2014 CFTCC Annual Symposium Andrew Ellington, PhD

1. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Coupling isothermal nucleic acid amplification
to glucometers for point-of-care cancer
biomarker testing
Prof. Andrew Ellington, The University of Texas at Austin

2. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Project Description
• We have developed isothermal nucleic acid
amplification assays for the detection of melanoma-
associated nucleic acid biomarkers.
• Engineered nucleic acid transducer modules were
integrated with the amplification system to:
– allow real-time sequence-specific amplicon validation and
BRAF V600E SNP distinction.
– transmogrify amplicon accumulation into signals measurable
by common glucometers.
• We are poised to validate our assay and detection
methodology on clinical surrogates and progress
toward fabrication of user friendly point-of-care
melanoma diagnostics.
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3. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Background and Significance
• Motivation:
• Melanoma was the 6th most common cancer in the US in 2010 and its annual
incidence is rising by 3 to 7%. Survival rates correlate to early detection, which
is challenging since most diagnoses are by visual inspection and biopsy.
Access to point-of-care (POC) nucleic acid diagnostic tool would improve early
melanoma diagnostic sensitivity as well as benefit patient management.
• Meeting an emerging need:
• Current molecular cancer diagnostics are relatively expensive, complicated,
non-portable and require trained personnel for operation. Therefore, we are
developing technologies that are suitable for POC applications during primary
care where such diagnostic advances will vastly improve patient management
and outcomes.
• Our learning experience with CFTCC:
• Has enabled us to focus our technologies for handling real world samples, and
to the improve sensitivity, speed and specificity of detection. We have also
geared our efforts towards co-opting widely used and accepted commercial
glucometers for cancer biomarker testing. We believe such technology is well-
suited to POC applications.
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4. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Clinical Needs if applicable
• The 5-year survival rate for patients with early melanoma is 94% versus less than
50% for those with melanomas greater than 3 mm in thickness.
• However, the sensitivity for early malignant melanoma detection was as low as
81% in dermatologists and only 41% in primary care physicians.
• Molecular diagnostic assays for melanoma biomarkers have the potential to fill in
the critical need. However, current technologies are unsuitable for POC
applications during primary care where such diagnostic advances will vastly
improve patient management and outcomes.
• Our technology enables genetic or gene expression biomarker testing on not only
diagnostics instruments such as real-time PCR machines but also on POC-
enabled platforms such as paperfluidics and the commercial glucometer.
• POC devices are relatively cheap and require minimal user intervention making
them ideally suited to the needs in varied healthcare settings including low income
urban and rural environments as well as improving the disease management for
both the healthcare provider and the patients by making early diagnostics more
accessible and predictive.
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5. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Current Diagnostics and Treatment Plan
(State of the art)
• Early melanoma detection is challenging since most diagnoses are by
simple visual inspection and biopsy.
• The ease of operation and quick time-to-result enable implementation of our
proposed POC technology during standard office visits to the primary care
physician or the dermatologist.
• Our minimally invasive molecular diagnostic tool should significantly
improve the sensitivity and specificity of early malignant melanoma
detection during regular doctor’s visits.
• The POC device is also applicable to intraoperative tissue analysis to aid
complete excision of tumors and affected lymph nodes.
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6. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Nucleic acid processors for
cancer biomarker detection
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NA analyte
Isothermal enzymatic NA
amplicon generation
Sequence-specific
amplicon validation/SNP
distinction
Signal transduction
Fluorimeter
Paperfluidic
Colorimetric
Electro-
chemical
Glucometer
Input Device Output

7. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Projected Milestones and Deliverables – July
1, 2012 through June 30, 2014
• As originally proposed we explored non-enzymatic nucleic acid amplifier circuits
for developing low-cost cancer molecular diagnostics. We achieved several
proposed milestones such as102-fold amplification of synthetic RNA inputs with
the catalytic hairpin assembly (CHA) circuit ,106-fold amplification of nucleic acid
inputs with layered CHA circuits and execution of circuits on paperfluidic devices.
However, standalone nucleic acid circuits did not provide sufficient amplification
for testing low concentrations of cancer biomarkers.
• Therefore we modified our approach by integrating the high signal amplitude of
enzymatic isothermal nucleic acid amplification with engineered nucleic acid
strand exchange modules for real-time signal validation and transduction to
varied detection platforms such as fluorimeters and commercial hand-held
glucometers.
• Our diagnostic assays allow sequence-validated detection of as few as 20
synthetic copies of melanoma biomarkers HELLS, NRP2, and the reference β-
actin and also provide unambigous distinction of the melanoma-associated BRAF
V600E SNP from the wildtype allele.
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8. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Catalyzed hairpin assembly
Fluorimetric detection

9. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Catalyzed hairpin assembly
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Electrochemical detection Colorimetric detection

10. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
RNA analytes can be detected by CHA
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11. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Stacked CHA circuits for high signal gain

12. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
4-layer CHA cascade yields 600,000-fold
signal gain
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13. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
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Enzymatic loop-mediated isothermal
amplification (LAMP) schematic
CHA-mediated end-point transduction of LAMP

14. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
CHA improves sensitivity and specificity of LAMP
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15. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
CHA-mediated real-time signal transduction
and SNP distinction in LAMP amplicons
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Copies
2 x 107 WT
2 x 107 SNP1
2 x 107 SNP2
0 WT
0 SNP1
0 SNP2

16. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Paperfluidic detection of LAMP amplicons
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17. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Versatile one-step strand exchange (OSD)
modules for signal transduction
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Fluorescent one-step strand displacement probes (OSD)
LAMP priming sites: asymmetric loop primer (LP)

18. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Sensitive melanoma biomarker detection
using LAMP-OSD assays
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Figure 2. Real-time sequence-specific detection of LAMP amplicons using one-step
strand displacement probes. The control gene β-actin and the melanoma-associated
LAMP amplicons of NRP2 and HELLS could be detected with a LOD of 20 molecules.

19. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Real-time BRAF V600E SNP distinction by
LAMP-OSD assays
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20. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Real-time BRAF V600E SNP distinction by
LAMP-OSD assays
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21. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
LAMP amplicon detection using a glucometer
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LAMP
amplicons

22. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Amplicon-specific signals measured using
commercial glucometers
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Agarose gel (A) and glucometer (B) measurements for samples with 0, 20, 2 x
104, 2 x 106 copies of target (T) DNA, and 7 x 105 copies of non-target (NT) DNA
in presence of target (T) LAMP primers and non-target (NT) LAMP primers.

23. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
LAMP transmogrification module is robust
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24. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Collaborative Efforts
• Our BU collaborators Drs. Alani and Ryu
have been instrumental in providing us
melanoma biomarker information and
materials for development of the
diagnostic assays.
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25. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Interactions with the CFTCC
• Working with our collaborators to obtain
additional funding, to better enable
clinical translation.
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26. CFTCC 2nd Annual Science Symposium – May 2014 NIH U54-EB-015403-02
Summary
• We have developed nucleic acid processor modules for sequence-specific
signal transduction of LAMP amplicons for measurement on a diversity of
platforms including the commercial glucometer.
• We have developed sensitive and specific assays for detection of
melanoma biomarkers and distinction of melanoma-associated SNPs.
• As we stride towards device fabrication we will further validate our assay
performance on clinical surrogates. Our BU collaborators Drs. Alani and
Ryu have guided the choice of appropriate surrogates.
• Our efforts during this program have empowered us with the capacity to
rapidly develop molecular diagnostic assays for almost any target of choice.
Such capability will be of tremendous use not only for cancer testing but
also for a wide array of diagnostic needs including but not limited to
detection of established and emerging infectious diseases.
• Although it is unfortunate that CFTCC funding was not renewed, we will
endeavor to pursue support from other organizations.
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