Solid-phase combinatorial chemistry and scintillation proximity assays

The Scintillation Proximity Assay (SPA) is a method that is frequently used to detect and quantify the strength of intermolecular interactions between a biological receptor and ligand molecule in aqueous media. This thesis describes the synthesis of scintillant-tagged-compounds for application in a novel cell-based SPA. A series of 4-functianlised-2,5-diphenyloxazole molecules were synthesised. These 4-functionalised-2,5-diphenyloxazoles were evaluated by Sense Proteomic Ltd. Accordingly, the molecules were evaluated for the ability to scintillate in the presence of ionising radiation. In addition, the molecules were incorporated into liposomal preparations which were subsequently evaluated for the ability to scintillate in the presence of ionising radiation. The optimal liposomal preparation was introduced into the membrane of HeLa cells that were used successfully in a cell-based SPA to detect and quantify the uptake of [14C]methionine. This thesis also describes the synthesis and subsequent polymerisation of novel poly(oxyethylene glycol)-based monomers to form a series of new polymer supports. These Poly(oxyethylene glycol)-polymer (POP) supports were evaluated for the ability to swell and mass-uptake in a variety of solvents, demonstrating that POP-supports exhibit enhanced solvent compatibilities over several commercial resins. The utility of POP-supports in solid-phase synthesis was also demonstrated successfully. The incorporation of (4’-vinyl)-4-benzyl-2,5-diphenyloxazole in varying mole percentage into the monomer composition resulted in the production of chemically functionalised scintillant-containing poly(oxyethylene glycol) polymer (POP-Sc) supports. These materials are compatible with both aqueous and organic solvents and scintillate efficiently in the presence of ionising radiation. The utility of POP-Sc supports in solid-phase synthesis and subsequent in-situ SPA to detect and quantify, in real-time, the kinetic progress of a solid-phase reaction was exemplified successfully.In addition, POP-Sc supports were used successfully both in solid-phase combinatorial synthesis of a peptide nucleic acid (PNA)-library and subsequent screening of this library for the ability to hybridise with DNA, which was labelled with a suitable radio-isotape. This data was used to identify the dependence of the number and position of complimentary codon pairs upon the extent of hybridisation. Finally, a further SPA was used to demonstrate the excellent compatibility of POP-Sc supports for use in the detection and quantification of enzyme assays conducted within the matrix of the POP-Sc support.

Solid-phase synthesis of an A-B loop mimetic of the C epsilon 3 domain of human IgE: macrocyclization by Sonogashira coupling

The solid-phase synthesis of a cyclic peptide containing the 21-residue epitope found in the A-B loop of the Cepsilon3 domain of human immunoglobulin E has been carried out. The key macrocyclization step to form the 65-membered ring is achieved in similar to15% yield via an "on-resin" Sonogashira coupling reaction which concomitantly installs a diphenylacetylene amino acid conformational constraint within the loop.

Microwave-assisted solid-phase peptide synthesis at 60 degrees C: alternative conditions with low enantiomerization

Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative, equimolar amount of DIC/HOBt (1:1)or TBTU/DIPEA(1:3), 25% DMSO/toluene, 60 degrees C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave-SPPS, peptides containing L-Ser, L-His, L-Cys and/or L-Met were manually synthesized traditionally, at 60 degrees C using conventional heating and at 60 degrees C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave-assisted coupling of L-Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 degrees C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave-assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS-ET and suggests alternative conditions for microwave-assisted SPPS. Copyright (C) 2009 European Peptide Society and John Wiley & Sons, Ltd.

