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September 2, 2005September 2, 2005
Integrated Micro/Nano Summer Undergraduate Research Experience
Combinatorial Bioassays in Droplet Arrays for Combinatorial Bioassays in Droplet Arrays for Monitoring Astronaut Health During Space TravelMonitoring 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
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Background & TerminologyBackground & 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 & Where:Who, What & Where:Goal of this ProjectGoal 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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Why:Why:Potential Applications of this ResearchPotential 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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How:How:Project OutlineProject Outline
I. Literature Searches for a Virus to Attempt to Detect in Astronaut Saliva
II. Hybridization Buffer Optimization
III. Microchannel DesignIV. Microchannel FabricationV. Droplet Generation Experiments
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Journal & Review Paper Search Journal & Review Paper Search Protocol & ResultsProtocol & 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 diagnosedspecificity: # well individuals (% of control group) in which virus
was not detected
• Most Convincing Papers
7 Y1997article37%gene (DNA)/ PCRHHV 8
Y1998article70%gene (RNA)/ PCRAdult Periodontal Disease
gene (DNA)/ PCR
N1994article71%gene (DNA)/ PCRHead & Neck Cancer
N1996article84%gene (DNA)/ PCRGastric H. Pylori Infection
N1994article48%gene (RNA)/ PCRHepatitis C
N1999article90%HHV-6 & 7
Y1996article95%100%elevated [creatinine] Renal Disease
Y1985article97%75%gene (DNA)/ PCREBV
SpecificitySensitivityCould Affect an
AstronautYear
Link to PDF file
Accuracy Method of Detection Disease
Y2002article72%gene (DNA)/ PCRVaricella-Zoster Virus
PBD
Y199081%anitbody (CA 125)Ovarian Cancer
HSV-1
EBV
Dengue
Parvovirus B 19
Gastric H. Pylori Infection
HIV
Neurocysticercosis
Rubella
Mumps
Measles
Hepatitis C
Hepatitis B
Hepatitis A
Y1986article97%55%antibody (IgG) / ELISA
article88%
Y1993article82%antibody
Y2005article90%antibody
N 1998article100%92%antibody
N1996article95%100%antibody
Y1997article94%97%antibody
N1997article97%97%antibody
N 1990article100%antibody
N"article98%98%antibody (IgG & IgM)
N "article94%94%antibody (IgG & IgM)
N 1994article100%97%antibody (IgG & IgM)
N"article100%100%antibody
N "article100%100%antibody
Y1992article98%100%antibody (IgG & IgM)
PBD
Y199081%antibody (CA 125)Ovarian Cancer
HSV-1
EBV
Dengue
Parvovirus B 19
Gastric H. Pylori Infection
HIV
Neurocysticercosis
Rubella
Mumps
Measles
Hepatitis C
Hepatitis B
Hepatitis A
Y1997article61%gene (DNA)/ PCRCMV
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Contribution to Monthly NASA ReportContribution to Monthly NASA Report
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Buffer Characterization:Buffer Characterization:an Overviewan 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
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Buffer CharacterizationBuffer Characterization
• Noteworthy EquipmentNoteworthy Equipment-Spectrofluorometer-RT-PCR machine-Autoclave -Automatic Delivery pipets-Eppendorf Tubes-Black Microwell Plates-pH meter
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Buffer CharacterizationBuffer Characterization
STNR as a function of MgCl2 after denaturing/cooling of sample
020000400006000080000
100000120000140000
1 10 50
[MgCl2]
Fluo
resc
ence
noise
signal
STNR as a function of [MgCl2] Hepatitis C
0
5000
10000
15000
0 0 1 5 5 10 20 20 50 100 100
[MgCl2]
Fluo
resc
ence
before
after
STNR as a function of pHHepatitis C
0100020003000400050006000700080009000
10000
7 7 7 8 8 8 8.5 8.5 8.5 9 9 9
pH
Fluo
resc
ence
before
after
STNR as a function of [KCl]Hepatitis C
0
2000
4000
6000
8000
10000
0 0 0 10 10 10 50 50 50 100 100 100
[KCl]
Fluo
resc
ence
noise
signal
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• 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.
Buffer CharacterizationBuffer Characterization
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PDMS Microchannel FabricationPDMS 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 -> 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
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Mask Design; Droplet Generation Mask Design; Droplet Generation ExperimentExperiment
Digital Syringe Pumps
L-edit rendition of µchannel incorporating Tim’s “Fusion Turns”
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Droplet Generation ExperimentDroplet Generation Experiment
Tim’s Experiment:
H20 and H20
Liana & George’s Experiment:
H20 and dye
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Future WorkFuture 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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AcknowledgementsAcknowledgements 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
Arroyo Vista IM-SURE house
IM-SURE voyage
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