Duy K. Pham

Effects of polymer properties on laser ablation behaviour

An investigation aiming to seek a correlation between ablation rates and various polymer thermal properties, based on experimental ablation data generated for 14 polymers commonly used in microfluidics, is presented. A statistical analysis was carried out for laser fluence against various polymer descriptors and/or their combinations. The results of the analysis show a relatively high correlation coefficient of 0.82 for polymer ablation data when we compare fluence against the product of ablation rate and the difference between the glass transition temperature and room temperature. The effects of polymer properties are also illustrated by an investigation of ablation behaviour of DNQ/novolak thin films, which had been exposed to different levels of UV radiation prior to laser ablation, using atomic force microscopy. The surface characteristics of the thin films following laser irradiation are discussed in terms of differences in laser absorption and the glass transition temperature of the films. The results are consistent with the glass transition temperature being a critical factor affecting laser/polymer interaction.

A comparative study between the adsorption and covalent binding of human immunoglobulin and lysozyme on surface-modified poly(tert-butyl methacrylate).

The adsorption and covalent immobilization of human immunoglobulin (HIgG) and lysozyme (LYZ) on surface-modified poly(tert-butyl methacrylate) PtBMA films have been evaluated using x-ray photoelectron spectroscopy (XPS), ellipsometry and atomic force microscopy (AFM). Surface modification of PtBMA (UV irradiation) afforded surfaces suitable for both the physical and covalent attachment of proteins. The XPS and ellipsometry results showed good correlation in terms of variable-dense/thickness protein layer formation between physisorbed and covalently bound proteins. The amount of physisorbed HIgG ranged from 23.0 +/- 1.6 ng mm(2) on PtBMA, with corresponding film thicknesses 17.0 +/- 1.2 nm. Covalent immobilization mediated through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) coupling chemistry, afforded 5.6-8 ng mm(2) of HIgG with a corresponding thickness of 5.9 +/- 0.6 nm on PtBMA. The attachment of LYZ to modified PtBMA surface was similarly translated, where adsorption yielded up to 15 ng mm(2), while covalent immobilization afforded typically 7-8 ng mm(2). The thickness of the adsorbed LYZ protein layer was 11.0 +/- 3.2 nm (PtBMA), suggesting the greater portion of protein adsorbs on surface-modified PtBMA.

Manipulation of the motility of protein molecular motors on microfabricated substrates

Heavy meromyosin (HMM), a proteolytically cleaved derivative of myosin has previously been shown to interact with actin in well-established in vitro motility assays on nitrocellulose surfaces. In this study, the assays were conducted to demonstrate that the motility of actin filaments is confined in the micron-sized channels fabricated via laser ablation in a layer of the photosensitive resist polymer O-acryloyloxime acetophenone oxime (AAPO). A solution containing myosin labeled with fluorophore 5-iodoacetamidofluorescein (5-IAF) was applied to the microfabricated AAPO surface and shown to bind specifically to the micron-size channels. In the motility assay, HMM, rhodamine-phalloidin labeled actin and ATP were sequentially added and the movement of the actin filaments was observed by fluorescence microscopy and recorded with a CCD camera. The experiments prove that although the actin filaments show an only-partial propensity for attachment in myosin-rich areas, their motility is confined to a large extent in micro-channels.

Effects of aqueous exposure on the mechanical properties of wool fibers - analysis by atomic force microscopy

Treated and untreated wool fibers are analyzed by methodologies based on atomic force microscopy. Untreated fibers are known to have an intact native surface lipid layer, while KOH treatment has the effect of removing this layer as well as disrupting the molecular substructure. The effective bulk Youngs modulus of the untreated fibers is measured in air and in situ during exposure to water. There is a nearly instantaneous drop by a factor of two from approximately 1.2 GPa. after onset of aqueous exposure. The surface stiffness of both untreated and KOH treated fibers is measured in situ during aqueous exposure for durations up to 12 X 103 seconds. A decrease by a factor of two is observed for the untreated surface, while there is a more rapid decrease by a factor of ten for the KOH treated surface. Measurements of an equivalent Youngs modulus for the near-surface layers obtained by transverse compression of less than 200 nm by the tip result in values of less than 1 MPa. Because wool is an inhomogeneous and anisotropic medium, the outcomes of the two different measurements of the modulus are complementary but not comparable. Tip-to-surface adhesion is also monitored during aqueous exposure, with the KOH treated surface exhibiting the lower value by a factor of approximately ten. There are also trends showing lower adhesion with duration of exposure. Rapid ingress of water into the bulk appears to be associated with decreased bulk stiffness, but has little immediate effect on surface and near-surface properties. The decrements in surface stiffness and observed effects on adhesion from aqueous exposure are then due to a much slower radial ingress of water, which is additionally rate-limited by the native lipid layer.

Predicting surface properties of proteins on the Connolly molecular surface

A novel approach has been developed to obtain surface properties of a protein to give a better interpretation of the surface related phenomena, in particular protein attachment on polymer surface. This is achieved by extending the concept of molecular surface to find out relevant surface characteristics determining the interaction behaviour. The Connolly molecular surface is useful in the modelling and computation of surface properties, which could be of fundamental importance to surface-based protein science and engineering. A methodology for obtaining electron charge, hydrophobicity as well as α-helix and β-sheet structural indices has been developed.

