ResonSense: simple linear fluorescent probes for quantitative homogeneous rapid polymerase chain reaction

Here we report a strand-specific fluorescent homogeneous assay format for rapid polymerase chain reaction (PCR). A number of similar assays are commonly used for research applications and are an ideal solution for a closed tube quantitative PCR. These assays use fluorescent resonant energy transfer (FRET) between donor and acceptor fluorescent moieties as the reporting mechanism. However, for different reasons these assays do not report amplification when very rapid cycling times are used. This is because current assays, such as TaqMan^®, are limited, in terms of assay speed, by the 5′-3′ exonuclease activity of Taq DNA polymerase. Other assays based on hybridisation require either a complex de-conformational event to occur, or require more than one probe to report amplification. Reducing the complexity of the experiment reduces costs in terms of design, optimisation and manufacture. Here, we describe ResonSense^® chemistries that use a simple linear fluorescent-labelled probe and a DNA minor-groove binding dye as either donor or acceptor moieties in a homogeneous assay format on the LightCycler^®. This assay format will provide for rapid analysis of samples and so it is particularly well suited to point-of-use testing.     

Nuclear fluorination of 2,4-diarylthiazoles with Accufluor

A series of 2,4-diphenylthiazole derivatives were synthesized and directly fluorinated at the 5-position by reaction with the N-F fluorinating reagent Accufluor^®. Although fluorination occurred selectively at the thiazole ring, it was always incomplete and thus yields for the novel fluorinated products were low to moderate (19-43%) following purification to remove starting material. Nonetheless, the target compounds were obtained in a convenient and straightforward manner. Selectfluor^® was not as effective as Accufluor^® as it gave a trace amount of the 5-chlorothiazole that was difficult to remove by chromatography.     

Tutorial: Simulating chromatography with Microsoft Excel Macros

Chromatography is one of the cornerstones of modern analytical chemistry; developing an instinctive feeling for how chromatography works will be invaluable to future generation of chromatographers. Specialized software programs exist that handle and manipulate chromatographic data; there are also some that simulate chromatograms. However, the algorithm details of such software are not transparent to a beginner. In contrast, how spreadsheet tools like Microsoft Excel™ work is well understood and the software is nearly universally available. We show that the simple repetition of an equilibration process at each plate (a spreadsheet row) followed by discrete movement of the mobile phase down by a row, easily automated by a subroutine (a “Macro” in Excel), readily simulates chromatography. The process is readily understood by a novice. Not only does this permit simulation of isocratic and simple single step gradient elution, linear or multistep gradients are also easily simulated. The versatility of a transparent and easily understandable computational platform further enables the simulation of complex but commonly encountered chromatographic scenarios such as the effects of nonlinear isotherms, active sites, column overloading, on-column analyte degradation, etc. These are not as easily simulated by available software. Views of the separation as it develops on the column and as it is seen by an end-column detector are both available in real time. Excel 2010™ also permits a 16-level (4-bit) color gradation of numerical values in a column/row; this permits visualization of a band migrating down the column, much as Tswett may have originally observed, but in a numerical domain. All parameters of relevance (partition constants, elution conditions, etc.) are readily changed so their effects can be examined. Illustrative Excel spreadsheets are given in the Supporting Information; these are easily modified by the user or the user can write his/her own routine.     

Spectroscopic characterization of the stabilising activity of migrating HALS in a pigmented PP/EPR blend

In previous work, the FT-IR maximum of absorption of a low molecular weight HALS containing a sebacate structure was reported to shift from 1738 to 1732 cm⁻¹ during the photo-aging of polypropylene. Previous studies have shown that the reduced mobility of the molecule caused the absorption to shift towards lower frequencies while decreasing its stabilizing activity. This paper presents further results evidencing the loss of activity of the low molecular weight HALS and proposes a novel mechanistic interpretation for the observed spectral shift.     

