New reactive polymer for protein immobilisation on sensor surfaces

Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ngmL(-1) and for PSA 0.01 ngmL(-1). The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors.     

Bacterial adsorption to thermoresponsive polymer surfaces

The ability of microorganisms to `recognise' a change in the hydrophobicity/hydrophilicity balance of the surface was demonstrated using thermoresponsive poly(N-isopropylacrylamide) co-polymers with different Lower Critical Solution Temperatures. The polymers were grafted onto hydrolysed glass under well controlled conditions and the adhesion was followed using ¹³C-labelled Listeria monocytogenes. Attachment of the bacteria was found to be directly affected by the polymer transition from a hydrophilic to a hydrophobic state but by less than one order of magnitude.     

Pretreatment of amphiphilic comb polymer surfaces dramatically affects protein adsorption

New applications in regenerative biotechnology require the ability to understand and control protein-surface interactions on micrometer and submicrometer length scales. Evidence presented here shows that micropatterned amphiphilic comb polymer films exhibit a pretreatment-dependent behavior with respect to protein adsorption for the proteins fibronectin, laminin, and for serum. A micropatterned surface, consisting of protein-reactive regions, separated by comb polymer, was created and tested for protein adsorption using the surface-sensitive imaging tool TOF-SIMS. Immersion of micropatterned surfaces in solutions of fibronectin or laminin resulted in uniform protein coverage on both the comb polymer and protein-reactive regions. However, preimmersion of similarly patterned surfaces in water for 2 h prior to protein incubation was found to dramatically improve the protein-resistant properties of the comb polymer regions. These results are consistent with poly(ethylene glycol) (PEG) side chain reorientation and/or hydration and poly(methyl methacrylate) (PMMA) backbone segregation away from the interface region.     

Role of polymer complexes in the formation of biofilms by corrosive bacteria on steel surfaces

The composition of exopolymer complexes (EPCs), synthesized by the monocultures Desulfovibrio sp. 10, Bacillus subtilis 36, and Pseudomonas aeruginosa 27 and by microbial associations involved in the corrosion of metal surfaces has been studied. An analysis of the monosaccharide composition of carbohydrate components, as well as the fatty acid composition of the lipid part of EPCs, was carried out by gas-liquid chromatography (GLC). It was found that bacteria in biofilms synthesized polymers; this process was dominated by glucose, while the growth of bacteria in a suspension was marked by a high rhamnose content. Hexouronic acids and hexosamine have been revealed as a part of B. subtilis 36 and P. aeruginosa 27 EPCs. Qualitative differences were revealed in the fatty acid composition ofexopolymers in biofilms and in a bacterial suspension. It was shown that the transition to a biofilm form of growth led to an increase in the unsaturation degree of fatty acids in the exopolymers of associative cultures. The results can be used to develop methods to control microbial corrosion of metal surfaces.     

Role of polymer complexes in the formation of biofilms by corrosive bacteria on steel surfaces

The composition of exopolymer complexes (EPCs), synthesized by the monocultures Desulfovibrio sp. 10, Bacillus subtilis 36, and Pseudomonas aeruginosa 27 and by microbial associations involved in the corrosion of metal surfaces has been studied. An analysis of the monosaccharide composition of carbohydrate components, as well as the fatty acid composition of the lipid part of EPCs, was carried out by gasliquid chromatography (GLC). It was found that bacteria in biofilms synthesized polymers; this process was dominated by glucose, while the growth of bacteria in a suspension was marked by a high rhamnose content. Hexouronic acids and hexosamine have been revealed as a part of B. subtilis 36 and P. aeruginosa 27 EPCs. Qualitative differences were revealed in the fatty acid composition of exopolymers in biofilms and in a bacterial suspension. It was shown that the transition to a biofilm form of growth led to an increase in the unsaturation degree of fatty acids in the exopolymers of associative cultures. The results can be used to develop methods to control microbial corrosion of metal surfaces.     

Thermodynamic aspects of plant cell adhesion to polymer surfaces

Summary A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm² which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm²) agree with the pattern of adhesion previously described.     

Protein-flexible chain polymer interactions to explain protein partition in aqueous two-phase systems and the protein-polyelectrolyte complex formation

Complexes formation between two model proteins (catalase and chymotrypsin) and polyelectrolytes (polyvinyl sulphonate and polyacrilic acid) and a non-charged flexible chain polymer (PCF) as polyethylene propylene oxide (molecular mass 8400) was studied by a spectroscopy technique combination: UV absorption, fluorescence emission and circular dichroism. All the polymers increase the protein surface hydrophobicity (S(0)) parameter value as a proof of the modification of the protein surface exposed to the solvent. Chymotrypsin showed an increase in its biological activity in polymer presence, which suggests a change in the superficial microenvironment. The decrease in the biological activity of catalase might be due to a competition between the polymer and the substrate. This result agrees with the polymer effect on the catalase superficial hydrophobic area. It was found that, when flexible chain polymers increase protein stability and the enzymatic activity they could be used to isolate this enzyme without inducing loss of protein enzymatic activity. Our findings suggest that the interactions are dependent on the protein physico-chemical parameters such as: isoelectric pH, hydrophobic surface area, etc.     

Conformational entropy of a polymer chain grafted to rough surfaces

A polymer molecule (represented by a statistical chain) end-grafted to a topologically rough surface was studied by static MC simulations. A modified self-avoiding walk on a cubic lattice was used to model the polymer in an athermal solution. Different statistical models of surface roughness were applied. Conformational entropies of chains attached to uncorrelated Gaussian, Brownian, and fractional Brownian surfaces were calculated. Results were compared with the predictions of a simple analytical model of a macromolecule end-grafted to a fractal surface.

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aequeous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm^2,† in excellent agreement with the value obtained from contact angles measured on layers of cells.     

Using structural motif templates to identify proteins with DNA binding function

This work describes a method for predicting DNA binding function from structure using 3-dimensional templates. Proteins that bind DNA using small contiguous helix–turn–helix (HTH) motifs comprise a significant number of all DNA-binding proteins. A structural template library of seven HTH motifs has been created from non-homologous DNA-binding proteins in the Protein Data Bank. The templates were used to scan complete protein structures using an algorithm that calculated the root mean squared deviation (rmsd) for the optimal superposition of each template on each structure, based on C_α backbone coordinates. Distributions of rmsd values for known HTH-containing proteins (true hits) and non-HTH proteins (false hits) were calculated. A threshold value of 1.6 Å rmsd was selected that gave a true hit rate of 88.4% and a false positive rate of 0.7%. The false positive rate was further reduced to 0.5% by introducing an accessible surface area threshold value of 990 Ų per HTH motif. The template library and the validated thresholds were used to make predictions for target proteins from a structural genomics project.     

Surface thermodynamics of leukocyte and platelet adhesion to polymer surfaces.

Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that a cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aqueous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm(2), in excellent agreement with the value obtained from contact angles measured on layers of cells.     

Adhesion of lactobacilli to polymer surfaces in vivo and in vitro

The ability of bacteria to attach to surfaces has been recognized as an important phenomenon, particularly for pathogenic organisms that utilize this capacity to initiate disease. The present investigation was undertaken to determine whether indigenous urogenital bacteria, lactobacilli, colonized prosthetic devices in vivo and in vitro and attached to specific polymer surfaces in vitro. Polyethylene intrauterine devices (IUDs) in place for 2 years were removed from six women who were asymptomatic and free of signs of cervical or uterine infection. Lactobacilli were found attached to the IUDs, as determined by culture, and fluorescent antibody and acridine orange staining techniques. This demonstrated that bacterial biofilms consisting of indigenous bacteria can occur on prosthetic devices without inducing a symptomatic infection. In vitro studies were then undertaken with well-documented lactobacilli strainsL. acidophilus T-13,L. casei GR-1, GR-2, and RC-17, andL. fermentum A-60. These organisms were found to adhere to IUDs and urinary catheters within 24 hours. A quantitative assay was designed to examine the mechanisms of adhesion ofL. acidophilus T-13 to specific polymer surfaces that are commonly used as prosthetic devices. The lactobacilli adhered optimally to fluorinated ethylene propylene when 10⁸ bacteria were incubated for 9 hours at 37°C in phosphate buffered saline, pH 7.1. Additional experiments verified that the lactobacilli adhered to polyethyleneterephthalate, polystyrene, and sulfonated polystyrene and to silkolatex catheter material. There was a linear relationship found between polymer hydrophobicity and bacterial adherence. These results demonstrate that lactobacilli bind to various surfaces in vivo and in vitro, and that the nature of the substratum can affect the colonization.     

