M. Allen Northrup, Stacy Lehew, Phoebe Landre, Peter Krulevitch, Bart Beeman, Dean Hadley, and Bill Bennet
Microtechnology Center, L-222, Lawrence Livermore National Laboratory, Livermore, CA 94551
Significant advantages can be attained by miniaturizing components of diagnostic instruments. Theory predicts huge gains can be made in efficiency and speed of analysis for chemical separation systems such as those used in chromatography and electrophoresis, and several research groups are taking advantage of these favorable scaling laws. Similar advantages are afforded by the miniaturization of chemical reactors allowing for new levels of performance and efficacy. We will show how these advantages are being used to build a miniature, low-cost, low-power, and high efficiency PCR instrument.
In this report we detail the design and development of a hand-held, low-power, feedback-controlled thermal cycling instrument for performing the polymerase chain reaction (PCR) that uses microfabricated, silicon-based reaction chambers. Several different reaction chamber designs have been modeled, built, and tested. Each design incorporates an integrated thin film heater, passive silicon cooling surfaces,.and optical windows for detection of the reaction. A highly efficient, battery-operated controller has been implemented that shows significant improvements over commercial thermal cycling instrumentation. Several different biological systems have been detected with the miniature PCR instrument including viral, bacterial and human genomic DNA targets. We have also performed amplification of human DNA targets that are specific for the disease, cystic fibrosis. This is a multiplex amplification system (i.e. amplifies 8 different sections of DNA simultaneously) and requires extremely precise temperature control. We have been able to provide the requisite control with the miniature system for the eight CF mutations on human DNA which were subsequently verified on simple test strips. The significance of these results are that for the first time a hand-held, battery-operated instrument along with simple test strips can be used to detect an important genetic disease. Recent results include low power, ultra-fast thermal cycling where we have been able to amplify DNA targets in 30 cycles in less than 7 minutes. We have also been able to monitor the reaction real-time in the miniature instrument using fluorescence monitoring with a miniature, low power optical system based on diodes. This system has also been able to detect less than 30 picomolar concentrations of fluorescently-label DNA primers. These results indicate a new ability to perform detailed studies of the reaction kinetics and improve the efficiency of this important diagnostic technique. Due to the use of microelectromechanical systems (MEMS) technology, we have shown that low-cost, high efficiency, biotechnological and clinical diagnostic instrumentation is a reality.
(This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract no. W-7405-ENG-48.) The authors acknowledge the support of the MEMS program of the Advanced Research Projects Agency. We would also like to acknowledge the collaboration of Roche Molecular Systems of Alameda, Ca.
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