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).

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.