Absolute quantification method for protein concentration.

A fast and accurate assay to determine the absolute concentration of proteins is described based on direct measurement of droplet entrapped oligomer formation in electrospray. Here we demonstrate the approach using electrospray differential mobility analysis (ES-DMA), which can distinguish monomers and dimers from higher order oligomers. A key feature of the method is that it allows determination of the absolute number concentration of proteins eliminating the need for protein-specific calibration.

Characterization of polydopamine thin films deposited at short times by autoxidation of dopamine.

Current interest in melanin films derived from the autoxidation of dopamine stems from their use as a universal adhesion layer. Here we report chemical and physical characterization of polydopamine films deposited on gold surfaces from stirred basic solutions at times ranging from 2 to 60 min, with a focus on times ≤10 min. Data from Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and electrochemical methods suggest the presence of starting (dopamine) and intermediate (C=N-containing tautomers of quinone and indole) species in the polydopamine films at all deposition times.

Competitive adsorption-desorption of IgM monomers-dimers on silica and modified silica surfaces.

Understanding competitive adsorption-desorption of proteins onto surfaces is an important area of research in food processing and biomedical engineering. Here, we demonstrate, how electrospray-differential mobility analysis that has been traditionally used for characterizing bionanoparticles, can be used for quantifying complex competitive adsorption-desorption of oligomeric proteins or multiprotein systems using monomers and dimers of IgM as a model example onto silica and modified silica surfaces. Using ES-DMA, we show that IgM dimers show a preference to stay adsorbed to different surfaces although monomers adsorb more easily and desorption rates of monomers and dimers of IgM are surface-type-dependent and are not significantly affected by shear.

Quantifying ligand adsorption to nanoparticles using tandem differential mobility mass analysis.

Although electrospray-differential mobility analyzers (ES-DMA) have been previously employed to characterize ligand binding to nanoparticles, absolute quantification of surface coverage can be inaccurate at times because of ligand conformational effects. In this Letter, we report a quantitative technique by in-flight coupling of a particle mass analyzer (APM) with ES-DMA, thus enabling a direct quantitative analysis of mass independent of particle size, material, morphology and conformation. We demonstrate the utility of ES-DMA-APM by studying two model complex systems (gold nanoparticle-bovine serum albumin and polystyrene bead-antibody) as a function of concentration and pH.

Protein adsorption-desorption on electrospray capillary walls--no influence on aggregate distribution.

Adsorbed proteins on walls of glass capillaries used for electrospray (ES) can desorb and potentially affect size distributions and, thus, quantification of aggregates of proteins. In this study we use differential mobility analysis (DMA) to investigate the size distribution of various proteins eluting from bare and passivated glass capillaries. We found no significant differences in aggregate distributions from unpassivated capillaries at 'steady state' when compared to aggregate distributions from passivated capillaries implying that desorbing proteins do not influence protein aggregate distribution.

A lectin-based gold nanoparticle assay for probing glycosylation of glycoproteins.

We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles.

Electrospray-differential mobility analysis of bionanoparticles.

Electrospray-differential mobility analysis (ES-DMA) is a versatile technique used to aerosolize bionanoparticles and measure their electrical mobility at ambient conditions. ES-DMA is similar to electrospray-mass spectrometry (ES-MS), but measures the effective particle size, rather than mass. It has a wide range of applications and nominally can be used to characterize biomolecules and nanoparticles ranging in size from a few nanometers (~3 nm) to several hundred nanometers, to obtain multimodal size distributions in minutes.

Electrospray-differential mobility analysis as an orthogonal tool to size-exclusion chromatography for characterization of protein aggregates.

The biopharmaceutical industry characterizes and quantifies aggregation of protein therapeutics using multiple analytical techniques to cross-validate results. Here, we demonstrate the use of electrospray-differential mobility analysis (ES-DMA), a gas-phase and atmospheric pressure ion-mobility method for characterizing protein aggregates. Two immunoglobulin Gs are systematically heat treated to induce aggregation and characterized using size-exclusion chromatography (SEC) and ES-DMA.

Method for determining the absolute number concentration of nanoparticles from electrospray sources.

