A Crosstalk- and Interferent-Free Dual Electrode Amperometric Biosensor for the Simultaneous Determination of Choline and Phosphocholine

Choline (Ch) and phosphocholine (PCh) levels in tissues are associated to tissue growth and so to carcinogenesis. Till now, only highly sophisticated and expensive techniques like those based on NMR spectroscopy or GC/LC- high resolution mass spectrometry permitted Ch and PCh analysis but very few of them were capable of a simultaneous determination of these analytes. Thus, a never reported before amperometric biosensor for PCh analysis based on choline oxidase and alkaline phosphatase co-immobilized onto a Pt electrode by co-crosslinking has been developed. Coupling the developed biosensor with a parallel sensor but specific to Ch, a crosstalk-free dual electrode biosensor was also developed, permitting the simultaneous determination of Ch and PCh in flow injection analysis. This novel sensing device performed remarkably in terms of sensitivity, linear range, and limit of detection so to exceed in most cases the more complex analytical instrumentations. Further, electrode modification by overoxidized polypyrrole permitted the development of a fouling- and interferent-free dual electrode biosensor which appeared promising for the simultaneous determination of Ch and PCh in a real sample

Electrophoretic protein deposition: a new enzyme immobilization method for the development of amperometric biosensors

Electrodeposition is an enzyme immobilization method based on the well-known electrophoretic phenomena of proteins under the influence of an electrical field. In the original method, the enzyme is mixed to a collagen dispersion at a pH value different from their isoelectric points to form macromolecular complexes which migrate to, and deposit on, an electrode surface held at an appropriate electric potential. In spite of the interest of such a method, quite similar to the enzyme entrapment in electrosynthesized polymers, the so-called "electrochemical immobilization", up to now few papers have been devoted on this subject. These studies do not deal with the understanding of both chemical and electrochemical processes involved in protein electrodeposition, which are particularly significant for the proper development of biosensors. For example, a study of a suitable electrochemical technique, able to control the protein deposition while minimizing the undesirable but collateral faradaic processes (i.e. O2 evolution), cannot be found in the relevant literature. More important, the realization of an useful biosensor, free of interference and fouling problems (which arise in real matrices analysis) has not yet been achieved with this approach. The electrodeposition method, herewith called "electrophoretic protein deposition" (EPD), has been investigated in our laboratory with the aim to develop a novel approach in amperometric biosensor realization. The influence of some chemical and electrochemical parameters on the protein deposition has been studied with several electrochemical methodologies. Galvanodynamic and potentiodynamic techniques have been compared in terms of membrane quality, thickness and spatial control of protein deposition. An electrochemical quartz crystal microbalance study permitted further insights about the growth of proteic deposit on the electrode surface. The enzyme electrodes so obtained have been further characterized to realize the feasibility of EPD procedure for the development of an useful biosensor. In this respect, the realization of amperometric biosensors using the hybrid approach has been drawn out to this novel enzyme immobilization procedure. In particular, EPD of co-crosslinked bovine serum albumin/glucose oxidase membranes coupled with electrosynthesized non-conducting films of poly-2-naphthol or poly-o-aminophenol permitted the realization of glucose biosensors with anti-interference and anti-fouling performances so interesting to assure a future employment for real sample analysis

An aceylcholinesterase/choline oxidase-based amperometric biosensor as a liquid chromatography detector for acetylcholine and choline determination in brain tissue

A liquid chromatography (LC) detector based on a fast response and sensitive bienzyme amperometric biosensor for acetylcholine (ACh) and choline (Ch) is described. The detector fabrication consisted of glutaraldehyde co-crosslinking of acetylcholinesterase and choline oxidase with bovine serum albumin on the Pt working electrode of a conventional thin-layer electrochemical flow cell. The influence of some experimental parameters (e.g. enzyme loading, thickness of the bienzyme layer, flow rate) on the detector characteristics has been studied in order to optimize the analyte response while minimizing band broadening and distortion. A mobile phase consisting of a phosphate buffer (I 0.1 M, pH 6.5) containing 5 mM sodium hexane sulphonate and 10 mM tetramethylammonium phosphate was found to give very satisfactory resolution and peak shape in ion-pair, reversed-phase LC. Linear responses were observed over at least four decades and absolute detection limits (at a signal to noise ratio of 3) were 12 and 27 fmole injected for Ch and ACh, respectively. After one month of intensive use in the LC system, the detector retained about 70% of its initial sensitivity. The potential of the described approach is demonstrated by the simultaneous determination of Ch and ACh in rat brain tissue homogenates

Assay of enzymes of clinical and biological significance by an interference free coline biosensor

