Initial evaluation of protein A modified capillary‐channeled polymer fibers for the capture and recovery of immunoglobulin G

A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.     

Preparation and Characterization of Prometryn Molecularly Imprinted Solid‐Phase Microextraction Fibers

Abstract

A novel reproducible solid‐phase microextraction (SPME) coating was prepared on the surface of silanized silica fibers by molecularly imprinted polymerization using prometryn as template molecule. The structure and extraction performance of molecularly imprinted polymer (MIP) coating was studied with the scanning electron microscope and high performance liquid chromatography (HPLC). Specific selectivity was found with the prometryn MIP‐coated fiber to prometry and its structural analogues such as atrazine, simetryn, terbutylazin, ametryn, propazine and terbutryn. In contrast, these triazines could not be selectively extracted by the non‐imprinted polymer fiber or commercial polydimethylsiloxane (PDMS), polydimethylsiloxane/divinylbenzene (PDMS/DVB), polyacrylate (PA) fibers.     

Small molecule adsorption on to polyester capillary‐channeled polymer fibers: Frontal analysis of naphthalene and naphthol (naphthalene and naphthol adsorption on capillary‐channeled polymer fibers)

Frontal analysis was carried out employing poly(ethylene‐terephthalate) capillary‐channeled polymer fibers as the stationary phase for the immobilization of low‐molecular‐weight polycyclic aromatic hydrocarbon compounds (naphthol and naphthalene) from 2% methanol/water solutions. The effects of several experimental parameters on the frontal profile, the breakthrough volume, and the equilibrium parameters were determined for each solute. The amount adsorbed at exhaustion of naphthalene and naphthol was also compared. The kinetics and thermodynamics were maintained at relatively fast flow rates/linear velocities (～6–18 mm/s). Comparisons of dynamic capacity revealed that naphthalene was more retained than naphthol, in most situations more than five times that of the naphthol adsorption. This increase in capacity is most likely due to the multilayering of naphthalene on the surface of the fibers through π–π interactions between the solute and the fiber surface and successive layering of solute molecules. The extent of layering is a function of the flow, with faster flow rates (and subsequent shear forces) reducing the extent of adsorbate–adsorbate interactions. Although the overall loading capacity of the capillary‐channeled polymer fibers is far below porous phases, there are a number of attractive attributes that support further development.     

Loading characteristics and chemical stability of headgroup‐functionalized poly(ethylene glycol)‐lipid ligand tethers on polypropylene capillary‐channeled polymer fibers

Polypropylene capillary‐channeled polymer fibers have been modified by adsorption of headgroup‐functionalized poly(ethylene glycol)‐lipids to generate a species‐specific stationary phase. In order to study ligand binding characteristics, a fluorescein‐labeled poly(ethylene glycol)‐lipid was used as a model system. Breakthrough curves and frontal analysis were employed to characterize the surface loading characteristics across a range of lipid concentrations and mobile phase flow rates. Efficient mass transfer and fluid transport yield a linear adsorption isotherm up to the maximum loading concentration of 3 mg/mL, at a linear velocity of 57.1 mm/s. Under these conditions, the dynamic binding capacity was found to be 1.52 mg/g of fiber support. Variation of the linear velocity from 8.6 to 57.1 mm/s showed only small changes in breakthrough volume. The maximum capacity of 1.8 mg/g is found under conditions of a load velocity of 34.2 mm/s and a concentration of 3 mg/mL lipid. Exposure of the lipid modified fibers to several challenge solvents reveals a chemically robust system, with only 50% acetonitrile and hexanes able to disrupt the lipid adsorption. The straightforward capillary‐channeled polymer fiber surface modification with headgroup‐functionalized lipids provides both a diverse yet practically robust ligand tethering system.     

Cellulose–water interactions during enzymatic hydrolysis as studied by time domain NMR

The different states and locations of water within the cellulose matrix can be studied by the use of time domain low field NMR. In this work we show how the state and location of water associated with cellulose in filter paper fibers are affected by enzymatic hydrolysis. Three locations of water were identified in the filter paper; (1) bound water associated with the microfibril surfaces and (2) water in the cell wall or cellulose matrix and (3) capillary water in the lumens and between fibers. The different mechanisms of cellulase enzymes can be seen in their effect on the cellulose–water interactions and the synergistic effects between endo- and exo enzymes can be easily detected by time domain NMR. An interesting observation is that it is possible to link the state and location of water within the cellulose fiber with structural changes upon enzymatic hydrolysis.     