Micropatterning of Myosin on O-Acryloyl Acetophenone Oxime (AAPO), Layered with Bovine Serum Albumin (BSA)

This paper describes a simple laser-based method for preparing microchannels in a bilayer system consisting of a UV sensitive polymer, O-acryloyl acetophenone oxime (AAPO), layered with a protein-blocking agent, bovine serum albumin (BSA). Patterned surfaces suitable for biomolecular attachment are achieved through the use of a computer-controled laser ablation system, comprising a research-grade inverted optical microscope, a pulsed nitrogen laser emitting at 337 nm and a programmable X-Y-Z stage. Exposed areas with diameters of 5–20 μm, 1–5 μm, and sub-micron widths are readily achieved by focussing through a 20×dry objective, a 40×dry objective, or a 100×oil immersion lens, respectively. When combined with a sub-micron resolution, high-speed, computer-controlled X-Y-Z stage, well-defined channels or arrays can be patterned in the AAPO, revealing either the underlying hydrophobic primed-glass surface, or pendant amino groups suitable for the covalent binding of biomolecules, depending on the amount of energy delivered to the surface. The subsequent removal of the attached BSA creates well-defined regions with high contrast. Myosin was physically adsorbed to the base of the channels, and fluorescently-labeled actin microfilaments were observed to selectively bind to the myosin following ATP hydrolysis, confirming retention of bioactivity.

Protein immobilisation on micro/nanostructures fabricated by laser microablation.

The performance of biomedical microdevices requires the accurate control of the biomolecule concentration on the surface, as well as the preservation of their bioactivity. This desideratum is even more critical for proteins, which present a significant propensity for surface-induced denaturation, and for microarrays, which require high multiplexing. We have previously proposed a method for protein immobilisation on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. This study investigates the relationship between the properties of the micro/nanostructured surface, i.e., topography and physico-chemistry, and protein immobilisation, for five, molecularly different proteins, i.e., lysozyme, myoglobin, α-chymotrypsin, human serum albumin, and human immunoglobulin. Protein immobilisation on microstructures has been characterised using quantitative fluorescence measurements and atomic force microscopy. It has been found that the sub-micrometer-level, combinatorial nature of the microstructure translates in a 3-10-fold amplification of protein adsorption, as compared to flat, chemically homogenous polymeric surfaces. This amplification is more pronounced for smaller proteins, as they can capitalize better on the newly created surface and variability of the nano-environments.

AFM analysis of the extracellular polymeric substances (EPS) released during bacterial attachment on polymeric surfaces

Extracellular polymeric substances (EPS) secreted by bacteria have a key role in adhesion and aggregation of bacterial cells on solid surfaces. In the present study, atomic force microscopy (AFM) has been used to study the adhesion propensity of bacterial strain St. guttiformis, and the ultrastructure and distribution of the EPS materials, on hydrophobic poly(tert-butylmethacrylate)(PtBMA) and hydrophilic polystyrene maleic acid (PSMA) surfaces. The results showed that bacterial attachment to the PSMA surface over incubation periods of 24-72 h was insignificant, whereas there was a strong propensity for the bacterial cells to attach to the PtBMA surface, forming multi-layered biofilms. For the PSMA surface, planktonic EPS adsorbed onto the polymeric surface and formed a continuous surface layer. For the PtBMA surface, non-contact mode imaging revealed that capsular EPS on the cell surface exhibited granular structures with the lateral dimensions of 30-50 nm and the vertical roughness of 7-10 nm. Lateral force imaging showed inter-connected elongated features which had lower frictional property compared to the surrounding EPS matrix, suggesting possible segregation of hydrophobic fractions of the EPS materials. The planktonic EPS adsorbed onto the PtBMA surface also showed similar nanometer-scale granular structures and could form stacks up to 150 nm in height. However, lateral force imaging did not show frictional differences, as in the case of capsular EPS. This is attributed to possible differences in the composition of the two EPS materials, and/or greater deformation of the planktonic EPS in the contact imaging mode which may obscure the fine surface features.

Controlled self-assembly of actin filaments for dynamic biodevices

We investigated the immobilization of actin filaments and its self-assembly in vitro in a continuous-flow system on poly(styrene-maleic acid) (PSMA), poly(methyl methacrylate) (PMMA), poly(t-butyl methacrylate) [P(tBuMA)] polymeric surfaces and along the linear channels. Among these polymeric surfaces, PSMA appeared to be more suitable for supramolecular manipulations as it lacked inherent fluorescence, had good biocompatibility with actin-myosin, and provided sufficient amounts of binding sites for the covalent immobilization of actin. Covalent attachment of G-actin on PSMA polymeric surfaces resulted in stable polymerization followed by alignment of filaments over 1.5 h, along with a greater surface density of the proteins. It is shown that electrostatic condensation of intact F-actin filaments and F-actin/gelsolin filaments with Ba2+ can be successfully used for progressive bundle formation and alignment in the constant flow. Actin bundles retained their ability to support HMM-anti-HMM bead translocation. Long-range cooperative transitions in actin induced by gelsolin represent a structural perturbation of the barbed end and presumably result in regularly organized bundles that secure directional movement. This simple technique for fabrication of self-assembled and aligned F-actin/gelsolin bundles provides a convenient experimental system for nanotechnological applications.

Study of atomic force microscopy force–distance curves by simulation using the Connolly surface for proteins

A simulation of the interaction between atomic force microscope tips and protein surfaces employing the concept of the Connolly molecular surface with a carbon probe has been investigated. A methodology has been developed to allow the computation of the Connolly surface for a protein, where numerous atoms are simultaneously interacting with each other. The van der Waals and electrostatic interactions between the probe and the relevant Connolly surface elements are integrated to obtain the total interaction, resulting in precise theoretical accounts for a variety of interaction components.