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We introduce rapid replica molding of ordered, high-aspect-ratio, thiol-ene micropillar arrays for implementation of microfluidic immobilized enzyme reactors (IMERs). By exploiting the abundance of free surface thiols of off-stoichiometric thiol-ene compositions, we were able to functionalize the native thiol-ene micropillars with gold nanoparticles (GNPs) and these with proteolytic α-chymotrypsin (CHT) via thiol-gold interaction. The micropillar arrays were replicated via PDMS soft lithography, which facilitated thiol-ene curing without the photoinitiators, and thus straightforward bonding and good control over the surface chemistry (number of free surface thiols). The specificity of thiol-gold interaction was demonstrated over allyl-rich thiol-ene surfaces and the robustness of the CHT-IMERs at different flow rates and reaction temperatures using bradykinin hydrolysis as the model reaction. The product conversion rate was shown to increase as a function of decreasing flow rate (increasing residence time) and upon heating of the IMER to physiological temperature. Owing to the effective enzyme immobilization onto the micropillar array by GNPs, no further purification of the reaction solution was required prior to mass spectrometric detection of the bradykinin hydrolysis products and no clogging problems, commonly associated with conventional capillary packings, were observed. The activity of the IMER remained stable for at least 1.5 h (continuous use), suggesting that the developed protocol may provide a robust, new approach to implementation of IMER technology for proteomics research. Graphical abstract [ABSTRACT FROM AUTHOR]

Interactions between Intralipid dispersion and local anesthetics (bupivacaine, prilocaine, and lidocaine) were investigated. The amount of bupivacaine (the most cardiotoxic analyte of the local anesthetics studied) entrapped in Intralipid in the presence of plasma was studied using an off-line filtration and solid phase extraction method combined with capillary zone electrophoresis for quantification of free unbound bupivacaine. To confirm interactions between the analytes and Intralipid at lower concentrations, direct injection mass spectrometry was used. The use of immobilized Intralipid chromatography-atmospheric pressure ionization–ion trap mass spectrometry in the study of interactions between drugs and Intralipid dispersion is demonstrated. Finally, interactions between Intralipid dispersion and local anesthetics were investigated by electrokinetic capillary chromatography. The electrophoretic mobility of the Intralipid dispersed phase was calculated using the iterative procedure and a homologous series of alkyl phenyl benzoates (C1–C6), and the retention factors for the analytes were determined. [ABSTRACT FROM AUTHOR]

Large unilamellar vesicles of egg-phosphatidylcholine (eggPC), a naturally occurring phospholipid, were used in capillary electrophoresis (CE) for semi-permanent coating of fused silica capillaries. The stability of the phospholipid coating was tested at different cetyltrimethylammonium bromide (CTAB) concentrations with and without CaCl2 present in the coating solution. The effect of physical factors influencing the coating stability (e.g. duration of the coating time, storage temperature of the coating solution) were also studied. Standing overnight in background electrolyte (BGE) solution did not alter the eggPC phospholipid coating noticeably. The performance of the coating was tested with a mixture of basic proteins (lysozyme, ribonuclease A and a-chymotrypsinogen A). Highest efficiencies (over 200,000 plates m-1) were achieved when the capillary was filled for 15 h with a liposome solution containing both CTAB and CaCl2. [ABSTRACT FROM AUTHOR]

The effect of calcium on phospholipid coatings in fused silica capillaries used in capillary electrophoresis was studied. The anionic liposomes used for the coating consisted of 3 mM 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine and phosphatidylserine in the ratio 80/20 mol%. Coating was performed as part of the preconditioning, and the capillaries could be used for several runs without the need for liposomes in the background electrolyte solution or for liposome rinses between runs. Phospholipids could easily be flushed away by rinsing with a chloroform-methanol (2:1 v/v) mixture, which made it possible to recoat and reuse the capillaries. A calcium:phospholipid ratio of approximately 3 gave the most stable coating. The stability of the coating and success of the coating procedure were studied by measuring the electroosmotic flow and by separating uncharged steroids, which were used as model compounds. Many parameters that affect the coating, such as preconditioning (with different acids and bases), buffer, temperature during coating, and the physical structures of liposomes, were studied, with and without calcium in the liposome solution. The separation of steroids was improved and was less dependent on coating conditions when calcium was present during the coating. Capillaries optimally coated with anionic phospholipids were applied in the separation of phenols. [ABSTRACT FROM AUTHOR]

The iridoid glycosides, methyl catalpol, asperuloside, verbenalin, cinnamoyl catalpol, catalpol and aucubin, were studied from both larvae and adults of butterfly Melitaea cinxia. Special emphasis in the study was put on finding a correlation between the iridoid glycoside content in butterflies and plants. An optimised partial filling micellar electrokinetic capillary chromatographic–electrospray ionisation mass spectrometric (PF-MECC-ESI-MS) method was employed for the separation and identification of the six iridoid glycosides. In this work, the isolation and determination of catalpol and aucubin from extracts of both larvae and adults of Melitaea cinxia butterflies is demonstrated. The PF-MECC-ESI-MS method, using the [M+Na]+, [M+Li]+ and/or [M+NH4]+ adducts in ESI-MS, was used for quantification of aucubin and catalpol in the insects. In addition, the identification of all analytes was attempted by direct infusion MS/MS analysis. LOQ values for the iridoid glycosides varied between 10 mg/l (for verbenalin) to 50 mg/l (for catalpol and aucubin) corresponding to 0.1% of the samplés dry mass. A correlation was noticed between the concentrations of iridoid glycosides in plants and the concentrations in larvae feeding on them. [ABSTRACT FROM AUTHOR]

