Dwhyte O. Barrett (M.S. Candidate) Advisor : Dr. Michael Murphy

1. DESIGN AND MICROFABRICATION OF A LIGASE DETECTION REACTION (LDR) DEVICE Dwhyte O. Barrett (M.S. Candidate) Advisor : Dr. Michael Murphy Department of Mechanical Engineering Louisiana State University May 3, 2003 ME Graduate Conference

2. Overview • Introduction • Background on LDR • Project Objective • Design Issues • LIGA Process and Polycarbonate • LDR Time Reduction • Mixing • LDR Fluidics • Temperature Profile, Modeling and Control • Future Work • Acknowledgements • Questions ME Graduate Conference

3. Introduction • The history of DNA can be traced back to 1865 when Gregor Mendel found that heredity is passed on in different units. • In 1953 Francis Crick and James Watson described the Double Helix structure of DNA and showed that each strand of DNA was a template for the other revealing a copying mechanism for the genetic material. • Since these discoveries scientists have studied, analyzed and characterized DNA not only as the building blocks for life but also as the starting place for certain diseases such as cancer. American Cancer Society ME Graduate Conference

4. Cancer cells develop because of mutations in DNA. People can inherit these mutated DNA, which accounts for inherited cancers. DNA can be damaged by exposure to radiation, smoking or pollutants. • It is the second leading cause of death in the United States. • Half of all men and one-third of all women in the US will develop cancer during their lifetimes. • 1,334,100 new cases of cancer will be diagnosed this year. • The cost to the economy will be $171.6 billion in treatments and loss wages. • The 5-year relative survival rate for people with cancers who had early detection is about 82%. If all Americans had early detection testing the 5-year relative survival rate would increase to about 95%. ME Graduate Conference American Cancer Society

5. Background on LDR • The LDR was developed and patented (pat# 5,494,810) by Francis Barany and his associates at Cornell University. • Barany has done extensive work in DNA analysis and gene mutations. • Used in conjunction with the Polymerase Chain Reaction (PCR) it is particularly useful for the detection of rare cancer-associated mutations. • The reaction uses PCR products and several primers that are mixed together with a buffer and heated up to 95oC. Ligase enzymes are then added and mixed after 90 seconds of heating. The resulting mixture goes through twenty cycles of 95oC and 65oC for 30 seconds and 4 minutes, respectively. After cycling the reaction is stopped at 0oC. Barany F., “The ligase chain reaction (LCR) in a PCR world”, PCR Methods Appl.1, 5-16,1991. ME Graduate Conference

6. 0.1 µM G12 V 1 µl (PCR Product) 1 µM czip 11 2 µl 1 µM com-2 2 µl 200 mM DTT 1 µl 10 mM NAD 1 µl 2 x buffer 12 µl Mix @ 95 °C Hold for 2 min Ligase 1 µl (Stored @ 0°C ) Mix @ 95 °C 95 °C 30 s 65 °C 4 min X 20 cycles Cool to 0 °C OUT LDR Schematic Layout ME Graduate Conference

7. Background • At 95°C denaturation (splitting) of DNA occurs • At 65°C the ligase anneals the DNA strands together Barany F., “The ligase chain reaction (LCR) in a PCR world”, PCR Methods Appl.1, 5-16,1991. ME Graduate Conference

8. 95ºC 72ºC 55ºC  t  t 4* t Polymerase Chain Reaction (PCR) • Common method used for creating copies of specific fragments of DNA. PCR rapidly amplifies a single DNA molecule into many billions of molecules. • Separate the two DNA chains in a double helix by heating the vial to 90-95°C for  t seconds. • At 55°C anneal to the end of the DNA strands, for about  t seconds. • Make a complete copy of the templates at around 72°C for 4* t seconds • Repeat 20 – 40 times Mitchell M., “Design and Microfabrication of a Molded Polycarbonate Continuous Flow Polymerase Chain Reaction Device”, LSU Master’s Thesis, 2002. ME Graduate Conference

9. Sample Prep PCR Amplification LDR Identification Project objective • Miniaturization of LDR • Incorporate in modular lab-on-a-chip technology. • Main aim of this technology is to minimize the time and sample volume for chemical and biological analyses, and reduce the cost of fabrication so that the instruments can be used clinically. ME Graduate Conference

10. Design issues • Manufacturing and Suitable material • LDR time reduction: • Current macroscale reaction takes over 2½ hours for 20 cycles • Current microscale PCR down to 10 minutes for 20 cycles from 1½ hours in the macroscale • All reagents must be kept on chip with temperature profile maintained: • Storage • Ligase kept at 0°C and reaction also stops at 0°C • Cycling done between 65°C and 95°C • Mixing the reagents • Fluid flow • System Control ME Graduate Conference

11. LIGA process • Efficient means of mass producing microchips • Use the mold insert for hot embossing • Extensive use of PMMA for hot embossing Mitchell M., “Design and Microfabrication of a Molded Polycarbonate Continuous Flow Polymerase Chain Reaction Device”, LSU Master’s Thesis, 2002. ME Graduate Conference