Intermolecular Communication on a Liposomal Membrane: Enzymatic Amplification of a Photonic Signal with a Gemini Peptide Lipid as a Membrane‐Bound Artificial Receptor

A supramolecular system that can activate an enzyme through photo‐isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self‐assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH‐dependent L ‐lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane‐bound receptor induced photo‐isomerization, which significantly altered the receptor’s metal‐binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu²⁺ ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.     

Phospholipid biotinylation of polydimethylsiloxane (PDMS) for protein immobilization

Polydimethylsiloxane (PDMS) surfaces can be functionalized with biotin groups by adding biotinylated phospholipids to the PDMS prepolymer before curing. The addition of beta-D-dodecyl-N-maltoside (DDM) in the solution blocks non-specific protein binding on these functionalized PDMS surfaces. We characterize the surface by measuring fluorescently labeled streptavidin binding. Single molecule tracking shows that the phospholipids are not covalently linked to PDMS polymer chains, but the surface functionalization is not removed by washing. We demonstrate the immobilization of biotinylated antibodies and lectins through biotin-avidin interactions.     

Synthesis and characterization of novel PDMS nanocomposites using POSS derivatives as cross‐linking filler

We report the synthesis and characterization of novel elastomeric nanocomposites containing polyhedral oligomeric silsesquioxanes (POSS) as both the cross‐linker and filler within a polydimethylsiloxane (PDMS) polymer matrix. These polymer composites were prepared through the reaction of octasilane‐POSS (OS‐POSS) with vinyl‐terminated PDMS chains using hydrosilylation chemistry. In addition, larger super‐POSS cross‐linkers, consisting of two pendant hepta(isobutyl)POSS molecules attached to a central octasilane‐POSS core, were also used in the fabrication of the PDMS composites. The chemical incorporation of these POSS cross‐linkers into the PDMS network was verified by solid‐state ¹H magic angle spinning NMR. Based on dynamic mechanical analysis, the PDMS nanocomposites prepared with the octafunctional OS‐POSS cross‐linker exhibited enhanced mechanical properties relative to polymer systems prepared with the tetrafunctional TDSS cross‐linker at equivalent loading levels. The observed improvements in mechanical properties can be attributed to the increased dimensionality of the POSS cross‐linker. The PDMS elastomers synthesized from the larger super‐POSS molecule showed improved mechanical properties relative to both the TDSS and OS‐POSS composites due to the increased volume‐fraction of POSS filler in the polymer matrix. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2589–2596, 2009     

溶菌酶蛋白在聚合物防污膜表面的吸附

采用分子动力学模拟方法比较了溶菌酶蛋白在两种典型聚合物防污材料聚乙二醇(PEG)和聚二甲基硅氧烷(PDMS)表面的吸附行为, 在微观上探讨了聚合物膜表面性质对蛋白质吸附的影响. 根据蛋白质与聚合物膜之间的相互作用、能量变化及表面水化层分子的动力学行为, 解释了PEG防污涂层相对于PDMS表面具有更佳防污效果的原因: (1) 相比PDMS涂层, 蛋白质与PEG涂层的结合能量较低, 使其结合更加疏松; (2) 蛋白质吸附到材料表面要克服表面水化层分子引起的能障, PEG表面与水分子之间结合紧密, 结合水难于脱附, 造成蛋白质在其表面的吸附需要克服更高的能量, 不利于蛋白质的吸附.     

药物甲氧苄氨嘧啶分子模板聚合物膜的选择性结合及透 过性质研究

用分子印迹技术制备了对甲氧苄氨嘧啶具有特异结合和透过性质的膜状分子模板聚合物,Scatchard分析表明,在分子模板聚合物膜中存在一类等价的可与甲氧苄氨嘧啶结合的位点,该结合位点的平衡离解常数为Kd=4.85×10^-2mmol/L,甲氧苄氨嘧啶分子模板聚合物膜的选择性透过实验表明,在该聚合物膜中存在着由形状和功能基团均与模板分子甲氧苄氨嘧啶相互补的孔穴组成的通道,该通道可有选择性地通过模板分子。     

溶菌酶蛋白在聚合物防污膜表面的吸附

采用分子动力学模拟方法比较了溶菌酶蛋白在两种典型聚合物防污材料聚乙二醇(PEG)和聚二甲基硅氧烷(PDMS)表面的吸附行为,在微观上探讨了聚合物膜表面性质对蛋白质吸附的影响.根据蛋白质与聚合物膜之间的相互作用、能量变化及表面水化层分子的动力学行为,解释了PEG防污涂层相对于PDMS表面具有更佳防污效果的原因:(1)相比PDMS涂层,蛋白质与PEG涂层的结合能量较低,使其结合更加疏松;(2)蛋白质吸附到材料表面要克服表面水化层分子引起的能障,PEG表面与水分子之间结合紧密,结合水难于脱附,造成蛋白质在其表面的吸附需要克服更高的能量,不利于蛋白质的吸附.     

