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Highly selective ion separations are vital for producing pure salts, and membrane-based separations are promising alternatives to conventional ion-separation techniques. Our previous work demonstrated that simple pressure-driven flow through negatively charged isoporous membranes can separate Li + and K + with selectivities as high as 70 in dilute solutions. The separation mechanism relies on spontaneously arising streaming potentials that induce electromigration, which opposes advection and separates cations based on differences in their electrophoretic mobilities. Although the separation technique is simple, this work shows that high selectivities are possible only with careful consideration of experimental conditions including transmembrane pressure, solution ionic strength, the K + /Li + ratio in the feed, and the extent of concentration polarization. Separations conducted with a rotating membrane show Li + /K + selectivities as high as 150 with a 1000 rpm membrane rotation rate, but the selectivity decreases to 1.3 at 95 rpm. These results demonstrate the benefits and necessity of quantitative control of concentration polarization in highly selective separations. Increases in solution ionic strength or the K + /Li + feed ratio can also decrease selectivities more than an order of magnitude.

Quantitation of therapeutic monoclonal antibodies (mAbs) in human serum could ensure that patients have adequate levels of mAbs for effective treatment. This research describes the use of affinity, glass-fiber membranes in a 96-well-plate format for rapid (<5 min) quantitation of the therapeutic mAb trastuzumab and a mAb against the SARS-CoV-2 spike protein. Adsorption of a poly(acrylic acid)-containing film in membrane pores and activation of the -COOH groups in the film enable covalent-linking of affinity peptides or proteins to the membrane. Passage of mAb-containing serum through the affinity membrane results in mAb capture within 1 min. Subsequent rinsing, binding of a secondary antibody conjugated to a fluorophore, and a second rinse yield mAb-concentration-dependent fluorescence intensities in the wells. Calibration curves established from analyses on different days have low variability and allow determination of mAb levels in separately prepared samples with an average error <10%, although errors in single-replicate measurements may reach 40%. The assays can occur in diluted serum with physiologically relevant mAb concentrations, as well as in undiluted serum. Thus, the combination of 96-well plates containing affinity membranes, a microplate reader, and a simple vacuum manifold affords convenient mAb quantitation in <5 min.

MALDI-TOF mass spectrometry imaging (MSI) is a powerful tool for studying biomolecule localization in tissue. Protein distributions in tissue provide important histological information; however, large proteins exhibit a high limit of detection in MALDI-MS when compared to their corresponding smaller proteolytic peptides. As a result, several techniques have emerged to digest proteins into more detectable peptides for imaging. Digestion is typically accomplished through trypsin deposition on the tissue, but this technique increases the complexity of the tissue microenvironment, which can limit the number of detectable species. This proof-of-principle study explores tryptic tissue digestion during electroblotting through a trypsin-containing membrane. This approach actively extracts and enzymatically digests proteins from mouse brain tissue sections while simultaneously reducing the complexity of the tissue microenvironment (compared to trypsin deposition on the surface) to obtain an increased number of detectable peptide fragments. The method does not greatly compromise spatial location or require expensive devices to uniformly deposit trypsin on tissue. Using electrodigestion through membranes, we detected and tentatively identified several tryptic peptides that were not observed after on-tissue digestion. Moreover, the use of pepsin rather than trypsin in digestion membranes allows extraction and digestion at low pH to detect peptides from a complementary subset of tissue proteins. Future studies will aim to further improve the method, including changing the substrate membrane to increase spatial resolution and the number of detected peptides.

Effective monoclonal antibody (mAb) therapies require a threshold mAb concentration in patient serum. Moreover, the serum concentration of the mAb Bevacizumab should reside in a specific range to avoid side effects. Methods for conveniently determining the levels of mAbs in patient sera could allow for personalized dosage schedules that lead to more successful treatments. This work utilizes microporous nylon membranes functionalized with antibody-binding peptides to capture Bevacizumab, Rituximab, or Panitumumab from diluted (25%) serum. Modification of the capture-peptide terminus is often crucial to creating the affinity necessary for effective binding. The high purity of eluted mAbs allows for their quantitation using native fluorescence, and membranes are effective in spin devices that can be used in any laboratory. The technique is effective over the therapeutic range of Bevacizumab concentrations. Future work aims at further modifications to develop rapid point-of-care devices and decrease detection limits.

Nanochannel-based fluidic diodes display ion selectivity and ion current rectification (ICR), which may prove to be important in energy-harvesting devices and biosensors. This paper reports asymmetric functionalization of the outer surface of a flexible nanochannel polymer membrane to create fluidic diodes that give ICR. Layer-by-layer (LbL) adsorption with cross-linking of only the underlying part of the polyelectrolyte nanofilm leads to a porosity step across the film. The combination of a high effective surface charge density and the porosity step in the film leads to a remarkable maximum ICR factor of ∼200 with a pH gradient across the film. Incorporation of pH-sensitive polyelectrolyte components enables the ICR factor to increase an order of magnitude on going from pH 8 to pH 3. Moreover, the coated membrane shows excellent anion selectivity. Thus, LbL adsorption with partial cross-linking provides a simple method for creating coated nanochannel membranes that serve as pH-responsive ionic diodes for potential chemical/biosensors.

Flow through negatively charged nanopores separates Li + and K + with selectivities of up to 70 and Li + passages from 20% to above 100%. Remarkably, both the Li + /K + selectivity and Li + passage initially increase with flow rate, breaking the permeability/selectivity trade-off. Modelling demonstrates that flow through the membranes creates electric fields that retard transport of cations. Selectivity increases with flow rate because the K + electromigration velocity exceeds its convective velocity, but for Li + electromigration is weaker than convection. Modelling also shows the importance of controlling concentration polarization. With further work, related separations might provide highly pure Li salts for battery manufacturing.

