the low resolution. The resolution of the AD converter is 14 bit for the full input range of 0.0–2.5 V. But the actual voltage range of the platinum sensor corresponding to the operating temperature range is very small compared to the full input range of the AD converter if we do not use the amplification circuit. Then, the net available resolution in reading the operating temperature range is seriously degraded, resulting in measurement noises due to the severe quantization errors. So, the actual voltage variation of the sensor should be amplified to the full input range of the AD converter to fully use the whole 14 bit resolution. Through the Pt heater connected to the external power supply, the heat is applied to the micro chamber. On the other hand, for rapid cooling, a heat sink/fan type cooling system is used. The external power supply gives the power to the platinum heater in proportion to the analog output of the data acquisition system. In this case, the automatic control algorithm adjusts the analog output to control the temperature as fast and precisely as possible. In our study, a nonlinear PI algorithm is used to control the chamber

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(b) Figure 2. Experimental setup of the chip PCR: (a) a schematic diagram for the interface between the chip and the control system; (b) a photograph of forced cooling using a fan and three heat sinks.

temperature by linearizing the nonlinear dynamics of the micro thermal cycling and by using an anti-windup technique [23]. The graphic user interface (GUI) is set up for the following experimental conveniences: easy real-time scheduling of the desired temperature profile; manual setting of the controller tuning parameters; real-time plotting of the temperature; saving the present user’s recipes and history; and reloading previous recipes. Conventional PCR and chip PCR were performed using the PCR Core System II (Promega Corp., Madison, USA). 815

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Figure 3. Two-dimensional temperature contours of a cross-sectional area along the centerline of the chip (a) without a groove and (b) with a groove.

3. Numerical analysis of the PCR chip Numerical simulation has been carried out in order to clarify the thermal diffusion characteristics of a PCR chip with grooves. We use a finite-volume based simulation package FLOW3D. The chip has two planes of symmetry, therefore only one quadrant of the chip domain is considered. Figure 3 shows the effects of the groove on the temperature field of an entire cross-sectional area along the centerline of the chip. The main action of the groove is to inhibit thermal conduction in the horizontal direction. Even though the same power of 8 W is applied, when the groove exists, there is a considerable increase of the temperature around the reaction chamber, as shown in figure 3(b). Denser isotherms appearing above the groove manifest good thermal isolation between the heater and the silicon substrate. Figure 4 shows the temperature variations measured beneath the chamber depending on the groove depth. The groove width is set to 1000 µm. The deeper 816

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The PCR mixture contains 10× PCR buffer (10 mM Tris–HCl (pH 9.0), 50 mM KCl and Triton(r)X-100), 1.5 mM MgCl2, 200 µM dNTP, 1 µM upstream and downstream control primer, 1 ng/50 µl positive control plasmid DNA (supplied by kit), and 1.25 units µl−1 Taq DNA polymerase. Then, each of the silicon-glass PCR chips was filled with 3.6 µl, and each conventional PCR tube was filled with 5 µl of the PCR mixture. The conventional PCR was amplified in a GeneAmp(r) tube (Perkin–Elmer) on the DNA thermal cycler (Eppendorf, Inc.). For chip PCR, approximately 5 µl of the PCR mixture was injected into the entry port of the PCR chips using a syringe. The chips were then sealed with silicon rubber gaskets. The PCR reaction mixture was initially heated to 95 ◦ C for 2 min and cycled for 30 cycles: 30 s at 95 ◦ C, 30 s at 55 ◦ C and 1 min at 72 ◦ C. A final extension was performed at 72 ◦ C for 7 min. The amplified mixtures were collected in polypropylene microcentrifuge tubes. Amplified products were detected using a 2% agarose gel (SeaKem LE; BioWhittaker Molecular Applications, Rackland, ME, USA) in 100 mM Tris, 90 mM boric acid, 1.0 mM EDTA (pH 8.0). The gel was stained with 1 µg ethidium bromide (Sigma Diagnostics) per 10 ml gel. The PCR products (323 bp) were run at 100 V for 30 min with a 100 bp DNA ladder (G210A; Promega).