Selective displacement chromatography in multimodal cation exchange systems

A library of displacer analogues with varying degrees of electrostatic, hydrophobic and hydrogen bonding moieties was evaluated for their ability to enhance the selectivity of multimodal (MM) chromatography under high loading conditions. The library was screened for displacement of model proteins using a robotic liquid handling system and selective batch separations were achieved for proteins that were inseparable with linear gradient chromatography. Trends in protein displacement were identified and displacers with higher hydrophobicity and net charge exhibited improved protein displacements. Proteins that interacted with the resins primarily via electrostatic interactions were more readily displaced than those that possessed a significant hydrophobic contribution to their binding. In addition, multimodal displacers were found to be more selective than single mode electrostatic displacers. Column chromatography studies were also carried out and baseline separations were achieved for model protein pairs using selective displacement. Finally, operation of these columns in the desorption mode resulted in baseline separation of model proteins which were not separable by selective displacement chromatography. This study indicates that the inherent selectivity of MM resins can be augmented by the selectivity of the displacer under non-linear competitive binding conditions, creating new opportunities for protein separations not possible using traditional gradient operations.     

Precise Size‐Selective Sieving of Nanoparticles Using a Highly Oriented Two‐Dimensional Supramolecular Polymer

The development of size‐selective membranes with well‐defined nanopores towards the precise separation of nanometer‐sized substances is a challenging task to achieve. Here a supramolecular membrane is presented that comprises a highly oriented, honeycomb‐like, 2D supramolecular polymer on a polycarbonate filter support. It enables precise size‐selective sieving of colloidal nanoparticles (NPs). Owing to the uniform parallel‐aligned nanocavities within the 2D supramolecular polymers, the composite membrane shows a high size‐selectivity with a sub‐nanometer accuracy in the cutoff size of about 4.0 nm. In principle, the species of size‐separable particles are unlimited, as demonstrated by quantum dots, noble metal, and metal oxide NPs. This supramolecular membrane combined with filtration advances the potential of NPs in terms of their monochromatic emission and size monodispersity, and also enables rapid removal of small magnetic NP adsorbents that are otherwise difficult to capture.     

Methods of modulating hydrogen bonded interactions in synthetic host-guest systems

Hydrogen bonded interactions are among the most important non-covalent interactions in supramolecular chemistry. The strength, selectivity and directionality inherent in hydrogen bonding processes have allowed the creation of complex and efficient molecular hosts capable of selective binding to a wide variety of complementary guests. Major advances in controlling host-guest complexation have occurred in the last decade, principally through systematic modification of the electrostatic properties and/or geometry of the hosts, thereby fine-tuning the molecular recognition event. More recently, systems have been developed which allow the effectiveness and selectively of hydrogen bonding interactions to be reversibly modulated by an external stimulus, more accurately mimicking biological systems and providing building blocks for the construction of novel advanced materials, sensors and devices. In this review, we highlight some of the methods available for modulating the strength and selectivity of hydrogen bonded interactions in synthetic host-guest systems.     

Reversible Supramolecular Surface Attachment of Enzyme–Polymer Conjugates for the Design of Biocatalytic Filtration Membranes

To be used successfully in continuous reactor systems, enzymes must either be retained using filtration membranes or immobilized on a solid component of the reactor. Whereas the first approach requires large amounts of energy, the second approach is limited by the low temporal stability of enzymes under operational conditions. To circumvent these major stumbling blocks, we have developed a strategy that enables the reversible supramolecular immobilization of bioactive enzyme–polymer conjugates at the surface of filtration membranes. The polymer is produced through a reversible addition–fragmentation transfer method; it contains multiple adamantyl moieties that are used to bind the resulting conjugate at the surface of the membrane which has surface‐immobilized cyclodextrin macrocycles. This supramolecular modification is stable under operational conditions and allows for efficient biocatalysis, and can be reversed by competitive host–guest interactions.     