Dynamic Solid Phase DNA Extraction and PCR Amplification in Polyester-Toner Based Microchip

A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with similar to 270 mu m, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 mu L of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/mu L., (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of lambda-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

Preconcentration and determination of Cu(II) in a fresh water sample using modified silica gel as a solid-phase extraction adsorbent

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Solid-phase peptide synthesis in highly loaded conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of sugar cane bagasse as solid phase extractor for cadmium determination by FAAS

The present paper describes the use of sugar cane bagasse as solid phase extractor for cadmium determination after complexation of the analyte with ammonium diethyldithiophosphate (ADDP) and sorption of the Cd-DDP complexes on the solid support. The concomitants were separated using a flow injection analysis (FIA) system coupled to flame atomic absorption spectrometry (FAAS) for determination. The main parameters such as ADDP concentration, acid medium, flow rate, reaction coil length, and reaction time were investigated.The results obtained with HNO3 showed good accuracy and precision. The enhancement factor was 20.5 times for a 120-second preconcentration time, and the analytical frequency was 25 determinations per hour. The calibration curve was linear over the concentration range of 1-40 mu g L-1 Cd with a LOD of 0.697 mu g L-1 Cd and a relative standard deviation of 0.96% after 12 successive measurements of 30 mu g L-1 Cd.The proposed method was evaluated for the FIA-FAAS analysis of certified reference materials (tomato leaves, spinach leaves, and bovine liver) and Cd-spiked foods (shrimp, sardine, tuna, chicken liver and bovine liver). Good recoveries (80.0-97.1%) for the Cd-spiked samples and certified reference materials were obtained. The results of bagasse-packed minicolumns were compared with Si-C,8 packed minicolumns. The F-test was applied between Si-C-18/Bagasse minicolumns, Si-C-18/certified values, and bagasse/certified values. It was found that the results were in agreement with the certified values at a 95% confidence level.

High precision and selectivity for quantitation of enrofloxacin and ciprofloxacin in five chicken tissues using solid phase extraction and ESI LC-MS/MS for application in monitoring residues

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Flow-injection spectrophotometric determination of captopril in pharmaceutical formulations using a new solid-phase reactor containing AgSCN immobilized in a polyurethane resin

A simple flow-injection analysis procedure was developed for determining captopril in pharmaceutical formulations employing a novel solid-phase reactor containing silver thiocyanate immobilized in a castor oil derivative polyurethane resin. The method was based on silver mercaptide formation between the captopril and Ag(I) in the solid-phase reactor. During such a reaction, the SCN- anion was released and reacted with Fe3+, which generated the FeSCN2+ complex that was continuously monitored at 480 nm. The analytical curve was linear in the captopril concentration range from 3.0 x 10(-4) mol L-1 to 1.1 x 10(-3) mol L-1 with a detection limit of 8.0 x 10(-5) mol L-1. Recoveries between 97.5% and 103% and a relative standard deviation of 2% for a solution containing 6.0 x 10(-4) mol L-1 captopril (n = 12) were obtained. The sample throughput was 40 h(-1) and the results obtained for captopril in pharmaceutical formulations using this procedure and those obtained using a pharmacopoeia procedure were in agreement at a 95% confidence level.

Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography for fatty acid profiling of cell wall phospholipids

The combination of solid-phase microextraction (SPME) with comprehensive two-dimensional gas chromatography is evaluated here for fatty acid (FA) profiling of the glycerophospholipid fraction from human buccal mucosal cells. A base-catalyzed derivatization reaction selective for polar lipids such as glycerophospholipid was adopted. SPME is compared to a miniaturized liquidliquid extraction procedure for the isolation of FA methyl esters produced in the derivatization step. The limits of detection and limits of quantitation were calculated for each sample preparation method. Because of its lower values of limits of detection and quantitation, SPME was adopted. The extracted analytes were separated, detected, and quantified by comprehensive two-dimensional gas chromatography with flame ionization detection (FID). The combination of SPME and comprehensive two-dimensional gas chromatography with FID, using a selective derivatization reaction in the preliminary steps, proved to be a simple and fast procedure for FA profiling, and was successfully applied to the analysis of adult human buccal mucosal cells.