Voltammetric assay of rifampicin and isoniazid drugs, separately and combined in bulk, pharmaceutical formulations and human serum at a carbon paste electrode

The electrochemical oxidation of both the rifampicin (RIF) and isoniazid (INH) drugs has been investigated by cyclic and square-wave voltammetry in Britton-Robinson buffers (pH 2-11) at a carbon paste electrode. The oxidation of rifampicin generated a well-defined pH-dependent quasi-reversible anodic-cathodic peak couple corresponding to a mechanism involving the transfer of two electrons/two protons, typical to that of hydroquinones, in addition to an irreversible anodic peak at a more positive potential which may be due to the oxidation of phenolic hydroxyl group. For the isoniazid, an irreversible anodic peak was observed, which may be attributed to the irreversible oxidation of the amide moiety of the drug molecule. A validated square-wave adsorptive anodic stripping voltammetric procedure was described to assay the two drugs separately or combined in pharmaceutical formulations and human serum. The recoveries of RIF in rimactane^® capsules (300 mg RIF) and INH in isocid^® tablets (200 mg INH) were found to be 98.57±0.81% and 100.57±0.74%, respectively. The proposed procedure was also successfully applied to simultaneous assay of rifampicin and isoniazid drugs combined in rimactazid tablets (150 mg INH+300 mg RIF) with recoveries of 98.79±0.97% and 99.54±0.74%, respectively, without the necessity for sample pretreatment or time-consuming extraction steps prior to the analysis. The results were favorably compared to those obtained by the reported USP method. Moreover, the proposed procedure was successfully applied to simultaneous assay of both drugs in human serum samples with limits of detection and quantitation of 5×10⁻⁸ and 1.7×10⁻⁷ M for RIF and 6.1×10⁻⁸ and 2×10⁻⁷ M for INH.     

A composite thin film optical sensor for dissolved oxygen in contaminated aqueous environments

A robust optical composite thin film dissolved oxygen sensor was fabricated by ionically trapping the dye ruthenium(II) tris(4,7-diphenyl-1,10-phenanthroline) dichloride in a blended fluoropolymer matrix consisting of Nafion^® and Aflas^®. Strong phosphorescence, which was strongly quenched by dissolved oxygen (DO), was observed when the sensor was immersed in water. The sensor was robust, optically transparent, with good mechanical properties. Fast response, of a few seconds, coupled with sensitivity of about 0.1 mg L⁻¹ (DO) over the range 0-30 mg L⁻¹ and resistance to leaching, were also exhibited by this system. The Stern-Volmer (SV) plot exhibited slight downward turning at all oxygen concentrations. A linear plot was obtained when the SV equation was modified to account for the varying sensitivity of dye molecules in the matrix to the quencher. Good long term stability was observed.     

Conversion of 1-Boc-indoles to 1-Boc-oxindoles

A facile synthesis of substituted oxindoles 2 from the corresponding indole is described. The reaction, which proceeds through the 2-(indolyl) borate intermediate, is general and applicable to several indoles.     

Simple and highly efficient synthesis of oxaziridines by tetrabutylammonium Oxone

Oxygenation of various aldimines with tetrabutylammonium monoperoxysulfate produced the corresponding E- or a mixture of E- and Z-oxaziridines with very high yields (?90%) and good to excellent selectivities (75-100%) within 20 min to 10 h in CH₃CN at room temperature (∼25 °C). The E/Z isomer ratio critically depends on the stereo-electronic nature of the substituents in the oxaziridines, solvent, and the presence of Lewis acids and bases.     

A spectrophotometric method for determination of the solubilizing activity of cellulase complexes

Summary A standardized spectrophotometric method for determination of the solubilizing activity of microbial cellulase complexes has been developed. It is based on the release of coloured compounds from microcrystalline cellulose (Avicel SF^®) dyed with Levafixbrillantrot E-2B^®.

---

Eine spektrophotometrische Methode zur Bestimmung der solubilisierenden Aktivität des Cellulase-Komplexes (Kurze Mitteilung)

Zusammenfassung Es wurde eine standardisierte spektrophotometrische Methode zur Bestimmung der Aktivität des Cellulase-Komplexes von Mikroorganismen ausgearbeitet. Die Methode beruht auf der Möglichkeit, mit Levafixbrillantrot E-2 B^® gefärbte mikrokristalline Cellulose (Avicel SF^®) als Substrat zu verwenden.

