Electrospray Differential Mobility Analysis Research Articles

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

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 reflectance-Fourier 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 × 10(6) to 1.65 × 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 α-helix content and more open structures (β-sheet, random, or expanded).

Physical Characterization of Icosahedral Virus Ultra Structure, Stability, and Integrity Using Electrospray Differential Mobility Analysis

We present a rapid and quantitative method to physically characterize the structure and stability of viruses. Electrospray differential mobility analysis (ES-DMA) is used to determine the size of capsomers (i.e., hexons) and complete capsids. We demonstrate how to convert the measured mobility size into the icosahedral dimensions of a virus, which for PR772 become 68.4 nm for vertex-to-vertex, 54.4 nm for facet-to-facet, and 58.2 nm for edge-to-edge lengths, in reasonable agreement with dimensions from transmission electron microscopy for other members of the family Tectiviridae (e.g., PRD1). These results indicate ES-DMA's mobility diameter most closely approximates the edge-to-edge length. Using PR772's edge length (36.0 nm) and the size of the major capsid hexon (≈8.4 nm) from ES-DMA with icosahedral geometry, PR772's T = 25 symmetry is confirmed and the number of proteins in the capsid shell is determined. We also demonstrate the use of ES-DMA to monitor the temporal disintegration of PR772, the thermal degradation of PP7, and the appearance of degradation products, essential to viral stability assays. These results lay groundwork essential for the use of ES-DMA for a variety of applications including monitoring of vaccine and gene therapy vector products, confirmation of viral inactivation, and theoretical studies of self-assembling macromolecular structures.

Probing the Nucleus Model for Oligomer Formation during Insulin Amyloid Fibrillogenesis

We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers—exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the β-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a β-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy.

Structural Analysis of Soft Multicomponent Nanoparticle Clusters

Quantitative techniques are essential to analyze the structure of soft multicomponent nanobioclusters. Here, we combine electrospray differential mobility analysis (ES-DMA), which rapidly measures the size of the entire cluster, with transmission electron microscopy (TEM), which detects the hard components, to determine the presence and composition of the softer components. Coupling analysis of TEM and ES-DMA derived data requires the creation and use of analytical models to determine the size and number of constituents in nanoparticle complexes from the difference between the two measurements. Previous ES-DMA analyses have been limited to clusters of identical spherical particles. Here, we dramatically extend the ES-DMA analysis framework to accommodate more challenging geometries, including protein corona-coated nanorods, clusters composed of heterogeneously sized nanospheres, and nanobioclusters composed of both nanospheres and nanorods. The latter is critical to determining the number of quantum dots attached to lambda (λ) phage, a key element of a rapid method to detect bacterial pathogens in environmental and clinical samples.

Packing and Size Determination of Colloidal Nanoclusters

Here we demonstrate a rapid and quantitative means to characterize the size and packing structure of small clusters of nanoparticles in colloidal suspension. Clustering and aggregation play important roles in a wide variety of phenomena of both scientific and technical importance, yet characterizing the packing of nanoparticles within small clusters and predicting their aerodynamic size remains challenging because available techniques can lack adequate resolution and sensitivity for clusters smaller than 100 nm (optical techniques), perturb the packing arrangement (electron microscopies), or provide only an ensemble average (light scattering techniques). In this article, we use electrospray-differential mobility analysis (ES-DMA), a technique that exerts electrical and drag forces on the clusters, to determine the size and packing of small clusters. We provide an analytical model to determine the mobility size of various packing geometries based on the projected area of the clusters. Data for clusters aggregated from nominally 10 nm gold particles and nonenveloped viruses of various sizes show good agreement between measured and predicted cluster sizes for close-packed spheres.

Competitive Adsorption of Thiolated Polyethylene Glycol and Mercaptopropionic Acid on Gold Nanoparticles Measured by Physical Characterization Methods

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 (intensity average) due to the formation of SH-PEG coatings on Au-NPs was 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 the 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 that 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.

