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We developed a generalized technique to characterize polymer-nanopore interactions via single channel ionic current measurements. Physical interactions between analytes, such as DNA, proteins or synthetic polymers, and a nanopore cause multiple discrete states in the ionic current. We modeled the transitions of the ionic current to individual states with an equivalent electrical circuit of the nanopore system, which allowed us to describe the system response. This enables the estimation of short-lived states in single-molecule nanopore data that are presently not characterized by existing analysis techniques. Our approach considerably improves the range and resolution of single-molecule characterization with nanopores. For example, we characterized the residence times of molecules in the nanopore that are three times shorter than those estimated with existing algorithms. Because the molecule’s residence time follows an exponential distribution, we recover nearly 20-fold more events per unit time that can be used for analysis. Furthermore, the measurement range was extended from 11 monomers to as few as 8. Finally, we apply this technique to recover a known sequence of single stranded DNA from previously published ion channel recordings, identifying discrete current states with sub-picoampere resolution.

We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5′-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2′-deoxyguanosine-5′-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

Molecular dynamics (MD) simulations were used to refine a theoretical model that describes the interaction of single polyethylene glycol (PEG) molecules with α-hemolysin (αHL) nanopores. The simulations support the underlying assumptions of the model, that PEG decreases the pore conductance by binding cations (which reduces the number of mobile ions in the pore) and by volume exclusion, and provide bounds for fits to new experimental data. Estimation of cation binding indicates that four monomers coordinate a single K+ in a crown-ether like structure, with, on average, 1.5 cations bound to a PEG 29-mer at a bulk electrolyte concentration of 4 M KCl. Additionally, PEG is more cylindrical and has a larger cross-section area in the pore than in solution, although its volume is similar. Two key experimental quantities of PEG are quantitatively described by the model: the ratio of single channel current in the presence of PEG to that in the polymer’s absence (blockade depth), and the mean residence time of PEG in the pore. The refined theoretical model is simultaneously fit to the experimentally determined current blockade depth and the mean residence times for PEGs with 15 – 45 monomers, at applied transmembrane potentials of -40 to -80 mV, and for three electrolyte concentrations. The model estimates the free energy of the PEG-cation complexes to be -5.3 kBT. Finally the entropic penalty of confining PEG to the pore is found to be inversely proportional to the electrolyte concentration.

Biological and solid-state nanometer-scale pores are the basis for numerous emerging analytical technologies for use in precision medicine. We developed Modular Single-Molecule Analysis Interface (MOSAIC), an open source analysis software that improves the accuracy and throughput of nanopore-based measurements. Two key algorithms are implemented: ADEPT, which uses a physical model of the nanopore system to characterize short-lived events that do not reach their steady-state current, and CUSUM+, a version of the cumulative sum statistical method optimized for longer events that do. We show that ADEPT detects previously unreported conductance states that occur as double-stranded DNA translocates through a 2.4 nm solid-state nanopore and reveals new interactions between short single-stranded DNA and the vestibule of a biological pore. These findings demonstrate the utility of MOSAIC and the ADEPT algorithm, and offer a new tool that can improve the analysis of nanopore-based measurements.

The ability to perturb large ensembles of molecules from equilibrium led to major advances in understanding reaction mechanisms in chemistry and biology. Here, we demonstrate the ability to control, measure, and make use of rapid temperature changes of fluid volumes that are commensurate with the size of single molecules. The method is based on attaching gold nanoparticles to a single nanometer-scale pore formed by a protein ion channel. Visible laser light incident on the nanoparticles causes a rapid and large increase of the adjacent solution temperature, which is estimated from the change in the nanopore ionic conductance. The temperature shift also affects the ability of individual molecules to enter into and interact with the nanopore. This technique could significantly improve sensor systems and force measurements based on single nanopores, thereby enabling a method for single molecule thermodynamics and kinetics.

Proteinaceous nanometer-scale pores are ubiquitous in biology. The canonical ionic channels (e.g., those that transport Na(+), K(+), Ca(2+), and Cl(-) across cell membranes) play key roles in many cellular processes, including nerve and muscle activity. Another class of channels includes bacterial pore-forming toxins, which disrupt cell function, and can lead to cell death. We describe here the recent development of these toxins for a wide range of biological sensing applications. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

for on-line monitoring of micro-spheres in an optical tweezers-based assembly cell. ASME Journal of Computing and Information Science in Engineering, 7(4):330-338, 2007. Readers are encouraged to get the official version from the journal's web site or by contacting Dr. S.K. Gupta (skgupta@umd.edu). AbstractOptical tweezers have emerged as a powerful tool for micro and nanomanipulation. Using optical tweezers to perform automated assembly requires on-line monitoring of components in the assembly workspace. This paper presents algorithms for estimating 3-dimensional positions of micro-spheres in the assembly workspace. Algorithms presented in this paper use images obtained by optical section microscopy. The images are first segmented to locate areas of interest and then image gradient information from the areas of interest is used to locate the positions of individual micro-spheres in the XY-plane. Finally, signature curves are computed and utilized to obtain the Z-locations of spheres. We have tested these algorithms with glass micro-spheres of two different sizes under different illumination conditions. Our experiments indicate that the algorithms described in this paper provide sufficient computational speed and accuracy to support the operation of optical tweezers.