Optimization of lipospheres production by factorial design and their performances on a dielectrophoretic lab-on-a-chip platform

Aim of this study was to investigate the effect of the preparation parameters on the characteristics of lipospheres that would optimally fit to a lab-on-a-chip platform. Lipospheres were produced by melt dispersion technique using different lipid mixture heated to 70 °C and then emulsified into an external aqueous phase. The initial part of the work was devoted to the selection of the best lipid composition by a classical intuitive approach while the optimization and the screening of the experimental parameters were conducted through a “design of experiments”. Once the best preparation parameters were selected, they were adopted also for the production of cationic lipospheres (CLS). The second part of the study describes the analysis of the lipospheres performances when applied to a DEParray™Chip. The loading, distribution, movement and separation of neutral and cationic lipospheres were investigated. The obtained data show that both neutral and cationic lipospheres can be efficiently used in association with DEParray™Chip.     

Inverted open microwells for cell trapping, cell aggregate formation and parallel recovery of live cells

The inverted open microwell is a novel microstructure supporting isolation and trapping of cells, analysis of cell-cell and cell-molecule interactions and functional cell sorting. This work introduces the inverted open microwell concept, demonstrating successful isolation of K562 cells in 75 μm microwells fabricated on a flexible printed circuit board substrate, and recovery of viable cells onto standard microtiter plates after analysis and manipulation. Dielectrophoresis (DEP) was used during the delivery phase to control cell access to the microwell and force the formation of cell aggregates so as to ensure cell-cell contact and interaction. Cells were trapped at the air-fluid interface at the bottom edge of the open microwell. Once trapped, cells were retained on the meniscus even after DEP de-activation and fluid was exchanged to enable perfusion of nutrients and delivery of molecules to the microwell, as demonstrated by a calcein-staining protocol performed in the microsystem. Finally, cell viability was assessed on trapped cells by a calcein release assay and cell proliferation was demonstrated after multiple cells had been recovered in parallel onto standard microtiter plates.     

The Eigenvalues of the Angular Spheroidal Wave Equation

Approximate relations are obtained between the eigenvalues λ and the ellipticity parameter c² of the angular spheroidal wave equation. Although based on WKBJ methods and the assumption that λ is large, the relations are useful throughout the complex c²-plane. They are exact at c² = 0, and reproduce the standard asymptotic formulas for λ when c² is large. At intermediate values of c², they provide approximations for the square-root branch points of the multivalued function λ(c²) in the complex c²-plane at which adjacent eigenvalues of the same class become equal in pairs. These branch points lie on an infinite sequence of distorted circular rings. Their exact locations have been computed for the first four rings for angular wavenumbers m = 0,…,4.     

Simultaneous determination of pseudouridine and creatinine in untreated urine by ion-pair liquid chromatography with diode-array ultraviolet detection

A simple procedure for the simultaneous determination of pseudouridine and creatinine in urine using ion-pair high-performance liquid chromatography with ultraviolet detection is described. It consists of simply diluting the filtered urine with mobile phase (1:20) followed by direct chromatographic injection. A single analysis takes only 10 min. This method has been applied to the analysis of urine samples from normal donors and patients with different types of cancer. The mean values, means, of the peak-area ratio of pseudouridine to creatinine were 61.79.10(-3) and 81.92.10(-3) for male and female normal donors, respectively. Out of twenty-five urine samples of patients with cancer examined, nineteen (all the forteen males included) had values higher than means + 2 sigma.     

Development and Validation of a Reversed-Phase HPLC Method with UV Detection for the Determination of L-Dopa in Vicia faba L. Broad Beans

L-Dopa (LD), a substance used medically in the treatment of Parkinson’s disease, is found in several natural products, such as Vicia faba L., also known as broad beans. Due to its low chemical stability, LD analysis in plant matrices requires an appropriate optimization of the chosen analytical method to obtain reliable results. This work proposes an HPLC-UV method, validated according to EURACHEM guidelines as regards linearity, limits of detection and quantification, precision, accuracy, and matrix effect. The LD extraction was studied by evaluating its aqueous stability over 3 months. The best chromatographic conditions were found by systematically testing several C₁₈ stationary phases and acidic mobile phases. In addition, the assessment of the best storage treatment of Vicia faba L. broad beans able to preserve a high LD content was performed. The best LD determination conditions include sun-drying storage, extraction in HCl 0.1 M, chromatographic separation with a Discovery C₁₈ column, 250 × 4.6 mm, 5 µm particle size, and 99% formic acid 0.2% v/v and 1% methanol as the mobile phase. The optimized method proposed here overcomes the problems linked to LD stability and separation, thus contributing to the improvement of its analytical determination.     

Surface and Electrochemical Characterization of a New Layered GC/Betaine/Pt Electrode and Investigation on its Performance as a Sensor for two B Complex Vitamins,B1 and B6: Preliminary Results

The electrochemical preparation of a novel composite glassy carbon/betaine/platinum (GC/BT/Pt) electrode with meso- nano Pt globules dispersed on the outer surface and betaine, as intermediate layer, electrodeposited on GC by Cyclic Voltammetry (CV) or Pulsed Potential (PP) techniques, is presented. Surface analysis by Scanning Electron Microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS) confirms the strategic role of grafted betaine in modulating GC/BT- interaction sites and the range of potentials for adequate platinum CV electrodeposition. The GC/BT/Pt performance, concerning sensitivity, linearity, work potentials and repeatability tests with Flow Injection Analysis (FIA), was ascertained for the detection of vitamins B1 and B6.     

A validated LC–MS/MS method for quantitative determination of L-dopa in Fagioli di Sarconi beans (Phaseolus vulgaris L.)

An analytical method based on ultrasound assisted extraction (UAE) and liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI/MS/MS) was validated and applied for determining L-dopa in four ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris L.), marked with the European label PGI (Protected Geographical Indication). The selectivity of the proposed method was ensured by the specific fragmentation of the analyte. Simple isocratic chromatographic conditions and mass spectrometric detection in multiple reaction monitoring (MRM) acquisition mode were used for sensitive quantification. The LC–ESI/MS/MS method was validated within a linear range of 0.001–5.000 μg/mL. Values of 0.4 and 1.1 ng/mL were obtained for the limits of detection and quantification, respectively. The repeatability, inter-day precision, and recovery values ranges were 0.6%–4.5%, 5.4%–9.9%, and 83%–93%, respectively. Fresh and dried beans, as well as pods, cultivated exclusively with organic methods avoiding any synthetic fertilizers and pesticides were analyzed showing an L-dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight.