The adsorption of five proteins with very different molecular characteristics, i.e. α-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin, has been characterized using quantitative fluorescence measurements and atomic force microscopy. It has been found that the 'combinatorial' nature of the micro/nano-channels surface allows for the increased adsorption of molecularly different proteins, comparing with the adsorption on flat surfaces. This amplification increases for proteins with lower molecular surface that can capitalize better on the newly created surface and nano-environments. Importantly, the adsorption on micro/nano-fabricated structures appears to be less dependent on the local molecular descriptors, i.e. hydrophobicity and charges, due to the combinatorialization of the nano-areas presented to the proteins. The amplification of adsorption is important, ranging from 3- to 10-fold, with a higher amplification for smaller, globular proteins.
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.
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.
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 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.
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.
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.
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.
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.
Ablation rate of polymers in laser ablation-based microfabrication depends on both laser parameters and polymer characteristics. This study aims to establish a scaling relationship linking the ablation rate and the properties of polymers commonly used in microfluidics. Ablation rate of polymers was determined experimentally using 193nm, 248nm and 308nm radiations. Polymer descriptors included thermal and surface properties. Statistical analysis was carried out for laser fluence against various polymer descriptors and/or their combinations. Analysis results show a relatively high correlation coefficient of 0.82 for the polymer ablation data when we compare fluence against the product of ablation rate and the difference between glass transition temperature and room temperature.
An attempt to simulate the interaction between an AFM tip and a protein surface by employing the concept of Connolly molecular surface with a carbon probe has been investigated. A methodology has been developed to permit the computation of the Connolly surface for a protein, where numerous atoms are simultaneously interacting 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 a precise theoretical account for a variety of interaction components. The simulation offers a meaningful opportunity for AFM scientists to interpret AFM surface mapping results more precisely or on a more general level the polymer surface-protein surface interactions.
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 labelled 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 labelled 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.
Microarray-like chips are based on effective immobilisation of surface biomolecules and preservation of their bioactivities. We have applied the concept of Connolly molecular surface for modelling and computation of the pure surface properties of proteins, which are of fundamental importance to surface-based protein science and engineering, especially for protein microarray chips. This is achieved by de-convoluting various molecular interaction components into single surface elements, and integrating a specific property of the atoms mostly close to the surface element to obtain the pure surface property of a protein. A methodology for obtaining electron charge, hydrophobicity as well as a-helix and b-pleated sheet structural indices has been developed. A parallel study shows that this technique is useful for modelling and computation of protein-protein and protein-polymer interactions, including protein attachment in molecularly confined spaces.
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