Immobilised peptide displaying phages as affinity ligands. Purification of lactoferrin from defatted milk

An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 microm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 microm) phage particles as ligands without any risk of blocking the monolithic column. Bound lactoferrin was eluted using 1M NaCl with a purity of >95%. The technique presents a good alternative to conventional immunoaffinity chromatography for purification of a protein of interest from complex samples due to (i) the robustness of the system in terms of recovery and ligand leakage and (ii) economical aspect in terms of low ligand cost.     

Targeted particulate adhesion to cellulose surfaces mediated by bifunctional fusion proteins

The adhesion of particles to surfaces is an integral element in many commercial and biological applications. In this article, we report on the direct measurements of protein-mediated deposition and binding of particles to model cellulose surfaces. This system involves a family of heterobifunctional fusion proteins that bind specifically to both a red dye and cellulose. Amine-coated particles were labeled with a red dye, and a fusion protein was attached to these particles at various number densities. The strength of adhesion of a single particle to a cellulose fiber was measured using micropipette manipulation as a function of the specificity of the protein and its surface density and contact time. The frequency and force of adhesion were seen to increase with contact time in fiber experiments. The dynamics of adhesion of the functionalized particles to cellulose-coated glass slides under controlled hydrodynamic flow was explored using a flow chamber for two scenarios: detachment of bound particles and attachment of particles in suspension as a function of the shear rate and surface density of protein. Highly specific adhesion was observed. The critical shear rate for particle detachment was an increasing function of cellulose binding domain (CBD) density on particle surface. A rapid irreversible attachment of particles to cellulose was observed under flow. Using a family of proteins that were divalent for binding either the red dye or cellulose, we found that particle detachment occurred because of the failure of the cellulose-CBD bond. A comparison of fiber binding and particle detachment results suggests that forces of adhesion of particles to cellulose of up to 2 nN can be obtained with this molecular system through multiple interactions. This study, along with the adhesion simulations currently under development, forms the basis of particulate design for specific adhesion applications.     

Size-Exclusion "capture and release" separations using surface-patterned poly(N-isopropylacrylamide) hydrogels

Micrometer-scale poly(N-isopropylacrylamide) (poly-NIPAAm) hydrogel monolith patterns were fabricated on solid surfaces using soft lithography. At sufficiently high aspect ratios, the hydrogel monoliths swell and contract laterally with temperature. The spaces between the monoliths form a series of trenches that catch, hold, and release appropriately sized targets. A series of poly-NIPAAm monoliths were fabricated with dry dimensions of 40 microm height, 12 microm diameter, and a spacing of 12 microm between monoliths. Above the lower critical solution temperature (LCST), the monoliths collapse to their dry dimensions and the spacing between monoliths is 12 microm. Below the LCST, the monoliths swell by 70% in the lateral direction, reducing the gap size between monoliths to 3 microm. The potential use of the hydrogel monoliths as size-selective "catch and release" structures was demonstrated with a mixture of 6 and 20 microm polystyrene microspheres, where the 6 microm diameter particles were selectively concentrated and separated from the larger particles.     

Mesoporous silica spheres from colloids

A novel method has been developed to synthesize mesoporous silica spheres using commercial silica colloids (SNOWTEX) as precursors and electrolytes (ammonium nitrate and sodium chloride) as destabilizers. Crosslinked polyacrylamide hydrogel was used as a temporary barrier to obtain dispersible spherical mesoporous silica particles. The influences of synthesis conditions including solution composition and calcination temperature on the formation of the mesoporous silica particles were systematically investigated. The structure and morphology of the mesoporous silica particles were characterized via scanning electron microscopy (SEM) and N2 sorption technique. Mesoporous silica particles with particle diameters ranging from 0.5 to 1.6 microm were produced whilst the BET surface area was in the range of 31-123 m2 g-1. Their pore size could be adjusted from 14.1 to 28.8 nm by increasing the starting particle diameter from 20-30 nm up to 70-100 nm. A simple and cost effective method is reported that should open up new opportunities for the synthesis of scalable host materials with controllable structures.     

