Anal. Chem. 1998, 70, 2997-3002
Capillary Tube Resistive Thermal Cycling Neal A. Friedman† and Deirdre R. Meldrum*
Department of Electrical Engineering, University of Washington, Box 352500, Seattle, Washington 98195-2500
A system that performs rapid thermal cycling of microliter and smaller liquid volumes inside glass capillary tubes that have an optically transparent thin film of indium-tin oxide (ITO) covering the exterior is described. The ITO film acts as both a heater and a temperature sensor, while cooling is accelerated with forced air. Unlike existing batch-mode thermal cycling systems, this system allows control over each sample’s temperature profile. Temperature transition rates of 44 degrees Celsius per second during heating and 15 degrees Celsius per second during cooling have been achieved, allowing successful polymerase chain reaction (PCR) experiments to be performed in 20 min. Capillary external temperature can be regulated typically to within (0.25 degrees Celsius, and peak temperatures more than 800 degrees Celsius have been demonstrated. Capillary internal (sample) temperatures at present are controllable typically to within 2 degrees Celsius. The resistive film can be used as a temperature sensor, and the optical transparency of the thin-film coating could permit fluorescent monitoring of the sample during thermal cycling, making this method well suited for real-time quantitative PCRs. Efforts underway to improve DNA processing methods used for the Human Genome Project are moving toward smaller sample volumes and automated processing.1-4 Ongoing research in the Genomation Laboratory at the University of Washington’s Department of Electrical Engineering has focused on automated sample processing using capillary tubes.5 The result of this effort is the high-throughput Acapella 1000 system, which automatically prepares 1-µL or smaller liquid sample volumes inside glass capillary tubes at rates up to 1000 capillaries per 8-h day. This machine is designed to perform polymerase chain reaction (PCR) and DNA sequencing reactions, both of which require DNA samples to be thermally cycled 30-40 times through a three-step temperature sequence. A follow-on system, now under development, is expected to achieve rates up to 5000 capillaries per 8-h day. In addition to desiring a fast thermal cycling method compatible with the capillary tube format, potential Acapella users have expressed interest in real-time fluorescence monitoring of DNA * To whom correspondence should be addressed. Tel.: (206) 685-7639. Fax: (206) 543-3842. E-mail:
[email protected]. † E-mail:
[email protected]. (1) Robinson, G. Electron. Eng. Times 1995, December 18, 33-35. (2) Hodgson, J. Bio/Technology 1995, 13, 231-233. (3) Service, R. F. Science 1995, 268, 26-27. (4) Hunkapiller, T.; Kaiser, R. J.; Koop, B. F.; Hood, L. Science 1991, 254, 5967. (5) Meldrum, D. R. IEEE/ASME Trans. Mechatron. 1997, 2 (2), 99-109. S0003-2700(97)01303-6 CCC: $15.00 Published on Web 06/17/1998
© 1998 American Chemical Society
reactions as they progress. Such monitoring is used to characterize the reaction dynamics of PCR and also as a diagnostic tool.6 An additional user requirement for Acapella is to allow samples to be processed with individualized thermal profiles, rather than thermal cycling many samples in a batch mode, as is typically done by commercially available thermal cycling systems. This is particularly true for medical diagnostic and genetic screening applications as well as reaction kinematics research using realtime fluorescence monitoring. The need for a thermal cycling method compatible with the capillary tube format and the desire to allow individual sample thermal profiles and real-time optical monitoring led to our experiments with transparent, thin-film heater elements being applied directly to the capillary surface. Glass capillaries are ideal containers for real-time quantitation because of their optical transparency. Also, their high surface-to-volume ratio and excellent thermal conductivity combine to ensure a rapid transfer of heat between the sample and the capillary. This paper presents a novel method to perform rapid thermal cycling of microliter-sized samples inside glass capillary tubes coated with a thin-film heater. Rapid temperature transitions are demonstrated, and temperature regulation and accuracy of the experimental apparatus are shown to be sufficient to perform PCRs. Adaptation of the experimental method for high-throughput environments such as those encountered by the Acapella system is discussed. Since the thin-film heater is tightly coupled to the capillary and contributes negligible thermal mass to the overall system, heat transfer to the sample is rapid, and there is little extraneous thermal mass to slow cooling. As implemented, the thin film heater is 3000 Å thick, with a total mass on the order of 10-5 g, while a 4-µL liquid sample has a mass of 4 × 10-3 g, and the glass capillary mass is 5.6 × 10-3 g. In this arrangement, the ratio of heater and container mass to sample mass is approximately 1.4: 1, unlike in other thermal cycling systems, where the combined mass of the heater, container, and heat transfer media can exceed that of the sample by orders of magnitude, thus limiting the thermal cycling speed. The transparent semiconductor, indium-tin oxide (ITO), was chosen for the heating film. ITO is commonly used as a transparent conductor, for antistatic and electromagnetic shielding, and as a heater and temperature sensor.7,8 ITO is a polyphase semiconductor material consisting of oxides of tin (SnO and SnO2), (6) Peccoud, J.; Jacob C. Biophys. J. 1996, 71 (1), 101-108. (7) Yust, M.; Kreider, K. G. Thin Solid Films 1989, 176, 73-78. (8) Thin Film Devices, Inc. Transparent Conductors Indium Tin Oxide ITO2000 Series product data sheet, 1996.
