Dmitri Vezenov

The challenges of sequencing by synthesis

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to

Experimental verification of the rainbow trapping effect in adiabatic plasmonic gratings

100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

Spontaneous Stepwise Self‐Assembly of a Polyoxometalate–Organic Hybrid into Catalytically Active One‐Dimensional Anisotropic Structures

We report the experimental observation of a trapped rainbow in adiabatically graded metallic gratings, designed to validate theoretical predictions for this unique plasmonic structure. One-dimensional graded nanogratings were fabricated and their surface dispersion properties tailored by varying the grating groove depth, whose dimensions were confirmed by atomic force microscopy. Tunable plasmonic bandgaps were observed experimentally, and direct optical measurements on graded grating structures show that light of different wavelengths in the 500–700-nm region is “trapped” at different positions along the grating, consistent with computer simulations, thus verifying the “rainbow” trapping effect.

Noncontact method for calibration of lateral forces in scanning force microscopy.

An inorganic-organic hybrid surfactant with a hexavanadate cluster as the polar head group was designed and observed to assemble into micelle structures, which further spontaneously coagulate into a 1D anisotropic structure in aqueous solutions. Such a hierarchical self-assembly process is driven by the cooperation of varied noncovalent interactions, including hydrophobic, electrostatic, and hydrogen-bonding interactions. The hydrophobic interaction drives the quick formation of the micelle structure; electrostatic interactions involving counterions leads to the further coagulation of the micelles into larger assemblies. This process is similar to the crystallization process, but the specific counterions and the directional hydrogen bonding lead to the 1D growth of the final assemblies. Since most of the hexavanadates are exposed to the surface, the 1D assembly with nanoscale thickness is a highly efficient heterogeneous catalyst for the oxidation of organic sulfides with appreciable recyclability.

Interaction of single-stranded DNA with curved carbon nanotube is much stronger than with flat graphite.

This paper describes a noncontact calibration procedure for lateral force microscopy in air and liquids. The procedure is based on the observation that the sensitivity of a force microscope may be calibrated using the raw thermal noise spectrum of the cantilever and its known spring constant, which can be found from the same uncalibrated thermal noise spectrum using Saders method (Rev. Sci. Instrum.1999, 70, 3967-3969). In addition to the power spectrum of the cantilever thermal noise, this noncontact calibration method only requires knowledge of the plan view dimensions of the cantilever that could be measured using an optical microscope. This method is suitable for in situ force calibration even in viscous fluids through a two-step calibration procedure, where the cantilever thermal spectra are captured both in air and in the desired liquid. The lateral calibration performed with the thermal noise technique agrees well with sensitivity values obtained by the wedge calibration procedure. The approach examined in this paper allows for complete calibration of normal and lateral forces without contacting the surface, eliminating the possibility for any tip damage or contamination during calibration.

Optical Response of Magnetic-Fluorescent Microspheres Used for Force Spectroscopy in the Evanescent Field

We used single molecule force spectroscopy to measure the force required to remove single-stranded DNA (ssDNA) homopolymers from single-walled carbon nanotubes (SWCNTs) deposited on methyl-terminated self-assembled monolayers (SAMs). The peeling forces obtained from these experiments are bimodal in distribution. The cluster of low forces corresponds to peeling from the SAM surface, while the cluster of high forces corresponds to peeling from the SWCNTs. Using a simple equilibrium model of the single molecule peeling process, we calculated the free energy of binding per nucleotide. We found that the free energy of ssDNA binding to hydrophobic SAMs decreases as poly(A) > poly(G) ≈ poly(T) > poly(C) (16.9 ± 0.1; 9.7 ± 0.1; 9.5 ± 0.1; 8.7 ± 0.1 kBT, per nucleotide). The free energy of ssDNA binding to SWCNT adsorbed on this SAM also decreases in the same order poly(A) > poly(G) > poly(T) > poly(C), but its magnitude is significantly greater than that of DNA-SAM binding energy (38.1 ± 0.2; 33.9 ± 0.1; 23.3 ± 0.1; 17.1 ± 0.1 kBT, per nucleotide). An unexpected finding is that binding strength of ssDNA to the curved SWCNTs is much greater than to flat graphite, which also has a different ranking (poly(T) > poly(A) > poly(G) ≥ poly(C); 11.3 ± 0.8, 9.9 ± 0.5, 8.3 ± 0.2, and 7.5 ± 0.8 kBT, respectively, per nucleotide). Replica-exchange molecular dynamics simulations show that ssDNA binds preferentially to the curved SWCNT surface, leading us to conclude that the differences in ssDNA binding between graphite and nanotubes arise from the spontaneous curvature of ssDNA.

