Developing a Microfluidic Thioaptamer
Diagnostic System for Use in Space
UTMB/JSC Aerospace Medicine Grand Rounds
November 24, 2009
Ross H. Durland, PhD, Director, Product Development
AM Biotechnologies, LLC
Disclosures
AM Biotechnologies and Sandia National Laboratories are
collaborating to develop a Microfluidic Thioaptamer Diagnostic
System suitable for use in space by NASA. This system and its
components are investigational and are not approved for any
clinical diagnostic use.
As an employee and stockholder of AM Biotechnologies, Dr.
Durland has a significant financial interest in the technology
presented herein.
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Diagnostic Testing During Spaceflight
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Critical to astronaut health research and medical
monitoring
Significant need for in-flight diagnostics
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time sensitivity
storage & transport limitations (Shuttle Retirement and
Planetary Exploration; ESMD RISK 3428)
Limited existing capabilities
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Portable Clinical Blood Analyzer (i-STAT)
blood gases, electrolytes, pH, hematocrit
protein biomarkers are a major unmet need
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Importance of Protein Biomarkers
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Ground and flight health research
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Bone demineralization – NTx, CTx, OC, BSAP
Immunological status – IFNγ, IL1β, IL6, IL8, TNFα
Radiation & Oxidative stress – NOS, SOD, GADD45α
Cardiovascular health – cTnI, CK-MB, CRP, BNP
Translatable to flight medical and research
diagnostics
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Objective
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Develop a Microfluidic Thioaptamer Diagnostic
System for use during space flight
Key components
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thioaptamer affinity reagents (AM)
microfluidic capillary electrophoresis device (Sandia)
Focus on protein biomarkers
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key unmet need
biomarker discovery (Goodwin/JSC)
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Aptamers
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Nucleic acids that bind a
specific molecular target
DNA or RNA
Synthetic or naturally
occurring
Affinity & specificity
comparable to antibodies
Targets include proteins,
carbohydrates, small
molecules, intact cells, virions
Applications in research,
diagnostics, imaging,
therapeutics
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Thioaptamers
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Aptamers containing phosphorodithioate modifications
phosphate
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phosphorodithioate
Developed by Dr. Gorenstein, AM
Features
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Increased binding affinity for proteins
High binding specificity
Nuclease resistance
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In Vitro Selection of Thioaptamers
Synthesize combinatorial DNA
or RNA library on beads
Select individual beads that
bind target protein
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Comparison of Thioaptamers and Antibodies
Attribute
Antibodies
Thioaptamers
Affinity
High
High
Specificity
High
High
Discovery
In Vivo
In Vitro
Refrigerated, Frozen
Ambient
Months
Years
Biological
Chemical
Low
High
Storage
Shelf Life
Production
Batch Reproducibility
=
=
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Microfluidic Capillary Electrophoresis Device
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Developed at Sandia National Laboratories
Key features
‰ microliter sample volumes
‰ short analysis times (minutes)
‰ high sensitivities (pM)
Selected applications
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‰ protein gel electrophoresis & isoelectric focusing
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‰ protein biotoxin detection
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‰ microfluidic immunoassays
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J. Han & A.K. Singh (2004), J Chromatogr A 1049:205-209; A.E. Herr & A.K. Singh (2004), Anal Chem 76:4727-4733.
J.A. Fruetel et al. (2005), Electrophoresis 26:1144-1154; R.J. Meagher et al. (2008), Lab on a Chip 8:2046-2053.
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A.E. Herr et al. (2005), Anal Chem 77:585-590; A.E. Herr et al. (2007), Proc Natl Acad Sci USA 104:5268-5273.
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Microfluidic Device
Fluidic
cartridge
with chip
Touch
screen
interface
Microfluidic
chip
Cartridge
reader
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Operating Principles
+
fluorescentlabeled TA
TA-protein
complex
target
protein
microfluidic channel
sieving matrix
Load TA + target
TA/protein
mixture
Apply voltage
LIF
0V
+V
complex
free TA
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Test System
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XBY-6 thioaptamer
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Selected at UTMB*
14 bp duplex containing 6 dithio modifications
binds NF-κB p50 with high affinity (KD ~ 150 pM)
NF-κB p50
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recombinant human, purified (Promega)
*X. Yang et al. (1999), Bioorg Med Chem Lett 9:3357-3362; D.E. Volk et al. (2002), Bioorg Chem 30:396-419.