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the grooves, the higher the heating rates. From figure 4, the heating rates are obtained by the temperature calculation for 2 s after the power 8 W is applied to the heater. Figure 5 shows the dependence of the heating rate on the groove geometry. To obtain a higher heating rate, one has to reduce the distance between the chamber and the groove, and increase the groove width. One can see, however, that the groove depth plays the most dominant role in increasing the heating rate. Changing the groove position from lower to upper does not make any difference in the thermal characteristics, because the thermally isolating actions of the upper and lower grooves are the same. In addition to the three-dimensional thermal simulation, a thermal resistance, R, and thermal capacitance, C, model [25] of a PCR chip reactor has also been developed in order to predict the thermal cycling behavior of the chip. We divide the quadrant of the chip into small finite thermal elements. In this study, the constructed equivalent R–C circuit of the PCR chip consists of 65 thermal elements of which the R–C values can be evaluated using the dimensions and material properties of the elements. Using this lumped R–C model, the cooling


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Figure 5. Dependence of the heating rate on the groove geometry: (a) lower groove; (b) upper groove.

4. Experimental results and discussion 4.1. Performance of thin-film heater and sensor in the PCR chip The experiment for the characteristics of the heater and sensor was conducted in a closed space (40 × 40 × 50 cm) to minimize the effects of air convection. For the stable operation of the PCR chip, the physical properties of the thin films for the heater and sensor should not deteriorate during the PCR cycle. Power corresponding to the temperature of 95 ◦ C is constantly applied to the micro heater of the PCR chip for 3 h. After this, the resistance values of heater and sensor are measured with respect to time. As shown in figure 6(a), the resistance is maintained over the entire range. From the results, the physical properties of thin film do not change and remain stable during the high-temperature heating. The resistance of the sensor is also measured at various steady-state temperatures using the data acquisition system (DAQ). From figure 6(b), the resistance of the sensor shows a good linearity in the entire temperature range. The linear property of the sensor can be represented by the following equation R = R0 × [1 + α(T − T0 )]

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where R is the resistance of the sensor () at temperature T (◦ C), R0 is the resistance () at reference temperature T0 (◦ C) and α is the temperature coefficient of resistance (TCR) of the Pt thin film. The TCR of the Pt thin film was estimated to be

(b) Figure 6. Thermal stability data of the thin-film sensor in the chip: (a) the stability of the heater and sensor with time; (b) the plot of sensor resistance with respect to the temperature.

2.48 × 10−3 ◦ C−1. Once calibrated, the instrument is capable of in situ temperature measuring for the reaction chamber temperature based on the sensor voltage. 4.2. Effects of the groove on the thermal characteristics of the PCR chip High heating and cooling rates are essential for the high-speed operation of the PCR chip. To increase the heating rate, one should lower either the total thermal mass of the chip or the heat conduction in the horizontal direction for an efficient energy accumulation. In this study, the grooves are fabricated around the reaction chamber to minimize the horizontal 817

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Figure 8. Heating profiles of the chip with different groove depths. Figure 7. Equilibrium temperature versus voltage plot of the chip with different groove depths. The resistance values of the heaters in the chips are 101.5 without a groove, 100.3 for a 100 µm groove, 102.7 for a 148 µm groove, 101.2 for a 224 µm groove and 103.1 for a 280 µm groove at room temperature. The same chips are used for figures 10, 11 and 13.

heat conduction. Grooves, 1 mm wide, with different depths are formed in the PCR chip and their effects on temperature responses and energy consumption are analyzed. Figure 7 shows the variation of equilibrium temperatures with respect to the depth of the groove around the chamber. Voltages of 6, 10, 14 and 18 V are applied to each chip for 10 min. When the chip reaches thermal equilibrium, the temperature of the center of the chamber is measured. As shown in figure 7, the equilibrium temperature increases with the depth of the groove. This means that a deeper groove around the chamber inhibits the horizontal heat conduction effectively and helps to concentrate the applied power into the reaction chamber. These thermal isolation effects are shown to be predominant as the applied voltage increases. In addition, the groove effect is more noticeable in dynamic situations such as heating and cooling cycles. Figure 8 shows the initial heating profiles according to the groove depth when 18 V is applied. For the cases of 100 µm and 146 µm, the heating rate is not significantly changed compared with the case of no groove. This indicates that the heat spreading into the silicon substrate is not restricted by the groove. Thus, the thermal blocking effect does not appear on heating the chip. However, for depths over 224 µm (i.e. approximately two-thirds of the thickness (300 µm) of the Si substrate), the rate at which the temperature rises increases rapidly. In this case, the thermal blocking in the horizontal direction begins to take effect and results in a very high heating rate. For the power for chamber heating, as shown in figure 9, the powers required for annealing (55 ◦ C), extension (72 ◦ C) and denaturation (95 ◦ C) are decreased as the groove depth is increased. In particular, for the case of a 280 µm groove, we find that, compared to the case with no groove, the 818

Figure 9. Consuming power variations of the chips with different groove depths at annealing, extension and melting temperatures.