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Figure 1. Schematic of capillary tube resistive thermal cycling experimental apparatus: 1, controlling computer; 2, 150-V, 75-mA power supply; 3, cooling fan with arrows indicating air flow; 4, thermocouple sensor; 5, thermocouple amplifier with optoisolation; 6, ITO-coated capillary tube; 7, voltage and current sensing resistors.
indium (In2O3), and possibly free tin and indium. The exact stoichiometric ratios depend on the method and conditions of manufacture and may change due to oxidation or other environmental conditions.9 The stoichiometry and geometric characteristics (such as thickness) affect the electrical and optical characteristics of the film. An additional layer of SiO2 protects the ITO from oxidation or reduction, helping to stabilize the ITO surface and, hence, the resistance. Without the SiO2 coating, atoms of indium (and, to a lesser degree, tin) are free to migrate along the surface. At elevated temperatures, and under the influence of high electrical current (both of which are expected during thermal cycling in this experimental apparatus), atomic mobility increases, and the resistance of the film becomes less predictable. EXPERIMENTAL SECTION Apparatus. The experimental apparatus, shown schematically in Figure 1, consists of a computer with analog inputs and outputs, a computer-controlled power supply and cooling fan, several voltage and temperature sensors connected to the computer, and a mechanical fixture to hold the sensors and capillary tubes. The mechanical fixture, shown in cross section in Figure 2, also acts as an air duct to direct the air from the fan past the capillary tube. Temperature sensors provide measurements of the ambient air as well as the capillary exterior. During system development, temperature sensors were also inserted inside capillary tubes to verify performance. Data collection and system operation were handled by custom software running on an ISA bus resident, PC-44 processor card from Innovative Integration (Westlake Village, CA) containing one 50-MHz TMS320C40 32-bit floating-point digital signal processor (DSP) from Texas Instruments (Dallas, TX) and 4 MB of DRAM. The PC-44 card also provides 16 analog voltage inputs and four analog voltage outputs. The host PC was used for data collection and software development but was not used for real-time activity. The heating power supply was built around PA-83 high-power op-amps from Apex Microtech (Tucson, AZ) rated for 150 V, 75 mA continuous duty, while cooling air was provided by a 12-V dc box fan (part no. 270-243B, Radio Shack, Fort Worth, TX). (9) Meng, L.; Macarico, A.; Martins, R. Mater. Res. Soc. Symp. Proc. 1995, 388, 379-384.
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Figure 2. Cross-sectional view of experimental apparatus: 1, cooling fan, 2, fan duct with adjustable louvre, with air flow indicated by arrows; 3, LM-45 air temperature sensor; 4, ITO-coated capillary tube; 5, copper alligator clips to hold capillary in position and provide electrical contact; 6, thermocouple; 7, thermocouple leads; 8, copper alligator clips; 9, AD595 thermocouple amplifier; 10, thermocouple holder; 11, plastic spacers; 12, OP-27 op-amp; 13, TIL-111 optoisolator; 14, three-degrees-of-freedom mount.
