Protein adsorption on novel acrylamido-based polymeric ion-exchangers. I. Morphology and equilibrium adsorption

The protein uptake equilibrium and particle morphology are determined for novel polymeric ion-exchange media based on acrylamido monomers with a high density of functional groups and a variety of morphological characteristics. The study considers two anion-exchangers and a cation-exchanger. Physical properties determined experimentally include particle density, ion-exchange capacity, particle size distribution, and equilibrium isotherms for model proteins. The pore structure was evaluated using size exclusion chromatography with neutral probe molecules and transmission electron microscopy. For the anion-exchangers, two types of structures were inferred. The first is comprised of particles that contain a low-density gel supported by denser polymer aggregates. This material had a very low size-exclusion limit for neutral probes, but exhibited an extremely high and reversible protein adsorption capacity (280-290 mg BSA/ml). The second structure is comprised of particles with large, open macropores. While the size-exclusion limit was very high, the protein adsorption capacity was low (60 mg BSA/ml). Moreover, the adsorption was nearly irreversible. The physical structure of the cation-exchanger appeared to be intermediate between those of the anion-exchangers, containing both large pores and smaller pores yielding an intermediate, but reversible, protein uptake capacity (120-130 mg alphaCHY/ml). The different behavior of these materials with regards to protein adsorption correlates well with their physical structure. For these ion-exchangers, high protein adsorption capacities are attained when a low-density polymer gel with a high concentration of functional groups is present.     

Protein adsorption on novel acrylamido-based polymeric ion-exchangers III. Salt concentration effects and elution behavior

The effect of salt concentration on the adsorption and desorption of BSA has been determined for a polymeric anion-exchanger based on acrylamido monomers. The material investigated possesses a high adsorption capacity at low salt concentration and the bound protein can be recovered quantitatively at high salt concentrations. The effects of salt on adsorption and desorption rates were evaluated from batch and shallow-bed experiments, and a model was developed to describe the data quantitatively. The adsorption capacity decreases as the salt concentration is increased, but both adsorption and desorption rates increase at higher salt concentrations. The predictability of the behavior of columns packed with this material was examined by comparing model predictions and experimental results obtained in laboratory columns. In general, a good agreement was obtained between predicted and experimental breakthrough and elution profiles, especially in shorter columns. Thus, the model allows a prediction of the effects of column length, mobile phase flow-rate, protein feed concentration, and salt concentration on dynamic capacity, productivity, and on the concentration of product fractions.     

Protein adsorption on novel acrylamido-based polymeric ion exchangers. II. Adsorption rates and column behavior

Uptake kinetics and breakthrough behavior were determined for bovine serum albumin (BSA) and alpha-chymotrypsinogen (alphaCHY) in new polymeric ion-exchange media based on acrylamido monomers. Two anion exchangers and a cation exchanger were investigated. As shown in Part I of this work, the two anion exchangers have different morphologies. The first one, BRX-Q, comprises a low-density gel with a matrix of denser polymeric aggregates. While this material has a very low size-exclusion limit for neutral probes, it exhibits an extremely high binding capacity for BSA. The second anion exchanger, BRX-QP, comprises large open pores but has a very low binding capacity. The cation exchanger, BRX-S, also comprises large open pores but exhibits an intermediate capacity; likely as a result of the presence of smaller pores. Dynamic protein uptake experiments showed that the highest mass transfer rates are obtained with BRX-Q. The apparent diffusivity is also highest for this material and increases substantially as the protein concentration is reduced. For these particles, the external film resistance is dominant at very low protein concentrations. Much lower rates and apparent diffusivities are obtained for BRX-QP. Finally intermediate rates and apparent diffusivities are found with BRX-S. The concentration dependence of the apparent pore diffusivity is much less pronounced in this case. The apparently paradoxical result that mass transfer rates are highest for the material with the smallest neutral-probe size-exclusion limit can be explained in terms of a general conceptual model where parallel pore and adsorbed-phase diffusion paths exist in these particles. In the first case, adsorbed phase diffusion in gel pores is dominant, while in the second transport is dominated by diffusion in a macroporous network. In the third case, both contributions are important. The conceptual model provides an accurate prediction of the breakthrough behavior of columns packed with these media using independently determined rate parameters. Dynamic binding capacities of 80-140 mg/ml were observed for BSA on BRX-Q in ca. 1.5 cm columns operated at 300-900 cm/h in agreement with theoretical predictions.     

