A method for the design, construction, and assembly of modular, polymer-based, microfluidic devices using simple micro-assembly technology was demonstrated to build an integrated fluidic system consisting of vertically stacked modules for carrying out multi-step molecular assays. As an example of the utility of the modular system, point mutation detection using the ligase detection reaction (LDR) following amplification by the polymerase chain reaction (PCR) was carried out. Fluid interconnects and standoffs ensured that temperatures in the vertically stacked reactors were within ± 0.2 C° at the center of the temperature zones and ± 1.1 C° overall. The vertical spacing between modules was confirmed using finite element models (ANSYS, Inc., Canonsburg, PA) to simulate the steady-state temperature distribution for the assembly. Passive alignment structures, including a hemispherical pin-in-hole, a hemispherical pin-in-slot, and a plate-plate lap joint, were developed using screw theory to enable accurate exactly constrained assembly of the microfluidic reactors, cover sheets, and fluid interconnects to facilitate the modular approach. The mean mismatch between the centers of adjacent through holes was 64 ± 7.7 μm, significantly reducing the dead volume necessary to accommodate manufacturing variation. The microfluidic components were easily assembled by hand and the assembly of several different configurations of microfluidic modules for executing the assay was evaluated. Temperatures were measured in the desired range in each reactor. The biochemical performance was comparable to that obtained with benchtop instruments, but took less than 45 min to execute, half the time.

would be possible. I would like to thank my major professor Dr. Michael Murphy, who offered me this extraordinary opportunity to do research in the Bio-MEMS group. His vision, knowledge, experience and encouragement was vital for me to go through and finish the work. I also would like to thank Dr. Steven Soper from the Chemistry Department and Dr. Dimitri Nikitopoulos from the Mechanical Engineering, without their input and knowledge this work would not have been possible. I would like to show my appreciation to fellow students Amit Maha, for significant contribution towards the mixer development, Dr. Yun Wang, Hashimoto, Daniel Pin-Chen, Michael Mitchell who helped with experiments and made insightful suggestions. To everybody in the Muset Lab, thank you for providing a good work atmosphere that enabled a person to strive not only academically but also socially. I thank the entire staff at CAMD for given the privilege to use the facility during the fabrication of my project. I would like to express my gratitude to Chenelle, family and friends who have always been a source of strength and encouragement.

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The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point mutations in deoxyribonucleic acid (DNA). A microscale Ligase Detection Reaction (LDR) device was designed and manufactured in polycarbonate. There are at least two mixing stages involved in the LDR identification process. Various micromixers were simulated in Fluent (v5.4, Lebanon, NH) and several test geometries were selected for fabrication. Passive diffusional micromixers were made with aspect ratios from 7 to 20. The mixers were made by SU-8 lithography, LIGA, laser ablation and micromilling to characterize each fabrication method. It was found that LIGA was best for making the micromixers, but was the longest process. The micromixers were fabricated and are being tested using fluorescent dyes. For a successful reaction temperatures of 0°C, 95°C and 65°C were needed. A stationary chamber method was used with thermal cycling in which the sample held while the temperature is cycled. Finite element analysis showed uniform temperatures in the rectangular 1.5 μl chambers and that air slits can effectively separate the thermal cycle zone from the 0°C cooling zone and the mixing region. A test device was laid out and micromilled with the temperature zones. A commercial thin film heater and a thermoelectric module were used with a PID controller to obtain the required process temperatures. Heating from 65°C to 95°C took 10 seconds, while cooling from 95°C to 65°C also took 10 seconds. The residence times at the required temperatures can adapt to changes in the LDR as parameters and reactant concentrations are varied.