power consumption is reduced by 24.0%, 23.3% and 25.6% for annealing, extension and denaturation, respectively. Figure 10 shows the cooling profiles of the chips during the natural air cooling. The power is cut off after the chips reach equilibrium at 95 ◦ C, which corresponds to the denaturation temperature of DNA. The temperature measurements for the cooling behaviors are conducted right after the power is turned off. The chips with 100 µm and 146 µm grooves are cooled down with the familiar pattern of the case of no groove. On the other hand, for chips with 224 µm and 280 µm grooves, the chip is cooled down more rapidly than that with no groove due to the low applied power. From the above experimental observations, it is explained that the heat transfer around the chamber is mainly affected by


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two factors. As shown in figure 11, one factor is the heat release from the silicon surface into the air in a vertical direction (I). The other factor is the heat conduction through the silicon substrate in a horizontal direction (II). For the cases of no groove, 100 µm or 146 µm grooves, the thermal gradient in the horizontal direction around the chamber is not too high because of high conductive silicon spreading. Accordingly, the temperature of the reaction chamber is mainly determined by the vertical heat convection into the air and thus all three cases exhibit a similar tendency. However, in the cases of 224 µm and 280 µm grooves, thermal blocking by groove limits the heat spreading in the horizontal direction. As a result, a high heating rate of the PCR chamber can be obtained and thus a decrease in the required power can also be possible. For further understanding, we make comparisons between experimental and numerical results. As shown in figure 12, the fast cooling profile in the presence of a deep groove is predicted from the numerical lumped model mentioned in

Figure 12. Predicted cooling characteristics of a PCR chip using a lumped R–C model compared with experimental measurements.

section 3. Even though predicted cooling profiles slightly differ from those of experiments, the similar enhanced cooling effect due to the presence of the groove can also be observed in the lumped model results. The use of an inaccurate value of the convective heat transfer coefficient in the lumped model may give rise to the difference of cooling profiles. We assume that the value is 12 W m−2 K. The reason why the grooved PCR chip has faster cooling than the PCR chip without a groove is that the lower energy is consumed to heat up the chamber to a reference temperature, e.g. 95 ◦ C, for the deeper groove. For example, power input is reduced by more than 25% with the 280 µm deep groove (see figure 9). So, the grooved chip cools faster after the power is turned off because the chip has a lower thermal budget than that of a chip without a groove. This lower thermal budget results in a faster convective cooling rate. When the cooling mechanism is conductive, for example, using a heat sink, the deep and wide groove acts as a large thermal resistance in the horizontal direction. So, the cooling mechanism for this grooved PCR chip should be designed as convective, for example, using a fan. Using the lumped model, we have conducted PCR thermal cycling numerically using a proportional controller [25]. The average power required for successful PCR reactions for different groove depths and widths is predicted and measured as shown in figures 13 and 6. The actual power for the entire chip cycling is four times the value in figure 13, because only the quadrant of the chip is considered. One can see that a considerable amount of power can be saved, up to 50%, by using a 280 µm deep and 4000 µm wide groove. However, such a deep and wide groove could inhibit the heat conduction in the horizontal direction during the cooling phase, resulting in slower cycling due to the increase of cooling time. So, more careful analyses should be conducted to optimize the groove geometry for a better control of PCR thermal cycling. Such an air blocking method has been seen in previous research. Daniel et al [6] constructed an independent air 819

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Figure 13. Average input power predicted using a lumped model for successful PCR thermal cycling for different groove depths and widths.

space by etching around the reaction chamber using the patterned silicon nitride as a mask. In their experiments, a heating rate of even 60 ◦ C s−1 was demonstrated with that structure. But, for the LOC with an integrated PCR part or for µ-TAS, their structure is difficult to apply to the system. The manufacture of independent air space around the chamber might be complicated and it is difficult to design electrodes and microchannel arrays, which are necessary for LOC or µ-TAS. As a result, our proposed thermal isolation method using grooves has advantages in electrode wiring and microchannel array design. In our method, the groove can be patterned on to the bonded surface or the opposite surface with a micro heater according to the design purpose of the chip. Moreover, the easy and simple groove patterning process would be appropriate for mass production of PCR chips. (b)