Protein adsorption and transport in polymer-functionalized ion-exchangers

A wide variety of stationary phases is available for use in preparative chromatography of proteins, covering different base matrices, pore structures and modes of chromatography. There has recently been significant growth in the number of such materials in which the base matrix is derivatized to add a covalently attached or grafted polymer layer or, in some cases, a hydrogel that fills the pore space. This review summarizes the main structural and functional features of ion exchangers of this kind, which represent the largest class of such materials. Although the adsorption and transport properties may generally be used operationally and modeled phenomenologically using the same methods as are used for proteins in conventional media, there are noteworthy mechanistic differences in protein behavior in these adsorbents. A fundamental difference in protein retention is that it may be portrayed as partitioning into a three-dimensional polymer phase rather than adsorption at an extended two-dimensional surface, as applies in more conventional media. Beyond this partitioning behavior, however, the polymer-functionalized media often display rapid intraparticle transport that, while qualitatively comparable to that in conventional media, is sufficiently rapid quantitatively under certain conditions that it can lead to clear benefits in key measures of performance such as the dynamic binding capacity. Although possible mechanistic bases for the retention and transport properties are discussed, appreciable areas of uncertainty make detailed mechanistic modeling very challenging, and more detailed experimental characterization is likely to be more productive.     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Analytical applications of Zr(IV) and Ti(IV) arsenophosphates as ion-exchangers

The distribution of 27 metal ions between zirconium and titanium arsenophosphate and demineralized water, perchloric acid and nitric acid has been studied. On the basis of the results, several binary and ternary separations can be designed. The data have been used in application of these materials to the analysis of certain alloys and rocks.     

Elucidation of protein adsorption behavior on polymeric surfaces: toward high-density, high-payload protein templates

The elucidation of protein adsorption behavior on polymeric surfaces is very important, since their use as arrays and carriers of biomolecules is ever growing for a wide variety of bioapplications. We evaluate protein adsorption characteristics on chemically homogeneous and heterogeneous polymeric surfaces by employing polystyrene-block-polymethylmethacrylate (PS-b-PMMA) diblock copolymer, PS homopolymer, PMMA homopolymer, and PS/PMMA blend as protein templates. We also investigate distance-dependent protein adsorption behavior on the interfacial region between PS and PMMA. We observe selective protein adsorption exclusively onto PS areas for the chemically heterogeneous PS-b-PMMA and PS/PMMA blend templates. On blend films, protein adsorption is highly favored on the PS regions located near the PS:PMMA interface over that on the PS areas situated away from the interface. Protein density on PS domains is inversely proportional to the separation distance between two neighboring PS:PMMA interfaces. We also observe a higher protein density on the PS-b-PMMA than on the PS or PMMA homopolymer templates. This effect is due to the fact that chemically heterogeneous PS-b-PMMA presents periodically spaced PS:PMMA interfaces on the nanometer scale, whereas no such interfaces are present on homopolymer films. The density of protein molecules on the heterogeneous PS-b-PMMA surface is approximately 3-4-fold higher than on the homogeneous PS surface for the identical experimental conditions. These results demonstrate that self-assembling, chemically heterogeneous, nanoscale domains in PS-b-PMMA diblock copolymers can be used as excellent, high-payload, high-density protein templates. The unique advantages of the diblock copolymer may prove the spontaneously constructed protein nanotemplates to be highly suitable as functional substrates in many proteomics applications.     

Ethane/ethylene adsorption on carbon nanotubes: temperature and size effects on separation capacity

We present the results of Monte Carlo simulations of the adsorption of single-component ethane and ethylene and of equimolar mixtures of these two gases on bundles of closed, single-walled carbon nanotubes. Two types of nanotube bundles were used in the simulations: homogeneous (i.e., those in which all the nanotubes have identical diameters) and heterogeneous (those in which nanotubes of different diameters are allowed). We found that at the same pressure and temperature more ethane than ethylene adsorbs on the bundles over the entire range of pressures and temperatures explored. The simulation results for the equimolar mixtures show that the pressure at which maximum separation is attained is a very sensitive function of the diameter of the nanotubes present in the bundles. Simulations using heterogeneous bundles yield better agreement with single-component experimental data for isotherms and isosteric heats than those obtained from simulations using homogeneous bundles. Possible applications of nanotubes in gas separation are discussed. We explored the effect of the diameter of the nanotubes on the separation ability of these sorbents, both for the internal and for the external sites. We found that substrate selectivity is a decreasing function of temperature.     

