ABSTRACT:The adsorption and conformation of bovine serum albumin (BSA) on gold nanoparticles (AuNPs) were interrogated both qualitatively and quantitatively via complementary physicochemical characterization methods. Dynamic light scattering (DLS), asymmetric-flow field flow fractionation (AFFF), fluorescence spectrometry, and attenuated total reflectanceFourier transform infrared (ATR-FTIR) spectroscopy were combined to characterize BSA-AuNP conjugates under fluid conditions, while conjugates in the aerosol state were characterized by electrospray-differential mobility analysis (ES-DMA). The presence of unbound BSA molecules interferes with DLS analysis of the conjugates, particularly as the AuNP size decreases (i.e., below 30 nm in diameter). Under conditions where the γ value is high, where γ is defined as the ratio of scattering intensity by AuNPs to the scattering intensity by unbound BSA, DLS size results are consistent with results obtained after fractionation by AFFF. Additionally, the AuNP hydrodynamic size exhibits a greater proportional increase due to BSA conjugation at pH values below 2.5 compared with less acidic pH values (3.4-7.3), corresponding with the reversibly denatured (E or F form) conformation of BSA below pH 2.5. Over the pH range from 3.4 to 7.3, the hydrodynamic size of the conjugate is nearly constant, suggesting conformational stability over this range. Because of the difference in the measurement environment, a larger increase of AuNP size is observed following BSA conjugation when measured in the wet state (i.e., by DLS and AFFF) compared to the dry state (by ES-DMA). Molecular surface density for BSA is estimated based on ES-DMA and fluorescence measurements. Results from the two techniques are consistent and similar, but slightly higher for ES-DMA, with an average adsorbate density of 0.015 nm -2 . Moreover, from the change of particle size, we determine the extent of adsorption for BSA on AuNPs using DLS and ES-DMA at 21°C, which show that increasing the concentration of BSA increases the measured change in AuNP size. Using ES-DMA, we observe that the BSA surface density reaches 90% of saturation at a solution phase concentration between 10 and 30 μmol/L, which is roughly consistent with fluorescence and ATR-FTIR results. The equilibrium binding constant for BSA on AuNPs is calculated by applying the Langmuir equation, with resulting values ranging from 0.51 Â 10 6 to 1.65 Â 10 6 L/mol, suggesting a strong affinity due to bonding between the single free exterior thiol on N-form BSA (associated with a cysteine residue) and the AuNP surface. Moreover, the adsorption interaction induces a conformational change in BSA secondary structure, resulting in less R-helix content and more open structures (β-sheet, random, or expanded).

Studies designed to investigate the environmental or biological interactions of nanoscale materials frequently rely on the use of ultrasound (sonication) to prepare test suspensions. However, the inconsistent application of ultrasonic treatment across laboratories, and the lack of process standardization can lead to significant variability in suspension characteristics. At present, there is widespread recognition that sonication must be applied judiciously and reported in a consistent manner that is quantifiable and reproducible; current reporting practices generally lack these attributes. The objectives of the present work were to: (i) Survey potential sonication effects that can alter the physicochemical or biological properties of dispersed nanomaterials (within the context of toxicity testing) and discuss methods to mitigate these effects, (ii) propose a method for standardizing the measurement of sonication power, and (iii) offer a set of reporting guidelines to facilitate the reproducibility of studies involving engineered nanoparticle suspensions obtained via sonication.

Novel physicochemistries of engineered nanomaterials (ENMs) offer considerable commercial potential for new products and processes, but also the possibility of unforeseen and negative consequences upon ENM release into the environment. Investigations of ENM ecotoxicity have revealed that the unique properties of ENMs and a lack of appropriate test methods can lead to results that are inaccurate or not reproducible. The occurrence of spurious results or misinterpretations of results from ENM toxicity tests that are unique to investigations of ENMs (as opposed to traditional toxicants) have been reported, but have not yet been systemically reviewed. Our objective in this manuscript is to highlight artifacts and misinterpretations that can occur at each step of ecotoxicity testing: procurement or synthesis of the ENMs and assessment of potential toxic impurities such as metals or endotoxins, ENM storage, dispersion of the ENMs in the test medium, direct interference with assay reagents and unacknowledged indirect effects such as nutrient depletion during the assay, and assessment of the ENM biodistribution in organisms. We recommend thorough characterization of initial ENMs including measurement of impurities, implementation of steps to minimize changes to the ENMs during storage, inclusion of a set of experimental controls (e.g., to assess impacts of nutrient depletion, ENM specific effects, impurities in ENM formulation, desorbed surface coatings, the dispersion process, and direct interference of ENM with toxicity assays), and use of orthogonal measurement methods when available to assess ENMs fate and distribution in organisms.