Identifying the target proteins of small-molecule drug candidates is important for determining their molecular mechanisms of action. Porous membranes derivatized with such small molecules may provide an attractive target-identification platform due to a high protein-capture efficiency during flow through membrane pores. This work employs carbonic anhydrase II (CAII) binding to immobilized 4-(2-aminoethyl)benzenesulfonamide (AEBSA) to examine the efficiency and selectivity of affinity capture in modified membranes. Selective elution of captured protein, tryptic digestion, tandem mass spectrometry analysis, and label-free quantification (LFQ) identify CAII as the dominant AEBSA target in diluted serum or cell lysate. CAII identification relies on determining the ratio of protein LFQ intensities in sample and control experiments, where free AEBSA added to the control loading solution limits CAII capture. Global proteomics shows that the spiked CAII is the only protein with a log 2 ratio consistently >2, and the detection limit for CAII identification is 0.004 wt % of the total protein in 1:4 diluted human serum or 0.024 wt % of the total protein from breast cancer cell lysates. The same approach also identifies native CAII in human kidney cell lysate as an AEBSA target. Comparison of affinity capture using membranes, Affi-Gel 10 resin or M-270 Dynabeads derivatized with AEBSA suggests that only membranes allow identification of low-abundance CAII as a target.

Hypotheses: Oil droplet stability and electrical charge, and membrane's affinity for oil govern droplet attachment to a membrane surface. Moderate droplet-surface affinity encourages surface coalescence and removal of droplets to help maintain the membrane relatively oil-free., Experiments: Droplet attachment onto model nanofiltration membranes was studied, in situ and in real time, using the Direct Observation Through the Membrane method. Optically transparent nanofiltration membranes were designed by forming polyelectrolyte multilayer films, with either positively or negatively charged surfaces, on Anopore ultrafilters. Crossflow across the membrane surface employed hexadecane-in-water emulsions stabilized by an anionic surfactant (sodium dodecylsulfate) in model sea water or aqueous solutions containing NaCl or MgSO 4 ., Findings: Moderate affinity between oil and the polyelectrolyte-coated surface promotes crossflow controlled coalescence to remove droplets larger than a critical size, d drop crit , in the crossflow shear. The torque balance on a sessile oil droplet in a linear shear field overpredicted d drop crit pointing to a need for more accurate estimates of lift and drag forces on a droplet. In the presence of divalent cations, lower electrostatic repulsion between droplets facilitated droplet-droplet adhesion and led to rapid coalescence that resulted in membrane fouling. The most significant fouling appeared in tests with positively charged and less oleophobic coatings., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ion passage through ion-exchange membranes is vital in electrodialysis desalination, batteries and fuel cells, and water splitting. Simplified models of ion transport through such membranes frequently assume complete exclusion of co-ions (ions with the same sign of charge as the fixed charge in the membrane) from the membrane. However, a second assumption of constant counterion electrochemical potentials across the membrane leads to simple analytical expressions for ion fluxes and transmembrane potentials. Moreover, linear corrections to account for a small membrane electrical resistance yield analytical expressions with a wider applicability. For bi-ionic potential measurements and current-induced concentration polarization at low salt concentrations, these analytical solutions match the fluxes and potentials obtained numerically without the limiting assumptions. This gives confidence in both the limiting assumptions (under appropriate conditions) and the numerical solutions. At low ion concentrations, the analytical solutions may enable rapid characterization of membrane coatings or boundary layers in solution, and such boundary layers are important in many applications of ion-exchange membranes. In fact, the assumption of complete co-ion exclusion is sometimes more limiting than the constraint of constant electrochemical potentials of counterions across the membrane. Remarkably, this limiting case readily yields the ion accumulation and depletion regions above "ion-exchange patches" that reside beneath a solution with an applied electric field. Such regions are important for sample preconcentration in microfluidic devices.

