A new titanium biofunctionalized interface based on poly(pyrrole-3-acetic acid) coating: proliferation of osteoblast-like cells and future perspectives.

In recent years, many procedures based on surface modification have been suggested to improve the biocompatibility and biofunctionality of orthopedic titanium-based implants. In this contest, the development of a new titanium-based biomaterial that could be covalently modified with biologically active molecules (i.e., RGD-peptides, growth factors, etc.) able to improve osteoblasts response was investigated. The strategy followed was based on a preliminary coating of the implant material by an adherent thin polymer film to which bioactive molecules could be grafted exploiting the polymer surface chemical reactivity. In this work, we focused our attention on pyrrole-3-acetic acid (Py-3-acetic), a pyrrole with carboxylic acid substituent, whose electrosynthesis and characterization on titanium substrates were already accomplished and whose potentialities in the design of new biocompatible surfaces are well evident. As first step, the biocompatibility of the electrochemically grown PPy-3-acetic films was investigated performing in vitro tests (adhesion and proliferation) with mouse bone marrow cells. Successively, the availability and reactivity of surface carboxylic groups were tested through the grafting of an aminoacidic residue to PPy-3-acetic films.

Identification of peptides in antimicrobial fractions of cheese extracts by electrospray ionization ion trap mass spectrometry coupled to a two-dimensional liquid chromatographic separation.

Electrospray ionization ion trap mass spectrometry (ESI-ITMS) coupled to a two-dimensional liquid chromatographic separation was applied to the identification of peptides in antimicrobial fractions of the aqueous extracts of nine Italian cheese varieties. In particular, the chromatographic fractions collected during a preliminary fast protein liquid chromatography (FPLC) separation on the cheese extracts were assayed for antimicrobial activity towards Lactobacillus sakei A15. Active fractions were subsequently analyzed by reversed-phase high-performance liquid chromatography electrospray ionization sequential mass spectrometry (HPLC/ESI)-ITMSn, with n up to 3. Peptide identification was then performed starting from a conventional proteomics approach based on tandem mass spectrometric (MS/MS) analysis followed by database searching. In many cases this strategy had to be integrated by a careful correlation between spectral information and predicted peptide fragmentation, in order to reach unambiguous identifications. When even this integrated approach failed, MS3 measurements provided decisive information on the amino acid sequence of some peptides, through fragmentation of pendant groups along the peptide chain. As a result, 45 peptides, all arising from hydrolysis of milk caseins, were identified in nine antimicrobial FPLC fractions of aqueous extracts obtained from five of the nine cheese varieties considered. Many of them corresponded to peptides already known to exhibit biological activity.

Biocompatible channels for field-flow fractionation of biological samples: correlation between surface composition and operating performance.

Biocompatible methods capable of rapid purification and fractionation of analytes from complex natural matrices are increasingly in demand, particularly at the forefront of biotechnological applications. Field-flow fractionation is a separation technique suitable for nano-sized and micro-sized analytes among which bioanalytes are an important family. The objective of this preliminary study is to start a more general approach to field-flow fractionation for bio-samples by investigation of the correlation between channel surface composition and biosample adhesion. For the first time we report on the use of X-ray photoelectron spectroscopy (XPS) to study the surface properties of channels of known performance. By XPS, a polar hydrophobic environment was found on PVC material commonly used as accumulation wall in gravitational field-flow fractionation (GrFFF), which explains the low recovery obtained when GrFFF was used to fractionate a biological sample such as Staphylococcus aureus. An increase in separation performance was obtained first by conditioning the accumulation wall with bovine serum albumin and then by using the ion-beam sputtering technique to cover the GrFFF channel surface with a controlled inert film. XPS analysis was also employed to determine the composition of membranes used in hollow-fiber flow field-flow fractionation (HF FlFFF). The results obtained revealed homogeneous composition along the HF FlFFF channel both before and after its use for fractionation of an intact protein such as ferritin.

Determination of choline in milk, milk powder, and soy lecithin hydrolysates by flow injection analysis and amperometric detection with a choline oxidase based biosensor.