Capillary temperatures were measured with 0.003- or 0.0005in.-diameter bare wire type K thermocouples (part nos. CHAL003 and CHAL-0005, respectively, Omega Engineering, Stamford, CT). Each thermocouple was directly wired to an AD595-C thermocouple amplifier chip (Analog Devices, Norwood, MA). Due to the high voltage (up to 150 V) present in the capillary heating film, electrical isolation was required for thermocouples placed on the capillary exterior; thus, the AD595 output was connected to the PC-44 analog input through an optoisolator (TIL-111, Texas Instruments). Ambient air temperature was measured with an LM-45 integrated temperature sensor from National Semiconductor (Santa Clara, CA), connected directly to an analog input. Thermal conductivity of the capillary external temperature sensor was enhanced with OmegaTherm 201 thermal contact paste (Omega Engineering). Reference DNA samples were processed in a commercially available capillary thermal cycler from Idaho Technology (Idaho Falls, ID). Capillary Tubes. Borosilicate glass capillary tubes, 55 mm long, 0.84-mm o.d., 0.39-mm i.d., with a 5 µL internal volume, were purchased from Drummond Scientific (Broomhall, PA). Capillaries were coated with a 3000-Å film of the semiconductor ITO by Thin Film Devices (Anaheim, CA) using their proprietary, index-matched ITO (IMITO) vacuum sputtering process. This ITO film is rated to be at least 80% transparent for wavelengths between 400 and 700 nm. For this application, a nominal resistance of 25 ( 5 Ω/square was specified, yielding a total resistance of approximately 525 Ω/capillary. To ensure a circumferentially uniform coating, the ITO was applied in three stages with a 120° rotation between each application. Gold contact rings were applied to the ITO near the capillary ends. These plated gold contact pads served to protect the underlying ITO as well as to provide a robust electrical connection. To stabilize the ITO and to protect it from environmental damage, the ITO was coated with 1500 Å of SiO2. Capillary End Seals. Other users of capillary tubes12-15 recommend using a butane flame to melt capillary ends closed to (10) Boysen, C.; Carlson, C.; Hood, E.; Hood, L.; Nickerson, D. A. Immunogenetics 1996, 44 (2), 121-127. (11) Wittwer, C. T.; Garling, D. J. BioTechniques 1991, 10 (1), 76-83 (12) Wittwer, C. T. The Rapid Cyclist 1992, 1 (1), 6-8.
Figure 3. (a) Capillary cassette: 1, capillary; 2, rubber pad; 3, slotted arms; 4, spring-loaded outer arms; 5, Delrin top. (b) Capillary sealing clip for ITO thermal cycling: 1, capillary; 2, 1/4-in. styrene I-beam; 3, 1-mm-thick rubber pad.
prevent sample loss during air thermal cycling. To remove the liquid samples after thermal cycling, the capillary tube is scored with a sapphire or similar glass cutter, and a few millimeters at each end are broken off. The capillary is disposed of after the sample is retrieved. The initial supply of ITO-coated capillaries was manufactured by hand as a special order item; therefore, they were limited in number during system development, providing a strong incentive for reuse. Instead of the destructive flame-sealing technique, ITO-coated capillaries were sealed with one of the two methods shown in Figure 3. When thermally cycled in an Idaho Technology air thermal cycler, up to 32 capillaries at a time were held in an aluminum fixture (Figure 3a). The fixture consists of two parallel arms, each 3.67 mm thick, fixed 43 mm apart. A series of 32 slots, milled across the “front” edge of the fixed arms, accommodate capillary tubes with both open ends of the capillary tubes overhanging the metal arms by 2.5 mm. A second pair of spring-loaded arms presses rubber pads against the open capillary ends, providing an airtight seal. The rubber pads are 50 durometer hardness, 1/ -in.-thick translucent silicon rubber (McMaster-Carr, Santa Fe 16 Springs, CA) glued in place with Dow Corning 732 RTV silicone (Dow Corning, Midland, MI). A Delrin top plate holds the entire fixture in the thermal cycler. This cassette can be automatically loaded with capillaries by the Acapella 1000 system. The cassette is manually placed into the air thermal cycler. When the capillaries were processed in the experimental apparatus using ITO thermal cycling, the ends were sealed with rubber pads held in place by a plastic clip constructed from 1/4in. polystyrene I-beams (Evergreen Scale Models, Kirkland, WA), (13) Idaho Technology, Inc. Idaho Technology Model 1600 Air Thermal Cycler manual, 1993. (14) Rasmussen, R., Hamerlynck, S., Eds. The Rapid Cyclist 1992, 1 (1), 12. (15) Ririe, K. The Rapid Cyclist 1994, 2 (1), 8.
