Harsh Bais, PhD



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  • Krishna Jayant, Jan J. Hirtz, Ilan Jen-La Plante, David M. Tsai, Wieteke D. A. M. De Boer, Alexa Semonche, Darcy S. Peterka, Jonathan S. Owen, Ozgur Sahin, Kenneth L. Shepard and Rafael Yuste. Targeted intracellular voltage recordings from dendritic spines using quantum-dot-coated nanopipettes Nature Nanotechnology, published online DOI: 10.1038/NNANO.2016.268

    Dendritic spines are the primary site of excitatory synaptic input onto neurons, and are biochemically isolated from the parent dendritic shaft by their thin neck. However, due to the lack of direct electrical recordings from spines, the influence that the neck resistance has on synaptic transmission, and the extent to which spines compartmentalize voltage, specifically excitatory postsynaptic potentials, albeit critical, remains controversial. Here, we use quantum-dot-coated nanopipette electrodes (tip diameters ∼15–30 nm) to establish the first intracellular recordings from targeted spine heads under two-photon visualization. Using simultaneous somato-spine electrical recordings, we find that back propagating action potentials fully invade spines, that excitatory postsynaptic potentials are large in the spine head (mean 26 mV) but are strongly attenuated at the soma (0.5–1 mV) and that the estimated neck resistance (mean 420 MΩ) is large enough to generate significant voltage compartmentalization. Nanopipettes can thus be used to electrically probe biological nanostructures.

  • Siddharth Shekar, David J. Niedzwiecki, Chen-Chi Chien, Peijie Ong, Daniel A. Fleischer, Jianxun Lin, Jacob K. Rosenstein, Marija Drndić, Kenneth L. Shepard. Measurement of DNA Translocation Dynamics in a Solid-State Nanopore at 100 ns Temporal Resolution NanoLetters DOI: 10.1021/acs.nanolett.6b01661

    Despite the potential for nanopores to be a platform for high-bandwidth study of single-molecule systems, ionic current measurements through nanopores have been limited in their temporal resolution by noise arising from poorly optimized measurement electronics and large parasitic capacitances in the nanopore membranes. Here, we present a complementary metal-oxide-semiconductor (CMOS) nanopore (CNP) amplifier capable of low noise recordings at an unprecedented 10 MHz bandwidth. When integrated with state-of-the-art solid-state nanopores in silicon nitride membranes, we achieve an SNR of greater than 10 for ssDNA translocations at a measurement bandwidth of 5 MHz, which represents the fastest ion current recordings through nanopores reported to date. We observe transient features in ssDNA translocation events that are as short as 200 ns, which are hidden even at bandwidths as high as 1 MHz. These features offer further insights into the translocation kinetics of molecules entering and exiting the pore. This platform highlights the advantages of high-bandwidth translocation measurements made possible by integrating nanopores and custom-designed electronics.

  • Delphine Bouilly, Jason Hon, Nathan S. Daly, Scott Trocchia, Sefi Vernick, Jaeeun Yu, Steven Warren, Ying Wu, Ruben L. Gonzalez, Jr., Kenneth L. Shepard, and Colin Nuckolls Single-Molecule Reaction Chemistry in Patterned Nanowells NanoLetters DOI: 10.1021/acs.nanolett.6b01657

    A new approach to synthetic chemistry is performed in ultraminiaturized, nanofabricated reaction chambers. Using lithographically defined nanowells, we achieve single-point covalent chemistry on hundreds of individual carbon nanotube transistors, providing robust statistics and unprecedented spatial resolution in adduct position. Each device acts as a sensor to detect, in real-time and through quantized changes in conductance, single-point functionalization of the nanotube as well as consecutive chemical reactions, molecular interactions, and molecular conformational changes occurring on the resulting single-molecule probe. In particular, we use a set of sequential bioconjugation reactions to tether a single-strand of DNA to the device and record its repeated, reversible folding into a G-quadruplex structure. The stable covalent tether allows us to measure the same molecule in different solutions, revealing the characteristic increased stability of the G-quadruplex structure in the presence of potassium ions (K+) versus sodium ions (Na+). Nanowell-confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time.