Molecular recognition. Design of new receptors for complexation and catalysis

Abstract

In recent years there has been intense activity in the design of synthetic molecules capable of enzyme-like recognition and binding of small substrates.¹ Two fundamental approaches have been taken. The first has generally involved non-directional binding forces (such as solvophobic, π-stacking and dispersion interactions) in water-soluble cyclophane frameworks.² This approach led to extremely important quantitative insights into the hydrophobic effect and the enthalpic and entropic contributions of solvent reorganization to binding.³ However, the weakly oriented nature of the binding interactions has resulted in only moderate substrate selectivity beyond the shape recognition permitted by the cavity. In nature such selectivity is a prerequisite for the chiral recognition and catalytic activity of enzymes and is achieved by hydrogen bonding and electrostatic interactions. The second major approach to artificial receptors makes use of these more directional interactions by incorporating several hydrogen bonding groups into a cleft or cavity of defined geometry.⁴ The resulting hosts form strong and selective complexes to those substrates with complementary shape and hydrogen bonding characteristics.⁵ In these cases, however, the binding free energy is solvent dependent, diminishing to zero as the polarity of the medium increases, due to the strong solvation of the hydrogen bonding sites. A central goal in contemporary molecular recognition research must be to develop receptors that effectively use directed hydrogen bonding interactions in competitive solvents. Success will probably require combining strong (possibly charged) hydrogen bonding groups with hydrophobic sites capable not only of effective apolar association with the substrate but also of protecting the polar sites from full solvation.     

药物甲氧苄氨嘧啶分子模板聚合物 结合作用及选择性研究

采用分子自组装印迹技术合成了对甲氧苄氨嘧啶有高选择性的模板聚合物受体.通过Scatchard分析、     

A Simple Method for Fabricating Patterned Curvilinear Microstructures in Poly(dimethylsiloxane) by Selective Wetting

The fabrication of patterned microstructures in poly(dimethylsiloxane) (PDMS) is a prerequisite for soft lithography. Herein, curvilinear surface relief microstructures in PDMS are fabricated through a simple three‐stage approach combining microcontact printing (μCP), selective surface wetting/dewetting and replica molding (REM). First, using an original PDMS stamp (first‐generation stamp) with linear relief features, a chemical pattern on gold substrate is generated by μCP using hexadecanethiol (HDT) as an ink. Then, by a dip‐coating process, an ordered polyethylene glycol (PEG) polymer‐dot array forms on the HDT‐patterned gold substrate. Finally, based on a REM process, the PEG‐dot array on gold substrate is used to fabricate a second‐generation PDMS stamp with microcavity array, and the second‐generation PDMS stamp is used to generate third‐generation PDMS stamp with microbump array. These fabricated new‐generation stamps are utilized in μCP and in micromolding in capillaries (MIMIC), allowing the generation of surface micropatterns which cannot be obtained using the original PDMS stamp. The method will be useful in producing new‐generation PDMS stamps, especially for those who want to use soft lithography in their studies but have no access to the microfabrication facilities.     

Rapid Bacterial Recognition over a Wide pH Range by Boronic Acid-Based Ditopic Dendrimer Probes for Gram-Positive Bacteria

We have developed a convenient and selective method for the detection of Gram-positive bacteria using a ditopic poly(amidoamine) (PAMAM) dendrimer probe. The dendrimer that was modified with dipicolylamine (dpa) and phenylboronic acid groups showed selectivity toward Staphylococcus aureus. The ditopic dendrimer system had higher sensitivity and better pH tolerance than the monotopic PAMAM dendrimer probe. We also investigated the mechanisms of various ditopic PAMAM dendrimer probes and found that the selectivity toward Gram-positive bacteria was dependent on a variety of interactions. Supramolecular interactions, such as electrostatic interaction and hydrophobic interaction, per se, did not contribute to the bacterial recognition ability, nor did they improve the selectivity of the ditopic dendrimer system. In contrast, the ditopic PAMAM dendrimer probe that had a phosphate-sensing dpa group and formed a chelate with metal ions showed improved selectivity toward S. aureus. The results suggested that the targeted ditopic PAMAM dendrimer probe showed selectivity toward Gram-positive bacteria. This study is expected to contribute to the elucidation of the interaction between synthetic molecules and bacterial surface. Moreover, our novel method showed potential for the rapid and species-specific recognition of various bacteria.