Factors affecting the behavior and effectiveness of phospholipid bilayer coatings for capillary electrophoretic separations of basic proteins

Phospholipid bilayer coatings can prevent adsorption of cationic proteins on the surface of fused silica capillaries used in capillary electrophoresis. However, the performance of such bilayer coatings is strongly dependent on solution conditions. The factors affecting the rate of formation of phospholipid bilayer coatings were investigated using the double-chained zwitterionic 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC, C(14)) as a model phospholipid. The effectiveness of these coatings for CE separations of model cationic lysozyme, cytochrome c, ribonuclease A, and alpha-chymotrypsinogen A was also assessed. Increasing the ionic strength of a 0.1 mM DMPC solution reduced capillary coat times from >2 hours in 2.5 mM Tris (pH 7.4) buffer to 3.4 min in 40 mM Tris and dramatically improved separation performance such that > or =1.4 x 10(5) plates/m were observed in capillaries coated for 5 min with 0.1 mM DMPC in 20 mM Tris-HCl (pH 7.4). The presence of Ca(2+) in the coating solution also increases the rate of formation of the phospholipid bilayer coating. The type of vesicle strongly affects its adsorption rate onto the silica surface. The time required to coat the capillary was 7.2 min for small unilamellar vesicles (SUVs) and 22.5 min for large unilamellar vesicles and excessively long for multilamellar vesicles. Highest efficiency protein separations were achieved with bilayer coatings prepared from SUVs. The coating rate was enhanced by using greater DMPC concentrations and unaffected by pH. The type of buffer present in the DMPC coating solution affects the coating behavior, with HEPES buffer yielding a faster coat time than either Tris or phosphate buffers. Histone H1 was separated on a 0.1 mM DMPC-coated capillary.     

Enhanced stability self-assembled coatings for protein separations by capillary zone electrophoresis through the use of long-chained surfactants

Semipermanent coatings were generated within fused-silica capillaries by flushing the capillary with a 0.1 mM solution of the double-chained cationic surfactants didodecyldimethylammonium bromide, dimethylditetradecylammonium bromide (2C(14)DAB), dihexadecyldimethylammonium bromide, and dimethyldioctadecylammonium bromide (2C(18)DAB) and the triple-chained surfactant tridodecylmethylammonium iodide. All of these coatings were semipermanent, whereby the coating remained intact after the unadsorbed surfactant was removed from the capillary. The separation efficiencies for four model cationic proteins ranged from 1.2 to 1.4 million plates/m for the 2C(14)DAB coating to 0.3-0.4 million plates/m for the 2C(18)DAB coatings. The stability of the coating increased with increasing hydrophobicity of the surfactant (i.e., increasing chain length and decreasing cmc). Over 60 successive separations were performed on a 2C(18)DAB-coated capillary over 12 days, without any regeneration of the coating. The migration times varied by less than 2.3% over this period with no loss in efficiency.     

Phospholipid bilayer coatings for the separation of proteins in capillary electrophoresis

The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) was investigated for its use as a wall coating for the prevention of protein adsorption in capillary electrophoresis. DLPC forms a semipermanent coating at the capillary wall, which allows excess phospholipid to be removed from the capillary prior to electrophoretic separation. A DLPC-coated capillary allowed for the separation of both cationic and anionic proteins with efficiencies as high as 1.4 million plates/m. Migration time reproducibility was less than 1.3% RSD from run to run and less than 4.0% RSD from day to day. Protein recovery was as high as 93%. Cationic and anionic proteins could be separated over a pH range of 3-10, all yielding good efficiencies (N up to 1 million plates/m). The chain length of the phospholipid affected the performance of the wall coating. The C10 analogue of DLPC (DDPC) did not form a coating on the capillary wall while the C14 analogue of DLPC (DMPC) formed a stable coating that prevented protein adsorption to the same extent as its C12 counterpart.     

