In Situ Synthesis of Horseradish Peroxidase Nanoflower@Carbon Nanotube Hybrid Nanobiocatalysts with Greatly Enhanced Catalytic Activity.

Organic-inorganic hybrid nanoflowers (NFs) consisting of horseradish peroxidase (HRP) and copper II (Cu2+) are successfully synthesized with the involvement of carbon nanotubes (CNTs) by in situ and post-modification methods. Catalytic activities of in situ synthesized HRP-NF@CNT (HRP-NF@CNT-Is) and post-modification-synthesized HRP-NF@CNTs (HRP-NF@CNT-Pm) are systematically examined. The 30 mg CNTs incorporated HRP-NF@CNT-Is (HRP-NF@CNT-30Is) exhibits greatly increased catalytic activity and stability toward 3,3',5,5'-tetramethylbenzidine (TMB), thanks to the synergistic effect between HRP-NF and CNTs and the peroxidase-like activity of CNTs in the presence of hydrogen peroxide (H2O2). While HRP-NF@CNT-30Is retains almost 85% of its initial activity even after 10 cycles, HRP-NF (without CNTs) loses half of its initial activity at the same experimental conditions. We study how two experimental parameters, the pH values and temperatures, influence the catalytic activity of HRP-NF@CNT-30Is, in addition to the fact that HRP-NF@CNT-30Is is employed to detect the presence of H2O2 and glutathione (GSH) with colorimetric and spectrophotometric readouts. For instance, HRP-NF@CNT-30Is is used to sensitively detect H2O2 in the range of 20 to 300 µM with an LOD of 2.26 µM. The catalytic activity of HRP-NF@CNT-30Is is suppressed in the presence of GSH, and then an obvious color change from blue to nearly colorless is observed. Using this strategy, GSH is also sensitively determined in the range of 20-200 µM with an LOD of 11.2 µM. We expect that HRP-NF@CNTs can be used as a promising and novel nanobiocatalyst for various biomedical and industrial applications in the near future.

One-Dimensional Poole-Frenkel Conduction in the Single Defect Limit.

A single point defect surrounded on either side by quasi-ballistic, semimetallic carbon nanotube is a nearly ideal system for investigating disorder in one-dimensional (1D) conductors and comparing experiment to theory. Here, individual single-walled nanotubes (SWNTs) are investigated before and after the incorporation of single point defects. Transport and local Kelvin Probe force microscopy independently demonstrate high-resistance depletion regions over 1.0 µm wide surrounding one point defect in semimetallic SWNTs. Transport measurements show that conductance through such wide depletion regions occurs via a modified, 1D version of Poole-Frenkel field-assisted emission. Given the breadth of theory dedicated to the possible effects of disorder in 1D systems, it is surprising that a Poole-Frenkel mechanism appears to describe defect scattering and resistance in this semimetallic system.

Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.

DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by τclosed. Under Vmax conditions, the average time of τclosed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by τopen, which includes molecular recognition of the incoming dNTP. All α-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair.

Electrochemical charge-transfer resistance in carbon nanotube composites.

Using a model system of single, isolated carbon nanotubes loaded with high-capacitance metal-oxide films, we have quantitatively investigated electrochemical composites on the single-nanotube scale. Electrochemical charging and discharging of a model MnO2 storage material was used to probe interfacial charge transfer and surface impedances at the nanotube interface. We found that one single-walled carbon nanotube has an apparent surface resistivity of 30 mΩ cm(2), approximately 4 times smaller than for a multiwalled carbon nanotube and 50 times smaller than the 1.5 Ω cm(2) resistivity of Pt or graphite films. The improvement originates in the electrochemical-transport properties of microelectrodes shrunk to a nanotube's dimensions rather than any unique nanotube property like curvature, bandstructure, or surface chemistry. In explaining the enhanced performance of certain nanotube-containing composites, the results overturn widely held assumptions about nanotubes' roles while also providing guidelines for optimizing effective composites.

Electronic measurements of single-molecule catalysis by cAMP-dependent protein kinase A.

Single-molecule studies of enzymes open a window into their dynamics and kinetics. A single molecule of the catalytic domain of cAMP-dependent protein kinase A (PKA) was attached to a single-walled carbon nanotube device for long-duration monitoring. The electronic recording clearly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Using recordings of a single PKA molecule extending over 10 min and tens of thousands of binding events, we determine the full transition probability matrix and conversion rates governing formation of the apo, intermediate, and closed enzyme configurations. We also observe kinetic rates varying over 2 orders of magnitude from one second to another. Anti-correlation of the on and off rates for PKA binding to the peptide substrate, but not ATP, demonstrates that regulation of enzyme activity results from altering the stability of the PKA-substrate complex, not its binding to ATP. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute useful for an enzyme with crucial roles in cell signaling.

Electronic measurements of single-molecule processing by DNA polymerase I (Klenow fragment).

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme's open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme's closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme's open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.

Hollow microneedle array fabrication using a rational design to prevent skin clogging in transdermal drug delivery.

Microneedle (MN) technology is promising to replace hypodermic needles for practical use and painless drug delivery. However, the complex top-down fabrication process of functional MN arrays is a bottleneck that hinders their widespread use. Here, we fabricate the tapered hollow MN array using a unique bi-level-tip by combining strain-engineering and capillary self-assembly of carbon nanotube (CNT) microstructures. Strain-engineering facilitated by the offset pattern of the catalyst enables the growth of bent, bi-level CNT microstructures while capillary self-assembly helps in constituting the tapered geometry of MNs. The bottom-up fabrication that consists of only two standard photolithography steps and CNT growth to form the scaffold of MNs followed by a polymer (polyimide) reinforcement step to impart mechanical stiffness to MNs provides scalable and fewer processing steps. The tapered shape of the MN allows an 8 times smaller force to pierce and penetrate the skin compared to the straight MN. The liquid delivery rate of the bi-level-tip MN is measured to be 26% better than the flat tip MN of the same lumen size as its geometry reduces skin clogging effect at the needle tip. In addition, cytotoxicity tests verify that the polyimide reinforced CNT-MNs are biocompatible for future in vivo applications.

Formation of functional nanobiocatalysts with a novel and encouraging immobilization approach and their versatile bioanalytical applications.

The discovery of functional organic-inorganic hybrid nanoflowers (FNFs) consisting of proteins/enzymes as the organic components and Cu(ii) ion as the inorganic component has made an enormous impact on enzyme immobilization studies. The FNFs synthesized by an encouraging and novel approach not only showed high stabilities but also much enhanced catalytic activities as compared to free and conventionally immobilized enzymes. A recent development demonstrated that FNF formation has moved beyond the initial discovery in which enzymes and Cu2+ ions used as the organic and inorganic parts, respectively, are replaced with new organic (chitosan, amino acid and plant extracts) and inorganic (Cu2+ and Fe2+) materials. The new organic materials incorporated into FNFs act as Fenton-like agents and then show peroxidase-like activity owing to the metal ions and the porous structure of FNFs in the presence of hydrogen peroxide (H2O2). All FNFs have been widely utilized in many different scientific and industrial fields due to their greatly enhanced activities and stabilities. This review focuses primarily on the preparation, characterization, and bioanalytical applications of FNFs and explains the mechanisms of their formation and enhanced activities and stabilities.

Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths.

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures.

Observing lysozyme's closing and opening motions by high-resolution single-molecule enzymology.

Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 µs allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 µs, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 µs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme's state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.