Choline (Ch) is widely distributed in nature since it is an important source of methyl groups, an essential component of certain lipids and, in the nervous tissue of most organisms, a precursor of acetylcholine, which is a major neurotransmitter. Ch can be selectively determined by detection of H2O2 generated by the choline oxidase (ChO) catalyzed reaction. Several amperometric methods, based on the this reaction, have been developed in order to detect choline or species which determine choline release such as cholinesterase (ChE), acetylcholine (AChE), phospholipase D (PLD) and choline containing phospholipids. Measurement of serum ChE is important to assess liver function and monitor excessive exposure to organophosphorus insecticides[1]. It is also useful in predicting susceptibility to prolonged apnea after the administration of succinylcholine[2]. There are many methods for the measurement of cholinesterase activity including manometric, titrimetric and photometric procedures. The reference procedure for ChE assay is the Ellman colorimetric method[3] in which haemoglobin and glutathione present in erythrocytes act as interfering substances. PLD is widespread in plants as well as in some microorganisms and mammalian tissues and can have multiple effects and significance on cellular functions, such as receptor signal transduction[4] and an important role in postharvest metabolism of plant tissue. Methods published[5] for PLD activity assay have mainly involved the determination of choline by bromothymol blu or by synthetic substrates of PLD. Titrimetric or pH-stat techniques have been described as well as radioassay procedure which is the fastest and most sensitive method but it requires the use of expensive and potentially health hazardous radiolabeled phospholipids. The fundamental role covered by ChE and PLD in clinical and biological fields justifies the increasing interest in developing assay methodologies able to assure sensitivity, accuracy without requiring expensive instrumentation or long procedure time. In this context amperometric biosensors based on ChO play a surely innovative and quite promising role. The techniques reported for immobilizing ChO on the electrode surface are quite laborious. Covalent immobilization on nylon net[6], often coupled to multimembranes assembly[7] in order to preserve the electrode from interference and fouling, besides being complex and time consuming, can cause a certain slowing down of the sensor response. On the contrary a fast response time is an essential requirement for the on line monitoring of analytes in real samples. The aim of the present study was to develop a choline amperometric biosensor easier to realize moreover assuring high enzyme stability, fast response time, anti-interferent and anti-fouling properties. This goal has been reached in our laboratory immobilizing choline oxidase by co-crosslinking on a platinum electrode previously modified by an overoxidized polypyrrole film. Such an immobilization procedure, already reported in the case of a bienzymic sensor based on choline oxidase and acetylcholinesterase co-immobilization on a platinum electrode[8,9], allowed to obtain an immobilized enzyme-layer characterized by high biocomponent stability and good mechanical properties. Moreover the employment of a bilayer made of co-crosslinked choline oxidase and overoxidised polypyrrole assures notable permselectivity[10] allowing the rejection of interferents usually present in real matrices. Such a Ch biosensor, being interference free, has been employed to assay ChE and PLD in real matrices. In order to optimize the sensor response towards the enzyme to be assayed, the influence of experimental variables such as pH of buffer solution, rotation rate of the electrode and the substrate concentration has been studied. The present method, upon optimization, allowed wide linear range up to 0.600 UI/ml in the case of serum ChE (referred to acetylcholine as enzyme substrate) and to 0.33 UI/ml in the case of PLD. Moreover it revealed suitable for assay ChE in serum samples and PLD in plant crude extracts at activities value respectively down to 5x10-4 UI/ml and to 8x10-5 UI/ml. [1] E. Silk, J. King, M. Whittaker, Ann. Clin. Biochem. 16 (1979) 57 [2] A. Dietz, HM Rubinstein, T. Lubrano, Clin. Chem. 19 (1973) 1309 [3] G. L. Ellman et al. Biochem.Pharmacol. 7 (1961) 88-95 [4] J. H. Exton J. Biol. Chem. 272 (1997) 15579 [5] A. J. Morris, A. M. Frohman, J. Engebrecht, Analytical Biochemistry 252 (1997) 1 [6] E. Vrbovà, I. Kroupovà, O. Valentovà, Z. Novotnnà, J. Kas, Analytica Chimica Acta 280 (1993) 43 [7] G. Palleschi, M. Lavagnini, D. Moscone, R. Pilloton, D. D’Ottavio, M. E. Evangelisti Biosensors & Bioelectronics 5 (1990) 27 [8] A. Guerrieri, G.E. De Benedetto, F.Palmisano, P.G. Zambonin, Analyst 120 (1995) 2731 [9] A. Guerrieri, F. Palmisano, Anal. Chem. 73 (2001) 2875 [10] A. Guerrieri, G. E. De Benedetto, F. Palmisano, P. G. Zambonin, Biosensors & Bioelectronics 13 (1) (1998) 10

