Sensitivity improvement in 19F NMR-based screening experiments: theoretical considerations and experimental applications

NMR-based binding and functional screening performed with FAXS (fluorine chemical shift anisotropy and exchange for screening) and 3-FABS (three fluorine atoms for biochemical screening) represents a potential alternative approach to high-throughput screening for the identification of novel potential drug candidates. The major limitation of this method in its current status is its intrinsic low sensitivity that limits the number of tested compounds. One approach for overcoming this problem is the use of a cryogenically cooled (19)F probe that reduces the thermal noise in the receiver circuitry. Sensitivity improvement in the two screening techniques achieved with the novel cryogenic (19)F probe technology permits an increased throughput, detection of weaker binders and inhibitors (relevant in a fragment-based lead discovery program), detection of slow binders, and reduction in protein and substrate consumption. These aspects are analyzed with theoretical simulations and experimental quantitative performance evaluation. Application of 3-FABS combined with the cryogenic (19)F probe technology to rapid screening at very low enzyme concentrations and the current detection limits reached with this approach are also presented.     

Electroanalytical sensors for nonconducting media based on electrodes supported on perfluorinated ion-exchange membranes

Electroanalytical sensors, suitable for the analysis and monitoring of electroactive analytes present in gaseous phase or low-conductive liquid media, and based on electrodes in close contact with perfluorinated ion-exchange polymers are reviewed. The basic operative mechanism of these sensors, in which ion-exchange polymers act as solid polymer electrolytes (SPE's), is thoroughly discussed, while stressing the fundamental reasons why their behavior differs from that of conventional membrane electrodes. The procedures for preparing composite working electrodes by coating one side of ion-exchange membranes with stable porous films of conductive materials are described, along with the most common strategies followed to assemble this type of sensors. Useful examples of measurements in electrolyte-free media of inorganic and organic electroactive species of interest mainly for environmental analysis are given. Future prospects for the development of these sensors are also discussed.     

Separation of capsular polysaccharide K4 and defructosylated K4 by high-performance capillary electrophoresis

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. The two polyanions, K4 and defructosylated K4, are separated and readily determined within 30 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm. A linear relationship was found for the two polysaccharides over a wide range of concentrations, from approximately 30 ng (0.5 microg/microL) to 210 ng (3.5 microg/microL). The described method was used to evaluate the defructosylation process of K4 under drastic acid conditions.     

A novel general route for the preparation of enantiopure imidazolines

A novel procedure for the preparation of enantiopure 1,4-disubstituted 2-imidazolines is reported. Enantiopure beta-amino alcohols are converted into N-hydroxyethylamides, which are reacted with excess thionyl chloride, or with thionyl chloride followed by phosphorus pentachloride to yield N-chloroethylimidoyl chlorides. These intermediates are treated with amines and anilines to produce N-chloroethylamidines, which are converted into imidazolines upon workup with aqueous hydroxide. The method is simple and efficient and has been used to prepare a wide variety of enantiopure imidazolines, in a modular fashion, from readily available amino alcohols.     

Solid-phase extraction system for Pb (II) ions enrichment based on multiwall carbon nanotubes coupled on-line to flame atomic absorption spectrometry

The present paper proposes the application of multiwall carbon nanotubes (MWCNTs) as a solid sorbent for lead preconcentration using a flow system coupled to flame atomic absorption spectrometry. The method comprises the preconcentration of Pb (II) ions at a buffered solution (pH 4.7) onto 30 mg of MWCNTs previously oxidized with concentrated HNO₃. The elution step is carried out with 1.0 mol L⁻¹ HNO₃. The effect of the experimental parameters, including sample pH, sampling flow rate, buffer and eluent concentrations were investigated by means of a 2⁴ full factorial design, while for the final optimization a Doehlert design was employed. Under the best experimental conditions the preconcentration system provided detection and quantification limits of 2.6 and 8.6 μg L⁻¹, respectively. A wide linear range varying from 8.6 up to 775 μg L⁻¹ (r > 0.999) and the respective precision (relative standard deviation) of 7.7 and 1.4% for the 15 and 200 μg L⁻¹ levels were obtained. The characteristics obtained for the performance of the flow preconcentration system were a preconcentration factor of 44.2, preconcentration efficiency of 11 min⁻¹, consumptive index of 0.45 mL and sampling frequency estimated as 14 h⁻¹. Preconcentration studies of Pb (II) ions in the presence of the majority foreign ions tested did not show interference, attesting the good performance of MWCNTs. The accuracy of the method was assessed from analysis of water samples (tap, mineral, physiological serum and synthetic seawater) and common medicinal herbs submitted to the acid decomposition (garlic and Ginkgo Biloba). The satisfactory recovery values obtained without using analyte addition method confirms the feasibility of this method for Pb (II) ions determination in different type of samples.     

Development of a laccase-based flow injection electrochemical biosensor for the determination of phenolic compounds and its application for monitoring remediation of Kraft E1 paper mill effluent

This paper describes the development of a new system for amperometric determination of phenolic compounds, and its application for monitoring these compounds in paper mill effluent. The method was based on a flow system, a dialysis sampler, and a laccase-based biosensor. The performance of this system was investigated with respect to pH, ionic strength, working potential, and flow-rate dependence. The biosensor showed an excellent long-term stability allowing measurements for over than 3 months. The sensitivity of laccase-based biosensor was tested for phenol, p-chlorophenol, guaiacol and chloroguaiacol; the detector presented selective measurements of micromolar concentration of these compounds. The integration of a dialysis membrane sampling in the system protected the biosensor surface from fouling and gave independence of sample conditions that commonly influence the biosensor performance. These favorable characteristics allowed its application for direct measurements in complex media with no sample pretreatment. This ability was confirmed employing this system in a continuous analysis of phenolic compounds during the remediation of paper mill effluent by ozonization process.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations: a statistical study of complex protein maps