Length Distribution of Single‐Walled Carbon Nanotubes in Aqueous Suspension Measured by Electrospray Differential Mobility Analysis

The first characterization of the length distribution of single-walled carbon nanotubes (SWCNT) dispersed in a liquid by electrospray differential mobility analysis (ES-DMA) is presented. Although an understanding of geometric properties of SWCNTs, including length, diameter, aspect ratio, and chirality, is essential for commercial applications, rapid characterization of nanotube length distributions remains challenging. Here the use of ES-DMA to obtain length distributions of DNA-wrapped SWCNTs dispersed in aqueous solutions is demonstrated. Lengths measured by ES-DMA compare favorably with those obtained from multiangle light scattering, dynamic light scattering, field flow fractionation with UV/vis detection, and atomic force microscopy, validating ES-DMA as a technique to measure SWCNTs of <250 nm in length. The nanotubes are previously purified and dispersed by wrapping with oligomeric DNA in aqueous solution and centrifuging to remove bundles and amorphous carbon. These dispersions are particularly attractive due to their amenability to bulk processing, ease of storage, high concentration, compatibility with biological and high-throughput manufacturing environments, and for their potential applications ranging from electronics and hydrogen-storage vessels to anticancer agents.

Quantitative characterization of quantum dot‐labeled lambda phage for Escherichia coli detection

We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (lambda) phage, and Escherichia coli are examined by several cross-validated methods. Flow and image-based cytometry clarify fluorescent labeling of bacteria, with image-based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image-based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4-17 QDs) and show that lambda phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal-biological conjugates.

Particle concentration measurement of virus samples using electrospray differential mobility analysis and quantitative amino acid analysis

Virus reference materials are needed to develop and calibrate detection devices and instruments. We used electrospray differential mobility analysis (ES-DMA) and quantitative amino acid analysis (AAA) to determine the particle concentration of three small model viruses (bacteriophages MS2, PP7, and ϕX174). The biological activity, purity, and aggregation of the virus samples were measured using plaque assays, denaturing gel electrophoresis, and size-exclusion chromatography. ES-DMA was developed to count the virus particles using gold nanoparticles as internal standards. ES-DMA additionally provides quantitative measurement of the size and extent of aggregation in the virus samples. Quantitative AAA was also used to determine the mass of the viral proteins in the pure virus samples. The samples were hydrolyzed and the masses of the well-recovered amino acids were used to calculate the equivalent concentration of viral particles in the samples. The concentration of the virus samples determined by ES-DMA was in good agreement with the concentration predicted by AAA for these purified samples. The advantages and limitations of ES-DMA and AAA to characterize virus reference materials are discussed.

Insulin oligomers: what and where are they?

Recent reports have suggested that (i) oligomers, rather than amyloid fibrils, are toxic and possibly cause disease, and (ii) disease progression occurs in the absence of amyloid fibrils. Using a model amyloid protein, the hormone insulin, we have studied the reaction kinetics pathway from folded native hexamers through oligomers to fibrils. Here we report on several experimental methods to detect and follow oligomers during the prefibrillation stage. Small angle x‐ray and neutron scattering, electrospray differential mobility analysis, ultracentrifugation, circular dichroism and size exclusion chromatography were used. Surprisingly, none of these methods detected measurable concentrations of oligomers. This finding suggests that fibrillogenesis may occur without successive transitions through protofibril nuclei or, more probably, with a nuclei‐induced reaction at very low nuclei concentrations. Other experiments have directly (cryo‐electron microscopy) and indirectly (seeding experiment) detected the presence of aggregated structures. The challenge now is to find a separation technique that will selectively isolate the different oligomers and aggregated structures, critical for determining the molecular basis of amyloid‐related diseases. This work was funded by the U.S. Department of Energy, DOE (DE‐FG02‐07ER46429).

