Lucia Cenci

BEL β-trefoil. A novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms

A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

Solvent-Responsive Molecularly Imprinted Nanogels for Targeted Protein Analysis in MALDI-TOF Mass Spectrometry

Molecular imprinted poly(acrylamido)-derivative nanogels have shown their selectivity to bind the protein human serum transferrin (HTR) and also showed their capability for instantaneous solvent-induced modification upon the addition of acetonitrile. Integrated to matrix-assisted laser desorption/ionization time-of-flight mass analysis the HTR-imprinted solvent-responsive nanogels permitted the determination of HTR straight from serum and offered novel perspectives in targeted protein analysis.

Molecularly imprinted polymers coupled to matrix assisted laser desorption ionization mass spectrometry for femtomoles detection of cardiac troponin I peptides

Molecularly imprinted polymers (MIPs) were combined to MALDI‐TOF‐MS to evaluate a selective enrichment (SE) method for the determination of clinically relevant biomarkers from complex biological samples. The concept was proven with the myocardial injury marker Troponin I (cTnI). In a first part, MIP materials entailed for the recognition of cTnI epitopes (three peptides selected) were prepared and characterized in dimensions (0.7–2μm), dissociation constants (58–817 nM), kinetics of binding (5–60 min), binding capacity (ca. 1.5 µg/mg polymer), imprinting factors (3 > IF > 5) and selectivity for the peptide epitope. Then, the MIPs, incubated with cTnI peptides and spotted on the target with the DHB matrix, were assayed for the desorption of the peptides in MALDI‐TOF‐MS. The measured detection limit was ca. 300 femtomols. Finally, the MIP‐SE MALDI‐TOF‐MS was tested for its ability to enrich in the cTnI peptides from a complex sample, mimic of serum (i.e. 81 peptides of digested albumin). The MIP‐SE MALDI‐TOF‐MS successfully enriched in cTnI peptides from the complex sample proving the technique could offer a flexible platform to prepare entailed materials suitable for diagnostic purposes. Copyright

Study on molecularly imprinted nanoparticle modified microplates for pseudo-ELISA assays

Nanosized Molecularly Imprinted Polymers (nanoMIPs) are designed artificial nanoreceptors with a predetermined selectivity and specificity for a given analyte, lately proposed as a replacement to antibodies in immunoassays. The nanoMIP-plate preparation based on nanoparticle adsorption was studied with the aim to rationally identify and discuss the critical points in the nanoMIP-assay development, in an example based on the iron homeostasis biomarker hepcidin and hepcidin-specific nanoMIPs (Kd = 9nM). Plates were prepared by deposition and drying of nanoMIP (0.5-4µg/well), or by nanoMIPs co-depositions (proteins, PVA). Rehydration (> 1h) of dry nanoMIP-plates showed the reconstitution of the imprinted binding sites. NanoMIP-plate mechanical stresses (several washings; pipetting) caused nanoMIP desorption (~90%). After 10 washes the quantity of nanoMIP was 0.2µg/well, the imprinted binding sites were ~270 fmol/well, their accessibility the 92%. Co-depositions resulted in higher amount of adsorbed nanomaterial (1.2µg/well), but low accessibility of the imprinted binding sites (2-47%). Tested in a competitive sequential assay, using as competitor horseradish peroxidase conjugate to hepcidin, the nanoMIP-plate permitted to determine hepcidin in serum samples, yet with a narrow dynamic range of response (0.9-10nM). Critical points in the assay were: the instability of the nanoMIP adsorption, which lead to the progressive loss of binding sites/well, and the affinity of the nanoMIP for the analyte (Kd = 9nM), which corresponds to kinetics dissociation constants on the time-scale of the washing lengths (minutes), thus compatible with the release of the bound hepcidin during the washings. The found limits set the conditions to develop a successful nanoMIP-assay: (i) stable microplate derivatization; (ii) maximized number of imprinted binding sites/well; (iii) nanoMIP/analyte equilibrium not perturbed on the time scale of the minutes (i.e. Kd ~ pM).

Development of an in-house mixed-mode solid-phase extraction for the determination of 16 basic drugs in urine by High Performance Liquid Chromatography-Ion Trap Mass Spectrometry

The aim of the present work was to develop a novel in-house mixed-mode SPE sorbent to be used for the HPLC-Ion TrapMS determination of 16 basic drugs in urine. By using a computational modelling, a virtual monomer library was screened identifying three suitable functional monomers, methacrylic acid (MAA), itaconic acid (IA) and 2-acrylamide-2-methylpropane sulfonic acid (AMPSA), respectively. Three different sorbents were then synthetized based on these monomers, and using as cross-linker trimethylolpropane trimethacrylate (TMPTMA). The sorbent characterization analyses brought to the selection of the AMPSA based phase. Using this novel in-house sorbent, a SPE-HPLC-Ion TrapMS method for drug analysis in urine was validated proving to be selective and accurate and showing a sensitivity adequate for toxicological urine analysis. The comparison of the in-house mixed-mode SPE sorbent with two analogous commercial mixed-mode SPE phases showed that the first one was better not only in terms of process efficiency, but also in terms of quality-price rate. To the best of our knowledge, this is the first time in which an in-house SPE procedure has been applied to the toxicological analysis of a complex matrix, such as urine.