as shown in Figure 3b. The pads were made from 1-mm-thick self-adhesive closed-cell rubber (Small Parts Inc., Miami Lakes, FL). Electrical Contacts. The power leads to the ITO film were attached using copper alligator clips with flat jaws from Radio Shack (part no. 270-373). The jaws were bent to encircle the capillary tubes. Alligator clips with teeth were found to scratch the plated metal contact rings. Reagents. CMOS grade, hydrofluoric acid in a 10:1 buffered oxide etch was purchased from Ashland Chemical (Dublin, OH). The human genomic DNA template (100 ng/µL) and associated primers (400 ng/µL) chosen for the PCR experiments were provided by University of Washington colleague, Dr. Deborah Nickerson. The PCR primers (Vβ23 G1 and Vβ23 G2) chosen select the sequence for the variable region of the β chain in the human T-cell receptor, producing a single PCR product 777 base pairs long.10 The PCR reaction buffer (10× medium purple), dNTPs (4 mM), and enzyme diluent (10×) were purchased from Idaho Technology. Unlike traditional PCR buffers, the Idaho Technology buffers contain a loading dye (Cresol Red and Sucrose, or Tartrazine and Ficol) and use nonacetylated bovine serum albumin (BSA) instead of gelatin. These dyes are known not to interfere with PCR reactions,11 and the BSA is substituted for gelatin to prevent the Thermus aquaticus (Taq) enzyme from adsorbing onto the capillary walls. Adsorption of Taq is a problem with capillaries due to their high surface-to-volume ratio.12 The Taq enzyme was from Promega (Madison, WI) in a 5 units/µL solution, and BSA was purchased as a 20 µg/µL solution from Boehringer-Mannheim (Indianapolis, IN). After thermal cycling, PCR products were separated by gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide (1.5 µL/100 mL) referenced to a Hi-Lo DNA marker from Minnesota Molecular (Minneapolis, MN). Procedures. Cleaning Capillaries. During the application of the ITO, SiO2 and/or gold, the capillary tubes were contaminated such that PCR reactions failed even when the capillaries were thermal cycled in a commercial air thermal cycler. Merely using one of these capillaries to aliquot from a tube containing a PCR mix was found to poison the remainder of that PCR mix. The contamination was not completely removed by either hydrochloric or nitric acid, nor by sodium hydroxide. The problem was eventually solved by etching the capillary ends and interior with hydrofluoric acid. The capillary interiors were cleaned by flushing with CMOS grade hydrofluoric acid in a 10:1 buffered oxide etch for 1 h, followed by a rinse with deionized, distilled water. The capillary ends were soaked in the same etching solution for 15 min, taking care to leave the gold contacts dry and unetched. Both the HF etch and water flush were performed using a 10 cm3 plastic syringe with a plastic (not rubber) plunger. A 4-mm length of Viton rubber tubing, 1/32-in. (0.794-mm) i.d. and 3/32-in. (2.38-mm) o.d. (Small Parts Inc., Miami Lakes, FL), was inserted into the syringe end, and the capillaries were then inserted into the Viton tubing. One syringe was used to fill the capillaries with the HF solution. The capillaries were then placed horizontally on a plastic rack for the 1-h soaking time. Afterward, 10 mL of deionized water was flushed through each capillary with another syringe. See the Safety Precautions section before working with HF! Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
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Table 1. PCR Master Mix Formulations amount in a 100-µL total volume reaction (µL) 10 1 1 4 44
reagent description
concn of stock solution
(a) Master Mix B, 3-µL Aliquot per Reaction Idaho buffer, medium purple 10× primer A, Vβ 23 g1 400 ng/µL primer B, Vβ 23 g2 400 ng/µL dNTPs 4 mM water
(b) Master Mix T, 1-µL Aliquot per Reaction Taq enzyme 5 units/µL bovine serum albumin 2.5 µg/µL Idaho Taq diluent comprised of Tris, pH 8.3 10 mN bovine serum albumin 2.5 µg/µL 7 water
1 2 10
4 16
(c) Master Mix D, 1-µL Aliquot per Reaction human genomic DNA template 100 ng/µL water
Master Reagent Mixes. As suggested elsewhere13,14 when working with the small, microliter volumes common for capillary PCR, master mixes were prepared ahead of time, and each sample was then prepared from aliquots from the master mixes. Three master mixes were prepared for PCRs: mix B contained the reaction buffer (10 µL of 10X stock, Idaho Technology medium purple), dNTPs (4 µL of 4 mM stock), 1 µL of each primer (400 ng/µL stock), and 44 µL of deionized water; mix T contained Taq enzyme (1 µL of 5 units/µL stock), BSA (2 µL of 2.5 µg/µL stock), 10 µL of the Idaho Technology enzyme stock diluent, and 7 µL deionized water; and mix D contained the human genomic DNA template diluted to 20 µg/µL. For reactions in the 5 µL capillaries, 3 µL of mix B and 1 µL each of mix T and mix D were pipetted onto a sterile paraffin sheet and mixed by several up-and-down strokes of the pipet, and then the sample was