4.3. Temperature distribution of the PCR chip Figures 14(a) and (b) show the transient temperature response at the upper and lower plates of the chip with and without the PCR buffer in the chamber. The transient temperature was measured at the center points of the upper and lower plates after applying a constant voltage of 14 V. As shown in figure 14(a), the temperature differences are obtained for the heating range 6–7 ◦ C, for the holding range 8–9 ◦ C and for the cooling range 1–3 ◦ C, when the reaction chamber is filled with air. This nonuniformity of temperature indicates that air in the reaction chamber serves as a thermal insulation layer to delay thermal conduction from the lower plate to the upper plate. On the other hand, it is known that the PCR buffer with DNA has much higher thermal conductivity than the air and also the silicon substrate [13]. Therefore, when the reaction chamber is filled with PCR buffer, we can expect that the temperature difference between the upper and the lower plate surfaces would be negligible. Figure 14(b) shows the expected temperature profiles. The temperature difference is 820

Figure 14. Temperature difference plots of the upper and the lower plates of the chip filled (a) with no PCR buffer, and (b) with PCR buffer.

less than 0.2 ◦ C in all regions. Thus, we can assume that the measured temperature from the thin film is the nearly same as that of the PCR buffer. This corresponds to the result of Lin et al [13]. However, during the PCR cycle, air bubbles can be occasionally generated in the chamber over 90 ◦ C. These bubbles may be generated by the evaporation of PCR buffer or the incomplete sealing in the fluid inlet and outlet passages. For this case, generated and growing bubbles create an insulation layer inside the chamber and result in a large temperature difference (4–5 ◦ C) between the upper and lower plates (figure 14(b)). Also, these bubbles may prevent the circulation of the PCR buffer in the reaction chamber by convection and inhibit PCR. Actually, we have observed that PCR does not take place when large bubbles are generated in the chamber.


(a) Figure 15. Temperature profiles of the chip for air cooling, air cooling with a heat sink, forced cooling with a fan only, and forced cooling with both a fan and a heat sink.

4.4. Temperature control for the PCR chip For a high performance thermal cycling, the temperature control system in figure 2(a) is used. The nonlinear PI algorithm controls the temperature efficiently by linearizing the nonlinear dynamics of the thermal cycler [23]. Experiments are performed for four cases: natural air cooling, air cooling with the heat sinks attached to the chip, forced cooling with only a fan, and forced cooling with the fan and the heat sinks. Figure 15 shows the cooling profiles of the four cases. For natural air cooling, it took approximately 30 s to cool from 95 ◦ C down to 55 ◦ C. A long cooling time is not desirable for acquiring rapid thermal cycling and retaining the activity of Taq DNA polymerase. So the cooling time needs to be reduced. For the other cases, the cooling times of the chip are 13 s, 2.5 s and 1.5 s, respectively. From the above results, it is revealed that the forced cooling method is necessary for rapid thermal cycling. In the case of the heat sink and/or the cooling fan, more heating power is required because of the additional heat release through the increased heat transfer area and heat transfer coefficient by the heat sink and the cooling fan, respectively. The heating power values for each cooling case were measured. The heating power of the grooved chip (280 µm) for air cooling is estimated to be 1.34 W. In comparison with the air cooling, there were heating power increases of 24% for the case of the heat sink, and 145% for the case of the fan. Furthermore, from the four heating experiments, we confirmed that the heat sinks and the fan did not nearly affect the heating rate. This indicates that the high performance nonlinear PI controller can efficiently make up for the additional heat release by adding more power. In contrast to the heating process, the PI controller cannot do anything for rapid cooling but enters just zero voltage because the voltage

(b) Figure 16. Thermal cycling of the PCR chip: (a) conventional thermal cycling; (b) rapid thermal cycling. It takes 3 min for 30 cycles.

cannot be negative. Then, it is evident that the additional heat release by the heat sink and/or the cooling fan can increase the cooling rate regardless of the effects of the nonlinear PI controller. Figure 16(a) shows a typical thermal cycling of the PCR chip. In this case, the self-made temperature control system and the forced cooling method are used. From the results, remarkable heating and cooling rates are obtained of approximately 36 ◦ C s−1 and 22 ◦ C s−1, respectively. Moreover, the overshoot is not over 0.7 ◦ C and the steady-state error is less than ±0.1 ◦ C. Also, as shown in figure 16(b), rapid thermal cycling that takes 6 s per cycle can be demonstrated. After all, for 30 cycles, only 3 min are required, which is about 20 times shorter than conventional thermal cycling. 821

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the surface adsorption of the enzyme and the primer, and to improve the PCR [7, 8, 10, 12, 14, 15, 22]. In our experiment, however, PCR band observation is possible without BSA or PEG. This is the first observation of a PCR band ever made with slab gel electrophoresis after PCR without adding BSA or PEG at the current capacity of a few microliters. Even though the additive for surface passivation is not added, microliter PCR detectable by gel electrophoresis is successfully performed just by using precise temperature control and silicon oxide passivation. Currently, the study of the optimization of PCR yield and the total required time is being carried out by choosing optimal adjustable parameters, such as temperature and dwell times. Also, the effects of additives and surface passivation on PCR are being investigated.

