Enhanced electrophoretic resolution of monosulfate glycosaminoglycan disaccharide isomers on poly(methyl methacrylate) chips

To improve the separation of monosulfate glycosaminoglycan disaccharide isomers by microchip electrophoresis, we found that addition of 1,4-dioxane (DO) dramatically improved analyte resolution, probably due to solvation effects. Methylcellulose (MC) was tested for the ability to suppress EOF and analyte adsorption to the chip. To improve analyte resolution, buffer pH, beta-CD, and DO were systematically investigated. Fast separation was achieved by increasing the electric field strength, and field-amplified sample stacking occurred with increasing buffer concentrations. Therefore, based on our findings, we describe an efficient method for the separation of monosulfate and trisulfate unsaturated disaccharides (DeltaDi-UA2S, DeltaDi-4S, DeltaDi-6S, and DeltaDi-triS) derivatized with 2-aminoacridone hydrochloride. A mixture of monosulfate disaccharide isomers (DeltaDi-UA2S, DeltaDi-4S, and DeltaDi-6S) was baseline-separated within 75 s on a poly(methyl methacrylate) chip using a mixed buffer (DO/running buffer 57:43 v:v), 0.5% MC, pH 6.81, with an E(sep) of 558 V/cm. The theoretical plate was in the range of 5 x 10(5) to 1 x 10(6) m-1.     

Dynamic modification of poly(methyl methacrylate) chips using poly(vinyl alcohol) for glycosaminoglycan disaccharide isomer separation

We describe a microchip electrophoresis (MCE) method for the assay of unsaturated disaccharides of chondroitin sulfates, dermatan sulfates, and hyaluronic acid (HA). Poly(vinyl alcohol) (PVA) could be irreversibly adsorbed onto poly(methyl methacrylate) (PMMA) substrates and this approach was applicable for dynamic coating. The characteristics of the PMMA surface with PVA coating were evaluated in terms of the wettability, EOF, and adsorption of 2-aminoacridone (AMAC)-labeled disaccharide. The water contact angle decreased from 73 degrees on a pristine PMMA surface to 37.5 degrees on a PVA-coated surface, indicating that the PVA coating increased hydrophilicity. EOF was reduced approximately twofold and was relatively stable. Scanning electron microscopy and fluorescence microscopy images showed that adsorption of AMAC-labeled disaccharides was dramatically suppressed. Using the PVA coating, baseline separation of two pairs of glycosaminoglycan (GAG) disaccharide isomers, DeltaDi-diS(B)/DeltaDi-diS(D) and DeltaDi-0S/DeltaDi-HA, was achieved in Tris-borate buffer within 130 s by MCE.     

Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).     

Propene polymerization with the MgCl2-supported dichlorobis(β-diketonato)titanium catalysts activated by ordinary alkylaluminums

Several kinds of dichlorobis(β-diketonato)titanium complexes, i.e., Ti(ace-tylacetonato)₂Cl₂, Ti(1-benzoylacetonato)₂Cl₂, Ti(2,2,6,6-tetramethyl-3,5-heptanedionato)₂Cl₂ and Ti(4,4,4-trifluoro-1-phenyl-1,3-butanedionato)₂Cl₂, were synthesized and the corresponding MgCl₂-supported catalysts were prepared by impregnation method. The test of them for propene polymerization revealed that those MgCl₂-supported catalysts could be activated not only by methylaluminoxane (MAO) but also by ordinary alkylaluminums as well. The effect of typical Lewis bases on the catalyst performance was investigated in some detail, which indicated that organic silanes are most effective for the improvement of isospecificity of those catalysts. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 129–135, 1998     

Risk Assessment of Passive Smoking Based on Analysis of Hair Nicotine and Cotinine as Exposure Biomarkers by In-Tube Solid-Phase Microextraction Coupled On-Line to LC-MS/MS

Passive smoking due to environmental tobacco smoke is a serious public health concern because it increases the risk of lung cancer and cardiovascular disease. However, the current status and effect of passive smoking in various lifestyles are not fully understood. In this study, we measured hair nicotine and cotinine levels as exposure biomarkers in non-smokers and assessed the risk from the actual situation of passive smoking in different lifestyle environments. Nicotine and cotinine contents in hair samples of 110 non-smoker subjects were measured by in-tube solid-phase microextraction with on-line coupling to liquid chromatography-tandem mass spectrometry, and self-reported lifestyle questionnaires were completed by the subjects. Nicotine and cotinine were detected at concentrations of 1.38 ng mg⁻¹ and 12.8 pg mg⁻¹ respectively in the hair of non-smokers, with levels significantly higher in subjects who reported being sensitive to tobacco smoke exposure. These levels were also affected by type of food intake and cooking method. Nicotine and cotinine in hair are useful biomarkers for assessing the effects of passive smoking on long-term exposure to environmental tobacco smoke, and our analytical methods can measure these exposure levels in people who are unaware of passive smoking. The results of this study suggest that the environment and places of tobacco smoke exposure and the lifestyle behaviors therein are important for the health effects of passive smoking.     

