Single Molecule Mass Spectrometry Using a Single Nanopore

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Single Molecule Mass Spectrometry Using a Single
Nanopore

• Dr. John J. Kasianowicz
• Colleagues
• NIST J. Robertson, V. Stanford
• Brazil O. Krasilnikov, C. Rodrigues

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NIST Nanobiotechnology Project
Targets

• Goals
• real-time electronic detection, identification,
  quantification of DNA, proteins other
  biomolecules
• potential impact early cancer detection, other
  health care measurement needs, Systems Biology,
  Homeland Security, ...

DNA
Proteins
Actual devices in the near term will be based on
measurements of molecules (e.g., cell signaling
agents, proteins, nucleic acids, metabolites) in
cytoplasm and in blood (e.g., like glucose
sensors).
Cell-signaling agents
Star Trek Tricorder Hollywoods rendition of
future Biomedical instrumentation.
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Why Electronics for Future Bio-Measurement Needs?

• Electronic measurements revolutionized biological
  research twice in the past 70 years
• 1940s-1950s Crucial for understanding how
  nerves work
• 1970s Single biological molecule measurements
• The need to obtain more detailed quantitative
  information about many small molecules
  simultaneously requires an approach that uses
  nano- micro-fabrication, systems integration,
  rapid measurement i.e., the realm of electronics.

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Biological Roles of Pore-Forming Proteins
Nerve activity Cell-cell communication Membrane
transport Toxins ...
Anthrax nanopore
Staph nanopore
from nerve
NIST research has shown that these nanopore
structures have the potential for use in
quantitative biosensing applications
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Electronic Detection of Single DNA Molecules with
a Nanopore

• Single DNA molecules can be driven through a
  nanopore by voltage ...

... different DNA molecule types cause different
signals
Can we electronically sequence DNA base by base?
Kasianowicz, et al., 1996. Proc. Natl Acad. Sci.
(USA) Kasianowicz, JJ., S.E. Henrickson, H.H.
Weetall, B. Robertson., 2001. Analytical
Chemistry Kasianowicz, J.J., J.W.F. Robertson,
E.R. Chan, J. E. Reiner, V.M Stanford. 2008.
Ann. Rev. Anal. Chem.
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Single Molecule Mass Spectrometry in Aqueous
Solution Using a Nanopore

• Neutral polymers can also enter leave a nanopore

Each peak in the mean blockade current
corresponds to a particular size polymer
Amount of ionic current blocked scales with the
polymer size
The method can easily resolve ALL the molecules
in the polydisperse sample and easily
distinguishes between polymers that differ by
only 0.6 nm in length!
Robertson,J.W.F., C.G. Rodrigues, V.M Stanford,
K.A. Rubinson, O.V. Krasilnkov, J.J.
Kasianowicz. 2007. Proc. Natl Acad. Sci.
(USA) Patent application pending.
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Opportunities

• Rapid/inexpensive genomic sequencing mass
  spectrometry measurements could help catalogue
  ALL life on the planet (impacts drug
  development/discovery ecology)
• New tools to electronically detect identify
  molecules could also be used to study the origins
  of life (e.g., instrumentation for interplanetary
  exploration exobiology)

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Other Electronic Biosensing Opportunities

• Electronic Detection of Anthrax Toxins

EF

• The binding of Anthrax Edema Factor (EF) to the
  Anthrax nanopore changes the pores electronic
  signature
• The measurement is rapid (2 seconds vs. many
  hours for cell-based assays)
• This technique has been shown to provide the
  basis for High-Throughput Screening of
  therapeutic agents against Anthax toxins

NIST Ft. Detrick (USAMRIID NCI)
collaboration Halverson, K.M, R.G. Panchal,
T.L. Nguyen, R. Gussio, S.F. Little, M. Misakian,
S. Bavari, J.J. Kasianowicz, 2005. J. Biol.
Chem.