Micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] brushes grown from photopatterned halogen initiators by atom transfer radical polymerization

Photolithographic techniques have been used to fabricate polymer brush micro- and nanostructures. On exposure to UV light with a wavelength of 244 nm, halogens were selectively removed from films of chloromethylphenyltrichlorosilane and 3-(2-bromoisobutyramido)propyl-triethoxysilane on silicon dioxide. Patterning was achieved at the micrometer scale, by using a mask in conjunction with the incident laser beam, and at the nanometer scale, by utilizing interferometric lithography (IL). Friction force microscopy images of patterned surfaces exhibited frictional contrast due to removal of the halogen but no topographical contrast. In both cases the halogenated surface was used as an initiator for surface atom-transfer radical polymerization. Patterning of the surface by UV lithography enabled the definition of patterns of initiator from which micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] bottle brushes were grown. Micropatterned brushes formed on both surfaces exhibited excellent resistance to protein adsorption, enabling the formation of protein patterns. Using IL, brush structures were formed that covered macroscopic areas (approximately 0.5 cm2) but exhibited a full width at half maximum height as small as 78 nm, with a period of 225 nm. Spatially selective photolytic removal of halogens that are immobilized on a surface thus appears to be a simple, rapid, and versatile method for the formation of micro- and nanostructured polymer brushes and for the control of protein adsorption.     

Plasma protein adsorption to zwitterionic poly (carboxybetaine methacrylate) modified surfaces: chain chemistry and end-group effects on protein adsorption kinetics, adsorbed amounts and immunoblots

Protein-surface interactions are crucial to the overall biocompatability of biomaterials, and are thought to be the impetus towards the adverse host responses such as blood coagulation and complement activation. Only a few studies hint at the ultra-low fouling potential of zwitterionic poly(carboxybetaine methacrylate) (PCBMA) grafted surfaces and, of those, very few systematically investigate their non-fouling behavior. In this work, single protein adsorption studies as well as protein adsorption from complex solutions (i.e. human plasma) were used to evaluate the non-fouling potential of PCBMA grafted silica wafers prepared by nitroxide-mediated free radical polymerization. PCBMAs used for surface grafting varied in charge separating spacer groups that influence the overall surface charges, and chain end-groups that influence the overall hydrophilicity, thereby, allows a better understanding of these effects towards the protein adsorption for these materials. In situ ellipsometry was used to quantify the adsorbed layer thickness and adsorption kinetics for the adsorption of four proteins from single protein buffer solutions, viz, lysozyme, α-lactalbumin, human serum albumin and fibrinogen. Total amount of protein adsorbed on surfaces differed as a function of surface properties and protein characteristics. Finally, immunoblots results showed that human plasma protein adsorption to these surfaces resulted, primarily, in the adsorption of human serum albumin, with total protein adsorbed amounts being the lowest for PCBMA-3 (TEMPO). It was apparent that surface charge and chain hydrophilicity directly influenced protein adsorption behavior of PCBMA systems and are promising materials for biomedical applications.     

The role of enamel proteins in protecting mature human enamel against acidic environments: a double layer force spectroscopy study

Characterisation of the electrostatic properties of dental enamel is important for understanding the interfacial processes that occur on a tooth surface and how these relate to the natural ability of our teeth to withstand chemical attack from the acids in many soft drinks. Whereas, the role of the mineral component of the tooth enamel in providing this resistance to acid erosion has been studied extensively, the influence of proteins that are also present within the structure is not well understood. In this paper, we report for the first time the use of double-layer force spectroscopy to directly measure electrostatic forces on as received and hydrazine-treated (deproteinated) enamel surfaces in solutions with different pH to determine how the enamel proteins influence acid erosion surface potential and surface charge of human dental enamel. The deproteination of the treated samples was confirmed by the loss of the amide bands (~1,300-1,700 cm(-1)) in the FTIR spectrum of the sample. The force characteristics observed were found to agree with the theory of electrical double layer interaction under the assumption of constant potential and allowed the surface charge per unit area to be determined for the two enamel surfaces. The values and, importantly, the sign of these adsorbed surface charges indicates that the protein content of dental enamel contributes significantly to the electrostatic double layer formation near the tooth surface and in doing so can buffer the apatite crystals against acid attack. Moreover, the electrostatic interactions within this layer are a driving factor for the mineral transfer from the tooth surface and the initial salivary pellicle formation.     

