Jonathan P. Wright

Staleya guttiformis attachment on poly(tert-butylmethacrylate) polymeric surfaces

The attachment behaviour of Staleya guttiformis DSM 11458(T) on poly(tert-butyl methacrylate) (P(tBMA)) polymeric surfaces has been studied. The electrostatic charge of the S. guttiformis cell surface (measured as zeta potential via microelectrophoresis) was -43.18 mV. S. guttiformis cells appeared weakly hydrophilic as the water contact angle measured on lawns of bacterial cells was found to be 55+/-4.9 degrees. It was found that while attaching on P(tBMA) surfaces, S. guttiformis cells produced extracellular polymeric substances (EPS) as observed from atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis. The AFM high resolution imaging revealed the nano-topography of the free (the EPS that is produced by the bacterial cells, but no longer directly attached to the cells) EPS associated on the cell surface and also found on P(tBMA) surface. The free EPS exhibited granular structure with lateral dimensions of 30-50 nm and a vertical nano-roughness of 7-10nm. Another type of the EPS secreted by S. guttiformis cells appeared as a hydrogel substance, presumably polysaccharide that formed a biopolymer network that facilitated bacterial attachment.

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.

Alignment of Plate-Like Particles in a Colloidal Dispersion under Flow in a Uniform Pipe Studied by High-Energy X-ray Diffraction

High-energy angle-dispersive X-ray diffraction has been used to study the alignment of colloidal suspension of kaolinite particles in water as they flow along a pipe. X-rays with energies above 25 keV have a major advantage, as they can penetrate through thick samples and walls of containers and permit investigation of samples under realistic flow conditions. As an example of the method, flow through a circular cross-section pipe with an internal diameter of 5 mm has been studied: this is typical of industrial applications. The angular distribution of intensities of peaks in the diffraction pattern as a function of the location of the pipe in the X-ray beam provides information about the alignment of particles under flow. Order parameters have been calculated to describe the alignment and direction of orientation. It is observed that the particles align in the direction of flow with their flat faces parallel to the flow. The experimental results are compared with the calculations of the local strain rate that help to explain the onset of alignment of the particles.

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.

AFM analysis of the polymer microstructures used for novel multianalyte protein microassay

We recently described a technique to fabricate shallow (< 50 nm) microstructures on PMMA surface for use in multianalyte protein micro-assay based on the ablation of a top thin gold layer using pulsed nitrogen laser (337 nm). In the present study, AFM has been used to investigate the surface characteristics and to provide physical insights into the formation of these complex microstructures. It has been shown that lateral diffusion of the heat generated during the gold ablation extended to ca. 3 μm on either side of the laser focal spot (ca. 5μmm wide), effectively ablated the gold layer and created shallow regions of ca. 20 nm. The heat also created a depression (ca. 5 μm wide) in the polymer region at the laser spot, and a hump, that increased in height with laser dose, at the center of the depression. It is suggested that volume shrinkage caused by stress relaxation and material redistribution, and volume expansion caused by fragmentation of the polymer are responsible mechanisms. Chemical changes also occurred resulting in the middle zone of the microstructure, which corresponds to the central hump, being hydrophobic, whereas the outer zone was hydrophilic. It is suggested that degraded hydrophilic products may be present in the outer zone, whereas the middle zone may contain smaller hydrophobic fragments due to more advanced fragmentation. The variation in the morphology and surface chemistry in the shallow microstructures effectively combinatorialize the surface properties of the microstructures, thus facilitating the patterning of different proteins.