Chelating ion-exchangers containing n-substituted hydroxylamine functional groups : Part IV. Column separations on a hydroxamic acid resin

Ion exchange separations on a new hydroxamic acid ion exchanger are described. Quantitative separation of iron(III) from various salts and from several analytical standards has been achieved, and sources of interference in the colorimetric determination of iron with thioglycollic acid can be eliminated. Quantitative separations of copper from iron and from cobalt and nickel are possible. Recoveries and separations of iron and uranium from simulated sea-water samples are demonstrated.     

Protein adsorption and transport in dextran-modified ion-exchange media. III. Effects of resin charge density and dextran content on adsorption and intraparticle uptake

Custom-synthesized variants of the commercial Capto S resin were used to examine the effects of resin charge density and dextran content on protein adsorption and intraparticle uptake. For the small protein lysozyme, resin charge density had the greatest effect on equilibrium capacity, consistent with calculations suggesting that lysozyme capacity should be limited by the available charge on the resin. Isocratic retention data and confocal microscopy imaging for this protein revealed a consistent ordering of the resins linking stronger protein-resin interactions with higher static capacities but slower intraparticle uptake rates over the range of properties studied. For the larger protein lactoferrin, it was found that increasing dextran content led to increased protein exclusion from the dextran layer, but that increasing resin charge density helped overcome the exclusion, presumably due to the increased electrostatic attraction between the resin and protein. Collectively examining the lysozyme and lactoferrin data along with information from previous studies suggests that a trade-off in maximizing dynamic capacities should exist between static capacities that increase to a finite extent with increased resin charge density and uptake rates that decrease with increased charge density. Column breakthrough data for lysozyme and lactoferrin appear to support the hypothesis, though it appears that whether a resin charge density is low or high must be considered in relation to the protein charge density. Using these trends, this work could be useful in guiding resin selection or design.     

Protein adsorption on nanoporous TiO2 films: a novel approach to studying photoinduced protein/electrode transfer reactions

We have investigated the use of nanoporous TiO2 films as substrates for protein immobilisation. Such films are of interest due to their high surface area, optical transparency, electrochemical activity and ease of fabrication. These films moreover allow detailed spectroscopic study of protein/electrode electron transfer processes. We find that protein immobilisation on such films may be readily achieved from aqueous solutions at 4 degrees C with a high binding stability and no detectable protein denaturation. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of up to 850 for an 8 microns thick film). We demonstrate that the redox state of proteins such as immobilised cytochrome-c (Cyt-c) and haemoglobin (Hb) may be modulated by the application of an electrical bias potential to the TiO2 film, without the addition of electron transfer mediators. The binding of Cyt-c on the TiO2 films is investigated as a function of film thickness, protein concentration, protein surface charge and ionic strength. We demonstrate the potential use of immobilised Hb on such TiO2 films for the detection of dissolved CO in aqueous solutions. We further show that protein/electrode electron transfer may be initiated by UV bandgap excitation of the TiO2 electrode. Both photooxidation and photoreduction of the immobilised proteins can be achieved. By employing pulsed UV laser excitation, the interfacial electron transfer kinetics can be monitored by transient optical spectroscopy, providing a novel probe of protein/electrode electron transfer kinetics. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of novel bioanalytical devices such as biosensors.     

Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake

Delivery and toxicity are critical issues facing nanomedicine research. Currently, there is limited understanding and connection between the physicochemical properties of a nanomaterial and its interactions with a physiological system. As a result, it remains unclear how to optimally synthesize and chemically modify nanomaterials for in vivo applications. It has been suggested that the physicochemical properties of a nanomaterial after synthesis, known as its "synthetic identity", are not what a cell encounters in vivo. Adsorption of blood components and interactions with phagocytes can modify the size, aggregation state, and interfacial composition of a nanomaterial, giving it a distinct "biological identity". Here, we investigate the role of size and surface chemistry in mediating serum protein adsorption to gold nanoparticles and their subsequent uptake by macrophages. Using label-free liquid chromatography tandem mass spectrometry, we find that over 70 different serum proteins are heterogeneously adsorbed to the surface of gold nanoparticles. The relative density of each of these adsorbed proteins depends on nanoparticle size and poly(ethylene glycol) grafting density. Variations in serum protein adsorption correlate with differences in the mechanism and efficiency of nanoparticle uptake by a macrophage cell line. Macrophages contribute to the poor efficiency of nanomaterial delivery into diseased tissues, redistribution of nanomaterials within the body, and potential toxicity. This study establishes principles for the rational design of clinically useful nanomaterials.     