Determination of protein aggregation with differential mobility analysis: Application to IgG antibody

Here we describe the use of electrospray differential mobility analysis (ES-DMA), also known as gas-phase electrophoretic mobility molecular analysis (GEMMA), as a method for measuring low-order soluble aggregates of proteins in solution. We demonstrate proof of concept with IgG antibodies. In ES-DMA, aqueous solutions of the antibody protein are electrosprayed and the various aerosolized species are separated according to their electrophoretic mobility using a differential mobility analyzer. In this way, complete size distributions of protein species present from 3 to 250 nm can be obtained with the current set up, including distinct peaks for IgG monomers to pentamers. The sizes of the IgG and IgG aggregates measured by DMA were found to be in good agreement with those calculated from simple models, which take the structural dimensions of IgG from protein crystallographic data. The dependence of IgG aggregation on the solution concentration and ionic strength was also examined, and the portion of aggregates containing chemically crosslinked antibodies was quantified. These results indicate that ES-DMA holds potential as a measurement tool to study protein aggregation phenomena such as those associated with antibody reagent manufacturing and protein therapeutics.

Quantitative characterization of virus‐like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy

Here we characterize virus-like particles (VLPs) by three very distinct, orthogonal, and quantitative techniques: electrospray differential mobility analysis (ES-DMA), asymmetric flow field-flow fractionation with multi-angle light scattering detection (AFFFF-MALS) and transmission electron microscopy (TEM). VLPs are biomolecular particles assembled from viral proteins with applications ranging from synthetic vaccines to vectors for delivery of gene and drug therapies. VLPs may have polydispersed, multimodal size distributions, where the size distribution can be altered by subtle changes in the production process. These three techniques detect subtle size differences in VLPs derived from the non-enveloped murine polyomavirus (MPV) following: (i) functionalization of the surface of VLPs with an influenza viral peptide fragment; (ii) packaging of foreign protein internally within the VLPs; and (iii) packaging of genomic DNA internally within the VLPs. These results demonstrate that ES-DMA and AFFFF-MALS are able to quantitatively determine VLP size distributions with greater rapidity and statistical significance than TEM, providing useful technologies for product development and process analytics.

Gas-Phase Ion-Mobility Characterization of SAM-Functionalized Au Nanoparticles

We present results of a systematic examination of functionalized gold nanoparticles (Au-NPs) by electrospray-differential mobility analysis (ES-DMA). Commercially available, citrate-stabilized Au colloid solutions (10-60 nm) were sized using ES-DMA, from which changes in particle size of less than 0.3 nm were readily discerned. It was found that the formation of salt particles and the coating of Au-NPs by salt during the electrospray process can interfere with the mobility analysis, which required the development of sample preparation and data correction protocols to extract correct values for the Au-NP size. Formation of self-assembled monolayers (SAMs) of alkanethiol molecules on the Au-NP surface was detected from a change in particle mobility, which could be modeled to extract the surface packing density of SAMs. A gas-phase temperature-programmed desorption (TPD) kinetic study of SAMs on Au-NPs found the data to be consistent with a second-order Arrhenius-based rate law, yielding an Arrhenius factor of 1.0 x 10 (11) s (-1) and an activation energy approximately 105 kJ/mol. For the size range of SAM-modified Au-NP we considered, the effect of surface curvature on the energetics of binding of carboxylic acid terminated SAMs is evidently negligible, with binding energies determined by TPD agreeing with those reported for the same SAMs on planar surfaces. This study suggests that the ES-DMA can be added to the tool set of characterization methods used to study the structure and properties of coated nanoparticles.

Quantifying the Surface Coverage of Conjugate Molecules on Functionalized Nanoparticles

Here we present a method to determine the surface coverage or surface density of biological molecules conjugated to nanoparticle surfaces. Electrospray-differential mobility analysis (ES-DMA) is used to determine a coating thickness by measuring the change in the size of gold nanoparticles before and after modification with thiol-derivatized single-stranded DNA. The DNA surface coverage is then obtained from the coating thickness through the use of a simple random coil model. The method requires neither fluorescent tagging nor calibration curves. We believe ES-DMA to be a broadly applicable nanometrology tool for the characterization of biologically conjugated nanoparticles.