Understanding the toxicity of ionic liquids (ILs) is crucial in the search of greener chemicals. By comparing in vivo toxicity and in vitro interactions determined between compounds and biomimetic lipid membranes, more detailed toxicity vs. structure relation can be obtained. However, determining the interactions between non-surface-active compounds and liposomes has been a challenging task. Organisational changes induced by ILs and IL-like spirocyclic compounds within 1,6-diphenyl-1,3,5-hexatriene-doped biomimetic liposomes was studied by steady-state fluorescence anisotropy technique. The extent of organisational changes detected within the liposome bilayers were compared to the toxicity of the compounds determined using Vibrio Fischeri bacteria. Four liposome compositions made of pure 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocoline (POPC) and mixtures of POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS), and cholesterol (Chol) were tested as biomimetic models. Changes observed within the POPC/POPS/Chol 55:20:25 bilayers correlated the best with the toxicity results: ten out of twelve compounds followed the trend of increasing bilayer disorder – increasing toxicity. The study suggests that the toxicity of non-surface-active compounds is dependent on their ability to diffuse into the bilayers. The extent of bilayer's organisational changes correlates rather well with the toxicity of the compounds. Highly sensitive technique, such as fluorescence anisotropy measurements, is needed for detecting subtle changes within the bilayer structures. [ABSTRACT FROM AUTHOR]

7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (mTBD) has useful catalytic properties and can form an ionic liquid when mixed with an acid. Despite its potential usefulness, no data on its thermodynamic and transport properties are currently available in the literature. Here we present the first reliable public data on the liquid vapor pressure (temperature from 318.23 K to 451.2 K and pressure from 11.1 Pa to 10 000 Pa), liquid compressed density (293.15 K to 473.15 K and 0.092 MPa to 15.788 MPa), liquid isobaric heat capacity (312.48 K to 391.50 K), melting properties, liquid thermal conductivity (299.0 K to 372.9 K), liquid refractive index (293.15 K to 343.15 K), liquid viscosity (290.79 K to 363.00 K), liquid–vapor enthalpy of vaporization (318.23 K to 451.2 K), liquid thermal expansion coefficient (293.15 K to 473.15 K), and liquid isothermal compressibility of mTBD (293.15 K to 473.15). The properties of mTBD were compared with those of other relevant compounds, including 1,5-diazabicyclo(4.3.0)non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and 1,1,3,3-tetramethylguanidine (TMG). We used the PC-SAFT equation of state to model the thermodynamic properties of mTBD, DBN, DBU, and TMG. The PC-SAFT parameters were optimized using experimental data. [ABSTRACT FROM AUTHOR]

Our study demonstrates that nanoplasmonic sensing (NPS) can be utilized for the determination of the phase transition temperature (Tm) of phospholipids. During the phase transition, the lipid bilayer undergoes a conformational change. Therefore, it is presumed that the Tm of phospholipids can be determined by detecting conformational changes in liposomes. The studied lipids included 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Liposomes in gel phase are immobilized onto silicon dioxide sensors and the sensor cell temperature is increased until passing the Tm of the lipid. The results show that, when the system temperature approaches the Tm, a drop of the NPS signal is observed. The breakpoints in the temperatures are 22.5 °C, 41.0 °C, and 55.5 °C for DMPC, DPPC, and DSPC, respectively. These values are very close to the theoretical Tm values, i.e., 24 °C, 41.4 °C, and 55 °C for DMPC, DPPC, and DSPC, respectively. Our studies prove that the NPS methodology is a simple and valuable tool for the determination of the Tm of phospholipids. [ABSTRACT FROM AUTHOR]

We investigated the toxicological effect of seven novel cholinium, guanidinium, and tetramethylguanidinium carboxylate ionic liquids (ILs) from an ecotoxicological point of view. The emphasis was on the potential structure-toxicity dependency of these surface-active ILs in aqueous environment. The median effective concentrations (EC50) were defined for each IL using Vibrio (Aliivibrio) fischeri marine bacteria. Dipalmitoylphosphatidylcholine (DPPC) liposomes were used as biomimetic lipid membranes to study the interactions between the surface-active ILs and the liposomes. The interactions were investigated by following the change in the DPPC phase transition behaviour using differential scanning calorimetry (DSC). Critical micelle concentrations for the ILs were determined to clarify the analysis of the toxicity and the interaction results. Increasing anion alkyl chain length increased the toxicity, whereas branching of the chain decreased the toxicity of the ILs. The toxicity of the ILs in this study was mainly determined by the surface-active anions, while cations induced a minor impact on the toxicity. In the DSC experiments the same trend was observed for all the studied anions, whereas the cations seemed to induce more variable impact on the phase transition behaviour. Toxicity measurements combined with liposome interaction studies can provide a valuable tool for assessing the mechanism of toxicity. [ABSTRACT FROM AUTHOR]