Effect of pH on protein adsorption capacity of strong cation exchangers with grafted layer

The effect of pH on the static adsorption capacity of immunoglobulin G, human serum albumin, and equine myoglobin was investigated for a set of five strong cation exchangers with the grafted tentacle layer having a different ligand density. A sharp maximum of adsorption capacity with pH was observed for adsorbents with a high ligand density. The results were elucidated using the protein structure and calculations of pK(a) of ionizable groups of surface basic residues. Inverse size-exclusion experiments were carried out to understand the relation between the adsorption capacity and pore accessibility of the investigated proteins.     

Influence of surface chemistry and protein concentration on the adsorption rate and S-layer crystal formation

Bacterial crystalline surface layers (S-layers) are the outermost envelope of prokaryotic organisms representing the simplest biological membranes developed during evolution. In this context, the bacterial protein SbpA has already shown its intrinsic ability to reassemble on different substrates forming protein crystals of square lattice symmetry. In this work, we present the interaction between the bacterial protein SbpA and five self-assembled monolayers carrying methyl (CH(3)), hydroxyl (OH), carboxylic acid (COOH) and mannose (C(6)H(12)O(6)) as functional groups. Protein adsorption and S-layer formation have been characterized by atomic force microscopy (AFM) while protein adsorption kinetics, mass uptake and the protein layer viscoelastic properties were investigated with quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicate that the protein adsorption rate and crystalline domain area depend on surface chemistry and protein concentration. Furthermore, electrostatic interactions tune different protein rate adsorption and S-layer recrystallization pathways. Electrostatic interactions induce faster adsorption rate than hydrophobic or hydrophilic interactions. Finally, the shear modulus and the viscosity of the recrystallized S-layer on CH(3)C(6)S, CH(3)C(11)S and COOHC(11)S substrates were calculated from QCM-D measurements. Protein-protein interactions seem to play a main role in the mechanical stability of the formed protein (crystal) bilayer.     

间氨基酚修饰超高交联吸附树脂的合成及吸附性能

合成了间氨基酚修饰超高交联吸附树脂(MMAR)。用红外光谱等进行表征,并用于吸附水溶液中2-氨基吡啶的研究。热力学研究结果表明,Freundlich吸附等温方程能够对静态吸附等温线进行很好的拟合。吸附焓变ΔH0,其绝对值小于46 KJ.mo-l1,表明以物理吸附为主以及该吸附剂容易脱附的特征;△G0,说明吸附是自发行为;△S0,表明吸附质分子在树脂表面上的运动受到了限制。动力学研究结果表明,吸附符合一级动力学方程,吸附速率随着温度的升高而增大,颗粒内扩散是速率控制步骤之一。表观活化能Ea10 KJ.mo-l1,说明吸附较容易进行。     

Kinetics of protein adsorption by nanoporous carbons with different pore size

Kinetics of bovine serum albumin and ovalbumin adsorption by nanoporous carbons with different main pore sizes (1.6, 5, 7.8 and 28 nm) was studied. Experimental kinetics curves were well described by multi-exponential equation with different number of exponents (from 1 to 4). Protein adsorption kinetics showed significant dependence on pore size of carbonaceous adsorbent. Correlation between pore size distribution and amount of protein adsorbed revealed threshold pore size 7.3 nm for BSA and 6.8 nm for OVA, which are close to hydrodynamic diameter of protein molecules. The fastest and the highest adsorption of proteins were observed in carbons having developed porosity with pore sizes larger than 15 nm.     

Protein adsorption on the mesoporous molecular sieve silicate SBA-15: effects of pH and pore size

A mesoporous molecular sieve silicate, SBA-15, with three pore sizes (38.1 A, 77.3 A, and 240 A) has been synthesized using a non-ionic, tri-block copolymer as a template in a sol-gel method. The effects of synthesis conditions on the pore size and pore-size distribution of this adsorbent have been described. The adsorption of proteins on these crystalline, ordered, materials has been studied. The kinetics of adsorption and equilibrium capacity have been probed with three proteins of different dimensions. The effects of electrostatic interactions and protein size are illustrated. It has been shown that SBA-15 materials can be tailored to show size selectivity for proteins, and very high capacities (450 mg/g) can be obtained. Furthermore, the rates of adsorption are shown to be dependent on the pore size, protein structure and solution pH.     