We have developed a simple, fast, and accurate method to measure the absolute number concentration of nanoparticles in solution. The method combines electrospray differential mobility analysis (ES-DMA) with a statistical analysis of droplet-induced oligomer formation. A key feature of the method is that it allows determination of the absolute number concentration of particles by knowing only the droplet size generated from a particular ES source, thereby eliminating the need for sample-specific calibration standards or detailed analysis of transport losses.

Evaluation of electrospray differential mobility analysis for virus particle analysis: Potential applications for biomanufacturing.

The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines.

Characterizing the adsorption of proteins on glass capillary surfaces using electrospray-differential mobility analysis.

We quantify the adsorption and desorption of a monoclonal immunoglobulin-G antibody, rituxamab (RmAb), on silica capillary surfaces using electrospray-differential mobility analysis (ES-DMA). We first develop a theory to calculate coverages and desorption rate constants from the ES-DMA data for proteins adsorbing on glass capillaries used to electrospray protein solutions. This model is then used to study the adsorption of RmAb on a bare silica capillary surface.

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.

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.

Characterization of gold nanoparticles modified with single-stranded DNA using analytical ultracentrifugation and dynamic light scattering.

We report the characterization of gold nanoparticles modified with thiol-terminated single stranded DNA (ssDNA) using analytical ultracentrifugation. Dynamic light scattering was used to measure the diameter of bare and ssDNA modified gold nanoparticles to corroborate the predictions of our models. Sedimentation coefficients of nominally 10 and 20 nm diameter gold nanoparticles modified with thiol-terminated thymidine homo-oligonucleotides, 5-30 bases in length, were determined with analytical ultracentrifugation.

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.

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.

Selective binding of RNase B glycoforms by polydopamine-immobilized concanavalin A.

Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules.

A consensus rating method for small virus-retentive filters. I. Method development.

Virus filters are membrane-based devices that remove large viruses (e.g., retroviruses) and/or small viruses (e.

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.

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.

Adsorption behavior of DNA-wrapped carbon nanotubes on self-assembled monolayer surfaces.

We have examined the adsorption of DNA-wrapped single-walled carbon nanotubes (DNA-SWNTs) on hydrophobic, hydrophilic, and charged surfaces of alkylthiol self-assembled monolayers (SAMs) on gold. Our goal is to understand how DNA-SWNTs interact with surfaces of varying chemical functionality. These samples were characterized using reflection absorption FTIR (RAIRS), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy.

Synthesis and structural characterization of glucopyranosylamide films on gold.

Self-assembled monolayers (SAMs) of glucose derivatives on gold have been prepared from alpha- and beta-glucopyranosylamide derivatives. The glucosyl conjugates were synthesized stereoselectively via the in situ generation of glucosyl isoxazolines followed by treatment with thiopyridyl esters. The resulting film structures were characterized by atomic force microscopy, reflection Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy.

Independent control of grafting density and conformation of single-stranded DNA brushes.

We describe self-assembly of ssDNA brushes that exploits the intrinsic affinity of adenine nucleotides (dA) for gold surfaces. The grafting density and conformation of these brushes is deterministically controlled by the length of the anchoring dA sequences, even in the presence of thymine nucleotides (dT). We produce and characterize brushes of model block-oligonucleotides, d(T(m)-A(n)), with systematically varied lengths m and n of the thymine and adenine blocks [denoted d(T(m)) and d(A(n)), respectively].

Alkanethiols on platinum: multicomponent self-assembled monolayers.

We have studied the formation of self-assembled monolayers (SAMs) of n-alkanethiols on platinum thin films using X-ray photoelectron spectroscopy (XPS), reflection-absorption infrared spectroscopy (RAIRS), spectroscopic ellipsometry (SE), and contact angle (CA) measurements. Specifically, SAMs of 1-hexanethiol, 1-dodecanethiol, and 1-octadecanethiol were grown on polycrystalline Pt films, and the effects of Pt surface preparation, deposition conditions, and solvent treatments on the initial quality and stability of the monolayer in air were investigated. The SAMs prepared under ambient conditions on piranha-cleaned and UV/ozone-cleaned substrates were compared to monolayers formed on template-stripped Pt in an inert atmosphere.