Molecularly imprinted polymer membranes and thin films for the separation and sensing of biomacromolecules.

This review describes recent advances associated with the development of surface imprinting methods for the synthesis of polymeric membranes and thin films, which possess the capability to selectively and specifically recognize biomacromolecules, such as proteins and single- and double-stranded DNA, employing "epitope" or "whole molecule" approaches. Synthetic procedures to create different molecularly imprinted polymer membranes or thin films are discussed, including grafting/in situ polymerization, drop-, dip-, or spin-coating procedures, electropolymerization as well as micro-contact or stamp lithography imprinting methods. Highly sensitive techniques for surface characterization and analyte detection are described, encompassing luminescence and fluorescence spectroscopy, X-ray photoelectron spectroscopy, FTIR spectroscopy, surface-enhanced Raman spectroscopy, atomic force microscopy, quartz crystal microbalance analysis, cyclic voltammetry, and surface plasmon resonance. These developments are providing new avenues to produce bioelectronic sensors and new ways to explore through advanced separation science procedures complex phenomena associated with the origins of biorecognition in nature.

Detection of Macromolecular Fractions in HCN Polymers Using Electrophoretic and Ultrafiltration Techniques.

Elucidating the origin of life involves synthetic as well as analytical challenges. Herein, for the first time, we describe the use of gel electrophoresis and ultrafiltration to fractionate HCN polymers. Since the first prebiotic synthesis of adenine by Oró, HCN polymers have gained much interest in studies on the origins of life due to the identification of biomonomers and related compounds within them. Here, we demonstrate that macromolecular fractions with electrophoretic mobility can also be detected within HCN polymers. The migration of polymers under the influence of an electric field depends not only on their sizes (one-dimensional electrophoresis) but also their different isoelectric points (two-dimensional electrophoresis, 2-DE). The same behaviour was observed for several macromolecular fractions detected in HCN polymers. Macromolecular fractions with apparent molecular weights as high as 250 kDa were detected by tricine-SDS gel electrophoresis. Cationic macromolecular fractions with apparent molecular weights as high as 140 kDa were also detected by 2-DE. The HCN polymers synthesized were fractionated by ultrafiltration. As a result, the molecular weight distributions of the macromolecular fractions detected in the HCN polymers directly depended on the synthetic conditions used to produce these polymers. The implications of these results for prebiotic chemistry will be discussed.

Characterization and diabetic wound healing benefits of protein-polysaccharide complexes isolated from an animal ethno-medicine Periplaneta americana L.

Periplaneta americana L. (PA), a type of animal medicine, has been widely used for wound healing in clinical settings. In order to further investigate the bioactive wound healing substances in PA, crude PA protein-polysaccharide complexes were further purified by cellulose DE-52 and Sephadex G100 chromatography in succession. Among these isolated fractions, two fractions eluted by 0.3 M and 0.5 M NaCl with the higher yield, respectively named PaPPc2 and PaPPc3 respectively, were chosen for the wound healing experiments. Mediated by HPGPC, amino acid and monosaccharide composition analysis, circular dichroism spectrum, glycosylation type, FT-IR, and 1H NMR analysis, the characterization of PaPPc2 and PaPPc3 was implemented. And then, the benefits of PaPPcs to promote cell proliferation, migration, and tube formation of HUVECs were determined in vitro, indicated these fractions would facilitate angiogenesis. Finally, as proof of concept, PaPPc2 and PaPPc3 were employed to accelerate the acute wounds of diabetic mice, involving in increase blood vessels and the amounts of angiogenesis-related cytokines (α-SMA, VEGF, and CD31). In short, this study provides an experimental basis to demonstrate the protein-polysaccharide complexes of Periplaneta americana L. as its wound healing bioactive substances.

Size exclusion chromatography--a blessing and a curse of science and technology of synthetic polymers.

Size exclusion chromatography, SEC is one of the most popular methods for the separation of different kinds of macromolecules. This critical review gives concise information about macromolecules and their behavior in solution, basic understanding about principles, instrumentation, and application possibilities of SEC, and more in detail discusses drawbacks and pitfalls of the method with the emphasis on synthetic polymers. Selected practical advices are included to help enhance the quality of SEC results.

Polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain reproducible relative mobility (Rf) values as physical-chemical constants. Application and extension of Ogston's (random fiber) model for a gel allows for calculation of molecular volume, surface area, or radius, free mobility, and valence from RJ measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking) with known operating characteristics for any pH at 0 degrees and 25 degrees C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative scale PAGE is operational for milligram loads and feasible for gram quantities.