5. Conclusion

Figure 17. A photograph of slab gel electrophoresis for conventional PCR and the chip PCR.

4.5. Chip PCR test and evaluation For chip PCR, the PCR buffer mixture is injected into the reaction chamber through the inlet using a syringe, and the inlet and outlet passages are sealed using acrylic plastic blocks and a VersaChem epoxy (ITW performance polymers, FL, USA). After the pre-heat treatment for perfect denaturation of DNA, 30 thermal cycles are performed for PCR. The PCR chip is heated to 95 ◦ C for 2 min and then cycled for 30 cycles: 30 s at 95 ◦ C, 30 s at 55 ◦ C and 1 min at 72 ◦ C. It takes about 62 min to complete 30 cycles. A final extension was performed at 72 ◦ C for 7 min. The bulk PCR using an e-tube is conducted by following the same thermal schedule as for the PCR chip. However, the temperature ramp rate (36 ◦ C s−1 for heating and 22 ◦ C s−1 for cooling) of the chip is much higher than that of the bulk PCR. Accordingly, it takes over 90 min to complete 30 cycles for the bulk PCR. After the PCR, the samples of both cases are extracted. Both the amplified sample and the 100 bp DNA ladder are mixed with loading dye and then undergo electrophoresis in 2% agarose gel. Then the fluorescent images of PCR products are obtained using a gel documentation system (Alpha Innotech Co.). Figure 17 is a fluorescent image of the amplified DNA obtained after PCR in the chip and the bulk e-tube. As shown in figure 17, DNA is successfully amplified on the chip. However, the amount of amplification is relatively small compared with PCR using the bulk e-tube. This observation may be due to the surface area difference between the reaction chamber in the chip and the e-tube. The surface area of the reaction chamber in the chip is many times larger than the e-tube under the same volume of reaction mixture. The large surface area of the reaction chamber in the chip allows Taq DNA polymerase and primer to be absorbed easily on the surface, causing the reaction efficiency to drop. Because of this, most researchers have adopted several supplementary additives such as bovine serum albumin (BSA) and polyethylene glycol (PEG) to hinder 822

We have fabricated a Si-based micromachined PCR chip integrated with a platinum thin-film heater and a temperature sensor. The volume of the PCR chamber in the chip is about 3.6 µl and the chip size is 17 mm × 40 mm. For thermal isolation of the PCR chamber, a groove of various depths is patterned around the chamber. The effects of the groove depth on temperature characteristics are analyzed experimentally. From the numerical analysis, the effects of groove geometry including width, depth and position on the thermal characteristics of PCR chip are also investigated. The required power consumption of the fabricated PCR chip is also investigated in terms of the various depths of the groove. From the results, it is revealed that the groove shows several advantages of power consumption, microfabrication and response time for heating and cooling. The heating rate is increased with the increase of groove depth. Especially, these effects are predominant at depths in the region of above 224 µm. Compared with the results for cases with no groove, the power consumption of the chip with a 280 µm groove is reduced by 24.0%, 23.3% and 25.6% for annealing, extension and denaturation, respectively. We also study the effects of the PCR buffer and the bubbles in the reaction chamber on the temperature uniformity. From these results, it is found that the temperature difference between the thin-film sensor (on the lower plate) and the PCR buffer can be neglected if there is no air bubble in the PCR buffer. By using a nonlinear feedback PI control scheme, high performance temperature control can be possible. The obtained heating and cooling rates were about 36 ◦ C s−1 and 22 ◦ C s−1, respectively. The overshoot and the steady-state error are less than 0.7 ◦ C and ±0.1 ◦ C, respectively. With such a high performance control scheme, we could implement a remarkable thermal cycling of conducting 30 cycles for 3 min. Finally, the chip PCR of plasmid DNA was successfully performed with no additives using the temperature control system.

Acknowledgments This work has been processed under the collaboration of several divisions of Samsung Advanced Institute of Technology. Contributors from each division are shown as authors of this article. We thank J C Lee, M H Chung and


Dr S H Kang for their valuable assistance. This work was partially supported by the Ministry of Commerce, Industry and Energy (MOCIE) of the Republic of Korea under the next generation new technology development project (00008069) through the Biochip Project Team at Samsung Advanced Institute of Technology.

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