aspirated into a capillary. Table 1 shows the formulations for each of the master mixes and the volumes of each stock reagent necessary for a 100 µL total reaction volume (enough for 20 individual 5-µL reactions). BSA is included in both the Taq master mix and the buffer master mix. The Idaho Technology buffer reflects the higher Mg2+ concentrations recommended for rapid thermal cycling in capillary tubes.15 Capillary Placement. The capillary ends were sealed with a plastic clip (Figure 3b) and held in position in the fan’s air stream by copper alligator clips. A small blob of thermal paste was applied to the capillary exterior, and the external capillary thermocouple sensor was manually positioned in the thermal paste. To adjust the thermocouple position, a fixed heating voltage, known to result in an equilibrium temperature of approximately 32 °C, was applied to the capillary. The thermocouple output was monitored as the voltage was switched between the 32 °C equilibrium value and a second voltage that produced a 40 °C equilibrium. The thermocouple position was adjusted until it properly reflected both temperature points. Thermal Cycling. The PCR reaction was originally optimized in a commercially available, capillary-based air thermal cycler from Idaho Technology, with a program consisting of a 20-s initial denature hold at 93 °C, followed by 35 three-step cycles of 1 s at 93 °C, 1 s at 55 °C, and 20 s at 72 °C, before a final extension of 3000
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30 s at 72 °C. The reaction was also successful with a 75 °C extension temperature and 0-s hold times at the 55 and 93 °C temperatures. The temperature cycling was controlled by software running on a PC-44 DSP board. At steady state, the heating current was regulated by a proportional-integral-derivative (PID) control law operating at 200 Hz, while the analog sensors were sampled at 1 kHz to reduce phase errors between sensors. The thermocouple held against the capillary exterior was used for temperature feedback to the temperature controller. Another thermocouple inserted into the capillary measured actual sample temperature during system validation. The voltage drop across and current flow through the ITO film were also measured and used to calculate the ITO film resistance in real-time. The fan was turned on only during cooling transitions. Additional logic operated during temperature transitions to reset the PID control law for each new temperature point, to limit maximum voltage, and to allow selection of arbitrary slew rates. This logic also permitted nontraditional thermal cycling profiles such as sinusoidal sweeps used during system development to analyze sensor performance and tune the PID control law. During operation, the PC-44 transmitted voltage and temperature data to the host PC at 20 Hz for off-line analysis. Safety Precautions. The capillary cleaning procedure using HF etch was performed under a fume hood. Personal protection equipment included wrap-around safety glasses under a full face shield, a chemical rubber apron with full-length arm coverage worn over a lab coat, and rubber gauntlets worn over disposable vinyl gloves. The capillaries were handled with plastic tweezers, and all containers and holding and drying racks were plastic. Safety instructors at the University of Washington stress that HF burns are always worse than they appear. HF burns are often asymptomatic for several hours and require different medical treatment than typical acid burns. Emergency medical personnel may not be familiar with proper HF treatment protocols.16-19 Ethidium bromide is a mutagen and should be handled prudently and disposed of according to local regulations. University of Washington safety officials suggest using nonporous vinyl gloves (not latex). The 1500 Å SiO2 coating is not sufficient to provide electrical insulation from the heating voltage, which ranges up to 150 V dc. Therefore the ITO capillaries and all electrical wires and connections should be properly insulated, and contact with the apparatus during operation should be avoided. RESULTS AND DISCUSSION Maximum Slew Rates. A 5 µL, 55-mm capillary was thermal cycled through a PCR profile (92 °C, 55 °C, 72 °C) using the ITO film as a heater, and the capillary exterior surface temperature was measured by a CHAL-003 thermocouple. Data are shown in Figure 4. The temperature transition from 55 to 72 °C was found to require approximately 1.2 s, yielding an average heating slew rate of 13.3 °C/s. External capillary temperature was regulated (16) Bracken, W. M.; Cuppage, F.; McLaury, R. L.; Kirwin, C.; Klaassen, C. D. J. Occup. Med. 1985, 27 (10), 733-739. (17) Vance, M. V.; Curry, S. C.; Kunkel, D. B.; Ryan, P. J.; Ruggeri, S. B. Ann. Emergency Med. 1986, 15 (8), 890-896. (18) Asvesti, C.; Guadagni, F.; Anastasiadis, G.; Zakopoulou, N.; Danopoulou, I.; Zographakis, L. Cutis 1997, 59 (6), 306-308. (19) Matsuno, K. Occup. Med. 1996, 46 (4), 313-317.