Emulsifying and surface properties of citric acid deamidated wheat gliadin

The properties of citric acid deamidated wheat gliadin (d-gliadin) and d-gliadin emulsions at concentrations of 2% and 0.5% at pH 7 and 3 were investigated. d-gliadin exhibited high solubility at neutral pH and had a different molecular weight distribution compared with control gliadin. Stability of d-gliadin emulsion increased after heat treatment. Heat treatment led to a red shift of the fluorescence emission maxima of emulsions, indicating the rearrangement of d-gliadin molecules and increase of the protein structure flexibility at the oil–water interface. The emulsion was sensitive to NaCl up to 150 mM due to electrostatic screening effects. d-gliadin concentration influenced the droplet size and the saturation surface load of emulsion. d-gliadin slowly adsorbed on the oil–water interface during the adsorption process. Higher d-gliadin concentration exhibited higher surface pressure. Surface adsorption properties confirmed that d-gliadin was a good emulsifier in oil–water emulsions.     

Reversible changes in cell morphology due to cytoskeletal rearrangements measured in real-time by QCM-D

The mechanical properties and responses of cells to external stimuli (including drugs) are closely connected to important phenomena such as cell spreading, motility, activity, and potentially even differentiation. Here, reversible changes in the viscoelastic properties of surface-attached fibroblasts were induced by the cytoskeleton-perturbing agent cytochalasin D, and studied in real-time by the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D is a surface sensitive technique that measures changes in (dynamically coupled) mass and viscoelastic properties close to the sensor surface, within a distance into the cell that is usually only a fraction of its size. In this work, QCM-D was combined with light microscopy to study in situ cell attachment and spreading. Overtone-dependent changes of the QCM-D responses (frequency and dissipation shifts) were first recorded, as fibroblast cells attached to protein-coated sensors in a window equipped flow module. Then, as the cell layer had stabilised, morphological changes were induced in the cells by injecting cytochalasin D. This caused changes in the QCM-D signals that were reversible in the sense that they disappeared upon removal of cytochalasin D. These results are compared to other cell QCM-D studies. Our results stress the combination of QCM-D and light microscopy to help interpret QCM-D results obtained in cell assays and thus suggests a direction to develop the QCM-D technique as an even more useful tool for real-time cell studies.     

Dye Adsorption from Aqueous Solution by Cellulose/Chitosan Composite: Equilibrium,Kinetics, and Thermodynamics

A novel eco-friendly porous adsorbent of cellulose (CE)/chitosan (CS) aerogel was prepared through sol-gel process and freeze-drying to remove Congo Red (CR). A series of aerogels were prepared by adjusting the mass ratios of CE and CS. Composite aerogels were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and X-ray diffraction (XRD). SEM images showed that it was possible to change the structure of the aerogel by adjusting the amount of chitosan. The effects of dosage of chitosan, initial pH, temperature, adsorbent dosage, contact time, and initial dye concentration on adsorption capacities for CR were studied in detail. Batch adsorption studies showed that aerogel exhibited maximum removal efficiency to CR at a composite ratio of 1:3 and dosage of 2.5 g/l. CE/CS aerogel had excellent adsorption capacities for CR at a pH range of 3-11, which indicated stability of the aerogel in both acidic and alkaline conditions. CR adsorption on the composite aerogel fitted pseudo-second-order kinetics and Langmuir isotherm. The Langmuir isotherm model revealed that the maximum theoretical adsorption capacity of this material for CR was 381.7 mg/g at pH 7.0 at 303 K for 24 h. The adsorption mechanism included electrostatic and chemical interactions. The results indicated that the adsorption capacity of CE/CS aerogels was higher than the other chitosan composites adsorbents.     