Hydrophobic,pellicular, monolithic capillary columns based on cross-linked polynorbornene for biopolymer separations

Monolithic capillary columns were prepared by transition metal-catalyzed ring-opening metathesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene inside a silanized 200-microm-i.d. fused-silica capillary using a mixture of toluene and 2-propanol as porogen and Cl2(PCy3)2Ru(=CHPh) as initiator. The synthesized columns allowed the rapid and highly efficient separation of single- and double-stranded nucleic acids by ion-pair reversed-phase high-performance liquid chromatography and of proteins by reversed-phase high-performance liquid chromatography. Compared to 3-mm-i.d. analytical columns synthesized from an identical polymerization mixture, a considerable improvement in the peak widths at half-height of oligonucleotides in the order of 60-80% was obtained. Significant differences in morphology between the capillary column, where the surface of the monolith was rather soft and rugulose, and the analytical column, where the surface was very sharp and smooth, were observed, most probably due to differences in polymerization kinetics. The synthesized monoliths were successfully applied to the separation of the diastereomers of phosphorothioate oligodeoxynucleotides. To confirm the identity of the eluting compounds on the basis of their intact molecular masses, the chromatographic separation system was on-line hyphenated to electrospray ionization mass spectrometry.     

Oligomerized phospholipid bilayers as semipermanent coatings in capillary electrophoresis

Double-chained surfactants form semipermanent coatings that prevent protein adsorption in capillary electrophoresis (CE). To make such coatings more permanent, vesicles of the unsaturated phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine were prepared and subjected to free-radical-initiated polymerization, both inside the capillary and in free solution. The latter generated oligomers of 2-5 units based on ESI-TOF MS, and formed the more stable coating in CE. Rinsing the capillary with a solution of the ex situ oligomerized DOPC suppressed EOF (0.8 x 10(-)(8) m(2)/V.s) for more than 20 h, whereas in situ oligomerized electroosmotic flow (EOF) suppressed the EOF for only 10 h. Mixtures of anionic and cationic proteins were separated under neutral pH and low ionic strength buffer with efficiencies of 480,000-930,000 plates/m and recoveries of 75-99%.     

Microscale exoglycosidase processing and lectin capture of glycans with phospholipid assisted capillary electrophoresis separations

Capillary electrophoresis separations of glycans labeled with 1-aminopyrene-3,6,8-trisulfonic acid were achieved with separation efficiencies ranging from 480,000 to 640,000 theoretical plates in a 60.2 cm, 25 μm inner diameter fused silica capillary. Under these separation conditions, the coefficient of variation in peak area is 10%, and if labeling efficiency is estimated at 100%, the limit of detection is 15 fM. The capillary electrophoresis method incorporated phospholipid additives to enhance the separation of glycans with slight differences in hydrodynamic volume. In addition, the phospholipid additives supported the integration of the lectin concanavalin A as well as the enzymes α1-2,3 mannosidase or β1-4 galactosidase to provide structural and compositional information about the glycans subject to separation. The use of in-capillary cleavage of terminal glycan residues with exoglycosidases offers a number of advantages over benchtop enzymatic sequencing, including reduced consumption of analyte, as well as enzyme. These methods were used to evaluate glycans derived from the glycoproteins α1-acid glycoprotein, fetuin, and ribonuclease B, as well as from glycoproteins collected from MCF7 cells.     

Preparation and characterization of biocompatible Nb-C coatings

Nb-C composite films, obtained by DC magnetron sputtering method, were investigated as possible candidates for the protective layers used in medical implants. Coatings of different carbon/niobium ratios were prepared and analyzed for elemental and phase composition, crystallographic structure, texture, corrosion behavior, and cell viability. The coating with the highest C/Nb ratio (≈ 1.9) was found to have a nanocomposite structure, in which NbC nanocrystalline phase coexists with an amorphous a-C one. The coated samples exhibited an improved corrosion resistance as compared with the Ti alloy. Cell viability measurements proved that human osteosarcoma cells are adherent to the coating surfaces, the highest viability being found for the film with the highest carbon content.     

