RAPID, INDEPENDENTLY CONTROLLED POLYMERASE CHAIN REACTION VIA MULTIPLEXED LASER RADIATION

We report the design and testing of an instrument implementing thermal multiplexing for rapid microfluidic amplification of multiple DNA targets with different optimal annealing temperatures. Through spatial modulation of an infrared laser, dis- tinct temperature profiles can be generated simultaneously in an array of microfluidic reaction chambers in a polymer device. For our dual-chamber system, temperature differences up to 15°C have been achieved. Such multiplexed temperature control has not been reported to date. Thermal multiplexed PCR relies on key optical and heat transfer properties. An infrared laser was selected for its ability to heat water without damaging the substrate of the sample handling device; therefore, we use a wavelength of 1450nm that matches an absorption peak of water and a minimal absorption by common polymers such as poly-methyl methacrylate (PMMA) and polycarbonate. The use of a polymer for sample handling is not only beneficial for its affordability but neces- sary for its low thermal conductivity (e.g., 0.2 W/m·K), which is critical to both minimizing heat loss by conduction to the sur- rounding substrate and enabling thermal isolation of each reaction volume, i.e. minimal thermal crosstalk. Independent con- trol of multiple reactions heated from a common source further requires the laser radiation to be divided and modulated. This modulation occurs during the annealing step, since reactions with different targets have unique primers with particular melting temperatures that correlate with Guanine-Cytosine (GC) content and length. The reaction with the highest annealing tempera- ture will determine the baseline temperature maintained by the laser during this step. The shutters will then operate at a cali- brated duty cycle to lower the other reactions to their unique annealing temperatures. EXPERIMENTAL The design of a prototype dual-chamber instrument was guided by optical and thermal modeling to predict the temperature response of aqueous reaction volumes in various substrates, geometries, and radiation sources (5). The core elements include a 600mW 1450nm infrared laser diode (Hi-Tech Optoelectronics), an aspheric collimating lens (Thorlabs), a lenslet array fab- ricated in-house (6), a solenoid-driven optical shutter (7), and a polymer microchip with an array of 1 µL chambers micro- milled from cyclic olefin copolymer (COC) (8). The configuration of the optical system and microchip is illustrated in Fig. 1. The microchip was designed to provide an adequate path length for absorbing radiation, as well as a relatively low surface- area-to-volume ratio to minimize both heat loss by conduction and adsorption of biomolecules to the walls of the device. The reaction chambers are spaced 1 mm center-to-center to align with the lenslets and air gaps are milled between the chambers for enhanced thermal isolation. The shutter is driven by a solenoid array salvaged from a dot matrix printhead. The solenoids are powered by an external 5V source, which is connected to a power transistor and triggered with a square-wave control sig- nal generated in software and provided through a data acquisition device (National Instruments). A thermocouple