Development of a real-time array platform for nucleic acid detection using microfluidics and microheaters.

摘要

DNA analysis is becoming an increasingly important tool in many fields. In addition to genotyping, applications such as food testing, forensic investigation, and disease detection have all benefited from the use of DNA testing methods. DNA testing offers a very specific detection method based on hybridization between complementary sequences and can be used to uniquely identify an organism. DNA testing also offers fast results compared to methods requiring bacteria or cell culture, and thus is positioned for use in molecular diagnostic systems where rapid results are needed. A penultimate goal of DNA analysis is personalized medicine---tailoring treatment to the specific genome of the patient and even acting to prevent disease.;With an eye toward enabling molecular diagnostics for genetic testing a micro-heater array device has been designed and developed. The device was designed and fabricated using the SwIFT-Lite(TM) process at Sandia National Laboratories. It contains 18 individually controllable microheaters in a 3 x 6 array on a silicon substrate. The microheater array device was designed for use as a real-time biosensor platform with a waveguide for evanescent excitation of fluorescently labeled DNA, as well as a microfluidic system for sample delivery. The design process including modeling, fabrication, and characterization of the heaters and waveguide is detailed. Enabling technologies developed in conjunction with the device and which are needed to complete it for operation are also presented. These are the development of a microfluidic system for sample delivery, surface chemistry and modifications for DNA immobilization, a DNA spotting system compatible with the microheater device, and a FRET system for real-time DNA mexperiments. Experiments on the effects of temperature on DNA hybridization systems are presented and discussed. The results of the experiments suggest that optimization of temperature for DNA hybridization reactions is necessary, and has application in molecular diagnostics where time is of the essence. A platform such as the one described herein that can provide individually controllable temperature conditions for a specific hybridization reaction would help enable the development of molecular diagnostic systems for DNA detection by increasing the reliability of results in a given time frame and promote expanded genetic testing.