Microfluidic capillary system for immunoaffinity separations of C-reactive protein in human serum and cerebrospinal fluid

A miniaturized system based on microfluidic capillaries is presented for point-of-care testing and clinical assessment. The approach relies on microsyringe pump-generated flow to deliver reagents and immunoaffinity chromatography to isolate the antigen from biological matrixes. Capillary sandwich immunoassays for C-reactive protein (CRP) were demonstrated in human serum and cerebrospinal fluid (CSF), which are relevant matrixes for cardiovascular disease risk and meningitis research, respectively. Capillaries packed with antibody-coated silica beads were used to capture CRP from the matrix and a second, dye-labeled antibody was introduced to form a sandwich complex. An acidic elution buffer dissociated the antibody-antigen complexes, and the labeled antibody was detected with diode laser-induced fluorescence. Four parameter logistic functions and % relative error plots were used to model and assess the data. The calibration ranges for CRP were 0.05-3.0 microg/mL in 1:10 diluted serum and 0.01-30 microg/mL in undiluted CSF. The microfluidic apparatus employed a flow rate of 2 microL/min and a sample injection volume of 250 nL. Since it was not necessary to reach antibody-antigen reaction equilibrium and the assay platform dimensions were minimal, run times were as short as 10 min.     

交联PS微球负载纳米Ni颗粒的制备及表征

采用种子聚合法制备出交联聚苯乙烯(PS)微球,经预处理在微球表面引入官能团,采用化学镀法在微球表面负载纳米级Ni颗粒,利用FI-IR、SEM、XRD等测试手段对其进行表征,结果表明:PS微球粒径均匀,平均粒径约为4.3μm,表面光滑;微球负载的纳米Ni颗粒分散性良好、平均晶粒度为9.41nm.利用负载微球对高氯酸铵(AP)进行催化实验,表明负载微球可使AP高温分解峰降低94.2℃左右.研究表明:采用PS微球负载可提高纳米Ni颗粒的分散性,进而提高了Ni颗粒的催化性能.     

Surface plasmon resonance detection for capillary electrophoresis separations

A miniaturized surface plasmon resonance sensor has been used as an on-line detector for capillary electrophoresis separations. The capillary was modified slightly to shield the sensor electronics from the high voltages applied during the separation. A three-component mixture of high refractive index materials was separated and detected at the millimolar level by an untreated gold-sensing surface. A simple protein immobilization procedure was used to functionalize the surface for selective protein detection. A hybrid buffer system was developed, in which both the deposition of immobilized protein layers and the electrophoretic delivery of protein analytes were optimized. The detection system has a reproducibility of 15%, a dynamic range of 3 orders of magnitude, and a detection limit for IgG of 2 fmol.     

Preparation and characterization of carbon and titanium carbide coatings

Carbon and titanium carbide coatings were deposited onto 304 and 316 stainless steel, Monel 400, molybdenum and copper substrates by the d.c. diode sputtering method. The former were prepared using a graphite target in an argon atmosphere whereas the latter were deposited using a titanium target in an argon-methane gas mixture. The coatings were characterized using Auger electron spectroscopy, X-ray photoelectron spectroscopy, Rutherford backscattering spectrometry, scanning electron microscopy and X-ray diffraction techniques. The adhesion of the coatings to the substrates and the effect of annealing on their crystallinity were studied.     

有机-无机杂化纳米复合材料合成及性能测试

采用溶胶-凝胶法,以甲基三乙氧基硅烷(MTEOS)和正硅酸乙酯(TEOS)及纳米氧化硅为原料,成功制备了有机-无机杂化纳米复合材料及涂层.以X射线衍射(XRD)、富立叶红外光谱(FTIR)、扫描电子显微镜(SEM)和原子力显微镜(AFM)等手段研究了杂化材料的工艺与结构及性能的关系,并对涂覆于铝合金基体上的纳米复合材料涂层的防腐蚀性能进行了实验检测.结果表明,有机-无机杂化纳米复合材料涂层具有优良的抗腐蚀性能.     

Effect of direct control of electroosmosis on peptide and protein separations in capillary electrophoresis.

The separations of peptide and protein mixtures in capillary zone electrophoresis (CZE) at various solution conditions were studied with the direct control of electroosmosis. The zeta potential at the aqueous/capillary interface and the resulted electroosmosis in the presence of an electric field were directly controlled by using an additional electric field applied from outside of the capillary. The controlled electroosmotic flow affected the migration time and zone resolution of peptide and protein mixtures. The changes in the magnitude and polarity of the zeta potential caused the various degrees of peptide and protein adsorption onto the capillary through the electrostatic interactions. The separation efficiencies of peptide and protein mixtures were enhanced due to the reduction in peptide and protein adsorption at the capillary wall. The direct manipulations of the separation efficiency and resolution of peptide and protein mixtures in CZE were demonstrated by simply controlling the zeta potential and the electroosmotic flow with the application of an external electric field.     