Modeling analytical ultracentrifugation experiments with an adaptive space-time finite element solution for multicomponent reacting systems.

We describe an extension of the adaptive space-time finite element method (ASTFEM) used in the solution of the Lamm equation to the case of multicomponent reacting systems. We use an operator splitting technique to decouple the sedimentation-diffusion process from the reaction process. The former is solved with an ASTFEM approach based on the Petrov-Galerkin method and on adaptive moving grids, and the latter is solved with the implicit midpoint Euler's method. Our solution can effectively eliminate the sedimentation errors for each component or species involved in the reaction, and it is free from oscillation near the cell bottom. It offers second-order accuracy, and guarantees conservation of mass without any additional postprocessing, and it permits modeling of multicomponent, equilibrating systems where the reaction rate can be kinetically controlled between an instantaneous reaction and a noninteracting mixture. The proposed ASTFEM solution provides improved efficiency and accuracy compared to classical approaches, especially when medium-sized and large molecules are modeled.

Improved antibody aggregate removal by hydroxyapatite chromatography in the presence of polyethylene glycol.

This paper reports a method for improving the effectiveness of aggregate removal from monoclonal antibodies by the use of hydroxyapatite (HA) in the presence of polyethylene glycol (PEG). PEG preferentially enhances aggregate retention, thereby increasing the degree of separation between aggregated and non-aggregated antibodies. PEG has a similar effect on ion exchangers but only half the enhancement observed with HA. The HA method is suitable for preparation of aggregate-free IgG and IgM monoclonal antibodies for research, diagnostic, or therapeutic applications.

Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies.

Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses. These applications leverage monoliths' inherent properties, such as fast operation and high capacity for large biomolecules. The height equivalent to a theoretical plate (HETP) and dynamic binding capacity do not change with velocity. This is explained by the convective transport through the channels with a diameter of above 1000 nm and has been experimentally verified and also supported by theoretical analyses. Despite low absolute surface area, these large channels provide enough area for adsorption of these large biomolecules, which cannot penetrate into conventional chromatography media designed for protein separation. Monoliths for preparative separations are mainly cast as polymethacrylate or polyacrylamide blocks and have been functionalized as ion exchangers or hydrophobic interaction chromatography media. So-called cryogels have channels more than 30 microm wide, enabling efficient processing of suspensions or even cell-chromatography. This review discusses the pressure drop characteristics, mass transfer properties, scale-up, and applications of monoliths in the context of conventional chromatography media.

Characterization of natural rubber using size-exclusion chromatography with online multi-angle light scattering. Study of the phenomenon behind the abnormal elution profile.

Natural and synthetic poly(cis-1,4-isoprene) were characterized by size-exclusion chromatography coupled with an online multi-angle light scattering detector (SEC-MALS). Unlike synthetic poly(cis-1,4-isoprene) (SR), natural rubber (NR) samples showed anomalous elution profiles. The beginning of elution was very similar to SR but, after a certain elution volume, the molar masses of the eluting macromolecules increased with elution volume instead of continuing to decrease, which resulted in an upturn curve profile. Adding tetrabutylammonium bromide (TBABr) to THF (solvent and mobile phase) removed this phenomenon. In addition, using different concentrations of TBABr showed that TBABr had two simultaneous actions. TBABr reduced the abnormal elution profiles and the quantity of aggregates (insoluble part or gel). These results mean that the main phenomenon involved in abnormal elution was delayed entities adsorbing on the column packing. Their delayed elution was responsible for the artificial increase in molar masses, especially at high elution volumes. The results obtained suggest that these entities are very compact and have a sphere-like structure.

Focusing manipulation of microalgae in a microfluidic device using self-produced macromolecules.

Extracellular polymeric substances (EPSs) are self-produced biosynthetic macromolecules that have a three-dimensional architecture in bacterial biofilms and are mainly composed of a mixture of polysaccharides, proteins and nucleic acids. Compared with synthetic polymers, EPSs can have a long relaxation time due to their structural complexity. We exploited the non-Newtonian rheological behavior of EPSs extracted from Chlorella vulgaris with the help of cell focusing and particle focusing in confined microchannels. The microalgae showed a 'self-ordering' behavior in the 'self-secreted' substances. The EPSs were characterized and analyzed chemically and rheologically. In a microfluidic device, they enable outstanding particle focusing over a wide range of flow rates. This study can open an effective, unique pathway for applications of biomass related resources such as EPSs.