This review critically examines current models for nanofiltration (NF) of electrolyte solutions. We start from linear irreversible thermodynamics, we derive a basic equation set for ion transfer in terms of gradients of ion electrochemical potentials and transmembrane volume flux. These equations are extended to the case of significant differences of thermodynamic forces across the membrane (continuous version of irreversible thermodynamics) and solved in quadratures for single salts and trace ions added to single salts in the case of macroscopically-homogeneous membranes. These solutions reduce to (quasi)analytical expressions in the popular Spiegler-Kedem approximation (composition-independent phenomenological coefficients), which we extend to the case of trace ions. This enables us to identify membrane properties (e.g. ion permeances, ion reflection coefficients, electrokinetic charge density) that control its performance in NF of multi-ion solutions. Further, we specify the phenomenological coefficients of irreversible thermodynamics in terms of ion partitioning, hindrance and diffusion coefficients for the model of straight cylindrical capillaries. The corresponding expressions enable assessment of the applicability of the popular nanopore model of NF. This model (based on the use of macroscopic approaches at nanoscale) leads to a number of trends that have never been observed experimentally. We also show that the use of the Born formula (frequently employed for the description of dielectric exclusion) hardly leads to meaningful values of solvent dielectric constant in membrane pores because this formula disregards the very solvent structure whose changes are supposed to bring about the reduction of dielectric permittivity in nanopores. We conclude that the effect should better be quantified in terms of ion excess solvation energies in the membrane phase. As an alternative to the nanopore description of NF, we review recent work on the development of an advanced engineering model for NF of multi-ion solutions in terms of a solution-diffusion-electromigration mechanism. This model (taking into account spontaneously arising transmembrane electric fields) captures several trends observed experimentally, and the use of trace ions can provide model parameters (ion permeances in the membrane) from experiment. We also consider a recent model (ultrathin barrier layers with deviations from local electroneutrality) that may reproduce observed feed-salt concentration dependences of membrane performance in terms of concentration-independent properties like excess ion solvation energies. Due to its complexity, practical modelling of nanofiltration will probably be performed with advanced engineering models for the foreseeable future. Although mechanistic studies are vital for understanding transport and developing membranes, future simulations in this area will likely need to depart from typical continuum models to provide physical insight. For enhancing the quality of modelling input, it is essential to improve the control of concentration polarization in membrane test cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cation-exchange membranes allow preferential passage of cations over anions, but they show minimal selectivity among cations, which limits their use in ion separations. Recent studies show that modification of cation-exchange membranes with polyelectrolyte multilayers leads to exceptional monovalent/divalent cation electrodialysis selectivities, but no studies report high selectivity among monovalent ions. This work demonstrates that adsorption of protonated poly(allylamine) (PAH)/poly(4-styrenesulfonate) (PSS) multilayers on Nafion membranes leads to high K + /Li + selectivities in Donnan dialysis, where K + and Li + ions in a source phase pass through the membrane and exchange with Na + ions in a receiving phase. Addition of 0.01 M HNO 3 to a source phase containing 0.01 M KNO 3 and 0.01 M LiNO 3 increases the K + /Li + selectivity from 8 to ∼60 through (PAH/PSS) 5 PAH-coated Nafion membranes, primarily because of a ≥fivefold increase in K + flux. These selectivities are much larger than the ratio of 1.9 for the aqueous diffusion coefficients of K + and Li + , and uncoated Nafion membranes give a K + /Li + selectivity <3. Bi-ionic transmembrane potential measurements at neutral pH confirm that the membrane is more permeable to K + than Li + , but this selectivity is less than in Donnan dialysis with acidic solutions. In situ ellipsometry data indicate that PAH/PSS multilayers (assembled at pH 2.3, 7.5, or 9.3) swell at pH 2.0, and this swelling may open cation-exchange sites that preferentially bind K + to enable highly selective transport. The coated membranes also exhibit modest selectivity for K + over H + , suggesting selective transport based on preferential partitioning of K + into the coatings. Selectivity declines when increasing the source-phase KNO 3 concentration to 0.1 M, perhaps because the discriminating transport pathway saturates. Moreover, selectivities are lower in electrodialysis than in Donnan dialysis, presumably because electrodialysis engages other transport mechanisms, such as electroosmosis and strong electromigration.

Rapid, convenient methods for monoclonal antibody (mAb) isolation are critical for determining the concentrations of therapeutic mAbs in human serum. This work uses porous nylon membranes modified with a HER2 peptide mimotope, KGSGSGSQLGPYELWELSH (KH19), for rapid affinity capture of Herceptin, a mAb used to treat breast cancer. Covalent linking of KH19 to poly(acrylic acid)-containing films in porous nylon leads to a Herceptin-binding capacity of 10 mg per mL of membrane and allows selective Herceptin capture from diluted (1:3) human serum in 5 min. Liquid chromatography-mass spectrometry demonstrates the high purity of eluted Herceptin. Moreover, the fluorescence intensity of the protein eluted from membranes increases linearly with the amount of Herceptin spiked in loading solutions containing diluted (1:3) human serum. These results demonstrate the promise of mimotope-modified membranes for Herceptin analysis that does not require secondary antibodies or derivatization with fluorescent labels. Thus, mimotope-containing membranes may form part of a simple benchtop analysis system for assessing the concentrations of therapeutic mAbs.

Proteolytic digestion is an important step in characterizing protein sequences and post-translational modifications (PTMs) using mass spectrometry (MS). This study uses pepsin- or trypsin-containing spin membranes for rapid digestion of single proteins or simple protein mixtures prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100 μL of pretreated protein solutions through the functionalized membranes requires less than 1 min and conveniently digests proteins into large peptides that aid in confirming specific protein sequence variations and PTMs. Peptic and tryptic peptides from spin digestion of apomyoglobin and four commercial monoclonal antibodies (mAbs) typically cover 100% of the protein sequences in direct infusion MS analysis. Increasing the spin rate leads to a higher fraction of large peptic peptides for apomyoglobin, and MS analysis of peptic and tryptic peptides reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal lysine clipping and glycosylation. Relative to overnight in-solution digestion of mAbs, spin digestion yields higher sequence coverages. Spin-membrane digestion followed by infusion MS readily differentiates a mAb to the Ebola virus from a related antibody that differs by addition of a single amino acid.

Proteolysis is often a critical step in protein characterization via mass spectrometry. Compared to complete digestion, limited proteolysis gives larger peptides, and the dominant cleavage sites may identify highly accessible, flexible protein regions. This paper explores controlled proteolysis in porous nylon membranes containing immobilized trypsin. Passage of protein solutions through ∼100 μm thick membranes provides reaction residence times as short as milliseconds to limit digestion. Additionally, variation of the membrane pore size and the protease-immobilization method (electrostatic adsorption or covalent anchoring to adsorbed polymer in membrane pores) affords control over the proteolysis rate. When digesting the highly labile protein β-casein, large membrane pores (5.0 μm) and covalent enzyme anchoring to adsorbed polymer lead to particularly long tryptic peptides. With the more trypsin-resistant proteins cytochrome c and apomyoglobin, in-membrane proteolysis with short residence times, 1.2 μm membrane pores, and trypsin electrostatically immobilized to an adsorbed polyanion cleaves the proteins after lysine residues in flexible regions. For both cytochrome c and apomyoglobin, cleavages in an interhelix region yield two particularly large peptides that cover the entire protein sequence.