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Electropherograms of XBY-6 + NF-κB p50
No NF-κB
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Fluorescence Intensity (AU)
10 nM NF-κB
5 nM NF-κB
80 nM NF-κB
TA
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4
TA:p50
complex
2
0
0
60
120 0
60
120
0
60
120
0
60
120
Time (sec)
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NF-κB p50 Standard Curve
1.0
Normalized Peak Area
0.8
0.6
0.4
0.2
0.0
0.1
1
10
100
1000
NF-κB Concentration (nM)
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NF-κB p50 Detection with Antibodies
0 nM NF-κB p50
2.0
Ab
1.5
Fluorescence Intensity (AU)
Fluorescence Intensity (AU)
2.0
10 nM NF-κB p50
IS
1.0
0.5
0.0
1.5
Ab:p50
complex
IS
Ab
1.0
0.5
0.0
0
100
Time(sec)
200
0
100
200
Time (sec)
IS – internal standard (AF647-labeled BSA)
Ab – AF647-labeled anti-NF-κB p50 antibody
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On-Chip Mixing and Preconcentration
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On-Chip Mixing and Preconcentration
NF-κB p50 Preconcentration Time
0s
30 s
90 s
180 s
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Fluorescent Signal (AU)
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10
8
6
4
2
0
0
30
60
0
30
60
0
30
60
0
30
60
Time (sec)
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Status
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NASA Phase I SBIR completed
Proof of principle demonstrated
Thioaptamers outperform antibodies
On-chip mixing & preconcentration
Good run-to-run reproducibility
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Phase II Plans
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Optimize quantitation in serum
Select TAs to relevant protein targets
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Optimize assay conditions
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BSAP, OC, NTx, CTx, CRP, Mb, SOD
sensitivity, specificity, accuracy, precision
Refine prototype device
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re-usable, replaceable test cartridges
multiple analytes
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Target Final System Characteristics
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Suitable for use in microgravity
Small, rugged form-factor (< 10 lb)
Low power consumption (battery operated)
Easy to use, self-contained, and automated
Rapid (<10 minutes) and sensitive (pg/mL)
Minimal sample volumes (microliters)
Compatible with biological fluids (serum, urine, saliva)
Simultaneous quantitation of multiple biomarkers (8-32)
Long-term reagent stability at ambient temperature
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Rotating Wall Vessel for Simulated Microgravity
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Assembly of 3D Tissues in RWVs
cell or tissue sample
cells
RWV
microscopic, genetic,
proteomic analysis
microcarrier beads
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Human Cells & Tissues Grown in RWV
Norman Human Neural Progenitor and Brain Glioma
Bone (Osteoblast/Osteoclast)
Human Cornea
Cardiac
Airway Smooth Muscle (ASM)
Human Breast CA
Normal Human Lung
Normal Human Thyroid
Normal Human Lymphoid
Normal Human Large Intestine
Human Small Intestine
Normal Intestinal epithelial
Normal Human Kidney
Skeletal Muscle
Adrenal medulla
Normal Human Liver
Cervical
Adipose
Human Pancreatic islet cells
Normal Human Renal proximal tubule
Normal Adult Stem Cells
Human Ovarian Ca
Human Peripheral Blood Lymphocytes
Human Colon Ca
Human Prostate Ca
Endocrine
Cartilage (chondrocytes)
Normal Human Skin
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Summary
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System is well suited for use during spaceflight
Thioaptamers
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Microfluidic Capillary Electrophoresis Device
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high affinity & specificity for protein biomarkers
selected in vitro
readily synthesized
robust and stable at ambient temperature
minimal sample requirements
rapid analysis
high sensitivity
potential to measure 8-32 analytes per sample
High value for NASA
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in-flight diagnostics for health research & monitoring
near-term opportunities for novel biomarker discovery, research on ISS
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Acknowledgments
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AM Biotechnologies
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Sandia National Laboratories
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Xianbin Yang, Johnnie Engelhardt, Nancy Ward, Ellen
Mierzejewski, Mark Shumbera
Anson Hatch, Ariel Hecht, Greg Sommer, Laura
Santos, Anup Singh
NASA
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Thomas Goodwin, Chuck Lloyd
This research was funded in part by a NASA Small Business Innovative
Research (SBIR) grant, #NNX09CE33P
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