Ion concentration of external solution as a characteristic of micro- and nanogel ionic reservoirs

Ion-sensitive hydrogel is regarded as an ionic reservoir, i.e., a system capable of changing the external pH or ionic strength by accumulating or releasing ions. The concept of a hydrogel ionic reservoir was demonstrated for hydrogel particles of three different size ranges: macrogel (1000-6000 microm), microgel (approximately 20-200 microm), and nanogel (approximately 0.2 microm). Ion sensitivity of poly(N-isopropylacrylamide-co-1-vinylimidazole) (PNIPA-VI) microgels with imidazolyl (ionizable) groups was confirmed by the pH dependence of their volume, while nanogels were characterized by dynamic light scattering. On the contrary, the volume of poly(N-isopropylacrylamide) (PNIPA) microgels without ionizable groups was pH independent in the whole range of pH from 10 to 2. Four distinct regions of pH-behavior were observed for PNIPA-VI hydrogel micro- and nanoparticles using potentiometric titration of their suspensions. Time-resolved measurements of ion concentrations in the suspension of hydrogel particles revealed a substantial difference in kinetics of pH equilibration for (i) ion-sensitive hydrogels (PNIPA-VI) vs hydrogels without ionizable groups (PNIPA) and (ii) PNIPA-VI hydrogels of different sizes. On the basis of the experimental observations, a two-step mechanism affecting the kinetics of proton uptake into the hydrogel particles with ionizable groups was proposed: (1) fast binding of ions to the immediate surface of each particle and (2) a slower successive diffusion of bound sites into the next inner layer of polymer network. In accord with the mechanism proposed, a quasi-chemical kinetic model of pH relaxation to equilibrium was developed to fit the experimental data for the time course of proton uptake by macro-, micro-, and nanogels into two exponentials with the characteristic times of tau(1) and tau(2). We believe the same kinetic model will be pertinent to describe phenomenological and molecular mechanisms controlling proton transport in/out bacteria, cells, organelles, drug delivery vehicles, and other natural or artificial multifunctional ionic containers. The approach can be easily extended for the other ions (e.g., Na(+), K(+), and Ca(2+)).     

Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.     

A modeling approach to describe the adhesion of rough, asymmetric particles to surfaces

A combined theoretical and experimental study of the adhesion of alumina particles and polystyrene latex spheres to silicon dioxide surfaces was performed. A boundary element technique was used to model electrostatic interactions between micron-scale particles and planar surfaces when the particles and surfaces were in contact. This method allows quantitative evaluation of the effects of particle geometry and surface roughness on the electrostatic interaction. The electrostatic interactions are combined with a previously developed model for van der Waals forces in particle adhesion. The combined model accounts for the effects of particle and substrate geometry, surface roughness and asperity deformation on the adhesion force. Predictions from the combined model are compared with experimental measurements made with an atomic force microscope. Measurements are made in aqueous solutions of varying ionic strength and solution pH. While van der Waals forces are generally dominant when particles are in contact with surfaces, results obtained here indicate that electrostatic interactions contribute to the overall adhesion force in certain cases. Specifically, alumina particles with complex geometries were found to adhere to surfaces due to both electrostatic and van der Waals interactions, while polystyrene latex spheres were not affected by electrostatic forces when in contact with various surfaces.     

Mechanical Properties of Bacterially Synthesized Nanocellulose Hydrogels

The mechanical characteristics of bacterially synthesized nano-cellulose (BNC) were studied with uniaxial compression and tensile tests. Compressive loads result in a release of water and the deformation of the water-saturated network corresponds approximately to the volume of released water. The BNC hydrogel exhibits a mainly viscous response under compression. The strain response under tensile loads has an elastic and a viscous component. This can be described with a Maxwell model, where the viscosity is strain rate-dependent. When the aqueous phase of the BNC hydrogel is stabilized with an additional alginate hydrogel matrix, the system exhibits an elastic response under compressive loads. The analysis of the ‘alginated’ BNC network with the Maxwell model shows that the alginate matrix increases the viscosity of the composite system. The results of the mechanical tests show that the water absorbed in the BNC hydrogel strongly influences its mechanical behavior.     