Using multiple templates to improve quality of homology models in automated homology modeling

When researchers build high-quality models of protein structure from sequence homology, it is today common to use several alternative target-template alignments. Several methods can, at least in theory, utilize information from multiple templates, and many examples of improved model quality have been reported. However, to our knowledge, thus far no study has shown that automatic inclusion of multiple alignments is guaranteed to improve models without artifacts. Here, we have carried out a systematic investigation of the potential of multiple templates to improving homology model quality. We have used test sets consisting of targets from both recent CASP experiments and a larger reference set. In addition to Modeller and Nest, a new method (Pfrag) for multiple template-based modeling is used, based on the segment-matching algorithm from Levitt's SegMod program. Our results show that all programs can produce multi-template models better than any of the single-template models, but a large part of the improvement is simply due to extension of the models. Most of the remaining improved cases were produced by Modeller. The most important factor is the existence of high-quality single-sequence input alignments. Because of the existence of models that are worse than any of the top single-template models, the average model quality does not improve significantly. However, by ranking models with a model quality assessment program such as ProQ, the average quality is improved by approximately 5% in the CASP7 test set.     

A direct observation of bacterial coverage and biofilm formation by Acidithiobacillus ferrooxidans on chalcopyrite and pyrite surfaces

To obtain a fundamental understanding of the population behaviour of Acidithiobacillus ferrooxidans at chalcopyrite and pyrite surfaces, the early stage attachment behaviour and biofilm formation by this bacterium on chalcopyrite (CuFeS₂) and pyrite (FeS₂) were studied by optical microscopy, Raman spectroscopy, time-of-flight secondary ion mass spectrometry (ToF-SIMS) and electron backscatter diffraction (EBSD). The results indicate there was no significant difference in selectivity of bacterial attachment between chalcopyrite and pyrite. However, the result of ToF-SIMS analysis suggests that the surface of the pyrite was covered more extensively by biofilm than that of the chalcopyrite, which may indicate more extracellular polymeric substances (EPS) formation by bacterial cells growing on pyrite. EBSD and optical image analysis indicated that selectivity of bacterial attachment to chalcopyrite was not significantly affected by crystal orientation. The results also suggest that the bacterial population in defective areas of chalcopyrite was significantly higher than on the polished surfaces.     

Microtubule protein preparations from C6 glial cells and their spontaneous polymer formation

C6 cell tubulin is indistinguishable from hog brain tubulin with respect to its molecular weight, amino acid composition, and colchicine-binding activity. Moreover, microtubule assembly systems from both sources form the same structures: rings, ribbons, tubules, and drug-induced polymers. There is, nevertheless, a difference between the cultured cell and brain systems which lies in the nature of their microtubule-associated accessory proteins. C6 microtubule preparations exhibit few rings at 0 degrees C, have low polymerization yield, and have a low content of accessory proteins. The addition of brain accessory proteins enhances the numbers of rings, and the yield of microtubules, to levels comparable with those of brain preparations. The polymerizing ability of C6 microtubule protein decays much faster than that of brain, but it can be restored by the addition of brain accessory protein. The results suggest that C6 accessory proteins are more labile than their brain counterparts.