Cinzia Tozzi

Molecularly imprinted solid-phase extraction sorbent for the clean-up of chlorinated phenoxyacids from aqueous samples.

A molecularly imprinted polymer (MIP) was synthesized using the herbicide 2,4,5-trichlorophenoxyacetic acid as a template, 4-vinylpyridine as an interacting monomer, ethylendimethacrylate as a cross-linker and a methanol-water mixture as a porogen. The binding properties and the selectivity of the polymer towards the template were investigated by frontal and zonal liquid chromatography. The polymer was used as a solid-phase extraction material for the clean-up of the template molecule and some related herbicides (2,4-dichlorophenoxyacetic acid, fenoprop, dichlorprop) from river water samples at a concentration level of ng/ml with quantitative recoveries comparable with those obtained with a traditional C18 reversed-phase column when analyzed by capillary electrophoresis. The results obtained show that the MIP-based approach to the solid-phase extraction is comparable with the more traditional solid-phase extraction with C18 reversed-phase columns in terms of recovery, but it is superior in terms of sample clean-up.

Binding properties of 2,4,5-trichlorophenoxyacetic acid-imprinted polymers prepared with different molar ratios between template and functional monomer.

Several molecularly-imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared with a molar ratio between the functional monomer and the template molecule in the pre-polymerisation mixture set between 1+2 and 20+1. The functional monomer used was 4-vinylpyridine (4-VP), the cross-linker was ethylene dimethacrylate, and the porogenic solvent was a mixture of methanol-water 3+1 (v/v). The polymers obtained were grinded, sieved and packed in 100 mm x3.9 mm HPLC columns. The effects of the mobile phase composition were evaluated by eluting the columns with acetonitrile-water mixtures. The results obtained indicate that column capacity, selectivity factor and the imprinting effect are controlled by ion-pair and hydrophobic interactions between the analyte and the stationary phase. In the full range of ratios considered, column capacity, selectivity factor and imprinting effect are inversely proportional to the molar ratio between the template molecule and the functional monomer.

Affinity chromatography techniques based on the immobilisation of peptides exhibiting specific binding activity.

Affinity chromatography is one of the powerful techniques in selective purification and isolation of a great number of compounds. New challenges in scientific research, such as high-throughput systems, isolation procedures that allow to obtain a single substance from a complex matrix in high degree of purity, low costs and wide availability, have led to the discovery of new tailor-made synthetic recognition systems. In this review the design, synthesis, purification and characterisation of peptides with recognition properties are discussed. Applications of peptide ligands are described and analytical tools mentioned.

Estradiol binding synthetic polypeptides

We have synthesised polypeptides that mimic the binding properties of natural receptors with high affinity and selectivity towards the steroid hormone estradiol, performing a template polymerisation in aqueous medium and without creating rigid structures.

Synthetic peptides as artificial receptors towards proteins from genetically modified organisms

The aim of this work was the preparation of peptide ligands with good affinity and selectivity towards proteins from genetically modified organisms, namely neomycin phosphotransferase II (Npt II) and the endotoxin Cry1A. A 12 x 12 combinatorial solid phase synthesis in aqueous medium was performed to prepare peptide libraries. From this library, two dipeptides with binding properties towards the chosen ligands (Pro-Lys for Npt II, K(eq) 7.59 x 10(4)M(-1); Trp-Gln for Cry 1A, K(eq) 4.35 x 10(4)M(-1)) were selected as scaffolds for the synthesis of new tetrapeptide libraries. The equilibrium constants of the newly selected tetrapeptides increased slightly respect to the dipeptides (Pro-Lys-His-Phe for Npt II, K(eq) 7.88 x 10(4)M(-1); Trp-Gln-Ala-Phe for Cry 1A, K(eq) 5.65 x 10(4)M(-1)), but selectivity towards other proteins (wheat gliadins, bovine gamma-globulins, bovine serum albumin and chicken ovalbumin) became higher. It was demonstrated that selected tetrapeptides recognised well the ligands also in presence of very complex mixtures of potentially interfering proteins, such as whole cell lysates. This approach can be considered as a general method to obtain tailor-made reagents with antibody-like binding properties towards biomacromolecules.

New immunochemical approach to low-molecular-mass analytes determination.

One of the goals of the new automated immunoassay analysers is to perform direct analysis in complex matrices thus overcoming traditional limits of homogeneous immunoassays: reduced sensitivity and ability to determine above all proteins and antibodies. The aim of this work was to demonstrate the feasibility of quantitative homogeneous immunoassay for small analytes in a complex matrix with a rapid and easy assay by exploiting an agglutination reaction. We studied the steroid hormone progesterone, and used new technology, the Copalistrade mark, which uses a special optical-sizing flow particle analysis and a semiconductor laser as a light source. We used polystyrene microbeads coated with the antigen as markers and put them in competition with the analyte in solution for the analytical antiserum. We synthesised and tested different conjugates of progesterone-bovine serum albumin, optimising all the experimental parameters. We performed a short pre-treatment of the serum and we obtained a detection limit of 0.011 ngcm(-3) and a working range between 0.026 and 43 ngcm(-3). We estimated human serum specimens and, with minor experimental revisions, the amounts of progesterone found agreed accurately with AutoDELFIAtrade mark analysis. The recoveries are good. All the experimental steps are easy, rapid and enable us to process many samples contemporarily.