Daniel Forciniti

On One-dimensional Self-assembly of Surfactant-coated Nanoparticles

Nanometer-sized metal and semiconductor particles possess novel properties. To fully realize their potential, these nanoparticles need to be fabricated into ordered arrays or predesigned structures. A promising nanoparticle fabrication method is coupled surface passivation and self-assembly of surfactant-coated nanoparticles. Due to the empirical procedure and partially satisfactory results, this method still represents a major challenge to date and its refinement can benefit from fundamental understanding. Existing evidences suggest that the self-assembly of surfactant-coated nanoparticles is induced by surfactant-modified interparticle interactions and follows an intrinsic road map such that short one-dimensional (1D) chain arrays of nanoparticles occur first as a stable intermediate before further assembly takes place to form higher dimensional close-packed superlattices. Here we report a study employing fundamental analyses and Brownian dynamics simulations to elucidate the underlying pair interaction potential that drives the nanoparticle self-assembly via 1D arrays. We find that a pair potential which has a longer-ranged repulsion and reflects the effects of surfactant chain interdigitation on the dynamics is effective in producing and stabilizing nanoparticle chain arrays. The resultant potential energy surface is isotropic for dispersed nanoparticles but becomes anisotropic to favor the growth of linear chain arrays when self-assembly starts.

Protein refolding using aqueous two-phase systems

Abstract We present a novel refolding technique in which aqueous two-phase systems are used to both dissociate protein aggregates (inclusion bodies) and to refold the protein in only one operation. Three denaturant salts (sodium thiocyanate, calcium chloride, and lithium bromide) were used as phase-forming salts with poly(ethylene glycol) (PEG). We have tested the technique by dissolving carbonic anhydrase II aggregates and refolding the denatured protein in PEG-NaCI-NaSCN systems.

STRUCTURAL PROPERTIES OF MIXTURES OF HIGHLY ASYMMETRICAL ELECTROLYTES AND UNCHARGED PARTICLES USING THE HYPERNETTED CHAIN APPROXIMATION

We have solved the Ornstein–Zernike equation in the hypernetted chain (HNC) approximation for several mixtures of electrolytes and uncharged hard spheres. The mixtures that we studied range from a simple restricted primitive model plus hard spheres of the same size to highly asymmetric electrolytes plus hard spheres of different sizes. We monitored the radial distribution function and thermodynamic properties. We find that the presence of neutral particles changes the nature of the interaction between the charged particles. We also find a strong correlation between nonelectrostatic and electrostatic contributions to the free energy of the mixtures. Potential applications of this approach to the study of protein solubility in solutions of nonionic polymers are discussed.

Purification of human antibodies from transgenic corn using aqueous two-phase systems.

A recombinant human antibody expressed in corn was purified using aqueous two‐phase extraction. The antibody was an immunoglobulin G fully unglycosylated. Using systems of different compositions and/or pHs in each of one or two partitioning stages followed by one more stage in which the antibody was precipitated at the liquid/liquid interface facilitated the removal of different impurities in each stage. The best system yields a product 72% pure (22‐fold purification) with a yield of 49%. The optimum extraction was done in two partitioning stages followed by an interfacial precipitation stage using poly(ethylene)glycol/potassium phosphate systems. NaCl was added to the first stage to eliminate large molecular weight impurities. The pH in the first stage was kept at 6 but a pH of 8 was used in the second stage and in the precipitation stage.

Monte Carlo simulations of peptide adsorption on solid surfaces (Monte Carlo simulations of peptide adsorption)

Monte Carlo simulations (MC) were used to study the adsorption of a negatively charged peptide (ASP-ASP-ILE-ILE-ASP-ASP-ILE-ILE) dissolved in water onto charged surfaces and in vacuum onto neutral surfaces. When the peptide was placed between two charged surfaces, it always adsorbed sideways onto the positively charged surface even when it was initially positioned at the negatively charged one. The structure of water around the peptide significantly changed upon adsorption. The peptide adsorbed on a neutral surface heads-on. The subsequent addition of water does not seem to affect the location and orientation of the peptide at the surface. Our results indicate that dehydration of the peptide upon adsorption always happens to the residues that are closest to the surface independently of the surface’s charge.

Distribution of Cells between Solid/Liquid and Liquid/Liquid Interfaces

The use of aqueous two‐phase systems (ATPSs) and each systemapos;s individual phase‐forming species to prevent Streptococcus sanguis attachment onto hydroxyapatite discs was explored. The strategy that we followed was to attach the cells to a solid surface in the presence of an additional interface. Conditions under which, simultaneously, the phase‐forming species form two phases and the cells proliferate were identified. Growth curves were constructed in the presence of various polymers and salts commonly used to prepare ATPSs. Several aqueous two‐phase systems were selected such that bacterial growth was comparable to that observed in pure medium. Cells were allowed to attach to hydroxyapatite discs for 7 days in the presence of varying concentrations of media, media with polymer, media with salt, and media with ATPS. Streptococcus sanguis attachment to the disks was evaluated by scanning electron microscopy. The addition of a PEG/Na2SO4 ATPS to high concentrations of yeast‐tryptone (YT) media (>65%) and of a PEG/MgSO4 ATPS to nutrient‐limited media reduces surface coverage of S. sanguis to less than 10%. Comparison of the attachment levels for the systems containing PEG/Na2SO4 to media containing the individual phase‐forming species and to the YT reference systems indicated that nutrient availability did not affect attachment.

Effect of glycosylation on the partition behavior of a human antibody in aqueous two-phase systems

Human proteins are expressed in some hosts wrongly glycosylated or nonglycosylated. Although it is accepted that glycosylation contributes to the stability of the protein in solution, the effect of glycosylation on the stability of human antibodies is not fully understood. In this work, we present solubility studies of two human antibodies that have the same primary structure but different glycosylation pattern. The studies were done by monitoring the partitioning behavior of both proteins in a series of aqueous two‐phase systems at and away the isoelectric point of the proteins and at different temperatures. Our studies show that in the absence of direct electrostatic forces, the partitioning behavior of the antibodies depends on the presence or absence of the polysaccharide chains. Overall, the nonglycosylated protein is less soluble than the glycosylated one. The potential of aqueous two‐phase systems for the separation of the glycosylated and nonglycosylated proteins was also explored. A simple series of extractions seems to be enough to separate the glycosylated variety from the nonglycosylated one at high purity but low yields.

Purification and Characterization of Crystallins by Aqueous Two-Phase Extraction

Crystallins are a family of water-soluble proteins that constitute up to 90% of the water-soluble proteins in mammalian eye lenses. We present in this paper an alternative purification method for these proteins using polyethylene glycol/dextran aqueous two-phase extraction. Under the appropriate conditions, we were able to recover the γ-crystallin fraction essentially free of the remaining proteins. High concentrations of salt at a neutral pH maximize the recovery of γ-crystallins in the top phase and minimize the contamination by the other proteins present in the lenses. The proposed protocol decreases the separation time by about 50% The complex partition behavior observed for these proteins reflects a delicate balance between protein/phase-forming species (various polymers and salts) and protein/protein interactions. This is evidenced in part, by the role played by the largest proteins in this group as a “pseudo” phase-forming species.

Protein Refolding Using Chaotropic Aqueous Two-Phase Systems

In this report we explore novel aqueous two-phase systems and their applications to the recovery of recombinant proteins, which are expressed as insoluble and inactive protein aggregates (inclusion bodies).