Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics—regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template–deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq’s fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq’s temperature sensitivity to its rate-determining open state.

Nanoscale materials and devices provide new opportunities to monitor and understand biomolecules. This presentation will review novel devices in which single molecules of DNA polymerase have been joined to single-walled carbon nanotube (SWNT) transistors. DNA polymerases are the enzymes that convert single-stranded DNA into double-stranded helices. This biochemical process is the primary step in DNA replication, amplification, and most sequencing technologies. When one of these polymerases is attached to a SWNT transistor, the enzyme’s native activity is simultaneously transduced into a high-resolution electrical signal [1]. Each time the enzyme works its way down a DNA strand, it generates a nucleotide-by-nucleotide signal that encodes the DNA sequence. This hybrid, biofunctional device is the key element for developing a new, electronic DNA sequencing technology. As a solid-state, manufacturable element, the SWNT transistors provide exciting possibilities for high-density, high-throughput, and low cost sequencing solutions. In addition, the technique is easily generalized to DNA polymerases with specialized properties. Proof-of-principle measurements have been accomplished with DNA polymerases from five different organisms, including the highly processive polymerase of φ29 phage and the high-temperature polymerase Thermus aquaticus (Taq), which is the foundation of the polymerase chain reaction (PCR) and commercial DNA amplification. The SWNT transistor technique operates equally well at room temperature and the elevated PCR temperature of 72 °C, which opens opportunities for novel, high-temperature sequencing strategies with Taq or similar thermostable polymerases. [1] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)

Single-molecule measurements of protein dynamics help unveil the complex conformational changes and transitions that occur during ligand binding and catalytic processes. Using high-resolution single-molecule nanocircuit techniques, we have investigated differences in the conformational dynamics and transitions of lysozyme interacting with three ligands: peptidoglycan substrate, substrate-based chitin analogue, and indole derivative inhibitors. While processing peptidoglycan, lysozyme followed one of the two mechanistic pathways for the hydrolysis of the glycosidic bonds: a concerted mechanism inducing direct conformational changes from open to fully closed conformations or a nonconcerted mechanism involving transient pauses in intermediate conformations between the open and closed conformations. In the presence of either chitin or an indole inhibitor, lysozyme was unable to access the fully closed conformation where catalysis occurs. Instead, lysozymes' conformational closures terminated at slightly closed, "excited" conformations that were approximately one-quarter of the full hinge-bending range. With the indole inhibitor, lysozyme reached this excited conformation in a single step without any evidence of rate-liming intermediates, but the same conformational motions with chitin involved three hidden, intermediate processes and features similar to the nonconcerted peptidoglycan mechanism. The similarities suggest that these hidden processes involve attempts to accommodate imperfectly aligned polysaccharides in the active site. The results provide a detailed glimpse of the enzyme-ligand interplay at the crux of molecular recognition, enzyme specificity, and catalysis.

Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

Nanoscale materials provide new opportunities to interface solid-state electronics with biomolecules and biochemical activity. For example, single-walled carbon nanotubes (SWNTs) have the special property of electronic resistance that is sensitive to single electrons. We have exploited this sensitivity to build nanoelectronic biosensors that monitor the biochemical activity of individual proteins [1]. As an attached protein moves, binds, or performs catalysis, its charged amino acid sidechains induce resistance fluctuations in the SWNT device that may be monitored with microsecond resolution [2]. Recently, we have used this measurement platform for single-molecule measurements of DNA polymerases [3]. Polymerases are the key enzymes for converting single-stranded DNA to double-stranded helices, the primary step in DNA replication, amplification, and most sequencing technologies. When a polymerase processing DNA is also attached to a SWNT device, the electrical signal provides a high-resolution readout of single-nucleotide incorporations and exciting possibilities for high-density, high-throughput electronic DNA sequencing. To investigate the feasibility of electronic DNA sequencing, we have compared single-molecule transduction by DNA polymerases from three different organisms. By working with multiple families of DNA polymerases, we have tested the applicability of the electronic technique while also generating detailed records of differences among the enzymes. For example, we observe an anomalous rate variability when measuring the polymerase from the bacillus phage φ29. Base incorporation rates average 20 s-1 for most the enzymes processing single-stranded DNA templates, but rates up to 200 and 400 s-1 occurred when φ29 encountered homopolymeric sequences of poly(dT) or poly(dC), respectively. When processing poly(dA) and poly(dG) sequences, on the other hand, φ29 had bursts of activity interrupted by pauses lasting 50 to 300 s. This sequence-dependent activity illustrates one way that single-molecule methods reveal information hidden in ensemble-based techniques. Another workhorse protein in DNA sequencing technologies is the DNA polymerase derived from the thermophilic bacteria Thermus aquaticus (Taq). Anomalous stability at high temperatures makes Taq a unique enzyme for the polymerase chain reaction (PCR) and commercial amplification of DNA. In a first for single-molecule biophysics, SWNT devices have recorded Taq activity over a wide temperature range from 22 to 94 °C, including the typical PCR operating temperature of 72 °C. Even operating at this high temperature, the technique resolved Taq testing incoming nucleotides for complementarity and incorporating correct matches in the base-by-base construction of Watson-Crick pairs. The detailed recordings reveal the similarities of Taq’s operation to other, room temperature polymerases. [1] Y. Choi, et. al., Science 335, 319 (2012). [2] M. V. Akhterov, et. al., ACS Chem. Biol. 10, 1495 (2015). [3] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)