Unlabelled: This paper examines the recovery of the enteric adenovirus human adenovirus 40 (HAdV 40) by cross-flow ultrafiltration and interprets recovery values in terms of physicochemical interactions of virions during sample concentration. Prior to ultrafiltration, membranes were either blocked by exposure to calf serum (CS) or coated with a polyelectrolyte multilayer (PEM). HAdV 40 is a hydrophobic virus with a point of zero charge between pH 4.0 and pH 4.3. In accordance with predictions from the extended Derjaguin-Landau-Verwey-Overbeek theory, the preelution recovery of HAdV (rpre) from deionized water was higher with PEM-coated membranes (rpre (PEM) = 74.8% ± 9.7%) than with CS-blocked membranes (rpre (CS) = 54.1% ± 6.2%). With either membrane type, the total virion recovery after elution (rpost) was high for both deionized water (rpost (PEM) = 99.5% ± 6.6% and rpost (CS) = 98.8% ± 7.7%) and tap water (rpost (PEM) = 89% ± 15% and rpost (CS) = 93.7% ± 6.9%). The nearly 100% recoveries suggest that the polyanion (sodium polyphosphate) and surfactant (Tween 80) in the eluent disrupt electrostatic and hydrophobic interactions between the virion and the membrane. Addition of EDTA to the eluent greatly improved the elution efficacy (rpost (CS) = 88.6% ± 4.3% and rpost (PEM) = 87.0% ± 6.9%) with surface water, even when the organic carbon concentration in the water was high (9.4 ± 0.1 mg/liter). EDTA likely disrupts cation bridging between virions and particles in the feed water matrix or the fouling layer on the membrane surface. For complex water matrices, the eluent composition is the most important factor for achieving high virion recovery., Importance: Herein we present the results of a comprehensive physicochemical characterization of HAdV 40, an important human pathogen. The data on HAdV 40 surface properties enabled rigorous modeling to gain an understanding of the energetics of virion-virion and virion-filter interactions. Cross-flow filtration for concentration and recovery of HAdV 40 was evaluated, with postelution recoveries from ultrapure water (99%), tap water (∼91%), and high-carbon-content surface water (∼84%) being demonstrated. These results are significant because of the very low adenovirus recoveries that have been reported, to date, for other methods. The recovery data were interpreted in terms of specific interactions, and the eluent composition was designed accordingly to maximize HAdV 40 recovery., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

This paper describes a convenient synthesis of nitrilotriacetate (NTA)-containing polymers and subsequent layer-by-layer adsorption of these polymers on flat surfaces and in membrane pores. The resulting films form NTA-metal-ion complexes and capture 2-3 mmol of metal ions per mL of film. Moreover, these coatings bind multilayers of polyhistidine-tagged proteins through association with NTA-metal-ion complexes. Inclusion of acrylic acid repeat units in NTA-containing copolymers promotes swelling to increase protein binding in films on Au-coated wafers. Adsorption of NTA-containing films in porous nylon membranes gives materials that capture ∼46 mg of His-tagged ubiquitin per mL. However, the binding capacity decreases with the protein molecular weight. Due to the high affinity of NTA for metal ions, the modified membranes show modest leaching of Ni(2+) in binding and rinsing buffers. Adsorption of NTA-containing polymers is a simple method to create metal- and protein-binding films and may, with future enhancement of stability, facilitate development of disposable membranes that rapidly purify tagged proteins.

Reverse osmosis and nanofiltration (NF) employ composite membranes whose ultrathin barrier layers are significantly more permeable to water than to salts. Although solution-diffusion models of salt transport through barrier layers typically assume ubiquitous electroneutrality, in the case of ultrathin selective skins and low ion partition coefficients, space-charge regions may occupy a significant fraction of the membrane barrier layer. This work investigates the implications of these deviations from electroneutrality on salt transport. Both immobile external surface charge and unequal cation and anion solvation energies in the barrier layer lead to regions with excess mobile charge, and the size of these regions increases with decreasing values of either feed concentrations or ion partition coefficients. Moreover, the low concentration of the more excluded ion in the space-charge region can greatly increase resistance to salt transport to enhance salt rejection during NF. These effects are especially pronounced for membranes with a fixed external surface charge density whose sign is the same as that of the more excluded ion in a salt. Because of the space-charge regions, the barrier-layer resistance to salt transport initially rises rapidly with increasing barrier thickness and then plateaus or even declines within a certain thickness range. This trend in resistance implies that thin, defect-free barrier layers will exhibit higher salt rejections than thicker layers during NF at a fixed transmembrane pressure. Deviations from electroneutrality are consistent with both changes in NF salt rejections that occur upon changing the sign of the membrane fixed external surface charge, and CaCl2 rejections that in some cases may first decrease, then increase and then decrease again with increasing CaCl2 concentrations in NF feed solutions.

This paper presents rapid protein purification and proteolysis methods that integrate membrane technology and pipet tips. Pushing a protein-containing solution through a protease-modified membrane at the end of a pipet tip digests proteins in 30 s or less, and the short proteolysis time avoids reformation of disulfide bonds to enable tryptic digestion without alkylation of cysteine residues. Moreover, proteolysis is more complete than digestion for 30 min in solution. Antibody digestion at the end of a pipet tip leads to 100% peptide coverage in MS analyses. Similarly, when membranes contain Ni(2+) complexes, pipetting aqueous polyhistidine-tagged protein through the membrane and subsequent rinsing and elution yield purified polyhistidine-tagged protein in 2 min. These applications demonstrate the potential for combining functional membranes and pipet tips for rapid sample purification and pretreatment.