     

Microwave assisted synthesis of the fragrant compound Calone 1951

Calone 1951^®, 7-methyl-benzo[b][1,4]dioxepin-3-one, possesses a strong marine, ozone note with floral nuances and is synthesised via a three-step procedure using microwave irradiation. High yields were obtained, and reaction times reduced to a few minutes, allowing for an efficient and inexpensive synthesis of Calone 1951^®.     

Thermodynamic approach for studying both the retention and complexation mechanisms with hydroxy-propyl-beta-cyclodextrin of a phenoxy-propionic acid herbicide series

The retention and complexation mechanisms of a herbicide series were studied from a chromatographic approach using a novel column called “Nautilus^®”. The effects of water fraction and the hydroxy-propyl-β-cyclodextrin (HP-β-CD) concentration in the mobile phase were analysed in relation to the column temperature. Two retention models of phenoxy-propionic acid (PPA) derivatives were investigated. It was shown that the retention mechanism was led by free PPA herbicide for low HP-β-CD concentrations and by the PPA/HP-β-CD complex for the highest ones. In addition, an enthalpy-entropy compensation study revealed that both the solute retention and complexation mechanisms were independent of the number of chlorine atoms in the structure. Also the thermodynamic results showed that (1) the retention process depended on the water fraction (X) in the mobile phase and (2) the PPA/HP-β-CD complexation mechanism was shown to be entropically controlled.     

Synthesis, characterisation and complex forming ability towards ferric ions of oligo(ether-amide)s of Jeffamines ED and chelidamic acid

New chelating oligo(ether-amide)s (CA-PE)s containing chelidamic acid residues in the main chain were prepared by reacting chelidamic acid with Jeffamines ED^® in the presence of N,N^′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine. A mixture of products having one or two polyether sequences with chelidamate end-groups was obtained. It was found spectrophotometrically that CA-PE polymers formed a complex with Fe³⁺ at pH 3-6 having a maximum absorbance in the 472-495 nm range. Fe³⁺ ion complexes of CA-PE were water soluble, except Fe³⁺-CA-PE₆₀₀. The stoichiometric ratio between chelidamic acid residues of oligo(ether-amide)s CA-PE and Fe³⁺ ions was found to be 2 at pH 5 by the method of shift of equilibrium. A hydroxypyridine structure of the chelidamic acid residues in the complex was suggested.     

A short synthesis of morachalcone A

Advanced C-prenylated intermediates for three aromatase inhibitors, including morachalcone A, can be synthesized through a Claisen-Schmidt condensation followed by Florisil^®-catalyzed [1,3]-sigmatropic rearrangement of a prenyl phenyl ether.     

The effect of spatial confinement of Nafion in porous membranes on macroscopic properties of the membrane