Surface-sensitive quantitative studies of competitive molecular adsorption on nanoparticles were conducted using a modified attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy method. Adsorption isotherms for thiolated poly(ethylene glycol) (SH-PEG) on gold nanoparticles (AuNPs) as a function of molecular mass (1, 5, and 20 kDa) were characterized. We find that surface density of SH-PEG on AuNPs is inversely proportional to the molecular mass (M(m)). Equilibrium binding constants for SH-PEG, obtained using the Langmuir adsorption model, show the binding affinity for SH-PEG is proportional to M(m). Simultaneous competitive adsorption between mercaptopropionic acid (MPA) and 5 kDa SH-PEG (SH-PEG5K) was investigated, and we find that MPA concentration is the dominant factor influencing the surface density of both SH-PEG5K and MPA, whereas the concentration of SH-PEG5K affects only SH-PEG5K surface density. Electrospray differential mobility analysis (ES-DMA) was employed as an orthogonal characterization technique. ES-DMA results are consistent with the results obtained by ATR-FTIR, confirming our conclusions about the adsorption process in this system. Ligand displacement competitive adsorption, where the displacing molecular species is added after completion of the ligand surface binding, was also interrogated by ATR-FTIR. Results indicate that for SH-PEG increasing M(m) yields greater stability on AuNPs when measured against displacement by bovine serum albumin (BSA) as a model serum protein. In addition, the binding affinity of BSA to AuNPs is inhibited for SH-PEG conjugated AuNPs, an effect that is enhanced at higher SH-PEG M(m) values.

Competitive adsorption kinetics between thiolated polyethylene glycol (SH-PEG) and mercaptopropionic acid (MPA) on gold nanoparticles (Au-NPs) were studied using a prototype physical characterization approach that combines dynamic light scattering (DLS) and electrospray differential mobility analysis (ES-DMA). The change in hydrodynamic particle size (intensityaverage) due to the formation of SH-PEG coatings on Au-NPs were measured by DLS in both two component (Au-NP + MPA or Au-NP + SH-PEG) and three component (Au-NP +MPA + SH-PEG) systems. ES-DMA was employed to quantify the surface coverage of SH-PEG and establish a correlation between surface coverage and the change in particle size measured by DLS. A change in equilibrium binding constant for SH-PEG on Au-NPs at various concentrations of SH-PEG and MPA showed that the presence of MPA reduced the binding affinity of SH-PEG to the Au-NP surface. Kinetic studies showed SH-PEG was desorbed from the Au-NP surface following a second-order desorption model after subsequently introducing MPA. The desorption rate constant of SH-PEG from the Au-NP surface by MPA displacement was strongly affected by the concentration of MPA and the excess SH-PEG in solution.

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Suspensions of natural graphite particles were prepared in an aqueous medium using carboxymethyl cellulose ͑CMC͒ and emulsified styrene-butadiene copolymer latex as part of an environmentally friendly fabrication process for graphite anodes ͑negative electrodes͒ intended for application in Li-ion batteries. Suspensions were characterized by adsorption isotherms, electroacoustic measurements, rheology and sedimentation tests, at two different degrees of carboxymethyl substitution ͑DS͒ on CMC. A lower DS value ͑0.7͒ resulted in greater uptake of CMC on graphite compared with a higher DS value ͑1.28͒. This was attributed to attractive hydrophobic interactions associated with the lower carboxymethyl substitution. The greater adsorption for DS = 0.7 correlates with lower relative viscosity in concentrated graphite suspensions, a higher adhesion strength with a copper substrate, and a greater retention of discharge capacity after cycling. The effect of DS is attributed to differences in the aqueous dispersion properties and stability of graphite suspensions. Based on these results, we fabricated high-capacity graphite negative electrodes characterized by gravimetric and volumetric energy densities of greater than 340 mAh/g and 560 mAh/cm 3 , respectively. This formulation also led to improved adhesion strength, giving the as-fabricated cell an attractive cycle life greater than 90% of initial discharge capacity after 200 cycles.

The role of poly(acrylic acid) (PAA) in the dispersion of silicon nitride suspensions was investigated experimentally. The effects of concentration, relative molecular mass, and suspension pH were evaluated. The ionization of PAA was characterized by potentiometric titration and indicated a pH-dependent conformational transition. The isoelectric point for silicon nitride decreased from pH 6.3 to pH 3 as the PAA concentration increased, roughly independent of relative molecular mass. A broadening of the stability region for silicon nitride was attributed to this effect. Redispersion in alkaline media, subsequent to destabilization by surface-charge neutralization, improved following preadsorption of PAA in acidic media. This effect was attributed to formation of a protective polymer adlayer on the surface, which prevented primary minimum aggregation; an electrosteric contribution also may be present. The influence of free polymer on the suspension properties also is discussed.