Arnold Vainrub

Resolution of 90 nm (λ/5) in an optical transmission microscope with an annular condenser

Resolution of 90 nm was achieved with a research microscope simply by replacing the standard bright-field condenser with a homebuilt illumination system with a cardioid annular condenser. Diffraction gratings with 100 nm width lines as well as less than 100 nm size features of different-shaped objects were clearly visible on a calibrated microscope test slide. The resolution increase results from a known narrower diffraction pattern in coherent illumination for the annular aperture compared with the circular aperture. This explanation is supported by an excellent accord of calculated and measured diffraction patterns for a 50 nm radius disk.

Thermodynamics of Association to a Molecule Immobilized in an Electric Double Layer

Abstract A thermodynamic theory of association to a molecule immobilized near a surface has been developed. Exact equations for the binding enthalpy, entropy and equilibrium reaction constant for an immobilized complex are derived. Using linear Poisson–Boltzmann theory of the electric double-layer interaction between an ion-penetrable sphere and a hard plate allows a closed form evaluation. We briefly discuss application of the theory to a DNA chip at high (1 M NaCl) and low (0.01 M NaCl) ionic strength for dielectric and metallic substrates. Predicted strong electrostatic effects suggest the feasibility of electronic control of DNA hybridization and design of chips avoiding the DNA folding problem.

A model for Structure and Thermodynamics of ssDNA and dsDNA Near a Surface: a Coarse Grained Approach

New methods based on surfaces or beads have allowed measurement of properties of single DNA molecules in very accurate ways. Theoretical coarse grained models have been developed to understand the behavior of single stranded and double stranded DNA. These models have been shown to be accurate and relatively simple for very short systems of 6-8 base pairs near surfaces. Comparatively less is known about the influence of a surface on the secondary structures of longer molecules important to many technologies. Surface fields due to either applied potentials and/or dielectric boundaries are not in current surface mounted coarse grained models. To gain insight into longer and surface mounted sequences we parameterized a discretized worm-like chain model. Each link is considered a sphere of 6 base pairs in length for dsDNA, and 1.5 bases for ssDNA (requiring an always even number of spheres). For this demonstration of the model, the chain is tethered to a surface by a fixed length, non-interacting 0.536 nm linker. Configurational sampling was achieved via Monte-Carlo simulation. Our model successfully reproduces end to end distance averages from experimental results, in agreement with polymer theory and all atom simulations. Our average tilt results are also in agreement with all atom simulations for the case of dense systems.

Accurate prediction of binding thermodynamics for DNA on surfaces

For DNA mounted on surfaces for microarrays, microbeads, and nanoparticles, the nature of the random attachment of oligonucleotide probes to an amorphous surface gives rise to a locally inhomogeneous probe density. These fluctuations of the probe surface density are inherent to all common surface or bead platforms, regardless of whether they exploit either an attachment of presynthesized probes or probes synthesized in situ on the surface. Here, we demonstrate for the first time the crucial role of the probe surface density fluctuations in the performance of DNA arrays. We account for the density fluctuations with a disordered two-dimensional surface model and derive the corresponding array hybridization isotherm that includes a counterion screened electrostatic repulsion between the assayed DNA and probe array. The calculated melting curves are in excellent agreement with published experimental results for arrays with both presynthesized and in situ synthesized oligonucleotide probes. The approach developed allows one to accurately predict the melting curves of DNA arrays using only the known sequence-dependent hybridization enthalpy and entropy in solution and the experimental macroscopic surface density of probes. This opens the way to high-precision theoretical design and optimization of probes and primers in widely used DNA array-based high-throughput technologies for gene expression, genotyping, next-generation sequencing, and surface polymerase extension.

A Non-Watson–Crick Motif of Base-pairing on Surfaces for Untethered Oligonucleotides

A structural view of DNA association/hybridization to a target oligonucleotide molecule near a surface has been developed. Recent experiments have showed a kinetically rapid hybridization between large target DNA fragments and oligonucleotides electrostatically immobilized (untethered) to a surface. Theory and computer simulations have been used to investigate the nature of the specificity and affinity in such a system. Simulations were performed for a modified silicon dioxide surface with positively charged groups at neutral pH. The dosing of a surface with unattached oligonucleotide was simulated. The oligonucleotide was found to associate with the surface in salt water in a way that some of the bases remained stacked, and most of the bases near the surface on average pointed preferentially toward the solution, away from the surface. Use of an analytic solution to the linear Poisson–Boltzmann (PB) theory of the electric double layer interaction between DNA and a hard surface predicts tight binding in this system. The simulation thus gives a mechanism for specificity and the theory a mechanism for affinity. The geometry is such that only non-helical base pairs would be accommodated with an irregular backbone.

