Capillary electrophoresis in pure nonaqueous solvents

The use of nonaqueous solvents in capillary electrophoresis (CE) was investigated. Nonaqueous solvents used included methanol, ethanol, acetonitrile, dimethylformamide, and molten salts. Both indirect, and direct UV detection as well as electrochemical detection were examined. The behavior of electroosmotic flow and Joule heating was examined as well as selectivity patterns for a variety of inorganic and organic ionic species. Analytes examined included inorganic anions, alkali and alkaline earth metal ions, alkanesulfonates, alkyl sulfates, linear alkylbenzensulfonates, derivatized and free amino acids, and fatty acids. Selectivity was dependent not only on the nature of the solvent, but also on the nature and concentration of the electrolyte. With many inorganic ions completely reversed separation order relative to aqueous systems was observed. Although the addition of metal ions caused changes in resolution, complete separation of alkanesulfonates (C2-C16) and alkyl sulfates (C8-C18) was possible by a change from protic (methanol) to aprotic conditions (addition of acetonitrile). The partial separation of positional isomers of linear alkyl benzensulfonate homologues (C10-C15) was achieved in methanol/acetonitrile mixtures. The complete separation of alkali and alkaline earth metal ions, which has not been reported to date, and separation of potassium and ammonium were observed in a methanol/imidazole electrolyte. Significantly different migration order was achieved for dansylated amino acids compared to aqueous systems. Changes in selectivity of free amino acids were possible in basic and acidic electrolytes. Complete separation of a wide range of fatty acids (C1-C20) was possible in less than 20 min. The unique selectivity patterns in nonaqueous solvents was related to changes in solvation and ion-interaction effects. The ability to easily adjust selectivity via choice of electrolyte and to inject aqueous samples directly into the nonaqueous electrolytes suggests that such systems may offer significant advantages for many analytical problems. The results illustrate that when selectivity, efficiency, quantitation, and detection limits are taken into consideration, nonaqueous CE approaches should, in many situations, offer a unique alternative to aqueous based CE analyses. Electroosmotic flow was appreciable, and was cathodal in basic solutions. Reversal of electroosmotic flow was observed in the presence of an excess of a strong acid (HCl or HClO4) in the separation electrolyte. The nature of electrolyte anion (chloride, perchlorate, and acetate) and solvent (methanol/acetonitrile mixtures) had significant effects on the electroosmotic flow in acidic conditions. Evidence was observed for ion adsorption (protons and anions) on to silica surfaces and ion-interaction in the electrolyte. Adsorption and ion-interaction could be used to control both the direction and magnitude of electroosmotic flow. Reproducibility of electroosmotic flow was good under appropriate experimental conditions (%RSD = 1.1)

Bone CLARITY reveals that anti-sclerostin antibody penetrates the lacunar canalicular system in mice under physiological loading conditions

Anti-sclerostin antibody (Scl-Ab) neutralizes sclerostin activity, a secreted protein from osteocytes, resulting in WNT mediated increases in bone formation and decreases in bone resorption; this dual effect on bone results in rapid increases in bone mass and strength

Pharmacokinetic comparison of a diverse panel of non-targeting human antibodies as matched IgG1 and IgG2 isotypes in rodents and non-human primates.

In this study we compared the pharmacokinetic profile of four unrelated antibodies, which do not bind to mammalian antigens, in IgG1 and IgG2 frameworks in both rats and non-human primates (NHP). This allowed for extensive cross comparison of the impact of antibody isotype, complementarity determining regions (CDR) and model species on pharmacokinetics without the confounding influence of antigen binding in the hosts. While antibody isotype had no significant impact on the pharmacokinetics, the CDRs do alter the profile, and there is an inverse correlation between the neonatal Fc receptor (FcRn) affinity and pharmacokinetic performance. Faster clearance rates were also associated with higher isoelectric points; however, although this panel of antibodies all possess basic isoelectric points, ranging from 8.44 to 9.18, they also have exceptional in vivo half-lives, averaging 369 hours, and low clearance rates, averaging 0.18 ml/h/kg in NHPs. This pattern of pharmacokinetic characteristics was conserved between rats and NHPs

\u3b2-galactosidase assays of single-cell lysates on a microchip: a complementary method for enzymatic analysis of single cells

The use of microfluidic glass chips for continuous single-cell lysis and assay of internal \u3b2-Galactosidase (\u3b2-Gal) content is described. Cells were transported single file toward a Y-shaped mixing junction at which lytic agents were introduced by suction. Flow velocities of 3c100 and 3c40 \u3bcm/s were used under protein denaturing [35 mM sodium dodecylsulfate (SDS)] and nondenaturing (0.1% Triton X-100) conditions, respectively. Complete and reproducible lysis of individual cells on-chip occurred within 30 s using Triton X-100 and 2 s when using SDS. Optimal concentrations of lysis and enzyme substrate reagents were determined using microtitre plate and chip-based procedures. Fluorescence peaks, due to the enzymatic product fluorescein mono-\u3b2-D-galactopyranoside (FMG), were detected downstream of the mixing and cell lysis point for the reaction of \u3b2-Gal with 200 \u3bcM of the fluorogenic substrate fluorescein-di-\u3b2-D-galactopyranoside (FDG). FMG fluorescence was observed from cells preincubated with FDG off-chip then subsequently lysed on-chip with SDS. Unincubated cells were mixed on-chip with both FDG and Triton X-100, each individual cell generating FMG fluorescence downstream of the mixing point detected within 2 min of mixing. In contrast, viable cells incubated with FDG required 1 h or more in order to generate significant signal in a flow cytometer.NRC publication: N