Stability of dynamic response of suspension bridges

The potential occurrence of internal parametric resonance phenomena has been recently indicated as a potential contributory cause of the appearance of critical dynamic states in long-span suspension bridges. At the same time, suspension bridges, in view of their flexibility, are prone to aeroelastic response, such as vortex shedding, torsional divergence and flutter. In this paper, a non-linear dynamic model of a suspension bridge is devised, with the purpose of providing a first attempt toward a unified framework for the study of aeroelastic and internal resonance instabilities. Inspired by the pioneering work of Herrmann and Hauger, the analyses have been based on a linearized formulation that is able to represent the main structural non-linear effects and the coupling given by aerodynamic forces. The results confirm that the interaction between aeroelastic effects and non-linear internal resonance leads to unstable conditions for wind speeds which can be lower than the critical threshold for standard aeroelastic predictions

Flavored tetraquark spectroscopy

The recent confirmation of the charged charmonium like resonance Z(4430) by the LHCb experiment strongly suggests the existence of QCD multi quark bound states. Some preliminary results about hypothetical flavored tetraquark mesons are reported. Such states are particularly amenable to Lattice QCD studies as their interpolating operators do not overlap with those of ordinary hidden-charm mesons

La Lettera dei 138 (2006). Bilancio di un tentativo di dialogo non (del tutto) riuscito

Contributo sottoposto a valutazioneSOMMARIO: 1. Premessa - 2. La nascita della Lettera - 3. Contenuto della Lettera e commenti - 4. La (flebile) risposta della Santa Sede - 5. Conclusioni.ABSTRACT: The paper describes the birth of the “Common Word Between Us and You” initiative (2006) – also called “Letter of the 138” by the Italian commentators – its motives, contents and limits, and how it was welcomed by christian religious leaders and christian or lay scholars. It also aims to put a light on the meaning and possibilites of the interreligious dialogue between christian churches/denominations and religious leaders of the muslim majority countries

La Responsabilita’ degli esponenti di societa’ a partecipazione pubblica e il giudice contabile

The analysis focused on the responsibility of the leaders of companies with public participation, with special attention to the profile of the jurisdiction, recently a significant change of direction by the Court of Cassation. Setting married by the Supreme Court opened the way for a new strand of case law of the courts accounts, aiming to extend the jurisdiction of public tax also to investee companies, on the assumption of their supposed subjectivity public due to the nature (public) of the resources entrusted. Is preferable that the extension of jurisdiction accounting companies can not participate regardless of recurrence, in individual cases, a relationship of service between the agent and administration damaged. From a strictly public tax, which must be observed is the inertia of the instruments of the activation reaction, since the damage is public tax only its origin in acts prejudicial to put in place at the head of society, it is consolidated only later, in Head to the property of the participant (institutional or regional) if and when that body fails, through his representative, to enable these tools to sort reaction that recognizes in its membership. So, always the interpretation of the overall system, it is possible to hypothesize a direct action by the prosecutor in civil accounting, in the form of social responsibility under art. 2392 bis civil law, time to restoration of assets and therefore to the protection of public participation

Le mani sulle donne. Islam, culture religiose, violenza sessuale dopo il capodanno di Colonia

Contributo sottoposto a valutazioneSOMMARIO. 1. La notte di san Silvestro - 2. Una finestra sulle banlieue - 3. Innovazioni giuridiche emergenziali: la modifica del codice penale tedesco - 4. Rotherham. Breve analisi della violenza sessuale interetnica e interculturale - 5. DallâEgitto al resto del mondo. Il maschilismo come fenomeno globale. Cenni sullâattuale situazione dei paesi di cultura islamica - 6. Precisazioni sulla misoginia europea (e statunitense) - 7. Le violenze sessuali nelle piscine pubbliche- 8. Ritorno alla violenza  âtribaleâ? - 9. Daoud e la âmiseria sessualeâ del mondo arabo - 10. Dinamiche di gruppo e cause socio-psicologiche della violenza sessuale. La sfida allo Stato - 11. Conclusioni.ABSTRACT: The study offers an analysis of the acts of violence taking place in Cologne during the new yearâs Eve of 2015. The goal is to shed light on the likely or at least possible causes of what happened that night through a comparison with similar events that occurred elsewhere, in order to avoid interpreting those violent acts through religious motivations only. Within a larger and multidisciplinary study framework, it should be given relevance to the cultural, social and psychological forces that direct and shape the individual and group behavior