Preparation and characterization of oxidized alginate covalently cross-linked galactosylated chitosan scaffold for liver tissue engineering

Liver tissue engineering (LTE) requires a perfect extracellular matrix (ECM) for hepatocytes culture to maintain high level of liver-specific functions. Here, we reported a LTE scaffold derived from oxidized alginate covalently cross-linked galactosylated chitosan via Schiff base reaction, without employing any extraneous chemical cross-linking agent. The structure of galactosylated chitosan (GC) and oxidized alginate was confirmed by Fourier transformed infrared (FTIR) spectra, proton nuclear magnetic resonance (¹H-NMR) spectroscopy, X-ray diffraction (XRD) or thermogravimetric (TG) analysis. The structure and properties of a series of the scaffolds were characterized by FTIR, XRD, scanning electron microscopy (SEM), porosity, equilibrium swelling, mechanical properties, thermal stability and in vitro degradation. FTIR spectra confirmed the characteristic peak of Schiff base groups in the scaffolds and XRD indicated the scaffolds could be amorphous. SEM analysis showed that the scaffolds displayed highly porous surfaces with average pore size of 50-150 μm and interconnected pore structure in the internal structure with average pore size of 100-250 μm. Porosity measurement suggested the scaffolds had a porosity of about 70%. The compressive modulus of the scaffolds (hydrated) was in the range of 4.2-6.3 kPa. Further studies showed that, with the increase of the oxidized alginate content, the equilibrium swelling and in vitro degradation rate of the scaffolds decreased and the thermal stability slightly increased, which might mainly attribute to the difference of the degree of cross-linking and the nature properties of the raw materials. Additionally, the biocompatibility of the scaffolds was evaluated in vitro. The results showed that the hepatocytes cultured on the scaffolds had a typical spheroidal morphology, formed multi-cellular aggregates and presented perfect integration with the scaffolds, which suggested that the scaffolds may be potential candidates for LTE strategies.     

Factors affecting the temporal stability of semipermanent bilayer coatings in capillary electrophoresis prepared using double-chained surfactants

Surfactants such as didodecyldimethylammonium bromide (DDAB) adsorb onto fused-silica capillaries to form semipermanent bilayer coatings. However, such coatings must be regenerated between runs to maintain efficiency and reproducibility. In this paper, chemical and physical factors affecting the stability of DDAB coatings are investigated. Chemical factors such as ionic strength and the nature of the buffer anion (e.g., from acetate to phosphate), which decrease the critical micelle concentration of DDAB, improve the coating stability. Increasing buffer pH also increases the coating stability. Finally, reducing the capillary diameter and reducing the volume of buffer flushed through the capillary enhance the coating stability. Using 50 mM acetate, pH 5.0, in a 25-microm-i.d. capillary, cationic proteins were separated with efficiencies of 1.05 million plates/m and a run-to-run migration time reproducibility of 0.6-0.8% RSD for 10 successive runs without regeneration of the DDAB coating between runs.     

Preparation and characterization of bioactive monolayer and functionally graded coatings

Hydroxyapatite powders were made by reacting orthophosphoric acid with calcium hydroxide and dense bioactive coatings were subsequently produced by the plasma spray technique. Three types of hydroxyapatite (flame spheroidized) monolayer coatings and three types of functionally graded coatings were manufactured. It was found that average microhardness values of monolayer coatings decreased as the indentation load increased. The relationship between indentation load and indent diagonal length observed Meyer's law. Microhardness and fracture toughness of coatings were affected by characteristics of feedstock powders for plasma spraying. The indentation fracture toughness of coatings could be significantly increased by incorporating a toughening phase. ©1999 Kluwer Academic Publishers     

