Single Molecule Mass Spectrometry Using a Single Nanopore Dr. John J. Kasianowicz Colleagues: NIST: J. Robertson, V. Stanford Brazil: O. Krasilnikov, C. Rodrigues

NIST Nanobiotechnology Project 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,... 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). DNA Proteins Cell-signaling agents Targets

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 NIST research has shown that these nanopore structures have the potential for use in quantitative biosensing applications Nerve activity Cell-cell communication Membrane transport Toxins... Anthrax nanopore Staph nanopore from nerve

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., Proc. Nat’l Acad. Sci. (USA); Kasianowicz, JJ., S.E. Henrickson, H.H. Weetall, & B. Robertson., Analytical Chemistry; Kasianowicz, J.J., J.W.F. Robertson, E.R. Chan, J. E. Reiner, & V.M Stanford Ann. Rev. Anal. Chem.

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 red: polydisperse blue: monodisperse 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! Amount of ionic current blocked scales with the polymer size Robertson,J.W.F., C.G. Rodrigues, V.M Stanford, K.A. Rubinson, O.V. Krasilnkov, & J.J. Kasianowicz 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 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, J. Biol. Chem. 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 EF