Atomic Force Microscopy of A-Gliadin Fibrils and in situ Degradation

Atomic force microscopy (AFM) has been used in air and in aqueous buffer to study the structure of fibrils formed by the self-assembly of A-gliadin protein molecules. The images showed fibrils with a diameter of between 15 and 30 nm and lengths ranging from about 100 nm to 2 μm. No branched fibrils were observed, and there was no indication of a strong lateral inter-fibril interaction that would result in side-by-side association. Disassembly of the fibrils occurred when the pH of the aqueous buffer was reduced. In contrast the reverse process of fibril assembly and adsorption to the mica surface was less readily observed in situ. Some short fibrils were observed to assemble, but the lengths and densities were considerably less than those obtained by external deposition and drying.     

Coloration and Decoloration of Textiles Using a TiO<Subscript>2</Subscript> Composite Pigment

A colorable pigment was prepared by dye adsorption onto titanium dioxide and subsequent silane coating. The effects of pH value, dye concentration, and adsorption times on dye adsorption were discussed. Large adsorption capacity of an anionic dye was obtained at pH value of 2 and the adsorption process was well described by the Langmuir isotherm model. Good dyeability and color fastness of pigment dyed fabric were achieved in the normal life cycle under sunlight. The decoloration of pigment was realized through photocatalytic degradation of dye molecules by titanium dioxide under ultraviolet irradiation when reusing the pigment dyed textiles after disposal. The new absorption peaks in the FTIR spectrum at 2924.95 cm^-1, 1714.91 cm^-1, 1461.17 cm^-1, and 1289 cm^-1 verified silane modification. Silane modification improved fixation of dyes onto the pigment and immobilization of pigments onto substrates. The close attachment of silane coating layer to titanium dioxide was conducive to photodegradation of dye molecules in the pigment.     

Kinetics of leucine-lysine peptide adsorption and desorption at -CH3 and -COOH terminated alkylthiolate monolayers

The kinetics of adsorption and desorption of two highly asymmetrical model peptides were studied at methyl- and carboxylic acid-terminated alkylthiolate self-assembled monolayer (SAM) surfaces on gold. The model peptides were leucine-lysine (LK), α-helical (LKα14), and β-strand (LKβ15) peptides that have a well-defined secondary structure with the leucines localized on one side and the lysines on the other side. These secondary structures were previously shown to be maintained after adsorption and to control LK peptide orientation on these surfaces. The kinetics of peptide adsorption were analyzed by surface plasmon resonance as a function of peptide solution concentrations at pH 7.4. Peptide desorption was measured by rinsing with a buffer at various times along the adsorption curve. Both peptides had a saturation coverage of approximately 1 ML (monolayer) on the carboxyl SAM. Both peptides exhibited mostly irreversible binding on both surfaces, but the LKα14 peptide showed some limited reversible binding. Reversibly bound peptides could be in the second adlayer interacting only with peptides in the first layer or peptides interacting with a partially covered adsorption site and therefore not able to fully bind to the SAM surface. The near complete lack of reversible binding for LKβ15 is possibly due to strong peptide-peptide hydrogen bonding in β-sheet structures within the adsorbed layer. For a given dose of either peptide, much less peptide adsorbed on the methyl SAMs. The adsorption rate of irreversibly bound LKα14 on carboxylic acid SAMs was first-order with respect to solution concentration. Both peptides showed nucleation-like adsorption kinetics on the carboxylic acid SAM, indicating that peptide-peptide bonding is needed to stabilize the adsorbed layer. Adsorption on the methyl SAM was much lower in quantity for both peptides and seemed to require prior aggregation of the proteins in solution, at least for LKβ15.     

