Florin Fulga

Microbeads on microposts: an inverted architecture for bead microarrays.

The rapid development of genomics and proteomics requires accelerated improvement of the microarrays density, multiplexing, readout capabilities and cost-effectiveness. The bead arrays are increasingly attractive because of their self-assembly-based fabrication, which alleviates many problems of top-down microfabrication. Here we present a simple, reliable, robust and modular technique for the fabrication of bead microarrays, which combines the directed assembling of beads in microstructures and PDMS-based replica molding. The beads are first self-assembled in pyramidal microwells fabricated by anisotropic etching of silicon substrates, then transferred on the apex of PDMS pyramids that replicate the silicon microstructures. The arrays are chemically and biochemically robust; they are spatially addressable and have the potential for being informationally addressable; and they appear to offer better readout capabilities than the classical microarrays.

Modeling of biological nanostructured surfaces

The paper presents a methodology using atom or amino acid hydrophobicities to describe the surface properties of proteins in order to predict their interactions with other proteins and with artificial nanostructured surfaces. A standardized pattern is built around each surface atom of the protein for a radius depending on the molecule type and size. The atom neighborhood is characterized in terms of the hydrophobicity surface density. A clustering algorithm is used to classify the resulting patterns and to identify the possible interactions. The methodology has been implemented in a software package based on Java technology deployed in a Linux environment.

Impact of Protein Adsorption on the Geometry of Microfluidics Devices

Abstract“Lab-on-a-chip” microfluidics devices manipulate biological fluids, which contain significant quantities of biomolecules, in particular proteins and DNA, and even living cells. As the dimensions of these devices continue to decrease and approach the sub-micron range, and as the trend towards “disposable” devices continues, the impact of the inevitable adsorption of biomolecules becomes more important. In this paper we estimated the protein-adsorption-related sensitivity of the geometry of a rectangular micron-sized channel. The estimation the thickness of the adsorbed protein layer versus processing parameters, i.e., protein concentration in the fluid; ionic strength of fluid; and surface tension of the walls, is based on a proposed semi-empirical model for protein adsorption. The model, derived from the data contained in a biomolecule adsorption database, uses the concept of a “generic protein”, i.e., a protein with molecular properties averaged over the range of data present in the database. The estimation of protein-adsorption-related impact on the geometry of a rectangular micron-sized channel, i.e., narrowing of the micro-channel, increases dramatically below a threshold value of approximately 1.5–2 μm.

Protein immobilisation on micro/nanostructures fabricated by laser microablation.

The performance of biomedical microdevices requires the accurate control of the biomolecule concentration on the surface, as well as the preservation of their bioactivity. This desideratum is even more critical for proteins, which present a significant propensity for surface-induced denaturation, and for microarrays, which require high multiplexing. We have previously proposed a method for protein immobilisation on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. This study investigates the relationship between the properties of the micro/nanostructured surface, i.e., topography and physico-chemistry, and protein immobilisation, for five, molecularly different proteins, i.e., lysozyme, myoglobin, α-chymotrypsin, human serum albumin, and human immunoglobulin. Protein immobilisation on microstructures has been characterised using quantitative fluorescence measurements and atomic force microscopy. It has been found that the sub-micrometer-level, combinatorial nature of the microstructure translates in a 3-10-fold amplification of protein adsorption, as compared to flat, chemically homogenous polymeric surfaces. This amplification is more pronounced for smaller proteins, as they can capitalize better on the newly created surface and variability of the nano-environments.

Multi-threading protein surface functional description

The paper presents an image-oriented description of artificial and biological nanostructured surfaces, with applicability to the functional characterization of atom neighborhoods at the surface of proteins. The property which is considered is the hydrophobicity around each surface atom. The actual hydrophobicity distribution on the atoms that form an atoms vicinity is replaced by an equivalent hydrophobicity density distribution, computed in a standardized hexagonal or octagonal pattern around the atom. The software implementation is a desktop multi-threading application, able to process a large number of atom properties, such as type, 3D coordinates, charge and hydrophobicity. The atoms at the surface of a molecule are divided among the execution threads and a feature vector is created for each of them. The purpose of this work is to create a database of molecular surfaces that will be used in several nanotechnology research fields.

