2014 CFTCC Annual Symposium Patrick Doyle, PhD

1. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
miRNA Gel Pads for the Point of
Care Detection of Cancer
Biomarkers
Rathi L. Srinivas, Hyewon Lee, and Patrick S. Doyle
Department of Chemical Engineering, MIT
Avrum Spira
Depts. of Medicine, and Pathology & Laboratory Medicine, BU
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2. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Project Description
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• Develop a point of care mfluidic chip
capable of detecting multiple miRNA
biomarkers for lung cancer
– Synthesize functional gel pads integrated
into microfluidic devices
– Demonstrate on-chip multiplexed miRNA
detection using universal labeling method
without complex external equipment for
POC application
– Develop novel sub-nanoliter oil-isolation for
ultrasensitive detection
– Profile low total RNA extracts in tissue
samples in chip

3. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
microRNA as a New Class of Biomarkers
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microRNA (miRNA): short non-coding RNA sequences that
are emerging as specific, stable, and robust biomarkers for
cancer diagnosis/prognosis
Main cancer-related cell functions targeted by miRNA in lung cancer
Hubaux et al., Metabolomics 2012

4. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
miRNA Profiling Considerations
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abundance varies by sample and are only ~0.01% of total RNA mass
Sample RNA Amount Features of miRNAs
Required
system
sensitivity
Single Cell 10-50 pg High-quality miRNA
~500
copies/cell
Tissue
(FNA)
1-10µg Cell-type heterogeneity ~ 1-10 amol
Serum
(100 µl)
5-10 ng
Typically requires
target amplification
~500 aM – 5 fM
Detection systems must have high sensitivity and high specificity
with discrimination of 1-2 nucleotide mismatch
Pritchard et al., Nature Reviews Genetics 2012

5. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Clinical Needs
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Lung Cancer kills
160,000 people
annually in USA
• 85% of these
cases are
NSCLC
80% of cases
occur due to
smoking
• 44 Million
smokers in
USA
If caught early,
NSCLC is highly
curative
• Only 30% of
cases are
diagnosed early
Unmet Clinical Need: Point-of-care testing to enable
minimally invasive early diagnosis of lung cancer
High rates of false positive screens for CT and chest x-ray (CXR) screens

6. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Microarrays
RT-PCR
Bead-Based
16-18h
reaction
times
Risk of
sequence
bias
RNA
Extraction
Required
Not POC
Current miRNA Diagnostics Have Limitations
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POC miRNA profiling requires a new approach
A recent study indicated vast inconsistency in
cross-platform miRNA measurements
Wan, Y.-W. et al. PLoS One 2014
Only FDA Approved miRNA cancer diagnostic
is not POC and requires >100 ng total RNA

7. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Competitive Landscape
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– Asuragen: Affy-chips screening miRNA for pancreatic cancer,
only miRNA FDA approved, large samples of total RNA (100ng)
and not POC
– Menssana BreathlinkTM: test of VOCs, not FDA approved,
scientific acceptance lacking
– Biodesix: Phase III, serum protein detection, personalized
drug choice
– Firefly Bioworks: early stage miRNA discovery platform,
not POC

8. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Hydrogel Substrates For Bioassays
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For sensitive & specific detection, gel substrates are
better than solid substrates
Levicky et al., Trends Biotech, 2005.
Commercial miRNA detection schemes often employ
solid substrates which are thermodynamically limited
Gel Detection: ~1000X higher
sensitivity than microarray
Sorokin et al., J Biomol Struct Dyn, 2006.
Srinivas et al, Analytical Chemistry 2011

9. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
microRNA Detection on Gel Particle Arrays
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SC Chapin, et al., Angewandte Chemie, Int. Ed., 2011
*Selective fluorescent labeling
of only those miRNA captured
on gel-embedded probes
Ligation Labeling Scheme:
No Target Bias
Encoded gel particles bearing graphical
bar-code and embedded with miRNA
probes
Particle Array Synthesis

10. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Limit of Detection: 20 fM
Assay Time: 4h
Direct Labeling Scheme
Sub-amol detection from 100 ng total
RNA from tissue through direct labeling
Addition of signal amplification
scheme based on amplification
of universal linker molecule
Direct detection of miR141
in raw prostate cancer
serum
Limit of Detection: 300 aM
Assay Time: 16 h
Signal Amplification
No amplification bias!
microRNA Detection on Gel Particle Arrays

11. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Designing Next-Generation Gel Assay
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Develop new scheme which provides similar sensitivity while
reducing assay time and running assay on-chip
Projection Lithography to
polymerize immobilized
hydrogel microstructures
• Same chemistries can be applied
• Covalently functionalized with biological
moeties
• Porosity-tuned to allow diffusion and
reaction of biomolecules through matrix
• Easy liquid manipulation inside a
channel for on-chip assay
Leverage aspects of hydrogels and
flow-through assays
Volume of each gel post: 100 – 500 pL

12. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Projected Milestones and Deliverables – July
1, 2012 through June 30, 2014
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Milestone Q3
2012
Q4
2012
Q1
2013
Q2
2013
Q3
2013
Q4
2013
Q1
2014
Q2
2014
gel pad synthesis
fluorescence assay on DNA
oil isolation
FCMI design review
fluorescence synthetic
miRNA detection
tissue sample
profiling
amplification scheme
for miRNA detection
FCMI Prototype Instrument

13. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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shape control
size control
composition control
Gel Posts are Robust and Versatile
Technical Results

14. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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R.L. Srinivas, S.D. Johnson, and P.S. Doyle, Analytical Chemistry 2013
Fill channel with aqueous reagents
Gel post is isolated within oil phase
Solute retention inside gel can be
explained by examining relative
hydrodynamic resistance inside
gel post (Rgel) relative to
surrounding channel (Rc)
hc = channel height
rpore = gel mesh size
Two-phase flow sweeps out
aqueous phase
Gel Posts as pL-sized Reaction Chambers

15. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Creation of size-specific
oil-isolated ompartments
in single channel
Yellow dye Oil flush Oil flushPBS flush Red dye
Load or unload same gel post with different solutes and confine
Surfactant-Free
Full Geometric and Fluidic Control
R.L. Srinivas, S.D. Johnson, and P.S. Doyle, Analytical Chemistry, 2013

16. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Post Shape Determines Water Retention
R.L. Srinivas, S.D. Johnson, and P.S. Doyle, Analytical Chemistry, 2013

17. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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FDG (substrate)
Higher Product
Concentration
Physical Confinement of Enzymatic Reactions

18. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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R.L. Srinivas, S.D. Johnson, and P.S. Doyle, Analytical Chemistry, 2013
Filed provisional patent, 2013
Molecules are physically bound to
gel post (rpost = 75 mm)
Substrate molecules are
not physically entrapped:
flush with oil to enable
retention
Key Requirement: reaction should
not start until gel compartment is
isolated within oil phase
Confined-Volume Enzymatic Assay

19. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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FDG is catalyzed into fluorescein via 2-step catalysis with first step
being rate limiting, providing natural delay in start of reaction
Posts contain high
(500 nM) biotin probe
Signal Generation in Oil Isolated Posts
R.L. Srinivas, S.D. Johnson, and P.S. Doyle, Analytical Chemistry, 2013

20. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
20R.L. Srinivas, S.D. Johnson, and P.S. Doyle, Analytical Chemistry 2013
Multiplexed reaction providing up to 57-fold increase
in sensitivity in nucleic acid detection relative to direct
labeling using SA-PE in 20 minute assay
Multiplexed Nucleic Acid Assay

21. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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PDMS is water permeable  Change chip to glass or plastic
miRNA hybridization temperature >37 ˚C
Gel posts evaporate in <20 minutes after oil isolation
Introduce ligation labeling
scheme onto gel posts
Optimize gel geometry for
robust oil isolation
Cylindrical posts sometimes
retain excess water
Moving from DNA to miRNA Detection

22. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Parameters
Pressure: 5 psi
Channel Width: 500 mm
Contact Angle of water
on gel post: 60 degrees
Simulations done using Open Foam
Optimizing Gel Post Shape Using Simulations

23. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Glass ChipsPlastic Chips (FCMI Collaboration)
Enables 1) miRNA hybridization @ 55 ˚C
2) negligible gel posts evaporation
Both materials are durable for our bioassay format
Translatability of Assay to Different Chip Chemistries

24. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Translating microRNA gel particle assay onto chip format
• Gel post polymerized and
immobilized on-chip
• Deliver solutions via
continuous flow
(no target depletion)
<4h Ultrasensitive microRNA assay

25. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Intrachannel Multiplexed miRNA Detection
(Direct Label)
Clinical Diagnosis Panels:
Typically 3-5 miRNA

26. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Platform LOD Target
Amplification?
Input
RNA(ng)
Assay
Time
Gel particles/ Firefly 20 fM No 100 - 250 3 h
Luminex beads 2 pM No 500 -
2000
3 h
Illumina bead array 20 fM Yes 100 – 200 6 h
Affymetrix Panomics 20 aM No 1 – 100 16 – 20 h
MicroArray 4 fM No 100 -1000 16 – 20 h
RT-PCR 1 aM Yes 0.1 - 500 6 h
Gel Post (Projected) 20 fM No 100 -250 3 h
Comparison to Other Systems

27. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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miR-21 is upregulated in
lung cancer tissue
Total RNA input = 1.25 ng/ml
Expression ratio (0.47) is close
with previous measurements
(0.32)
Chapin et al, Angewandte Chemie 2011
Interrogating Real Samples: Tissue RNA Extract

28. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Modified Enzymatic Amplification Scheme

29. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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10 minute amplification time: ~50X boost in net signal
Pritchard et al., Nature Reviews Genetics 2012
Projected LoD ~1fM (<5ng total RNA input required)
Close to serum profiling requirements
Enzymatic Amplification: Preliminary Data

30. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Collaborative Efforts
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• Strategizing with Spira lab (BU) and Rong
Fan (Yale) regarding miRNA panel.
• Single cell collaborations with Yaffe (MIT)
and Fan (Yale) labs.
physical isolation of
mouse lung cancer cell

31. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Interactions with the CFTCC
Fraunhofer Beta Core Prototype
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LED and
photodiode
based optics
Disposable fluid
inputs for reagents
Microfluidic
chip interface
Controls inside
instrument housing
• Microfluidic Chips
– Successfully designed and
tested thermoplastic chips in
cyclic olefin copolymer (Zeonex
690R) to avoid evaporation loss
from PDMS design
• Prototype Instrument Design
– Controls fluid, temperature,
optics to automate assay
– Current status: detailed design
finalization
– Expected completion by
6/30/14

32. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Summary
• Developed on-chip multiplexed miRNA assay with
instrument for POC applications
• Designed and implemented novel amplification scheme
for ultrasensitive detection
• Profiled low total RNA input tissue samples
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Publications:
Srinivas R.L., Johnson S.D., and Doyle P.S., Oil-Isolated Hydrogel Microstructures for Sensitive Bioassays
On Chip, Analytical Chemistry, 2013
Lee H, Srinivas R.L. and Doyle P.S., miRNA detection using POC Gel Pads, to be submitted 2014
Patents:
Provisional patent filed on gel pad synthesis and oil isolation technique