Figure 4. External capillary temperature response during a single PCR step. Temperature was measured with a CHAL-003 thermocouple during a single PCR cycle (55, 72, and 92 °C, indicated by horizontal lines).
Figure 5. External and internal capillary temperature tracking. Temperatures were measured with CHAL-003 thermocouples during a single PCR cycle (55, 72, and 92 °C, indicated by horizontal lines).
to within (0.25 °C at all times. The cooling slew rate from 92 to 55 °C averaged 15 °C/s using ambient air at 22 °C. Using a more aggressive control law, peak heating slew rates of 44 °C/s have been measured, a limiting factor being the 75-mA current rating of the PA-83 power amplifier circuit. Internal Temperature. Figure 5 shows simultaneous measurements using CHAL-003 thermocouples both inside and outside a dry 5 µL, 55-mm capillary during a PCR thermal cycle (92 °C, 55 °C, 72 °C). The internal (sample) temperature lags the external temperature by less than 0.1 s during temperature transitions. Steady-state internal temperature fluctuated by no more than (0.5 °C but displays a typical steady-state error offset approximately 2 °C relative to the external temperature. The steady-state offset error is not unexpected, as the AD595 chips are rated to (1 °C accuracy, and the external thermocouple is partially affected by the external environment. Larger offsets can occur if the external thermocouple is not properly positioned against the capillary surface. With practice, users can routinely obtain 2 °C repeatability with the experimental apparatus. Peak Temperature. In an unscheduled destructive experiment, a short circuit resulted in runaway heating of an ITO-coated
Figure 6. Capillary resistance and temperature measured during PCR. (a) ITO resistance data after low-pass filtering; (b) capillary external temperature from the same PCR cycles.
capillary. The experiment terminated itself when the capillary softened enough to bend (borosilicate glass melts at approximately 850 °C), the ITO film formed an open circuit, and current flow ceased. ITO Resistance. Figure 6a shows the measured, low-passfiltered, ITO film resistance as the temperature is changed during a typical PCR thermal cycling profile. Figure 6b shows the measured external capillary temperature during the same PCR profile. The ITO film displays a total resistance change of approximately 15 Ω over the 70 °C PCR temperature range, yielding a temperature sensitivity of 4.7 °C/Ω and a temperature change of resistance (TCR) of 579 × 10-6 Ω/Ω/°C. Operating the film as a sensor requires a minimum current flow even during cooling, which slows the cooling rate slightly and prevents temperatures much below 30 °C from being achieved. Driving the film as a heater introduces additional resistance fluctuations, which further limit its measurement accuracy to (10 °C. PCR Results. After the capillaries were cleaned with HF, PCR reactions conducted in ITO capillaries produced yields comparable to those of normal capillaries when thermal cycled together in an Idaho Technology air thermal cycler. This indicates that the contamination had been eliminated. PCR reactions were thermal cycled using the ITO film as a heater and electrophoresed on an agarose gel along with commercially prepared Hi-Lo DNA reference ladders (see Figure 7). All samples contain the same reagent mixes and were compared to the same PCR reagent mix thermal cycled in an Idaho Technology air thermal cycler. PCRs performed in the ITO capillaries produced the expected 777 base pair PCR product, but with weaker product bands than those of the reference samples processed in the Idaho Technology air thermal cycler. This indicates that PCR product yield is lower using the ITO thermal cycling. No nonspecific bands were evident, indicating that the annealing temperature was accurate enough to yield a specific reaction. The reduced PCR yield is attributed to the previously observed temperature uncertainty of 2 °C between internal and external capillary temperatures, which is sufficient to adversely affect the reaction dynamics. The ability to control the internal temperature is limited by how well the external temperature sensor reflects the internal temperature. Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
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Figure 7. PCR results. Lanes 2-6 contain PCR reactions thermal cycled with ITO-coated capillaries; lane 8 is the same PCR reaction mix thermal cycled in an Idaho Technology air thermal cycler in an untreated capillary. A 777 base pair product is visible in all PCR lanes. Lanes 1 and 7 contain Hi-Lo DNA reference (first eight bands are 50, 100, 200, 300, 400, 500, 750, and 1000 bases long).
