Post on 23-Jan-2016
description
May 2003 ME Graduate Conference 1
Dwhyte O. Barrett (M.S. Candidate)Advisor : Dr. Michael MurphyDepartment of Mechanical
EngineeringLouisiana State University
May 3, 2003
DESIGN AND MICROFABRICATION OF A
LIGASE DETECTION REACTION (LDR) DEVICE
May 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
May 2003 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
May 2003 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%.
American Cancer Society
May 2003 ME Graduate Conference 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.
May 2003 ME Graduate Conference 6
LDR Schematic Layout
0.1 µM G12 V1 µl
(PCR Product)
1 µM czip 11 2 µl 1 µM com-2
2 µl200 mM DTT
1 µl10 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 s65 °C 4 minX 20 cycles
Cool to 0 °C
OUT
May 2003 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.
May 2003 ME Graduate Conference 8
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.
1. Separate the two DNA chains in a double helix by heating the vial to 90-95°C for t seconds.
2. At 55°C anneal to the end of the DNA strands, for about t seconds.
3. Make a complete copy of the templates at around 72°C for 4* t seconds
4. Repeat 20 – 40 times
t 4* t t
95ºC
72ºC
55ºC
Mitchell M., “Design and Microfabrication of a Molded Polycarbonate Continuous Flow Polymerase Chain Reaction Device”,
LSU Master’s Thesis, 2002.
May 2003 ME Graduate Conference 9
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.
Identification
LDR
PCR Amplification
Sample Prep
May 2003 ME Graduate Conference 10
Design issues
Manufacturing and Suitable material LDR time reduction:
1. Current macroscale reaction takes over 2½ hours for 20 cycles
2. 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:
1. Storage2. Ligase kept at 0°C and reaction also stops at 0°C 3. Cycling done between 65°C and 95°C
Mixing the reagents Fluid flow System Control
May 2003 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
Resist
Plating Base
SubstrateX-Ray Radiation
Mask MembraneMask Absorber
Exposed Resist
Developed Resist
Electrodeposited Metal
Metal Microstructures
Mold Insert
Plastic Microstructures/Mold
Mitchell M., “Design and Microfabrication of a Molded Polycarbonate Continuous Flow Polymerase Chain Reaction Device”,
LSU Master’s Thesis, 2002.
May 2003 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
May 2003 ME Graduate Conference 13
LDR Time Reduction
LDR product DNA (50-70 bp) longer than normal DNA (20-50 bp)
Different detection methods:1) Zip Code gene array2) Commercial DNA sequencer
Zip Code: PMMA microchips that are UV- exposed to spot proteins to capture LDR product
Proteins may not always attach
May 2003 ME Graduate Conference 14
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:
1) Large tubes : reduce time- sequencer2) Large tubes : reduce volume – normal time – sequencer3) Large tubes : reduce volume- time- sequencer 4) Chip: normal time – gel electrophoresis5) Chip: reduce time- gel electrophoresis 6) LDR CHIP w/mixing
Experiments
May 2003 ME Graduate Conference 15
Example test matrix
RUN 1 RUN 2 RUN 3 RUN 4 RUN 5 RUN 6
0.1 µM G12 V 1 µl1 µM czip 11 2µl1 µM com-2 2 µl200 mM DTT 1 µl10 mM NAD 1µl2 x buffer 12µlLigase 1 µl
0.1 µM G12 V 1 µl1 µM czip 11 2 µl1 µM com-2 2 µl200 mM DTT 1 µl10 mM NAD 1 µl2 x buffer 12µlLigase 1 µl
0.1 µM G12 V 1 µl1 µM czip 11 2 µl1 µM com-2 2 µl200 mM DTT 1 µl10 mM NAD 1 µl2 x buffer 12µlLigase 1 µl
0.1 µM G12 V 1 µl1 µM czip 11 2 µl1 µM com-2 2 µl200 mM DTT 1 µl10 mM NAD 1 µl2 x buffer 12µlLigase 1 µl
0.1 µM G12 V 1 µl1 µM czip 11 2 µl1 µM com-2 2 µl200 mM DTT 1 µl10 mM NAD 1 µl2 x buffer 12µlLigase 1 µl
0.1 µM G12 V 1 µl1 µM czip 11 2 µl1 µM com-2 2 µl200 mM DTT 1 µl10 mM NAD 1 µl2 x buffer 12µlLigase 1 µl
94 C 30 s65 C 4minX 20 cycles
94 C 30 s65 C 2minX 20 cycles
94 C 20 s65 C 1minX 20 cycles
94 C 20 s65 C 4minX 20 cycles
94 C 20 s65 C 2minsX 20 cycles
94 C 20 s65 C 1minX 20 cycles
Results: Good Results: Faint Results: None Results:Good Results: None Results: None
May 2003 ME Graduate Conference 16
May 2003 ME Graduate Conference 17
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
µm µm µm
w1 25 50 50
w2 12.5 25 25
wm 20 20 20
wt 5 12.5 12.5
α 17.06°
15.52°
15.52°
β 5.01° 6.55° 6.55°
Øc 22.07°
22.07°
22.07°
W2
W1
Wm
Wt
α
β
Wm
W1
Wm
W2
W1
W2
W2
W1
W2
Øc
May 2003 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 :1. Electroosmotic : bulk movement of a solution past a
stationary surface due to an externally applied electric field.
2. Electrophoresis : motion of a charged particle surface submerged in a fluid under the action of an electric field.
May 2003 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.
sPa
VmE
V
mCV
msU
EU
x
EO
xEO
viscosity
field electric axial
potential zeta oticelectroosm
ty permittivi
velocity oticelectroosm
1
11
1
Probstein, R.F, Physicochemical Hydrodynamics,2nd Edition, Wiley & Sons, New York, 1995.
May 2003 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
sPa
VmE
V
mCV
msU
EU
x
EP
xEP
viscosity
field electric axial
potential zeta reticelectropho
ty permittivi
velocity reticelectropho
1
11
1
Probstein, R.F, Physicochemical Hydrodynamics,2nd Edition, Wiley & Sons, New York, 1995.
May 2003 ME Graduate Conference 21
Plug-V +V
The Chemistry Department of Louisiana State University is set up to run electrokinetic transport with up to sixteen
different reservoirs with each individually controlled.
-V
+VPlug
-V+V Plug
Test Geometry
May 2003 ME Graduate Conference 22
May 2003 ME Graduate Conference 23
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
94 ºC
65 ºC
30 s 4 min
May 2003 ME Graduate Conference 24
3000µm
9500µm 9500µm
5000µm
3000µm
4000µm
4000µm
5000µm
3000µm
1000µm
65°C 95°C
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
Thermal Model of LDR Device
May 2003 ME Graduate Conference 25
Polycarbonate
Heater
Air Gap
Channel
75µm
800µm
400µm
300µm
300µm
400µm
300µm
Thermal Model of LDR Device
May 2003 ME Graduate Conference 26
Temperature distribution shown for chip over a length of 1 cm
Steady State Temperature Distribution
May 2003 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
May 2003 ME Graduate Conference 28
Block dimensions : 1cm x 1cmx .5cm
Qload =0.9W Module:
1. I=1.8 A2. V= 2.2 V3. Q = 4.4W4. 1cm x 1cm x 3mm
Heating rate 6°C/s Cooling rate 3°C/s
I, V
Block
TEC
Thermoelectric Parameters
May 2003 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
May 2003 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
May 2003 ME Graduate Conference 31
QUESTIONS?