Direct measurement of the binding force between microfabricated particles and a planar surface in aqueous solution by force-sensing piezoresistive cantilevers

We propose a force measurement method for evaluating the binding force between microscale flat surfaces in an aqueous solution. Using force-sensing piezoresistive cantilevers with sub-nanonewton force resolution, we have directly measured binding forces between SiO2-SiO2 microcontacts, which were created by gravity-driven random collision between microfabricated SiO2 cylindrical particles and a planar SiO2 substrate in a HCl solution. First, to examine our method we measured the pH dependence of the binding force. The binding forces were 12 and 5.8 nN at pH 1.0 and 2.0, respectively. As the pH increased, the binding force decreased and became zero at pH greater than 3.0. We confirmed that the bindings were based on the van der Waals' (VDW) force at pH 2.0 or less whereas a repulsive double-layer force acted between the surfaces at pH 3.0 or more. Second, the binding forces were categorized into a friction force or an adhesion force between the particles and the substrate. In the measurement, the friction force between the particle and the substrate was measured in the case when the particle slid on the substrate. On the contrary, the adhesion force was measured when the particle came off the substrate. Whether the particle slid or came off depended on the aspect ratio of the particle. We fabricated cylindrical particles with an aspect ratio of 0.03-2.0 and distinguished the friction force from the adhesion force by changing the aspect ratio of the particles. As a result, the friction force per unit contact area between SiO2-SiO2 flat surfaces was found to be 330 pN/microm2 +/- 20% when we used particles with a low aspect ratio (<0.1), and the adhesion force per unit contact area was 90 pN/microm2 +/- 20% for particles with a high aspect ratio (>0.4). For fluidic self-assembly that utilizes microscale surface contact in a liquid, our measurement method is an effective tool for studying and developing systems.     

聚富马酸丙二醇酯/羟基磷灰石复合多孔水凝胶的制备及其矿化性能研究

采用二步法合成了可降解的聚富马酸丙二醇酯(poly(propylene fumarate),PPF),并和N-乙烯基吡咯烷酮(N-vinyl pyrrolidone,NVP)共聚,以1,4-二氧六环为溶剂,通过改变溶剂的量制备了溶胀性能不同的PPF水凝胶.采用万能力学测试仪和扫描电子显微镜分别表征了水凝胶的压缩模量和形貌结构.选择20％ PPF和10％ NVP的聚合体系,预掺3％的纳米羟基磷灰石(hydroxyapatite,HA),以氯化钠粒子为致孔剂,制备了孔径在280～450 μm的纳米复合多孔水凝胶,使其压缩模量提高了61％.模拟体液矿化10天的结果显示,磷灰石成核位点的存在和良好的与外界液体环境物质交换的能力,促进了多孔水凝胶表面磷灰石的沉积,说明HA的复合可以有效提高PPF多孔水凝胶的成骨活性.     

Size segregation in a fluid-like or gel-like suspension settling under gravity or in a centrifuge

We investigate size segregation effects in a bidisperse concentrated suspension when slowly settling under gravity or when submitted to a centrifugal field. Experiments are carried out with PMMA spheres of two different mean diameters (190 and 25 microm) suspended in a hydrophobic index-matched fluid. Spatial repartitions of both small and large spheres and velocity fluctuations of particles are measured using fluorescently labeled PMMA spheres and a particle-image-velocimetry method. Large particles behave as hard spheres in purely hydrodynamic interactions, while small spheres interact through weakly attractive forces. For a small amount of small spheres among large ones, the suspension remains fluid during settling and the organization of the velocity field of particles into finite-sized structures also called "blobs" promotes size segregation. A larger proportion of weakly attractive small spheres in the bidisperse suspension causes a considerable slowdown of the settling process under gravity and the occurrence of a large-scale collective behavior together with a loss of size segregation. When centrifuging the gel-like bidisperse suspension, a shear-induced melting of the particle network induces a spectacular segregation of species. As a consequence, aging tests of soft yielding materials using centrifugation methods are not representative of the shelf-life stability of the products. A tentative model based on the competition between viscous stresses acting upon particles and adhesive stresses gives a correct estimate of the critical stationary acceleration for the destabilization of the particle network and the onset of size segregation in a gel-like suspension.     

Synthesis of spherical polymer and titania photonic crystallites

The fabrication of small structured spherical particles that are essentially small photonic crystals is described. The particles are 1-50 microm in diameter and are porous with nearly close-packed monodisperse pores whose size is comparable to the wavelength of light. The solid matrix of the particles is titania, which provides a large refractive index contrast between the particle matrix and pores. The particles are made by encapsulating polymer colloidal particles in emulsion droplets of hexanes in which a titanium alkoxide precursor is dissolved. Subsequent osmotic removal of the hexanes from the droplets and condensation of the alkoxide precursor leads to spherical aggregates of polymer spheres with titania filling the spaces between the polymer spheres. The polymer particles are then burned out leaving behind the desired porous titania particles. The size and structure of the pores and high refractive index of the titania matrix are expected to produce particles that are very efficient scatterers of light, making them useful as pigments.     

