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Dive into the research topics where Kevin J. Freedman is active.

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Featured researches published by Kevin J. Freedman.


Scientific Reports | 2013

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field

Kevin J. Freedman; S. Raza Haq; Joshua B. Edel; Per Jemth; Min Jun Kim

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106 V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.


Analytical Chemistry | 2011

Chemical, thermal, and electric field induced unfolding of single protein molecules studied using nanopores.

Kevin J. Freedman; Maike Jürgens; Anmiv S. Prabhu; Chi Won Ahn; Per Jemth; Joshua B. Edel; Min Jun Kim

Single-molecule experimental techniques have recently shown to be of significant interest for use in numerous applications in both the research laboratory and industrial settings. Although many single-molecule techniques exist, the nanopore platform is perhaps one of the more popular techniques due to its ability to act as a molecular sensor of biological macromolecules. For example, nanopores offer a unique, new method for probing various properties of proteins and can contribute to elucidating key biophysical information in conjunction with existing techniques. In the present study, various forms of bovine serum albumin (BSA) are detected including thermally refolded BSA, urea-denatured BSA, and multiple forms of BSA detected at elevated electric field strengths (with and without urea). We also provide excluded volume measurements for each of these states that normally are difficult to obtain due to unknown and unstable protein conformations.


Analytical Chemistry | 2013

Single-molecule studies of intrinsically disordered proteins using solid-state nanopores.

Deanpen Japrung; Jakob Dogan; Kevin J. Freedman; Achim Nadzeyka; Sven Bauerdick; Tim Albrecht; Min Jun Kim; Per Jemth; Joshua B. Edel

Partially or fully disordered proteins are instrumental for signal-transduction pathways; however, many mechanistic aspects of these proteins are not well-understood. For example, the number and nature of intermediate states along the binding pathway is still a topic of intense debate. To shed light on the conformational heterogeneity of disordered protein domains and their complexes, we performed single-molecule experiments by translocating disordered proteins through a nanopore embedded within a thin dielectric membrane. This platform allows for single-molecule statistics to be generated without the need of fluorescent labels or other modification groups. These studies were performed on two different intrinsically disordered protein domains, a binding domain from activator of thyroid hormone and retinoid receptors (ACTR) and the nuclear coactivator binding domain of CREB-binding protein (NCBD), along with their bimolecular complex. Our results demonstrate that both ACTR and NCBD populate distinct conformations upon translocation through the nanopore. The folded complex of the two disordered domains, on the other hand, translocated as one conformation. Somewhat surprisingly, we found that NCBD undergoes a charge reversal under high salt concentrations. This was verified by both translocation statistics as well as by measuring the ζ-potential. Electrostatic interactions have been previously suggested to play a key role in the association of intrinsically disordered proteins, and the observed behavior adds further complexity to their binding reactions.


Nature Communications | 2016

Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping.

Kevin J. Freedman; Lauren M. Otto; Aleksandar P. Ivanov; Avijit Barik; Sang Hyun Oh; Joshua B. Edel

Single-molecule techniques are being developed with the exciting prospect of revolutionizing the healthcare industry by generating vast amounts of genetic and proteomic data. One exceptionally promising route is in the use of nanopore sensors. However, a well-known complexity is that detection and capture is predominantly diffusion limited. This problem is compounded when taking into account the capture volume of a nanopore, typically 108–1010 times smaller than the sample volume. To rectify this disproportionate ratio, we demonstrate a simple, yet powerful, method based on coupling single-molecule dielectrophoretic trapping to nanopore sensing. We show that DNA can be captured from a controllable, but typically much larger, volume and concentrated at the tip of a metallic nanopore. This enables the detection of single molecules at concentrations as low as 5 fM, which is approximately a 103 reduction in the limit of detection compared with existing methods, while still maintaining efficient throughput.


Journal of Physics: Condensed Matter | 2010

Chemically modified solid state nanopores for high throughput nanoparticle separation

Anmiv S. Prabhu; Talukder Z. Jubery; Kevin J. Freedman; Rafael Mulero; Prashanta Dutta; Min Jun Kim

The separation of biomolecules and other nanoparticles is a vital step in several analytical and diagnostic techniques. Towards this end we present a solid state nanopore-based set-up as an efficient separation platform. The translocation of charged particles through a nanopore was first modeled mathematically using the multi-ion model and the surface charge density of the nanopore membrane was identified as a critical parameter that determines the selectivity of the membrane and the throughput of the separation process. Drawing from these simulations a single 150 nm pore was fabricated in a 50 nm thick free-standing silicon nitride membrane by focused-ion-beam milling and was chemically modified with (3-aminopropyl)triethoxysilane to change its surface charge density. This chemically modified membrane was then used to separate 22 and 58 nm polystyrene nanoparticles in solution. Once optimized, this approach can readily be scaled up to nanopore arrays which would function as a key component of next-generation nanosieving systems.