12. Polycarbonate • Need higher temperature capability than that offered by PMMA for devices PCR • Moldable via hot embossing or injection molding • Can do thermal bonding of layers • Compatible with fluid actuation ME Graduate Conference

13. LDR Time Reduction • LDR product DNA (50-70 bp) longer than normal DNA (20-50 bp) • Different detection methods: • Zip Code gene array • Commercial DNA sequencer • Zip Code: PMMA microchips that are UV- exposed to spot proteins to capture LDR product • Proteins may not always attach ME Graduate Conference

14. Experiments • Currently using small plastic tubes with large commercial thermal cycler • Tubes have to be closed to prevent evaporation • NEN™ DNA sequencer is used • Operates on the principle of electrophoresis • Holds up to 30 samples • Systematic progression: • Large tubes : reduce time- sequencer • Large tubes : reduce volume – normal time – sequencer • Large tubes : reduce volume- time- sequencer • Chip: normal time – gel electrophoresis • Chip: reduce time- gel electrophoresis • LDR CHIP w/mixing ME Graduate Conference

15. Example test matrix ME Graduate Conference

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17. W1 W2 W1 W2 Wm Øc α Wt β W2 W2 W1 Wm W1 W2 Mixing • Two mixing stages • Can make a bulk mixture of the reagents off chip • Minimize moving parts • Diffusion mixing with aspect ratios 10-20 • Test geometries laid out on a microchip with multiplexing Wm ME Graduate Conference

18. LDR Fluidics • Two main methods of fluid transport are pressure driven and electrokinetics • Pressure driven flow results in a parabolic velocity profile and will cause dispersion for small samples • Electrokinetics results in “plug like” flow resulting in minimal sample dispersion • Electrokinetic transport refers to a combination of : • Electroosmotic : bulk movement of a solution past a stationary surface due to an externally applied electric field. • Electrophoresis : motion of a charged particle surface submerged in a fluid under the action of an electric field. ME Graduate Conference

19. Electroosmotic flow • The Helmholtz-Smoluchowski equation for electroosmotic velocity: • This equation when solved predicts a plug like profile. The electroosmotic velocity is approximately 0.1mm/sec, when ς=0.1V, Ex = 100 V/cm for water. Probstein, R.F, Physicochemical Hydrodynamics,2nd Edition, Wiley & Sons, New York, 1995. ME Graduate Conference

20. Electrophoretic Flow • The same equation holds for the electrophoretic flow • Here the zeta potentials are different : in electroosmosis it is a property of the stationary surface while in electrophoresis it is a property of the moving surface Probstein, R.F, Physicochemical Hydrodynamics,2nd Edition, Wiley & Sons, New York, 1995. ME Graduate Conference

21. +V Plug -V -V +V +V -V Plug Plug Test Geometry • The Chemistry Department of Louisiana State University is set up to run electrokinetic transport with up to sixteen different reservoirs with each individually controlled. ME Graduate Conference

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23. 94 ºC 65 ºC 30 s 4 min Temperature Profile Modeling • Steady-state • Outer surface at ambient temperature (25°C) • 2-D heat flow • Convection negligible compared to conduction and radiation • Constant flux heaters • Models run using ANSYS 5.5 ME Graduate Conference

24. 5000µm 3000µm 65°C 95°C 4000µm 4000µm 5000µm 3000µm 9500µm 9500µm 3000µm 1000µm Thermal Model of LDR Device Density PC = 5.7e-15 (kg/µm3) Air = 1.1614e-18 (kg/ µm3) Water = 1e-15 (kg/ µm3) Thermal conductivity PC=2.2e6 (kg µm/s3K) Air= 26.3e3 (kg µm/s3K) Water=613e3 (kg µm/s3K) H= 15 W/m2K ME Graduate Conference

25. Polycarbonate 800µm Air Gap 400µm Heater 300µm 75µm 300µm 400µm Channel 300µm Thermal Model of LDR Device ME Graduate Conference

26. Steady State Temperature Distribution • Temperature distribution shown for chip over a length of 1 cm ME Graduate Conference

27. Thermoelectric Module • Uses Peltier effect to transfer heat when an electric current passes through. • Compact with no moving parts • Switching direction of current changes the module from a cooling to heating mode • Ranges between -50°C and 150°C ME Graduate Conference

28. I, V Thermoelectric Parameters • Block dimensions : 1cm x 1cmx .5cm • Qload =0.9W • Module: • I=1.8 A • V= 2.2 V • Q = 4.4W • 1cm x 1cm x 3mm • Heating rate 6°C/s • Cooling rate 3°C/s Block TEC ME Graduate Conference

29. Future Work • Maximize time reduction • Validate models and simulations with experiments • Build and test prototype • Assemble total system : sample preparation device,PCR device ,LDR device and detection device ME Graduate Conference

30. Acknowledgements This work is funded by : • Bioengineering Research Partnership (NIH R24-CA84625-03) through the National Human Genome Research Institute (NHGRI) and the National Cancer Institute (NCI) of the National Institutes of Health (NIH) • PCR group • LSU Chemistry Department ME Graduate Conference

31. QUESTIONS? ME Graduate Conference