Time‐Controllable Lipophilic‐Drug Release System Designed by Loading Lipid Nanoparticles into Polysaccharide Hydrogels

A hybrid hydrogel composed of solid lipid nanoparticles (LNPs) entrapped within chemically cross‐linked carboxymethylcellulose (CMC) is developed to achieve localized and sustained release of lipophilic drugs. The analysis of LNP stability as well as the hydrogel swelling and mechanical properties confirm the successful incorporation of particles up to a concentration of 50% w/w_CMC. The initial LNP release rate can be prolonged by increasing the particle diameter from 50 to 120 nm, while the amount of long‐term release can be adjusted by tailoring the particle surface charge or the cross‐linking density of the polymer. After 30 d, 58% of 50 nm diameter negatively charged LNPs escape from the matrix while only 17% of positively charged nanoparticles are released from materials with intermediate cross‐linking density. A mathematical diffusion model based on Fick's second law is efficient to predict the diffusion of the particles from the hydrogels.     

Macroporous ZrO2 ceramics prepared from colloidally stable nanoparticles building blocks and organic templates

ZrO2 macroporous materials with well-ordered structures were prepared using nano-ZrO2 particles as the building materials and polystyrene spheres as the organic templates. A well-dispersed nano-ZrO2 suspension with a narrow particle size distribution was prepared by deagglomeration of as-received nano-ZrO2 powders via ultrasonication, and then centrifugation was performed to remove agglomerated bigger particles. Negatively charged polystyrene spheres were uniformly coated with positively charged nano-ZrO2 particles by means of electrostatic attraction at pH 4. Green samples were prepared by slip casting from colloidally stable suspension of nano-ZrO2 coated polystyrene spheres. ZrO2 macroporous materials with well-ordered microstructure derived from the nano-ZrO2 coated polystyrene spheres.     

Pull-off force measurements between rough surfaces by atomic force microscopy

Direct measurements of the pull-off (adhesion) forces between pharmaceutical particles (beclomethasone dipropionate, a peptide-type material, and lactose) with irregular geometry and rough polymeric surfaces (series of polypropylene coatings, polycarbonate, and acrylonitrile-butadiene-styrene) were carried out using the atomic force microscope. These measurements showed that roughness of the interacting surfaces is the significant factor affecting experimentally measured pull-off forces. A broad distribution of pull-off force values was noted in the measurements, caused by a varying adhesive contact area for a particle located on rough substrate. The possibility of multiple points of contact between irregularly shaped pharmaceutical particles and substrate surfaces is demonstrated with nanoindentations of the particle in a fluoro-polymer film. Force-distance curves showing the "sawtooth" pattern are additional evidence that particles make contact with substrates at more than one point. Reduced adhesion of 10- to 14-microm-diameter lactose and peptide material particles to the polypropylene coatings with a roughness of 194 nm was found in this study. Similar pull-off force versus roughness relationships are also reported for the model spherical particles, silanized glass particle with a size of 10 microm and polystyrene particle with a diameter of 9 microm, in contact with polypropylene coatings of varying roughness characteristics. It was found that the model recently proposed by Rabinovich et al. (J. Colloid Interface Sci. 232, 1-16 (2000)) closely predicts the pull-off forces for glass and lactose particles. On the other hand, the adhesion of the peptide material and polystyrene particle to polypropylene is underestimated by about an order of magnitude with the theoretical model, in which the interacting substrates are treated as rigid materials. The underestimate is attributed to the deformation of the peptide material and polystyrene particles.     