Journal of Laboratory Automation | 2010

Nanopore-Based Devices for Bioanalytical Applications

Rafael Mulero; Anmiv S. Prabhu; Kevin J. Freedman; Min Jun Kim

With over a decade passed since the first reported use of a Staphylococcal α-hemolysin pore to study single molecules of single-stranded DNA, research in the field of nanopores has advanced rapidly. We discuss the technological progression of nanopore-based devices from the initial use of α-hemolysin pores to the advent of solid-state nanopores to the burgeoning of organic-inorganic hybrid pores driven by the desire to achieve fast and inexpensive DNA sequencing. Additional nanopore-based efforts are also discussed that study other classes of molecules, such as proteins. We discuss the use of nanopores for protein folding and binding analysis. In addition to single-molecule analysis, we report on the introduction of nanopore arrays on thin film membranes for ultrafiltration. Owing to their reduced spatial dimensionality, such membranes offer greater control over how the pores interact with analytes thus leading to very efficient separation. With several technical hindrances yet to be overcome, the devices we report are still works in progress. The realization of these devices will enhance laboratory processes by permitting superior spatial and temporal analytical resolution at the single-molecule level resulting in laboratory capacities of great impact.


ACS Nano | 2013

Detection of long and short DNA using nanopores with graphitic polyhedral edges.

Kevin J. Freedman; Chi Won Ahn; Min Jun Kim

Graphene is a unique material with a thickness as low as a single atom, high in-plane conductivity and a robust lattice that is self-supporting over large length scales. Schematically, graphene is an ideal solid-state material for tuning the properties of a nanopore because self-supported sheets, ranging from single to multiple atomic layers, can create pores with near-arbitrary dimensions which can provide exquisite control of the electric field drop within the pore. In this study, we characterize the drilling kinetics of nanopores using a thermionic electron source and various electron beam fluxes to minimize secondary hole formation. Once established, we investigated the use of multilayer graphene to create highly tailored nanostructures including nanopores with graphite polyhedral crystals formed around the nanopore edge. Finally, we report on the translocation of double stranded and single stranded DNA through such graphene pores and show that the single stranded DNA translocates much slower allowing detection of extremely short fragments (25 nucleotides in length). Our findings suggest that the kinetic and controllable properties of graphene nanopores under sculpting conditions can be used to further enhance the detection of DNA analytes.


Analytical Chemistry | 2013

Gold Nanoparticle Translocation Dynamics and Electrical Detection of Single Particle Diffusion Using Solid-State Nanopores

Gaurav Goyal; Kevin J. Freedman; Min Jun Kim

This paper describes the use of gold nanoparticles to study particle translocation dynamics through silicon nitride solid-state nanopores. Gold nanoparticles were dispersed in 20 mM KCl solution containing nonionic surfactant Triton X-100 and their translocation was studied at different applied voltages. The use of low electrolyte concentration resulted in current enhancement upon particle translocation. The counterion cloud around the nanoparticles is proposed to be the reason for current enhancement phenomena because associated counterion cloud is believed to increase the ion density inside the pore during particle translocation. Further, single particle diffusion events were also recorded at 0 mV voltage bias and 0 pA background ionic current with high signal-to-noise ratio as the particles moved down their concentration gradient. The ability of nanopore sensors to detect single particle diffusion can be extended to field-free analysis of biomolecules in their native state and at or near physiological salt concentrations.


ACS Nano | 2014

High Precision Fabrication and Positioning of Nanoelectrodes in a Nanopore

Aleksandar P. Ivanov; Kevin J. Freedman; Min Jun Kim; Tim Albrecht; Joshua B. Edel

A simple and versatile method for the direct fabrication of tunneling electrodes with controllable gap distance by using electron-beam-induced deposition (EBID) is presented. We show that tunneling nanogaps smaller than the minimum feature size realizable by conventional EBID can be achieved with a standard scanning electron microscope. These gaps can easily be embedded in nanopores with high accuracy. The controllability of this fabrication method and the nanogap geometry was verified by SEM and TEM imaging. Furthermore, tunneling spectroscopy in a group of solvents with different barrier heights was used to determine the nanogap functionality. Ultimately, the presented fabrication method can be further applied for the fabrication of arrays of nanogap/nanopores or nanogap electrodes with tunable electrode materials. Additionally, this method can also offer direct fabrication of nanoscale electrode systems with tunable spacing for redox cycling and plasmonic applications, which represents an important step in the development of tunneling nanopore structures and in enhancing the capabilities of nanopore sensors.


Nanotechnology | 2011

SEM-induced shrinking of solid-state nanopores for single molecule detection

Anmiv S. Prabhu; Kevin J. Freedman; Joseph W. F. Robertson; Zhorro Nikolov; John J. Kasianowicz; Min Jun Kim

We have investigated the mechanism by which the diameter of solid-state nanopores is reduced by a scanning electron microscope. The process depends on beam parameters such as the accelerating voltage and electron flux and does not involve simple electron-beam-induced deposition of hydrocarbon contaminants. Instead, it is an energy-dependent process that involves material flow along the surface of the nanopore membrane. We also show that pores fabricated in this manner can detect double stranded DNA.

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Min Jun Kim

Southern Methodist University

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