Atom transfer radical polymerization of ionic monomers from membrane surfaces yields polyelectrolyte brushes that swell in water and repel oil droplets to resist fouling during filtration of oil-in-water emulsions. However, surfactant adsorption to polyelectrolyte brushes may overcome this fouling resistance. This work examines adsorption of cationic and anionic surfactants in polyanionic brushes and the effect of these surfactants on emulsion filtration. In situ ellipsometry with films on flat surfaces shows that brushes composed of poly(3-sulfopropyl methacrylate salts) (pSPMK) swell 280% in water and do not adsorb sodium dodecyl sulfate (SDS). pSPMK-modified microfiltration membranes reject >99.9% of the oil from SDS-stabilized submicron emulsions, and the specific flux through these modified membranes is comparable to that through NF270 nanofiltration membranes. Moreover, the brush-modified membranes show no decline in flux over a 12 h filtration, whereas the flux through NF270 membranes decreases by 98.7%. In contrast, pSPMK brushes adsorb large quantities of cetyltrimethylammonium bromide (CTAB), and at low chain densities the brushes collapse in the presence of this cationic surfactant. Filtration of CTAB-stabilized emulsions through pSPMK-modified membranes gives minimal oil rejection, presumably due to the brush collapse. Thus, the fouling resistance of polyelectrolyte brush-modified membranes clearly depends on the surfactant composition in a particular emulsion.

Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs, because of their high specificities to target cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-containing nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultrahigh-resolution Orbitrap MS analysis. Variation of the residence times (from 3 ms to 3 s) of antibody solutions in the membranes yields "bottom-up" (1-2 kDa) to "middle-down" (5-15 kDa) peptide sizes within less than 10 min. These peptides cover the entire sequences of Trastuzumab and a Waters antibody, and a proteolytic peptide comprised of 140 amino acids from the Waters antibody contains all three complementarity determining regions on the light chain. This work compares the performance of "bottom-up" (in-solution tryptic digestion), "top-down" (intact protein fragmentation), and "middle-down" (in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for "bottom-up", "top-down", and "middle-down" analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to "bottom-up" and "top-down" approaches for antibody characterization, in-membrane digestion uses minimal sample preparation time, and this technique also yields high peptide and sequence coverage for the identification of PTMs.

Electrodialysis (ED) membranes typically exhibit modest selectivities between monovalent and divalent ions. This paper reports a dramatic enhancement of the monovalent/divalent cation selectivities of Nafion 115 membranes through coating with multilayer poly(4-styrenesulfonate) (PSS)/protonated poly(allylamine) (PAH) films. Remarkably, K(+)/Mg(2+) ED selectivities reach values >1000, and similar monovalent/divalent cation selectivities occur with feed solutions containing K(+) and Ca(2+). For comparison, the corresponding K(+)/Mg(2+) selectivity of bare Nafion 115 is only 1.8 ± 0.1. However, with 0.01 M KNO3 and 0.01 M Mg(NO3)2 in the source phase, as the applied current density increases from 1.27 to 2.54 mA cm(-2), the K(+)/Mg(2+) selectivities of coated membranes decrease from >1000 to 22. Water-splitting at strongly overlimiting current densities may lead to a local pH increase close to the membrane surface and alter film permeability or allow passage of Mg(OH)x species to decrease selectivity. When the source phase contains 0.1 M KNO3 and 0.1 M Mg(NO3)2, the K(+) transference number approaches unity and the K(+)/Mg(2+) selectivity is >20,000, presumably because the applied current is below the limiting value for K(+) and H(+) transport is negligible at this high K(+) concentration. The high selectivities of these membranes may enable electrodialysis applications such as purification of salts that contain divalent or trivalent ions.

Membrane adsorbers rapidly capture tagged proteins because flow through membrane pores efficiently conveys proteins to binding sites. Effective adsorbers, however, require membrane pores coated with thin films that bind multilayers of proteins. This work employs adsorption of polyelectrolytes that chelate metal ions to create functionalized membranes that selectively capture polyhistidine-tagged (His-tagged) proteins with binding capacities equal to those of high-binding commercial beads. Adsorption of functional polyelectrolytes is simpler than previous membrane-modification strategies such as growth of polymer brushes or derivatization of adsorbed layers with chelating moieties. Sequential adsorption of protonated poly(allylamine) (PAH) and carboxymethylated branched polyethylenimine (CMPEI) leads to membranes that bind Ni(2+) and capture ∼60 mg of His-tagged ubiquitin per mL of membrane. Moreover, these membranes enable isolation of His-tagged protein from cell lysates in <15 min. The backbone amine groups in CMPEI likely increase swelling in water to double protein binding compared to films composed of PAH and the chelating polymer poly[(N,N-dicarboxymethyl)allylamine] (PDCMAA), which has a hydrocarbon backbone. Metal leaching from PAH/CMPEI- and PAH/PDCMAA-modified membranes is similar to that from GE Hitrap FF columns. Eluates with 0.5 M imidazole contain <10 ppm of Ni(2+).

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Layer-by-layer adsorption of protonated poly(allylamine) (PAH) and deprotonated poly(N,N-dicarboxymethylallylamine) (PDCMAA) yields thick films with a high density of iminodiacetic acid (IDA) ligands that bind metal ions. When film deposition occurs at pH 3.0, PAH/PDCMAA bilayer thicknesses reach 200 nm, and Cu(2+) binding capacities are ~2.5 mmol per cm(3) of film. (PAH/PDCMAA)10 films deposited at pH 3.0 are 4-8-fold thicker than films formed at pH 5.0, 7.0, or 9.0, presumably because of the low charge density on PDCMAA chains at pH 3.0. However, with normalization to film thickness, all films bind similar amounts of Cu(2+) from pH 4.1 solutions of CuSO4. In micrometer-thick films, equilibration of binding sites with Cu(2+) requires ~4 h due to a low Cu(2+) diffusion coefficient (~2.6 × 10(-12) cm(2)/s). Sorption isotherms determined at several temperatures show that Cu(2+) binding is endothermic with a positive entropy (binding constants increase with increasing temperature), presumably because metal-ion complexation involves displacement of both a proton from IDA and water molecules from Cu(2+). (PAH/PDCMAA)10 films retain their binding capacity over four absorption/elution cycles and may prove useful in metal-ion scavenging, catalysis, and protein binding.

