2014 CFTCC Annual Symposium Poster Session http://www.bu.edu/cftcc/multi-media/2014cftccsymposiummedia/

1. Capture and Release Nanopore-Nanofiber Mesh:
pH Triggered Nucleic Acid Detection
Joseph S. Hersey, Allison Squires, Amit Meller, and Mark W. Grinstaff
Departments of Biomedical Engineering, Physics, and Chemistry, Boston University, Boston Massachusetts
Introduction NFM Fabrication Concept: Capture and Release NP-NFM
Nanopore Concept: Sub 10 nm diameter solid-state nanopores (NP) detect
single molecules of nucleic acids by electrophoretically drawing these highly
anionic biopolymers through the NP and measuring the translocation event
as a transient drop in current.1
Nanopore sensitivity:
• NPs detect DNA at the single molecule level (detect attomoles of DNA).1
• However, NPs struggle to distinguish an unmodified target sequence
from other sequences within a complex sample.2
Nanopore-Nanofiber mesh: We have recently developed a novel NP
modification by electrospinning a polymeric nanofiber mesh (NFM) onto the
surface of a NP chip to affect the analyte translocation dynamics.3
Nanofiber mesh specificity:
• Chemically modified NFMs can selectively capture target molecules.
Sensitive + specific DNA detection: A proof-of-concept NP-NFM has been
created to demonstrate the selective capture of thiol-functionalized double-
stranded DNA (dsDNA) and pH induced release of the complementary non-
functionalized single-stranded DNA (ssDNA) into a sensitive DNA detector.
Figure 1. Schematic cross-section of a solid-state nanopore with a
nanofiber mesh (orange) coating. Target DNA (blue) is captured
and detected while non-specific DNA (red) is removed.
Fabrication steps
• Synthesize a poly(glycerol-co-ε-caprolactone) (PGC-OH) polymer4
• Functionalize PGC-OH with a PEG-Maleimide side chain
• Fabricate solid-state nanopore devices5
• Electrospin NFM onto NP chips in parallel (up to 50 devices per
minute)3
• Characterize NFM morphology using SEM
Goal: Design a NP-NFM that specifically binds a target nucleic
acid sequence. Non-specifically adsorbed nucleic acids will be
washed from the surface before a release mechanism is used to
liberate the target nucleic acid for subsequent single-molecule
detection with a nanopore. This device will provide a label-free
and amplification-free DNA diagnostic tool.
Polymer: PGC-PEG-Maleimide
Capture Mechanism: Maleimide-Thiol reaction
Release Mechanism: pH 11 wash melts dsDNA to ssDNA
ssDNA detection on NP:

2. Capture and Release Nanopore-Nanofiber Mesh:
pH Triggered Nucleic Acid Detection
Joseph S. Hersey, Allison Squires, Amit Meller, and Mark W. Grinstaff
Departments of Biomedical Engineering, Physics, and Chemistry, Boston University, Boston Massachusetts
Proof of Concept: SYBR I Detection Capture and Release NP-NFM Capture and Release NP-NFM: Results
Experimental Design:
• Create 500 bp dsDNA with or without a thiol modification on
one of the strands.
• Electrospin maleimide functionalized NP-NFM.
• Apply 4 µL drop of 50 nM dsDNA onto the NFM, wait 18 hours,
and image on a gel scanner (excitation 497 nm, emission 520
nm).
• Thiol-functionalized DNA that covalently binds to the NP-NFM
will not wash away at pH 7 but non-specifically bound DNA will.
• A pH 11 wash melts the dsDNA releasing ssDNA causing the
SYBR I fluorescence to decrease.
Sybr I Fluorescence
Low High
Experimental Design:
• Use the same protocol as the Sybr I experiments except use 2.5
nM dsDNA and wait only 30 minutes.
• Apply a voltage (500 mV) across the nanopore and detect DNA
as transient drops in current after each wash step.
• Wash 1: Remove any unbound material using a pH 7 wash.
Wash 2: Confirm unbound DNA was removed. Wash 3: pH 11
buffer melts any remain DNA releasing ssDNA into the
nanopore.
• dsDNA only:
• No dsDNA is captured onto the mesh.
• All of the dsDNA is removed during pH 7 wash 1.
• Thiol-dsDNA:
• No dsDNA is observed in either pH 7 wash 1 or 2 indicating
the DNA has been captured.
• The pH 11 wash shows signs of translocations; however, this
wash also causes the pore to block.
Conclusions and Future Work
A proof of concept capture and release NP-NFM was developed
• Demonstrated the ability to selectively binding a model thiol
functionalized dsDNA
• Confirmed DNA binding to the NP-NFM optically
• Utilized a nanopore system to detect pico-molar double-
stranded and single-stranded DNA after each wash step.
• pH 11 release washes likely cause the polymer to degrade
resulting in nanopore blockage. However, the blockage profile
contains translocation events and blockage occurs more
quickly if DNA is captured onto the NP-NFM
Future work:
• Develop a new release mechanism to avoid blockages
Literature Cited
1. Wanunu, M. Phys. Life Rev. 2012, 9, 125.
2. Branton, D.; Deamer, D.; et al. Nat. Biotechnol. 2008, 26, 1146.
3. Squires, A. H.; Hersey, J. S.; Grinstaff, M. W.; Meller, A. J. Am. Chem. Soc. 2013, 135,
16304.
4. Wolinsky, J. B.; Ray, W. C.; et al. Macromolecules 2007, 40, 7065.
5. Kim, M. J.; Wanunu, M.; Bell, D. C.; Meller, A. Adv. Mater. 2006, 18, 3149.
Acknowledgements
The authors gratefully acknowledge the National Institutes of Health for funding support
of this project (R21 EB017377) and the Boston University Division of Materials Science
and Engineering.
For more information visit: http://people.bu.edu/mgrin