CP/MAS 13C NMR analyses of hydrogen bonding and the chain conformation in the crystalline and noncrystalline regions for poly(vinyl alcohol) films

The chain conformation and hydrogen bonding in the crystalline and noncrystalline regions have been characterized for atactic poly(vinyl alcohol) (PVA) films prepared under different conditions by CP/MAS ¹³C NMR analyses developed recently. The CH resonance lines of the crystalline and noncrystalline components split in different ways, depending significantly on casting solvents and annealing. These lines are found to be successfully resolved into 3–7 constituent lines by the least‐squares curve fitting. In this analysis, nine lines with different chemical shifts are prepared as elementary lines for the curve fitting by assuming the upfield shifts due to the γ‐gauche effect and the downfield shifts due to the formation of the intramolecular hydrogen bonds. The integrated intensities of the constituent lines thus obtained are also interpreted in terms of statistical calculations, assuming the random distribution of the trans and gauche conformations along PVA chains and the statistical distribution of the intramolecular and intermolecular hydrogen bonds between appropriate adjacent OH groups. On the basis of probabilities f_t and f_a for the trans conformation and the intramolecular hydrogen bond obtained through these analyses, the effects of casting solvents and annealing are discussed for both crystalline and noncrystalline components. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 1–9, 2000     

Microfluidic baker's transformation device for three-dimensional rapid mixing

We developed a new passive-type micromixer based on the baker's transformation and realized a fast mixing of a protein solution, which has lower diffusion constant. The baker's transformation is an ideal mixing method, but there is no report on the microfluidic baker's transformation (MBT), since it is required to fabricate the complicated three-dimensional (3D) structure to realize the MBT device. In this note, we successfully fabricate the MBT device by using precision diamond cutting of an oxygen-free copper substrate for the mould fabrication and PDMS replication. The MBT device with 10.4 mm mixing length enables us to achieve complete mixing of a FITC solution (D = 2.6 × 10(-10) m(2) s(-1)) within 51 ms and an IgG solution (D = 4.6 × 10(-11) m(2) s(-1)) within 306 ms. Its mixing speed is 70-fold higher for a FITC solution and 900-fold higher for an IgG solution than the mixing speed by the microchannel without MBT structures. The Péclet number to attain complete mixing in the MBT device is estimated to be 6.9 × 10(4).     

A touch-and-go lipid wrapping technique in microfluidic channels for rapid fabrication of multifunctional envelope-type gene delivery nanodevices

Multifunctional envelope-type gene delivery nanodevices (MENDs) are promising non-viral vectors for gene therapy. Though MENDs remain strong in prolonged exposure to blood circulation, have low immunogenic response, and are suitable for gene targeting, their fabrication requires labor-intensive processes. In this work, a novel approach has been developed for rapid fabrication of MENDs by a touch-and-go lipid wrapping technique in a polydimethylsiloxane (PDMS)/glass microfluidic device. The MEND was fabricated on a glass substrate by introduction of a condensed plasmid DNA core into microfluidic channels that have multiple lipid bilayer films. The principle of the MEND fabrication in the microfluidic channels is based on electrostatic interaction between the condensed plasmid DNA cores and the coated lipid bilayer films. The constructed MEND was collected off-chip and characterized by dynamic light scattering. The MEND was constructed within 5 min with a narrow size distribution centered around 200 nm diameter particles. The size of the MEND showed strong dependence on flow velocity of the condensed plasmid DNA core in the microfluidic channels, and thus, could be controlled to provide the optimal size for medical applications. This approach was also proved possible for fabrication of a MEND in multiple channels at the same time. This on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. Our results strongly indicated that MENDs fabricated with our microfluidic device have a good potential for medical use. Moreover, MENDs fabricated by this microfluidic device have a great potential for clinical use because the devices are autoclavable and all the fabrication steps can be completed inside closed microfluidic channels without any external contamination.