Adsorption and Conformation of Serum Albumin Protein on Gold Nanoparticles Investigated Using Dimensional Measurements and in Situ Spectroscopic Methods

Abstract

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).

Electrospray–differential mobility analysis of bionanoparticles

“…In this dissertation, most bionanoparticles dealt with will be singly charged. The variation of this slip factor as a function of particle diameter is well established [20]. For particles smaller than the mean free path of gas (~ 66 nm at 293 K and 100 kPa) it is inversely proportional to D analyte , and for larger particles it decreases linearly with increasing particle diameter.…”

“…This work was followed over the next decade by several reports from Allmaier et al [11], Wick et al [12] and de la Mora et al [13] related to characterization of several other proteins, viruses and polymers. Starting in 2006 as seen in Figures 1.3A and 1.3B a dramatic increase in the number of publications and citations related to ES-DMA occurred with contributions from Biswas et al [14], Zachariah et al [15], Loo et al [16], Hogan et al [17], Pergantis et al [18], Pease et al [19] and Hackley et al [20]. Although developed primarily for size determination, with an increasing pool of users, several orthogonal utilities have been found for this technique a detailed discussion of which will be appear later in Chapter 3.…”

“…when sheath flow equals excess flow and analyte or aerosol flow in, equals monodispersed size selected flow out) the ratio of the sheath to the analyte flow in provides an ultimate measure of the theoretical resolution [20]. The transfer function can be further broadened by Brownian diffusion and the equations can get extremely complicated.…”

“…This study found that the coverage of SAMs was independent of the size of the AuNPs. BSA was recently used as a model protein to study AuNP-protein interactions as a function of concentration and pH [20]. To further model the complexity inside human plasma where multicomponent species may be present, competitive adsorption -desorption of SAMs and PEGs on AuNPs has been successfully studied [66,67].…”

Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity

“…These disulfide bridges are the basis for the compact heart-shaped structure at physical pH [25]. From acidic to basic conditions, BSA is known to undergoe several transitions including the extended (E), fast (F), normal (N), basic (B) and aged (A) forms.…”