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Investigation of protein-polymeric surface interaction requires reliable practical techniques for evaluation of the efficiency of protein immobilization. In this study the efficiency of protein immobilization was evaluated using three different techniques: (1) protein-binding assay with fluorescent detection and (2) quantification, and (3) atomic force microscopy. This approach enables us to rapidly analyse the adsorption properties of different proteins. The comparative physico-chemical adsorption of α-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin in the micro-wells fabricated via a localized laser ablation of a protein-blocked thin gold layer (50 nm) deposited on a Poly(methyl ethacrylate) film has been studied. Correlations were observed between the quantitative and qualitative differences depending on both protein and polymeric surface hydrophobicity.

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.

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.

The immobilization efficiency of the complexes of oligonucleotide/poly(L-lysine) onto Poly(Styrene/Maleic Acid), PSMA, and Poly(Styrene/Maleic Anhydride), PSMAA, has been investigated using X-ray photoelectron spectroscopy and atomic force microscopy (AFM) in conjugation with fluorescence-based measurements of DNA attachment. A mono-molecularly thin layer of either electrostatically or covalently (via amide bond) coupled poly(L-lysine) (PL) allows the "switching" of the chemistry from a COOH-based to NH2-based one. The COOH-based chemistry has the advantage of a high yield of reaction but the disadvantage of a low surface concentration of DNA molecules (negative-negative electrostatic exclusion) whereas the NH2-based chemistry provides a higher surface concentration (positive-negative electrostatic attraction) but has a lower yield of covalent binding reaction. The immobilization efficiency of covalently coupled 26-mer oligonucleotides/poly(L-lysine) to polymeric surfaces was estimated as 0.3-0.5 x1012 molecules/mm2 for both polymeric surfaces studied. The electrostatic adsorption of poly(L-lysine)/oligonucleotides onto PSMA and functionalized PSMAA surfaces yielded 0.5 x 1011 and 0.1 x 1010 molecules/mm2, respectively. Although this mode of attachment is not "covalent binding" per se , the evidence is provided that this attachment is strong enough to withstand PCR cycles. The properties of these oligonucleotide/poly(L-lysine) complexes make them promising candidates for DNA-DNA hybridisation assays and PCR.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

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 10 12 molecules / cm 2 ) compared to the latter (3.3 x 10 12 molecules / cm 2 ). 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.

Diazonaphthoquinone/novolak (DNQ) photoresist have the property of changing physical-chemical properties during exposure to UV light, which reflects in a change of the polymer hydrophobicity. A combinatorial surface having different exposed area was fabricated, in order to study the influence of hydrophobicity over protein adsorption and EDC-mediated covalent attachment. The results indicate two different behaviours, reflecting a substantial different mechanism of interaction. While protein adsorption decreased following the hydrophobicity decrease, covalent attachment increased, thus reflecting the effectiveness of the covalent mediator, which cross-links the protein to the carboxylic groups that form during exposure. Based on the results of the present work, a combinatorial microarray will be fabricated, to be used in the biosensor field.

Our earlier papers showed that laser gain is possible on the innershell 2s-2p transitions of Ne-like ions. This result was confirmed by more comprehensive calculations. In more recent works it was shown that laser gain is also possible on transitions between highly excited states. We summarize these results and present atomic-kinetic calculations for gain values along the Ne-like sequence for argon, iron, krypton, silver and xenon. For these ions the optimum plasma conditions are found for all possible laser transitions. We have also found some unknown laser transition in Ni-like tantalum and the optimum plasma parameters for laser action on these transitions.

Within accurate atomic kinetic model it is shown that the inversion state of heavy Ne-like ions in plasma is possible at electron densities ne greater than 1023 cm-3 if electron temperature is high enough (Te greater than Eioniz/2). Near optimum plasma conditions are found for Ne-like silver lasers. New effective lasing transitions in Ne- like silver are found. Large ASE effect (gL greater than 100) is predicted for Ne-like silver plasma produced by a powerful short-pulse drive laser.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Energy level populations and spectral line intensities in plasmas are studied using an advanced uniform approach for the calculation of ion energy levels and elementary processes rate coefficients in plasmas. The spectrum of Ne-like argon for the uniform steady-state plasma is calculated for a wide variation of electron temperature and density. Hence the most efficient lasing at 5 3p - 3s transitions with wavelengths 697.6, 860.6, 727.5, 725.7, 670.7 angstroms should be expected at 1019 < ne < 1020 cm-3 and Te approximately 60 eV. It is shown that at electron densities of 1015 < ne < 1021 cm-3 ratios of the strongest 3p - 3s line intensities are sensitive to electron density; their dependence on electron temperature is much less pronounced.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.