High-resolution light microscopy of nanoforms

We developed a high resolution light imaging system. Diffraction gratings with 100 nm width lines as well as less than 100 nm size features of different-shaped objects are clearly visible on a calibrated microscope test slide (Vainrub et al., Optics Letters, 2006, 31, 2855). The two-point resolution increase results from a known narrowing of the central diffraction peak for the annular aperture. Better visibility and advanced contrast of the smallest features in the image are due to enhancement of high spatial frequencies in the optical transfer function. The imaging system is portable, low energy, and battery operated. It has been adapted to use in both transmitting and reflecting light. It is particularly applicable for motile nanoform systems where structure and functions can be depicted in real time. We have isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1-2nm metallic nanoclusters containing 40-300 atoms. Proteons are capable of spontaneous assembling into higher nanoform systems assuming structure of complicated topology. The arrangement of complex proteon system mimics the structure of a small biological cell. It has structures that imitate membrane and nucleolus or nuclei. Some of these nanoforms are motile. They interact and divide. Complex nanoform systems can spontaneously reduce to simple proteons. The physical properties of these nanoforms could shed some light on the properties of early life forms or forms at extreme conditions.

Acoustic Wave (TSM) Biosensors: Weighing Bacteria

This chapter is focused on the development and use of acoustic wave biosensor platforms for the detection of bacteria, specifically those based on the thickness shear mode (TSM) resonator. We demonstrated the mechanical and electrical implications of bacterial positioning at the solid-liquid interface of a TSM biosensor and presented a model of the TSM with bacteria attached operating as coupled oscillators. The experiments and model provide an understanding of the nature of the signals produced by acoustic wave devices when they are used for testing bacteria. The paradox of “negative mass” could be a real threat to the interpretation of experimental results related to the detection of bacteria. The knowledge of the true nature of “negative mass” linked to the strength of bacteria attachment will contribute significantly to our understanding of the results of “weighing bacteria.” The results of this work can be used for bacterial detection and control of processes of bacterial settlement, bacterial colonization, biofilm formation, and bacterial infection in which bacterial attachment plays a role.

Free Energy Considerations for Nucleic Acids with Dangling Ends Near a Surface: a Coarse Grained Approach

A coarse grained model for the thermodynamics of nucleic acid hybridization near surfaces has been extended and parameterized to consider the contribution of unpaired dangling ends. The parameters of the model differ when representing a double stranded DNA section or a single stranded DNA section. The thermodynamic effects of the possibility of different dangling end combinations were considered in the presence of different types of surfaces. Configurational sampling was achieved by the Metropolis Monte Carlo method. To gain a more complete picture of the free energy changes, an estimation of the conformational entropy was included. We find a strong thermodynamic effect for dangling mismatches due to sequence requirements when they are nearer the surface as opposed to being held away from the surface.

Predicting DNA duplex stability on oligonucleotide arrays.

DNA duplex stability on oligonucleotide microarray was calculated using recently developed electrostatic theory of on-array hybridization thermodynamics. In this method, the first step is to finding the enthalpy and entropy of duplex formation in solution. This standard calculation was done with nearest-neighbor scheme and on-line software. Next the defined parameters and the arrays single characteristic, the surface density of probes, are used to predict on-array duplex melting behavior. Reasonable accords of calculated and experimental melting curves for in situ synthesized microfluidic array were observed. The proposed method could be useful in microarray design and hybridization optimization. However, lack of melting curve measurements for different microarray platforms makes more experiments desirable to determine the methods accuracy.

Precise measurement of the resolution in light microscopy using Fourier transform

The resolution power of light microscope has been accurately measured (+/-5%) by Fourier transform of various object images and further evaluation of the highest spatial frequency in Fourier spectrum. Any unknown shape plane object with a shape features size smaller than the resolution to be measured was shown to provide a reliable resolution test. This simple method gives a direct measurement of the resolution power as defined by Abbe [Archiv. F. Mikroskopische Anat. 9, 413 (1873)]. The results have been justified by comparison to a standard resolution measurement by using calibrated periodic line patterns. Notably, the approach is applicable in super-resolution light microscopy (transmission, reflection, and fluorescence), where calibrated resolution targets do not occur. It was conveniently implemented by using a compact disk as a test object and free IMAGEJ imaging software.