Michael J. Tarlov

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. A uniform overlayer analysis of the XPS data indicates that film thickness increased linearly at short deposition times of ≤10 min. At deposition times ≥10 min, the films appeared largely continuous with surface roughness ≈ ≤ 2 nm, as determined by atomic force microscopy (AFM). Pinhole-free films, as determined by anionic redox probe measurements, required deposition times of 60 min or greater.

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(Tm-An), with systematically varied lengths m and n of the thymine and adenine blocks [denoted d(Tm) and d(An), respectively]. The hairpin conformation, dominant for self-complementary d(Tm-An) oligos in solution, is disrupted by the high preferential affinity of dA for gold surfaces. As a result, the d(Tm-An) oligos adsorb as a brush of d(T) strands immobilized via the d(A) blocks. Quantitative analysis by FTIR spectroscopy and x-ray photoelectron spectroscopy (XPS) reveals a unique feature of DNA immobilization via d(A) blocks: The surface density of dA nucleotides is close to saturation and is nearly independent of d(A) block length. Accordingly, the lateral spacing (grafting density) of the d(T) blocks is determined by the length of the d(A) blocks. The d(T) blocks extend away from the surface in a brush-like conformation at a lateral spacing 2–3 times larger (a grafting density 5–10 times lower) than in analogous films immobilized via standard thiol linkers. This combination of brush-like conformation and low saturation grafting density is expected to increase the efficiency of DNA hybridization at surfaces. Therefore, immobilization via d(A) blocks offers a method of producing DNA brushes with controlled properties for applications in biotechnology and nanotechnology.

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. Biotechnol. Bioeng. 2008;101: 1214–1222.

Sputtered thin-film pH electrodes of platinum, palladium, ruthenium, and iridium oxides

Abstract Thin-film metal oxides have been investigated for use as pH electrodes. These materials may have potential for measuring pH under conditions that are not favorable for glass electrodes. Reactive sputtering of platinum, palladium, ruthenium, and iridium metal targets in argon-oxygen atmospheres is used to produce 1 μm thick electrodes on alumina and silicon substrates. The structure of the deposits is determined and compared to the fully crystalline structure developed after annealing at 420 °C. The pH response is measured for pH 2-11 versus a glass electrode. Exposures of up to 24 h at pH 2–11 are used to study the stability under harsh conditions. Palladium and platinum oxides are found to be less stable than ruthenium oxide. X-ray photoelectron spectroscopy studies are used to determine the surface chemical state and verify the stoichiometry of the sensing surface. The stability of thin-film RuO 2 indicates the usefulness of further testing at high temperatures and wider pH ranges.

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. Biotechnol. Bioeng. 2009; 102: 845–855.

Mechanistic and response studies of iridium oxide pH sensors

Abstract Results are presented on the pH-potential response of iridium oxide films prepared by d.c. magnetron reactive sputtering. Freshly deposited films exhibit a nearly Nernstian response to pH and little hysteresis. The redox sensitivity of films prepared by sputtering in water-saturated oxygen and annealed in an oxygen atmosphere has been examined as well. In addition, methods are described for preparing model iridium oxide sensor surfaces for ultrahigh vacuum surface analytical studies. Stoichiometric IrO 2 -like surfaces are shown to be relatively inert to gas phase water. Hydroxylation of IrO 2 -like surfaces can be induced, however, by r.f. water plasma treatment.

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. Although both the ES and the DMA are mature technologies, they are finding increased use in combination to characterize particles in liquids. In this paper, we review ES-DMA, and how it has recently been used to characterize bionanoparticles such as polymers, proteins, viruses, bacteriophages and nanoparticle-biomolecule conjugates.

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. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the alpha-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the alpha-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms.

Surface plasmon microscopy of biotin-streptavidin binding reactions on UV-photopatterned alkanethiol self-assembled monolayers

We report surface plasmon imaging of streptavidin binding to photopatterned biotinylated alkanethiol self-assembled monolayers (SAMs) on gold. Micrometer-scale patterns of a mixed biotin- and hydroxyl-terminated monolayer were formed in an inert, hydroxy-terminated alkanethiol monolayer using a UV-photopatterning procedure. Using surface plasmon microscopy, contrast is readily observed between the mixed biotin- and hydroxy-terminated SAM region after specific binding of streptavidin has occurred and the pure hydroxy-terminated region where nonspecific binding of streptavidin is negligible. Surface plasmon microscopy was also able to monitor in situ and in real time the binding of streptavidin to the patterned SAMs. The ability of surface plasmon microscopy to detect and spatially resolve 2-dimensional monolayer binding events may prove useful in diagnostic applications involving the parallel interrogation at surface biomolecular arrays.

Atmospheric pressure microplasmas for modifying sealed microfluidic devices

A dc microdischarge technique for the chemical modification of microchannel walls is reported. In this method, an atmospheric pressure plasma is ignited directly in the channels of assembled microfluidic devices. Our results show that strongly hydrophilic or hydrophobic properties are imparted to the walls of polystyrene microchannels by the appropriate selection of a plasma gas. This localized tuning of the surface and wetting properties is expected to be useful in the manufacture of microfluidic channels in a variety of substrates. Small area x-ray photoelectron spectroscopy was used to identify oxygen and fluorine on polystyrene surfaces resulting from the plasma treatments.