  • Tarun Chari, Rebeca Ribeiro-Palau, Cory R. Dean, and Kenneth Shepard Resistivity of Rotated Graphite−Graphene Contacts NanoLetters DOI: 10.1021/acs.nanolett.6b01657

    Robust electrical contact of bulk conductors to two-dimensional (2D) material, such as graphene, is critical to the use of these 2D materials in practical electronic devices. Typical metallic contacts to graphene, whether edge or areal, yield a resistivity of no better than 100 Ω μm but are typically >10 kΩ μm. In this Letter, we employ single-crystal graphite for the bulk contact to graphene instead of conventional metals. The graphite contacts exhibit a transfer length up to four-times longer than in conventional metallic contacts. Furthermore, we are able to drive the contact resistivity to as little as 6.6 Ω μm2 by tuning the relative orientation of the graphite and graphene crystals. We find that the contact resistivity exhibits a 60° periodicity corresponding to crystal symmetry with additional sharp decreases around 22° and 39°, which are among the commensurate angles of twisted bilayer graphene.

  • Hassan Sakhtah, Leslie Koyama, Yihan Zhang, Diana K. Moralesa, Blanche L. Fields, Alexa Price-Whelan, Deborah A. Hogan, Kenneth Shepard, and Lars E. P. Dietricha The Pseudomonas aeruginosa efflux pump MexGHIOpmD transports a natural phenazine that controls gene expression and biofilm development PNAS, Early Edition, June 6, 2016.

    Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHIOpmD in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHIOpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based selfresistance, controlled by a simple circuit, in a Gram-negative pathogen.

  • S. B. Warren, S. Vernick, E. Romano, and K. L. Shepard Complementary Metal-Oxide-Semiconductor Integrated Carbon Nanotube Arrays: Toward Wide-Bandwidth Single-Molecule Sensing Systems Nano Letters DOI: 10.1021/acs.nanolett.6b00319

    There is strong interest in realizing genomic molecular diagnostic platforms that are label-free, electronic, and single-molecule. One attractive transducer for such efforts is the single-molecule field-effect transistor (smFET), capable of detecting a single electronic charge and realized with a point-functionalized exposed-gate one-dimensional carbon nanotube field-effect device. In this work, smFETs are integrated directly onto a custom complementary metaloxide-semiconductor chip, which results in an array of up to 6000 devices delivering a measurement bandwidth of 1 MHz. In a first exploitation of these high-bandwidth measurement capabilities, point functionalization through electrochemical oxidation of the devices is observed with microsecond temporal resolution, which reveals complex reaction pathways with resolvable scattering signatures. High-rate random telegraph noise is detected in certain oxidized devices, further illustrating the measurement capabilities of the platform.

  • D. L. Bellin, H. Sakhtah, Y. Zhang, A. Price-Whelan, L. E.P. Dietrich & K. L. Shepard Electrochemical camera chip for simultaneous imaging of multiple metabolites in biofilms. Nat. Commun. 7:10535 doi: 10.1038/ncomms10535 (2016).

    Monitoring spatial distribution of metabolites in multicellular structures can enhance understanding of the biochemical processes and regulation involved in cellular community development. Here we report on an electrochemical camera chip capable of simultaneous spatial imaging of multiple redox-active phenazine metabolites produced by Pseudomonas aeruginosa PA14 colony biofilms. The chip features an 8mm8mm array of 1,824 electrodes multiplexed to 38 parallel output channels. Using this chip, we demonstrate potential-sweepbased electrochemical imaging of whole-biofilms at measurement rates in excess of 0.2 s per electrode. Analysis of mutants with various capacities for phenazine production reveals distribution of phenazine-1-carboxylic acid (PCA) throughout the colony, with 5-methylphenazine-1-carboxylic acid (5-MCA) and pyocyanin (PYO) localized to the colony edge. Anaerobic growth on nitrate confirms the O2-dependence of PYO production and indicates an effect of O2 availability on 5-MCA synthesis. This integrated-circuit-based technique promises wide applicability in detecting redox-active species from diverse biological samples.