Selective ion exclusion from charged nanopores in track-etched membranes allows separation of ions with different charges or mobilities. This study examines pressure-driven transport of dissolved ions through track-etched membranes modified by adsorption of poly(styrene sulfonate) (PSS)/protonated poly(allylamine) (PAH) films. For nominal 30 nm pores modified with a single layer of PSS, Br(-)/SO4(2-) selectivities are ∼3.4 with SO4(2-) rejections around 85% due to selective electrostatic exclusion of the divalent anion from the negatively charged pore. Corresponding membranes containing an adsorbed PSS/PAH bilayer are positively charged and exhibit average K(+)/Mg(2+) selectivities >10 at 8 mM ionic strength, and Mg(2+) rejections are >97.5% at ionic strengths <5 mM. The high rejection of Mg(2+) compared to SO4(2-) likely results from both a smaller pore size after deposition of the PAH layer and higher surface charge because of Mg(2+) adsorption. Simultaneous modeling of K(+) and Mg(2+) rejections using the nonlinearized Poisson-Boltzmann equation gives an average modified pore diameter of 8.4 ± 2.1 nm, which does not vary significantly with ionic strength. This diameter is smaller than that calculated from hydraulic permeabilities and estimated pore densities, suggesting that narrow regions near the pore entrance control ion transport. In addition to simple electrostatic exclusion, streaming potentials lead to differing rejections of Br(-) and acetate in PSS/PAH-modified pores, and of Li(+) and Cs(+) in PSS-modified pores. For these cases, electrical migration of ions toward the feed solution results in higher rejection of the more mobile ion.

Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of nonphosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment from 5 mL of 1 fmol/μL phosphoprotein digests and concentration into small-volume (tens of microliters) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer's disease.

In principle, incorporation of comb-like block copolymers in multilayer polyelectrolyte films can both increase film thickness relative to coatings containing linear polymers and provide more swollen films for increased sorption of proteins. In the absence of added salt, alternating adsorption of 5 bilayers of protonated poly(allylamine) (PAH) and comb-like poly(2-hydroxyethyl methacrylate)-graft-poly(acrylic acid) (PHEMA-g-PAA) leads to ∼2-fold thicker coatings than adsorption of PAH and linear PAA, and the difference in the thicknesses of the two coatings increases with the number of bilayers. Moreover, the (PAH/PHEMA-g-PAA)n films sorb 2- to 4-fold more protein than corresponding films prepared with linear PAA, and coatings deposited at pH 3.0 sorb more protein than coatings adsorbed at pH 5.0, 7.0, or 9.0. In fact changes in deposition pH and addition of 0.5 M NaCl to polyelectrolyte adsorption solutions alter protein sorption more dramatically than variations in the constituent polymer architecture. When deposited from 0.5 M NaCl at pH 3.0, both (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 films increase in thickness more than 400% upon adsorption of lysozyme. These films contain a high concentration of free -COOH groups, and subsequent deprotonation of these groups at neutral pH likely contributes to increased protein binding. Lysozyme sorption stabilizes these films, as without lysozyme films deposited at pH 3.0 from 0.5 M NaCl desorb at neutral pH. Films deposited at pH 9.0 from 0.5 M NaCl are more stable and also bind large amounts of lysozyme. The high binding capacities of these films make them attractive for potential applications in protein isolation or immobilization of enzymes.

Layer-by-layer polyelectrolyte adsorption is a simple, convenient method for introducing ion-exchange sites in porous membranes. This study demonstrates that adsorption of poly(acrylic acid) (PAA)-containing films at pH 3 rather than pH 5 increases the protein-binding capacity of such polyelectrolyte-modified membranes 3-6-fold. The low adsorption pH generates a high density of -COOH groups that function as either ion-exchange sites or points for covalent immobilization of metal-ion complexes that selectively bind tagged proteins. When functionalized with nitrilotriacetate (NTA)-Ni(2+) complexes, membranes containing PAA/polyethylenimine (PEI)/PAA films bind 93 mg of histidine(6)-tagged (His-tagged) ubiquitin per cm(3) of membrane. Additionally these membranes isolate His-tagged COP9 signalosome complex subunit 8 from cell extracts and show >90% recovery of His-tagged ubiquitin. Although modification with polyelectrolyte films occurs by simply passing polyelectrolyte solutions through the membrane for as little as 5 min, with low-pH deposition the protein binding capacities of such membranes are as high as for membranes modified with polymer brushes and 2-3-fold higher than for commercially available immobilized metal affinity chromatography (IMAC) resins. Moreover, the buffer permeabilities of polyelectrolyte-modified membranes that bind His-tagged protein are ~30% of the corresponding permeabilities of unmodified membranes, so protein capture can occur rapidly with low-pressure drops. Even at a solution linear velocity of 570 cm/h, membranes modified with PAA/PEI/PAA exhibit a lysozyme dynamic binding capacity (capacity at 10% breakthrough) of ~40 mg/cm(3). Preliminary studies suggest that these membranes are stable under depyrogenation conditions (1 M NaOH).