Polyelectrolytes were incorporated into porous reinforcing materials to study the properties of ionomers in confined spaces and to determine the effect of the porous material on the behaviour of the membranes. Nafion^® was imbibed into porous polypropylene (Celgard^®), ultra-high-molecular weight polyethylene (Daramic^®), and polytetrafluoroethylene (PTFE) films. Through the use of reinforcing materials, it is possible to prepare membranes that are thinner, but stronger than pure ionomer membranes. Thin reinforced membranes have advantages such as lower areal resistance (as low as 0.14 Ω cm² for 57 μm CG3501 + Nafion^® compared to 0.34 Ω cm² for 89 μm cast Nafion^®) and lower dimensional changes due to swelling (as low as a 4% change in length and width for WDM + Nafion^® compared to 13% for cast Nafion^®). Using reinforcing materials results in a reduction in important membrane properties compared to bulk Nafion^®, such as proton conductivity (as low as 0.016 S cm⁻¹ for CG3401 + Nafion^® compared to 0.076 S cm⁻¹ for cast Nafion^®), effective proton mobility (as low as 3.2 × 10⁻⁴ cm² V⁻¹ s⁻¹ CG3401 + Nafion^® compared to 7.6 × 10⁻⁴ cm² V⁻¹ s⁻¹ for cast Nafion^®), and water vapour permeance (as low as 0.036 g h⁻¹ Pa⁻¹ m⁻² for WDM + Nafion^® compared to 0.056 g h⁻¹ Pa⁻¹ m⁻² for cast Nafion^®). By normalizing the membrane properties with respect to ionomer content, it was possible to examine the properties of the Nafion^® inside the pores of the membranes. The proton conductivity (as low as 0.032 S cm⁻¹ for CG3401 + Nafion^®), effective proton mobility (as low as 3.6 × 10⁻⁴ cm² V⁻¹ s⁻¹ for CG3401 + Nafion^®), and water vapour permeability (as low as 2.7 × 10⁻⁶ g h⁻¹ Pa⁻¹ m⁻¹ for PTFE MP 0.1 + Nafion^®) of the ionomer in the membrane are also diminished compared to bulk Nafion^® due to decreased connectivity of the ionomer and a restriction in macromolecular motions caused by the pore walls. A series of porous materials with increasing pore were also examined. As the pore size of the PTFE MP materials increased from 0.1 μm to 10 μm, the proton conductivity (0.022 S cm⁻¹ to 0.041 S cm⁻¹), effective proton mobility ((4.1 to 5.6) × 10⁻⁴ cm² V⁻¹ s⁻¹), and water vapour permeability ((2.4 to 4.3) × 10⁻⁶ g h⁻¹ Pa⁻¹ m⁻¹) of the reinforced membranes improved with increasing pore size and the properties of the ionomer inside the membranes approached the value of bulk Nafion^®.     

Thermal analysis of Vectran® fibers and films

Vectra^® liquid crystalline polymers (LCP's) were introduced as commercial products in the mid-1980's. The first of these (Vectra A130) was a wholly aromatic thermotropic copolyester ofp-hydroxybenzoic acid and 6-hydroxy-2-naphthoic acid. Vectra A130 is a thermotropic LCP that can be melt spun into filaments that on heat treatment are characterized by high strength and high modulus. Vectra resin can also be extruded into films. In the fiber or film form this material is commercially known as Vectran^®. Heat treatment enhances the tensile strength of Vectran fiber variants. Because of this, the elucidation of the physical transformations taking place in the internal structure of the material during heating has always been an important subject. Several thermal techniques are used to indicate clearly that what is observed as a glass transition is unlike the conventional glass transition in typical semicrystalline polymers. There is also an indication of the presence of multiple states of mesophase aggregation that collapse into a single state when taken to high enough temperatures.     

New superporous hydrogels composites based on aqueous Carbopol solution (SPHCcs): synthesis, characterization and in vitro bioadhesive force studies

In this investigation new superporous hydrogels composites based on aqueous Carbopol^® solution (SPHCcs) were prepared. SEM images indicated that the inner surface of SPHCcs contained a lot of pores connected each other and the outer surface of them was non-porous. The swelling ratio decreased with increasing the content of aqueous Carbopol^® solution, and the final swelling ratio was similar to that of the SPH although the initial swelling ratio was lower. The density measurement revealed that the porosity increased when aqueous Carbopol^® solution was incorporated. It was observed from in vitro bioadhesive force study that SPHCcs adhered to the intestinal mucosal more quickly and exhibited higher mucoadhesion as compared with SPH. It is evident that the hydrogels synthesized in this study could be a potential candidate for transmucosal drug delivery system.     