Designing the next generation of high performance energy storage devices requires a deeper understanding of structural degradation. To this end, a wide variety of scanning probe microscopy techniques can help characterize charge transport and aging mechanisms in Li-ion battery materials and their interfaces. For example, we use in-situ probe microscopy and Kelvin probe force microscopy (KPFM) to investigate the mechanical and potentiostatic changes in MnO2 cathodes as they undergo charge cycling. Topography monitors mechanical expansion during charging, and the dependence of expansion on scan rate reveals the separate roles of Mn3+/4+ redox pseudocapacitance and faster, double-layer surface capacitance. On the same electrodes, KPFM observes the evolution of surface potentials with lateral resolution as low as 40 nm. At this length scale, inhomogeneous lithiation is clearly observed as a highly nonuniform, fractal growth or shrinkage of Mn3+ and Mn4+ phases. Via surface potential, KPFM also reveals "dead" zones that do not participate in charging, "hot" zones that charge or discharge most readily, and the evolution of each type as an electrode is repeatedly cycled. This work was supported as part of the Nanostructures for Electrical Energy Storage (NEES), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science.

Electronic conduction in one dimension is uniquely sensitive to single point perturbations. As quasi-one-dimensional conductors, single-walled carbon nanotubes are ideal examples of this sensitivity, and they can be exploited as single-molecule sensors in air, liquid, or vacuum. Even in complex physiological solutions, dynamic biochemical activity has been monitored with single-bond and microsecond resolution. However, past work along these lines was achieved on individual devices with low success rates of 10-20%. Here, we describe simultaneous progress on three fronts that has produced large-scale arrays of single-molecule devices. First, precise control of catalytic chemical vapor deposition has dramatically increased our yield of usable single-nanotube devices across 4” substrates. Second, contamination control methods have reduced charge-trap fluctuations that previously disqualified many transistors from single-molecule sensing experiments. Thirdly, new surface modification protocols have brought predictability and control to single-molecule biofunctionalization of the nanotube surfaces. When combined, these techniques scale up single-molecule dynamic sensing from single devices to large arrays. New research directions with these arrays include massively parallel sensing applications such as drug discovery and DNA sequencing.

Bioelectronic devices built with single molecules of a protein, enzyme, or aptamer represent a new class of hybrid electronics. When biofunctionalization of nanoscale conductors is reduced to one molecule, that molecule's dynamic activity can be transduced into a large amplitude, high bandwidth electronic output. Using DNA polymerase I as an example, we show that single-molecule bioelectronics reveal biochemical activity with bond-by-bond resolution.

Designing the next generation of high performance energy storage devices requires a deeper understanding of structural degradation. To this end, a wide variety of scanning probe microscopy techniques can help characterize charge transport and aging mechanisms in Li-ion battery materials and their interfaces. For example, we use in-situ probe microscopy and Kelvin probe force microscopy (KPFM) to investigate the mechanical and potentiostatic changes in MnO2 cathodes as they undergo charge cycling. Topography monitors mechanical expansion during charging, and the dependence of expansion on scan rate reveals the separate roles of Mn3+/4+ redox pseudocapacitance and faster, double-layer surface capacitance. On the same electrodes, KPFM observes the evolution of surface potentials with lateral resolution as low as 40 nm. At this length scale, inhomogeneous lithiation is clearly observed as a highly nonuniform, fractal growth or shrinkage of Mn3+ and Mn4+ phases. Via surface potential, KPFM also reveals "dead" zones that do not participate in charging, "hot" zones that charge or discharge most readily, and the evolution of each type as an electrode is repeatedly cycled. This work was supported as part of the Nanostructures for Electrical Energy Storage (NEES), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science.