Pore water (SO4, CH4, alkalinity, HS-, Ca, Sr, Mg, Ba) and solid phase (Ca, Sr, Mg, Ba) data measured in pockmark sediments of the Northern Congo Fan

This study focuses on the vertical distribution of authigenic carbonates (aragonite and high Mg-calcite) in the form of finely disseminated precipitates as well as massive carbonate concretions present in and above gas hydrate bearing sediments of the Northern Congo Fan. Analyses of Ca, Mg, Sr and Ba in pore water, bulk sediments and authigenic carbonates were carried out on gravity cores taken from three pockmark structures (Hydrate Hole, Black Hole and Worm Hole). In addition, a background core was retrieved from an area not influenced by fluid seepage. Pore water Sr/Ca and Mg/Ca ratios are used to reveal the current depths of carbonate formation as well as the mineralogy of the authigenic precipitates. The Sr/Ca and Mg/Ca ratios of bulk sediments and massive carbonate concretions were applied to infer the presence and depth distribution of authigenic aragonite and high Mg-calcite, based on the approach presented by Bayon et al. [Bayon et al. (2007). Sr/Ca and Mg/Ca ratios in Niger Delta sediments: Implications for authigenic carbonate genesis in cold seep environments. Marine Geology 241(1-4), 93-109, doi:10.1016/j.margeo.2007.03.007]. We show that the approach developed by Bayon et al. (2007) for sediments of cold seeps of the Niger Delta is also suitable to identify the mineralogy of authigenic carbonates in pockmark sediments of the Congo Deep-Sea Fan. We expand this approach by combining interstitial with solid phase Sr/Ca and Mg/Ca ratios, which demonstrate that high Mg-calcite is the predominant authigenic carbonate that currently forms at the sulfate/methane reaction zone (SMRZ). This is the first study which investigates both solid phase and pore water signatures typical for either aragonite or high Mg-calcite precipitation for the same sediment cores and thus is able to identify active and fossil carbonate precipitation events. At all investigated pockmark sites fossil horizons of the SMRZ were deduced from high Mg-calcite located above and below the current depths of the SMRZ. Additionally, aragonite enrichments typical for high seepage rates were detected close to the sediment surface at these sites. However, active precipitation of aragonite as indicated by pore water characteristics only occurs at the Black Hole site. Dissolved and solid phase Ba concentrations were used to estimate the time the SMRZ was fixed at the current depths of the diagenetic barite fronts. The combined pore water and solid phase elemental ratios (Mg/Ca, Sr/Ca) and Ba concentrations allow the reconstruction of past changes in methane seepage at the investigated pockmark sites. At the Hydrate Hole and Worm Hole sites the time of high methane seepage was estimated to have ceased at least 600 yr BP. In contrast, a more recent change from a high flux to a more dormant stage must have occurred at the Black Hole site as evidenced by active aragonite precipitation at the sediment surface and a lack of diagenetic Ba enrichments.

Porewater and solid-phase phosphorus geochemistry in surface sediments of sediment core GeoB9510-3, GeoB9518-4 and GeoB9519-6

Despite intensive research on the different domains of the marine phosphorus (P) cycle during the last decades, frequently discussed open questions still exist especially on controlling factors for the benthic behaviour of P and its general distribution in sediment-pore water systems. Steady state or the internal balance of all relevant physical and (bio)geochemical processes are amongst the key issues. In this study we present and discuss an extended data set from surface sediments recovered from three locations on the NW African continental slope. Pore water data and results from sequential sediment extractions give clear evidence to the well-known close relationship between the benthic cycles of P and iron. Accordingly, most of the dissolved phosphate must have been released by microbially catalyzed reductive dissolution of iron (oxhydr)oxides. However, rates of release and association of P and iron, respectively, are not directly represented in profiles of element specific sediment compositions. Results from steady-state based transport-reaction modelling suggest that particle mixing due to active bioturbation, or rather a physical net downward transport of P associated to iron (oxyhydr)oxides, is an essential process for the balance of the inspected benthic cycles. This study emphasizes the importance of balancing analytical data for a comprehensive understanding of all processes involved in biogeochemical cycles.