Evaluation and application of Diffusive Gradients in Thin Films (DGT) technique using Chelex-100, Metsorb™ and Diphonix binding phases in uranium mining environments

A new resin- Diphonix^® in Diffusive Gradients in Thin Films (DGT) technique for the determination of uranium was investigated and compared with previously used binding phases for uranium, Chelex^®-100 and Metsorb™. The DGT gel preparation and the elution procedure were optimized for the new resin. The U uptake on Diphonix^® resin gel was 97.4 ± 1.5% (batch method; [U] = 20 μg L⁻¹; 0.01 M NaNO₃; pH = 7.0 ± 0.2). The optimal eluent was found to be 1 M 1-hydroxyethane-1, 1-diphosphonic acid (HEDPA) with an elution efficiency of 80 ± 4.2%. Laboratory DGT study on U accumulation using a DGT samplers with Diphonix^® resin showed a very good performance across a wide range of pH (3–9) and ionic strength (0.001–0.7 M NaNO₃). Diffusion coefficients of uranium at different pH were determined using both, a diffusion cell and the DGT time-series, demonstrating the necessity of the implementation of the effective diffusion coefficients into U-DGT calculations. Diphonix^® resin gel exhibits greater U capacity than Chelex^®-100 and Metsorb™ binding phase gels (a Diphonix^® gel disc is not saturated, even with loading of 10.5 μmol U). Possible interferences with Ca²⁺ (up to 1.33 × 10⁻² M), PO₄³⁻${{\text{PO}}_4}^{3\mathrm{-}}$ (up to 1.72 × 10⁻⁴ M), SO₄²⁻${{\text{SO}}_4}^{2\mathrm{-}}$ (up to 4.44 × 10⁻³ M) and −HCO₃${{\text{HCO}}_3}^{\mathrm{-}}$ (up to 8.20 × 10⁻³ M) on U-DGT uptake ([U] = 20 μg L⁻¹) were investigated. No effect or minor effect of Ca²⁺, PO₄³⁻${{\text{PO}}_4}^{3\mathrm{-}}$, SO₄²⁻${{\text{SO}}_4}^{2\mathrm{-}}$, and −HCO₃${{\text{HCO}}_3}^{\mathrm{-}}$ on the quantitative measurement of U by Diphonix^®-DGT was observed. The results of this study demonstrated the DGT technique with Diphonix^® resin is a reliable and robust method for the measurement of labile uranium species under laboratory conditions.     

PEG modified poly(amide-b-ethylene oxide) membranes for CO2 separation

In the present work, membranes from commercially available Pebax^® MH 1657 and its blends with low molecular weight poly(ethylene glycol) PEG were prepared by using a simple binary solvent (ethanol/water). Dense film membranes show excellent compatibility with PEG system up to 50 wt.% of content. Gas transport properties have been determined for four gases (H₂, N₂, CH₄, CO₂) and the obtained permeabilities were correlated with polymer properties and morphology of the membranes. The permeability of CO₂ in Pebax^®/PEG membrane (50 wt.% of PEG) was increased two fold regarding to the pristine Pebax^®. Although CO₂/N₂ and CO₂/CH₄ selectivity remained constant, an enhancement of CO₂/H₂ selectivity (∼11) was observed. These results were attributed to the presence of EO units which increases CO₂ permeability, and to a probable increase of fractional free-volume. Furthermore, for free-volume discussion and permeability of gases, additive and Maxwell models were used.     

A facile one-pot synthesis of α-fluoro-α,β-unsaturated esters from alkoxycarbonylmethyltriphenylphosphonium bromides

A convenient one-pot synthesis of α-fluoro-α,β-unsaturated esters from ethoxy- and tert-butoxycarbonylmethyltriphenylphosphonium bromide was developed. The fluorinated phosphoranes, generated in situ from alkoxycarbonylmethyltriphenylphosphonium bromides and 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Selectfluor^®), undergo a Wittig reaction with aldehydes to yield α-fluoro-α,β-unsaturated esters with (Z)-selectivity.     

A new odorless one-pot synthesis of thioesters and selenoesters promoted by Rongalite

Rongalite^® promotes cleavage of diaryl disulfides generating chalcogenolate anions that then undergo facile acylation with anhydrides in the presence of CsF to afford thioesters (3) with good to excellent yields. By using the present protocol, 5-arylthio-5-oxopentanoic acid (4) can be facilely prepared. The important features of the methodology are broad substrate scope, simple operation, and no requirement for metal catalysts. It is noteworthy that acylations of diphenyl diselane with anhydrides are also conducted smoothly to afford selenoesters (5) in good yields under the standard conditions.