Multitechnique study on a recombinantly produced Bacillus halodurans laccase and an S-layer/laccase fusion protein

Methods for organizing functional materials at the nanometer scale are essential for the development of novel fabrication techniques. One of the most relevant areas of research in nanobiotechnology concerns technological utilization of self-assembly systems, wherein molecules spontaneously associate into reproducible supramolecular structures. For this purpose, the laccase of Bacillus halodurans C-125 was immobilized on the S-layer lattice formed by SbpA of Lysinibacillus sphaericus CCM 2177 either by (i) covalent linkage of the enzyme to the natural protein self-assembly system or (ii) by construction of a fusion protein comprising the S-layer protein and the laccase. The laccase and the S-layer fusion protein were produced heterologously in Escherichia coli. After isolation and purification, the properties of the proteins, as well as the specific activity of the enzyme moiety, were investigated. Interestingly, the S-layer part confers a much higher solubility on the laccase as observed for the sole enzyme. Comparative spectrophotometric measurements of the enzyme activity revealed similar but significantly higher values for rLac and rSbpA/Lac in solution compared to the immobilized state. However, rLac covalently linked to the SbpA monolayer yielded a four to five time higher enzymatic activity than rSbpA/Lac immobilized on a solid support. Combined quartz crystal microbalance with dissipation monitoring (QCM-D) and electrochemical measurements (performed in an electrochemical QCM-D cell) revealed that rLac immobilized on the SbpA lattice had an approximately twofold higher enzymatic activity compared to that obtained with the fusion protein.     

Adsorption of Graphene Oxide onto Synthetic Fibers: Experimental Conditions

Comparative studies on the adsorption capacity of two synthetic fibers, polyamide (PA 66) and polyester (PET) pre-treated with N-cetylpyridinium chloride (PET-NCPCl), towards graphene oxide (GO) have been carried out. The fiber samples were characterized by scanning electron microscopy (FE-SEM) and Raman spectroscopy. The results of adsorption isotherms, kinetics, and zeta potential determinations as a function of the GO concentration, pH, and temperature show that at a low pH of 2.5 and a high temperature of 323 K, almost 99 % of the 75 mg/l GO solution is adsorbed onto PA 66 and 70 % onto the PET-NCPCl fibers. The interaction should be first attributed to electrostatic forces, also the adsorption data exhibited a good fit to the Freundlich isotherm model and the free energy value of 10 kJ/mol was in the range of physical adsorption, which could suggest that the interaction is driven mainly by physical forces. Due to the increasing development of wastewater treatments based on the GO reactivity with metals and cationic contaminants, synthetic fibers coated with GO could be considered an adsorbent for environmental applications.     

The binding of bile salts by vegetable fibre

The binding of bile salts by dietary fibre plays an important role in cholesterol metabolism in man. In most of the adsorption studies of bile salts in vitro, the chemical composition of the fibre preparations has not been described, even broadly, nor has the effect of co-precipitated compounds (e.g. proteins) been considered. Hence, it seemed useful to investigate the adsorption of Na cholate (NaC) and Na taurocholate (NaTC) by well-defined cell wall material (CWM) from parenchymatous, lignified and cutinised tissues of mature runner bean pods as well as leeks under a variety of experimental conditions. This study was to identify the groups of polymers which are responsible for adsorption. The results showed dramatic differences in the adsorption characteristics of the wall preparations at different pH values. Some of the findings are reported below. The CWM from the various tissues was prepared by sequentially extracting the wet ball-milled tissues with 1% aq. Na deoxycholate and phenol/acetic acid/water (2:1:1, w/v/v). Experiments with labelled deoxycholate showed that the final preparations contained negligible amounts of adsorbed deoxycholate. Since the amount of residual starch in the preparations was small, no attempt was made to remove it; however, if required, this could be completely removed by extraction with 90% aq. dimethyl sulphoxide. The particle size of the preparations varied from 25–50 μm. For adsorption studies the following preparations were used: (1) whole-, depeetinated-and delignified-CWM from parenchymatous and lignified tissues of runner beans, together with the H and Na forms; (2) CWM of runner beans at different stages of maturity; (3) CWM of whole-and decutinised-leaves and cutinised tissues of leeks and (4) carboxymethyl cellulose and amberlite resin. The binding of bile salts was measured by a modified isotope-dilution procedure. The neutral sugars from the polysaccharides were determined as alditol acetates by GLC and an estimate of the uronic acid content of the preparations was obtained by a modified carbazole method. Experiments on the effect of pH on the binding of cholate by the various preparations showed that the adsorption capacity was very much dependent on the pH. The binding increased as the pH decreased. These experiments were complicated by the precipitation cholic acid at a pH value of <4. Nevertheless, an interaction between cholate and CWM persisted in acid solutions. Removal of pectic substances and lignin from the runner bean preparations resulted in a decrease of the adsorption capacity. The results suggested that the adsorption is greatest under conditions in which the ionization of cholic acid and the acidic groups of the cell wall polymers is at its lowest. Following these studies, determinations were made of the adsorption of cholate from an aqueous solution (without buffer) by wall preparations in the H and Na forms. With all the preparations the adsorption was greatest when these were in the H form. Adsorption studies with the preparations from leeks showed that the adsorption capacity of the parenchymatous tissues was considerably more than that of cutinised tissues. Hence, pectic substances in the H form (an not lignin or cutin) are probably the principal binding agents of cholate. The adsorption of Na taurocholate by the various preparations was independent of pH value. The implications of these results are discussed.     

