Arvind Balijepalli

Disease Detection and Management via Single Nanopore-Based Sensors

Sensors Joseph E. Reiner,*,† Arvind Balijepalli,‡,§ Joseph W. F. Robertson,‡ Jason Campbell,‡ John Suehle,‡ and John J. Kasianowicz‡ †Department of Physics, Virginia Commonwealth University, 701 W. Grace Street, Richmond, Virginia 23284, United States ‡Physical Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8120, United States Laboratory of Computational Biology, National Heart Lung and Blood Institute, Rockville, Maryland 20852, United States

Algorithms for On-Line Monitoring of Micro Spheres in an Optical Tweezers-Based Assembly Cell

Optical 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 positions and orientations of microscale and nanoscale components in the 3-Dimensional 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 generate probable locations and orientations of components in the XY-plane. Finally, signature curves are computed and utilized to obtain component locations and orientations in 3-D space. We have tested these algorithms with silica micro-spheres as well as metallic nanowires. We believe that the algorithms described in this paper will provide the foundation for realizing automated assembly operations in optical tweezers-based assembly cells.Copyright

Temperature Sculpting in Yoctoliter Volumes

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 in 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.

Theory of Polymer-Nanopore Interactions Refined Using Molecular Dynamics Simulations

Molecular dynamics 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 described by the model: the ratio of single channel current in the presence of PEG to that in the polymers 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 to 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.

Quantifying short-lived events in multistate ionic current measurements.

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 current. We modeled the transitions of the current to individual states with an equivalent electrical circuit, which allowed us to describe the system response. This enabled the estimation of short-lived states 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 synthetic polymers 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 applied this technique to recover a known sequence of single-stranded DNA from previously published ion channel recordings, identifying discrete current states with subpicoampere resolution.

Generating Simplified Trapping Probability Models From Simulation of Optical Tweezers System

This paper presents a radial basis function based approach to generate simplified models to estimate the trapping probability in optical trapping experiments using offline simulations. The difference form of Langevins equation is used to perform physically accurate simulations of a particle under the influence of a trapping potential and is used to estimate trapping probabilities at discrete points in the parameter space. Gaussian radial basis functions combined with kd-tree based partitioning of the parameter space are then used to generate simplified models of trapping probability. We show that the proposed approach is computationally efficient in estimating the trapping probability and that the estimated probability using the simplified models is sufficiently close to the probability estimates from offline simulation data.

Analytical applications for pore-forming proteins.

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.

The effects of diffusion on an exonuclease/nanopore-based DNA sequencing engine

Over 15 years ago, the ability to electrically detect and characterize individual polynucleotides as they are driven through a single protein ion channel was suggested as a potential method for rapidly sequencing DNA, base-by-base, in a ticker tape-like fashion. More recently, a variation of this method was proposed in which a nanopore would instead detect single nucleotides cleaved sequentially by an exonuclease enzyme in close proximity to one pore entrance. We analyze the exonuclease/nanopore-based DNA sequencing engine using analytical theory and computer simulations that describe nucleotide transport. The available data and analytical results suggest that the proposed method will be limited to reading <80 bases, imposed, in part, by the short lifetime each nucleotide spends in the vicinity of the detection element within the pore and the ability to accurately discriminate between the four mononucleotides.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

A Flexible System Framework for a Nanoassembly Cell Using Optical Tweezers

The optical tweezers instrument is a unique tool for directed assembly of nanocomponents. In order to function as a viable nanomanufacturing tool, a software architecture is needed to run the optical tweezers hardware, provide an effective user interface, and allow automated operation. A flexible software system framework is described to utilize the optical tweezers hardware to its full potential. Initially we lay out the requirements for the system framework and define the broad architectural choices made while implementing the different modules. Implementation details of key system modules are then described. The flexible nature of the architecture is demonstrated by showing how a simulation module can be seamlessly included into the framework to replace the optical tweezers hardware as necessary. Finally, we show some representative assembly operations to demonstrate the capabilities of the system.Copyright