Nanoscale Chemical Features of the Natural Fibrous Material Wood.

Peak force infrared (PFIR) microscopy is a recently developed approach to acquire multiple chemical and physical material properties simultaneously and with nanometer resolution: topographical features, infrared (IR)-sensitive maps, adhesion, stiffness, and locally resolved IR spectra. This multifunctional mapping is enabled by the ability of an atomic force microscope tip in the peak force tapping mode to detect photothermal expansion of the sample. We report the use of the PFIR to characterize the chemical modification of bio-based native and intact wooden matrices, which has evolved into an increasingly active research field. The distribution of functional groups of wood cellulose aggregates, either in native or carboxylated states, was detected with a remarkable spatial resolution of 16 nm. Furthermore, mechanical and chemical maps of the distinct cell wall layers were obtained on polymerized wooden matrices to localize the exact position of the modified regions. These findings shall support the development and understanding of functionalized wood materials.

Differences in surface chemistry of regenerated lignocellulose fibers determined by chemically sensitive scanning probe microscopy.

Tuning the composition of regenerated lignocellulosic fibers in the production process enables targeting of specific material properties. In composite materials, such properties could be manipulated by controlled heterogeneous distribution of chemical components of regenerated fibers. This attribute requires a visualization method to show their inherent chemical characteristics. We compared complementary microscopic techniques to analyze the surface chemistry of four differently tuned regenerated lignocellulosic fibers. Adhesion properties were visualized with chemical force microscopy and showed contrasts towards hydrophilic and hydrophobic atomic force microscopy tips. Fibers containing xylan showed heterogeneous adhesion properties within the fiber structure towards hydrophilic tips. Additionally, peak force infrared microscopy mapped spectroscopic contrasts with nanometer resolution and provided point infrared spectra, which were consistent to classical infrared microscopy data. With this setup, infrared signals with a spatial resolution below 20 nm reveal chemical gradients in specific fiber types.

Yield strength of glued Langmuir-Blodgett films determined by friction force microscopy.

We used friction force microscopy measurements to determine the yield strength of several structurally similar Langmuir-Blodgett (LB) bilayer films deposited on a hydrophobic substrate. Film failure was initiated by increasing the load applied by the probe of the atomic force microscope in the course of continuous scanning at nominally the same location on the sample. This film failure was readily detected in friction versus load curves, as well as by imaging of trenches created due to removal of the film. The depths of the trenches formed in the course of these yield strength experiments were consistent with complete removal of these bilayer films, as evidenced by comparisons to film thicknesses measured by ellipsometry. The structure of the LB bilayer was modified by replacing a tetra-chain amphiphile bearing four quaternary ammonium groups with a polymeric surfactant resulting in little change in the yield strength. On the other hand, the addition of a polyanionic gluing layer at the central interface of the bilayers almost doubled the yield strength of the films. To uncover any possible structural effects created by changes in the terminal functionality, the hydrocarbon top layer of the bilayer was replaced with a perfluorinated capping layer. In spite of the changes in frictional properties, the yield strength of this film also appeared to be dominated by the presence of the glued interface.

Optical response of magnetic fluorescent microspheres used for force spectroscopy in the evanescent field.

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 system's 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.

Heterobifunctional modification of DNA for conjugation to solid surfaces.

Many biosensors, DNA arrays, and next-generation DNA sequencing technologies need common methods for end modification of random DNA sequences generated from a sample of DNA. Surface immobilization of chemically modified DNA is often the first step in creating appropriate sensing platforms. We describe a simple technique for efficient heterobifunctional modification of arbitrary double-stranded DNA fragments with chosen chemical groups. The modification requires the use of short (10-20 base pairs) synthetic adaptors having desired terminal functional groups and installs known sequences, which can be used for hybridization of primers in the sequencing-by-synthesis approaches. The method, based on ligation under optimized conditions, is selective and provides high yields of the target heterobifunctional DNA product. An additional two-step procedure can be applied to select further for the desired bifunctionalized product using PCR amplification with a chemically modified primer. Both functional groups in the modified DNA are chemically active and can be used in surface immobilization of the DNA strands to create the surface of a biosensor or sequencing chip.

Quantitative modeling of forces in electromagnetic tweezers.

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.

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 $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.

Peeling single-stranded DNA from graphite surface to determine oligonucleotide binding energy by force spectroscopy.

We measured the force required to peel single-stranded DNA molecules from single-crystal graphite using chemical force microscopy. Force traces during retraction of a tip chemically modified with oligonucleotides displayed characteristic plateaus with abrupt force jumps, which we interpreted as a steady state peeling process punctuated by complete detachment of one or more molecules. We were able to differentiate between bases in pyrimidine homopolymers; peeling forces were 85.3 - 4.7 pN for polythymine and 60.8 +/- 5.5 pN for polycytosine, substantially independent of salt concentration and the rate of detachment. We developed a model for peeling a freely jointed chain from the graphite surface and estimated the average binding energy per monomer to be 11.5 +/- 0.6 k(B)T and 8.3 +/- 0.7 k(B)T in the cases of thymine and cytosine nucleotides, respectively. The equilibrium free-energy profile simulated using molecular dynamics had a potential well of 18.9 k(B)T for thymidine, showing that nonelectrostatic interactions dominate the binding. The discrepancy between the experiment and theory indicates that not all bases are adsorbed on the surface or that there is a population of conformations in which they adsorb. Force spectroscopy using oligonucleotides covalently linked to AFM tips provides a flexible and unambiguous means to quantify the strength of interactions between DNA and a number of substrates, potentially including nanomaterials such as carbon nanotubes.