High-resolution pore-water and solid-phase analyses of sediment cores GeoB6520-3 (Hydrate Hole) and GeoB6521-2 (Worm Hole) of the northern Congo Fan

The geochemical cycling of barium was investigated in sediments of pockmarks of the northern Congo Fan, characterized by surface and subsurface gas hydrates, chemosynthetic fauna, and authigenic carbonates. Two gravity cores retrieved from the so-called Hydrate Hole and Worm Hole pockmarks were examined using high-resolution pore-water and solid-phase analyses. The results indicate that, although gas hydrates in the study area are stable with respect to pressure and temperature, they are and have been subject to dissolution due to methane-undersaturated pore waters. The process significantly driving dissolution is the anaerobic oxidation of methane (AOM) above the shallowest hydrate-bearing sediment layer. It is suggested that episodic seep events temporarily increase the upward flux of methane, and induce hydrate formation close to the sediment surface. AOM establishes at a sediment depth where the upward flux of methane from the uppermost hydrate layer counterbalances the downward flux of seawater sulfate. After seepage ceases, AOM continues to consume methane at the sulfate/methane transition (SMT) above the hydrates, thereby driving the progressive dissolution of the hydrates "from above". As a result the SMT migrates downward, leaving behind enrichments of authigenic barite and carbonates that typically precipitate at this biogeochemical reaction front. Calculation of the time needed to produce the observed solid-phase barium enrichments above the present-day depths of the SMT served to track the net downward migration of the SMT and to estimate the total time of hydrate dissolution in the recovered sediments. Methane fluxes were higher, and the SMT was located closer to the sediment surface in the past at both sites. Active seepage and hydrate formation are inferred to have occurred only a few thousands of years ago at the Hydrate Hole site. By contrast, AOM-driven hydrate dissolution as a consequence of an overall net decrease in upward methane flux seems to have persisted for a considerably longer time at the Worm Hole site, amounting to a few tens of thousands of years.

Preparation of an Immunosorbent and its use in the Solid Phase Extraction of Benzodiazepines

The use of capillary electrophoresis (CE) has been restricted to applications having high sample concentrations because of its low sensitivity caused by small injection volumes and, when ultraviolet (UV) detection is used, the short optical path length. Sensitivity in CE can be improved by using more sensitive detection systems, or by preconcentration techniques which are based on chromatographic and/or electrophoretic principles. One of the promising strategies to improve sensitivity is solid phase extraction (SPE). Solid Phase Extraction utilizes high sample volumes and a variety of complex matrixes to facilitate trace detection. To increase the specificity of the SPE a selective solid phase must be chosen. Immunosorbents, which are a combination of an antibody and a solid support, have proven to be an excellent option because of high selectivity of the antibody. This thesis is an exploratory study of the application of immunosorbent-SPE combined with CE for trace concentration of benzodiazepines. This research describes the immobilization and performance evaluation of an immunosorbent prepared by immobilizing a benzodiazepine-specific antibody on aminopropyl silica. The binding capacity of the immunosorbent, measured as µg of benzodiazepine/ gram of immunosorbent, was 39 ± 10. The long term stability of the prepared immunosorbent has been improved by capping the remaining aminopropyl groups by reaction with acetic anhydride. The capped immunosorbent retained its binding capacity after several uses.

Assessment Of Robustness On Analysis Using Headspace Solid-phase Microextraction And Comprehensive Two-dimensional Gas Chromatography Through Experimental Designs.

Plackett-Burman experimental design was applied for the robustness assessment of GC×GC-qMS (Comprehensive Two-Dimensional Gas Chromatography with Fast Quadrupolar Mass Spectrometric Detection) in quantitative and qualitative analysis of volatiles compounds from chocolate samples isolated by headspace solid-phase microextraction (HS-SPME). The influence of small changes around the nominal level of six factors deemed as important on peak areas (carrier gas flow rate, modulation period, temperature of ionic source, MS photomultiplier power, injector temperature and interface temperature) and of four factors considered as potentially influential on spectral quality (minimum and maximum limits of the scanned mass ranges, ions source temperature and photomultiplier power). The analytes selected for the study were 2,3,5-trimethylpyrazine, 2-octanone, octanal, 2-pentyl-furan, 2,3,5,6-tetramethylpyrazine, and 2-nonanone e nonanal. The factors pointed out as important on the robustness of the system were photomultiplier power for quantitative analysis and lower limit of mass scanning range for qualitative analysis.