Supramolecular Assembly of Conjugated Polymers Containing Polyoxometalate Terminal Side Chains in Polar and Nonpolar Solvents

Force spectroscopy based on magnetic tweezers is a powerful technique for manipulating single biomolecules and studying their interactions. The resolution in magnetic probe displacement, however, needs to be commensurate with molecular sizes. To achieve the desirable sensitivity in tracking displacements of the magnetic probe, some recent approaches have combined magnetic tweezers with total internal reflection fluorescence microscopy. In this situation, a typical force probe is a polymer microsphere containing two types of optically active components: a pure absorber (magnetic nanoparticles for providing the pulling force) and a luminophore (semiconducting nanoparticles or organic dyes for fluorescent imaging). To assess the systems capability fully with regard to tracking the position of the force probe with subnanometer accuracy, we developed a body-of-revolution formulation of the method of auxiliary sources (BOR-MAS) to simulate the absorption, scattering, and fluorescence of microscopic spheres in an evanescent electromagnetic field. The theoretical formulation uses the axial symmetry of the system to reduce the dimensionality of the modeling problem and produces excellent agreement with the reported experimental data on forward scattering intensity. Using the BOR-MAS numerical model, we investigated the probe detection sensitivity for a high numerical aperture objective. The analysis of both backscattering and fluorescence observation modes shows that the total intensity of the bead image decays exponentially with the distance from the surface (or the length of a biomolecule). Our investigations demonstrate that the decay lengths of observable optical power are smaller than the penetration depth of the unperturbed excitation evanescent wave. In addition, our numerical modeling results illustrate that the expected sensitivity for the decay length changes with the angle of incidence, tracking the theoretical penetration depth for a two-media model, and is sensitive to the bead size. The BOR-MAS methodology developed in this work for near-field modeling of bead-tracking experiments fully describes the fundamental photonic response of microscopic BOR probes at the subwavelength level and can be used for future improvements in the design of these probes or in the setup of bead-tracking experiments.

Quantitative modeling of forces in electromagnetic tweezers

Conjugated polymers are important materials with unique electrical and optoelectronic properties, and have found applications in a variety of molecular electronic devices including light-emitting diodes, solar cells, field effect transistors, photodetectors, sensors, and nonlinear optics. Polyoxometalates (POMs), a large group of metal oxide clusters consisting of early-transition-metal cations and oxo ligands, are also considered electrically active materials and have attracted increasing interest in recent years due to their outstanding properties in catalysis, medicine, and photoelectronic responses. Incorporating POM clusters covalently with conjugated polymers may lead to hybrid materials with not only value-adding properties, but also synergistic effects. For example, POM-containing hybrid polymers have been found to be good candidates for photovoltaic cells, photocatalytic materials, and catalytic nanoparticles. While it is known that the device performance of hybrid materials depends strongly on their aggregated microor nanostructures, there has been very little research on the aggregation of POM-containing conjugated polymers in both solution and solid states. Such studies are essential if POM-based controllable ordered structures are to be realized. On the other hand, understanding the aggregation behavior of polyelectrolyte solutions has always been an important goal in polymer and biological sciences, since it may help explain the behaviors of DNA , RNA, and proteins in living cells and guide the design of polyelectrolyte-based functional materials. However, due to the coexistence of complex interand intrachain electrostatic interactions, polyelectrolyte solutions are still poorly understood. Herein, we report the self-assembly behavior of conjugated polymers containing POM terminal side chains in polar and nonpolar solvents. As shown in Figure 1, such polymers have rigid rodlike structures due to the phenylene–ethynylene backbone. The POM cluster, an organoimido-derivatized hexamolybdate approximately 1 nm in size and carrying two negative charges with two tetrabutyl ammonium (TBA) as the counterions, is covalently linked to the polymer backbone as the terminal of their side chains.

Metabolic Profiling of Bacteria by Unnatural C‐terminated D‐Amino Acids

This paper discusses numerical simulations of the magnetic field produced by an electromagnet for generation of forces on superparamagnetic microspheres used in manipulation of single molecules or cells. Single molecule force spectroscopy based on magnetic tweezers can be used in applications that require parallel readout of biopolymer stretching or biomolecular binding. The magnetic tweezers exert forces on the surface-immobilized macromolecule by pulling a magnetic bead attached to the free end of the molecule in the direction of the field gradient. In a typical force spectroscopy experiment, the pulling forces can range between subpiconewton to tens of piconewtons. In order to effectively provide such forces, an understanding of the source of the magnetic field is required as the first step in the design of force spectroscopy systems. In this study, we use a numerical technique, the method of auxiliary sources, to investigate the influence of electromagnet geometry and material parameters of the magnetic core on the magnetic forces pulling the target beads in the area of interest. The close proximity of the area of interest to the magnet body results in deviations from intuitive relations between magnet size and pulling force, as well as in the force decay with distance. We discuss the benefits and drawbacks of various geometric modifications affecting the magnitude and spatial distribution of forces achievable with an electromagnet.

Heterobifunctional modification of DNA for conjugation to solid surfaces

Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross-link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus amidated D-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.