Preparation,characterization, and blood compatibility of polylactide-based phospholipid polymer

l-α-glycerophosphorylcholine (GPC) was obtained by hydrolysis of lecithin extracted from eggs. FT-IR and ¹H-NMR analysis indicated a successful preparation of GPC. Polylactide-based phospholipid polymer (PLLA-PC) was synthesized by ring-opening polymerization of l-lactide in the presence of GPC to improve the cell/material interfacial reaction of PLLA for tissue engineering applications. The yield of the reaction strongly depended on the reaction time. Values above 80% were obtained which are much higher than those reported in literature. Copolymers with the largest molecular weights were obtained at 122 °C for 48 h. The properties and biocompatibility of the PLLA-PC copolymers were characterized. Surface rearrangement was detected due to the dynamic molecular motion according to X-ray photoelectron spectroscopy data. Besides, increase in hydrophilicity and decreases in fibrinogen adsorption and platelet adhesion were observed due to the hydrophilic phosphorylcholine moieties in the copolymer.     

Development of a conductivity-based photothermal absorbance detector for capillary separations

A contactless conductivity-based absorbance detector has been developed for use with capillary separations. Detection is based on a photothermal process. As analytes pass through the detector they absorb light, producing a thermal perturbation. This thermal event results in a change in the solution conductivity. The measured change in conductivity is directly related to the absorption of light. The major advantage to this type of detector is that the measured absorbance is, to a first approximation, independent of optical path length, allowing small-diameter capillaries to be used. This approach combines the optical simplicity of traditional transmission-based instruments with the path length independence of similar refraction-based photothermal detectors. In addition to the initial development and characterization of the photothermal absorbance detector, multiphysical modeling of the heat transfer within the conductivity cell was performed.     

Preparation and characterization of plasticized gelatin films cross-linked with low concentrations of Glutaraldehyde

Thermal and mechanical properties as well as moisture resistance and water vapor barrier properties of films from bovine gelatin added with d-sorbitol (30 wt%) as plasticizer and cross-linked with low amounts of glutaraldehyde (GTA, from 0 to 2 wt%) were investigated to determine their suitability as barrier layers for flexible packaging materials. Results revealed that free amino side chain groups of gelatin decreased with GTA, confirming the occurrence of cross-linking between GTA and gelatin. The extent of cross-linking reaction in the presence of d-sorbitol was lower compared with the unplasticized counterpart suggesting that plasticizer hampers GTA to react. The glass transition temperature (T _g) as measured from differential scanning calorimetry (DSC) increased with GTA concentration owing to the formation of more reticulated materials while the incorporation of d-sorbitol led to a small reduction in this parameter due to plasticization. Increasing GTA concentration from 0 to 1 wt% provoked the enhancement of elastic modulus from 3.7 ± 0.2 to 4.9 ± 0.2 GPa. These values reduced significantly by the addition of d-sorbitol, whereas elongation at break improved in about 150%. The optimum formulation for the intended purpose was that containing 1 wt% GTA and 30 wt% d-sorbitol since it exhibited the best set of properties: total soluble mater reduced from 100 to 16%, moisture absorption decreased from 1854.1 ± 85 to 210.4 ± 8%, water vapor permeability at 65% relative humidity improved from 2.42 ± 0.27 to 0.94 ± 0.06 × 10⁻¹⁴ kg m Pa⁻¹ s⁻¹ m⁻², with minor reduction in opacity and with the additional benefit of releasing only 5% of the initial GTA content.     

水性塑料涂料用聚氨酯-苯乙烯核壳交联乳液的合成研究

通过引入双功能团单体甲基丙烯酸-β-羟乙酯(HEMA),将双键引入聚氨酯(PU)链中,再与苯乙烯(St)单体乳液聚合合成了交联型聚氨酯-聚苯乙烯(PUS)核壳复合乳液。研究了核壳质量比、HEMA用量对乳液及涂膜性能的影响,并用FTIR和TEM对聚合物结构和胶粒形态进行了表征。研究表明,随着核壳比的增大,乳液粒径和MFT先增大后减小,涂膜对高抗冲击聚苯乙烯(HIPS)塑料基材的附着力增加;HEMA用量增加,涂膜的耐水性和附着力提高。当核壳质量比为4/5,HEMA/PU为6%时,涂膜对HIPS塑料基材附着良好。