Parallel and multiplexed bead-based assays and encoding strategies

Advances in high throughput screening (HTS), together with the rapid progress in combinatorial chemistry, genomic and proteomic sciences have dramatically stimulated the development of a variety tools to enable the drug discovery process to become more efficient. Major future challenges in HTS include obtaining high density and good quality data based on assays that are rapid, reliable, inexpensive, sensitive, simple and miniaturised. This paper reviews the development and role of bead-based assays for HTS including DNA and single nucleotide polymorphism (SNP) assays, particularly from a multiplex perspective and evaluating the recent advances in bead-based arrays. The encoding strategies that are commonly used in bead-based assays are highlighted, while the importance of magnetic beads in genomic and proteomic purifications is discussed. In conclusion, bead-based assays offer a powerful promising approach for many aspects of drug discovery.     

1,4-dioxane as a solvent in spectroscopy and photochemistry

Commercially available 1,4-dioxane, even of spectroscopic grade, contains various impurities that arise mostly from reaction of the solvent with oxygen. Aspects relevant to the spectroscopy and photochemistry of the dioxane/oxygen system are discussed. Methods of purification and storage of the solvent are presented.     

Neutron activation analysis of microgram amounts of thorium in uranium ores and process liquors by means of thorium-233

A method is described for neutron activation analysis of thorium by employing thorium-233. Decontamination steps include anion exchange in 12.5 N HCL, precipitation of fluoride, extraction with mesityl oxide and thorium oxalate precipitation. The time spent is less than 1 h; spurious activities in counting samples amount only to 2% and can easily be corrected for. The sensitivity of the method is ca. 0.02 μg of thorium in a neutron flux of 10¹²n/cm²/sec with an irradiation time of 5 min.     

Concentration effects in gel permeation chromatography : IV. Contribution of viscosity phenomena and macromolecular expansion

The changes in elution volumes with the changes in the concentration of an injected polymer solution are caused by several contributing effects. Under model conditions, it is possible to assume only the effect of a viscosity gradient in a zone moving along the column and the effect of the concentration dependence of the hydrodynamic volume of a macromolecular coil. The non-Gaussian shape of the zone and the dependence of the width of the elution curve on concentration are factors that complicate the theoretical treatment of concentration effects. The described physico-chemical model allows to evaluate the ratio of mentioned two contributions in the concentration dependence of elution volumes. According to this model, the contribution of the concentration dependence of the hydrodynamic volume does not exceed about 20% of the total change in elution volume with the varying concentration under real experimental conditions. The efficiency of the columns used and the total injected volume of the polymer solution affect this ratio only negligibly. It is obvious from a comparison with earlier results that these conclusions are not fundamentally changed even by the revised model of the concentration dependence of the swelling factor.     

On-line filtration system for determining total chromium and chromium in the soluble fraction of industrial effluents by flow injection flame atomic absorption spectrometry

Two manifolds were assessed for the purpose of determining both the total chromium content and that present as a soluble form in industrial effluents by flow injection flame atomic absorption spectrometry (FI-FAAS). To determine the chromium content in the soluble fraction the samples were used without additional treatment, a 0.45 microm filter being included in the FI system. To determine the total chromium content, the samples were acidified with nitric acid 20% (v/v) and heated for 30 s in a microwave oven (temperatures of about 70 degrees C were reached). The problem posed by the very different concentration range in which total and soluble chromium are present was overcome by using programmed flow rate methodology and by only partially emptying the sample loop. A personal computer controlled both the rotation speed of a peristaltic pump and the volume of sample injected into the system, thus obtaining the dispersion degree required. Using the manifold proposed, the chromium content in the soluble fraction can be determined in the 0.5-20 microg mL(-1) range using a 10 microg mL(-1) single standard for calibration. To determine the total chromium content, a calibration line in the 20-200 microg mL(-1) range was obtained using a single 50 microg mL(-1) chromium standard solution. The reliability of the semi-automatic devices was verified by comparing the results obtained with those found by treating the samples and using both FAAS in a conventional way and a spectrophotometric method using diphenylcarbazide at the 95% confidence level (ANOVA test). The proposed procedures showed a RSD lower than +/-3%.     

Electrodeposition of cobalt on stainless steel foils. Preparation of57Co sources for Mössbauer experiments

Conditions for the electrodeposition of⁵⁹Co and⁵⁷Co on stainless steel foils have been studied. A set of electrolytic parameters has been determined to get a reasonably good electrodeposited sample. The⁵⁷Co electrodeposited foil was then introduced in a quartz tube to be annealed in an argon flow. The final product was a 11 Ci Mössbauer source for college experiments which was succesfully tested by obtaining spectra of stainless steel and sodium nitroprusside.     

Effects of genesis and ionizing radiation on the kinetics of reduction of NiO-CeO2 mixed oxides with hydrogen

The reduction of mixed oxides NiO-CeO₂ of various composition with hydrogen was studied at 290–470° by means of thermogravimetry. Some physicochemical properties of the system were also investigated and compared with earlier studied analogous samples of different “origin”. The degree of mutual interaction of the two components, the reduction kinetics and the sensitivity to the different sorts of radiation (gamma-rays, fast neutrons and electrons), applied in different doses, are strongly affected by the “pre-history” and by the composition in both compared series of mixed oxides.     

Determination of iron at ng/ml level by solid phase spectrophotometry after preconcentration on cation exchange filters

A cationic exchanger paper is used to retain analytes in solution and, after drying, to analyze directly by measuring the UV-Vis absorbance of the paper. The method was applied to determination of iron using its known 1,10-phenanthroline complex. Using 100 ml of sample the applicable concentration range was between 1.0 and 10.0 ng/ml with a detection limit of 0.2 ng/ml and a RSD around 2%. The method was applied to determination of iron total in snow, human serum and wine.     

Nucleocyclitols. Synthesis of substituted purinyl-muco-inositol derivatives

Several carbocyclic nucleoside analogs possessing a 6-substituted purine linked to a mesylated muco-inositol were synthesized. The coupling of triethylamine-activated 6-chloropurine with 2,3-anhydro-l,5,6-tri-O-(methanesulfonyl)-epi-inositol gave a 6-chloro purinyl muco-inositol amenable to further synthetic transformations in the heterocyclic moiety by substitution of the chlorine atom by nitrogen nucleophiles such as methylamino, diethylamino, benzylamino, hydrazino, morpholino, hydroxylamino, piperidino, and glycyl groups.     

Some applications of gel-permeation chromatography in investigations of structure and the solution properties of radical-initiated poly(vinyl chloride)

The applicability of the published universal calibration parameters for gel-permeation chromatography on polystyrene standards and poly(vinyl chloride) samples with a defined structure has been compared. It was shown experimentally that of several theoretically possible molecular weight averages attributed to the elution volume at the position of the peak maximum, the root mean-square average molecular weight M _Rms = (M _wM _n)^0.5 shows the best accordance. The molecular weights obtained by gel-permeation chromatography were compared with those determined by viscometry, osmometry, and the light-scattering method. The reproducibility of gel-permeation chromatography measurements is 3%, and the average variance of the results as compared with results obtained by the above methods is about 8%. It was also found that the gel-permeation chromatography does not involve any anomalies interfering with results obtained by other methods.