Microporous membranes are attractive for protein purification because convection rapidly brings proteins to binding sites. However, the low binding capacity of such membranes limits their applications. This work reports a rapid, aqueous procedure to create highly permeable, polymer brush-modified membranes that bind large amounts of protein. The synthetic method includes a 10-min adsorption of a macroinitiator in a hydroxylated nylon membrane and a subsequent 5-min aqueous atom transfer radical polymerization of 2-(methacryloyloxy)ethyl succinate from the immobilized initiator to form poly(acid) brushes. This procedure likely leads to more swollen, less dense brushes than polymerization from silane initiators, and thus requires less polymer to achieve the same binding capacity. The hydraulic permeability of the poly(acid) membranes is 4-fold higher than that of similar membranes prepared by growing brushes from immobilized silane initiators. These brush-containing nylon membranes bind 120 mg/cm(3) of lysozyme using solution residence times as short as 35 ms, and when functionalized with nitrilotriacetate (NTA)-Ni(2+) complexes, they capture 85 mg/cm(3) of histidine(6)-tagged (His-tagged) Ubiquitin. Additionally the NTA-Ni(2+)-functionalized membranes isolate His-tagged myo-inositol-1-phosphate synthase directly from cell extracts and show >90% recovery of His-tagged proteins.

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.

Layer-by-layer polyelectrolyte adsorption in porous polymeric membranes provides a simple way to create ion-exchange sites without greatly decreasing hydraulic permeability (<20% reduction in permeability). At 80% breakthrough, membranes coated with 3-bilayer poly(styrene sulfonate) (PSS)/polyethyleneimine (PEI) films bind 37±6 mg of negatively charged Au colloids per mL of membrane volume. The binding capacity of membranes coated with 1-bilayer films decreases in the order PSS/PEI>PSS/poly(diallyldimethyl ammonium chloride)>PSS/poly(allylamine hydrochloride). Films terminated with a polyanion present cation-exchange sites that bind lysozyme, and the lysozyme-binding capacities of (PSS/PEI)(3)/PSS films increase with the ionic strength of the solution from which the last PSS layer is deposited. Charge screening during deposition of the terminal PSS layer gives rise to a larger number of ion-exchange sites and lysozyme binding capacities as high as 16 mg per mL of membrane. At 10% breakthrough, a stack of 3 membranes binds 3 times as much lysozyme as a single membrane, showing that stacking is an effective way to increase capacity.

The properties of poly(styrene sulfonate) (PSS)/poly(diallyldimethylammonium chloride) (PDADMAC) films vary dramatically with the number of polyelectrolyte layers deposited. Attenuated total reflectance infrared spectroscopy of (PDADMAC/PSS)n films deposited on a Ge crystal shows that coatings with fewer than 7 PDADMAC/PSS bilayers do not absorb significant amounts of SCN- or Ni(CN)4(2-) but coatings with more than 7 bilayers exhibit an ion-exchange capacity of about 0.5 mol/L of film. Consistent with ion-exchange, Ni(CN)4(2-) is the anion that is predominantly absorbed from equimolar mixtures of SCN- and Ni(CN)4(2-), even though SCN- initially exchanges into the film more rapidly than Ni(CN)4(2-). For silicon-supported PSS/PDADMAC films terminated with PSS, zeta potentials change from negative to positive as the number of adsorbed bilayers increases, presumably because of a high number of anion-exchange sites inside the film. These changes in film properties dramatically affect ion transport through (PSS/PDADMAC)nPSS-coated alumina membranes. The Cl-/SO4(2-) selectivities of these membranes are >30 with (PSS/PDADMAC)4PSS films but only 3 with (PSS/PDADMAC)6PSS films. Trends in zeta potentials and selectivities with increasing numbers of bilayers are consistent with the exponential growth mechanism, where a polycation absorbs throughout the film to create large numbers of anion-exchange sites, and during polyanion deposition, some of the polycation diffuses to the surface of the film to complex with polyanions from solution. Apparently, not all of the charge on the polycation is compensated by the polyanion; therefore, anion-exchange sites remain in the film, and the presence of this positive charge yields decreased Cl-/SO4(2-) selectivity.

Layer-by-layer adsorption of poly(acrylic acid)-Pd(II) complexes and poly(ethylenimine) on alumina powder followed by reduction of Pd(II) with NaBH(4) yields Pd-nanoparticle catalysts embedded in multilayer polyelectrolyte films. The use of different ratios of poly(acrylic acid) to Pd(II) in deposition solutions gives a series of films with Pd nanoparticles whose average diameters range from 2.2 to 3.4 nm, and the catalytic selectivities of these nanoparticles vary dramatically with their size. Turnover frequencies (TOFs) for the hydrogenation of monosubstituted unsaturated alcohols increase with decreasing average nanoparticle size, whereas multisubstituted unsaturated alcohols show the opposite trend. Hence, the ratio of TOFs for the hydrogenation of allyl alcohol and crotyl alcohol is 39 with average particle diameters of 2.2 nm and only 1.3 with average particle diameters of 3.4 nm. Ratios of TOFs for hydrogenation of allyl alcohol and beta-methallyl alcohol are as high as 240 with the smallest nanoparticles, but substantial isomerization of beta-methallyl alcohol complicates this comparison. Increasing selectivity with decreasing average particle size occurs with both films deposited on alumina powder and nanoparticles stabilized by polyelectrolytes in solution. Presumably, high selectivities occur on the smallest nanoparticles because the active sites on the smallest Pd nanoparticles are less available for binding and hydrogenation of multisubstituted double bonds than are active sites on larger particles.

