Synthesis and characterization of NiSn dendrimer-encapsulated nanoparticles.

We report the synthesis and characterization of NiSn dendrimer-encapsulated nanoparticles (DENs) with sizes in the range of approximately 1.2 nm. These types of materials have potential applications in energy storage, and particles in the 1-3 nm size range are particularly attractive for this use. The NiSn DENs described here contain an average of 147 atoms and are encapsulated within hydrophobic, sixth-generation poly(amidoamine) dendrimers. DENs prepared using four different Ni/Sn ratios, along with monometallic Ni and Sn DENs, are described. To prevent oxidation, the synthesis was carried out under dry conditions in toluene. These bimetallic DENs were characterized by UV-vis spectroscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. The results indicate that the compositions of the nanoparticles reflect the ratio of Ni(2+) and Sn(2+) used in the first step of the synthesis; the NiSn nanoparticles remain encapsulated within the dendrimers, and when dry they have a degree of stability even after a short exposure to air.

Lysine-based zwitterionic molecular micelle for simultaneous separation of acidic and basic proteins using open tubular capillary electrochromatography.

In this work, a zwitterionic molecular micelle, poly-epsilon-sodium-undecanoyl lysinate (poly-epsilon-SUK), was synthesized and employed as a coating in open tubular capillary electrochromatography (OT-CEC) for protein separation. The zwitterionic poly-epsilon-SUK containing both carboxylic acid and amine groups can be either protonated or deprotonated depending on the pH of the background electrolyte; therefore, either an overall positively or negatively charged coating can be achieved. This zwitterionic coating allows protein separations in either normal or reverse polarity mode depending on the pH of the background electrolyte. The protein mixtures contained four basic proteins (lysozyme, cytochrome c, alpha-chymotrypsinogen A, and ribonuclease A) and six acidic proteins (myoglobin, deoxyribonuclease I, beta-lactoglobulin A, beta-lactoglobulin B, alpha-lactalbumin, and albumin). Protein separations were optimized specifically for acidic (reverse mode) and basic (normal mode) pH values. Varying the polymer thickness by changing the polymer and salt concentration had a great influence on protein resolution, while nearly all peaks were also baseline resolved in both modes using the optimized poly-epsilon-SUK coating concentration of 0.4% (w/v). Proteins in human sera were separated under optimized acidic and basic conditions in order to demonstrate the general utility of this coating. Nanoscale characterizations of the poly-epsilon-SUK micellar coatings on silicon surfaces were accomplished using atomic force microscopy (AFM) to gain insight into the morphology and thickness of the zwitterionic coating. The thickness of the polymer coating ranged from 0.9 to 2.4 nm based on local measurements using nanoshaving, an AFM-based method of nanolithography.

Mechanistic investigation of N-homocysteinylation-mediated protein-gold nanoconjugate assembly.

Herein we report the use of protein-gold nanoconjugate (PGNs) as probes for elucidating mechanistic events involved in protein homocystamide detection with gold nanoparticles (GNPs), as was previously reported by our laboratory. Three different PGN probes are synthesized by direct adsorption of cytochrome c, albumin, or human serum onto citrate-capped GNPs. The PGNs are subsequently purified and treated to confer N-homocysteinylation. Individual PGN systems are evaluated to assess the effect of modification on (1) surface plasmon resonance (SPR), (2) protein structural conformation, and (3) assembly-association. The degree of PGN assembly and colorimetric signal observed postmodification varies based on the type of conjugated protein. For example, results of time-resolved dynamic light scattering studies indicate that modification of cytochrome c-PGNs yields rapid formation of macroscopic nanoparticle assemblies that eventually precipitate from solution. In contrast, albumin and human serum PGNs exhibit higher stability toward modification. Additionally, findings from circular dichroism studies indicate significant modification-induced denaturation, which is what may initiate assembly via electrosteric destabilization of PGNs. The results of electrophoretic studies appear to confirm that the process of N-homocysteinylation-mediated PGN assembly culminates in covalent interparticle association by disulfide cross-linking among modified proteins.

Gold nanoparticle sensor for homocysteine thiolactone-induced protein modification.

Homocysteine thiolactone-induced protein modification (HTPM) is a unique post-translational protein modification that is recognized as an emergent biomarker for cardiovascular disease. HTPM involves the site-specific acylation of proteins at lysine residues by homocysteine thiolactone (HTL) to produce protein homocystamide, which has been found at elevated levels in patients with coronary heart disease. Herein, we report the development of a novel gold nanoparticle (GNP) biochemical sensor for detection of protein homocystamide in an in vitro serum protein-based model system. Human serum albumin (HSA) and human sera were subjected to HTPM in vitro to produce HSA-homocystamide or serum protein homocystamide, respectively, which was subsequently treated with citrate-capped GNPs. This GNP sensor typically provided instantaneous visual confirmation of HTPM in the protein model systems. Transmission electron microscopy images of the GNPs in the presence of HSA-homocystamide suggest that modification-directed nanoparticle assembly is the mechanism by which the biochemical sensor produces a colorimetric signal. The resultant nanoparticle-protein assembly exhibited excellent thermal and dilutional stability, which is expected for a system stabilized by chemisorption and intermolecular disulfide bonding. The sensor typically provided a linear response for modified human sera concentrations greater than approximately 5 mg/mL. The calculated limit of detection and calibration sensitivity for the method in human sera were 5.2 mg/mL and 13.6 AU . (microg/mL)-1, respectively.

Capillary electrophoretic screening for the inhibition of homocysteine thiolactone-induced protein oligomerization.

We report the first demonstration of rapid electrophoretic monitoring of homocysteine thiolactone-induced protein oligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovascular diseases. HTPO of the model protein bovine cytochrome c was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures were determined following separation using capillaries coated with the cationic polymer, poly(diallyldimethylammonium chloride). UV detection provided adequate sensitivity for the monitoring of higher order species, which exist at relatively low concentrations in the protein reaction mixture as compared to the monomeric species. Separations performed under standard injection conditions were optimized on the basis of applied voltage and sample denaturation conditions. Separations performed using short-end injection allowed for more rapid analyses, typically in less than 70 s. Relative errors for run-to-run migration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitro method to screen therapeutic agents for their ability to inhibit HTPO. Changes in peak area for monomer and aggregate species were used to assess HTPO inhibition as a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro. Rapid analysis times of approximately 1.5 min were achieved for inhibition screening.