Effects of toluene on thiophene adsorption over NaY and Ce(IV)Y zeolites

Zeolites NaY and Ce(IV)Y were employed as adsorbents to remove organic sulfur compounds from model gasoline(MG) solutions with and without toluene in static adsorption experiments at room temperature(RT) and atmospheric pressure.The adsorbents were characterized by XRD,XRF and pyridine infrared spectrum(IR).The adsorption experiments show that the desulfurization performance of Ce(IV)Y is much better than that of NaY.The sulfur removal over both NaY and Ce(IV)Y decreases with the increase of toluene concentration in MG,however,the decline tendency on Ce(IV)Y is smooth,and it is steep on NaY.FT-IR spectra of thiophene adsorption indicate that thiophene molecules are mainly adsorbed on NaY via π electron interaction,but on Ce(IV)Y,in addition to the π electron interaction,both Ce4+-S direct interaction and protonation of thiophene also play important roles.Toluene molecules are adsorbed on NaY also via π electron interaction.Although the amount of Brnsted acid sites is increased due to the introduction of Ce4+ ions into NaY zeolite,it is not found to influence the adsorption mode of toluene over Ce(IV)Y.Compared with NaY zeolite,the improved desulfurization performance over Ce(IV)Y for removing organic sulfur compounds from MG solution,especially those containing large amount of aromatics,may be ascribed to the direct Ce(IV)-S interaction,which is much resistant to the influence resulted from toluene adsorption.     

Effects of pyrophosphate ions on protein adsorption onto calcium hydroxyapatite

The effects of pyrophosphate ions (PP: P2O7(4-)) on the adsorption of proteins onto calcium hydroxyapatite (Hap) were examined using typical proteins of bovine serum albumin (BSA: isoelectric point (iep) = 4.7, molecular mass (M(s)) = 67 200 Da, acidic protein), myoglobin (MGB: iep = 7.0, M(s) = 17 800 Da, neutral protein), and lysozyme (LSZ: iep = 11.1, M(s) = 14,600 Da, basic protein). The UV and CD measurements determined that both the secondary and the tertiary structures of protein molecules do not vary in the presence of PP. The adsorption of BSA was strongly depressed by the addition of PP in all the methods with changing the order of PP addition. Even if BSA was pre-adsorbed on the Hap surface, PP replaced BSA molecules by strong preferential adsorption onto Hap to reduce the amounts of adsorbed BSA. A similar effect was observed with the adsorption of MGB. On the other hand, the amount of adsorbed LSZ (n(LSZ)) was increased with an increase in the concentration of PP, and the n(LSZ) value showed a maximum point in each adsorption isotherm. This fact was explained by a compression of the electric double layer (EDL) around each LSZ molecule by PP. This compression of the EDL induced the reduction of lateral electrostatic repulsions between charged LSZ molecules on the Hap surface and enhanced the formation of closed-packed monolayers to raise the n(LSZ) value. However, since the number of PPs around a LSZ molecule is decreased by an increase in the LSZ concentration in each system, the thickness of the EDL may be increased. Hence, n(LSZ) was reduced again after the maximum point in each system. Tripolyphosphate (TPP: P3O10(5-)) ions exhibited similar effects on the adsorption behaviors of all proteins, but a much more pronounced effect was observed on the LSZ system. TPP with a higher eletronegativity shielded the EDL more highly than PP to increase the n(LSZ) value. The results of the zeta potential for all the protein systems supported the modes of protein adsorption discussed.     

Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations

The effects of the column length, the particle size, the gradient length and the flow rate of a nanoLC system on peptide peak capacity were investigated and compared. Columns packed with 1.7 microm and 3 microm C(18) materials into pieces of 75 microm capillary tubing of various lengths were tested with different gradient lengths and flow rates. While increasing the length of a column packed with the 1.7 microm material helped improve peptide peak capacity at the whole range of the tested gradient lengths (24-432 min), little improvement in peak capacity was observed with the increase of the length of a column packed with the 3 microm material unless a gradient longer than 50 min was carried out. Up to 30% of peak capacity increase was observed when a column's length is doubled, with little reduction in the throughput. In most cases, more than 50% of the increase in peak capacity was obtained with the reduction in the particle size from 3 microm to 1.7 microm. With the same backpressure generated, a shorter 1.7-microm-particle column outperformed a longer column packed with the 3 microm material. In a flow rate range of 100-700 nl/min, increasing the flow rate improved peak capacity for columns packed with 1.7 microm and 3 microm materials.