Optical waveguiding in suspensions of dielectric particles.

An optical waveguide formed by a suspension of dielectric nanoparticles in a microchannel is described. The suspensions, chosen for their guiding and scattering properties, are silica and polystyrene particles that have diameters of 30-900 nm and are dispersed in water with volume fractions up to 10%. Changing the diameter and concentration of the particles causes the suspensions to transition from Rayleigh to Mie scattering and from single to multiple scattering. The threshold for optical guiding in a waveguide core composed of these suspensions is set by the numerical aperture of the effective refractive-index difference introduced by the suspension and not by the average interparticle distance.

A low-threshold, high-efficiency microfluidic waveguide laser.

This communication describes a long (1 cm), laser-pumped, liquid core-liquid cladding (L2) waveguide laser. This device provides a simple, high intensity, tunable light source for microfludic applications. Using a core solution of 2 mM rhodamine 640 perchlorate, optically pumped by a frequency-doubled Nd:YAG laser, we found that the threshold for lasing was as low as 22 muJ (16-ns pulse length) and had a slope efficiency up to 20%. The output wavelength was tunable over a 20-nm range by changing the ratio of solvent components (dimethyl sulfoxide and methanol) in the liquid core.

Arrays and cascades of fluorescent liquid-liquid waveguides: broadband light sources for spectroscopy in microchannels.

This paper describes the fabrication and operation of fluidic broadband light sources for use "on-chip" in integrated microanalytical systems. These light sources consist of liquid-core, liquid-cladding (L2) microchannel waveguides with liquid cores containing fluorescent dyes, excited by incident light from an external halogen bulb. Simultaneous use of multiple fluorophores in a common solution, in a single L2 light source, is not possible, because energy transfer from fluorophores emitting at shorter wavelength to fluorophores emitting at longer wavelength is essentially complete. Two approaches circumvent this problem of energy transfer; both use spatial separation of the fluorophores in different streams. The first setup uses a cascade (series) of single-core, single-dye light sources of increasing absorption energy to generate a combined broadband output. The second approach uses a parallel array of single-core, single-dye light sources. The spectral content of the light output for both cascade and array light sources can be controlled through choice of flow rates and dyes. Output intensity from these light sources is comparable to standard fiber-optic spectrophotometer light sources. The paper also discusses the efficiency of energy transfer between parallel liquid cores as a function of the fluid medium (index of refraction, path length, and rate of flow).

Approaching zero: using fractured crystals in metrology for replica molding.

This report presents a simple and convenient method to generate nanoscale fractures (cracks) in smooth, single-crystalline Si substrates. The cracks propagated as approximately straight lines along the {100} crystal planes with controllable length defined by a stabilizing backlayer. Close to its tip, the crack presented a vertical offset of the two planes as step of smoothly decreasing height, ranging from the microscale to the atomic scale. The edges of a crack were in close contact at the tip of the crack but were separated at the edge where the crack was initiated. These steps served as ideal test features for probing the limits of the replication of soft lithography. Analysis of topography of original and replicated features (in "hard" poly(dimethylsiloxane and polyurethane) by atomic force microscopy demonstrated that steps down to 0.4 nm could be reproduced; these features approach the dimensions of atoms.

Chemical force spectroscopy in heterogeneous systems: intermolecular interactions involving epoxy polymer, mixed monolayers, and polar solvents.

We used chemical force microscopy (CFM) to study adhesive forces between surfaces of epoxy resin and self-assembled monolayers (SAMs) capable of hydrogen bonding to different extents. The influence of the liquid medium in which the experiments were carried out was also examined systematically. The molecular character of the tip, polymer, and liquid all influenced the adhesion. Complementary macroscopic contact angle measurements were used to assist in the quantitative interpretation of the CFM data. A direct correlation between surface free energy and adhesion forces was observed in mixed alcohol-water solvents. An increase in surface energy from 2 to 50 mJ/m(2) resulted in an increase in adhesion from 4-8 nN to 150-300 nN for tips with radii of 50-150 nm. The interfacial surface energy for identical nonpolar surface groups of SAMs was found not to exceed 2 mJ/m(2). An analysis of adhesion data suggests that the solvent was fully excluded from the zone of contact between functional groups on the tip and sample. With a nonpolar SAM, the force of adhesion increased monotonically in mixed solvents of higher water content; whereas, with a polar SAM (one having a hydrogen bonding component), higher water content led to decreased adhesion. The intermolecular force components theory was used for the interpretation of adhesion force measurements in polar solvents. Competition between hydrogen bonding within the solvent and hydrogen bonding of surface groups and the solvent was shown to provide the main contribution to adhesion forces. We demonstrate how the trends in the magnitude of the adhesion forces for chemically heterogeneous systems (solvents and surfaces) measured with CFM can be quantitatively rationalized using the surface tension components approach. For epoxy polymer, inelastic deformations also contributed heavily to measured adhesion forces.