May 2003 ME Graduate Conference1 Dwhyte O. Barrett (M.S. Candidate) Advisor : Dr. Michael Murphy Department of Mechanical Engineering Louisiana State University

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


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


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


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


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.


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


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.


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.


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


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


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.


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


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


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


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





94 C 30 s65 C 4minX 20 cycles




94 C 20 s65 C 2minsX 20 cycles


Results: Good Results: Faint Results: None Results:Good Results: None Results: None



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


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.


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.


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.


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



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


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


Polycarbonate

Heater

Air Gap

Channel

75µm

800µm

400µm

300µm

300µm

400µm

300µm

Thermal Model of LDR Device


Temperature distribution shown for chip over a length of 1 cm

Steady State Temperature Distribution


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


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


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


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


QUESTIONS?

Documents

May 2003 ME Graduate Conference1 Dwhyte O. Barrett (M.S. Candidate) Advisor : Dr. Michael Murphy Department of Mechanical Engineering Louisiana State University