Integrated Micro Nano Summer Undergraduate Research Experience Combinatorial Bioassays

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 1

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. • 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 siliconbased 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 nonflammable. 2

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. 3

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 4

How: Project Outline I. Literature Searches for a Virus to Attempt to Detect in Astronaut Saliva II. Hybridization Buffer Optimization III. IV. V. Microchannel Design Microchannel Fabrication Droplet Generation Experiments 5

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 6

Accuracy Disease Method of Detection Sensitivity Specificity Hepatitis A antibody (Ig. G & Ig. M) 100% 98% Hepatitis B antibody 100% Hepatitis C antibody Measles Link to PDF file Year Could Affect an Astronaut article 1992 Y 100% article " N antibody (Ig. G & Ig. M) 97% 100% article 1994 N Mumps antibody (Ig. G & Ig. M) 94% article " N Rubella antibody (Ig. G & Ig. M) 98% article " N Neurocysticercosis antibody article 1990 N HIV antibody 97% article 1997 N Gastric H. Pylori Infection antibody 97% 94% article 1997 Y Parvovirus B 19 antibody 100% 95% article 1996 N Dengue antibody 92% 100% article 1998 N EBV antibody 90% article 2005 Y HSV-1 antibody 82% article 1993 Y Ovarian Cancer anitbody (CA 125) antibody 81% 88% article 1990 Y PBD antibody (Ig. G) / ELISA 55% 97% article 1986 Y HHV-6 & 7 gene (DNA)/ PCR 90% article 1999 N Gastric H. Pylori Infection gene (DNA)/ PCR 84% article 1996 N EBV gene (DNA)/ PCR 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 CMV gene (DNA)/ PCR 61% gene (RNA)/ PCR 48% 1997 1994 Y Hepatitis C article HHV 8 gene (DNA)/ PCR 37% article 1997 Y 7 Renal Disease elevated [creatinine] article 1996 Y 100% 75% 100% 97% 95% N

Contribution to Monthly NASA Report 8

Buffer Characterization: an Overview • Point: Maximize Signal to Background Ratio (SNR) of the MB-c. DNA hybridization • Varied [Mg. Cl 2], p. H, & [KCl] in that order – Ranges: • 0, 1, 5, 10, 20, 50, 100 m. M Mg. Cl 2 concentrations • p. H 7, 8, 8. 5, 9 • 0, 10, 50, 100 m. M KCl concentrations • Smaller vs. Higher Concentrations of MB and c. DNA -expensive! • Our best buffer vs. IDT’s working buffer • Hepatitis C vs. Breast Cancer MB & c. DNA • Albert’s c. DNA vs. IDT’s c. DNA 9

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

Buffer Characterization 11

Buffer Characterization • Trends observed in Hepatitis C: – 50 m. M = optimal [Mg. Cl 2] 0 -100 m. M – p. H 7 = optimal p. H 7, 8, 8. 5, 9 – 50 m. M = optimal [KCl] 0 -100 m. M …w/ both small (0. 05: 0. 25) and high (0. 5: 2. 5 m. M) MB: c. DNA concentrations. 12

PDMS Microchannel Fabrication • • Si wafer Spin coat SU-8 Bake Photoresist Expose Post-expose bake Develop Rinse and Dry • • Pour PDMS Vacuum to eliminate air bubbles Bake Peel off PDMS channel; cut & punch holes • Use N 2 gun to blast away PDMS crumbs to avoid intra-channel blockage • O 2 plasma (CH 3 -> OH) render glass slide and PDMS hydrophobic, so droplets won’t stick to channel • Seal PDMS channel to glass slide 13

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

Droplet Generation Experiment Tim’s Experiment: H 20 and H 20 Liana & George’s Experiment: H 20 and dye 15

Future Work • Optimize flow rates for fusion turn channels – H 20 + 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 16

Acknowledgements v Graduate Students v Albert Tsung-Hsi Hsieh v Tim Wei-Yu Tseng v Jason Lung-Hsin Hung v Jeff Fisher IM-SURE voyage v Joe Harris & Grace, Dr. Jim Brody’s lab group v Wajeeh, Dr. Noo Li Jeon’s lab group v Undergraduate Students v v Adam Yuh Lin Patrick Pan George Yung-Chieh Chen Alok Vij v UROP Staff v Said Shokair v Jerry Mc. Millan v INRF Staff v Goran Matijasevic Arroyo Vista IM-SURE house 17