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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
Targets
NIST Nanobiotechnology Project
•Goals:
DNA
– 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, ...
Proteins
Cell-signaling agents
Star Trek Tricorder: Hollywood’s
rendition of future Biomedical
instrumentation.
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).
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.
Biological Roles of Pore-Forming
Proteins
Nerve activity
Cell-cell communication
Membrane transport
Toxins
...
from nerve
Staph
nanopore
Anthrax
nanopore
NIST research has shown that these nanopore
structures have the potential for use in quantitative
biosensing applications
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. Nat’l 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.
Single Molecule Mass Spectrometry in
Aqueous Solution Using a Nanopore
•Neutral polymers can also enter & leave a nanopore
red: polydisperse
blue: monodisperse
Amount of ionic current blocked
scales with the polymer size
Each peak in the mean blockade current
corresponds to a particular size polymer
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. Nat’l Acad. Sci. (USA)
Patent application pending.
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)
Other Electronic Biosensing
Opportunities
–Electronic Detection of Anthrax Toxins
EF
•
The binding of Anthrax Edema Factor (EF) to the Anthrax nanopore changes
the pore’s 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.