Bioactive glass 45S5 powders: effect of synthesis route and resultant surface chemistry and crystallinity on protein adsorption from human plasma

Despite its medical applications, the mechanisms responsible for the osseointegration of bioactive glass (45S5) have yet to be fully understood. Evidence suggests that the strongest predictor for osseointegration of bioactive glasses, and ceramics, with bone tissue as the formation of an apatitic calcium phosphate layer atop the implanted material, with osteoblasts being the main mediator for new bone formation. Most have tried to understand the formation of this apatitic calcium phosphate layer, and other bioresponses between the host and bioactive glass 45S5 using Simulated Body Fluid; a solution containing ion concentrations similar to that found in human plasma without the presence of proteins. However, it is likely that cell attachment is probably largely mediated via the adsorbed protein layer. Plasma protein adsorption at the tissue bioactive glass interface has been largely overlooked. Herein, we compare crystalline and amorphous bioactive glass 45S5, in both melt-derived as well as sol-gel forms. Thus, allowing for a detailed understanding of both the role of crystallinity and powder morphology on surface ions, and plasma protein adsorption. It was found that sol-gel 45S5 powders, regardless of crystallinity, adsorbed 3-5 times as much protein as the crystalline melt-derived counterpart, as well as a greater variety of plasma proteins. The devitrification of melt-cast 45S5 resulted in only small differences in the amount and variety of the adsorbed proteome. Surface properties, and not material crystallinity, play a role in directing protein adsorption phenomena for bioactive glasses given the differences found between crystalline melt-cast 45S5 and sol-gel derived 45S5.     

Direct molecular weight determination in the evaluation of dissolution methods for unreduced glutenin

A comparison of two dissolution methods, gentle stirring and sonication, for solubilising the ultra-large wheat protein glutenin was made based on a direct determination of the molecular weight of dissolved glutenin. The possible physical breakdown of glutenin by sonication was evaluated. Glutenin was isolated by extraction with hydrochloric acid and freeze dried. The extracted glutenin was dissolved either by gentle stirring at 4 °C in phosphate buffer pH 6.8 containing sodium dodecyl sulphate (SDS) or by ultrasonication in the same SDS containing buffer immersed in an ice-bath. The weight average molecular weight and the molecular weight distribution of the dissolved material were determined using asymmetrical flow field-flow factionation using a multiangle light scattering detector coupled on-line with a refractive index detector. These determinations showed that sonication caused physical breakdown of the ultra-large glutenin proteins. The average molecular weight was found to be 5×10⁶ after sonication and 2×10⁷ after gentle stirring.     

Assessment of protein entrapment in hydroxyapatite scaffolds by size exclusion chromatography

Although it is well known that the textural properties of scaffolds play an important role in the process of tissue regeneration, the investigation of such effects remain difficult especially at the micro/nano level. Texture confers the material the additional ability to entrap/concentrate molecules circulating in the body fluid regardless of their binding affinity to the material. The goal of the present work is to isolate protein entrapment from protein adsorption phenomena in two macroporous hydroxyapatite scaffolds with identical chemical structure, similar macroporosity but different micro/nanoporosity using proteins of different sizes. This was achieved implementing size exclusion chromatography and using the scaffolds as chromatographic columns. The results showed that the larger the crystal size and the lower the packing density of the crystals composing the scaffold increased protein retention but decreased the protein dwelling time in the column. Differences in the amount of protein retained depended on the protein type.     