Microstructured layers of spherical biofunctional core-shell nanoparticles provide enlarged reactive surfaces for protein microarrays

Nanostructured core-shell particles with tailor-made affinity surfaces were used to generate microstructured affinity surfaces by microspotting the particles to form densely packed amorphous nanoparticle layers. These layers provided a large reactive surface for the specific binding of protein ligands from aqueous solution. Biofunctional core-shell particles were synthesized for this purpose that consisted of a silica core with a diameter of 100 nm and an organic shell a few nm thick. The nanoparticle core was prepared by sol-gel chemistry and the shell formed in suspension by organosilane chemistry. The shell provided amino groups or carbonyl groups at its outer surface for subsequent covalent immobilization of streptavidin, rabbit IgG antibodies or goat IgG antibodies. AlexaFluor 647-conjugated and biotinylated cytochrome C and CyDye-labeled anti-rabbit IgG and anti-goat IgG were probed as model analytes. The core-shell nanoparticles were spotted using a pin-ring micro-arrayer onto microscope glass slides that were coated with a polycation monolayer by dip-coating prior to nanoparticle deposition. Amorphous particle layers of well-defined thicknesses in the range of 100 nm to 2 microm were obtained by printing aqueous particle suspensions containing 5-500 mg/mL (0.5-50 wt%) of silica particles. The specific affinity of the plotted nanoparticulate capture surface was demonstrated by binding Cy3-labeled donkey anti-rabbit IgG and Cy5-labeled mouse anti-goat IgG to immobilized rabbit IgG and goat IgG particles. The signal intensity per spot increased for any given analyte concentration when the amount of particles per spot was augmented. This was attributed to the increasing integration of receptor molecules per surface footprint, which shifted the binding equilibrium towards the formation of the receptor-ligand complex. Additionally, the locally-increased supply of receptor molecules at the nanoparticulate microchip surface resulted in a wide dynamic range of 4 fM-20 nM (covering six orders of magnitude).     

Computational and experimental investigations of mono-septanoside binding by Concanavalin A: correlation of ligand stereochemistry to enthalpies of binding

Structure-energy relationships for a small group of pyranose and septanose mono-saccharide ligands are developed for binding to Concanavalin A (ConA). The affinity of ConA for methyl "manno"β-septanoside 7 was found to be higher than any of the previously reported mono-septanoside ligands. Isothermal titration calorimetry (ITC) in conjunction with docking simulations and quantum mechanics/molecular mechanics (QM/MM) modeling established the specific role of binding enthalpy in the structure-energy relations of ConA bound to natural mono-saccharides and unnatural mono-septanosides. An important aspect in the differential binding among ligands is the deformation energy required to reorganize internal hydroxyl groups upon binding of the ligand to ConA.     

Comparison of cysteine and penicillamine ligands in a Co(II) maquette

l-Penicillamine (Pen) has been investigated as a ligand for metalloprotein design by examining the binding of Co(II) to the sequence NH(2)-KL(Pen)EGG.(Pen)IG(Pen)GA(Pen).GGW-CONH(2). For comparison, we have studied Co(II) binding to the analogous sequence with Cys ligands, the ferredoxin maquette ligand IGA that was originally designed to bind a [4Fe-4S] cluster. The Co(II) affinity and UV-vis spectroscopic properties of IGA indicate formation of a pseudotetrahedral tetrathiolate ligated Co(II). In contrast, IGA-Pen showed formation of a pseudotetrahedral complex with Co(II) bound by three Pen ligands and an exogenous H(2)O. EXAFS data on both Co(II) complexes confirms not only the proposed primary coordination spheres but also shows six Co(II)-C(beta) methyl group distances in Co(II)-IGA-Pen. These results demonstrate that ligand sterics in simple peptides can be designed to provide asymmetric coordination spheres such as those commonly observed in natural metalloproteins.     

Programmable fluidic production of microparticles with configurable anisotropy

We report a technique for continuous production of microparticles of variable size with new forms of anisotropy including alternating bond angles, configurable patchiness, and uniform roughness. The sequence and shape of the anisotropic particles are configured by exploiting a combination of confinement effects and microfluidics to pack precursor colloids with different properties into a narrow, terminal channel. The width and length of the channel relative to the particle size fully specify the configuration of the anisotropic particle that will be produced. The precursor spheres packed in the production zone are then permanently bonded into particles by thermal fusing. The flow in the production zone is reversed to release the particles for collection and use. Particles produced have linear chain structure with precisely configured, repeatable bond angles. With software programmable microfluidics, sequence and shape anisotropy are combined to yield synthesized homogeneous (type "A"), surfactantlike (type "A-B") or triblock (type "A-B-A") internal sequences in a single device. By controlling the dimensions of the microfluidic production zone, triangular prisms and particles with controlled roughness and patchiness are produced. The fabrication method is performed with precursors spheres with diameter as small as 3.0 microm.