A fast-response and interference-free amperometric biosensor based on choline oxidase immobilized onto an electropolymerized polypyrrole film for flow injection determination of choline in milk, milk powder, and soy lecithin hydrolysates is described. The sensor displayed an Imax value of 1.9 +/- 0.2 microA and an apparent Michaelis-Menten constant, k'M, equal to 1.75 +/- 0.07 mM. Detection limits of 0.12 microM could be obtained. Because even a slight deterioration of the anti-interference membrane can adversely affect measurement accuracy, a real time monitoring of the biosensor selectivity has been achieved by a dual Pt electrode flow-through cell where the enzyme modified electrode is coupled to an enzyme-free electrode in a parallel configuration. Finally, bracketing technique (alternate injections of sample and standards) allows a two-point calibration to be performed in real-time, correcting for any drift in sensor response.

Analytical characterization of poly(pyrrole-3-carboxylic acid) films electrosynthesised on Pt, Ti and Ti/Al/V substrates.

With the aim of developing a polymeric multilayer film for application in advanced biomaterials, as a first step poly(pyrrole-3-carboxylic acid) films (abbreviated as PPy-3-carbox) were electropolymerised from pyrrole-3-carboxylic acid solutions by cyclic voltammetry and chronoamperometry on platinum, titanium and Ti90Al6V4 substrates and characterised both electrochemically (cyclic voltammetry) and spectroscopically (X-Ray Photoelectron Spectroscopy, XPS). Electrochemical experiments showed that the potential range adopted for electropolymerization affects the polymer electroactivity, by analogy with unsubstituted polypyrrole. The combination of conventional and chemical derivation-XPS provided information on PPy-3-carbox surface structure, showing no significant difference between films grown on different substrates and an increase of the COOH groups amount (one group over three pyrrole rings, as an average) with respect to unsubstituted polypyrrole (PPy), as expected. Finally, a preliminary Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) investigation was performed in order to get further information on the polymer structure and electroactivity.

Electroanalytical and spectroscopic characterization of poly(o-phenylenediamine) grown on highly oriented pyrolytic graphite.

The polymerization of ortho-phenylenediamine (o-PD) on Highly Oriented Pyrolytic Graphite (HOPG) at different pH (1,3,5,7) was investigated by electroanalytical and spectroscopic methods. Cyclic voltammetry was used both to polymerize o-PD and to study the electroactivity of the resulting poly(ortho-phenylenediamine) (PPD) film. A redox couple associated to the PPD electroactivity, deeply influenced by the pH adopted during polymerization, was recorded. A correlation between this feature and the electrochemistry shown by the oligomers of o-PD, generated in solution during the polymer synthesis, was also found. A comparison between the absorption spectra, in the visible region, of the soluble oligomers and of the PPD films was also performed, suggesting that changes in both the polymer and the oligomer structure occur and are highly related to the polymerization pH. In particular, a higher degree of conjugation is exhibited by the PPD films electrosynthesised at lower pH and this likely explains the higher conductivity as well as the higher electroactivity shown by the material obtained in these conditions.

A disposable, reagentless, third-generation glucose biosensor based on overoxidized poly(pyrrole)/tetrathiafulvalene-tetracyanoquinodimethane composite.

A disposable glucose biosensor based on glucose oxidase immobilized on tetrathiafulvalene-tetracyanoquinodimethane (ITF-TCNQ) conducting organic salt synthesized in situ onto an overoxidized poly(pyrrole) (PPy(ox).) film is described. The TIF-TCNQ crystals grow through the nonconducting polypyrrole film (ensuring electrical connection to the underlying Pt electrode) and emerge from the film forming a treelike structure. The PPy(ox) film prevents the interfering substances from reaching the electrode surface. The sensor behavior can be modeled by assuming a direct reoxidation of the enzyme at the surface of the TTF-TCNQ crystals. A heterogeneous rate constant around 10(-6) - 10(-7) cm s(-1) has been estimated. The biosensor is nearly oxygen- and interference-free and when integrated in a flow injection system displays a remarkable sensitivity (70 nA/mM) and stability.