We are applying the unique capabilities of our recently developed sub-nanometer precision transmission electron beam ablation lithography (TEBAL) to demonstrate that the precise integration of solid-state nanopores with nanoelectrodes, nanochannels and microfluidics will address key obstacles that must be overcome to achieve nanopore-based low-cost high-speed single molecule analysis of DNA and proteins.
Nanopore-based analysis is a single-molecule technique that promises to carry out a range of analyses orders of magnitude faster and more economically than current methods, including length measurement, specific sequence detection, single-molecule dynamics and ultimately de novo sequencing. As shown to the right, the concept involves using an applied voltage to drive charged molecules, such as DNA or proteins, through a narrow pore which separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the pore they block the flow of ions, and thus their structure and length can be determined based on the degree and duration of the resulting current reductions.
For further background information, see:
Benefits of Nanoelectrodes
Sensing across the nanopore aperture instead of between macroscopic electrodes has several advantages. The thickness of the membrane containing the nanopore is no longer a limitation – a certain number of monomers are contained within the nanopore at any one time, so the measured current will be an average due to each one. With nanoelectrodes, there will be a similar effect related to the electrode thickness, but we can make the electrodes thinner than the membrane. Also, in the traditional nanopore-based analysis system described above, the capacitance between the macroscopic electrodes is large due to the very thin membrane. This has meant that only DC excitation signals could be used, otherwise transient current through the capacitance would swamp the measurement long enough for the molecule to exit the pore. The nanoelectrode configuration addresses this with far lower capacitance. Finally, with nanoelectrodes, the path length between the electrodes is reduced by several orders of magnitude, which will eliminate any solution related noise sources such as convection.
Due to their small size, nanoelectrodes are also capable of exerting forces large enough to manipulate individual molecules. Currently, nanopore-based analysis resolution is limited by the high speed at which molecules pass through the pore. Reducing the driving force does not help because the molecules experience unchanged perturbing forces due to Brownian fluctuations. We are working to slow down molecules, while also reducing the impact of perturbations, by applying opposing force via nanoelectrodes.
Nanopore and Nanoelectrode Fabrication
We fabricate nanopores in silicon nitride membranes using the well-established method of drilling with the focused electron beam in a field-emission transmission electron microscope (TEM).
Nanoelectrodes are created by first using a process of electron beam lithography (EBL), metal evaporation and lift-off to define bond pads, nanowires and interconnecting traces. Subsequently, using the focused electron in the TEM, material is then selectively ablated from the nanowires with sub-nanometer precision to form the desired shapes. See our research on nanofabrication and TEBAL for further information.
Integration with nano- and microfluidics
In traditional nanopore-based analysis experiments, billions of copies of the molecule to be analyzed are required to achieve an acceptable frequency of molecules passing through the pore, because molecules are only captured when they diffuse close to the pore. (Typical rates are on the order of 1 per second). This is clearly a disadvantage for practical applications of nanopore-based analysis, because significant time and cost is required to amplify the analyte molecules to the required concentration.
We are working to address this problem by integrating nanopores and nanoelectrodes into PDMS and SU-8 micro- and nanofluidic devices. This will enable molecules to be actively transported to the nanopore for analysis, eliminating the need for high concentrations.
NIH NHGRI August 2008 press release announcing funding grants for revolutionary DNA sequencing technologies, of which we are one of the recipients.