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

Integrated Micro/Nano Summer Undergraduate Research Experience . Combinatorial Bioassays in Droplet Arrays for Monitoring Astronaut Health During Space Travel. Liana Alston, Department of Biochemistry, UC Riverside Faculty Mentor:

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

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  1. Integrated Micro/Nano Summer Undergraduate Research Experience Combinatorial Bioassays in Droplet Arrays for Monitoring Astronaut Health During Space Travel 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. 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. Background & Terminology 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 & Where:Goal 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. Why:Potential 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. How:Project 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. Disease Method of Detection Accuracy Link to PDF file Year Could Affect an Astronaut Hepatitis A Hepatitis A antibody (IgG & IgM) 100% 98% article 1992 Y Hepatitis B Hepatitis B antibody 100% 100% article " N Hepatitis C Hepatitis C antibody 100% 100% article " N Measles Measles antibody (IgG & IgM) 97% 100% article 1994 N Mumps Mumps antibody (IgG & IgM) 94% 94% article " N Rubella Rubella antibody (IgG & IgM) 98% 98% article " N Neurocysticercosis Neurocysticercosis antibody 100% article 1990 N HIV HIV antibody 97% 97% article 1997 N Gastric H. Pylori Infection Gastric H. Pylori Infection antibody 97% 94% article 1997 Y Parvovirus B 19 Parvovirus B 19 antibody 100% 95% article 1996 N Dengue Dengue antibody 92% 100% article 1998 N EBV EBV antibody 90% article 2005 Y HSV-1 HSV-1 antibody 82% article 1993 Y Ovarian Cancer Ovarian Cancer anitbody (CA 125) antibody (CA 125) 81% 81% 88% article 1990 1990 Y Y PBD PBD antibody (IgG) / ELISA 55% 97% article 1986 Y HHV-6 & 7 gene (DNA)/ PCR 90% article 1999 N N Gastric H. Pylori Infection gene (DNA)/ PCR 84% article 1996 EBV gene (DNA)/ PCR 75% 97% article 1985 Y Varicella-Zoster Virus gene (DNA)/ PCR 72% article 2002 Y Head & Neck Cancer gene (DNA)/ PCR 71% article 1994 N Adult Periodontal Disease gene (RNA)/ PCR 70% article 1998 Y article 1994 Hepatitis C gene (RNA)/ PCR 48% N CMV gene (DNA)/ PCR 61% article 1997 Y HHV 8 gene (DNA)/ PCR 37% article 1997 Y Renal Disease elevated [creatinine] 100% 95% article 1996 Y Sensitivity Specificity

  8. Contribution to Monthly NASA Report

  9. Buffer Characterization:an 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. IDT’s working buffer • Hepatitis C vs. Breast Cancer MB & cDNA • Albert’s cDNA vs. IDT’s cDNA

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

  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 (0.5:2.5mM) MB:cDNA concentrations.

  13. 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 -> OH) render glass slide and PDMS hydrophobic, so droplets won’t 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 PDMS Microchannel Fabrication

  14. Mask Design; Droplet Generation Experiment L-edit rendition of µchannel incorporating Tim’s “Fusion Turns” Digital Syringe Pumps

  15. Droplet Generation Experiment Tim’s Experiment: H20 and H20 Liana & George’s 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 Brody’s lab group • Wajeeh, Dr. Noo Li Jeon’s 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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