Fractionation of lignin macromolecules by sequential organic solvents systems and their characterization for further valuable applications.

Lignin solvent fractionation is one of the promising methods for homogenizing and utilizing lignin commercially. In this work, fractionation characteristics of two lignin fractions were compared to investigate the potential of utilization of fractionated lignin. Two lignins [milled wood lignin(MWL) and organosolv lignin(OL) from yellow poplar] were sequentially fractionated with ethyl acetate(F1), 2-butanone(F2), methanol(F3), acetone(F4), and dioxane/water(F5). Yields of five MWL fractions F1 to F5 were 11.7%, 11.7%, 15.3%, 11.8%, and 49.6%, respectively, and yields of OL fractions were 26.2%, 26.1%, 18.7%, 3.7% and 25.4%. Average molecular weight of F1 (lowest molecular weight fraction) ranged from 1000 to 2400Da, whereas that of F5 (highest molecular weight fraction) was above 10000Da. According to functional group analysis, contents of phenolic hydroxyl groups and methoxyl groups decreased gradually with increasing molecular weight. DFRC analysis was performed to investigate the frequency of ß-O-4 linkages and it revealed that the higher molecular weight fractions yielded larger amounts of DFRC monomers, indicating that those fractions more frequently contain aryl ether linkages. TG/DTG showed that the low molecular weight fractions generally have lower initial thermal stability. Tg of the fractions ranged from 126°C to 156°C, increasing as the molecular weight of the lignin fraction increased.

Reaction-Mediated Desorption of Macromolecules: Novel Phenomenon Enabling Simultaneous Reaction and Separation.

Combining chemical reaction with separation offers several advantages. In this work possibility to induce spontaneous desorption of adsorbed macromolecules, once being PEGylated, through adjustment of the reagent composition is investigated. Bovine serum albumin (BSA) and activated oligonucleotide, 9T, are used as the test molecules and 20 kDa linear activated PEG is used for their PEGylation. BSA solid-phase PEGylation is performed on Q Sepharose HP. Distribution coefficient of BSA and PEG-BSA as a function of NaCl is determined using linear gradient elution (LGE) experiments and Yamamoto model. According to the distribution coefficient the selectivity between BSA and PEG - BSA of around 15 is adjusted by using NaCl. Spontaneous desorption of PEG - BSA is detected with no presence of BSA. However, due to a rather low selectivity, also desorption of BSA occurred at high elution volume. A similar procedure is applied for activated 9T oligonucleotide, this time using monolithic CIM QA disk monolithic column for adsorption. Selectivity of over 2000 is obtained by proper adjustment of PEG reagent composition. High selectivity enables spontaneous desorption of PEG-9T without any desorption of activated 9T. Both experiments demonstrates that reaction-mediated desorption of macromolecules is possible when the reaction conditions are properly tuned.

Superficially porous particles with 1000Å pores for large biomolecule high performance liquid chromatography and polymer size exclusion chromatography.

To facilitate mass transport and column efficiency, solutes must have free access to particle pores to facilitate interactions with the stationary phase. To ensure this feature, particles should be used for HPLC separations which have pores sufficiently large to accommodate the solute without restricted diffusion. This paper describes the design and properties of superficially porous (also called Fused-Core®, core shell or porous shell) particles with very large (1000Å) pores specifically developed for separating very large biomolecules and polymers. Separations of DNA fragments, monoclonal antibodies, large proteins and large polystyrene standards are used to illustrate the utility of these particles for efficient, high-resolution applications.

Separation of macromolecular proteins and removal of humic acid by cellulose acetate modified UF membranes.

Surface modifying macromolecules (SMMs) were synthesized with various polyurethane pre polymers end-capped with different groups and blended into the casting solution of cellulose acetate (CA) to prepare surface modified ultra-filtration (UF) membranes for water filtration applications. The surface modification of the CA membranes was confirmed by the FTIR and static contact angle (SCA) measurements. The membranes so prepared had the typical characteristics of UF membranes as confirmed by scanning electron microscopy (SEM). Membrane properties were studied in terms of membrane compaction, percentage water content (%WC), pure water flux (PWF), membrane hydraulic resistance (Rm), molecular weight cut-off (MWCO), average pore size and porosity. The result showed that PWF, %WC, MWCO and pore size increased whereas the Rm decreased by the addition of SMMs. The significant effect of SMMs on the fouling by humic acid (HA) was also observed. It was found that the cSMM-3 membrane, in which SMM was synthesized with diethylene glycol (DEG) and hydroxyl benzene sulfonate (HBS) was blended, had the highest flux recovery ratio FRR (84.6%), as well as the lowest irreversible fouling (15.4%), confirming their improved antifouling properties. Thus, the SMM modified CA membranes had proven, to play an important role in the water treatment by UF.

