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

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


1
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
2
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.
3
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.

4
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

5
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

6
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

7
Could Affect an Astronaut
Year
Link to PDF file
Accuracy
Method of Detection
Disease
Specificity
Sensitivity
8
Contribution to Monthly NASA Report
9
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

10
Buffer Characterization
  • Noteworthy Equipment
  • -Spectrofluorometer
  • -RT-PCR machine
  • -Autoclave
  • -Automatic Delivery pipets
  • -Eppendorf Tubes
  • -Black Microwell Plates
  • -pH meter

11
Buffer Characterization
12
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.

13
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

14
Mask Design Droplet Generation Experiment
L-edit rendition of µchannel incorporating Tims
Fusion Turns
Digital Syringe Pumps
15
Droplet Generation Experiment
Tims Experiment H20 and H20
Liana Georges Experiment H20 and dye
16
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

17
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
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