Polymer surface properties control the function of heavy meromyosin in dynamic nanodevices.

The actin-myosin system, responsible for muscle contraction, is also the force-generating element in dynamic nanodevices operating with surface-immobilized motor proteins. These devices require materials that are amenable to micro- and nano-fabrication, but also preserve the bioactivity of molecular motors. The complexity of the protein-surface systems is greatly amplified by those of the polymer-fluid interface; and of the structure and function of molecular motors, making the study of these interactions critical to the success of molecular motor-based nanodevices. We measured the density of the adsorbed motor protein (heavy meromyosin, HMM) using quartz crystal microbalance; and motor bioactivity with ATPase assay, on a set of model surfaces, i.e., nitrocellulose, polystyrene, poly(methyl methacrylate), and poly(butyl methacrylate), poly(tert-butyl methacrylate). A higher hydrophobicity of the adsorbing material translates in a higher total number of HMM molecules per unit area, but also in a lower uptake of water, and a lower ratio of active per total HMM molecules per unit area. We also measured the motility characteristics of actin filaments on the model surfaces, i.e., velocity, smoothness and deflection of movement, determined via in vitro motility assays. The filament velocities were found to be controlled by the relative number of active HMM per total motors, rather than their absolute surface density. The study allowed the formulation of the general engineering principles for the selection of polymeric materials for the manufacturing of dynamic nanodevices using protein molecular motors.

Simulation of connected image reversal and DESIRE techniques for submicron lithography

The paper deals with the simulation of DESIRE, for which purpose a mathematical model is built which emphasizes the role of Tg in diffusion. Taking into consideration that Image Reversal amplifies the changes of the Tg and additionally improves the lithographic characteristics of the process, the authors propose two routes for connecting ImRe with DESIRE. The simulations results are presented.

Optimum time and space resolution for tracking motile nano-objects

Motility assays are the tools of choice for the studies regarding the motility of protein molecular motors in vitro. Despite their wide usage, some simple, but fundamental issues still need to be specifically addressed in order to achieve the best and the most meaningful motility analyses. An analysis of the errors in the calculation of the average velocity and of the fluctuations of velocity due to pixel size is presented here. The magnitude of the fluctuations is correlated with the resolution of the objective lens used in observing the motility assays and with the parameters of the camera used for digitizing the images. Also, the errors in the angular distribution of velocities due to pixel size are characterized and discussed.

Interrogation of the dynamics of magnetic microbeads on the meso-scale via electromagnetic detection

Hybrid devices based on wholly bio-organic systems being interfaced with wholly inorganic systems are now being conceived of and constructed. A hypothetical device is likely to have some dynamic attributes and its dimensions will optimally be comparable with those of the current state of the art in microfabrication. While there are many established methods for interrogating the organic system in the laboratory, and thus extract information, few of those are compatible with micro/nano-technological integration. If magnetic dipoles can be incorporated into the biosystem, then there are a number of methods for non-intrusive interrogation (i.e. compatible with device functionality). Several such methods are discussed, and typical signal strengths are estimated for generic configurations. The most promising avenues arise either from detection of multiple parallel events, or from deployment of a scaled-down version of the well known vibrating loop method.

The Protein Surface Properties Calculator

The interactions of large molecules with surfaces and with each other are strongly dependent upon their surface, rather than their bulk properties. In addition, the local properties of biomolecular surfaces are very important in their own right in biomedicine and other areas, for example for locating binding sites. Following to previous work, we have developed a program to compute to compute amino acid and atom-based surface descriptors, and used it to generate a small database of charge and hydrophobicity-related surface properties for a set of proteins. The program requires the user to input two text files: one assigning a real number to each atom of each amino acid, and one assigning a real number to each amino acid. Although we have so far only computed surface charge (atom-based) and surface hydrophobicity (amino acid-based), we note that this program could be used to compute any surface parameter whatsoever, since the user can assign arbitrary atom-by-atom and amino acid properties.We discuss possible applications of this program and describe one current application, the Biomolecular Adsorption Database.