Title: Combinatorial Bioassays in Droplet Arrays for Monitoring Astronaut Health During Space Travel
1Combinatorial 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
2Background 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.
3Who, 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.
4WhyPotential 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
5HowProject 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
6Journal 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
7Could Affect an Astronaut
Year
Link to PDF file
Accuracy
Method of Detection
Disease
Specificity
Sensitivity
8Contribution to Monthly NASA Report
9Buffer 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
10Buffer Characterization
- Noteworthy Equipment
- -Spectrofluorometer
- -RT-PCR machine
- -Autoclave
- -Automatic Delivery pipets
- -Eppendorf Tubes
- -Black Microwell Plates
- -pH meter
11Buffer Characterization
12Buffer 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.
13PDMS 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
14Mask Design Droplet Generation Experiment
L-edit rendition of µchannel incorporating Tims
Fusion Turns
Digital Syringe Pumps
15Droplet Generation Experiment
Tims Experiment H20 and H20
Liana Georges Experiment H20 and dye
16Future 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
17Acknowledgements
- 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