Combinatorial Bioassays in Droplet Arrays for Monitoring Astronaut Health During Space Travel

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Combinatorial Bioassays in Droplet Arrays for
Monitoring Astronaut Health During Space Travel
Integrated Micro/Nano Summer Undergraduate
Research Experience

• Liana Alston, Department of Biochemistry, UC
  Riverside
• Faculty Mentor
• Dr. Abraham P. Lee, Department of Biomedical
  Engineering, UC Irvine
• Graduate Mentor
• Tsung-Hsi Albert Hsieh, Department of Biomedical
  Engineering, UC Irvine

September 2, 2005
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Background Terminology

• Microfluidics is a multidisciplinary field
  comprising physics, chemistry, engineering and
  biotechnology that studies the behavior of fluids
  at the microscale
• Polydimethylsiloxane (PDMS) is the most widely
  used silicon-based organic polymer, and is
  particularly known for its unusual flow
  properties. It is optically clear, and is
  generally considered to be inert, non-toxic and
  non-flammable.

Phloem The tree equivalent to veins of the human
body. Essentially it is a system of tubes that
transport sugar and other organic nutrients
throughout the plant.
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Who, What WhereGoal of this Project

• The development of a new diagnostic tool, that
  will require only a small salivary sample to
  perform rapid DNA analysis with a molecular
  beacon detector, resulting in an immediate
  qualitative verification of viral infection.

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WhyPotential Applications of this Research

• Non-invasive diagnosis of astronauts with
  reactivated and potentially symptomatic
  herpesvirus infection
• Non-invasive diagnosis of individuals with viral
  infections, in a domestic setting, to perhaps
  stop the spread of pandemic diseases

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HowProject Outline

• Literature Searches for a Virus to Attempt to
  Detect in Astronaut Saliva
• Hybridization Buffer Optimization
• III. Microchannel Design
• IV. Microchannel Fabrication
• V. Droplet Generation Experiments

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Journal Review Paper Search Protocol Results

• Criteria
• a VIRUS that can be diagnosed via a salivary
  sample
• a VIRUS that can be detected by DNA/RNA sequence
  in saliva
• a VIRUS with a high accuracy (80)
• sensitivity ill individuals correctly
  diagnosed
• specificity well individuals ( of control
  group) in which virus was not detected
• Most Convincing Papers

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Could Affect an Astronaut
Year
Link to PDF file
Accuracy
Method of Detection
Disease
Specificity
Sensitivity
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Contribution to Monthly NASA Report
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Buffer Characterizationan Overview

• Point Maximize Signal to Background Ratio (SNR)
  of the MB-cDNA hybridization
• Varied MgCl2, pH, KCl ? in that order
• Ranges
• 0,1,5,10,20,50,100 mM MgCl2 concentrations
• pH 7, 8, 8.5, 9
• 0, 10, 50, 100 mM KCl concentrations
• Smaller vs. Higher Concentrations of MB and cDNA
• -expensive!
• Our best buffer vs. IDTs working buffer
• Hepatitis C vs. Breast Cancer MB cDNA
• Alberts cDNA vs. IDTs cDNA

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Buffer Characterization

• Noteworthy Equipment
• -Spectrofluorometer
• -RT-PCR machine
• -Autoclave
• -Automatic Delivery pipets
• -Eppendorf Tubes
• -Black Microwell Plates
• -pH meter

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Buffer Characterization
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Buffer Characterization

• Trends observed in Hepatitis C
• 50mM optimal MgCl2 0-100mM
• pH 7 optimal pH 7,8,8.5,9
• 50mM optimal KCl 0-100mM
• w/ both small (0.05 0.25) and high (0.52.5mM)
  MBcDNA concentrations.

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PDMS Microchannel Fabrication

• Pour PDMS
• Vacuum to eliminate air bubbles
• Bake
• Peel off PDMS channel cut punch holes
• Use N2 gun to blast away PDMS crumbs to avoid
  intra-channel blockage ?
• O2 plasma (CH3 -gt OH) render glass slide and PDMS
  hydrophobic, so droplets wont stick to channel
• Seal PDMS channel to glass slide

• Si wafer
• Spin coat SU-8
• Bake
• Photoresist
• Expose
• Post-expose bake
• Develop
• Rinse and Dry

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Mask Design Droplet Generation Experiment
L-edit rendition of µchannel incorporating Tims
Fusion Turns
Digital Syringe Pumps
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Droplet Generation Experiment
Tims Experiment H20 and H20
Liana Georges Experiment H20 and dye
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Future Work

• Optimize flow rates for fusion turn channels
• H20 dye
• Using Metamorph to analyze the mixing within
  droplets in 0 vs. 90 vs. 180 degree channels
• Continue buffer characterization for more
  conclusive data supporting an optimal working
  buffer (esp. important for detecting reactivation
  of a latent virus
• Finding unique EBV sequence
• Unique to EBV (relative to other
  gammaherpesvirinae
• Unique to reactivated EBV relative to latent EBV

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Acknowledgements

• Graduate Students
• Albert Tsung-Hsi Hsieh
• Tim Wei-Yu Tseng
• Jason Lung-Hsin Hung
• Jeff Fisher
• Joe Harris Grace, Dr. Jim Brodys lab group
• Wajeeh, Dr. Noo Li Jeons lab group
• Undergraduate Students
• Adam Yuh Lin
• Patrick Pan
• George Yung-Chieh Chen
• Alok Vij
• UROP Staff
• Said Shokair
• Jerry McMillan
• INRF Staff
• Goran Matijasevic

IM-SURE voyage
Arroyo Vista IM-SURE house