Surface characterization of oligonucleotides immobilized on polymer surfaces

The immobilization and hybridization of amino-terminated oligonucleotide strands to cyclo-olefin-copolymer (COC) and polycarbonate (PC) surfaces have been investigated for potential application in micro-PCR devices. The oligonucleotides were covalently bound to the plasma-treated COC and PC surfaces via an N-hydroxy-sulfosuccinimide (NHSS) intermediate. Analysis by AFM showed that the oligonucleotides were present on the surfaces as lumps, and that the size, both vertically and laterally, of these lumps on the COC surface was larger compared to the PC surface. The immobilization efficiency of the former was also higher (15.8 x 1012 molecules / cm2) compared to the latter (3.3 x 1012 molecules / cm2). The higher efficiency of the COC surface is attributed to the more effective NHSS-functionalization and its higher surface roughness. Subsequent hybridization doubled the height of the lumps, while the lateral dimensions remained essentially unchanged. This is explained in terms of organization of the long probe strands used on the surface as flexible, coil-like polymer chains, which allow the complementary oligonucleotides to bind and increase the height of the lumps. The AFM frictional images showed that the hybridization had the effect of reversing hydrophilicity of the oligonucleotide lumps from being more hydrophilic to more hydrophobic, consistent with the hydrophilic bases of the probe strands being shielded as a result of hybridization.

Nanolithography of polymer surfaces by Atomic Force Microscopy

Laterally differentiated chemistry and structure of surfaces are commonly employed in a variety of devices/components (e.g., biosensors, array devices). At present such devices are based on macroscopic technologies. Future applications of differentiated surfaces are expected to place considerable demands on down-sizing technologies, i.e. enable meso/nanoscopic manipulation. The atomic force microscope (AFM) has emerged as an ideal platform for manipulation, visualization and characterisation of surface structures on the nano-scale1-14. Controlled AFM-based tip-induced lithography on P(tBuMA) thin film polymer surfaces has been obtained, at line widths down to tens of nanometres and depths in the sub-nanometre range. Parameters giving rise to production of nano-structures can in principle be defined for different polymers (lever-induced out-of-plane loading and in-plane shear forces, linear tip speed, tip shape and chemistry, polymer surface chemistry and mechanical properties). However, those sets of parameters, and their relationship to lithographic outcomes, cannot be derived from the currently accepted models for wear between macroscopic objects in sliding contact.

Computer-controlled laser ablation: A novel tool for biomolecular patterning

This paper describes a novel laser-based method for preparing microchannels in a bilayer system consisting of a UV sensitive polymer, acetophenone O-acryloyloxime (AAPO), layered with bovine serum albumin (BSA); BSA acts as a common blocking agent to prevent biomolecular attachment to the unexposed regions. The focus of the paper is on the use of a computer-controlled 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. By using a 100x oil immersion objective, channels of 1micrometers width and ca. 1 mm depth can be etched into the BSA-coated polymer. The precise width of the channel can be controlled by simply adjusting both the laser power and focusing. The addition of myosin to the base of these channels provides tracks on which actin filaments can move. By adjusting the width of the tracks, it is possible to regulate the direction of motion of the actin filaments.

Interactions of poly(amino acids) in aqueous solution with charged model surfaces- analysis by colloidal probe

Biomolecules in a confined solution environment may be subject to electrostatic forces with a range up to 100 nm, while the van der Waals interaction will account for shorter-range forces. The response of two model poly(amino acids) - poly-L-lysine and poly-L-glutamic acid - has been investigated for a number of model surfaces at pH 6 - including silica/Si-oxide. The model amino acids were adsorbed, or covalently coupled, to colloidal probes consisting of a microsphere attached to a force-sensing lever. The methodology was based on sensing of an interaction between the probe and a flat surface through carrying out force versus distance analysis with an atomic force microscope. The results were analysed within the framework of the conventional DLVO theory. The outcomes illustrate both repulsive and attractive long-range interactions that will hinder, or promote, colloidal biospecies in solution from entering the region of short-range force-fields at the physical interface. Accordingly the results have implications for the efficacy of methods and devices that seek to exploit the properties of micro/nano-fluidic systems. Large snap-on distances were observed for some systems and were ascribed to compression of the soft functionalized layers. Those observations and measurements of adhesion provided insights into conformation of the adsorbed species and strength of attachment.