Simply and reliably integrating micro heaters/sensors in a monolithic PCR-CE microfluidic genetic analysis system.

A novel fabrication process was presented to construct a monolithic integrated PCR-CE microfluidic DNA analysis system as a step toward building a total genetic analysis microsystem. Microfabricated Titanium/Platinum (Ti/Pt) heaters and resistance temperature detectors (RTDs) were integrated on the backside of a bonded glass chip to provide good thermal transfer and precise temperature detection for the drilled PCR-wells. This heater/RTD integration procedure was simple and reliable, and the resulting metal layer can be easily renewed when the Ti/Pt layer was damaged in later use or novel heater/RTD design was desired. A straightforward "RTD-calibration" method was employed to optimize the chip-based thermal cycling conditions. This method was convenient and rapid, comparing with a conventional RTD-calibration/temperature adjustment method. The highest ramping rates of 14 degrees C/s for heating and 5 degrees C/s for cooling in a 3-microL reaction volume allow 30 complete PCR cycles in about 33 min. After effectively passivating the PCR-well surface, successful lambda-phage DNA amplifications were achieved using a two- or three-temperature cycling protocol. The functionality and performance of the integrated microsystem were demonstrated by successful amplification and subsequent on-line separation/sizing of lambda-phage DNA. A rapid assay for Hepatitis B virus, one of the major human pathogens, was performed in less than 45 min, demonstrating that the developed PCR-CE microsystem was capable of performing automatic and high-speed genetic analysis.