With software limiting the heating slew rate to 5 °C/s, comparable PCR yields were obtained with denature hold times of 5 s at 90 °C, 1 s at 92 °C, or 0 s at 94 °C and extension temperatures of either 72 °C for 20 s or 75 °C for 15 s. The particular PCR primers chosen appear to be insensitive to annealing temperature in the range from 55 to 59 °C. All samples were processed for 35 cycles, with an initial denature time of 20 s and a final extension time of 30 s. The demonstration of a 0-s hold time for denaturing, coupled with the demonstrated rapid transition times of 2 s, promises the possibility of 35 PCR cycles requiring as few as 12 min. For comparison, commercially available thermal cyclers using plastic microtubes or 96-well trays typically require 2 h to perform 30 PCR cycles, while their capillary-based counterparts do so in 10-20 min with slew rates of 4-5 °C/s.11-15 Experimental microfabricated (“chip-based”) thermal cyclers report performance similar to that of the capillary air thermal cycler; although one reports a faster slew rate (10 °C/s) during heating, its cooling rate is only 2.5 °C/s.20-21 CONCLUSIONS AND FUTURE WORK The experimental system has been demonstrated to perform successful PCR reactions, while achieving higher temperature slew rates (44 °C/s heating, 15 °C/s cooling) than other reported methods. Although external capillary temperature regulation of (20) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A., Northrup, M. A. Anal. Chem. 1996, 68, p 4081. (21) Wilding, P.; Pfahler, J.; Bau, H. H.; Zemel, J. H.; Kricka, L. J. Clin. Chem. 1994, 40 (1), 43-37.
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(0.25 °C and internal capillary (sample) temperature regulation of (0.5 °C have been demonstrated, at present sensor limitations result in a sample temperature steady-state error uncertainty of 2 °C typically. The temperature uncertainty is the likely cause of the reduced PCR yields. The ITO film functions very well as a heater, but as a temperature sensor its accuracy is limited in this demonstration system to approximately (10 °C. The optically transparent glass capillary coated with ITO offers opportunities for real-time fluorescent monitoring during reactions. The high slew rates promise very rapid PCRs and, coupled with real-time monitoring, should prove useful for studying the reaction dynamics of PCR and DNA sequencing reactions and for tuning parameters of these and other reactions. The demonstrated ability to achieve high temperatures (greater than 800 °C) makes this technique useful for other, nonbiological reactions as well. Applying the ITO, SiO2, and gold films to the capillary, combined with the need to perform an HF cleaning step, makes the capillaries too expensive to be disposable. Therefore, it is likely that this method will find more limited use as a research tool, where the need to clean and sterilize the capillaries for reuse could be tolerated. However, for mass production, such as a thermal cycler being fed by an Acapella 1000 system, the cassette can be made entirely from plastic, with electrical contacts built into each of the milled capillary slots. An entire cassette could be thermal cycled in 10-20 min under computer control, with the option of real-time fluorescent monitoring and individual temperature profiles if desired. The feasibility of using ITO-coated capillary tubes for rapid thermal cycling of small liquid samples has been proven. Work is ongoing to decrease the sample temperature uncertainty, to improve the PCR yield, and to incorporate real-time fluorescent sample monitoring. ACKNOWLEDGMENT The authors thank Dr. Peter Wiktor for assistance with the system controller design and Ronald Seubert of Applied Precision Inc., who was inspired by the work of D. C Focht at Bioptechs Inc. to suggest the use of ITO-coated capillaries to us. Human genomic DNA, primers, and PCR advice were supplied by UW colleague Dr. Deborah Nickerson. This research was funded by the Washington Technology Center under Grant No. B39, and the National Institutes of Health Grant R01-HG01497. Received for review December 2, 1997. Accepted April 20, 1998. AC971303N
