Abstract
The rapid developments in nanotechnology have led to exciting progress in the investigation of biological events at the single-molecule level. When studying systems in which molecular individuality matters, single-molecule experiments offer several important advantages over ensemble measurements. First, single-molecule measurements provide important information that is “averaged out” in ensemble results. Second, by conducting many sequential measurements, they allow one to determine the distribution of molecular properties and investigate the inhomogeneous systems. Finally, they permit observation of rarely populated transients that are difficult or impossible to capture using bulk measurements. Traditional single-molecule detection techniques can be roughly grouped into two categories: applied optical spectroscopies, which provide optical and spectroscopic information, and scanning probe microscopies, which yield force and electrical current data. High-speed and reliable genome sequencing is one of the grand challenges in the 21st century. Recently, a number of groups have started to use nanopores for rapid detection of single DNA molecules and their sequences. Two types of nanopores have been used for this purpose: nanopores embedded in an insulating membrane, for example, biological nanopores such as a-hemolysin protein nanopores in lipid membranes; or solid-state nanopores in Si3N4 [9,10] and SiO2. [11,12] While most of the research has focused on double-stranded DNA (dsDNA) translocations, rapid DNA sequencing with nanopores requires single-stranded DNA (ssDNA) molecules to pass through the nanopores and be detected with single-base resolution. Solid-state nanopores possess advantageous features for robust DNA detection and sequencing. They are chemically, thermally, and mechanically stable, which makes it possible to denature DNA under conditions of high pH and/or increased temperature. It is also possible to incorporate solid-state nanopores with local electrical and optical probes to form an integrated circuit. In a recent paper, Li and co-workers demonstrated an exciting experimental investigation for detecting ssDNA using voltage-biased solid-state Si3N4 nanopores. A single nanopore was fabricated using focused ion-beam (FIB) milling followed by feedback-controlled ion-beam sculpting in an insulating Si3N4 membrane. [9] The membrane was then immersed in an ionic solution, which was then divided into two isolated reservoirs with a Ag/AgCl electrode inserted in each of the two reservoirs. By applying a bias voltage over the two electrodes, an electric field was established in the area between the electrodes, ideally, in the nanopore and near it. When DNA molecules were introduced to the reservoir with the negative electrode they diffused toward the nanopore and were captured by the local electric field near it. The electric field within the nanopore then forced the DNA molecules to pass through the pore and translocate into the positive electrode reservoir. The ionic current through the nanopore during the translocation event was monitored to study the history of the molecule=s interaction with the nanopore (Figure 1A). In the design by Li and co-workers, they studied both single dsDNA and ssDNA molecule translocations by performing the experiments under different pH values at room temperature. Figure 1B and C are two-dimensional (translocation time and mean ionic current blockage) histograms of the translocation event density at pH 7 (dsDNA, Figure 1B) and pH 13 (ssDNA, Figure 1C) environments. The DNA molecules used in the experiments are the same length (3kilobase long) and the bias voltage applied was fixed at 120 mV. At pH 7, as Figure 1B shows, a well-defined peak reveals a translocation time of about 170 ms and a current [*] Prof. H. Yan Department of Chemistry and Biochemistry and The Biodesign Institute Arizona State University, Tempe, AZ 85287 (USA) Fax: (+1)480-965-2747 E-mail: hao.yan@asu.edu
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.