Pradeep Waduge

Nanopore-Based Measurements of Protein Size, Fluctuations, and Conformational Changes

Proteins are structurally dynamic macromolecules, and it is challenging to quantify the conformational properties of their native state in solution. Nanopores can be efficient tools to study proteins in a solution environment. In this method, an electric field induces electrophoretic and/or electro-osmotic transport of protein molecules through a nanopore slightly larger than the protein molecule. High-bandwidth ion current measurement is used to detect the transit of each protein molecule. First, our measurements reveal a correlation between the mean current blockade amplitude and the radius of gyration for each protein. Next, we find a correlation between the shape of the current signal amplitude distributions and the protein fluctuation as obtained from molecular dynamics simulations. Further, the magnitude of the structural fluctuations, as probed by experiments and simulations, correlates with the ratio of α-helix to β-sheet content. We highlight the resolution of our measurements by resolving two states of calmodulin, a canonical protein that undergoes a conformational change in response to calcium binding.

Direct and Scalable Deposition of Atomically Thin Low-Noise MoS2 Membranes on Apertures.

Molybdenum disulfide (MoS2) flakes can grow beyond the edge of an underlying substrate into a planar freestanding crystal. When the substrate edge is in the form of an aperture, reagent-limited nucleation followed by edge growth facilitate direct and selective growth of freestanding MoS2 membranes. We have found conditions under which MoS2 grows preferentially across micrometer-scale prefabricated solid-state apertures in silicon nitride membranes, resulting in sealed membranes that are one to a few atomic layers thick. We have investigated the structure and purity of our membranes by a combination of atomic-resolution transmission electron microscopy, elemental analysis, Raman spectroscopy, photoluminescence spectroscopy, and low-noise ion-current recordings through nanopores fabricated in such membranes. Finally, we demonstrate the utility of fabricated ultrathin nanopores in such membranes for single-stranded DNA translocation detection.

Distance-dependent energy transfer between CdSe/CdS quantum dots and a two-dimensional semiconductor

Atomically thin semiconductors, such as the transition metal dichalcogenides, show great potential for nanoscale photodetection, energy harvesting, and nanophotonics. Here, we investigate the efficiency of energy transfer between colloidal quantum dots with a cadmium selenide core and cadmium sulfide shell and monolayer molybdenum diselenide (MoSe2). We show that MoSe2 effectively quenches the fluorescence of quantum dots when the two materials are in contact. We then separate the MoSe2 and quantum dots by inserting variable thickness hexagonal boron nitride (h-BN) spacers and show that the efficiency at which the MoSe2 quenches fluorescence decreases as the h-BN thickness is increased. For distances d, this trend can be modeled by a 1/d4 decay, in agreement with theory and recent studies involving graphene.

Programmed Synthesis of Freestanding Graphene Nanomembrane Arrays

Freestanding graphene membranes are unique materials. The combination of atomically thin dimensions, remarkable mechanical robustness, and chemical stability make porous and non-porous graphene membranes attractive for water purification and various sensing applications. Nanopores in graphene and other 2D materials have been identified as promising devices for next-generation DNA sequencing based on readout of either transverse DNA base-gated current or through-pore ion current. While several ground breaking studies of graphene-based nanopores for DNA analysis have been reported, all methods to date require a physical transfer of the graphene from its source of production onto an aperture support. The transfer process is slow and often leads to tears in the graphene that render many devices useless for nanopore measurements. In this work, we report a novel scalable approach for site-directed fabrication of pinhole-free graphene nanomembranes. Our approach yields high quality few-layer graphene nanomembranes produced in less than a day using a few steps that do not involve transfer. We highlight the functionality of these graphene devices by measuring DNA translocation through electron-beam fabricated nanopores in such membranes.

Differential Enzyme Flexibility Probed Using Solid-State Nanopores

Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.