Spray deposition in “tendone” vineyards when using a pneumatic electrostatic sprayer

The objective of this study was to evaluate the effect of electrostatic charge on foliar spray deposition in an Apulian “tendone” vineyard using an innovative pneumatic electrostatic sprayer. The sprayer was fitted with nozzles that linked the pneumatic atomization of the liquid, obtained using compressed air, to the electrostatic induction charge, thereby producing a stream of charged fine droplets. Furthermore, the sprayer was designed for low volume treatments, and the experimentation was carried out during a phenological stage with high leaf density to evaluate the performance of the machine under particularly challenging operative conditions.The sprayer was studied at three forward speeds (4, 5, and 6 km h⁻¹), and gave poor deposition inside the canopy, whether or not the electrostatic system was activated. Forward speed did not significantly affect the mean foliar spray deposition, whereas activation of the electrostatic system significantly increased the deposit only on the layer of foliage nearest to the sprayer (lower layer), but had no effect on deposition on the layer of foliage inside the canopy (upper layer). The ratio between the deposits on the two layers (lower:upper) was 6.5:1 when the electrostatic system was switched off, and 9.0:1 when it was switched on.However, this behaviour may allow targeted treatments on grapes, such as with Plant Protection Products (PPP) or bio growth stimulants. Furthermore, the small droplets produced by the machine are suitable for table grape protection because the droplets do not mark the grapes, which would reduce the quality of the product and its commercial value.     

Composition and Functional Properties of <Emphasis Type="Italic">Lupinus campestris</Emphasis> Protein Isolates

Protein isolates from L. campestris and soybean seeds were prepared using isoelectric precipitation (PI) and micellization (MI) procedures. The amount of protein recovered was considerably higher with the isoelectric precipitation than with the micellization procedure (60% and 30%, respectively). Protein contents were higher than 90% in protein isolates. Antinutritional factors content (alkaloids, lectins, and tannins) were reduced to innocuous levels after protein isolate preparation. Minimum protein solubility for the precipitated lupin protein isolate (LPI) was at pH 4.0, and between pH 4 and 6 for the micellized lupin protein isolate (LMI), increasing at both extremes of the pH scale. Water absorption for the LMI was 1.3 ml/g of protein and its oil absorption 2.2 ml/g of protein. The LPI had 1.7 ml/g of protein in both water and oil absorption. Foaming capacity and stability was pH-dependent. Foaming capacity was higher at pH 2 and lower near the protein isoelectric points. Minimum protein concentration for gelation in LMI was 8% w/v at pH 4, while for LPI was 6% at pH 4 and 6. Amino acid composition in L. campestris flour and protein isolates was high in lysine and low in methionine. Most of the essential amino acids in lupin protein isolates were at acceptable levels compared to a reference pattern for infants and adults. The electrophoretic pattern of both protein isolates showed three bands with different mobilities, suggesting that the protein fractions belong to alpha-conglutin (11S-like protein), beta-conglutin (7S-like protein) and gamma-conglutin. It is proven that some of the functional properties of L. campestris protein isolates are similar to those soybean protein isolates recovered under equal conditions.     

水稻穗顶部小穗退化突变体paa1-2的表型与等位基因序列分析

水稻穗顶部小穗退化在水稻生产中普遍存在,严重影响了水稻产量。本文对水稻穗顶部小穗退化突变体paa1-2进行表型观察,同时测序分析突变体paa1-2中已报道的TUTOU1和PAA1基因序列。结果表明,突变体paa1-2穗顶部退化表型是在幼穗发育6期后产生的。突变体paa1-2和野生型的TUTOU1基因序列一致,然而其PAA1基因存在突变,在第1512~1515 bp处存在4个碱基缺失,导致基因移码突变并使得蛋白翻译提前终止,PAA1-2可能是已报道PAA1基因的新等位突变基因。