Metal affinity complexes were chemically grafted onto the surface of gold matrix-assisted laser desorption/ionization (MALDI) plates by coupling a derivative of nitrilotriacetate (NTA) to immobilized poly(acrylic acid) (PAA) and subsequently forming the Fe(III)-NTA complex. The immobilized complexes can adsorb phosphorylated peptides preferentially from protein digests; deposition of digests on these surface-modified plates, followed by rinsing with an acetic acid solution, addition of matrix, and subsequent analysis by MALDI MS, resulted in mass spectra dominated by peaks corresponding to phosphopeptides. In the case of analyzing a tryptic digest of beta-casein, conventional MALDI MS revealed only one monophosphopeptide, while use of the Fe(III)-NTA-PAA-modified plate resulted in strong signals due to two additional tetraphosphorylated species. The diminution or elimination of signals due to nonphosphorylated species also greatly simplified the identification of phosphopeptides during analysis of ovalbumin digests and myoglobin digests spiked with an equimolar mixture of angiotensin and phosphoangiotensin. The matrix 2',4',6'-trihydroxyacetophenone mixed with diammonium hydrogen citrate proved to be much better than alpha-cyano-4-hydroxycinnamic acid for the detection of phosphorylated peptides from digests of beta-casein and ovalbumin.

Coating of substrates with polyelectrolyte multilayers terminated with poly(acrylic acid) (PAA) followed by activation of the free -COOH groups of PAA provides a surface that readily reacts with amine groups to allow covalent immobilization of antibodies. The use of this procedure to prepare arrays of antibodies in porous alumina supports facilitates construction of a flow-through system for analysis of fluorescently labeled antigens. Detection limits in the analysis of Cy5-labeled IgG are 0.02 ng/mL because of the high surface area of the alumina membrane, and the minimal diameter of the substrate pores results in binding limited by kinetics, not mass transport. Moreover, PAA-terminated films resist nonspecific protein adsorption, so blocking of antibody arrays with bovine serum albumin is not necessary. These microarrays are capable of effective analysis in 10% fetal bovine serum.

Synthesis of poly(2-hydroxyethyl methacrylate) (PHEMA) brushes from the inside of silica capillaries by surface-initiated atom transfer radical polymerization (ATRP) yields unique stationary phases for open-tubular capillary electrochromatography (OT-CEC). Although PHEMA brushes have only a small effect on the separation of a set of phenols and anilines, derivatization of PHEMA with ethylenediamine (en) allows baseline resolution of several anilines that co-elute from bare silica capillaries. Derivatization of PHEMA with octanoyl chloride (C8-PHEMA films) affords even better resolution in the separation of a series of phenols and anilines. Increasing the thickness of C8-PHEMA coatings by a factor of 2 enhances resolution for several solute pairs, presumably because of an increase in the effective stationary phase to mobile phase volume ratio. Thus, this work demonstrates that thick polymer brushes provide a tunable stationary phase with a much larger phase ratio than is available from monolayer wall coatings. Through appropriate choice of derivatizing reagents, these polymer brushes should allow separation of a wide range of neutral molecules as well as compounds with similar electrophoretic mobilities.

The use of sample probe surfaces patterned with 200-microm-diameter spots of hydrophilic, charged polymers significantly enhances the analysis of protein digests and DNA by MALDI-MS. Selective adsorption on these polymer-modified surfaces allows collection of specific proteolytic peptides, while subsequent rinsing of the deposited sample removes contaminants. In the case of partially digested myoglobin, the mass spectrum obtained using a sample probe modified with polyanionic functionalities permits detection of 22 proteolytic fragments, while analysis using a stainless steel MALDI sample probe gives only 11 detectable fragments. Similarly, during the analysis of bovine serum albumin digests, the use of several different surface-modified MALDI sample probes increases sequence coverage from 61.3 to 74.5%. Detection of phosphorylated peptides can be quite challenging during analyses of phosphoprotein digests by MALDI-MS because these anionic proteolytic fragments have low ionization efficiencies. However, MALDI signals from the phosphorylated proteolytic fragments sometimes increase dramatically when using a sample probe surface modified by a polycation (polyethylenimine or poly(acrylic acid) complexed with Fe(3+)). The signal enhancement apparently occurs because the positive surface selectively binds the phosphorylated peptides. The use of patterned, polycationic surfaces also shows great promise for selective adsorption and decontamination of DNA samples; a simple water rinse diminishes or eliminates the formation of multi-ion adducts, thereby improving mass resolution during subsequent analysis by MALDI-MS.

This paper describes a surface science/mass spectrometry effort to develop and characterize a patterned gold surface that serves as a MALDI sample platform capable of concentrating and purifying proteins. Using microcontact printing, small (200-microm diameter) hydrophilic spots of bare gold or chemically anchored poly(acrylic acid) (PAA) are patterned at 5-mm intervals in a hydrophobic field consisting of a self-assembled monolayer of hexadecanethiol. Building on recent innovations by others, the small hydrophilic spots concentrate the sample to achieve good reproducibility and high sensitivity in the MALDI signal. One of the key features in this work is the combination of the high density of carboxylate groups in PAA with a small spot size to afford both concentration and purification of proteins via ionic interactions. This translates into detection limits for salt-contaminated proteins that are 20-100 times lower (low femtomole) than those reported for previous polymer- or monolayer-modified MALDI probes (using proteins in the 3-15-kDa range). Reflectance FT-IR spectroscopy and ellipsometry were used to determine the amount of protein adsorbed to a PAA-modified sample plate as a function of pH and salt concentration. Amide absorbances in IR spectra correlate well with MALDI-MS signals measured after addition of 2,5-dihydroxybenzoic acid as a matrix.

Alternating adsorption of polycations and polyanions on permeable supports provides a convenient and versatile method for preparing composite membranes with selective, ultrathin polyelectrolyte skins. Control over charge and composition in the polyelectrolyte skin allows highly selective separation of ions according to charge, size, or hydration energy.

Ambient-temperature atom transfer radical polymerization of ethyleneglycol dimethacrylate from a surface yields homogeneous, cross-linked polymer films. The absence of polymerization in solution in this "living" procedure avoids physisorption and gives uniform, cross-linked films with controlled thicknesses from 3 to 300 nm., (© 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)