Selective extraction of proteins and other macromolecules from biological samples using molecular imprinted polymers.

The accurate determination of intact macromolecules in biological samples, such as blood, plasma, serum, urine, tissue and feces is a challenging problem. The increased interest in macromolecules both as candidate drugs and as biomarkers for diagnostic purposes means that new method development approaches are needed. This review charts developments in the use of molecularly imprinted polymers first for small-molecular-mass compounds then for proteins and other macromolecules. Examples of the development of molecularly imprinted polymers for macromolecules are highlighted. The two main application areas to date are sensors and separation science, particularly SPE. Examples include peptides and polypeptides, lysozyme, hemoglobin, ovalbumin, bovine serum albumin and viruses.

Molecularly imprinted polymers for separating and sensing of macromolecular compounds and microorganisms.

The present review article focuses on gathering, summarizing, and critically evaluating the results of the last decade on separating and sensing macromolecular compounds and microorganisms with the use of molecularly imprinted polymer (MIP) synthetic receptors. Macromolecules play an important role in biology and are termed that way to contrast them from micromolecules. The former are large and complex molecules with relatively high molecular weights. The article mainly considers chemical sensing of deoxyribonucleic acids (DNAs), proteins and protein fragments as well as sugars and oligosaccharides. Moreover, it briefly discusses fabrication of chemosensors for determination of bacteria and viruses that can ultimately be considered as extremely large macromolecules.

Serial polymers in the epidermis of Drosophila melanogaster larvae.

The paper reports the existence of peculiar polymers (e-polymers) obtained from the epidermis of Drosophila melanogaster larvae. E-polymers result from the assembly of two components held together by alkali-labile bonds. Such components can be separated by CsCl density gradients and by DEAE-cellulose chromatography after controlled alkaline hydrolysis. One of the components contains predominantly neutral sugars and a phenolic substance (S-fraction). The other contains predominantly amino acids, aminosugars and a phenolic substance. This fraction can be visualized as serial multimers of a monomer subunit. It is suggested that e-polymers are continuous tridimensional structures which might have morphogenetic significance.

Recent advances in bioprocessing application of membrane chromatography.

Compared to traditional chromatography using resins in packed-bed columns, membrane chromatography is a relatively new and immature bioseparation technology based on the integration of membrane filtration and liquid chromatography into a single-stage operation. Over the past decades, advances in membrane chemistry have yielded novel membrane devices with high binding capacities and improved mass transfer properties, significantly increasing the bioprocessing efficiency for purification of biomolecules. Due to the disposable nature, low buffer consumption, and reduced equipment costs, membrane chromatography can significantly reduce downstream bioprocessing costs. In this review, we discuss technological merits and disadvantages associated with membrane chromatography as well as recent bioseparation applications with a particular attention on purification of large biomolecules.

In vivo self-assembly of TMV-like particles in yeast and bacteria for nanotechnological applications.

Heterologous expression of tobacco mosaic virus coat protein and in vivo assembly of rod-shaped TMV-like particles encapsidating viral or host RNA were compared between Escherichia coli and Schizosaccharomyces pombe. TMV-like particles were produced in both hosts, irrespective of whether the TMV origin of assembly was present. The additional plasmid providing an OAS-containing RNA was able to alter the length distribution of the TMV-like particles. Plant and yeast-expressed CP behaved similarly upon isoelectric focusing, whereas CP expressed in bacteria migrated differently. After purification by buoyant density centrifugation, the encapsidated nucleic acids were determined to be of host origin as well as of viral origin. OAS-containing mRNA was packaged preferentially in yeast to some extent (8%). In consequence, the majority of TMV-like particles showed the same length distribution similar to those in the absence of OAS-containing mRNA, likely due to host RNA being primarily encapsidated. Notwithstanding this limitation for tailoring particle sizes, the heterologous expression system provides a new avenue to deliver versatile nucleoprotein scaffolds for a diversity of nanotechnological applications, without the need for an infectious virus. The results are discussed with reference to the competition of translation and packaging as well as to the selective decay of TMV RNA.