A statistical approach able to extract the information contained in a two-dimenisional polyacrylamide gel electrophoresis (2-D PAGE) separation is here reported. The method is based on the quantitative theory of peak overlapping, a procedure previously developed by the authors and here extended to 2-D separations. The whole map is divided into many strips in order to obtain 1-D separations on which the statistic procedure is applied: the developed algorithms, on the basis of spot experimental data (intensity and spatial coordinates) permit to estimate the intrinsic number of components and to single out the specific order present in spot positions. The procedure was validated on computer-simulated maps. Its applicability to real samples was tested on maps obtained from literature sources. The following important information on protein mixtures can be extracted: (i) the number of proteins can be accurately estimated, on the basis of the spatial coordinates and intensities of spots detected in the 2-D PAGE map; (ii) the model describing distribution of interdistance between adjacent spots can be identified in both the separation dimensions; (iii) the presence of repeated interdistances in spot positions in the maps can be easily singled out: these regularities suggest specific protein modifications.     

Hydrogen-bonded networks in ethanol proton wires: IR spectra of (EtOH)qH+-Ln clusters (L = Ar/N2, q < or = 4, n < or = 5)

Isolated and microsolvated protonated ethanol clusters, (EtOH)qH+-Ln with L = Ar and N2, are characterized by infrared photodissociation (IRPD) spectroscopy in the 3 microm range and quantum chemical calculations. For comparison, also the spectrum of the protonated methanol dimer, (MeOH)2H+, is presented. The IRPD spectra carry the signature of H-bonded (EtOH)qH+ chain structures, in which the excess proton is either strongly localized on one or (nearly) equally shared between two EtOH molecules, corresponding to Eigen-type ion cores (EtOH2+ for q = 1, 3) or Zundel-type ion cores (EtOH-H+-HOEt for q = 2, 4), respectively. In contrast to neutral (EtOH)q clusters, no cyclic (EtOH)qH+ isomers are detected in the size range investigated (q < or = 4), indicative of the substantial impact of the excess proton on the properties of the H-bonded ethanol network. The acidity of the two terminal OH groups in the (EtOH)qH+ chains decreases with the length of the chain (q). Comparison between (ROH)qH+ with R = CH3 and C2H5 shows that the acidity of the terminal O-H groups increases with the length of the aliphatic rest (R). The most stable (EtOH)qH+-Ln clusters with n < or = 2 feature intermolecular H-bonds between the inert ligands and the two available terminal OH groups of the (EtOH)qH+ chain. Asymmetric microsolvation of (EtOH)qH+ with q = 2 and 4 promotes a switch from Zundel-type to Eigen-type cores, demonstrating that the fundamental structural motif of the (EtOH)qH+ proton wire sensitively depends on the environment. The strength of the H-bonds between L and (EtOH)qH+ is shown to provide a rather sensitive probe of the acidity of the terminal OH groups.     

Determination of Phenolic Compounds Based on Co‐Immobilization of Methylene Blue and HRP on Multi‐Wall Carbon Nanotubes

This work evaluated an amperometric biosensor based on multi‐wall carbon nanotubes (MWCNT), chemically modified with methylene blue (Met) and horseradish peroxidase (HRP), for detection of phenolic compounds. The dependences of the biosensor response due to the enzyme immobilization procedure, HRP amounts, pH and working potential were investigated. The amperometric response for catechol using the proposed biosensor showed a very wide linear response range (1 to 150 μmol L^?1), good sensitivity (50 nA cm^?2 μmol^?1 L), excellent operational stability (after 300 determinations the response remained at 97%) and very good storage stability (lifetime>3 months). Based on all these characteristics, it is possible to affirm that the material is promising for phenol detection due to its good electrochemical response and enzyme stabilization. The biosensor response for various phenolic compounds was investigated.     

A complete family of isostructural cluster compounds with cubane-like M(3)S(4)M' cores (M = Mo,W; M' = Ni,Pd, Pt): comparative crystallography and electrochemistry

By reaction of the geometrically incomplete cubane-like clusters [(eta(5)-Cp')(3)Mo(3)S(4))][pts] and [(eta(5)-Cp')(3)W(3)S(4)][pts] (Cp' = methylcyclopentadienyl; pts = p-toluenesulfonate) with group 10 alkene complexes, three new heterobimetallic clusters with cubane-like cluster cores were isolated: [(eta(5)-Cp')(3)W(3)S(4)M'(PPh(3))][pts] ([5][pts], M' = Pd; [6][pts], M' = Pt); [(eta(5)-Cp')(3)Mo(3)S(4)Ni(AsPh(3))][pts] ([7][pts]). The compounds [5][pts]-[7][pts] are completing the extensive series of clusters [(eta(5)-Cp')(3)M(3)S(4)M'(EPh(3))][pts] (M = Mo, W; M' = Ni, Pd, Pt; E = P, As) which allows the consequences of replacing a single type of atom on structural and NMR and UV/vis spectroscopic as well as electrochemical properties to be determined. Single-crystal X-ray structure determinations of [5][pts]-[7][pts] revealed that [5][pts] was not isomorphous to the other members of the series [(eta(5)-Cp')(3)M(3)S(4)M'(EPh(3))][pts] due to distinctly different cell parameters, which in the molecular structure of [5](+) is reflected in a slightly different orientation of the PPh(3) ligand. Electrochemical measurements on the series showed that the Mo-based clusters were more difficult to oxidize than their W-based analogues. The Pd-containing clusters underwent two-electron oxidation processes, whereas the Ni- and Pt-containing clusters underwent two separated one-electron oxidation processes.