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.

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).

Micro- versus nano-sized molecularly imprinted polymers in MALDI-TOF mass spectrometry analysis of peptides.

The integration of molecularly imprinted polymers (MIPs) with MALDI-TOF mass spectrometry (MS) combines MIP selectivity with MS sensitivity. Whether the size of the MIP material-micro versus nano-has an effect on the MS analysis was the object of the study. MIPs, targeting respectively the epitope peptide NR11 of cardiac troponin I and the peptide CK13 of human serum transferrin, were synthesized and characterized. The size-related performance of the MIP materials hyphenated with MALDI-TOF-MS analysis was studied by the incubation of the target peptide with the respective micro- or nano-MIP, followed by rinsing to remove non-specific deposition of the MIP to the MALDI target plate, co-crystallization with the organic matrix, and mass analysis. The quality of the MS analysis was assessed comparing the S/N of the mass peaks of the MIP-bound peptide to that of the same quantity of free peptide. Sweet spots and lower S/N (~ 1 order of magnitude) were observed for micro-MIP materials, while in the case of nano-MIP-bound peptide, the S/N was comparable to that of the free peptide, indicating higher compatibility of the nano-MIPs to MALDI-TOF-MS. The nano-MIP/MALDI-TOF-MS permitted the selective determination of the target peptide in real serum samples. Graphical abstract ᅟ.

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.

Screening of the binding properties of molecularly imprinted nanoparticles via capillary electrophoresis.

In response to the need for straightforward analytical methods to assess the affinity of molecularly imprinted nanoparticles (MIP NPs) for ligands, capillary electrophoresis (CE) was exploited using MIP NPs targeting the iron-regulating hormone hepcidin. In this work, MIP NPs were challenged with their template peptide, i.e., the N-terminal 5-mer of hepcidin, in comparison to unrelated ligand peptides. A CE separation method was developed ex novo achieving, after optimization of the background electrolyte (150 mM sodium phosphate pH 7.4) and of the running temperature (35 °C), the full separation of the free ligand from the complexed MIP NPs. The CE binding isotherm allowed the estimation of a micromolar dissociation constant for the 5-mer template-MIP NPs complex, in agreement with independent measurements. The CE offered the advantages of a direct injection of the MIP NPs/ligand incubation mix, without preliminary fractionation steps, requiring only minimal sample volumes and short analysis times. In conclusion CE proved to be a valid technique for characterizing the interactions of MIP NP libraries for selected target compounds.

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.

Surface plasmon resonance based on molecularly imprinted nanoparticles for the picomolar detection of the iron regulating hormone Hepcidin-25.

BACKGROUND: Molecularly imprinted polymer (MIP) technique is a powerful mean to produce tailor made synthetic recognition sites. Here precipitation polymerization was exploited to produce a library of MIP nanoparticles (NPs) targeting the N terminus of the hormone Hepcidin-25, whose serum levels correlate with iron dis-metabolisms and doping. Biotinylated MIP NPs were immobilized to NeutrAvidin™ SPR sensor chip. The response of the MIP NP sensor to Hepcidin-25 was studied. FINDINGS: Morphological analysis showed MIP NPs of 20-50 nm; MIP NP exhibited high affinity and selectivity for the target analyte: low nanomolar Kds for the interaction NP/Hepcidin-25, but none for the NP/non regulative Hepcidin-20. The MIP NP were integrated as recognition element in SPR allowing the detection of Hepcidin-25 in 3 min. Linearity was observed with the logarithm of Hepcidin-25 concentration in the range 7.2-720 pM. LOD was 5 pM. The response for Hepcidin-20 was limited. Hepcidin-25 determination in real serum samples spiked with known analyte concentrations was also attempted. CONCLUSION: The integration of MIP NP to SPR allowed the determination of Hepcidin-25 at picomolar concentrations in short times outperforming the actual state of art. Optimization is still needed for real sample measurements in view of future clinical applications.

BEL ß-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 ß-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) ß-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, Galß1-3GalNAc, was examined in detail. All the three potential binding sites present in the ß-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.