This article examines the physical consequences of defects and disorder in carbon nanotubes (CNTs). It begins with a pedagogical categorization of the types of defects and disorder found in CNTs, including lattice vacancies and bond rotations, and goes on to discuss considers two primary sources of disorder: the environment surrounding a CNT and the substrate supporting it. It then considers various experimental methods for locating defects in CNTs, including atomic-resolution scanning tunnelling microscopy, transmission electron microscopy, electrochemical and chemoselective labelling, optical spectroscopy, and electrical conductance. The article concludes with a review of the long-range consequences of defects and disorder on the physical properties of CNTs such as chemical reactivity, electrical transport, and mechanical effects.

Large numbers of high quality graphene transistors with mobility approximately 5000 cm(2)/V*s were fabricated by chemical vapor deposition and packaged into ceramic carriers with an open cavity design. The ceramic carrier is compatible with standard electronics assembly, enabling the readout of graphene properties on the benchtop without large, expensive probing systems. After chemical functionalization, these sensors demonstrate sensitivity in the pM range and selectivity to many classes of biomolecules as a three terminal liquid-gated field effect transistor. High precision measurements of protein kinetics captured using this technology, commercially known as AGILE R100, are comparable and can exceed the capabilities of state-of-the-art biomolecule characterization tools. (C) 2016 Published by Elsevier B.V.

Recent work has demonstrated single-electron sensitivity in carbon nanotube transistors under a variety of conditions, including room temperature. In noncovalent cases like charged adsorbates or SiO2 imperfections, two-level fluctuators near the nanotube perturb the current electrostatically. In other cases, a sidewall defect or other covalent modification directly on the nanotube sidewall sensitizes a particular site. Comparative research has helped reveal differences in the transduction mechanisms of the two cases and provides design rules for maximizing reliable signals for electronic sensing. For example, noncovalent sensitization generally produces a smaller signal amplitude in a background of low-energy fluctuators at varying distances from the nanotube. Covalent modifications are far more sensitive than noncovalent perturbations, but the new degrees of freedom that accompany covalent disorder often have energy scales similar to the signal of interest, leading to multiple independent fluctuations that degrade the overall signal-to-noise for sensing. An outstanding compromise involves short, noncovalent linkers, which can produce highly predictable signal amplitudes without degradation of the device characteristics. Furthermore, noncovalent fabrication methods are scalable, so that wafer-scale arrays of molecular sensors are most likely to follow this path.

The faster kinetics of nanostructured charge-storage materials are often accompanied by accelerated degradation and failure, compromising the potential advantages of nanostructure and nanoporosity. Understanding and controlling the exact mechanisms of this degradation is a complex materials characterization challenge. The recent availability of in situ and in operando techniques allows new fundamental studies of these issues, including the failure of nanoscale porosity to fully accommodate the mechanical strains of charge insertion. Here, we use atomic force microscopy in liquid electrolytes to experimentally measure the mechanical expansion of porous, nanostructured Li-ion cathodes during charge cycling. Using thin films of LixMnO2 on Pt collectors, we analyze expansion as a function of scan rate, voltage window, and porosity. Comparative measurements in aqueous and nonaqueous electrolytes confirm the role of Li-ion species in the results. The rate dependent measurements of expansion can be separated into diffusion-limited, bulk components driven by Mn3+/4+ charging and faster, surface processes, in the same way that these contributions to capacitance are normally separated. Consequently, we have extracted the electrochemical-mechanical coupling coefficients ∂V/∂Q for surface and bulk processes separately. While the bulk coupling coefficient provides an excellent match to values predicted from first-principles theory, the surface coupling is surprisingly large. In fact, surface capacitance is responsible for a majority of the mechanical expansion at high rates, even when most of this capacitance would normally be attributed to double-layer charging mechanisms. The result demonstrates the importance of Mn3+/4+ species at the electrolyte interface and the inability of these species to take advantage of the film’s nanoporosity for accommodating expansion. In other words, fast surface charging processes do not involve mechanical relaxation of the material into adjacent pores, but rather drive expansion of the solid matrix.