Title page for ETD etd-08262004-121210


Type of Document Master's Thesis
Author Barrett, Dwhyte Omar
Author's Email Address dbarre1@lsu.edu
URN etd-08262004-121210
Title Design of a Microfabricated Device for Ligase Detection Reaction (LDR)
Degree Master of Science in Mechanical Engineering (M.S.M.E.)
Department Mechanical Engineering
Advisory Committee
Advisor Name Title
Michael Murphy Committee Chair
Steven Soper Committee Member
Warren Waggenspack Committee Member
Keywords
  • liga
  • pcr
  • thermoelectric
  • micromilling
  • ldr
  • thermal cycle
  • laser ablation
  • micromixers
  • su-8 lithography
Date of Defense 2004-08-04
Availability unrestricted
Abstract
The Ligase Detection Reaction (LDR) is a mutation detection technique used to identify point mutations in deoxyribonucleic acid (DNA). Developed by Francis Barany and associates at Cornell University it is used to find specific low abundant point mutations that may lead to colorectal cancer in the early stages of disease development.

The research objective was to design and manufacture a microscale Ligase Detection Reaction (LDR) device in polycarbonate. The LDR module will be incorporated with other microdevices such as: Continuous Flow Polymerase Chain Reaction (CFRCR) and Capillary Electrophoresis (CE) in modular lab-on-a-chip technology. In making the microdevice, the duration of original reaction had to be scaled down from the current 2 hours for 20 cycles for the macroscale reaction. It was found that an excess of primers in relation to PCR product was needed for efficient ligation. By changing the concentrations, volumes and time for the process the current time is down to 40 minutes for 20 cycles with indications that further time reductions are possible on the microscale.

There are two mixing stages involved in the reaction. Micromixers were simulated in Fluent (v5.4, Lebanon, NH) and several test geometries selected for fabrication. Passive diffusion mixing was used based on obtaining high aspect ratios, 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 are fabricated and tested using chemi-luminescence technique.

For a successful reaction, temperatures of 0C, 95C and 65C were needed. A stationary chamber was used for thermal cycling in which the sample sits while the temperature is cycled. Finite element analysis showed uniform temperatures in the rectangular 1.5μl chambers and that air slits can effectively separate the thermal cycle zone from the 0C cooling zone and also isolate the mixing region. A test device was laid out and micromilled with the temperature zones maintained and fluid flow controlled. A commercial thin film heater and a thermoelectric module were used with PID controls to obtain the required process temperatures. Heating from 65C to 95C took 10 seconds, while cooling from 95C to 65C also took 10 seconds. The residence times at the required temperatures can be adapted to changes in the LDR.

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